Method for producing natural sweeteners

Abstract

Provided are methods of modifying a cucurbitacine biosynthetic pathway by inhibiting cucurbitacine biosynthetic pathway enzymes in plants and plant cells, the production of plants and plant cells of the family Cucurbitacine having modified cucurbitacine and cucurbitacine derivatives, the plants and plant cells thus modified, and propagation thereof, and compositions comprising the modified plants and plant cells.

Description

[0001] Related applications

[0002] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 525,322, filed July 6, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence list declaration

[0004] The XML file titled 100786 Sequence List.XML was created on July 4, 2024, and comprises 256,208 bytes. The XML file, which was filed concurrently with this application, is incorporated herein by reference. Technical Field

[0005] In some embodiments, the present invention relates to methods for producing modified cucurbitane-type triterpenoids and compositions comprising said compounds, and uses thereof, said modified cucurbitane-type triterpenoids being used to produce a sweet glucosylated triterpenoid compound called mogroside. Background Technology

[0006] mogrosides are a compound of Cucurbitaceae (Cucurbitaceae family). Cucurbitaceaea family) plant monk fruit ( Siraitia grosvenorii These are specialized secondary metabolites derived from triterpenes found in the fruit of *Luo Han Guo*. Their biosynthesis in the fruit involves the synthesis of mogroside (a tetrahydroxycucurbitane triterpenoid compound), followed by a series of glucosylation reactions of the aglycone mogroside to yield the final sweet products mogroside IV and mogroside V. Figure 6 ).

[0007] The parent aglycone, mogroside, is derived from the successive hydroxylation of cucurbitacinol, which is the starting product of a stereospecific triterpenoid synthase, cucurbitacinol synthase. Cucurbitacinol is subsequently hydroxylated at C24 and C25 by epoxidases and epoxide hydrolases, and further hydroxylated at C11 by cytochrome P450 enzymes, ultimately yielding mogroside, as described by Itkin et al., 2016. Figure 1As described above, during fruit development, mogrosides undergo glucosylation at C3 and C24 positions to varying degrees, ranging from 1 to 6 glucosides. Glucosylated mogroside compounds are called mogrosides. With increasing glucose moiety, the sweetness of mogrosides increases, with M6 (containing 6 glucosides) being sweeter than M5, and M5 being sweeter than M4 (Kasai R. et al., Sweet cucurbitane glycosides from fruits of...). Siraitia siamensis (chi-ziluo-han-guo), a Chinese folk medicine. Agric Biol Chem 1989, 53(12):3347-3349). Purified mogroside V has been approved in Japan as a high-intensity sweetener (Jakinovich, W., Jr., Moon, C., Choi, YH, & Kinghorn, AD 1990. Evaluation of plant extracts for sweetness using the Mongolian gerbil. Journal of Natural Products, 53, 190–195), and its extract has been granted Generally Recognized As Safe (GRAS) status in the United States as a non-nutritive sweetener and flavor enhancer.

[0008] Mogroside V is known in the food industry as a natural non-sugar food sweetener, with a sweetening capacity approximately 250 times that of sucrose (Kasai R. et al., Sweet cucurbitane glycosides from fruits of...). Siraitia siamensis (chi-zi luo-han-guo), a Chinese folk medicine. Agric Biol Chem 1989, 53(12):3347-3349.). In addition, recent studies have revealed other health benefits of mogrosides (Li et al., Chemistry and pharmacology of Siraitia grosvenorii : a review.Chin J NatMed.2014 12(2):89-102.).

[0009] From the genus *Siraitia* ( SiraitiaExtracting mogrosides from the fruit of *Siraitia* can produce products of varying purities, often accompanied by an unpleasant aftertaste. Furthermore, due to the low yield and specific cultivation requirements of cultivated *Siraitia* species... Siraitia The yield of mogrosides from fruits is limited. Therefore, it would be significantly advantageous to produce sweet mogroside compounds using other plant varieties that are easy to cultivate at high yields.

[0010] The fruits of other species in the Cucurbitaceae family accumulate members of the cucurbitane-type triterpenoid family, such as cucurbitacin. However, cucurbitacin is bitter. Unglucosylated mogroside and unglucosylated cucurbitacin differ from each other in the number and position of oxidized substituents (hydroxyl or carbonyl groups) due to the dehydrogenation and acetylation of the hydroxyl groups.

[0011] Monk fruit alcohol is synthesized from squalene precursors. First, through a series of epoxidation reactions by squalene epoxidase, squalene is converted into diepoxysqualene. Diepoxysqualene (each squalene molecule with an epoxy group at the penultimate terminal position of 30 carbon atoms) is further converted into monk fruit alcohol as described above.

[0012] Cucurbitacins belong to the triterpenoid family, comprising over 20 members, varying in the number of hydroxyl, carbonyl, and acetyl groups on the cucurbitadienol skeleton. Squalene epoxides cyclize to form the cucurbitadienol skeleton under the action of cucurbitadienol synthase. The cucurbitadienol skeleton can undergo further chemical modifications, including various hydroxylation, acetylation, dehydrogenation, and reduction reactions, the combination of which determines the final cucurbitacin compound. For example, cucurbitacin E, the main cucurbitacin in wild bitter watermelon, such as... Figure 2 As shown, various oxygen atoms are present, along with acetylation at C25, dehydrogenation at C1-C2, reduction of the hydroxyl group to a carbonyl group, and possible glucosylation at C2, resulting in cucurbitacin E-glucoside. Cucurbitacin C is the main cucurbitacin in wild bitter cucumber, differing in that it has a hydroxyl group at C19 instead of C2, a hydroxyl group at C3 replacing the carbonyl group, and hydrogenation at C1-C2 replacing the double bond. The main cucurbitacin in wild bitter melon (Cucumis melo) is cucurbitacin B, which differs from cucurbitacin E only in the hydrogenation at C1-C2. Chemical modifications of members of the cucurbitacin family are as follows... Figure 1 As shown (from Ielciu, II et al., 2016). Farmacia , 64 (3).)

[0013] While mogrosides have garnered significant attention due to their value as a low-calorie sweetener, interest in cucurbitacin has primarily focused on its ability to activate or inhibit pro-apoptotic or anti-apoptotic proteins by inhibiting JAK / STAT, regulating the MAPK pathway, PARP cleavage, caspase-3 expression, and its effects on other downstream STAT3 targets (Alghasham, Int J Health Sci 2013, 7:77-89; Dai et al., Pharm Res 2023, 187, US20230000924), for use as anti-inflammatory and anticancer drugs (see, e.g., US20220000872, US20200390786, US20200282051, US20230001193, and US20170106003), for treating infections (US 20210268103), and for use in Traditional Chinese Medicine to treat liver diseases (Yang et al., Basic and Clin Pharm 2020, 127:371-379) and as a feed attractant and natural insecticide in agriculture (US20230044077, US Patent No. 11553703 and US20160309720).

[0014] Other relevant publications include Shang, Y et al., 2014 Science 346(6213); 1084-1088; Zhou, Y et al., 2016, Nature Plants 2(12), 1-8; Che, G. et al., 2019, Curr OpinionPlant Biol 47: 38-46; Kim, Y et al., 2020 Commun Biol 3(1) 444; Chen JC et al., Cucurbitacins and cucurbitane glycosides: structures and biological activities. Natural Product Reports. 2005.6;22(3):386-399.DOI: 10.1039 / b418841c.PMID: 16010347; Miro, M., 1995. Cucurbitacins and their pharmacological effects. Phytotherapy research , 9(3), pp. 159-168; and Dong, L. et al., 2021. An independent evolutionary origin for insect deterrentcucurbitacins in Iberis amara. Molecular Biology and evolution , 38 (11), pp. 4659-4673; PCT Publications WO2024 / 064695, WO2024 / 064694 and WO2021 / 202513. Summary of the Invention

[0015] According to one aspect of some embodiments of the present invention, a method is provided for producing plants or plant cells with modified cucurbitacin content, the method comprising downregulating the expression of at least one gene for a cucurbitacin biosynthesis pathway in the plant or plant cells, thereby altering cucurbitacin expression in the plant or plant cells.

[0016] According to one aspect of some embodiments of the present invention, a method is provided for producing plants or plant cells with modified cucurbitacin content, the method comprising culturing plants or plant cells expressing at least one gene modification of a cucurbitacin biosynthetic pathway.

[0017] According to one aspect of some embodiments of the present invention, the downregulation is achieved through genome editing.

[0018] According to one aspect of some embodiments of the present invention, a plant or plant cell modified to reduce the expression of at least one gene in the cucurbitacin biosynthesis pathway is provided, wherein the plant or plant cell is obtainable by the method according to the present invention.

[0019] According to one aspect of some embodiments of the present invention, the plant or plant cell of the present invention is an elite plant or plant cell.

[0020] According to one aspect of some embodiments of the present invention, the plant or plant cell is a hybrid plant or plant cell.

[0021] According to one aspect of some embodiments of the present invention, the plant or plant cell is an inbred plant or plant cell.

[0022] According to one aspect of some embodiments of the present invention, an inbred plant or plant cell is provided in which the nucleic acid sequence of at least one gene in the cucurbitacin biosynthesis pathway is altered.

[0023] According to one aspect of some embodiments of the present invention, a superior plant or plant cell is provided in which the nucleic acid sequence of at least one gene in the cucurbitacin biosynthesis pathway is altered.

[0024] According to one aspect of some embodiments of the present invention, a hybrid plant or plant cell is provided in which the nucleic acid sequence of at least one gene in the cucurbitacin biosynthesis pathway is altered.

[0025] According to one aspect of some embodiments of the present invention, the plant or plant cell contains at least one tetracyclic triterpenoid that can be glucosylated by UDP-glucuronyltransferase (UGT).

[0026] According to one aspect of some embodiments of the present invention, the UGT is a plant-based UGT.

[0027] According to one aspect of some embodiments of the present invention, the UGT is selected from S. grosvenorii UGT, which is selected from: UGT74-345-2, UGT73-348-2, UGT94-289-1, UGT73-327-2, UGT73-251-5, UGT73-251-6, UGT75-281-2, UGT85-269-4, UGT85-269-1, UGT94-289-2, and UGT94-289-3.

[0028] According to one aspect of some embodiments of the present invention, the UGT is a UGT having an amino acid sequence selected from SEQ ID NO. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 and 149.

[0029] According to one aspect of some embodiments of the present invention, the UGT is encoded by a polynucleotide having a nucleotide sequence selected from SEQ ID NO. 128, 129, 130, 131, 132, 133, 135 and 137.

[0030] According to one aspect of some embodiments of the present invention, an extract of the plant or plant cell of the present invention is provided, which contains at least one tetracyclic triterpenoid capable of being glucosylated by UGT.

[0031] According to one aspect of some embodiments of the present invention, the plant or plant cell is a species of bitter cucurbitaceae plant.

[0032] According to one aspect of some embodiments of the present invention, the plant or plant cell is a species that naturally expresses at least one gene for the cucurbitacin biosynthesis pathway.

[0033] According to one aspect of some embodiments of the present invention, the plant or plant cell is a species that has been genetically modified to express at least one gene for the cucurbitacin biosynthesis pathway.

[0034] According to one aspect of some embodiments of the invention, the plant or plant cell is an Iberisamara plant or plant cell.

[0035] According to one aspect of some embodiments of the present invention, the plant is selected from cultivated bitter cucurbitaceae plants and uncultivated cucurbitaceae plants.

[0036] According to one aspect of some embodiments of the present invention, the cultivated bitter cucurbitaceae plant is selected from common watermelon (Citrillus vulgaris) (Hawkesbury watermelon) or zucchini (Cucurbita pepo).

[0037] According to one aspect of some embodiments of the present invention, the non-cultivated bitter cucurbitaceae plants are selected from non-cultivated bitter melons, non-cultivated bitter cucumbers, and non-cultivated bitter watermelons.

[0038] According to one aspect of some embodiments of the present invention, the uncultivated bitter cucurbitaceae plant is selected from species of the genera Cucumis, Citrullus, Momordica, and Cucurbita.

[0039] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a gene selected from the genes in Table 2.

[0040] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a (2OG) and Fe(II)-dependent oxygenase gene.

[0041] According to one aspect of some embodiments of the present invention, the nucleic acid sequences of the (2OG) and Fe(II)-dependent oxygenase genes are selected from SEQ ID NO: 64, 67 and 70.

[0042] According to one aspect of some embodiments of the present invention, the downregulation of the (2OG) and Fe(II)-dependent oxygenase genes is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 66, 69 and 72.

[0043] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a cytochrome P450 gene.

[0044] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the cytochrome p450 gene is selected from SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61.

[0045] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the cytochrome p450 gene is selected from SEQ ID NO: 7, 28, 31, 34, 37 and 49.

[0046] According to one aspect of some embodiments of the present invention, the downregulation of the cytochrome p450 gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63.

[0047] According to one aspect of some embodiments of the present invention, the downregulation of the cytochrome p450 gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 9, 30, 33, 36, 39 and 51.

[0048] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is an FAD-binding berberine gene.

[0049] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the FAD-binding berberine gene is shown in SEQ ID NO: 85.

[0050] According to one aspect of some embodiments of the present invention, the downregulation of the FAD-binding berberine gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 87.

[0051] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is an NAD(P)-binding Rossmann folding gene.

[0052] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the NAD(P)-binding Rossmann fold gene is shown in SEQ ID NO: 88 or 91.

[0053] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the NAD(P)-binding Rossmann fold gene is shown in SEQ ID NO: 88.

[0054] According to one aspect of some embodiments of the present invention, the downregulation of the NAD(P)-binding Rossmann fold gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 91 or 94.

[0055] According to one aspect of some embodiments of the present invention, the downregulation of the NAD(P)-binding Rossmann fold gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 91.

[0056] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is an HXXXD-type acyl-transferase-like protein gene.

[0057] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the HXXXD type acyltransferase-like protein gene is selected from SEQ ID NO: 118, 121 and 124.

[0058] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the HXXXD type acyltransferase-like protein gene is shown in SEQ ID NO: 124.

[0059] According to one aspect of some embodiments of the present invention, the downregulation of the HXXXD type acyltransferase-like protein gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 120, 123 and 126.

[0060] According to one aspect of some embodiments of the present invention, the downregulation of the HXXXD type acyltransferase-like protein gene is achieved by targeting the nucleic acid sequence of SEQ ID NO: 126.

[0061] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a polyketide cyclase / hydratase gene.

[0062] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the polyketide cyclase / hydratase gene is shown in SEQ ID NO: 109.

[0063] According to one aspect of some embodiments of the present invention, the downregulation of the polyketide cyclase / hydratase gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 111.

[0064] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a proline dehydrogenase gene.

[0065] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the proline dehydrogenase gene is shown in SEQ ID NO: 112.

[0066] According to one aspect of some embodiments of the present invention, the downregulation of the proline dehydrogenase gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 114.

[0067] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a short-chain dehydrogenase / reductase gene.

[0068] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the short-chain dehydrogenase / reductase gene is shown in SEQ ID NO: 115.

[0069] According to one aspect of some embodiments of the present invention, the downregulation of the short-chain dehydrogenase / reductase gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 117.

[0070] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is an alcohol dehydrogenase gene.

[0071] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the alcohol dehydrogenase gene is selected from SEQ ID NO: 73, 76, 79 and 82.

[0072] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the alcohol dehydrogenase gene is selected from SEQ ID NO: 73 and 79.

[0073] According to one aspect of some embodiments of the present invention, the downregulation of the alcohol dehydrogenase gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 75, 78, 81 and 84.

[0074] According to one aspect of some embodiments of the present invention, the downregulation of the alcohol dehydrogenase gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 75 and 81.

[0075] According to one aspect of some embodiments of the present invention, the at least one cucurbitacin biosynthesis pathway gene is a peroxidase gene.

[0076] According to one aspect of some embodiments of the present invention, the nucleic acid sequence of the peroxidase gene is selected from SEQ ID NO: 94, 97, 100, 103 and 106.

[0077] According to one aspect of some embodiments of the present invention, the downregulation of the peroxidase gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 96, 99, 102, 105 and 108.

[0078] According to one aspect of some embodiments of the present invention, a method for generating glucosylated tetracyclic triterpenes is provided, comprising glucosylating at least one tetracyclic triterpenes capable of being glucosylated by contacting at least one tetracyclic triterpenes with UGT, thereby generating glucosylated tetracyclic triterpenes.

[0079] According to one aspect of some embodiments of the present invention, the glucosylated tetracyclic triterpenoid is a non-bitter glucosylated tetracyclic triterpenoid.

[0080] According to one aspect of some embodiments of the present invention, the glucosylated tetracyclic triterpenoid is mogroside.

[0081] According to one aspect of some embodiments of the present invention, the glucosylation is achieved in cells.

[0082] According to one aspect of some embodiments of the present invention, the cell is a plant cell.

[0083] According to one aspect of some embodiments of the present invention, the cell is not a plant cell.

[0084] According to one aspect of some embodiments of the present invention, the glucosylation is performed in a cell-free system.

[0085] According to one aspect of some embodiments of the present invention, the UGT is a plant-based UGT.

[0086] According to one aspect of some embodiments of the present invention, the UGT is selected from S. grosvenorii UGT, and the S. grosvenorii UGT is selected from: UGT74-345-2, UGT73-348-2, UGT94-289-1, UGT73-327-2, UGT73-251-5, UGT73-251-6, UGT75-281-2, UGT85-269-4, UGT85-269-1, UGT94-289-2, and UGT94-289-3.

[0087] According to one aspect of some embodiments of the present invention, the UGT is a UGT having an amino acid sequence selected from SEQ ID NO. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 and 149.

[0088] According to one aspect of some embodiments of the present invention, the UGT is encoded by a polynucleotide having a nucleotide sequence selected from SEQ ID NO. 128, 129, 130, 131, 132, 133, 135 and 137.

[0089] According to one aspect of some embodiments of the present invention, the UGT is a recombinant UGT.

[0090] According to one aspect of some embodiments of the present invention, a composition is provided comprising a plant or plant cell having modified cucurbitacin biosynthesis pathway gene expression, said plant or plant cell containing a tetracyclic triterpenoid capable of being glucosylated by UGT.

[0091] According to one aspect of some embodiments, the composition is rich in tetracyclic triterpenoid glucosides.

[0092] Unless otherwise specified, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to impose any necessary limitations. Attached Figure Description

[0093] The following description, with reference to the accompanying drawings, illustrates some embodiments of the invention by way of example only. A detailed description will now be provided in conjunction with the drawings. It should be emphasized that the details shown are merely illustrative and intended to provide an illustrative discussion of embodiments of the invention. In this regard, the description in conjunction with the accompanying drawings will make it clear to those skilled in the art how to practice embodiments of the invention.

[0094] In the attached diagram: Figure 1 The basic structure of cucurbitacin is shown, as well as specific modifications of individual cucurbitacin variants; Figure 2 The structure of an exemplary cucurbitacin (cucurbitacin E) is shown, and the target sites (circles) for modification by downregulating genes in the cucurbitacin biosynthesis pathway are shown. Figure 3 The structural diagram of the aglycone of mogroside, also known as mogroside, is shown. Figure 4 The structural formulas of cucurbitacin E glycoside (1) and cucurbitacin I glycoside (2) with glucosylation at C2 are shown; Figure 5 This demonstrates the different biosynthetic pathways by which cucurbitacin and mogroside are synthesized from cucurbitadienol (a common precursor of mogroside and cucurbitacin). Figure 6 The detailed biosynthetic pathway of mogrosides is shown, from squalene precursors to the aglycone mogroside, and then mogroside glucosylation to generate sweet mogrosides IV and V (from Itkin et al., 2016). Figures 7A-7D This is a chromatogram showing the silencing results of the C1G06g001610 gene expression in transgenic hairy roots, in which a new peak (arrow) of non-acetylated cucurbitacin I (7A) appears. Figure 7B and 7C This is a mass spectrum showing the presumed new peak of non-acetylated C30H42O7. Figure 7D It shows the natural C-25 acetylated cucurbitacin I and a novel deacetylated form of cucurbitacin; Figure 8 This is a schematic diagram of a plasmid vector used to silence candidate genes, including the C1G06g001600 gene encoding cucurbita dienol synthase (the first key step in the synthesis of cucurbitane-type triterpenoids) and the C1G06g001610 gene encoding acetyltransferase in hairy roots. Figures 9A-9B A mutant bitter watermelon hairy root-tissue culture model is shown (9A), stained with red fluorescent protein (RFP), a marker of transgenic cells / tissues (9B); Figure 10 This is a graph showing the cucurbitacin content in the hairy root cultures of the transformed bitter watermelon mutant, displaying the results for unsilenced (WT) and silenced cucurbitacin synthase gene (CLCG06g001600). CLCG06g001600 encodes the first key step in the synthesis of triterpenoid cucurbitanes. Figure 11 The images show the fruits of bitter watermelon (top) and non-bitter watermelon (bottom), as well as the cucurbitacin content (µg / g fresh weight) and the expression level of bHLH transcription factor CICG01G003370 (in FPKM) in the fruits during the flowering period. Figures 12A-12E This is a chromatogram showing the silencing results of the acyltransferase C1G06g001610 gene expression in transgenic hairy roots, with a new peak (12A, arrow) appearing in the co-elution with unacetylated cucurbitacin I (C30H42O7). Figure 12B-12D It is cucurbitacin E ( Figure 12B Cucurbitacin I Figure 12C ) and the cucurbitacin peak that appears during co-elution with cucurbitacin I, which is a modified cucurbitacin peak with acetyltransferase silence. Figure 12D The mass spectrum of ). Figure 12E The chromatogram shows the natural C-25 acetylated cucurbitacin E and the deacetylated form of cucurbitacin I. (Bottom chromatogram) Figure 12AThe image shows peaks of a purified mixture of cucurbitacin B, D, E, and I. Figures 13A-13H are chromatograms showing the silencing results of the expression of three dehydrogenase genes ClCG01G018250 (13B), ClCG03G002490 (13C), and ClCG09G009760 (13D) in transgenic hairy roots. The chromatograms show an intensified peak (13B-13D, arrows) of the modified, undehydrocucurbitacin (C32H46O8) when co-eluted with cucurbitacin B. Figure 13E-13H The mass spectrum of cucurbitacin E (13E) and the spectrum of modified cucurbitacin peaks that appear when dehydrogenase is silenced in hairy roots show the enhanced peak of undehydrogenated cucurbitacin (C32H46O8) when co-eluted with cucurbitacin B. Figures 14A-14L are chromatograms showing the silencing results of the expression of five cytochrome P450 genes ClCG06G001590 (14B), ClCG10G012530 (14C), ClCG06G001580 (14D), ClCG06G001620 (14E) and ClCG01G014540 (14F) in transgenic hairy roots, in which modified cucurbitacins (C30H46O5, C30H46O4, C30H44O3 and C30H46O2) appear. Figure 14G-14L The mass spectra of peaks 1, 2, 3, 4, 5 and 6 separated by chromatography are identified as modified cucurbitacin C30H46O4 (peak 1, 14G and peak 3, 14H), cucurbitacin C30H46O5 (peak 2, 14I), cucurbitacin C30H44O3 (peak 4, 14J and peak 5, 14K) and cucurbitacin C30H46O2 (peak 6, 14L). Figures 15A-15F are chromatograms showing the results of overexpression of cytochrome P450 ClCG06G001570 in yeast expressing the precursor cucurbitadienol (Cuc, C30H50O). Chromatograms 15A and 15B show that expression of ClCG06G001570 (15A) leads to the production of hydroxylated cucurbitadienol products (peaks 1, 2, and 3 overlap). Figure 15C-15E Mass spectra of cucurbitadienol (15C) and its hydroxylated products C30H48O3 (peak 1, 15D), C30H50O2 (peak 2, 15E), and C30H48O2 (peak 3, 15F) are shown. Detailed Implementation

[0095] In some embodiments, the present invention relates to a method for modifying the cucurbitacin biosynthesis pathway in a plant species by downregulating the expression of cucurbitacin biosynthesis genes, to modified plant species having tetracyclic triterpenes that are glucosylated by UDP-glucuronyltransferase (UGT) to produce sweet rather than bitter cucurbitane secondary metabolites, and to a method for producing glucosylated tetracyclic triterpenes by glucosylating tetracyclic triterpenes from the modified plant species or extracts thereof.

[0096] Before explaining at least one embodiment of the present invention in detail, it should be understood that the application of the present invention is not necessarily limited to the details set forth in the following specification or the examples illustrated therein. The present invention may have other embodiments and may be implemented or carried out in various ways.

[0097] Bitter tetracyclic triterpenoid cucurbitacins and sweet tetracyclic triterpenoid mogrosides share a common precursor, cucurbitadienol, a stereospecific product of cyclization of squalene epoxide molecules. Cucurbitadienol is converted into bitter cucurbitacins in bitter melon, bitter cucumber, bitter squash, and other cucurbitaceous plants, as well as cucurbitacins in non-cucurbitaceous plants (such as *Iberis amara*), through multiple hydroxylation, acetylation, and dehydrogenation reactions. These cucurbitacins may help them defend against herbivorous wildlife. Another set of enzymatic reactions converts cucurbitadienol to mogroside, which, although also bitter, can be glucosylated by UGT to sweet mogroside IV-V, a currently popular sugar substitute. However, from *Monk Fruit* (… Siraitia grosvenorii Extracting stable mogrosides from ( ) is inefficient and costly.

[0098] Although the biosynthetic pathway for the synthesis of mogrosides and mogrosides from cucurbitacin is well understood, little is known about the enzymatic reactions responsible for the production of cucurbitacin.

[0099] While conceiving and simplifying the implementation schemes of this invention, the inventors have identified target genes for modifying the cucurbitacin biosynthesis pathway in plants. By inhibiting the expression of candidate enzymes in bitter plants (such as bitter watermelon), analyzing the emergence of novel tetracyclic triterpenes, and correspondingly reducing the end product cucurbitacin, the inventors have begun to reveal the compositional enzymatic reactions of cucurbitacin biosynthesis (Examples 1 and 2), and redirected this pathway to new substrates for final glucosylation (Examples 3-6).

[0100] The inventors have identified candidate genes for the cucurbitacin biosynthetic pathway that can be modified, and have demonstrated that targeted silencing of some of these candidate genes results in actual modifications to the profiles of cucurbitacins and cucurbitacin derivatives in bitter species. Silencing of acetyltransferases in bitter species produces nonacetylated derivatives of cucurbitacin E (see Example 4), silencing of dehydrogenases in bitter species produces cucurbitacin derivatives with an extra proton (see Example 5), and silencing of the cytochrome P450 gene in bitter species produces oxidatively reduced modified cucurbitacin derivatives (see Example 6).

[0101] It should be understood that the teachings of this invention are intended to produce cucurbitacin-producing plants with modified cucurbitacins and cucurbitacin derivatives, including cucurbitacins and cucurbitacin derivatives with reduced bitterness due to modification of the cucurbitacin biosynthetic pathway.

[0102] Simultaneously, it reduces the bitterness of modified bitter cucurbitaceous plants and other cucurbitacin-producing plants, and identifies tetracyclic triterpenoids that can be glucosylated into sweet molecules rather than bitter molecules, which could provide new sources of useful non-bitter plants and plant products, as well as methods for synthesizing tetracyclic triterpenoid-based molecules with useful industrial and pharmacological properties that were previously difficult to obtain, including glucosylated tetracyclic triterpenoids.

[0103] Therefore, according to one aspect of the present invention, a method is provided for producing a plant or plant cell with a modified cucurbitacin content, the method comprising downregulating the expression of at least one gene for a cucurbitacin biosynthesis pathway in the plant or plant cell, thereby modifying cucurbitacin expression in the plant or plant cell.

[0104] definition

[0105] To make the invention easier to understand, some terms are first defined.

[0106] As used herein, the term "plant" refers to the whole plant, its organs (i.e., leaves, stems, roots, flowers, etc.), seeds, plant cells, and their progeny. The term "plant cell" includes, but is not limited to, cells within seeds, suspension cultures, embryos, meristematic regions, callus, leaves, buds, gametophytes, sporophytes, pollen, and microspores. Depending on the specific implementation plan, a plant is a plant strain.

[0107] According to the specific implementation plan, the plant or plant cell is a superior plant or plant cell.

[0108] According to the specific implementation plan, the plant or plant cell is a hybrid plant or plant cell.

[0109] Depending on the specific implementation plan, the plant or plant cell is an inbred plant or plant cell. As used herein, the term "inbred" refers to a relatively pure line obtained through controlled self-pollination or backcrossing to the same recurrent parent for at least five consecutive generations, and selection or equivalent selection of specific traits.

[0110] The phrase "plant part" refers to a portion of a plant, including single-celled and cellular tissues, such as intact plant cells, cell masses, and tissue cultures from which plants can regenerate. Examples of plant parts include, but are not limited to, single-celled and cellular tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, branches, and seeds; as well as scions, rhizomes, protoplasts, callus, etc.

[0111] The present invention also envisions modifying hairy root cultures of Cucurbitaceae plants or other cucurbitacin-producing plants for screening and producing components of the cucurbitacin biosynthetic pathway.

[0112] Any technique that can induce the formation of hairy roots in plants can be used in this invention.

[0113] Hairy roots are a type of plant formed by the Gram-negative soil bacterium Agrobacterium rhizogenes (Agrobacterium tumefaciens). Agrobacterium rhizogenes This causes proliferating roots to appear at the wound site of the plant after infection. The hairy root phenotype is characterized by rapid hormone-independent growth, lateral branching, genetic stability, and lack of geotropism.

[0114] It should be noted that, due to the presence of *Agrobacterium* and *Rhizobium* families... Rhizobiaceae Taxonomic changes of Agrobacterium rhizogenes (Agrobacterium rhizogenes) Agrobacterium rhizogenes ) has been renamed Rhizobium brevicornum ( Rhizobium rhizogenes Rhizobium rhizobia ( ) Rhizobium rhizogenes It can also be called Agrobacterium rhizogenes ( Agrobacterium rhizogenes ).

[0115] Hairy roots were initially identified as a plant disease caused by *Rhizobium rhizogenes*, a bacterium that can be isolated from soil. This Gram-negative bacterium transfers DNA from its root-inducing (Ri) plasmid into the genome of infected plant cells, leading to root formation. Specifically, the *rol* gene (see White et al., 1983), containing *rolA*, *rolB*, and *rolC* genes, is present in the T-DNA of the *Rhizobium rhizogenes* Ri plasmid, and the expression of these genes induces hairy root formation.

[0116] In a specific implementation, hairy root formation is induced by transforming plants with a bacterial strain containing the rol gene, wherein the bacterial strain is capable of infecting the plant.

[0117] As used herein, the term "rol gene" has its general meaning in this field. It refers to a group of bacterial genes capable of inducing the formation of hairy roots (Schmiilling et al., 1988; Bulgakov et al., 2008). Typically, the rol gene is carried by a plasmid such as the pRi plasmid.

[0118] In specific implementation schemes, bacterial strains naturally contain the rol gene in their genome, or are modified by introducing a heterologous rol gene.

[0119] In a specific implementation plan, the bacterial strain belongs to the genus Rhizobium.

[0120] In a preferred embodiment, a strain of Rhizobium rhizogenes is used.

[0121] Several strains of *Rhizobium rhizogenes* can be used to carry out this invention. Suitable strains include, but are not limited to, *Rhizobium rhizogenes* strain TR7, also known as ATCC 25818, and strains LBA9402, A4T, A4, LBA1334, ATCC 11325, ATCC 15834, LMG 155, HRI, TR105, ATCC 39207, R1000, LBA 9422, strain 1072, BL311, R1600, R1601, C58C1, A4RS, MSU440, ARqual, 8194, TR101, 2659, LBA8490, NIAES1724, C8 (MAFF03-10268), and DC-AR2.

[0122] In a specific implementation plan, the rhizobium ( Rhizobium rhizogenes The strain is either strain K599 or GV3101.

[0123] In the specific implementation plan, Rhizobium rhizobia ( Rhizobium rhizogenes The strain is strain K599.

[0124] In another implementation scheme, Agrobacterium tumefaciens (Agrobacterium tumefaciens) is used. Agrobacterium tumefaciens ) strain. In the specific implementation plan, the Agrobacterium tumefaciens strain used is... Agrobacterium tumefaciensThe strain has been modified to introduce the rol gene into its genome. In a specific implementation, Agrobacterium tumefaciens (Agrobacterium tumefaciens) is modified by transformation with a pRi plasmid containing the rol gene. Agrobacterium tumefaciens ).

[0125] In another embodiment, the Agrobacterium tumefaciens used is... Agrobacterium tumefaciens The strain does not contain the rol gene. In this embodiment, Agrobacterium tumefaciens (…) is used. Agrobacterium tumefaciens The transformation of plants induces callus formation. After the addition of one or more hormonal substances, the callus subsequently differentiates in hairy roots. In a specific embodiment, the hormonal substance is a hormone from the auxin family, such as 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA).

[0126] Agrobacterium tumefaciens ( Agrobacterium tumefaciens Multiple strains of *Agrobacterium tumefaciens* can be used to carry out this invention. Suitable strains include, but are not limited to, *Agrobacterium tumefaciens*. A. tumefaciens ) C58, C58C1, LBA4404, GV2260, GV3100, A136, GV3101, GV3850, EHA101, EHA105, AGL-1.

[0127] Through bacterial strains such as Rhizobium rhizobia ( Rhizobium rhizogenes ) and / or Agrobacterium tumefaciens ( Agrobacterium tumefaciens The transformation of plants is a technique known in the art. Those skilled in the art are familiar with the various techniques commonly used to perform the transformation steps. Depending on the plant species to be transformed, different parts of the plant may be used for infection. Such plant parts may include, for example, but not limited to, seeds, plant stems, leaves, petioles, cotyledonary nodes, hypocotyls, or other plant parts or cells.

[0128] Typically, by using rhizobium ( Rhizobium rhizogenes ) and / or Agrobacterium tumefaciens ( Agrobacterium tumefaciens Inoculum is applied to previously injured plant tissue to promote the growth of root nodules (rhizobium). Rhizobium rhizogenes ) and / or Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) infection.

[0129] In the specific implementation plan, the hairy root culture is a watermelon hairy root culture, which uses decoated watermelons (…). Citrullus lanatus Seed establishment. Sterilize the seeds (e.g., by hypochlorous acid), rinse, dry, and incubate for 10 days on plates containing ½ Murashige and Skog medium (MS) agar supplemented with 1.5% sucrose.

[0130] Rhizobium rhizobia transformed by electroporation with relevant constructs ( Rhizhobium rhizogenesStrain K599 was cultured, transformants were selected, and grown in liquid medium. Watermelon cotyledons were then transformed by puncturing them with a sterile syringe needle dipped in a suspension of Rhizobium rhizobium. The inoculated cotyledons were cultured on agar, and the transformed hairy roots that appeared after three weeks of tissue culture were excised from the cotyledons and passaged every two weeks.

[0131] According to specific implementation schemes, plants, plant parts, or plant cells may have modified cucurbitacin content. The term "modified cucurbitacin content" as used herein refers to a change in the types and amounts of cucurbitacins in a plant, plant part, or plant cell relative to the cucurbitacin profile of the same plant, plant part, or plant cell, without downregulating the genes involved in the cucurbitacin biosynthesis pathway of this invention. It should be understood that "modified cucurbitacin content" may also include the term "modified cucurbitacin expression," because cucurbitacins are catalytically active products of enzymes in the cucurbitacin biosynthesis pathway.

[0132] According to other alternative implementation schemes, at least one gene in the cucurbitacin biosynthesis pathway of plants, plant parts or plant cells undergoes nucleic acid alteration.

[0133] As used herein, the phrase "nucleic acid alteration" refers to any mutation in the DNA sequence of a plant (e.g., plant cells) that can lead to a downregulation of the expression level and / or activity of isolated polypeptides (e.g., polypeptides encoded by polynucleotides isolated by the present invention) according to some embodiments of the invention. Non-limiting examples of such nucleic acid alterations include: missense mutations, i.e., mutations in which one amino acid residue in a protein is altered by another amino acid residue, thereby eliminating the protein's enzymatic activity; nonsense mutations, i.e., mutations that introduce a stop codon into a protein, such as an early stop codon resulting in a shorter protein lacking enzymatic activity; frameshift mutations, i.e., mutations typically involving the deletion or insertion of nucleic acids that alter the protein reading frame and can lead to early termination (e.g., truncated proteins lacking enzymatic activity) by introducing a stop codon into the reading frame, or mutations in longer amino acid sequences (e.g., readthrough proteins) that affect the secondary or tertiary structure of the protein and result in nonfunctional proteins lacking the enzymatic activity of "wild-type" or non-mutated polypeptides; and readthrough mutations resulting from frameshift mutations or modified stop codon mutations (i.e., when the stop codon is mutated to an amino acid codon), affecting the enzyme's enzymatic activity. Loss of activity; promoter mutation, i.e., a mutation in the promoter sequence, usually located at the 5' of the transcription start site, which leads to downregulation of a specific gene product; regulatory mutation, i.e., a mutation in the upstream or downstream or internal region of a gene that affects the expression of the gene product; deletion mutation, i.e., a mutation in the gene sequence that removes the coding nucleic acid, which may lead to frameshift mutations or reading frame-compliant mutations (the deletion of one or more amino acid codons within the coding sequence); insertion mutation, i.e., a mutation that inserts coding or non-coding nucleic acid into the gene sequence, which may lead to frameshift mutations or reading frame-compliant insertion of one or more amino acid codons; inversion, i.e., a mutation that causes an inversion of the coding or non-coding sequence; splicing mutation, i.e., a mutation that causes aberrant or poor splicing; and duplication mutation, i.e., a mutation that causes duplication of the coding or non-coding sequence, which may be reading frame-compliant or may cause a frameshift.

[0134] According to one specific implementation plan, the plant part is a seed. According to another specific implementation plan, the plant part is a hybrid seed.

[0135] As used herein, the phrase "progeny plant" refers to any plant produced by asexual or sexual reproduction of one or more parent plants or their offspring. For example, a progeny plant can be obtained by cloning or self-pollinating a parent plant or by hybridizing two parent plants, and includes self-pollination as well as F1 or F2 or further generations. F1 is the first generation of offspring produced for the first time by at least one parent as a trait donor, while the offspring of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples produced by self-pollination, intercrossing, backcrossing, or other hybridization of F1s, F2s. Thus, F1 can be (in some embodiments) a hybrid produced by crossing two purebred parents (i.e., each purebred parent is homozygous for the trait of interest or its alleles, for example, in this case, a male-sterile and restorer line with long stigmas as described herein), while F2 can be (in some embodiments) an offspring produced by self-pollination of an F1 hybrid.

[0136] As used herein, “cultivated” refers to plant species that have undergone a domestication process (e.g., cucurbitacin-producing species, gourd species, non-cucurbitacin-producing species) and therefore possess agriculturally desirable characteristics, such as higher yields, resistance to biotic / abiotic stresses, and reproductive capacity. As used herein, the term “cultivated” refers to a plant whose origin or selection is primarily due to intentional human activity, while “non-cultivated” refers to a plant grown without intentional human intervention. Some non-limiting examples of human activities that contribute to plant cultivation include tillage and land preparation, planting and cultivating seeds or seedlings, selecting and breeding plants, pollinating and grafting plants. In specific embodiments, producing plants or plant cells with modified cucurbitacin content includes culturing plants or plant cells with modified cucurbitacin content. As used herein, the term “growing” refers to the cultivation of plants or plant cells, including but not limited to planting, cultivating, selecting, and breeding plants or plant cells.

[0137] As used in this article, the term "cucurbit" refers to any member of the Cucurbitaceae family, including cultivated genera and species such as gourd (e.g., snake gourd, wax gourd, yellow gourd), cucumber (e.g., prickly cucumber, musk cucumber, cucumber, small cucumber), purslane, medicinal watermelon, watermelon, cantaloupe, dense zucchini, squash, wild squash (calabazilla) and loofah.

[0138] According to the specific implementation plan, the plant belongs to the Cucurbitaceae family. Exemplary species are provided below.

[0139] Subfamily Zanonioideae (Small-striped pollen grains) Zanonieae (family Conical) o Fevilleinae: Fevillea genus ( Fevillea ) o Subtribe of Zanoniinae: Genus *Calamus* ( Alsomitra ), Conical fruit genus ( Zanonia ), Siolmatra Gerald's genus ( Gerrardanthus ), Trichoderma genus ( Zygosicyos ), genus *Calamus* Xerosicyos ), New Winged Cucurbita ( Neoalsomitra ), o Gynostemma pentaphyllum subtribe: Gynostemma pentaphyllum genus ( Hemsleya ), Gynostemma pentaphyllum ( Gomphogyne ), Conical fruit genus ( Gynostemma ) o Subtribe of Actinostemmatinae: Genus *Fritillaria* ( Bolbostemma ), Box Grass ( Actinostemma ) o Sicydiinae subfamily: Sicydium, Chalema genus *Cypripedium* ( Pteropepon ), Pseudosicydium, Cyclantheropsis Subfamily Cucurbitoideae (styles merged into a single row) Melothrieae Dendrosicyinae: Genus *Gentiana* Kedrostis ), genus *Gnaphalium* ( Dendrocyos ), Coral melon ( Corallocarpus ), Bags ( Iberville ) 、Tumamoca、 Halosicyos, Ceratosanthes, Doyerea, Trochomeriopsis, Seyrigia, Dieterlea, genus Cucurbita ( Cucurbita ) 、Apodanthera、Guraniopsis、Melothrianthus、Wilbrandia o Guraninae: Helmontia, Psiguria, genus *Grania* Gurania ) o Cucumerinae: Melancium, Cucumeropsis, Posadaea, genus Cucurbita ( Melothria ) 、Muellargia、 genus Equisetum ( Zehneria Cucumber ( ) Cucumber ) 、 (Including: genus *Cephalotaxus*) Mukia ), genus *Cypripedium* ( Dicaelospermum ), Cucumber genus ( Cucumber ), Cucumber genus ( Oreosyce ) and the genus *Ipomoea* ( Myrmecosicyos )).

[0140] o Trochomeriinae subtribe: Trochomeriinae genus ( Solemn ), Trochomeria, genus *Cypripedium* Dactyliandra ), Ctenolepsis Schizopeponeae tribe: Schizopepone genus (Schizopepon) Joliffieae o Thradianthinae: *Calamus* genus ( Indofevillea ), Monk Fruit ( Syriac ), genus *Ceratophyllum* ( Thladiantha ), bitter melon genus ( Momordica charantia ) o Telfairiinae subfamily: Telfaria Trichosantheae o Hodgsoniinae: Hodgsoniinae (Genus: Hodgsoniinae) Hodgson) o Ampelosicyinae subfamily: Ampelosicyos Peponium o Trichosanthinae: Genus Trichosanthinae ( Gymnopetalum ), Trichosanthes genus ( Trichosanthes ), Tricyclandra o Herpetosperminae: *Herpetosperminae* (*Herpetosperminae* genus) Cephalopentandra ), Trifolium ( Biswarea ), genus Cucurbita ( Herpetospermum ), Trichosanthes genus ( Edgar ) Benincaseae (a tribal group) o Benincasinae: Cogniauxia, Ruthalicia, genus Cucurbita ( Lagenaria ), Winter melon ( Benincasa ), Very small Watermelon genus ( Lemon ), genus *Hymenoplastrus* Acanthosicyos ), Eureiandra、Bambekea、Nothoalsomitra、 Red cucumber genus ( Red ), genus *Cypripedium* ( Diplocyclos ), Raphidiocystis、Lemurosicyos、Zombitsia、 Cucurbita ( ) Ecballium ), European melon ( Bryonia ) o Luffinae: Genus *Luffina* Loofah ) Cucurbitae (with diffuse-porous, spiny pollen): Cucurbita ( Pumpkin ), Sicana, Tecunumania、Calycophysum、Peponopsis、Anacaona、Polyclathra、 genus *Cypripedium* ( Schizocarp ), Penelopeia, Cionosicyos, Trichosanthes kirilowii ( ) Japan ), Selysia, Abobra Sicyeae (hairy nectaries, pollen grains with 4 to 10 pores) o Cyclantherinae subfamily: Hanbury genus Cucurbita ( Echinopepon ), Marah, genus Cucurbita ( Echinocystis ), Vaseyanthus、Brandegea、Apatzingania、Cremastopus、Elateriopsis、 False Spicy Cucumber ( ) Pseudocyclanthera ) 、 Spicy Cucumber ( ) Cyclanthera), Rhytidostylis o Sicyinae subtribe: *Cypripedium* ( Sicyos ), Sicyosperma、Parasicyos、Microsechium、 genus Chayote ( Sechium), Sechiopsis, Pterosicyos Status undetermined: Odosicyo List of genera in alphabetical order: Abobra, genus *Hypophthalmonia* Acanthosicyos ), genus Zygophyllum (Actinostemma ), genus *Calamus* ( Alsomitra ), Ampelosycios、Anacaona、Apatzingania、 Apodanthera, Bambekea, Winter melon (genus) Benincasa ), Trifolium ( Biswarea ), genus *Fritillaria* Bolbostemma ), Brandywine genus *Prunus* ( Bryonia ), Calycophysum, Trichosanthes kirilowii ( ) Japan ), genus *Cephalotaxus* ( Cephalopentandra ), Ceratosanthes、Chalema、Cionosicyos、 genus Citrus ( Lemon ), Cucurbita ( Red ), Cognition, genus Coral Melon ( Corallocarpus ), Cremastopus, Ctenolepis, Cucumber genus ( Cucumber ), African cucumber ( Cucumber Cucumber ( ) Cucumber ), Cucurbita ( Pumpkin ), Cucurbita ( Cucurbita ), genus *Calamus* ( Cyclamen ), genus *Cypripedium* ( Dactyliandra ), genus *Gnaphalium* ( Dendrocyos ), genus *Cypripedium* ( Dicaelospermum ), Dieterle , 、 genus *Cypripedium* Diplocyclos ), Doyerea , 、 Cucurbita ( ) Ecballium ), Cucurbita genus ( Echinocystis ), genus Cucurbita ( Echinopepon ), 、 Triangularis (genus) Edgar ), Elateriopsis, Eureiandra, Fevillea, Gerald's genus ( Gerrardanthus ), Conical fruit genus ( Gomphogenus ), Gurania, Guraniopsis, Cucurbita (Genus) Gymnopetalum ), Gynostemma pentaphyllum ( Gynostemma ), Halosicios、Hanburia、Helmontia、 Genus *Gentiana* Hemsley ), genus Cucurbita ( Herpetospermum ), genus *Pyrus* (oil residue fruit) Hodgson ), Bags ( Iberville ), Cucurbita genus ( Indofevillea ), Kedrostis, genus Cucurbita ( Lagenaria ), Lemuricyos, genus Luffa ( Loofah ), Marah, Melancium, genus Cucurbita ( Melothria ), Melothrianthus、Microsechium、 bitter melon genus ( Momordica charantia ), Muellerargia, genus *Cinnamomum* (Red Butterfly) Mukia ), genus *Ipomoea* ( Myrmecosicyos ), genus Cucurbita ( Neoalsomitra ), Nothoalsomitra, Odosicyos, genus Cucumber ( Oreosyce ), 、Parasicyos、Penelopeia、Peponium、Peponopsis、 Polyclathra, Posadaea, Praecitrullus, False Spicy Cucumber ( ) Pseudocyclanthera ) 、 Pseudosicydium, Psiguria, genus *Cypripedium* ( Pteropepon ), Pterosicyos、Raphidiocystis、 Ruthalicia, Rhytidostylis, genus *Cypripedium* ( Schizocarp ), genus *Cypripedium* Schizopepon)、 Sechiopsis,genus Chayote ( Sechium ), Selysia、Seyrigia、Sicana、Sicydium、 genus Cucurbita ( Sicyos ), Sicyosperma, Siolmatra, Monk fruit genus ( Syriac ), Cucurbita ( Solemn ), Tecumseh, Telfair, genus *Gastrodia* ( Thladiantha ), Trichosanthes genus ( Trichosanthes ), Tricyclandra, Trochomeria、Trochomeriopsis、Tumacoca、Vaseyanthus、Wilbrandia、 Bilei Gu ( genus) Xerocyos ), Conical fruit genus ( Zanonia ), genus Equisetum ( Zehneria ), Zombies, genus Cucurbita ( Zygosicyo ).

[0141] The genus *Cucurbita* refers to a genus within the family Cucurbitaceae, native to the Andes Mountains and Central America, where it was first cultivated. *Cucurbita* species can be domesticated or non-domesticated.

[0142] Exemplary species include, but are not limited to: o Silver-seeded pumpkin (C. argyrosperma, synonym C. mixta) —pipian Pumpkin (cushaw pumpkin Origin: Panama, Mexico o Kelly Pumpkin ( C. kellyana Origin: Western Pacific coast of Mexico o Palmer pumpkin ( C. palmeri Origin: Northwestern Mexico, Pacific coast o closely related pumpkins ( C. sororia Origin: Pacific coast of Mexico to Nicaragua, northeastern Mexico o finger-leaf squash ( C. digitata Origin: Southwestern United States, Northwestern Mexico California squash ( C. californica ) o Heart-shaped pumpkin ( C. cordata ) o Cylindrical pumpkin ( C. cylindrata ) o Palm-leaf pumpkin ( C. palmata ) o Ecuadorian squash ( C. ecuadorensis Origin: Pacific coast of Ecuador o C. ficifolia—Black gourd, chilacayote; Origin: Mexico, Panama, northern Chile, and Argentina o Stinky Pumpkin ( C. foetidissima) —Stink gourd, buffalo gourd; Origin: Mexico o Rough-leaf pumpkin ( C. scabridifolia It is likely a natural hybrid of *Cypripedium foetidissima* and *Cypripedium pedatifolia*. o Galeott Pumpkin ( C. galeottii — Little is known about it; Origin: Oaxaca, Mexico o Rendel squash ( C. lundelliana Origin: Mexico, Guatemala, Belize o Big Pumpkin ( C. maxima —Winter squash, pumpkin; Origin: Argentina, Bolivia, Ecuador o Andrei Pumpkin ( C. andreana Origin: Argentina o Musk squash ( C. muskrat — Butter squash, Dickinson squash, golden squash; Origin: Bolivia, Colombia, Ecuador, Mexico, Panama, Puerto Rico, Venezuela o Okechobi Pumpkin ( C. okeechobeensis Origin: Florida o Martinez pumpkin ( C. martinezii Origin: Gulf Coast and foothills o Feathered-leaf pumpkin ( C. pedatifolia Origin: Queretaro, Mexico o Mur squash ( C. moorei ) o Zucchini ( C. watermelon —Field squash, summer squash, densely packed zucchini, zucchini, small cucumber, acorn-shaped squash; Origin: Mexico, USA o closely related pumpkins ( C. fraternal Origin: Tamaulipas and Nuevo León, Mexico o Texas squash ( C. texana Origin: Texas, USA o creeping pumpkin ( C. radicans —calabacilla, calabaza de coyote; Origin: Central Mexico o Slender pumpkin ( C. slender ) The genus Cucumber refers to twining, tendril-bearing plants, including cucumber (Cucumis sativus), melon (Cucumismelo), horned melon (Cucumis metuliferus), and acerola cucumber (Cucumis anguria). Species of the genus Cucumber can be wild or domesticated.

[0143] Exemplary species of the Cucurbita genus include, but are not limited to: prickly cucumber ( Cucumber prickly Cogn.) Membrane cucumber ( Cucumber aetheocarpus ) African cucumber ( African cucumber Lf) Hollyhock leaves cucumber ( Cucumber althaeoides (Ser.) West Indian cucumber ( Cucumber watermelon L.) Silver-backed cucumber ( Silver cucumber (Domin)) Hairy cucumber ( Sour cucumber Cogn.) Ballard cucumbers ( Cucumber baladensis Thulin) gourd leaf cucumber ( Cucumis bryoniifolius (Merxm.)) Canok cucumber ( Cucumber canoxy Thulin) Carolina cucumber ( Cucumber carolina JHKirkbr.) Gray-haired cucumber ( Grey cucumber (Cogn.)) Club-shaped petiole cucumber ( Cucumber clavipetiolate ) Rib cucumber Cucumber ribbed ) Delicate cucumber ( Weak cucumber ) Dips cucumber ( Cucumber dipsacus ) Engler cucumbers English cucumber ) Fig leaf cucumber ( Cucumber fig leaf ) Spherical cucumber ( Globose cucumber ) Slender cucumber ( Cucumber slender ) Halberd-shaped cucumber ( Speared cucumber ) Seven-finger cucumber ( Cucumber heptadactyl ) Hairy cucumber ( Hairy cucumber Sond. Ground cucumber ( Cucumber Stent) (e.g.) Cucumber ) Wild cucumber ( Porcupine cucumber ) Indian cucumber ( Indian cucumber ) Significant cucumber ( Cucumber insignis ) Java cucumber ( Javanese cucumber ) Jeffrey Cucumber ( Cucumber jeffreyanus ) Kalahari cucumber ( Kalahari cucumber ) Keller cucumber ( Cucumber kelleri (Cogn.)) Kirkbridge cucumbers Cucumber kirkbridei ) Smooth-seeded cucumbers ( Cucumber ) Madras cucumbers Cucumis maderaspatanus L.) Milos cucumber ( Cucumber meusei ) melon( Cucumber melon L.) Mixed cucumbers ( Harvest cucumber (C.Jeffrey) Horned melon ( Cucumber metulifera E.Mey. ex Naudin) Multi-fruit cucumber ( Cucumber myriocarpus Naudin) Fruit cucumber ( Cucumber dropped Thulin) Bitter cucumber ( Cucumber picrocarpus F.Muell.) Prophet Cucumber ( Cucumber of the prophets L.) Cucumber with hairy bumps ( Cucumber pubic tuberculatus Thulin) Pustular cucumbers Cucumber pustule Naudin ex Hook.f.) Queensland cucumbers Queensland cucumber I.Telford) Quintanilla cucumber Cucumis quintanilhae R.Fern. & A.Fern.) Netted cucumber ( Cucumber reticulata (A.Fern. & R.Fern.)) Hairy cucumber ( Cucumber rigid E.Mey. ex Sond.) Richie cucumber ( Cucumber ritchiei (CBClarke)) beak-shaped cucumber ( Cucumber beaked JHKirkbr.) Lempf cucumber ( Cucumber (Scheff.)) Sacramento cucumbers Cucumber Paill. & Bois) Arrow-shaped cucumber ( Arrowhead cucumber Wawra & Peyr.) cucumber( Cucumber L., cucumber) bristly cucumber ( Cucumber bristle Cogn.) Silent Valley cucumbers Cucumber silentvalleyi ) Tulin cucumber ( Cucumber thulinianus ) Umbrella cucumber ( Cucumber umbrella ) Versatile Cucumber Cucumber variable ) Zambian cucumbers Zambian cucumber ) Cai He cucumber ( Cucumber zeyheri So) The genus *Citrullus* refers to a variety of desert vines, including cultivated watermelon (*Citrullus lanatus*). Species in the genus *Citrullus* can be wild or domesticated.

[0144] Exemplary species and subspecies of the genus *Citrus* include, but are not limited to: Bitter orange Schrad. — Citron Watermelon Citrullus colocynthis (L.) Schrad. —Medicinal Watermelon Citrullus cirrhosus Cogn. — A vineless melon Citrullus woolly —Desert Watermelon The desert watermelon subspecies, a variant of the Kordofan watermelon ( Citrullus woolly subsp. common var. Cordophanian (Ter-Avan.)Fursa) Original variant desert watermelon ( Citrullus woolly var. woolly ) Mucilaginous Seed Watermelon Citrullus mucosospermus (Fursa) Fursa) — gourd seed melon (egusimelon) Notting watermelon Citrullus naudinianus (Sond.)) Rem Watermelon ( Citrullus rehmii ) According to the specific implementation plan, cucurbitaceous plants are cultivated cucurbitaceous plants.

[0145] According to other implementation schemes, the cucurbitaceae plant is a bitter gourd, and in specific implementation schemes, it is a cultivated bitter gourdaceae plant. As used herein, a bitter gourdaceae plant is a cucurbitaceae species in which the cucurbitacin biosynthesis pathway functions to produce cucurbitacin. The amount of cucurbitacin and the degree of bitterness can vary from species to species.

[0146] Cucurbitaceae plants that accumulate cucurbitacin may be bitter, and usually non-bitter cucurbitaceae plants may accumulate cucurbitacin due to hybridization, stress, unfavorable growth conditions, or insect or other pest infestation.

[0147] It should be understood that bitter cucurbitaceae plants may be the product of interspecific hybridization.

[0148] In the specific implementation plan, "cultivated cucurbitaceae plants" refers to bitter cultivars of watermelon, including bitter Hawkesbury watermelons obtainable via USDA PI673137 or PI274035, and cultivated as a source of bitter cucurbitacins (Chambliss, OL and Jones, CM, 1966. Cucurbitacins: specific insect attractants in Cucurbitaceae). Science , 153 (3742), Pages 1392-1393.

[0149] In the specific implementation plan, "cultivated cucurbitaceae plants" refers to bitter melon ( Momordica charantia L.). As used herein, the term bitter melon is interchangeable with bitter gourd, bitter squash, Goya melon, karela, and balsam pear.

[0150] In other implementation schemes, the cucurbitaceous plant cultivated is the common watermelon ( Common lemon (A bitter form of Hawkesbury watermelon) or zucchini ( Cucurbita pepo (Pumpkin) or cucumber (Cucumissativus) or melon (Cucumis melo). In some implementations, bitter cucurbitaceae plants can be the product of intragenus interspecific hybridization.

[0151] In some embodiments, the non-cultivated cucurbitaceous plants are selected from non-cultivated bitter melons, non-cultivated bitter cucumbers, and non-cultivated bitter watermelons. In other embodiments, the non-cultivated bitter cucurbitaceous plants may be at least one species from the genera *Cucumis*, *Citrullus*, *Momordica*, and *Cucurbita*.

[0152] In some implementations, the cultivated cucurbitaceae plants are not naturally occurring mogroside-accumulating cucurbitaceae plants, for example, not *Siraitia grosvenorii* (monk fruit). Siraitia grosvenorii (monkfruit).

[0153] Although cucurbitacins were initially isolated from plants in the Cucurbitaceae family, they have since been found in plants of the Brassicaceae, Scrophulariaceae, Begoniaceae, Elaeocarpaceae, Datisacaceae, Desfontainiaceae, Polemoniaceae, Primulaceae, Rubiaceae, Sterculiaceae, Rosaceae, and Thymelaeaceae families. They have also been detected in several mushroom genera, including Russula and Hebeloma, and even in shell-less marine mollusks (Dorid nudibranchs) (Chen et al., Natural Product Reports 2005). 22:386-399). For a detailed list of all cucurbitacin-producing species and their respective cucurbitacins, see Miro et al., Phytotherapy Research, 1995. Therefore, in other embodiments, the plant is a naturally occurring cucurbitacin-producing non-cucurbitaceous plant. In specific embodiments, the cucurbitacin-producing non-cucurbitaceous plants are selected from plants of the Brassicaceae, Scrophulariaceae, Begoniaceae, Elaeocarpaceae, Datischaceae, Desfontainiaceae, Polemoniaceae, Primulaceae, Rubiaceae, Sterculiaceae, Rosaceae, and Thymelaeaceae families.

[0154] It should be understood that the present invention also envisions plants modified to produce cucurbitacin or other cucurbitacin-like (non-sweet) tetracyclic triterpenes, which can be modified by the method of the present invention to be glucosylated.

[0155] Each subspecies and type has many cultivars, each favored for its specific use or region.

[0156] In the context of this invention, the term "hybrid" or "hybrid" refers to a gamete fusion that produces offspring (i.e., cells, seeds, or plants) via pollination. This term includes sexual hybridization (one plant being pollinated by another) and self-pollination (self-pollination, i.e., when pollen and ovules come from the same plant or from genetically identical plants).

[0157] "Backcrossing" refers to the process by which breeders repeatedly cross hybrid offspring with one of the parents. For example, crossing the first-generation F1 hybrid with one of the parent genotypes of the F1 hybrid. The parent with which this hybrid is backcrossed is called the "recurrent parent".

[0158] Marker-assisted selection can be used to enhance or replace phenotypic selection.

[0159] As used herein, "outcrossing" refers to cross-pollination between plants with different genetic makeups, the opposite of self-pollination. Preferably, the two plants are the same species or subspecies, such as bitter melon, or cultivated bitter melon of the same subspecies. However, intercrossing between different plant species can also be considered.

[0160] As used herein, the term "heterosis" refers to the enhancement of heterosis or cross-pollination, that is, the functional improvement or enhancement of any biological quality in hybrid offspring. Heterosis is observed if the offspring's trait is enhanced due to the genetic contributions of its parents.

[0161] "Yield" describes the amount of fruit produced by a single plant or a group of plants or crops. Yield can be measured in several ways, such as by the amount of fruit produced. -1 And the average fruit yield per plant, measured in grams.

[0162] As used herein, "introgression" refers to the movement of one or more genes or a set of genes from one plant variety to another through breeding methods (such as crossbreeding). Introgression also refers to the movement of traits encoded by one or more genes or a set of genes from one plant variety to another.

[0163] "Transformed" refers to a plant whose genes have been incorporated into the traits of another plant.

[0164] A plant that has “virtually all the physiological and morphological characteristics” of a particular plant refers to a plant that has the same general physiological and morphological characteristics except for those characteristics derived from a specific transforming gene or gene group (e.g., long stigma).

[0165] In the context of this invention, the terms "associated with" or "associated" refer to, for example, a QTL and a phenotypic trait (e.g., bitterness) being in linkage disequilibrium, meaning that the QTL and the trait co-occur in progeny plants more frequently than when nucleic acids and phenotypes are segregated independently.

[0166] The terms “marker” or “molecular marker” or “genetic marker” refer to a genetic locus (“marker locus”) used as a reference point in identifying genetically linked loci such as QTLs.

[0167] A "probe" is an isolated nucleic acid with a conventionally detectable label or reporter molecule attached to it, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. This probe is complementary to one strand of the target nucleic acid; in the case of this invention, it is complementary to one strand of the genomic DNA of a gene representing the cucurbitacin biosynthesis pathway, said gene being derived from a target plant (e.g., a cucurbitaceous plant or other cucurbitacin-producing plant) or from a sample containing plant DNA. The probes of this invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0168] "Primer" refers to isolated nucleic acids that undergo hybridization with a complementary target DNA strand and anneal to form a hybrid of the primer and the target DNA strand, which is then extended along the target DNA strand by a polymerase (e.g., DNA polymerase). The primer pairs of this invention refer to primer pairs used to amplify target nucleic acid sequences, for example, through polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0169] The probes and primers are typically 11 nucleotides or longer, preferably 18 nucleotides or longer, more preferably 24 nucleotides or longer, and most preferably 30 nucleotides or longer. These probes and primers specifically hybridize to the target sequence under highly stringent hybridization conditions. According to some embodiments, the probes and primers of the present invention have complete sequence similarity to the target sequence, and although probes different from the target sequence can be designed using conventional methods, they still retain the ability to hybridize to the target sequence.

[0170] Methods for preparing and using probes and primers are described in, for example, *Molecular Cloning: A Laboratory Manual*, 2nd edition, Volumes 1–3, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (hereinafter referred to as "Sambrook et al., 1989"); *Current Protocols in Molecular Biology*, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (regularly updated) (hereinafter referred to as "Ausubel et al., 1992"); and *PCR Protocols: A Guide to Methods and Applications*, Academic Press: San Diego, 1990, by Innis et al. PCR primer pairs can be derived from known sequences, for example, by using computer programs for this purpose, such as Primer (version 0.5, COPYRGT. 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).

[0171] The term "specificity to (target sequence)" means that a probe or primer hybridizes only with the target sequence in a sample containing the target sequence under strict hybridization conditions.

[0172] As used in this article, “amplified DNA” or “amplifier” refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.

[0173] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a complex polynucleotide sequence (e.g., a combination of the above).

[0174] The term "separated" means at least partially separated from the natural environment, such as from plant parts or plant cells.

[0175] As used herein, a “homologous” or “orthologous” sequence refers to a naturally occurring or synthetic nucleic acid sequence (or a polypeptide encoded by it) that contains at least the functional portion of the polynucleotide / polypeptide of the present invention and is capable of imparting modified cucurbitacin content to the plant.

[0176] Such homologs or orthologs can be, for example, nucleotide sequences corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, or 83, or sequences encoding SEQ ID NO: The amino acid sequence of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, or 84 has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% % Homologous, such as when using the BestFit software with the Wisconsin sequence analysis package, determined by the Smith and Waterman algorithms and default parameters.

[0177] As used herein, the term cucurbitacin refers to any of a class of triterpenoid compounds produced by certain plants (particularly squash and gourd) that function as a defense against herbivores. Cucurbitacins are chemically classified as triterpenoids, formally derived from cucurbitane, a triterpenoid hydrocarbon—specifically, from the unsaturated variant cucurbit-5-ene, or 19(10→9β)-abeo-10α-lanostane-5-ene. Most cucurbitacins are tetracyclic triterpenoids, but some (e.g., cucurbitacins S and T) have an additional ring cyclized between C-16 and C-24. In one embodiment, cucurbitacin is a cucurbitacin glycoside. Cucurbitacins vary in the number and position of their ketone, hydroxyl, and acetyl groups.

[0178] Non-limiting examples of cucurbitacins include cucurbitacins AL and OS, and their glycoside derivatives. Table 1 shows a non-limiting list of cucurbitacins and their chemical formulas. Figure 1 The table provides details of the differences in ketone, hydroxyl, and acetyl groups between the different cucurbitacins AL and OS.

[0179] Table 1:

[0180] According to some embodiments of the present invention, the expression of one or more cucurbitacins selected from cucurbitacin AL and OS is reduced in plants or plant cells. In a specific embodiment, the expression of one or more cucurbitacins selected from cucurbitacin B, cucurbitacin I, and cucurbitacin E is reduced in plants or plant cells.

[0181] According to the present invention, cucurbitacin modification caused by downregulation of gene expression in the cucurbitacin biosynthesis pathway may include, but is not limited to, any one or more reductions in the hydroxylation of carbons C2, C16, C20 and C23 in tetracyclic triterpenoid molecules, reductions in the oxidation and / or dehydrogenation of carbons C3, C11, C21, C1-C2 and C22-C23 in tetracyclic triterpenoid molecules, and reductions in the glycosylation of carbon C2 (in cucurbitacin E-glucoside).

[0182] As used herein, the phrase "cucurbitacin biosynthetic pathway" or "cucurbitacin biosynthesis pathway" refers to at least one or more enzymatic reactions that catalyze the synthesis of cucurbitacin from precursor molecules (e.g., cucurbitadienol). The phrase "cucurbitacin biosynthetic pathway gene" refers to at least one or more genes encoding enzyme proteins that catalyze the production of cucurbitacin from cucurbitacin precursor molecules.

[0183] The inventors have collected a set of candidate watermelon genes that encode enzyme proteins that may catalyze one or more steps in the cucurbitacin biosynthesis pathway. Table 2 below is a non-limiting list of watermelon genes, grouped according to the annotated catalytic activity of the enzyme proteins they encode, and which, according to the method of the present invention, can serve as targets for downregulating the cucurbitacin biosynthesis pathway.

[0184] Table 2:

[0185] Therefore, in some embodiments, the at least one cucurbitacin biosynthesis pathway gene is a gene having a coding sequence selected from SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 112, 115, 118, 121, and 124, or a functional orthologous gene from different Cucurbitaceae species. In a specific embodiment, the at least one cucurbitacin biosynthesis pathway gene is a (2OG) and Fe(II)-dependent oxygenase gene. In some embodiments, the (2OG) and Fe(II)-dependent oxygenase genes are selected from ClCG01G014610, ClCG01G014610a, and ClCG01G014610b, having coding sequences selected from SEQ ID NO: 64, 67, and 70 that encode an enzyme protein having an amino acid sequence selected from SEQ ID NO: 65, 68, and 71.

[0186] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is a cytochrome p450 gene. In some embodiments, the cytochrome p450 gene is selected from ClCG01G003400, ClCG01G003790, ClCG01G014540, ClCG01G014560, ClCG01G017130, ClCG01G024550, ClCG02G019010, ClCG05G019420, ClCG05G019890, and ClCG06G001570. C1CG06G001580, C1CG06G001590, C1CG06G001620, C1CG09G012200, C1CG09G012210, C1CG10G005830, C1CG10G012530, C1CG03G015220, C1CG10G015150, C1CG11G002820, and C1CG11G017020, which have the following characteristics selected from SEQ The coding sequences of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61 encode an enzyme protein having an amino acid sequence selected from SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59 and 62. In a specific implementation scheme, the cytochrome p450 gene is selected from ClCG01G014540, ClCG06G001570, C1CG06G001580, C1CG06G001590, C1CG06G001620, and C1CG10G012530, which has a coding sequence selected from SEQ ID NO: 7, 28, 31, 34, 37, and 49, which encodes an enzyme protein having an amino acid sequence selected from SEQ ID NO: 8, 29, 32, 35, 38, and 50.

[0187] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is a FAD-binding berberine gene (a member of the dehydrogenase-oxidoreductase family). In some embodiments, the FAD-binding berberine gene is C1CG03G002490, which has a coding sequence as shown in SEQ ID NO: 85, which encodes an enzyme protein having an amino acid sequence selected from that shown in SEQ ID NO: 86.

[0188] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is an NAD(P)-binding Rossmann fold gene (a member of the dehydrogenase-oxidoreductase family). In some embodiments, the NAD(P)-binding Rossmann fold gene is selected from C1CG01G014570 and C1CG01G01004750, having coding sequences as shown in SEQ ID NO: 88 and 91, which encode an enzyme protein having an amino acid sequence selected from SEQ ID NO: 89 and 92. In a specific embodiment, the NAD(P)-binding Rossmann fold gene is C1CG01G014570, having a coding sequence as shown in SEQ ID NO: 88, which encodes an enzyme protein having the amino acid sequence of SEQ ID NO: 89.

[0189] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is an HXXD-type acyltransferase-like protein gene. In some embodiments, the HXXD-type acyltransferase-like protein gene is selected from C1CG01G014530, C1CG05G020040, and C1CG06G001610, having a coding sequence selected from SEQ ID NO: 118, 121, and 124, which encodes an enzyme protein having an amino acid sequence selected from SEQ ID NO: 119, 122, and 125, respectively. In a specific embodiment, the HXXD-type acyltransferase-like protein gene is C1CG06G001610, which has a coding sequence shown in SEQ ID NO: 124, which encodes an enzyme protein having an amino acid sequence of SEQ ID NO: 125.

[0190] In other embodiments, at least one cucurbitacin biosynthesis pathway gene is a polyketide cyclase / dehydratase gene (a member of the dehydrogenase-oxidoreductase family). In some embodiments, the polyketide cyclase / dehydratase gene is C1CG07G007250, having a coding sequence as shown in SEQ ID NO: 109, which encodes an enzyme protein having an amino acid sequence as shown in SEQ ID NO: 110.

[0191] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is a short-chain dehydrogenase / reductase gene (a member of the dehydrogenase-oxidoreductase family). In some embodiments, the short-chain dehydrogenase / reductase gene is C1CG03G016930, which has a coding sequence as shown in SEQ ID NO: 115, which encodes an enzyme protein having an amino acid sequence selected from that shown in SEQ ID NO: 116.

[0192] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is a proline dehydrogenase gene (a member of the dehydrogenase-oxidoreductase family). In some embodiments, the proline dehydrogenase gene is C1CG11G005180, which has a coding sequence as shown in SEQ ID NO: 112, which encodes an enzyme protein having an amino acid sequence selected from SEQ ID NO: 113.

[0193] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is an alcohol dehydrogenase gene (a member of the dehydrogenase-oxidoreductase family). In a specific embodiment, the alcohol dehydrogenase gene is selected from C1CG09G009760, C1CG01G014590, C1CG01G018250, and C1CG01G009780, having a coding sequence selected from SEQ ID NO: 73, 76, 79, and 82, which encodes an enzyme protein having an amino acid sequence selected from SEQ ID NO: 74, 77, 80, and 83. In a specific embodiment, the alcohol dehydrogenase gene is selected from C1CG09G009760 and C1CG01G018250, having a coding sequence selected from SEQ ID NO: 73 and 79, which encodes an enzyme protein having an amino acid sequence selected from SEQ ID NO: 74 and 80.

[0194] In other embodiments, the at least one cucurbitacin biosynthesis pathway gene is a peroxidase gene (a member of the dehydrogenase-oxidoreductase family). In a specific embodiment, the peroxidase gene is selected from C1CG01G017400, C1CG02G023760, C1CG02G023770, C1CG02G023780, and C1CG03G004740, having a coding sequence selected from SEQ ID NO: 94, 97, 100, 103, and 106, which encodes an enzyme protein having an amino acid sequence selected from SEQ ID NO: 95, 98, 101, 104, and 107.

[0195] Therefore, in some embodiments of the present invention, the plant or plant cell with modified cucurbitacin content is a plant cell of a bitter cucurbitaceae plant, wherein the expression of any one or more of the following genes is reduced: (2OG) and Fe(II) dependent oxygenase gene, cytochrome p450 gene, FAD-binding berberine gene, NAD(P)-binding Rossmann fold gene, HXXD type acyltransferase-like protein gene, polyketide cyclase / hydratase gene, short-chain dehydrogenase / reductase gene, proline dehydrogenase gene, alcohol dehydrogenase gene, and peroxidase gene.

[0196] In a specific implementation scheme, the at least one cucurbitacin biosynthesis pathway gene is a functional orthologous gene of bitter cucurbitaceous plants identified in watermelon. In a specific implementation scheme, the functional orthologous gene is a functional orthologous gene of bitter melon from watermelon cucurbitacin biosynthesis pathway.

[0197] Cucurbitacins include, but are not limited to, common cucurbitacins isolated from plant families (including Brassicaceae, Cucurbitaceae, Scrophulariaceae, Begoniaceae, Elaeocarpaceae, Datischaceae, Desfontainiaceae, Polemoniaceae, Primulaceae, Rubiaceae, Sterculiaceae, Rosaceae, and Thymelaeaceae), fungi (including Russula and Hebeloma), and some marine mollusks. In a specific embodiment, the cucurbitacin is derived from plants of the Cucurbitaceae family.

[0198] It should be understood that known bioinformatics tools such as BLAST (NIH) and UniProt BLAST (Expasy.org) can be used to identify homologs and orthologs of genes involved in the target cucurbitacin biosynthesis pathway in any species.

[0199] Table 2a is a compilation of non-restrictive examples of orthologous genes of the cucurbitacin biosynthesis pathway from melon (Cucumis melo), cucumber (Cucumis sativus), and zucchini (Cucurbitapepo) (pumpkin, squash) and watermelon (Citrullis lanatus, Charleston Grey Watermelon).

[0200] Table 2a

[0201] This invention envisions the production of plants or plant cells with modified cucurbitacin content, the modification being achieved by downregulating the expression of at least one gene involved in the cucurbitacin biosynthesis pathway. Downregulation of at least one gene involved in the cucurbitacin biosynthesis pathway can further modify the hydroxylation, redox, dehydrogenation, acetylation, and / or glucosylation of cucurbitacin or cucurbitacin precursors. As used herein, the phrase “downregulation” refers to the use of various molecules that interfere with transcription and / or translation (e.g., RNA silencing agents) to downregulate the expression of proteins (e.g., cucurbitacin biosynthesis pathway enzymes) at the genomic level (e.g., homologous recombination and site-specific endonucleases) and / or transcriptional level.

[0202] Under the same cultivation conditions, the expression level is usually compared with the expression level of the same plant species or plant cells that have not been exposed to the agent or solvent (also known as the control).

[0203] The downregulation of expression can be instantaneous or permanent.

[0204] According to the specific implementation plan, downregulated expression refers to the lack of mRNA and / or protein detected by RT-PCR or Western blot, respectively.

[0205] According to other specific implementation schemes, downregulation of expression refers to a reduction in the levels of mRNA and / or protein detected by RT-PCR or Western blot, respectively. The reduction may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0206] Non-limiting examples of agents that can downregulate gene expression in the cucurbitacin biosynthesis pathway are described in detail below.

[0207] Downward adjustment of nucleic acid levels

[0208] Downregulation of nucleic acid levels is typically achieved using nucleic acid agents, which have a nucleic acid backbone, DNA, RNA, their analogs, or combinations thereof. Nucleic acid agents can be encoded by DNA molecules or provided to the cell itself.

[0209] According to the specific implementation plan, the downregulating agent is a polynucleotide.

[0210] According to the specific implementation plan, the downregulator is a polynucleotide that can hybridize with the gene or mRNA encoding the enzyme in the cucurbitacin biosynthesis pathway.

[0211] According to the specific implementation plan, the downregulator directly interacts with the enzyme genes of the cucurbitacin biosynthesis pathway.

[0212] According to the specific implementation plan, the agent directly binds to the enzyme gene of the cucurbitacin biosynthesis pathway.

[0213] According to the specific implementation scheme, the agent indirectly binds to the cucurbitacin biosynthesis pathway enzyme gene (e.g., an effector that binds to the cucurbitacin biosynthesis pathway enzyme gene).

[0214] Depending on the specific implementation plan, the downregulator is either an RNA silencing agent or a genome editing agent.

[0215] Therefore, RNA silencing can be used to downregulate the expression of genes involved in the cucurbitacin biosynthesis pathway. As used herein, the phrase “RNA silencing” refers to a set of regulatory mechanisms mediated by RNA molecules [e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression], which result in the suppression or “silencing” of the expression of the corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0216] As used herein, the term "RNA silencer" refers to RNA capable of specifically inhibiting or "silencing" the expression of a target gene. In some embodiments, RNA silencers are able to prevent the complete processing (e.g., complete translation and / or expression) of mRNA molecules through post-transcriptional silencing mechanisms. RNA silencers include non-coding RNA molecules, such as RNA duplexes containing paired strands, and precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencers include dsRNAs, such as siRNA, miRNA, and shRNA.

[0217] In one implementation, the RNA silencing agent can induce RNA interference.

[0218] In another implementation, RNA silencing agents can mediate translational repression.

[0219] According to one embodiment of the invention, the RNA silencing agent is specific to the target RNA (e.g., the transcript of the cucurbitacin biosynthesis pathway enzyme gene) and does not cross-inhibit or silence other targets or splice variants that exhibit 99% or less overall homology with the target gene, for example, less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81% overall homology with the target gene, as determined by PCR, Western blotting, immunohistochemistry, and / or flow cytometry.

[0220] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNAs) in animals and plants.

[0221] The following is a detailed description of RNA silencing agents that can be used according to specific embodiments of the present invention.

[0222] DsRNA, siRNA, and shRNA —The presence of long dsRNA in cells can stimulate the activity of ribonuclease III called dicer. Dicer is involved in processing dsRNA into dsRNA fragments called short interfering RNA (siRNA). The siRNA derived from dicer activity is typically about 21 to 23 nucleotides in length and contains a double strand of about 19 base pairs. RNAi reactions are also characterized by an endonuclease complex, often called the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNA with a sequence complementary to the antisense strand of the siRNA double strand. Cutting of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA double strand.

[0223] Therefore, some embodiments of the present invention consider using dsRNA to downregulate the protein expression of mRNA.

[0224] According to one implementation scheme, dsRNA longer than 30 bp is used. Various studies have demonstrated that long dsRNAs can be used to silence gene expression without inducing stress responses or causing significant off-target effects—see, for example, [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13, 3803–3810; Bhargava A et al., Brain Res. Protoc. 2004; 13: 115–125; Diallo M. et al., Oligonucleotides. 2003; 13: 381–392; Paddison PJ et al., Proc. Natl Acad. Sci. USA. 2002; 99: 1443–1448; Tran N. et al., FEBS Lett. 2004; 573: 127–134].

[0225] The term "siRNA" refers to a small, repressive RNA duplex (typically between 18 and 30 base pairs) that induces RNA interference (RNAi) pathways. Typically, siRNA is a chemically synthesized 21-mer RNA with a central 19 bp duplex region and symmetrical 2-base overhangs at both ends; however, recent studies have shown that chemically synthesized 25-30 bp RNA duplexes are up to 100 times more potent at the same position than 21-mer RNA. The increased potency observed with longer RNA triggering RNAi is thought to result from providing the substrate (27-mer) to Dicer instead of the product (21-mer), and this increases the rate or efficiency of siRNA duplex entry into RISC.

[0226] It has been found that the position of the 3'-protrusion affects the potency of siRNA, and asymmetric duplexes with a 3'-protrusion on the antisense strand are generally more effective than those with a 3'-protrusion on the sense strand (Rose et al., 2005). This can be attributed to the loading of the asymmetric strand into RISC, as the opposite pattern of potency has been observed when targeting the antisense transcript.

[0227] The strands of double-stranded interfering RNA (e.g., siRNA) can be linked to form hairpin or stem-loop structures (e.g., shRNA). Therefore, as described above, the RNA silencing agent in some embodiments of the present invention can also be short hairpin RNA (shRNA).

[0228] As used herein, the term "shRNA" refers to an RNA agent having a stem-loop structure comprising a first and second region of complementary sequences, the complementarity and orientation of which are sufficient to allow base pairing to occur between the regions, the first and second regions being connected by a loop region resulting from the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between 3 and 23, or 5 and 15, or 7 and 13, or 4 and 9, or 9 and 11, and includes these numbers. Some nucleotides in the loop may participate in base pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'-CAAGAGA-3' and 5'-UUACAA-3' (International Patent Applications WO2013126963 and WO2014107763). Those skilled in the art will recognize that the resulting single-stranded oligonucleotides form a stem-loop or hairpin structure containing a double-stranded region capable of interacting with the RNAi mechanism.

[0229] The synthesis of RNA silencing agents suitable for some embodiments of the present invention can be performed as follows. First, the AA dinucleotide sequence in the cucurbitacin biosynthesis pathway gene or gene transcript (e.g., mRNA) sequence is scanned downstream of the AUG start codon. The presence of each AA and the 19 adjacent 3' nucleotides is recorded as potential siRNA target sites. Preferably, the siRNA target sites are selected from open reading frames, as the untranslated region (UTR) is rich in regulatory protein binding sites. UTR-binding proteins and / or translation initiation complexes may interfere with the binding of the siRNA endonuclease complex [Tuschl ChemBiochem.2:239-245]. However, it should be understood that siRNAs targeting the untranslated region are also effective, as demonstrated with GAPDH, where siRNA targeting the 5' UTR mediates a reduction of approximately 90% in cellular GAPDH mRNA and completely eliminates protein levels (www.ambion.com / techlib / tn / 91 / 912.html).

[0230] Secondly, using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov / BLAST / ), potential target sites are compared with appropriate genomic databases (e.g., Cucurbitaceae). Proposed target sites with significant homology to other coding sequences are filtered out.

[0231] Select a suitable target sequence as a template for siRNA synthesis. Preferred sequences are those with low G / C ratios, as these have been shown to be more effective in mediating gene silencing compared to sequences with G / C ratios higher than 55%. To better evaluate the selected siRNA, it is preferable to select multiple target sites along the length of the target gene. For more accurate evaluation of the selected siRNA, it is preferable to use a negative control simultaneously. The negative control siRNA preferably contains the same nucleotide composition as the siRNA but lacks significant homology with the genome. Therefore, it is preferable to use a disordered nucleotide sequence of the siRNA, provided that the sequence does not have significant homology with any other gene.

[0232] For example, suitable siRNAs for the expression of cucurbitacin biosynthesis pathway enzyme genes can be as described by Fusaro AF et al., EMBO Rep. 2006.11; 7(11):1168-75. doi: 10.1038 / sj.embor.7400837.Epub2006.10.13.PMID: 17039251; PMCID: PMC1679793. In short, an expression construct containing the target sequence (i.e., the coding sequence of the cucurbitacin biosynthesis pathway enzyme gene or a portion thereof) and the reverse form of the target sequence (reverse orientation) can be used. This construct is linked to a 5' promoter sequence via a 308 bp Arabidopsis polyubiquitin intron (SEQ ID NO: 127). When expressed in host cells, this construct produces hairpin transcripts (hpRNAs), which are subsequently digested by DICER into short double-stranded siRNA fragments (see, Figure 8 As shown, Tripathi, PK et al., Plant Growth Regul (2023). www.doi.org / 10.1007 / s10725-023-01013-0). Suitable expression constructs can be synthesized using target sequences from any cucurbitacin biosynthesis pathway enzyme gene or its functional homologs. In specific embodiments, the target sequence is selected from any gene sequence or portion thereof from the genes in Table 2. In other embodiments, the target sequence is selected from any of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126.

[0233] In a specific implementation scheme, the target gene is the cytochrome p450 gene selected from C1CG01G014540, C1CG06G001570, C1CG06G001580, C1CG06G001590, C1CG06G001620, and C1CG10G012530, and the target sequence is selected from SEQ ID NO: 9, 30, 33, 36, 39, and 51.

[0234] In other embodiments, the target gene is the FAD-binding berberine gene (a member of the dehydrogenase-oxidoreductase family), and the target sequence is shown in SEQ ID NO: 87.

[0235] In the specific implementation scheme, the target gene is an NAD(P)-binding Rossmann fold gene, and the target sequence is shown in SEQ ID NO: 90.

[0236] In the specific implementation scheme, the target gene is the HXXD type acyltransferase-like protein gene, and the target sequence is shown in SEQ ID NO: 126.

[0237] In other specific embodiments, the target gene is an alcohol dehydrogenase gene selected from C1CG09G009760 and C1CG01G018250, and the target sequences are selected from SEQ ID NO: 75 and 81, respectively.

[0238] In specific implementation schemes, the target sequences for silencing cucurbitacin biosynthesis pathway enzyme genes include, but are not limited to, the sequences listed in Table 3 of this document, which correspond to the target sequences of cucurbitacin biosynthesis pathway enzyme genes in Table 2.

[0239] Table 3:

[0240] It should be understood, and as described above, that the RNA silencing agents of some embodiments of the present invention are not limited to molecules containing only RNA, but also include chemically modified nucleotides and non-nucleotides.

[0241] miRNA and miRNA The mimic—according to another implementation scheme, the RNA silencing agent can be miRNA.

[0242] The terms “microRNA,” “miRNA,” and “miR” are synonyms referring to a collection of non-coding single-stranded RNA molecules, approximately 19–28 nucleotides in length, that regulate gene expression. miRNAs are present in a variety of organisms (viruses, especially humans) and have been shown to play roles in development, homeostasis, and disease etiology.

[0243] Below is a brief description of the miRNA activity mechanism.

[0244] Transcription of genes encoding miRNAs results in the production of miRNA precursors called pri-miRNAs. pri-miRNAs are typically part of a polycistronic RNA group containing multiple pri-miRNAs. pri-miRNAs can form hairpins with stems and loops. The stem may contain mismatched bases.

[0245] The hairpin structure of pri-miRNA is recognized by Drosha, an RNase III endonuclease. Drosha typically recognizes the terminal loop of pri-miRNA and cleaves approximately two helical loops at the stem, producing a 60-70 nucleotide precursor called pre-miRNA. Drosha cleaves pri-miRNA using the typical staggered cleavage pattern of RNase III endonucleases, producing a pre-miRNA stem loop with a 5' phosphate group and a ~2 nucleotide 3' overhang. It is estimated that the stem extending approximately one helical loop (~10 nucleotides) after the Drosha cleavage site is crucial for efficient processing. The pre-miRNA is then actively transported from the nucleus to the cytoplasm via Ran-GTP and the export receptor Ex-Portin-5.

[0246] The pre-miRNA double-stranded stem is then recognized by Dicer (or Dicer-like protein (DCL)), an RNase III endonuclease. Dicer also recognizes the 5' phosphate and 3' overhang at the base of the stem-loop. Subsequently, Dicer removes the terminal loop from the two turns of the helix at the base of the stem-loop, leaving an additional 5' phosphate and a 3' overhang of approximately 2 nucleotides. The resulting siRNA-like double-stranded structure (potentially containing mismatches) contains the mature miRNA and a similarly sized fragment (called miRNA). miRNA and miRNA It can originate from the opposite arms of pri-miRNA and pre-miRNA. miRNA The sequence can be found in cloned miRNA libraries, but it is usually less frequent than miRNAs.

[0247] Although initially associated with miRNA in a double-stranded form The miRNA binds to the RNA, but ultimately integrates as a single-stranded RNA into a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). The RISC can be composed of different proteins, leading to miRNA / miRNA interactions. The specificity of the double helix, the binding site of the target gene, the activity of the miRNA (inhibition or activation), and the miRNA / miRNA ratio. There is a difference in which chain of the double helix is ​​loaded into the RISC.

[0248] miRNA: miRNA When the miRNA in the double helix is ​​loaded into RISC, the miRNA... It will be removed and degraded. miRNA loaded into RISC: miRNA The strand of a double helix is ​​the one with the weaker 5' pairing. If miRNA:miRNA If the 5' ends of the two ends are roughly the same, then miRNA and miRNA They may all have gene silencing activity.

[0249] RISC recognizes target nucleic acids based on the high level of complementarity between miRNA and mRNA, especially nucleotides 2-7 of the miRNA.

[0250] Numerous studies have explored the base pairing requirements between miRNAs and their mRNA targets to achieve effective translation repression (Bartel 2004 review, Cell 116-281). In mammalian cells, the first eight nucleotides of the miRNA are likely crucial (Doench & Sharp 2004 GenesDev 2004-504). However, other parts of the microRNA can also participate in mRNA binding. Furthermore, sufficient base pairing at the 3' end can compensate for insufficient pairing at the 5' end (Brennecke et al., 2005 PLoS 3-e85). Computational studies analyzing miRNA binding across the entire genome have shown that bases 2-7 at the 5' end of the miRNA play a specific role in target binding, but the role of the first nucleotide (usually "A") has also been acknowledged (Lewis et al., 2005 Cell 120-15). Similarly, Krek et al. (2005, Nat Genet 37-495) used nucleotides 1-7 or 2-8 to identify and validate targets.

[0251] Target sites in mRNA can be located in the 5'UTR, 3'UTR, or coding region. Interestingly, multiple miRNAs can regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets suggests that the synergistic effect of multiple RISCs provides the most effective translational repression.

[0252] miRNAs can downregulate gene expression through either of two mechanisms that guide RISC: mRNA cleavage or translational repression. If the mRNA and miRNA have a certain degree of complementarity, the miRNA can direct the cleavage of the mRNA. When miRNA directs cleavage, the cleavage site is typically located between nucleotides that pair with residues 10 and 11 of the miRNA. Alternatively, if the miRNA-miRNA complementarity is insufficient, the miRNA can repress translation. Translational repression may be more common in animals because the complementarity between the miRNA and its binding site may be lower in animals.

[0253] It should be noted that in any pair of miRNAs and miRNAs The 5' and 3' ends may exhibit variability. This variability may be due to the variability regarding the cleavage site during enzymatic processing of Drosha and Dicer. miRNA and miRNA The variability at the 5' and 3' ends may also be due to mismatches in the stem structures of pri-miRNA and pre-miRNA. Stem mismatches can lead to different hairpin structure populations. Stem structure variability can also cause variability in the cleavage products of Drosha and Dicer.

[0254] The term "microRNA mimic" or "miRNA mimic" refers to a synthetic non-coding RNA capable of entering the RNAi pathway and regulating gene expression. miRNA mimics the function of endogenous miRNAs and can be engineered as a mature double-stranded molecule or a mimic precursor (e.g., pre-miRNA). miRNA mimics can consist of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid compounds (e.g., LNA or 2'-O,4'-C-ethylidene-bridged nucleic acid (ENA)). For mature double-stranded miRNA mimics, the length of the double-stranded region can vary between 13–33, 18–24, or 21–23 nucleotides. miRNAs can also contain a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. The sequence of a miRNA can be the first 13-33 nucleotides of the pre-miRNA. Alternatively, the sequence of a miRNA can be the last 13-33 nucleotides of the pre-miRNA.

[0255] The preparation of miRNA mimics can be achieved by any method known in the art, such as chemical synthesis or recombination methods.

[0256] As can be understood from the above explanation, cell-miRNA contact can be achieved by transfecting cells with, for example, mature double-stranded miRNA, pre-miRNA, or pri-miRNA.

[0257] The pre-miRNA sequence can contain 45-90, 60-80, or 60-70 nucleotides.

[0258] pri-miRNA sequences can contain 45-30,000, 50-25,000, 100-20,000, 1,000-1,500, or 80-100 nucleotides.

[0259] Antisense —Antisense molecules are single-stranded RNAs designed to block or suppress gene expression by specifically hybridizing with the mRNA of a gene. Downregulation of cucurbitacin biosynthetic enzymes can be achieved using antisense polynucleotides capable of specifically hybridizing with mRNA transcripts encoding enzymes in the cucurbitacin biosynthetic pathway.

[0260] When designing antisense molecules that can effectively downregulate the expression of enzymes in the cucurbitacin biosynthesis pathway, two important aspects of antisense technology must be considered. The first aspect is delivering the oligonucleotide to the cytoplasm of the appropriate cell, and the second aspect is designing an oligonucleotide that can specifically bind to a specific mRNA in the cell and inhibit its translation.

[0261] Existing technologies have taught numerous delivery strategies that can be used to efficiently deliver oligonucleotides to a variety of cell types [see, for example, Jääskeläinen et al., Cell Mol Biol Lett. (2002) 7(2):236-7; Gait, Cell Mol Life Sci. (2003) 60(5):844-53; Martino et al., J Biomed Biotechnol. (2009) 2009:410-260; Grijalvo et al., Expert Opin Ther Pat. (2014) 24(7):801-19; Falzarano et al., Nucleic Acid Ther. (2014) 24(1):87-100; Shilakari et al., Biomed Res Int. (2014) 2014: 526-391; Prakash et al., Nucleic Acids Res. (2014) 42(13):8796-807 and Asseline et al., J Gene Med. (2014) 16(7-8):157-65]

[0262] Furthermore, based on thermodynamic cycles that take into account the energy of structural changes in target mRNA and oligonucleotides, algorithms can be used to identify sequences with the highest predicted binding affinity to target mRNA [see, for example, Walton et al., Biotechnol Bioeng 65: 1-9 (1999)]. Such algorithms have been successfully used to implement antisense techniques in cells.

[0263] In addition, several techniques for designing and predicting specific oligonucleotide efficiencies using in vitro systems have been disclosed (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)).

[0264] Therefore, the development of high-precision antisense design algorithms and various oligonucleotide delivery systems enables ordinary technicians to design and implement antisense techniques suitable for downregulating the expression of known sequences without having to conduct excessive trial and error experiments.

[0265] Suitable antisense oligonucleotides can be constructed to target gene transcripts (e.g., mRNA) and / or fragments of any of the cucurbitacin biosynthesis pathway genes detailed in Table 2 above.

[0266] Nucleic acid agents can also be manipulated at the DNA level, as outlined below.

[0267] Downregulation of any cucurbitacin biosynthesis pathway gene can also be achieved by inactivating the gene (e.g., target genes listed in Table 2, including but not limited to those listed in Table 2) by introducing targeted mutations (e.g., point mutations, deletions, and insertions) involving loss-of-function alterations into the gene structure.

[0268] As used herein, the phrase "loss-of-function alteration" refers to any mutation in the DNA sequence of a gene (e.g., a gene for the cucurbitacin biosynthesis pathway) that leads to a downregulation of the expression level and / or activity of the expression product (i.e., mRNA transcript and / or translated protein). Non-limiting examples of such loss-of-function alterations include: missense mutations, i.e., mutations in which one amino acid residue in a protein is altered by another amino acid residue, thereby eliminating the protein's enzymatic activity; nonsense mutations, i.e., mutations that introduce a stop codon into a protein, such as an early stop codon in a shorter protein that results in a lack of enzymatic activity; frameshift mutations, i.e., mutations typically involving the deletion or insertion of nucleic acids that alter the protein's reading frame and can lead to early termination (e.g., truncated proteins lacking enzymatic activity) by introducing a stop codon into the reading frame, or mutations in longer amino acid sequences (e.g., readthrough proteins) that affect the protein's secondary or tertiary structure and result in nonfunctional proteins lacking the enzymatic activity of non-mutated polypeptides; and readthrough mutations resulting from frameshift mutations or modified stop codon mutations (i.e., when the stop codon is mutated into an amino acid codon), affecting the enzyme activity. Disappearance; promoter mutation, i.e., a mutation in the promoter sequence, usually located at the 5' of the transcription start site, which leads to downregulation of a specific gene product; regulatory mutation, i.e., a mutation in the upstream or downstream or internal region of a gene that affects the expression of the gene product; deletion mutation, i.e., a mutation in the gene sequence that removes the coding nucleic acid, which may lead to frameshift mutations or reading frame-compliant mutations (the deletion of one or more amino acid codons within the coding sequence); insertion mutation, i.e., a mutation that inserts coding or non-coding nucleic acid into the gene sequence, which may lead to frameshift mutations or reading frame-compliant insertion of one or more amino acid codons; inversion, i.e., a mutation that causes an inversion of the coding or non-coding sequence; splicing mutation, i.e., a mutation that causes aberrant or poor splicing; and duplication mutation, i.e., a mutation that causes duplication of the coding or non-coding sequence, which may be reading frame-compliant or may cause a frameshift.

[0269] Depending on the specific implementation plan, loss of gene function alteration may include at least one allele of the gene.

[0270] As used herein, the term "allelic gene" refers to any one or more alternative forms of a locus, all of which are associated with a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0271] According to other specific embodiments, the loss-of-function alteration of a gene involves both alleles of the gene. In this case, for example, a modified cucurbitacin biosynthesis pathway gene may be homozygous or heterozygous. According to this embodiment, homozygosity is a state in which the two alleles at, for example, the modified cucurbitacin biosynthesis pathway locus have the same nucleotide sequence. Heterozygosity refers to the different states of a gene at, for example, the modified cucurbitacin biosynthesis pathway locus.

[0272] Methods for introducing nucleic acid alterations into target genes are well known in the art [see, for example, Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244:1288-1292; Santiago et al., ProcNatl Acad Sci USA (2008) 105:5809-5814; International Patent Applications Nos. WO 2014085593, WO2009071334 and WO 2011146121; U.S. Patents Nos. 8771945, 8586526, 6774279 and UP Patent Application Publications Nos. 20030232410, 20050026157, US20060014264, the contents of which are incorporated herein by reference in their entirety], and include targeted homologous recombination, site-specific recombinases, PB transposases, and genome editing via engineered nucleases. Agents used to introduce nucleic acid alterations into target genes can be of publicly available design sources or commercially available from Transposagen, Addgene, and Sangamo Biosciences.

[0273] The following is a description of various exemplary methods for introducing nucleic acid alterations into a target gene, which can be used according to specific embodiments of the present invention, and agents for carrying out said methods.

[0274] Genome editing using engineered nucleases—this technique refers to a reverse genetics approach that uses engineered nucleases to cut at desired locations in the genome and create specific double-strand breaks, which are then repaired by endogenous cellular processes such as homology-directed repair (HDR) and non-homologous end joining (NFfEJ). NFfEJ directly joins DNA ends with double-strand breaks, while HDR uses homologous sequences as templates to regenerate the missing DNA sequence at the break point. For a specific nucleotide modification to be introduced into the genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases because most restriction endonucleases recognize multiple base pairs on the DNA as their targets, and the probability of recognizing a combination of base pairs being found at many locations in the genome is very high, resulting in multiple cuts not limited to the desired location. To overcome this challenge and generate site-specific single-strand or double-strand breaks, several different types of nucleases have been discovered and bioengineered to date. These nucleases include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system.

[0275] MeganucleaseMacronucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having one or two copies of a conserved LAGLIDADG motif. The four macronuclease families differ significantly from each other in terms of conserved structural elements, and therefore, in terms of DNA recognition sequence specificity and catalytic activity. Macronucleases are typically found in microbial species and possess the unique property of having very long recognition sequences (>14 bp), thus naturally exhibiting specificity for cleavage at target sites. This can be used to generate site-specific double-strand breaks in genome editing. These naturally occurring macronucleases are available to those skilled in the art; however, the number of such naturally occurring macronucleases is limited. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to generate macronuclease variants that recognize unique sequences. For example, various macronucleases have been fused to generate hybrid enzymes that recognize novel sequences. Alternatively, the DNA-interacting amino acids of the macronuclease can be altered to design sequence-specific macronucleases (see, for example, U.S. Patent 8,021,867). Macronucleases can be designed using methods such as those described below: Certo, MT et al., Nature Methods (2012) 9:073-975; U.S. Patent Nos. 8,304,222; 8,021,867; 8,119,381; 8,124,369; 8,129,134; 8,133,697; 8,143,015; 8,143,016; 8,148,098; or 8,163,514, the contents of which are incorporated herein by reference in their entirety. Alternatively, macronucleases with site-specific cleavage features can be obtained using commercially available technologies, such as Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0276] ZFN and TALEN —Two different types of engineered nucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been shown to be effective in generating targeted double-strand breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

[0277] Essentially, ZFN and TALEN restriction endonuclease technologies utilize non-specific DNA-cutting enzymes linked to specific DNA-binding domains (a series of zinc finger domains or TALE repeat sequences, respectively). Restriction endonucleases are typically chosen where the DNA recognition and cleavage sites are separate from each other. The cleavage portion is isolated and then linked to the DNA-binding domain, resulting in an endonuclease with very high specificity for the desired sequence. An exemplary restriction enzyme with this property is Fokl. Furthermore, Fokl has the advantage of requiring dimerization to acquire nuclease activity, meaning a significant increase in specificity, as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered to function only as heterodimers and possess increased catalytic activity. Heterodimer-functionalized nucleases avoid the possibility of unwanted homodimer activity, thereby improving the specificity for double-strand breaks.

[0278] Therefore, for example, to target specific sites, ZFN and TALEN are constructed as nuclease pairs, each member of which is designed to bind an adjacent sequence at the target site. Upon transient expression in cells, the nucleases bind to their target sites (in some embodiments, target sites within the sequence of any gene in the cucurbitacin biosynthesis pathway, such as those detailed in Table 2), and the FokI domain heterodimerizes to produce double-strand breaks. Repair of these double-strand breaks via the non-homologous end joining (NHEJ) pathway most commonly results in small deletions or small sequence insertions. Because each repair performed by NHEJ is unique, using a single nuclease pair can generate a series of alleles with a range of different deletions at the target site. These deletions are typically in lengths ranging from a few to several hundred base pairs, but larger deletions have been successfully generated in cell cultures by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). Furthermore, when a DNA fragment homologous to the target region is introduced together with a nuclease pair, double-strand breaks can be repaired through homology-directed repair to produce specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).

[0279] Although the nuclease portions of both ZFN and TALEN have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFN relies on the Cys2-His2 zinc finger, while TALEN relies on TALE. Both DNA recognition peptide domains are characterized by their natural presence in their proteins in combinatorial form. The Cys2-His2 zinc finger is typically found in repeating sequences spaced 3 bp apart and in various combinations of nucleic acid-interacting proteins. TALE, on the other hand, is found in repeating sequences where the recognition ratio between amino acids and recognized nucleotide pairs is 1:1. Because both zinc fingers and TALE occur in repeating patterns, different combinations can be explored to produce a variety of sequence-specificities. Techniques for preparing site-specific zinc finger endonucleases include, for example, modular assembly (where zinc fingers associated with triplet sequences are sequentially linked to cover the desired sequence), OPEN (in bacterial systems, low-strength selection is performed first on peptide domains and triplet nucleotides, followed by high-strength selection on peptide combinations and the final target), and bacterial one-hybrid screening of zinc finger libraries. ZFNs can also be designed and commercially available, for example, from Sangamo Biosciences™ (Richmond, CA).

[0280] Methods for designing and obtaining TALENs are described, for example, in Reyon et al., Nature Biotechnology 2012.5;30(5):460-5; Miller et al., Nat Biotechnol. (2011) 29: 143-148; Cermak et al., NucleicAcids Research (2011) 39(12): e82; and Zhang et al., Nature Biotechnology (2011) 29(2): 149-53. Mayo Clinical has introduced a recently developed web-based program called Mojo Hand for designing TAL and TALEN constructs for genome editing applications (accessible via www(dot)talendesign(dot)org). TALENs can also be designed and commercially available from sources such as Sangamo Biosciences™ (Richmond, CA).

[0281] CRISPR-CasSystems—Many bacteria and archaea possess endogenous RNA-based adaptive immune systems that can degrade the nucleic acids of invading bacteriophages and plasmids. These systems consist of clustered, regularly spaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR-associated (Cas) genes that encode protein components. CRISPR RNA (crRNA) contains short sequence segments homologous to specific viruses and plasmids and acts as a guide for Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogens. *Streptococcus pyogenes* (… Streptococcus pyogenes Studies of the type II CRISPR / Cas system have shown that three components form an RNA / protein complex and work together to exert sequence-specific nuclease activity: Cas9 nuclease, crRNA containing 20 base pairs homologous to the target sequence, and trans-activating crRNA (tracrRNA) (Jinek et al., Science (2012) 337: 816–821). Further evidence shows that a synthetic chimeric guide RNA (gRNA) composed of a fusion of crRNA and tracrRNA can guide Cas9 to cleave DNA targets complementary to crRNA in vitro. Transient expression of Cas9 with synthetic gRNA has also been demonstrated to generate double-strand break-targeting guide RNAs in various species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a,b; Jinek et al., 2013; Mali et al., 2013).

[0282] The CRIPCR / Cas system used for genome editing contains two distinct components: gRNA and a nuclease, such as Cas9.

[0283] gRNA is typically a 20-nucleotide sequence encoding a combination of a target homologous sequence (crRNA) and endogenous bacterial RNA, which links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA / Cas9 complex is recruited to the target sequence via base pairing between the gRNA sequence and the complement genomic DNA. For successful Cas9 binding, the genomic target sequence must also contain the correct prespacer adjacent motif (PAM) sequence immediately following the target sequence. The binding of the gRNA / Cas9 complex positions Cas9 to the genomic target sequence, allowing Cas9 to cleave both strands of DNA, resulting in a double-strand break. Like ZFN and TALEN, the double-strand breaks generated by CRISPR / Cas can undergo homologous recombination or NHEJ.

[0284] The Cas9 nuclease has two functional domains: RuvC and HNH, each cleaving a different DNA strand. When both domains are active, Cas9 causes double-strand breaks in genomic DNA.

[0285] A significant advantage of CRISPR / Cas is that the system's high efficiency, coupled with its ability to readily generate synthetic gRNA, allows for the simultaneous targeting of multiple genes. Furthermore, most cells carrying mutations exhibit biallelic mutations in the target genes.

[0286] However, the apparent flexibility of base pairing interactions between the gRNA sequence and the genomic DNA target sequence allows for an imperfect match with the target sequence that will be cleaved by Cas9.

[0287] Modified forms of Cas9 enzymes containing a single inactive catalytic domain, RuvC- or HNH-, are called "nicks." Because they contain only one active nuclease domain, Cas9 nicks cleave only one strand of the target DNA, producing a single-strand break or "nick." Single-strand breaks or nicks are typically repaired rapidly via the HDR pathway using an intact complementary DNA strand as a template. However, two adjacent nicks on opposing strands introduced by a Cas9 nick are considered double-strand breaks, a situation commonly referred to as a "double-nick" CRISPR system. Double nicks can be repaired via NHEJ or HDR, depending on the desired effect on the gene target. Therefore, if specificity and reduced off-target effects are critical, double nicks can be generated using a Cas9 nick by designing two gRNAs with closely adjacent target sequences on opposing strands of the genomic DNA, thus reducing off-target effects, since either gRNA alone would produce a nick that does not alter the genomic DNA.

[0288] Modified forms of the Cas9 enzyme containing two inactive catalytic domains (inactive Cas9 or dCas9) lack nuclease activity but can still bind to DNA specifically based on gRNA. dCas9 can be used as a platform for DNA transcription regulators, activating or repressing gene expression by fusing the inactive enzyme with known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

[0289] Currently, there are many publicly available tools that can help select and / or design target sequences, as well as bioinformatically determined unique gRNA lists for different genes in different species, such as Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes, and the CRISPR Optimal Target Finder.

[0290] Therefore, in some implementations, the downregulation of genes involved in the cucurbitacin biosynthesis pathway is achieved through genome editing, particularly via CRISPR.

[0291] To use the CRISPR system, both gRNA and Cas9 need to be expressed in target cells. The insert vector can contain two cassettes located on a single plasmid, or the two cassettes can be expressed by two separate plasmids. CRISPR plasmids are commercially available, such as the px330 plasmid from Addgene.

[0292] "Hit and run" or "in-out"—this involves a two-step recombination approach. In the first step, an insert vector containing dual positive / negative selection marker cassettes is used to introduce the desired sequence change. This insert vector contains a single, contiguous region homologous to the target locus and is modified to carry the desired mutation. The targeting construct is linearized at a site within the homologous region using a restriction enzyme, electroporated into cells, and positively selected to isolate homologous recombinants. These homologous recombinants contain a locally repetitive sequence separated by an intermediate vector sequence (including the selection cassette). In the second step, the target clone is negatively selected to identify cells that have lost the selection cassette between the repetitive sequences through intrachromosomal recombination. The locally recombinant event removes the repetitive sequence, and depending on the recombination site, the allele retains the introduced mutation or reverts to the wild type. The end result is the introduction of the desired modification without retaining any exogenous sequence.

[0293] The "double-replacement" or "tag and exchange" strategy involves a two-step selection process similar to hit-and-run techniques, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homologous arms is used to insert a double positive / negative selection cassette near the site where the mutation is to be introduced. After electroporation and positive selection, clones with homologous targeting are identified. Next, a second targeting vector containing a region homologous to the desired mutation is electroporated into the targeting clone, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

[0294] Site-specific recombinases—Cre recombinase derived from P1 phage and recombinase derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae Flp recombinases are site-specific DNA recombinases, each recognizing a unique 34-base-pair DNA sequence (referred to as "Lox" and "FRT," respectively). Sequences flanking either Lox or FRT sites can be easily removed during Cre or Flp recombinase expression via site-specific recombination, respectively. For example, the Lox sequence consists of an asymmetric eight-base-pair spacer region flanked by 13-base-pair inverted repeats. Cre recombines the 34-base-pair Lox DNA sequence by binding to the 13-base-pair inverted repeats and catalyzing strand cleavage and rejoining within the spacer region. The spacer region, created by the staggered DNA cleavage produced by Cre, is separated by 6 base pairs to produce an overlapping region, which acts as a homology sensor to ensure that recombination only occurs at recombination sites with identical overlapping regions.

[0295] Essentially, site-specific recombinase systems provide a means of removing the selection cassette after homologous recombination. This system also allows for the generation of conditionally altered alleles that can be inactivated or activated in a temporary or tissue-specific manner. Notably, Cre and Flp recombinases leave behind a 34-base-pair Lox or FRT “scar.” The retained Lox or FRT sites typically remain within introns or 3' UTRs of the modified locus, and current evidence suggests that these sites generally do not significantly interfere with gene function.

[0296] Therefore, Cre / Lox and Flp / FRT recombination involves the introduction of a targeting vector with 3' and 5' homologous arms containing the target mutation, two Lox or FRT sequences, and a selection cassette typically located between the two Lox or FRT sequences. Positive selection is applied, and homologous recombinants containing the targeting mutation are identified. Transient expression of Cre or Flp, combined with negative selection, results in the excision of the selection cassette, selecting cells that have lost this cassette. The final targeting allele contains a Lox or FRT scar containing the exogenous sequence.

[0297] Transposase—As used herein, the term “transposase” refers to an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome.

[0298] As used herein, the term "transposon" refers to a mobile genetic element containing a nucleotide sequence that can move to different locations within the genome of a single cell. In this process, transposons can cause mutations and / or alter the amount of DNA in the cell's genome.

[0299] Many transposon systems that can also transpose in cells, such as vertebrates, have been isolated or engineered, such as “Sleeping Beauty” [Izsvák and Ivics, Molecular Therapy (2004) 9, 147–156], piggyBac [Wilson et al., Molecular Therapy (2007) 15, 139–145], Tol2 [Kawakami et al., PNAS (2000) 97 (21): 11403–11408], or “Frog Prince” [Miskey et al., Nucleic Acids Res.12.1, (2003) 31(23): 6873–6881]. Typically, DNA transposons move from one DNA site to another in a simple cut-and-paste manner. Each of these elements has its own advantages; for example, Sleeping Beauty is particularly suitable for region-specific mutagenesis, while Tol2 has the highest tendency to integrate into expressed genes. The hyperactive system can be used for both Sleeping Beauty and piggyBac. Most importantly, these transposons have different target site preferences, thus allowing the introduction of sequence alterations into overlapping but distinct gene sets. Therefore, using more than one element is particularly preferred to achieve the best possible gene coverage. The basic mechanisms are the same across different transposases, so we will use piggyBac (PB) as an example for description.

[0300] PB is a 2.5 kb insect transposon, originally isolated from the white armyworm (Trichoplusia ni). The PB transposon consists of the transposase PBase and asymmetric terminal repeats flanking it. PBase recognizes the terminal repeats and induces transposition via a cut-and-paste mechanism, preferentially transposing the tetranucleotide sequence TTAA into the host genome. After insertion, the TTAA target site is repeated, resulting in the PB transposon flanking this tetranucleotide sequence. During migration, PB typically precisely excises itself to reconstruct a single TTAA site, restoring the host sequence to its pre-transfection state. After excision, PB can transpose to a new location or be permanently lost from the genome.

[0301] Typically, transposase systems provide an alternative means of removing the selection cassette after homologous recombination has ceased, similar to the use of Cre / Lox or Flp / FRT. Thus, for example, a PB transposase system comprises introducing a targeting vector with 3' and 5' homologous arms containing the target mutation, two PB terminal repeats at an endogenous TTAA sequence site, and a selection cassette located between the PB terminal repeats. Positive selection is applied, and homologous recombinants containing the target mutation are identified. Transient removal of PBase expression combined with negative selection results in the excision of the selection cassette, selecting cells that have lost it. The final targeting allele contains the introduced mutation and no exogenous sequence.

[0302] In order for PB to be used to introduce sequence changes, there must be a natural TTAA site relatively close to the location where the specific mutation is to be inserted.

[0303] Genome editing using a recombinant adeno-associated virus (rAAV) platform—this genome editing platform is based on rAAV vectors, which enable the insertion, deletion, or substitution of DNA sequences in the genome of living mammalian cells. The rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule that is positively or negatively sensed and is approximately 4.7 kb in length. These single-stranded DNA viral vectors have high transduction rates and the unique property of stimulating endogenous homologous recombination in the absence of double-stranded DNA breaks in the genome. Those skilled in the art can design rAAV vectors to target desired genomic loci and perform overall and / or subtle endogenous genetic alterations in cells. The advantage of rAAV genome editing is that it targets individual alleles and does not result in any off-target genomic changes. rAAV genome editing technologies are commercially available, such as the rAAV GENESIS™ system from Horizon™ (Cambridge, UK).

[0304] It should be understood that the agent may be a mutagen that induces random mutations and may be selectively applied to cells that exhibit downregulated expression levels and / or activity of genes involved in cucurbitaceous biosynthetic pathways.

[0305] Mutagens can be, but are not limited to, genetic agents, chemical agents, or radiation agents. For example, a mutagen can be ionizing radiation, such as, but not limited to, ultraviolet light, gamma rays, or alpha particles. Other mutagens can include, but are not limited to: base analogs that can cause replication errors; deamination agents such as nitrous acid; intercalating agents such as ethidium bromide; alkylating agents such as bromouracil; transposons; natural and synthetic alkaloids; bromine and its derivatives; sodium azide; psoralen (e.g., in combination with ultraviolet radiation). Mutagens can be chemical mutagens, such as, but not limited to: ICR191, 1,2,7,8-diepoxyoctane (DEO), 5-aza-C, N-methyl-N-nitrosoguanidine (MNNG), or ethyl methanesulfonate (EMS).

[0306] Methods for qualitative analysis and detection of sequence alterations are well known in the art, including but not limited to DNA sequencing, electrophoresis, enzyme-based mismatch detection assays and hybridization assays, such as PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blot hybridization, Northern blot hybridization and dot blot hybridization analysis.

[0307] Sequence alterations in specific genes can also be identified at the protein level using methods such as chromatography, electrophoresis, immunoassays such as ELISA, as well as Western blot analysis and immunohistochemistry.

[0308] Furthermore, those skilled in the art can readily design knock-in / knock-out constructs that include positive and / or negative selection markers for the efficient selection of transformed cells that have undergone homologous recombination events with the construct. Positive selection provides a means of enriching clonal populations that have already absorbed exogenous DNA. Non-limiting examples of such positive markers include glutamine synthase, dihydrofolate reductase (DHFR), and markers conferring antibiotic resistance, such as neomycin, hygromycin, puromycin, and blastcin S resistance cassettes. Negative selection markers are necessary for selection against random integration and / or elimination marker sequences (e.g., positive markers). Non-limiting examples of such negative markers include herpes simplex virus thymidine kinase (HSV-TK), which converts ganciclovir (GCV) into cytotoxic nucleoside analogs, hypoxanthine phosphoribosyltransferase (HPRT), and adenine phosphoribosyltransferase (ARPT).

[0309] It should be understood that the RNA silencing agents in some embodiments of the present invention are not limited to molecules containing only RNA, but also include chemically modified nucleotides and non-nucleotides.

[0310] In some embodiments, the RNA silencing agents provided herein may be functionally associated with a “cell-penetrating peptide.” As used herein, a “cell-penetrating peptide” is a peptide comprising a short (about 12-30 residues) amino acid sequence or functional motif that confers energy-independent (i.e., non-endocytic) transport properties associated with the transport of membrane-permeable complexes across the cytoplasmic and / or nuclear membranes. The cell-penetrating peptide in the membrane-permeable complex used in some embodiments of the invention preferably comprises at least one nonfunctional cysteine ​​residue, which is free or derived to form a disulfide bond with a double-stranded ribonucleic acid that has been modified for such a linkage. Representative amino acid sequences conferring such properties are found in U.S. Patent No. 6,348,185, the contents of which are incorporated herein by reference. The cell-penetrating peptide in some embodiments of the invention preferably includes, but is not limited to, penetratin, transporters, pIsl, TAT (48-60), pVEC, MTS, and MAP.

[0311] As taught herein, reducing the expression of genes involved in the cucurbitacin biosynthesis pathway in bitter cucurbitaceous plants can lead to the accumulation of tetracyclic triterpenes lacking chemical motifs (hydroxyl, acyl, carbonyl, etc.) that can interfere with the glucosylation of UDP-glucuronyltransferase (UGT), thereby allowing UGT to glucosylate them. Therefore, in some embodiments of the invention, a plant or plant cell (e.g., a bitter cucurbitaceous plant or plant cell, or a non-cucurbitaceous plant or plant cell expressing genes involved in the cucurbitacin biosynthesis pathway) is provided, wherein the expression of at least one gene in the cucurbitacin biosynthesis pathway is reduced, and the plant or plant cell contains at least one tetracyclic triterpenes capable of being glucosylated by UDP-glucuronyltransferase (UGT). In some embodiments, the UGT is a plant UGT. In a specific embodiment, the plant UGT is a cucurbitaceous plant UGT. In a specific embodiment, the cucurbitaceous plant UGT is the Siraitia grosvenori UGT. PCTIl2015 / 050933 discloses a non-limiting UGT suitable for glucosylating tetracyclic triterpenes capable of glucosylation.

[0312] Table 3a provides a non-restrictive list of amino acid sequences of the UGT enzyme proteins from S. grosvenorii and the nucleic acid sequences encoding them.

[0313] Table 3a:

[0314] Therefore, in some embodiments, the cucurbitaceous plant or plant cell of the present invention contains at least one tetracyclic triterpenoid capable of being glucosylated by UGT, wherein the UGT is selected from the group consisting of UGTs having an amino acid sequence as shown in any one of SEQ ID NO. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 and 149 or their functional equivalents. In other embodiments, the UGT is selected from the group consisting of UGT enzymes encoded by polynucleotides having a nucleic acid sequence of any one of SEQ ID NO. 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 and 138. In a specific embodiment, the UGT is a UGT having an amino acid sequence selected from SEQ ID NO. 139, 140, 141, 142, 143, 144, 147 and 149.

[0315] It is understood that plants with endogenous UGT enzyme activity can catalyze the glucosylation of newly generated glucosylated tetracyclic triterpenes produced by the method of the present invention. In the case of such glucosylation, previously bitter plants (e.g., bitter cucurbitaceae plants) can be transformed into non-bitter plants (e.g., non-bitter cucurbitaceae plants) by means of the accumulation of non-bitter tetracyclic triterpenoid glucosides.

[0316] Similarly, it can be understood that transforming cells (plant or non-plant cells) to express or overexpress UGT enzyme activity may also catalyze the glucosylation of newly generated glucosylated tetracyclic triterpenes produced by the method of the present invention. Therefore, plant UGT can be endogenous to the plant or plant cells, or it can be heterologous UGT expressed in the transformed plant or plant cells.

[0317] Therefore, in some embodiments, a method for generating glucosylated tetracyclic triterpenes is provided, comprising glucosylating at least one glucosylating tetracyclic triterpenes newly generated by the method of the present invention by contacting at least one tetracyclic triterpenes with UGT. When glucosylated by UGT, the tetracyclic triterpenes become glucosylated tetracyclic triterpenes.

[0318] Some glucosylated tetracyclic triterpenes retain the bitterness associated with cucurbitacin-like molecules—for example, although glucosylated, the mogroside precursor mogroside alcohol, as well as mogrosides I and II, remain bitter. Further glucosylation of the mogroside precursor (e.g., generating mogrosides IV, V, or VI) produces a sweet, rather than bitter, glucosylated tetracyclic triterpenes. Therefore, in some embodiments, the glucosylated tetracyclic triterpenes of the present invention are non-bitter glucosylated tetracyclic triterpenes. In specific embodiments, the glucosylated tetracyclic triterpenoid of the present invention is mogroside, such as any one of mogroside I, II, III, IV, V, and VI, including but not limited to single or combined mogrosides, including but not limited to: mogroside I-A1, mogroside I-E1, mogroside IIA, mogroside IIB, mogroside HE, 7-oxomogroside HE, 11-oxomogroside A1, mogroside III, mogroside III A1, mogroside III A2, mogroside IIIx, 11-deoxymogroside III, mogroside IV, mogroside IV-A, 11-oxomogroside IV-A, symmonoside I, mogroside V, 7-oxomogroside V, 11-oxomogroside V, mogroside VI, and mogroside VII.

[0319] In some embodiments of the method, the one or more mogrosides are selected from mogroside I-A1, mogroside I-E1, mogroside IIA, mogroside IIB, mogroside HE, 7-oxomogroside HE, 11-oxomogroside A1, mogroside III, mogroside III A1, mogroside III A2, mogroside IIIx, 11-deoxymogroside III, mogroside IV, mogroside IV-A, 11-oxomogroside IV-A, symmonoside I, mogroside V, 7-oxomogroside V, 11-oxomogroside V, mogroside VI, and mogroside VII.

[0320] In some embodiments of the method, the one or more mogrosides are mogrool.

[0321] In some embodiments of the method, the one or more mogrosides are mogroside I-Al.

[0322] In some embodiments of the method, the one or more mogrosides are mogroside I-E1.

[0323] In some embodiments of the method, the one or more mogrosides are mogroside IIA.

[0324] In some embodiments of the method, the one or more mogrosides are mogroside IIB.

[0325] In some embodiments of the method, the one or more mogrosides are mogroside HE.

[0326] In some embodiments of the method, the one or more mogrosides are 7-oxomogroside HE.

[0327] In some embodiments of the method, the one or more mogrosides are 11-oxomogroside A1.

[0328] In some embodiments of the method, the one or more mogrosides are mogroside III.

[0329] In some embodiments of the method, the one or more mogrosides are mogroside III Al.

[0330] In some embodiments of the method, the one or more mogrosides are mogroside III A2. In some embodiments of the method, the one or more mogrosides are mogroside IIIx.

[0331] In some embodiments of the method, the one or more mogrosides are 11-deoxymogroside III.

[0332] In some embodiments of the method, the one or more mogrosides are mogroside IV.

[0333] In some embodiments of the method, the one or more mogrosides are mogroside IV-A.

[0334] In some embodiments of the method, the one or more mogrosides are 11-oxomogroside IV-A.

[0335] In some embodiments of the method, the one or more mogrosides are symmenidine I.

[0336] In some embodiments of the method, the one or more mogrosides are mogroside V.

[0337] In some embodiments of the method, the one or more mogrosides are 7-oxomogroside V.

[0338] In some embodiments of the method, the one or more mogrosides are 11-oxomogroside V.

[0339] In some embodiments of the method, the one or more mogrosides are mogroside VI.

[0340] In some embodiments of the method, the one or more mogrosides are mogroside VII.

[0341] The plants or plant cells of the present invention can be propagated. Plant propagation is carried out after the downregulation of the cucurbitacin biosynthesis pathway in the plants or plant cells of the present invention is identified. The most common method of plant propagation is through seeds. However, regeneration through seed propagation has drawbacks, namely, the lack of uniformity in crops due to heterozygosity, as seeds are produced by plants through genetic variation controlled by Mendel's laws. Essentially, each seed is genetically different, and each seed will develop its unique traits. Therefore, it is preferable to produce plants that have the same traits and characteristics as the modified parent plants. Therefore, it is preferable to regenerate modified plants through micropropagation, which can rapidly and stably propagate the transformed plants.

[0342] Micropropagation is the process of cultivating a new generation of plants from a single tissue excised from a selected parent plant or cultivar. This method enables the mass propagation of plants with preferred tissues expressing fusion proteins. The resulting new generation of plants is genetically identical to the original plant and possesses all its characteristics. Micropropagation allows for the rapid production of high-quality plant material in a short period and provides rapid propagation of selected cultivars while preserving the original modified plant characteristics. The advantages of cloning plants lie in the speed of plant proliferation and the quality and uniformity of the resulting plants.

[0343] Micropropagation is a multi-stage process that requires changes in culture medium or growth conditions between stages. Therefore, the micropropagation process comprises four basic stages: Stage 1, initial tissue culture; Stage 2, tissue culture proliferation; Stage 3, differentiation and plant formation; and Stage 4, greenhouse cultivation and hardening-off. In Stage 1, the initial tissue culture stage, a tissue culture system is established and certified as pollution-free. In Stage 2, the initial tissue culture is proliferated until a sufficient number of tissue samples are obtained to meet production targets. In Stage 3, the tissue samples cultured in Stage 2 are divided and cultivated into individual seedlings. In Stage 4, the transformed seedlings are transferred to a greenhouse for hardening-off, where the plants' light tolerance is gradually increased, enabling them to grow in a natural environment.

[0344] While stable modifications are currently preferred, some embodiments of the present invention also envision transient modifications of leaf cells, meristematic cells, or the entire plant.

[0345] In some embodiments, modified plant or plant cell lysates are provided. Such cell lysates may contain the modified cucurbitacin biosynthetic pathway enzymes of the present invention, the tetracyclic triterpenes of the present invention, and / or products of further enzymatic reactions of the tetracyclic triterpenes (e.g., via UGT glucosylation to tetracyclic triterpenoid glucosides). Therefore, the cell lysates can be used to recover products of the modified cucurbitacin biosynthetic pathway, or to recover enzyme peptides of the modified cucurbitacin biosynthetic pathway. Methods for extracting active enzyme peptides and tetracyclic triterpenes are well known in the art.

[0346] The glucosylation of the tetracyclic triterpenes of the present invention, which are capable of glucosylation, can be carried out in plants or other cells that naturally express one or more UGTs that catalyze the glucosylation of tetracyclic triterpenes, or in plants or other cells modified to express one or more UGTs that catalyze glucosylation. Modification of cells to express UGTs suitable for glucosylating tetracyclic triterpenoid substrates in the mogroside biosynthesis pathway is described in detail in PCT IL2015 / 050933 et al.

[0347] Furthermore, the glucosylation of the tetracyclic triterpenes of the present invention, which can be glucosylated by UGT, can be carried out in a cell-free synthetic system. In some embodiments, glucosylation is carried out in multiple steps (e.g., enzymatic reactions) and can be achieved entirely in plants or other cells, entirely in a cell-free system, or in a combination of enzymatic reactions occurring in plants or plant cells and cell-free reactions.

[0348] Therefore, in some embodiments, the tetracyclic triterpenes of the present invention are glucylated in cells, such as plant cells, or in non-plant (e.g., yeast, bacteria, insect, or animal cells). In other embodiments, the tetracyclic triterpenes of the present invention are glucylated in a cell-free system.

[0349] The cell lysates of the present invention can also be used alone or in combination with other suitable substrates or enzymes for the cell-free synthesis of the modified tetracyclic triterpenes of the present invention.

[0350] In addition, tetracyclic triterpenes produced by downregulation of gene expression in the cucurbitacin biosynthesis pathway can be identified, and their desired biological or chemical properties, in addition to serving as glucosylation substrates for UGT, can be determined.

[0351] Therefore, a composition rich in tetracyclic triterpenoids is provided, said tetracyclic triterpenoids being produced by downregulation of gene expression in the cucurbitacin biosynthesis pathway according to the present invention. In some embodiments, the composition is rich in tetracyclic triterpenoid glucosides produced by downregulation of gene expression in the cucurbitacin biosynthesis pathway according to the present invention. In other embodiments, the composition is rich in tetracyclic triterpenoid glucosides produced by contact with UGT enzyme according to the method of the present invention.

[0352] In some embodiments, the composition can be used as a sweetener and flavor modifier.

[0353] The sweeteners and flavor modifiers disclosed herein may have concentrations ranging from 0.1% to up to 99%. In some embodiments, the sweeteners and / or flavor modifiers comprise compositions disclosed herein at concentrations of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, etc. 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99%, as well as intermediate concentrations.

[0354] Compounds and compositions (e.g., sweeteners and flavor enhancers) can be obtained from any part of a plant, such as fruit, leaves, or roots, which can be harvested and sorted by hand or mechanically, preserved (refrigerated, pH altered, dried), or processed immediately. Compositions and compounds can also be obtained from cell or tissue cultures and then processed as detailed herein.

[0355] The following are typical processing methods, used as a single process or in various combinations, aimed at producing specific ingredients (e.g., sweeteners and / or flavor enhancers) suitable for the desired application: The plant or its parts are crushed, juiced, pressed, dried, powdered, or extracted (water, water-ethanol mixture, ultrasonic treatment, Soxhlet extraction, supercritical fluid, etc.), and then subjected to at least one of the following treatments using an optimized combination of methods known in the industry: coarse separation / filtration, fine separation (microfiltration / ultrafiltration / nanofiltration, centrifugation, decantation, etc.), concentration (membrane, porous column, evaporation, etc.), separation and purification (affinity chromatography, pH adjustment, etc.), concentration, stabilization, formulation (including drying (thermal drying, vacuum drying, freeze drying, powdering, etc.)) or wet formulation.

[0356] In some embodiments, the compositions of the present invention are consumable compositions.

[0357] Consumer products include all food items, including but not limited to: cereal products, rice products, cassava products, sago products, baked goods, biscuit products, pastry products, bread products, confectionery products, dessert products, chewing gum, bubble gum, chocolate, ice, honey products, molasses products, yeast products, baking powder, salt and spice products, savory products, mustard products, vinegar products, sauces (condiments), tobacco products, cigars, cigarettes, processed foods, cooked fruits and vegetables, meat and meat products, jelly, jam, fruit sauce, egg products, milk and dairy products. Yogurt, cheese products, butter and butter substitutes, milk substitutes, soy products, edible oils and fats, pharmaceuticals, beverages, carbonated beverages, alcoholic beverages, beer, soft drinks, mineral water, soft drinks and other non-alcoholic beverages, fruit juices, fruit juices, coffee, artificial coffee, tea, cocoa (including forms requiring reconstruction), food extracts, plant extracts, meat extracts, flavorings, sweeteners, nutritional supplements, gelatin, pharmaceutical and non-pharmaceutical gums, tablets, lozenges, drops, emulsions, elixirs, syrups and other beverage preparations and combinations thereof.

[0358] The compositions of this invention can be used in a variety of consumer products, including but not limited to water-based consumer products, solid-dried consumer products, and dairy products, dairy-derived products, and dairy product substitutes. In some embodiments, the compositions are food products.

[0359] Water-based consumer products include, but are not limited to, beverages, water, aqueous beverages, fortified / slightly sweetened water beverages, mineral water, carbonated beverages, non-carbonated beverages, carbonated water, still water, soft drinks, non-alcoholic beverages, beer, wine, spirits, fruit juice beverages, fruit juice, vegetable juice, broth beverages, coffee, tea, black tea, green tea, oolong tea, herbal tea, cocoa (water-based), tea-based beverages, coffee-based beverages, cocoa-based beverages, syrups, frozen fruit, frozen fruit juice, water-based ice, fruit ice, ice cream, sauces, salad dressings, condiments, soups, and beverage plant-based ingredients (whole or ground) or instant powders for reconstitution (coffee beans, ground coffee, instant coffee, cocoa beans, cocoa powder, instant cocoa, tea leaves, instant tea powder). In some embodiments, the composition may be a beverage, such as Coca-Cola®, etc.

[0360] Solid dry consumer products include, but are not limited to, cereals, baked goods, biscuits, bread, breakfast cereals, cereal bars, energy bars / nutrition bars, granola, cakes, cookies, crackers, donuts, muffins, pastries, sweets, chewing gum, chocolate, fondant, hard candy, marshmallows, compressed sheets, snack foods and plant materials (whole or ground), as well as instant powders used for reconstitution as described above.

[0361] In some products, higher concentrations of sweeteners are often required to achieve a similar sweetness intensity, such as in dairy products, dairy-derived products, and dairy substitutes. Dairy-derived foods contain milk or milk proteins. Dairy substitutes contain proteins from plant sources (soybeans, rice, and other protein-rich plant materials) instead of milk proteins derived from mammalian milk. Dairy products, dairy-derived products, and dairy substitutes include, but are not limited to, milk, liquid milk, fermented dairy products, fermented and non-fermented milk-based beverages, lactic acid bacteria fermented dairy products, yogurt, yogurt-based beverages, smoothies, lassi, milkshakes, acidified milk, acidified milk beverages, buttermilk, kefir, milk-based beverages, milk / juice blends, fermented milk beverages, ice cream, desserts, sour cream, dips, salad dressings, farm cheese, frozen yogurt, soy milk, rice milk, soy milk beverages, and rice milk beverages.

[0362] Milk includes, but is not limited to, whole milk, skim milk, condensed milk, lightly condensed milk, reduced-fat milk, low-fat milk, and milk solids (which may be fat or non-fat).

[0363] The compositions of the present invention may also include one or more additional flavoring ingredients, such as additional sweeteners. A non-limiting list of suitable flavoring ingredients that can be used in the compositions of the present invention includes: sucrose, fructose, glucose, high fructose corn syrup, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharin, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), stevia glycosides, rebaudioside A, steviol glycosides, steviol glycosides, and trilobtain.

[0364] As used in this article, the term "about" refers to 10%.

[0365] The terms “comprises, comprising,” “includes, including,” “having,” and their variations refer to “including but not limited to.”

[0366] The term "composed of" means "including and limited to".

[0367] The term "consistently of" means that a composition, method, or structure may include additional ingredients, steps, and / or portions, but only if the additional ingredients, steps, and / or portions do not substantially alter the fundamental and novel characteristics of the claimed composition, method, or structure.

[0368] As used herein, unless the context clearly indicates otherwise, the singular forms “a” and “the” include plural indicators. For example, the terms “compound” or “at least one compound” can include a variety of compounds, including mixtures thereof.

[0369] Throughout this application, various embodiments of the invention may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the invention. Therefore, the scope description should be considered to have specifically disclosed all possible sub-scopes and individual numerical values ​​within those scopes. For example, a scope description such as 1 to 6 should be considered to have specifically disclosed sub-scopes, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those scopes, such as 1, 2, 3, 4, 5, 6. This applies regardless of the breadth of the scope.

[0370] In this document, any reference to a numerical range refers to all numbers (fractions or integers) cited within that range. The phrases “ranging / ranges between the first and second indicator numbers” and “ranging / ranges from the first indicator number” are used interchangeably in this document and are intended to include the first and second indicator numbers and all fractions and integers in between.

[0371] As used herein, the term “method” refers to the manner, means, techniques and steps for accomplishing a given task, including but not limited to those manner, means, techniques and steps known to or readily developed from known manner, means, techniques and steps by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0372] When referring to a particular sequence listing, it should be understood that such reference also includes sequences that substantially correspond to their complementary sequences, such as minor sequence variations caused by, for example, sequencing errors, cloning errors, or other alterations resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 / 50 nucleotide, or less than 1 / 100 nucleotide, or less than 1 / 200 nucleotide, or less than 1 / 500 nucleotide, or less than 1 / 1000 nucleotide, or less than 1 / 5,000 nucleotide, or less than 1 / 10,000 nucleotide.

[0373] It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually or in any suitable sub-combination or as suitably as in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment does not function without those elements.

[0374] Each of the various embodiments and aspects of the invention described above and claimed in the claims section is experimentally supported in the following examples.

[0375] Example

[0376] The following embodiments, together with the above description, illustrate some implementations of the invention in a non-limiting manner.

[0377] Example I: Elucidation of genes involved in the cucurbitacin biosynthesis pathway

[0378] Experimental Methods: A novel bitter watermelon cultivar was identified through candidate gene testing (PI673173, USDA). This cultivar exhibited similar yield and other horticultural characteristics to cultivated watermelon varieties, but its fruit was extremely bitter. This was attributed to the activation of the cucurbitacin metabolic pathway due to the expression of the bHLH transcription factor, a characteristic absent in cultivated sweet watermelons. Silencing of genes encoding the bHLH transcription factor is crucial for the evolution of edible cultivated watermelon (C. lanatus) and is considered a major domestication event in the evolution of edible watermelons (Zhou, Y. et al., 2016). Figure 11 The image shows bitter Hawkesbury watermelon fruit (top) and cultivated non-bitter Hawkesbury sweet watermelon. Expression levels (FPKM, Fragments Per Kilobase of Transcript per Million Mapped Reads, a simple expression level normalization method that normalizes reads based on gene length and the total number of mapped reads) are also shown, indicating that the controlling transcription factor (encoded by ClCG01G003370) determining cucurbitacin synthesis is expressed in bitter Hawkesbury watermelon but not in the sweet variety.

[0379] To identify the gene responsible for the enzymatic step in the production of bitter cucurbitacin E (the main cucurbitacin in bitter watermelon), gene expression was analyzed using RNaseq technology. Gene expression was studied in fruits of two watermelon lines (sweet Hawkesbury watermelon PI636601 and a bitter Hawkesbury watermelon mutant PI673137). Furthermore, the two lines were crossed, and the F1 hybrids were self-pollinated to produce an F2 population. Fruits were sampled approximately 30 days after flowering. Cucurbitacin content in the fruit samples was analyzed. In short, cucurbitacin was extracted from plant tissues in methanol, passed through a 45-micron filter, and analyzed by HPLC, as described by Itkin, M. et al. (2016). Proceedings of the National Academy of Sciences , 113 (47), pp. E7619-E7628) or as Matsuo, K. et al. (1999). Journal of agricultural and food chemistry , 47 (7), as described on pages 2755-2759.

[0380] RNA was extracted from fruits of F2 generation plants with cucurbitacin E content >50 μg / g fresh weight (“high cucurbitacin”) and fruits of F2 generation plants with undetectable cucurbitacin E content (“low cucurbitacin”), as well as fruits from both parental lines. RNA was sampled from fruits of at least six isolates. Fruit samples used for gene expression analysis included: 1. Flesh tissue of Hawkesbury watermelon (PI635601) during the flowering and development period and approximately 30 days after flowering; 2. Flesh tissue of bitter Hawkesbury watermelon (PI673173) during the flowering and development period and about 30 days after flowering; 3. Pulp tissue of non-bitter F2 isolates approximately 30 days after flowering; 4. Pulp tissue of bitter F2 isolates approximately 30 days after flowering, and 5. In addition, wild bitter watermelons ( Citrullus amarus Fruit samples were taken during the flowering period of species (PI 596653).

[0381] Using Spectrum TMRNA was extracted using a plant RNA extraction kit (Merck, product number STRN50) and an RNaseq library, and Illumina sequencing was performed by Macrogen (Seoul, South Korea). Standard bioinformatics tools were used to analyze the sequencing reads and align them to two watermelon reference genomes: the watermelon (Charleston Grey) reference genome v2.5 (ClCG#) listed in Table 2 and the watermelon 97103 reference genome v2.5 (Cla#) listed in Table 2 (both available at www.cucurbitgenomics.org / v2 / ). In some cases, the gene sequences listed in the two reference genomes were not identical, and the actual listed sequences were the product of manual annotation, taking into account the RNA sequences derived from gene expression analysis.

[0382] Extensive transcriptomic analysis was performed on bitter watermelon fruits that accumulate cucurbitacin and on non-bitter cultivated watermelon fruits to screen for a very limited number of candidate genes responsible for characterizing additional hydroxylation, oxidation, dehydrogenation, and acetylation of molecular modifications that distinguish cucurbitacin from the less modified precursor of mogrol—a sweet mogroside. These candidate genes were selected from a large gene family present in the watermelon genome and, along with brief annotations predicting gene function and the gene expression levels (FPKM) in the different non-bitter and bitter fruits listed above, are shown in Table 4.

[0383] Based on differential expression between non-bitter and bitter watermelon fruits (Table 4), only 21 genes encoding cytochrome P450 enzymes were selected from a family size of over 200 annotated cyp450 genes in the watermelon genome. Similarly, based on differential expression (see Table 4, candidate genes are shown in bold), three oxygenase genes were screened from a gene family of over 30 members, while the oxidoreductase gene family, including dehydrogenases and related enzyme families, contains over 600 members. Furthermore, some candidate genes were screened based on their overall expression rather than their differential expression between non-bitter and bitter watermelon fruits. Chromosomal localization, whether within a metabolic cluster or tandemly with other candidate genes, was also a basis for screening some candidate genes. The screening criteria for each gene are listed in the "Comments" section of Table 4: genes screened based on differential expression are shown in bold and categorized as "Expressed, highly expressed in bitter tissues." Genes screened based on general expression are shown in italics and categorized as "Expressed, likely housekeeping enzymes involved in cucurbitacin synthesis." Genes selected based on chromosome location are underlined and categorized as "part of a possible metabolic gene cluster" or marked with capital letters and categorized as "not expressed but linked to a candidate gene".

[0384] Using this strategy, a concise and focused list of candidate genes for functional characterization and silencing screening was constructed, representing approximately 5% of gene families that may be involved in this pathway and ~0.1% of genes in the watermelon genome (Table 4).

[0385] Table 4:

[0386] Note: "Watermelon genes" refers to candidate cucurbitacin biosynthesis pathway genes based on the watermelon (Charleston Grey) genome v2.5, which can be obtained from the website "cucurbitgenomics(dot)org".

[0387] “RNA expression (FPKM)” refers to the relative abundance of transcripts of each watermelon gene in the flesh of low-cucurbitacin (non-bitter) and high-cucurbitacin (bitter) Hawkesbury melon fruits at flowering time (“flowering”) or flowering + 30 days (“medium size”), as well as the relative abundance of transcripts at flowering + 30 days from bitter and non-bitter F2 (hybrids between bitter and non-bitter Hawkesbury watermelons) isolates and from wild bitter watermelon (C. amarus).

[0388] For example, the expression pattern of ClCG01G014540, a candidate encoding cytochrome P450, clearly shows that the gene's expression level is undetectable in non-bitter parents or non-bitter F2 isolates, but highly expressed in bitter parents, bitter F2 isolates, and bitter wild watermelon (C. amarus). Therefore, this technique can be used to identify promising candidate genes that may downregulate cucurbitacin biosynthesizers.

[0389] Example 2: Modification of genes involved in the cucurbitacin biosynthesis pathway

[0390] Different methods were used to study gene modifications in the cucurbitacin biosynthesis pathway to determine phenotypes, particularly gene function and their effects on metabolite accumulation: a. Cucurbitaceae varieties with high cucurbitacin accumulation in their roots transformed by *Agrobacterium rhizogenes* resulted in the formation of transgenic hairy roots. This could be, for example, the bitter watermelon PI673137, but could also include other watermelon varieties with non-bitter fruits (because cucurbitacin can accumulate in their root tissues). Control (unmodified hairy roots) accumulate cucurbitacin E—a 25-acetylated cucurbitane compound (see...). Figure 1 (Table in the table).

[0391] b. Transformation of high cucurbitacin-accumulating varieties (e.g., bitter watermelon PI673137) with Agrobacterium tumefaciens resulted in the formation of transgenic modified watermelon plants. Analysis of the cucurbitacin composition revealed modifications in the transgenic fruit, roots, and other plant tissues, with control non-transgenic plants and their respective tissues accumulating native cucurbitacins.

[0392] c. The function of the candidate gene can also be determined by overexpressing the candidate gene in a novel yeast strain engineered to synthesize the precursor cucurbitacinol (see [link to article]). Figure 5 This yeast strain and the same strategy were successfully used by Itkin et al. (2016) to identify *Siraitia grosvenorii* (monk fruit). Siratia grosvenorii The C11 Cyp450 hydroxylase of cucurbitadienol was described in Davidovich-Rikenati et al. (Yeast, 2015). Briefly, yeast cells were transformed with plasmids expressing cucurbitadienol and the target gene. Single transformed yeast colonies were cultured in SC-URA medium. After induction with 2% w / v galactose for 2 days, cells were collected, the precipitate was destroyed with hot 20% w / v KOH and 50% v / v EtOH, and extracted twice with an equal volume of n-hexane. The hexane extract was evaporated and resuspended in 1 ml MeOH for analysis of the cucurbitadienol product using LC–TOF–APCIMS.

[0393] Hairy root culture: In a 50 ml Falcon tube, apply 2% sodium hypochlorite to a peeled watermelon. Citrullus lanatus The seeds were sterilized and shaken for 20 min. Then, the seeds were rinsed 3-4 times in ddH2O. The wet seeds were placed on sterile petri dishes in a sterile workbench to allow the surface to dry. The air-dried seeds were placed on plates containing ½ Murashige and Skoog (MS) agar supplemented with 1.5% sucrose, sealed with sealing film (Merck KGaA, Darmstadt, Germany), and placed in a controlled climate chamber: irradiated for 10 days at 79 μE, 25°C, 80% humidity, and a 16-hour photoperiod.

[0394] Transformation of Rhizobium rhizobia using plasmids containing relevant silencing constructs via electroporation. Rhizhobium rhizogenesThe strain K599 was recovered on LB medium at 28°C for 4 hours and then plated on LB agar supplemented with spectinomycin (50 μg / ml). In the examples below, the silencing construct of cucurbitacin synthase was contained in the following plasmid pOmega1:ClCG06g001600-RFP. Single colonies were selected for each transformation and grown overnight at 28°C with constant stirring (240 rpm) in liquid LB supplemented with spectinomycin. Subsequently, 50 mL of Rhizobium rhizogenes ( R. rhizogenes The suspension was placed on solid YEB medium supplemented with spectinomycin and incubated at 28°C for two days. The colonies were then resuspended in MS buffer supplemented with acetylsyl syringone to a concentration of 100 mM. Finally, the OD600 of the suspension was adjusted to 0.6.

[0395] Ten days after the watermelon cotyledons germinate, use root nodules (Bacillus rhizobium) to promote root growth. R. rhizogenes Transformation. The cotyledons are soaked in rhizobium (a fungus that promotes root growth). R. rhizogenes Inoculation was performed by puncturing the cotyledon with a sterile syringe needle. Four incisions were made perpendicular to the midrib on the abaxial surface of the cotyledon. The cotyledons were then placed abaxially face down on a ½ MS (3% sucrose) agar plate without antibiotics and incubated in the dark for two days. The inoculated cotyledons were then transferred to a ½ MS (3% sucrose) agar plate supplemented with cefotaxime (400 μg / ml) and kanamycin (100 μg / ml) and incubated under light for one week. The cotyledons were subsequently transferred to a new plate with half the cefotaxime concentration.

[0396] Transformed hairy roots expressing mRFP began to appear after three weeks of tissue culture. These roots were excised from the cotyledons and subcultured every two weeks with cefotaxime and kanamycin. In each subculture step, the concentration of cefotaxime was reduced by 50% until the antibiotic was completely discontinued, while the concentration of kanamycin remained constant.

[0397] result:Each of the 42 genes was silenced using a siRNA strategy, and the accumulated metabolites were then measured. siRNA constructs were developed using a hairpin silencing strategy and constructed using standard GoldenBraid technology (as described by Fusaro et al., EMBORep. 2006). In short, this method uses type II restriction endonucleases and T4 DNA ligases to assemble multiple DNA sequences in a single reaction. Type II restriction endonucleases such as BsaI and BsmBI cleave outside their recognition sites, producing unique non-palindromic overhangs. DNA fragments can be assembled in a specific order, where fragments with annotated overhangs are ligated together. The siRNA sequences are constructed by ligating a promoter sequence to approximately 300 bp of a sequence corresponding to a portion of the gene to be silenced, followed by an intron sequence, and finally the inverse complementary sequence of the gene fragment preceding the intron. Gene-specific target sequences were synthesized based on the gene coding sequences shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 112, 115, 118, 121, and 124 (corresponding to the sequences listed in Table 3 of this document).

[0398] Example 3: Modifying cucurbitacin yield using hairy root cultures: silencing cucurbitacin synthase

[0399] To develop a protocol for silencing candidate genes in the hairy roots of bitter watermelon, the effect of silencing the first key step in cucurbitacin synthesis was tested via cucurbitacin synthase encoded by ClCG06g001600 (SEQ ID NO: 150). The silencing construct contained a unique gene target sequence (SEQ ID NO: 152) and was constructed as described above for *Agrobacterium rhizhogenes*. Hairy roots were cultured essentially as described above, and after ~6 weeks of growth, roots were extracted in methanol, and cucurbitacin content was analyzed by LCMS, as described by Itkin et al. (2016).

[0400] Using a hairy root culture experiment, modifications to the cucurbitacin biosynthesis pathway were observed. Silencing the cucurbitacin synthase gene (ClCG06g001600) significantly reduced the cucurbitacin content accumulated in transgenic hairy roots. Figure 10 This study confirmed the effectiveness of hairy root cultures in studying gene silencing in the cucurbitacin biosynthesis pathway.

[0401] Example 4: Modifying cucurbitacin yield using hairy root cultures: silencing candidate acetyltransferase genes

[0402] Using a unique target gene sequence SEQ ID NO: 126 to silence the candidate acetyltransferase gene (ClCG06g001610, SEQ ID NO: 124) resulted in the emergence of a previously undetected non-acetylated form of cucurbitacin I, namely cucurbitacin E. Figures 7A-7D (and 12A-12E).

[0403] Example 5: Modifying cucurbitacin yield using hairy root cultures: silencing candidate dehydrogenase genes

[0404] Using unique target gene sequences SEQ ID NO: 81, SEQ ID NO: 87 and SEQ ID NO: 75 to silence candidate alcohol dehydrogenase genes (ClCG01g018250, SEQ ID NO: 79; ClCG03G002490, SEQ ID NO: 85 and C1CG09G009760, SEQ ID NO: 73) resulted in enhancement of the previously poorly performing product (C32H46O8), which corresponds to cucurbitacin with two additional hydrogen atoms and was co-eluted with cucurbitacin B (Fig. 13B-13H), which is the hydrogenated form of cucurbitacin E.

[0405] Example 6: Improving Cucurbitacin Yield Using Hairy Root Culture: Silencing the Candidate Cytochrome P450 Gene

[0406] Using unique target gene sequences SEQ ID NO: 9, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 51 to silence candidate cytochrome P450 genes (ClCG01G014540, SEQ ID NO: 7; ClCG06G001580, SEQ ID NO: 31; ClCG06G001590, SEQ ID NO: 34; ClCG06G001620, SEQ ID NO: 37 and ClCG10G012530, SEQ ID NO: 49), respectively, previously poorly performing cucurbitacin compounds were produced with altered quality, indicating the loss of oxygen atoms (presumed molecular formulas C30H46O5, C30H46O3, C30H44O3 and C30H46O2) (Figures 14B-14L).

[0407] Example 7: Detection of cytochrome P450 gene function using yeast

[0408] Overexpression of the candidate cytochrome P450 gene (ClCG06G001570, SEQ ID NO: 28) in a yeast strain that produces cucurbitadienol resulted in the production of oxidized cucurbitadienol products, whose mass spectrometry showed the putative molecular formulas as C30H50O2 and C30H48O3 (Figures 15A-15F).

[0409] Example 8: Modification of Cucurbitacin Production in Bitter Watermelon

[0410] Using a hairy root model, an effective silencing method for cucurbitacin biosynthesis pathway genes was demonstrated. Watermelon varieties accumulating cucurbitacin were transformed with silencing constructs targeting cucurbitacin biosynthesis pathway genes. These genes included, for example, cytochrome P450 genes, dehydrogenase-oxidoreductase family genes (alcohol dehydrogenase, FAD-binding berberine enzyme family genes, NAD(P)-binding Rothman folding enzyme genes, peroxidase, polyketide dehydrase genes, proline dehydrogenase genes, and short-chain dehydrogenase / reductase genes), and HXXXD-type acyltransferase-like enzyme genes.

[0411] Gene silencing can be achieved by introducing a construct containing a gene-coding siRNA sequence into wounded plant tissue using Agrobacterium tumefaciens or other transformation technologies, thereby producing transgenic watermelon fruits. The gene is shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 112, 115, 118, 121, and 124, corresponding to the sequences listed in Table 3, as shown above. Furthermore, other silencing techniques, including genome editing strategies, can be used to silence the expression of target genes.

[0412] Gene silencing using CRISPR technology was achieved by designing guide RNAs targeting specific genes. Guide RNAs were designed to target regions within the gene's promoter or coding sequence. Plasmids containing appropriate guide RNAs and the Cas9 gene were transformed into watermelon plants in different combinations. RNA-seq of the transformed tissues was performed to verify the silencing of the target gene.

[0413] Preliminary research focuses on silencing genes involved in the cucurbitacin biosynthesis pathway, which have been successfully silenced in watermelon tissues using a hairy root model system. Therefore, silencing constructs targeting the following genes were initially designed: watermelon cytochrome P450 genes ClCG01G014540 (SEQ ID NO: 7), ClCG06G001570 (SEQ ID NO: 28), ClCG06G001580 (SEQ ID NO: 31), ClCG06G001590 (SEQ ID NO: 34), ClCG06G001620 (SEQ ID NO: 37) and ClCG10G012530 (SEQ ID NO: 49); watermelon alcohol dehydrogenase genes ClCG09G009760 (SEQ ID NO: 73) and ClCG03G002490 (SEQ ID NO: 79); watermelon FAD-binding berberine family gene ClCG03G002490 (SEQ ID NO: 85); and NAD(P)-binding Rossmann folding gene ClCG01G014570. (SEQ ID NO: 88); HXXD type acyltransferase-like gene ClCG06G001610 (SEQ ID NO: 124).

[0414] After transformation, watermelon tissues were cultured and analyzed to identify modifications to the gene function of the cucurbitacin biosynthetic pathway (e.g., chromatography and spectroscopy of tissue extracts) and to characterize the profiles of cucurbitacin and cucurbitacin derivatives.

[0415] Then, the successfully transformed watermelon tissues were cultured to obtain candidate plants and fruits that retained gene silencing and displayed a profile of modified cucurbitacin and cucurbitacin derivatives.

[0416] Then, plant tissues with the desired modified cucurbitacin biosynthesis pathway genes were further modified using one or more additional silencing constructs, and the effects of silencing multiple cucurbitacin biosynthesis pathway genes were evaluated to identify the profiles of modified cucurbitacins and cucurbitacin derivatives, providing potential substrates for the glucosylation of UGT enzymes.

[0417] Example 9: Modification of cucurbitacin production in other Cucurbitaceae and non-Cucurbitaceae plants

[0418] The silencing constructs were designed for orthologous genes of the target genes in species other than watermelon, as listed in Table 2. Examples of orthologous genes from melon (Cucumis melo), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo), listed in Table 2a, were used to design silencing constructs for these species. Silencing in other species can be achieved using the method described in Example 8, i.e., via Agrobacterium tumefaciens (…). A. tumefaciens Transformation using other transformation technologies, and / or gene editing.

[0419] Therefore, the results presented herein demonstrate that key genes involved in the cucurbitacin biosynthesis pathway can be identified and downregulated using the methods of this invention, leading to alterations in the tetracyclic triterpenoid profile of Cucurbitaceae plants or plant cells, specifically reduced expression of these genes. Silencing key functional genes in the cucurbitacin biosynthesis pathway can provide tools for modifying tetracyclic triterpenoid molecules, thereby generating novel substrates suitable for multiple glycosylation processes.

[0420] The applicant intends to incorporate, by reference, all publications, patents, and patent applications mentioned herein, in their entirety, as if each publication, patent, or patent application had been expressly acknowledged as incorporated herein by reference. Furthermore, any reference or designation of any reference in this application shall not be construed as an admission that the reference is prior art. Section headings used in this application shall not be construed as necessarily limiting. In addition, any priority documents of this application are incorporated herein by reference in their entirety.

Claims

1. A method for producing a plant or plant cell with a modified cucurbitacin content, the method comprising downregulating the expression of at least one gene in the plant or plant cell that facilitates the biosynthesis of cucurbitacin, thereby modifying the expression of cucurbitacin in the plant or plant cell.

2. A method for producing a plant or plant cell with a modified cucurbitacin content, the method comprising culturing the plant or plant cell of claim 1, wherein the plant or plant cell has the expression of at least one modified gene for a cucurbitacin biosynthesis pathway.

3. The method according to claim 1 or 2, wherein the downregulation is achieved through genome editing.

4. A plant or plant cell modified to reduce the expression of at least one gene in the cucurbitacin biosynthesis pathway, wherein the cucurbitaceous plant or plant cell is obtainable according to the method of claim 1.

5. The plant or plant cell according to claim 4, wherein the plant or plant cell is a superior plant or plant cell.

6. The plant or plant cell according to claim 4, wherein the plant or plant cell is a hybrid plant or plant cell.

7. The plant or plant cell according to claim 4, wherein the plant or plant cell is an inbred plant or plant cell.

8. An inbred plant or plant cell having a nucleic acid sequence alteration in at least one gene in the cucurbitacin biosynthesis pathway.

9. A superior plant or plant cell having a nucleic acid sequence alteration in at least one gene in the cucurbitacin biosynthesis pathway.

10. A hybrid plant or plant cell having a nucleic acid sequence alteration in at least one gene in the cucurbitacin biosynthesis pathway.

11. The plant or plant cell according to any one of claims 4-10, wherein the plant or plant cell comprises at least one tetracyclic triterpenoid capable of being glucosylated by UDP-glucuronyltransferase (UGT).

12. The plant or plant cell according to claim 11, wherein the UGT is a plant UGT.

13. The plant or plant cell according to claim 11, wherein the UGT is selected from Siraitia grosvenorii UGT, and the Siraitia grosvenorii UGT is selected from: UGT74-345-2, UGT73-348-2, UGT94-289-1, UGT73-327-2, UGT73-251-5, UGT73-251-6, UGT75-281-2, UGT85-269-4, UGT85-269-1, UGT94-289-2, and UGT94-289-3.

14. The plant or plant cell according to claim 11, wherein the UGT is a UGT having an amino acid sequence selected from SEQ ID NO. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 and 149.

15. The plant or plant cell according to claim 11, wherein the UGT is encoded by a polynucleotide having a nucleotide sequence selected from SEQ ID NO. 128, 129, 130, 131, 132, 133, 135 and 137.

16. An extract of a plant or plant cell according to any one of claims 4-11, comprising at least one tetracyclic triterpenoid capable of being glucosylated by UGT.

17. The method according to any one of claims 1-3, or the plant or plant cell according to any one of claims 4-15, or the extract according to claim 16, wherein the plant or plant cell is a bitter cucurbitaceae plant species.

18. The method according to any one of claims 1-3, or the plant or plant cell according to any one of claims 4-15, or the extract according to claim 16, wherein the plant or plant cell is a species that naturally expresses the at least one cucurbitacin biosynthesis pathway gene.

19. The method according to any one of claims 1-3, or the plant or plant cell according to any one of claims 4-15, or the extract according to claim 16, wherein the plant or plant cell is a species genetically modified to express at least one gene for the cucurbitacin biosynthesis pathway.

20. The method according to any one of claims 1-3, or the plant or plant cell according to any one of claims 4-15, or the extract according to claim 16, wherein the plant or plant cell is Iberisamara plant or plant cell.

21. The method according to any one of claims 1-3, or the plant or plant cell according to any one of claims 4-15, or the extract according to claim 16, wherein the plant is selected from cultivated bitter cucurbitaceae plants and uncultivated cucurbitaceae plants.

22. The method or plant or plant cell according to claim 21, wherein the cultivated bitter cucurbitaceae plant is common watermelon (Citrullus vulgaris), bitter Hawkesbury watermelon, cucumber (Cucumis sativus), or zucchini (Cucurbita pepo).

23. The method, plant, or plant cell according to claim 21, wherein the uncultivated bitter cucurbitaceae plant is selected from uncultivated bitter melon, uncultivated bitter cucumber, and uncultivated bitter watermelon.

24. The method, plant, or plant cell according to claim 17, wherein the uncultivated bitter cucurbitaceae plant is selected from species of the genera *Cucumis*, *Citrullus*, *Momordica*, and *Cucurbita*.

25. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is a gene selected from the genes in Table 2.

26. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-25, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is an (2OG) and Fe(II) dependent oxygenase gene.

27. The method, plant, or plant cell according to claim 26, wherein the (2OG) and Fe(II)-dependent oxygenase genes have nucleic acid sequences selected from SEQ ID NO: 64, 67, and 70.

28. The method, plant, or plant cell of claim 26, wherein the downregulation of the (2OG) and Fe(II)-dependent oxygenase genes is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 66, 69, and 72.

29. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is a cytochrome P450 gene.

30. The method or plant or plant cell according to claim 29, wherein the cytochrome p450 gene has a nucleic acid sequence selected from SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61.

31. The method, plant, or plant cell according to claim 29, wherein the cytochrome p450 gene has a nucleic acid sequence selected from SEQ ID NO: 7, 28, 31, 34, 37, and 49.

32. The method or plant or plant cell of claim 29, wherein the downregulation of the cytochrome p450 gene is achieved by targeting nucleic acid sequences selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63.

33. The method, plant, or plant cell of claim 29, wherein the downregulation of the cytochrome p450 gene is achieved by targeting a nucleic acid sequence selected from SEQ ID NO: 9, 30, 33, 36, 39, and 51.

34. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is an FAD-binding berberine gene.

35. The method, plant, or plant cell according to claim 34, wherein the FAD-binding berberine gene has a nucleic acid sequence as shown in SEQ ID NO:

85.

36. The method, plant, or plant cell of claim 34, wherein the downregulation of the FAD-binding berberine gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO:

87.

37. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is an NAD(P)-binding Rossmann folding gene.

38. The method, plant, or plant cell according to claim 37, wherein the NAD(P)-binding Rossmann fold gene has a nucleic acid sequence as shown in SEQ ID NO: 88 or 91.

39. The method, plant, or plant cell according to claim 37, wherein the NAD(P)-binding Rossmann fold gene has a nucleic acid sequence as shown in SEQ ID NO:

88.

40. The method, plant, or plant cell of claim 37, wherein the downregulation of the NAD(P)-binding Rossmann fold gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO: 91 or 94.

41. The method, plant, or plant cell of claim 37, wherein the downregulation of the NAD(P)-binding Rossmann fold gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO:

91.

42. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is an HXXD type acyltransferase-like protein gene.

43. The method, plant, or plant cell according to claim 42, wherein the HXXD type acyltransferase-like protein gene has a nucleic acid sequence selected from SEQ ID NO: 118, 121, and 124.

44. The method, plant, or plant cell according to claim 42, wherein the HXXD type acyltransferase-like protein gene has a nucleic acid sequence as shown in SEQ ID NO:

124.

45. The method, plant, or plant cell of claim 42, wherein the downregulation of the HXXD type acyltransferase-like protein gene is achieved by targeting a nucleic acid sequence selected from SEQ ID NO: 120, 123, and 126.

46. ​​The method, plant, or plant cell of claim 42, wherein the downregulation of the HXXD type acyltransferase-like protein gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO:

126.

47. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is a polyketide cyclase / hydratase gene.

48. The method, plant, or plant cell of claim 47, wherein the polyketide cyclase / hydrase gene has a nucleic acid sequence as shown in SEQ ID NO:

109.

49. The method, plant, or plant cell of claim 47, wherein the downregulation of the polyketide cyclase / hydrase gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO:

111.

50. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is a proline dehydrogenase gene.

51. The method, plant, or plant cell according to claim 50, wherein the proline dehydrogenase gene has a nucleic acid sequence as shown in SEQ ID NO:

112.

52. The method or plant or plant cell of claim 50, wherein the downregulation of the proline dehydrogenase gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO:

114.

53. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is a short-chain dehydrogenase / reductase gene.

54. The method, plant, or plant cell according to claim 53, wherein the short-chain dehydrogenase / reductase gene has a nucleic acid sequence as shown in SEQ ID NO:

115.

55. The method or plant or plant cell of claim 53, wherein the downregulation of the short-chain dehydrogenase / reductase gene is achieved by targeting a nucleic acid sequence as shown in SEQ ID NO:

117.

56. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is an alcohol dehydrogenase gene.

57. The method, plant, or plant cell according to claim 56, wherein the alcohol dehydrogenase gene has a nucleic acid sequence selected from SEQ ID NO: 73, 76, 79, and 82.

58. The method, plant, or plant cell according to claim 56, wherein the alcohol dehydrogenase gene has a nucleic acid sequence selected from SEQ ID NO: 73 and 79.

59. The method, plant, or plant cell of claim 56, wherein the downregulation of the alcohol dehydrogenase gene is achieved by targeting a nucleic acid sequence selected from SEQ ID NO: 75, 78, 81, and 84.

60. The method, plant, or plant cell of claim 56, wherein the downregulation of the alcohol dehydrogenase gene is achieved by targeting a nucleic acid sequence selected from SEQ ID NO: 75 and 81.

61. The method according to any one of claims 1-3, the plant or plant cell according to any one of claims 4-15 and 21-24, or the extract according to claim 16, wherein the at least one cucurbitacin biosynthesis pathway gene is a peroxidase gene.

62. The method, plant, or plant cell according to claim 61, wherein the peroxidase gene has a nucleic acid sequence selected from SEQ ID NO: 94, 97, 100, 103, and 106.

63. The method, plant, or plant cell of claim 61, wherein the downregulation of the peroxidase gene is achieved by targeting a nucleic acid sequence selected from SEQ ID NO: 96, 99, 102, 105, and 108.

64. A method for producing glucosylated tetracyclic triterpenes, the method comprising glucosylating at least one glucosylating tetracyclic triterpenes of the plant or plant cells of claim 11 by contacting the at least one tetracyclic triterpenes with UGT, thereby producing glucosylated tetracyclic triterpenes.

65. The method of claim 64, wherein the glucosylated tetracyclic triterpenoid is a non-bitter glucosylated tetracyclic triterpenoid.

66. The method according to claim 64, wherein the glucosylated tetracyclic triterpenoid is mogroside.

67. The method according to any one of claims 64-66, wherein the glucosylation is achieved in the cell.

68. The method of claim 67, wherein the cell is a plant cell.

69. The method of claim 67, wherein the cell is not a plant cell.

70. The method according to any one of claims 64-66, wherein the glucosylation is performed in a cell-free system.

71. The method according to any one of claims 64-70, wherein the UGT is plant UGT.

72. The method according to claim 71, wherein the UGT is selected from Luo Han Guo UGT, and the Luo Han Guo UGT is selected from: UGT74-345-2, UGT73-348-2, UGT94-289-1, UGT73-327-2, UGT73-251-5, UGT73-251-6, UGT75-281-2, UGT85-269-4, UGT85-269-1, UGT94-289-2, and UGT94-289-3.

73. The method of claim 72, wherein the UGT is a UGT having an amino acid sequence selected from SEQ ID NO. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 and 149.

74. The method of claim 71, wherein the UGT is encoded by a polynucleotide having a nucleotide sequence selected from SEQ ID NO. 128, 129, 130, 131, 132, 133, 135 and 137.

75. The method according to any one of claims 71-74, wherein the UGT is a recombinant UGT.

76. A composition comprising a plant or plant cell having modified cucurbitacin biosynthesis pathway gene expression, said plant or plant cell containing a tetracyclic triterpenoid capable of being glucosylated by UGT.

77. The composition according to claim 76, which is rich in tetracyclic triterpenoid glucosides.

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