Expression system and use method therefor and use thereof

Abstract

Provided are an expression system and a use method therefor and a use thereof. The expression system comprises a polynucleotide for increasing the yield of mogroside. The polynucleotide comprises at least one of a plurality of nucleotides encoding proteins for increasing the yield of mogroside, wherein the proteins comprise enzymes and / or functional proteins in a mogroside synthesis pathway, and are selected from at least one of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO.

Description

Expression system and method of use thereof, and use thereof

[0001] This application claims the priority of Chinese application No. 202410467492.9, filed on April 17, 2024, entitled "Expression system and method of use thereof, and use thereof", which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology and plant genetic engineering technology, in particular to an expression system and method of use thereof, and use thereof. BACKGROUND

[0003] With the improvement of people's living standards, metabolic diseases such as diabetes and obesity related to high-sugar diet have become an important threat to human physical health, so high-intensity, low-calorie artificial sweeteners are widely used in the food industry to replace sucrose. In recent years, with the popularity of artificial synthetic sweeteners, their safety has been questioned. Studies have shown that the intake of artificial synthetic sweeteners can lead to people's desire and dependence on sugar, impaired heat compensation leading to appetite stimulation, increased intake, increased body weight, and glucose intolerance. In 1983, a healthy natural non-sugar sweetener, mogroside, was discovered in the fruit of Momordica grosvenori, which is about 300 times sweeter than sucrose and is considered one of the natural, high-intensity, low-calorie sweeteners, and has a variety of pharmacological activities, such as anti-tumor, anti-cancer, anti-diabetic, anti-oxidative, anti-hypoglycemic, and anti-inflammatory effects. In the 1990s, mogroside was approved by the U.S. Food and Drug Administration (FDA) as a food sweetener additive and a substitute for sugar for diabetes and obesity patients, and these characteristics have greatly increased the global demand for mogroside, with good application prospects in the food, beverage, and pharmaceutical industries. However, as the demand for mogroside increases, there is a shortage of supply and high prices in the market. Therefore, there is a great market value in improving the synthetic capacity of mogroside.

[0004] Momordica grosvenori is the fruit of a perennial vine of the Cucurbitaceae family, mainly produced in Guangxi, and is a unique economic and medicinal plant in China. With the increasing demand for mogroside, the demand for Momordica grosvenori will also increase, but currently there are many varieties of Momordica grosvenori in use, and the overall quality of the germplasm is not ideal, the content of mogroside in Momordica grosvenori is not high, and the content of mogroside varies greatly in different varieties and regions. As the main source of mogroside, improving the yield of mogroside in Momordica grosvenori or other plants is the most direct and effective means to solve the shortage of mogroside in the market and the high price.

[0005] In summary, in order to meet the market demand for mogroside, there is an urgent need in the art to develop a method for increasing the yield of mogroside in momordica grosvenori. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide an expression system and a method and use thereof, so as to at least alleviate or even solve at least one of the problems raised in the background art.

[0007] In one aspect of the present application, the present application provides an expression system, comprising a polynucleotide for increasing the yield of mogroside, the polynucleotide comprising at least one of the coding nucleotides of a plurality of proteins for increasing the yield of mogroside; the proteins comprising enzymes and / or functional proteins of the mogroside synthesis pathway, the proteins being selected from at least one of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94 and FTO.

[0008] In some embodiments, the polynucleotide comprises the coding nucleotides of the proteins for increasing the yield of mogroside, the proteins comprising at least one of the following:

[0009] (a) at least CDS and / or FTO;

[0010] (b) at least CDS, EPH1 and CYP87D18.

[0011] In some embodiments, the coding nucleotide of the CDS is as shown in SEQ ID NO: 4, the coding nucleotide of the FTO is as shown in SEQ ID NO: 44, the coding nucleotide of the EPH1 is as shown in SEQ ID NO: 7, and the coding nucleotide of the CYP87D18 is as shown in SEQ ID NO: 8.

[0012] Further, the polynucleotide comprises the coding nucleotides of the proteins for increasing the yield of mogroside, the proteins comprising at least SQE, EPH, DREB1c, CDS, CYP87D18, UGT720 and UGT94.

[0013] Alternatively, the SQE comprises SQE1, SQE2 or a combination thereof, and the EPH comprises EPH1, EPH3 or a combination thereof.

[0014] Further, the polynucleotide comprises SEQ ID NOs: 1-9.

[0015] Further, the polynucleotide comprises at least one of the coding nucleotides of DREB1c, CDS, UGT720, UGT94 and FTO.

[0016] Further, the polynucleotide further comprises at least one of a 5' UTR, a 3' UTR, a transcription termination region, and a polyadenylation region; wherein the 5' UTR is located between a promoter sequence initiating a translation leader sequence and a coding nucleotide.

[0017] Further, the expression system further comprises an expression cassette, and the expression cassette comprises a control expression sequence; wherein the control expression sequence is operably linked to the polynucleotide.

[0018] Further, the control expression sequence comprises a promoter; the promoter is selected from at least one of a constitutive promoter, an inducible promoter, and a tissue-specific promoter; wherein the tissue-specific promoter comprises a fruit-specific promoter.

[0019] Further, the expression cassette is located in a plant transformation vector, the plant transformation vector comprises at least one of a Ti plasmid, a Ri plasmid, a Ti-derived plasmid, and a Ri-derived plasmid of a bacterium; wherein the bacterium comprises Agrobacterium.

[0020] Further, the plant transformation vector has at least one of a selectable marker and a screenable marker.

[0021] Further, the selectable marker comprises at least one of an antibiotic resistance marker and a herbicide resistance marker; the screenable marker comprises at least one of a fluorescent protein gene, a beta-glucuronidase gene, an amylase gene, a luciferase gene, a Xyle gene, and a beta-lactamase gene.

[0022] In another aspect of the present application, the present application provides a use of the above-mentioned expression system in increasing the production of mogrosides in Momordica grosvenori.

[0023] Further, the Momordica grosvenori comprises an entirety or a part of Momordica grosvenori; the variety of the Momordica grosvenori comprises at least one of Qingpiguo, Changtan Guo, Lajiangguo, Dongguaguo, Chashanguo, and Hongmaoguo.

[0024] In yet another aspect of the present application, the present application provides a method for increasing the production of mogrosides in a plant, the method comprising the step of using the above-mentioned expression system.

[0025] Further, the method comprises the step of applying the expression system to a plant, wherein the polynucleotide is expressed in the plant and is codon-optimized.

[0026] Further, the method further comprises: transforming the expression system into a plant cell by a transformation treatment to produce a transformed plant cell; cultivating the transformed plant cell to produce a transformed plant, the plant comprising Momordica grosvenori.

[0027] Further, the variety of the momordica grosvenori includes at least one of Qingpi fruit, Changtan fruit, Lajiang fruit, Dongguafa fruit, Chashan fruit, and Hongmao fruit.

[0028] Further, the transformation treatment includes at least one of protoplast transfection, pollen tube pathway transformation, Agrobacterium-mediated transformation, and biolistic transformation.

[0029] Further, the transformation treatment includes Agrobacterium-mediated transformation, and the Agrobacterium includes a plant transformation vector.

[0030] Further, the content of momordica grosvenori glycosides in the transformed plant is changed; and the momordica grosvenori glycosides include at least one of momordica grosvenori glycoside I, momordica grosvenori glycoside II, momordica grosvenori glycoside III, momordica grosvenori glycoside IV, siamenoside I, and momordica grosvenori glycoside V.

[0031] In another aspect, the present application provides a plant or plant part produced by the above method for increasing the yield of momordica grosvenori glycosides in a plant.

[0032] The present application has at least one of the following beneficial effects:

[0033] 1. The expression system of the present application can be stably expressed in a plant, thereby increasing the content of momordica grosvenori glycosides in the plant;

[0034] 2. The method for increasing the yield of momordica grosvenori glycosides in a plant of the present application can economically and at a high yield produce healthy natural sweeteners (momordica grosvenori glycosides) to replace high-calorie or artificially synthesized sweeteners on the market;

[0035] 3. The method for increasing the yield of momordica grosvenori glycosides in a plant of the present application can obtain a plant with high yield of momordica grosvenori glycosides, and industrial planting of the plant can greatly increase the yield of momordica grosvenori glycosides, reduce the investment in technology, equipment and planting conditions, and reduce the production cost.

[0036] The above summary is intended to illustrate the present application and is not intended to be limiting thereof. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features will be readily apparent to those skilled in the art by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0037] In the drawings, like reference numerals refer to same or similar functionalities throughout the several views. The drawings are not necessarily to scale. It is to be understood that the drawings only depict several embodiments of the disclosure and are not to be considered as limiting the scope thereof.

[0038] Figure 1 is a schematic diagram of the biosynthesis pathway of mogroside in plants;

[0039] Figure 2 is an expression system constructed in Example 1 of the present application;

[0040] Figure 3 is a PCR detection result of each encoding nucleotide of the expression system in Example 1 of the present application;

[0041] Figure 4 is a process of obtaining transformed momordica grosvenori in Example 2 of the present application;

[0042] Figure 5 is a PCR detection result of each encoding nucleotide of the transformed momordica grosvenori in Example 2 of the present application;

[0043] Figure 6 is a qPCR detection of the relative expression amount of nucleotide in the transformed momordica grosvenori in Example 2 of the present application;

[0044] Figure 7 is a detection result of the content of mogroside V in the transformed momordica grosvenori in Example 2 of the present application;

[0045] Figure 8 is an expression system constructed in Example 3 of the present application;

[0046] Figure 9 is a detection result of the content of mogroside V in the transformed momordica grosvenori in Example 3 of the present application. DETAILED DESCRIPTION

[0047] In order to more clearly understand the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be further described in detail. In the following, only certain exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be exemplary in nature rather than limiting.

[0048] In the present application, unless otherwise defined, all technical and scientific terms have the same meaning as understood by one of ordinary skill in the art (such as biology, chemistry, medicine, biochemistry, pharmacy, food and nutrition science).

[0049] In this document, "comprise", "comprising", "include", "including" should be interpreted as inclusive rather than exclusive. The word "by" and its variants should be interpreted exclusively, not inclusively.

[0050] In this document, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents.

[0051] Mogroside

[0052] "Mogroside" is a triterpene saponin compound of cucurbitane type, which is a natural sweetener. More than 60 mogrosides have been identified, which can be classified into mogroside I, mogroside II, mogroside III, mogroside IV, mogroside V and other mogrosides according to the number of sugar groups contained. The central structure of mogrosides is a lantane type of tetracyclic triterpenoid structure, which forms different types of glycosides by connecting different numbers of sugar groups and other groups (such as halogen groups, hydroxyl groups, carboxyl groups, amino groups, nitro groups, ester groups, aldehyde groups, carbonyl groups, carbon-carbon unsaturated bonds). The mogroside described in the present application contains sugar groups of triterpene saponins of cucurbitane type, and common mogrosides include mogroside V (88901-36-4), 11-oxo-mogroside V (126105-11-1), isomogroside V (1126032-65-2), 11-epi-mogroside V (HY-N7605), mogroside IIe (88901-38-6), mogroside III A2 (88901-43-3), mogroside IVe (89590-95-4), mogroside I E1 (88901-39-7), mogroside III (130567-83-8), mogroside IVe (88915-64-4), siamenoside I (126105-12-2).

[0053] Momordica cochinchinensis

[0054] It includes the whole or part of Momordica cochinchinensis plants, such as tissues, cells, protoplasts, cultures, calli, cell masses, grafts, seedlings, clones, micropropagations, cuttings, embryos, pollen, ovules, flowers, leaves, fruits, seeds, asexually propagated plants, roots, stems, root tips, etc., or their derivatives. Their genetic composition should be the same or similar to that of the plants from which they are obtained. In addition, it also includes any developmental stage, such as seedlings, cuttings before and after rooting, mature or immature plants or leaves.

[0055] Control or control Momordica cochinchinensis

[0056] "Control" or "control Momordica cochinchinensis" refers to a reference Momordica cochinchinensis used for comparison of phenotypic changes of Momordica cochinchinensis. The control can include the following types: (a) wild type or natural Momordica cochinchinensis of the same genotype as the subject; (b) Momordica cochinchinensis of the same genotype as the subject, but transformed with an irrelevant construction vector, such as a construction vector containing a marker gene; (c) untransformed Momordica cochinchinensis in the offspring of the subject; (d) the subject itself, but the expression system introduced does not express.

[0057] Expression cassette

[0058] "Expression cassette" refers to a polynucleotide encoding a polypeptide of interest or a variant or fragment thereof, operably linked to at least one control sequence. The expression cassette includes, in the 5'-3' direction, a promoter, a coding region, and a termination region. The regulatory region and / or the coding region can be endogenous or heterologous to the host cell. The expression cassette can contain other coding sequences (e.g., genes) for co-transformation with the organism. Alternatively, the other coding sequences (e.g., genes) can be provided in a separate expression cassette. The expression cassette has multiple restriction sites and / or recombination sites for insertion of the coding polynucleotide of interest or a variant or fragment thereof, under the transcriptional control of the regulatory region such as a promoter.

[0059] Operably linked

[0060] "Operably linked" refers to linkage of two or more elements in a manner that enables the elements to function. For example, operable linkage between a polynucleotide of interest and a promoter is linkage that enables expression of the polynucleotide of interest. Elements that are operably linked need not be contiguous, and can be separated by intervening nucleic acid sequences. When used in reference to the linkage of two protein coding regions, operably linked means that the coding regions are in the same reading frame.

[0061] Control sequence

[0062] "Control sequence" refers to a nucleic acid fragment that affects the transcription or translation of a polynucleotide of interest. Control sequences include promoters, transcription terminators, a variety of transcriptional and translational control elements, and the like. A terminator can be the natural terminator of a transcription initiation region, or a natural or heterologous polynucleotide or variant or fragment thereof, which can be derived from a plant host or other source.

[0063] Promoter

[0064] "Promoter" refers to a nucleic acid sequence that is capable of controlling the transcription of a nucleic acid in a cell. The promoter can be derived from a plant, microorganism (e.g., bacteria, virus), or animal, or can be a synthetic or modified nucleic acid sequence.

[0065] SQE

[0066] "SQE" refers to squalene epoxidase, which catalyzes the epoxidation of squalene to form cyclo-2,3-oxosqualene and 2,3;22,23-dioxygenated squalene.

[0067] CDS

[0068] "CDS" refers to cucurbitadienol synthase, which catalyzes the production of cyclo-24,25-cucurbitadienol from 2,3;22,23-dioxygenated squalene.

[0069] EPH

[0070] "EPH" refers to epoxy hydrolase, which catalyzes the production of 24,25-dihydroxycucurbitadienol from 24,25-cucurbitadienol.

[0071] DREB1c

[0072] "DREB1c" refers to a transcription factor dehydration responsive element binding protein 1c.

[0073] CYP87D18

[0074] "CYP87D18" refers to a cytochrome P450 oxidase, which can oxidize 24,25-dihydroxy-mogroside to mogroside (11,24,25-trihydroxy-mogroside).

[0075] UGT720

[0076] "UGT720" refers to a glucuronosyltransferase, which can catalyze mogroside I to mogroside II.

[0077] UGT94

[0078] "UGT94" refers to a glucuronosyltransferase, which can catalyze mogroside II to mogroside III; mogroside III to mogroside IV or siamenoside I; and mogroside IV and siamenoside I to mogroside V.

[0079] FTO

[0080] "FTO" refers to an AlkB family a-ketoglutarate-dependent hydroxylase, which also has the function of RNA demethylation.

[0081] In one aspect of the present application, the present application provides an expression system comprising a polynucleotide for increasing the yield of mogrosides, and the polynucleotide comprises at least one of the coding nucleotides of a plurality of proteins for increasing the yield of mogrosides; wherein the proteins comprise enzymes and / or functional proteins of the mogroside synthesis pathway, specifically, the proteins are selected from SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, FTO or a combination thereof. That is, the expression system comprises one or more nucleotides encoding enzymes of the mogroside synthesis pathway, and / or one or more nucleotides encoding functional proteins. And the expression system has at least one of the following beneficial effects: increasing the content of mogrosides in momordica fruit, economically and high-yield preparing mogrosides, and reducing production cost.

[0082] By optimizing the synthesis pathway of mogroside in plants, it is a direct and effective method to improve the yield of mogroside in plants. The biosynthesis pathway of mogroside in plants can be divided into two steps, namely the synthesis of mogrol and the glycosylation of mogrol. The synthesis of mogrol includes: acetyl-CoA generates squalene through the mevalonate pathway, squalene is catalyzed by squalene epoxidase (SQE), cucurbitadienol synthase (CDS), and epoxy hydrolase (EPH) to generate 24, 25-dihydroxy cucurbitadienol, and then catalyzed by CYP87D18 of cytochrome P450 superfamily to complete the hydroxylation to generate mogrol. In the glycosylation process of mogrol, mogrol is continuously glycosylated, and mogrol first generates bitter mogroside I and mogroside II under the action of glycosyltransferase UGT720, and then generates sweet mogroside III, mogroside IV, and siamenoside I under the action of glycosyltransferase UGT94, and finally generates mogroside V. Specifically, FIG. 1 shows a schematic diagram of the biosynthesis pathway of mogroside in plants. In addition, by improving the variety of momordica and increasing the yield of momordica, the yield of mogroside can also be indirectly improved. DREB1c or FTO has been confirmed to significantly improve the yield of rice, wheat or potato. Therefore, DREB1c and FTO also have the potential to improve the yield and quality of momordica, thereby indirectly improving the yield of mogroside.

[0083] According to embodiments of the present application, the polynucleotide used in the present application can encode a protein that increases the yield of mogrosides, preferably encode enzymes and / or functional proteins of the mogrosides synthesis pathway. As previously described, the enzymes that encode the mogrosides synthesis pathway include squalene epoxidase (SQE), cucurbitadienol synthase (CDS), epoxy hydrolase (EPH), cytochrome P450 oxidase (CYP87D18), glucuronosyltransferase (UGT720), glucuronosyltransferase (UGT94). And the kind of functional proteins mentioned herein is also not particularly limited, specifically, the functional proteins preferably include proteins that can promote plant growth or increase biological yield. In a specific embodiment, the functional proteins can be selected from the group consisting of dehydration-responsive element binding protein (DREB1c), MYB transcription factor, alpha-ketoglutarate-dependent hydroxylase of AlkB family (FTO) or a combination thereof. That is, the polynucleotide of the present application preferably includes at least one of the coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. And more preferably includes at least one of the coding nucleotides of DREB1c, CDS, UGT720, UGT94, FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides of the CDS protein sequence. As in some embodiments, the polynucleotide includes at least the coding nucleotides of the FTO protein sequence.

[0084] In a specific example, SQE includes SQE1, SQE2 or a combination thereof, and EPH includes EPH1, EPH3 or a combination thereof.

[0085] As in some embodiments, the polynucleotide includes one of the coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. In some embodiments, the polynucleotide includes the coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94 or FTO protein sequences. In some embodiments, the polynucleotide includes the coding nucleotides of EPH protein sequence. In some embodiments, the polynucleotide includes the coding nucleotides of CDS protein sequence. In some embodiments, the polynucleotide includes the coding nucleotides of CYP87D18 protein sequence. In some embodiments, the polynucleotide includes the coding nucleotides of FTO protein sequence. In some embodiments, the polynucleotide includes the coding nucleotides of DREB1c, UGT720 or UGT94 protein sequence.

[0086] As in some embodiments, the polynucleotide includes at least two of the coding nucleotides for SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1 and CYP87D18 protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for CDS and FTO protein sequences.

[0087] As in some embodiments, the polynucleotide includes at least three of the coding nucleotides for SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for CDS, EPH1 and CYP87D18 protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1, CYP87D18 and FTO protein sequences.

[0088] As in some embodiments, the polynucleotide includes at least four of the coding nucleotides for SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1, CYP87D18, CDS and FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1, CYP87D18 and CDS, and SQE, DREBlc, UGT720 or UGT94 protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1, EPH3, CYP87D18 and CDS protein sequences.

[0089] As in some embodiments, the polynucleotide includes at least five of the coding nucleotides for SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1, EPH3, CYP87D18 and CDS, and SQE or DREBlc protein sequences.

[0090] As in some embodiments, the polynucleotide includes at least six of the coding nucleotides for SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequences. As in some embodiments, the polynucleotide includes at least the coding nucleotides for EPH1, EPH3, SQE1, SQE2, SCYP87D18 and CDS protein sequences.

[0091] As in some embodiments, the polynucleotide comprises at least seven of the coding nucleotides of SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequence. As in some embodiments, the polynucleotide comprises at least the coding nucleotides of EPH1, EPH3, SQEl, SQE2, SCYP87D18 and CDS, and UGT720 or UGT94 protein sequence.

[0092] As in some embodiments, the polynucleotide comprises the coding nucleotides of SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequence.

[0093] And the sources of SQE, EPH, DREBlc, CDS, CYP87D18, UGT720, UGT94, FTO protein sequence are not particularly limited, and can each independently be derived from an animal, a plant or a microorganism, but in order to be better expressed in a plant, the above protein sequences are preferably derived from a plant, and the kind of the plant is not particularly limited, and can be selected from the group consisting of Asteraceae, Rosaceae, Caryophyllaceae, Poaceae, Solanaceae, Leguminosae, Vitaceae, Moraceae, Cucurbitaceae or a combination thereof, and specifically can be selected from the group consisting of Momordica grosvenori, Cucumis sativus, Citrullus lanatus, Momordica charantia, Luffa cylindrica, Benincasa cerifera, Cucurbita pepo, Cucurbita maxima or a combination thereof.

[0094] According to the embodiments of the present application, in addition to the coding nucleotides of the above protein sequences, the polynucleotide can further comprise at least one of 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), transcription termination region and polyadenylation region, so as to enable stable expression of the coding nucleic acid. In a specific embodiment, the polynucleotide preferably comprises 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), transcription termination region and polyadenylation region, wherein the 5'UTR is located between the promoter sequence which functions as a starting translation leader sequence and the protein coding nucleotide, the 3'UTR is located after the transcription termination region, and the polyadenylation region is located after the 3'UTR.

[0095] According to embodiments of the present application, the expression system further comprises an expression cassette, and the expression cassette comprises a control expression sequence, which refers to a nucleotide fragment that affects the expression level of a polypeptide encoded by the expression cassette, and the control expression sequence is operably linked to the polynucleotide for better expression of the polynucleotide. In specific embodiments, the control expression sequence comprises, in order from 5'-3' direction, a promoter, a transcription regulatory region, and a termination region, so that the coding sequence of the transcription regulatory region is expressed under the control of the promoter. Specifically, the sequence after the control expression sequence is operably linked to the polynucleotide is, in 5'-3' direction, promoter-polynucleotide-termination region. In a specific example, the transcription regulatory region can be driven by different types of promoters, that is, the type of the promoter in the control expression sequence is not particularly limited, for example, the promoter can be a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In specific embodiments, the promoter preferably refers to a promoter functional in plant cells, wherein the constitutive promoter includes but is not limited to the core promoter of rsyn7 promoter, the core promoter of caulimovirus promoter, plant ubiquitin protein promoter, plant actin promoter, and the promoters modified for weak expression / strong expression. In addition, most of the tissue-specific promoters are expressed in fruits, so the tissue-specific promoter preferably includes a fruit-specific promoter, and in a specific example, the tissue-specific promoter is known in the art, which includes but is not limited to polygalacturonase gene promoter (CkPGA), CFSP-1, HyPRP, E8S, 2A12, E4, PG promoter, or a combination thereof.

[0096] According to embodiments of the present application, in addition to the control expression sequence, the expression cassette can further comprise other coding sequences. Alternatively, the other coding sequences can be provided in multiple expression cassettes. In a specific example, the expression cassette has multiple restriction sites and / or recombination sites for inserting the coding polynucleotide of interest or its variants or fragments, so that it is under the transcriptional regulation of the regulatory region such as the promoter.

[0097] According to embodiments of the present application, the expression cassette can be located in a plant transformation vector for being transferred into a plant by the plant transformation vector. Specifically, the plant transformation vector refers to a DNA molecule for delivering a polynucleotide into a plant cell, and a plurality of exogenous genes or expression cassettes can exist in one transformation vector. Specifically, the type of the plant transformation vector is not particularly limited as long as it can integrate the polynucleotide sequence into the plant genomic DNA, and more specifically, the plant transformation vector includes, but is not limited to, at least one of a Ti plasmid, a Ri plasmid, a Ti-derived plasmid, and a Ri-derived plasmid, and in a preferred example, the bacteria include Agrobacterium. In a specific embodiment, the plant transformation vector of the present application is a binary expression vector that can transfect or transform a plant cell, and specifically can be a plant expression vector, and the plant expression vector is preferably a pCAMBIA vector (selected from pCAMBIA1300, pCAMBIA2300, pCAMBIA3301, or a combination thereof). Any polynucleotide in the expression system of the present application can constitute a plant transformation vector for transferring genetic material (e.g., a polynucleotide or an expression cassette) into a plant. The method of transformation includes, but is not limited to, electroporation, a gene gun, Agrobacterium-mediated, and protoplast transfection.

[0098] According to embodiments of the present application, the plant transformation vector can have at least one of a selectable marker and a screenable marker to facilitate screening of plants that are successfully transformed. Specifically, the type of the selectable marker is not particularly limited, for example, it includes, but is not limited to, an antibiotic resistance marker and a herbicide resistance marker. The type of the screenable marker is also not particularly limited, for example, it includes, but is not limited to, a fluorescent protein gene, a beta-glucuronidase gene, an amylase gene, a luciferase gene, a Xyle gene, and a beta-lactamase gene.

[0099] Specifically, the present application discloses an expression system having a polynucleotide and an expression cassette related to increasing the production of mogrosides, and after the expression system is transformed into a plant, the transformed plant that can express the polypeptide from the expression system is obtained by screening and selecting the type of selectable marker and / or screenable marker on the plant transformation vector, and the production of mogrosides in the transformed plant is significantly increased, for example, the production of at least one of mogroside IV, siamenoside I, and mogroside V is significantly increased. That is, using the expression system of the present application, a healthy natural sweetener can be economically and simultaneously produced at a high yield to replace high-calorie or artificially synthesized sweeteners on the market.

[0100] In another aspect of the present application, the present application provides the use of the above-mentioned expression system in increasing the production of mogrosides in Momordica grosvenori.

[0101] According to embodiments of the present application, the variety of Momordica grosvenori of the present application is not particularly limited, but preferably includes at least one of Qingpi fruit, Changtan fruit, Lajiang fruit, Dongguafa fruit, Chashan fruit, and Hongmao fruit. In addition, the Momordica grosvenori referred to in the present application includes the whole or part of Momordica grosvenori, specifically including the whole or part of any developmental stage, such as the whole Momordica grosvenori or the part (such as tissue, cell, protoplast, culture, callus, cell mass, graft, seedling, clone, micropropagation, cutting, embryo, pollen, ovule, flower, leaf, fruit, seed, vegetative reproduction plant, root, stem, root tip, or derivative thereof) of any developmental stage. It should be noted that the genetic composition of the above-mentioned part of Momordica grosvenori should be the same or similar to that of the Momordica grosvenori from which it is obtained.

[0102] In yet another aspect of the present application, the present application provides a method for increasing the yield of Momordica grosvenori in plants, which comprises the step of using the above-mentioned expression system. Specifically, the present application specifically provides a method for increasing the yield of Momordica grosvenori in plants by means of biotechnology and genetic engineering. That is to say, another object of the present application is to provide a method for obtaining a transgenic plant with high yield of Momordica grosvenori, which can be used to produce a transgenic plant with increased yield of Momordica grosvenori.

[0103] The types and compositions of polynucleotides, expression cassettes, and plant transformation vectors in the expression system have been described in detail above and will not be repeated here.

[0104] According to embodiments of the present application, the method can optionally comprise the step of preparing the expression system. Specifically, the step comprises: first selecting the encoding nucleotide that needs to be expressed; cloning the encoding nucleotide sequence; splicing the obtained encoding nucleotide sequence to obtain a polynucleotide; constructing an expression cassette, and recombining the polynucleotide and the expression cassette into a plant transformation vector to obtain the expression system. That is to say, the expression system of the present application is actually a modified plant transformation vector, which has the encoding nucleotide of the protein that can increase the yield of Momordica grosvenori. In specific embodiments, cloning the encoding nucleotide sequence comprises using the cDNA of the selected protein source species as a template to perform PCR amplification to obtain the corresponding encoding nucleotide, and then connecting the obtained encoding nucleotide fragment to a transformation vector (such as a T vector), followed by transformation into E. coli competent cells for expression, and the encoding nucleotide sequence with correct sequencing results is used for subsequent operations. Splicing the obtained encoding nucleotide sequence comprises fusing the obtained single encoding nucleotide into a long polynucleotide sequence by Fusion-PCR method, and the connection order is not particularly limited and can be arranged arbitrarily, but in specific embodiments, in order to ensure that each protein can independently exercise its function after expression, it is preferred that the adjacent two encoding nucleotides are connected by a spacer P2A polypeptide. The way of recombining the polynucleotide and the expression cassette into a plant transformation vector includes but is not limited to homologous recombination.

[0105] According to embodiments of the present application, the method comprises the step of administering the expression system described above into a plant, wherein the polynucleotide is expressed in the plant and is codon-optimized to obtain a plant with high mogroside production. The plant comprises Siraitia grosvenorii. The Siraitia grosvenorii can be of at least one of Qingpiguo, Changtan Guo, Lajiangguo, Dongguaguo, Chashanguo, and Hongmaoguo. Specifically, the method comprises the step of obtaining a transformed plant cell by transforming the expression system into a plant cell through a transformation process, and then cultivating the obtained transformed plant cell to obtain a transformed plant. The transformed plant has increased production of mogrosides, such as at least one of Mogroside I, Mogroside II, Mogroside III, Mogroside IV, Siamenoside I, and Mogroside V. That is, the method of the present application increases the synthesis of mogrosides in the plant by transforming the expression system capable of promoting the synthesis of mogrosides into the plant, thereby increasing the production of mogrosides. In a specific example, the transformed plant obtained by the method has a fresh mogroside content of greater than 100 mg / kg. In addition, the transformed plant obtained by the method can be continuously propagated to obtain a transgenic plant with high mogroside production.

[0106] According to embodiments of the present application, the method further comprises the step of transforming the expression system or the expression cassette into a plant cell through a transformation process to obtain a transformed cell; cultivating the transformed cell, and screening and selecting the transformed cell by the type of selectable marker and / or screenable marker on the plant transformation vector to obtain a positive transformed cell capable of encoding the polypeptide from the expression system or the expression cassette, and developing the positive transformed cell into a plant cell culture, which can be further cultivated into a transgenic plant, i.e., a transformed plant. In specific embodiments, the transformed cell can be "stably transformed" or "transiently transformed". Specifically, "stably transformed" refers to the fusion of the polynucleotide with the chromosome or the nucleotide of the plastid (such as chloroplast, chromoplast, and leucoplast) of the plant, which can be inherited by the offspring. "Transiently transformed" refers to the temporary entry of the polynucleotide into the plant, which does not bind to the genome of the plant and cannot be inherited by the offspring. The stably transformed cell can be cultivated into a transformed plant for propagation to obtain a stable strain with high mogroside production.

[0107] According to embodiments of the present application, the treatment mode of the transformation treatment is not particularly limited, and the transformation treatment can be performed by at least one of protoplast transfection, pollen tube channel transfer, Agrobacterium-mediated transformation, and biolistic transformation. In a preferred example, the transformation treatment comprises Agrobacterium-mediated transformation, which is simple to operate and has a high success rate of transformation, and is easy to obtain stably transformed plants. More specifically, the Agrobacterium used for performing the transformation treatment is preferably Agrobacterium comprising a plant transformation vector. In a specific embodiment, the Agrobacterium-mediated transformation can effectively transform the plant transformation vector into plants, the plant transformation vector preferably comprises a Ti plasmid or a Ri plasmid, and the plant transformation vector can be replicated in Agrobacterium and Escherichia coli, contains an insertion site to facilitate integration of a polynucleotide or an expression cassette, comprises a control expression sequence, contains a selectable marker, and the transformation vector contains a screenable marker.

[0108] Plants or plant parts produced according to the above aspects of the present application. As produced Momordica grosvenori include whole or parts of Momordica grosvenori, and specifically include whole or parts at any developmental stage, such as whole Momordica grosvenori or parts at any developmental stage (e.g., tissues, cells, protoplasts, cultures, calli, cell masses, grafts, seedlings, clones, micropropagations, cuttings, embryos, pollen, ovules, flowers, leaves, fruits, seeds, asexually propagated plants, roots, stems, root tips, or derivatives thereof). It should be noted, however, that the genetic makeup of the above-mentioned parts of Momordica grosvenori should be the same or similar to that of the Momordica grosvenori from which they are obtained.

[0109] EMBODIMENTS

[0110] The method according to the present application is described in detail below through specific examples. The following examples are only for illustrating the present application, and do not limit the scope of the present application in any way. Moreover, the methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0111] EMBODIMENT 1

[0112] In this embodiment, an expression system according to the present application is constructed by a molecular biology method. Specifically, the following steps are included: selecting a coding nucleotide that needs to be expressed; cloning the coding nucleotide sequence; splicing the coding nucleotide sequence to obtain a polynucleotide; constructing an expression cassette; and recombining the polynucleotide and the expression cassette into a plant transformation vector to obtain the expression system.

[0113] 1. Selecting the coding nucleotide sequence to be expressed: In this embodiment, the coding nucleotide sequences of mogroside synthetase SQE1, SQE2, EPH1, EPH3, CDS, CYP87D18, UGT720, UGT94 and the transcription factor DREB1c for increasing plant yield are selected as the nucleotide sequences to be expressed, wherein the coding nucleotide sequences of mogroside synthetase SQE1, SQE2, EPH1, EPH3, CDS, CYP87D18, UGT720 and UGT94 are derived from Siraitia grosvenorii, and the coding nucleotide sequence of the transcription factor DREB1c is derived from rice.

[0114] The coding nucleotide sequence is shown in the sequence listing. Specifically, the coding nucleotide sequence of SQE1 of Siraitia grosvenorii is shown in SEQ ID NO: 1. The coding nucleotide sequence of SQE2 of Siraitia grosvenorii is shown in SEQ ID NO: 2. The coding nucleotide sequence of EPH3 of Siraitia grosvenorii is shown in SEQ ID NO: 3. The coding nucleotide sequence of CDS of Siraitia grosvenorii is shown in SEQ ID NO: 4. The coding nucleotide sequence of UGT720 of Siraitia grosvenorii is shown in SEQ ID NO: 5. The coding nucleotide sequence of UGT94 of Siraitia grosvenorii is shown in SEQ ID NO: 6. The coding nucleotide sequence of EPH1 of Siraitia grosvenorii is shown in SEQ ID NO: 7. The coding nucleotide sequence of CYP87D18 of Siraitia grosvenorii is shown in SEQ ID NO: 8. The coding nucleotide sequence of DREB1c of rice is shown in SEQ ID NO: 9.

[0115] 2. Cloning the coding nucleotide sequence: The coding nucleotide sequences of SQE1, SQE2, EPH1, EPH3, CDS, CYP87D18, UGT720 and UGT94 are amplified from the cDNA of Siraitia grosvenorii using a high-fidelity PCR enzyme, and the coding nucleotide sequence of DREB1c is amplified from the cDNA of rice; the amplified sequences are respectively connected to separate T vectors, and then transformed into E. coli for replication and propagation. The primer sequences used for amplifying each coding nucleotide sequence are shown in Table 1 (Note: F in Table 1 refers to the upstream primer, and R refers to the downstream primer).

[0116] Table 1 Primer sequence

[0117] 3. Splicing the coding nucleotide sequences: the cloned nucleotides are fused into two long polynucleotide sequences by the method of Fusion-PCR to form the sequence structures of UGT720-UGT94-CYP87D18 and CDS-EPH3-SQE1-SQE2-EPH1-DREB1c; the different coding nucleotides are connected by the spacer P2A polypeptide to ensure that each enzyme can independently perform functions after expression.

[0118] 4. Constructing the expression cassette: a constitutive promoter (35S promoter) is integrated in front of the fused UGT720-UGT94-CYP87D18 sequence structure, and a transcription terminator NOS-polyA is integrated behind the sequence structure to construct the expression cassette of 35S::UGT720-UGT94-CYP87D18::NOS-polyA; a constitutive promoter (corn UBI promoter) is integrated in front of the fused CDS-EPH3-SQE1-SQE2-EPH1-DREB1c sequence structure, and a transcription terminator NOS-polyA is integrated behind the sequence structure to construct the expression cassette of UBI::CDS-EPH3-SQE1-SQE2-EPH1-DREB1c::NOS-polyA; the promoter and the coding nucleotide are directly connected by the spacer AAA to improve the translation efficiency.

[0119] 5. Recombining the polynucleotide and the expression cassette into a plant transformation vector: the constructed 35S::UGT720-UGT94-CYP87D18::NOS-polyA is recombined into the PmeI of the plant transformation vector by the homologous recombination kit; the constructed UBI::CDS-EPH3-SQE1-SQE2-EPH1-DREB1c::NOS-polyA is recombined into the HindIII and SacI sites of the plant transformation vector, and then the obtained recombinant expression vector is transformed into E. coli for replication and propagation, wherein the map of the constructed recombinant expression vector is shown in FIG. 2.

[0120] 6. Culturing the transformed E. coli described above using an antibiotic-containing medium, and extracting the plasmid for PCR and sequencing detection, wherein the primer sequences used for PCR identification and sequencing detection are shown in Table 1 above. Specifically, the PCR detection result is shown in FIG. 3, and the amplification bands of each gene in the electrophoretogram are single, and no non-specific amplification bands are generated. Then each amplification band in FIG. 3 is individually recovered, and the obtained recovered sequences are sequenced, and each gene sequence is correct. That is, it can be seen from the above results that all the nucleotides in the present embodiment are accurately cloned into the plasmid vector, and the obtained plasmid is the expression system.

[0121] Example 2

[0122] The transformed momordica charantia prepared by the method of the present application refers to a transgenic momordica charantia plant. It specifically includes the following steps: preparing momordica charantia explants, preparing agrobacterium, transforming and co-culturing explants, screening and culturing, differentiation and rooting, and identifying transformed momordica charantia. As shown in Figure 4, pictures of each growth stage of the transformed momordica charantia obtained in this example are shown, and the specific preparation steps are as follows:

[0123] 1. Preparation of momordica charantia explants: the leaves of female momordica charantia plants were selected as the material for preparing explants. The top healthy first or (and) second leaves of momordica charantia seedlings were cut off, first rinsed with 75% alcohol for 1 min, then disinfected with 2% sodium hypochlorite solution containing 0.1% Triton X-100 for 15 min, and then rinsed with sterile water for 5 times, 1 min for the first 4 times, and 5 min for the last time. The rinsed leaves were cut into 0.5x0.5cm leaf discs on sterile filter paper moistened with sterile water, which were the prepared explants.

[0124] 2. Preparation of agrobacterium: Agrobacterium GV3101 was selected as the infection agrobacterium. The constructed expression system (i.e. the plasmid in Example 1) was transformed into agrobacterium competent cells by liquid nitrogen freeze-thaw method, and the specific steps were as follows:

[0125] The agrobacterium competent cells were taken out from -80℃, slightly thawed with the temperature of the palm of the hand, and then placed on ice immediately. Then 100 ng of verified expression system was added to the competent cells, mixed gently, and placed on ice for 20 minutes. Then it was placed in liquid nitrogen for 1 minute, then in a 37℃ water bath for 2 minutes. Then 600 μL of LB medium was added, and the culture was incubated at 28℃ for 1-3 hours. Finally, an appropriate amount of bacterial solution was spread on solid LB medium containing antibiotics, and incubated at 28℃, and colonies were observed after two days. Positive colonies containing the expression system were identified by colony PCR.

[0126] 3. Transformation and co-cultivation of explants: First, cut the Siraitia grosvenorid explants onto the pre-culture medium and dark culture for one day; at the same time, pick the identified positive colonies and add them to 5 mL of LB medium containing antibiotics for overnight shaking culture; the next day, when the bacterial liquid culture is very turbid, take 1-2 mL to 50 mL of LB medium containing antibiotics and shake culture at 28°C until the OD600 is about 0.5; then, centrifuge the Agrobacterium at 3200g at 4°C for 10 min to collect the bacteria, and pour off the supernatant culture medium; then, add the infection medium (liquid MS medium containing 20 mg / L acetyl-syringone) to resuspend the Agrobacterium, and adjust the OD600 to 0.1; then, mix the pre-cultured Siraitia grosvenorid explants with the resuspended Agrobacterium, and place them in a shaking incubator at 28°C, 80 rpm for 15 min; after the Siraitia grosvenorid explants are taken out, place them on sterile filter paper, and after the excess culture medium on the Siraitia grosvenorid explants is absorbed, transfer the Siraitia grosvenorid explants to the co-culture medium, and dark culture at 22°C for 4 days to complete the co-cultivation.

[0127] 4. Screening and culture: First, after the co-cultivation is completed, gently transfer the leaf discs from the culture medium to empty sterile tissue culture bottles, and wash them with sterile water for 5 times, 2 minutes each time for the first four times, and 5 minutes for the last time; then, transfer the washed leaf discs to sterile filter paper and dry them; then, transfer the leaf discs to the screening medium, about 10 pieces per dish; place the dishes in a light incubator for screening culture, 28°C for 16 hours of light, and 26°C for 8 hours of darkness; after two to three weeks of screening culture, the leaf discs gradually turn yellow, and then some of them gradually turn green again; take out the re-greened leaf discs and transfer them to new screening medium for secondary screening.

[0128] 5. Differentiation and rooting: After about four weeks of secondary screening, the resistant leaf discs gradually grow larger and differentiate visible bud points; transfer the leaf discs with bud points to new screening medium again; after a period of culture, the bud points gradually grow to form rootless sprouts; cut the sprouts that have grown to more than 1 cm from the original mother and insert them into the rooting medium; place the culture bottles in a light incubator for rooting culture, 28°C for 12 hours of light, and 24°C for 12 hours of darkness, and after about 3-4 weeks, roots will gradually differentiate.

[0129] 6. Identification of transformed Siraitia grosvenorid: Use wild-type Siraitia grosvenorid as control Siraitia grosvenorid, extract the genomic DNA of the transformed Siraitia grosvenorid leaves that have completed rooting, and use it as a template for PCR identification, with the genomic DNA of the control Siraitia grosvenorid as a negative control. The specific steps are as follows:

[0130] Firstly, a small amount of leaves of transformed momordica cochinchinensis / control momordica cochinchinensis were placed in 1.5 mL centrifuge tubes, frozen with liquid nitrogen, and then rapidly ground into powder with a pestle; 500 μL of genomic DNA extraction solution (Tris-HCl, 100 mM; EDTA, 50 mM; NaHSO4, 0.38%; SDS, 1.25%; NaOH, 8.3 mM; pH = 8.0) was added and mixed well with a vortex shaker; after mixing, it was allowed to stand at room temperature for 5 minutes; after standing, an equal volume of DNA extraction solution was added and mixed well by inverting, and after mixing, it was centrifuged at 12,000 rpm for 5 minutes at room temperature; then, the supernatant was transferred to a new 1.5 mL centrifuge tube, an equal volume of isopropanol was added to the new centrifuge tube and mixed well by inverting, and then it was centrifuged again at 12,000 rpm for 5 minutes at room temperature; the supernatant was discarded, the precipitate was rinsed twice with 600 μL of 75% ethanol, and finally the precipitate was air-dried and dissolved in 100 μL of double-distilled water; finally, the dissolved genomic DNA was used as a template to design primers on the expression cassette of the expression system (the sequences of the primers used are shown in Table 1 above), and PCR was performed. The results are shown in Figure 5, and it can be clearly seen that the coding genes of the various proteins introduced into the transformed momordica cochinchinensis obtained in this example were successfully detected, that is, the various coding nucleotides in the expression system were integrated into the momordica cochinchinensis plant.

[0131] Secondly, after PCR identification, the RNA of the momordica cochinchinensis leaves was further extracted, and after reverse transcription, the obtained cDNA was used as a template for qPCR detection. The primer sequences used in the qPCR detection are shown in Table 2 below (Note: F in Table 2 refers to the upstream primer, and R refers to the downstream primer). The detection results are shown in Figure 6, and it can be clearly seen that the expression levels of the various coding nucleotides of the expression system in the transformed momordica cochinchinensis obtained in this example were significantly higher than those in the control momordica cochinchinensis.

[0132] Table 2 qPCR primer sequences

[0133] 7. Momordica cochinchinensis glycoside determination: The transformed momordica cochinchinensis plants, which were grown under the same conditions as the control momordica cochinchinensis and had the same degree of maturity, were harvested, and the momordica cochinchinensis glycoside content was measured. Specifically, liquid chromatography was used to measure and analyze the content of momordica cochinchinensis glycoside V in the transformed momordica cochinchinensis and the control momordica cochinchinensis, and the results are shown in Figure 7. The results show that the content of momordica cochinchinensis glycoside V in the transformed momordica cochinchinensis was significantly higher than that in the control momordica cochinchinensis.

[0134] Example 3

[0135] In this example, the coding nucleotide sequence of the momordica cochinchinensis glycoside synthase CDS (SEQ ID NO: 4) was selected as the nucleotide sequence to be expressed, and a recombinant expression vector was constructed by a method similar to that of Example 1, and the map is shown in Figure 8.

[0136] The recombinant expression vector constructed in this example was transformed into Momordica grosvenori by a method similar to that of Example 2. The content of mogroside V in the transformed Momordica grosvenori and the control Momordica grosvenori was measured and analyzed by liquid chromatography, and the results are shown in Figure 9. The results show that the content of mogroside V in the transformed Momordica grosvenori is significantly higher than that in the control Momordica grosvenori.

[0137] In addition, in another embodiment of the present application, the coding nucleotide sequences of the following three groups of proteins FTO, FTO and CDS, CDS, EPH1 and CYP87D18 were respectively used as the nucleotide sequences to be expressed, and the recombinant expression vectors were constructed according to the method and sequence of Example 1, and then the transformed Momordica grosvenori was constructed. The coding nucleotide sequence of FTO is shown in SEQ ID NO: 44. It was detected that the content of mogroside V in the transformed Momordica grosvenori was significantly higher than that in the control Momordica grosvenori.

[0138] In general, the expression system and its use method and purpose proposed in the present application allow high-yield production of Momordica grosvenori, which can be used for large-scale, low-cost and scalable production of Momordica grosvenori. After the expression system of the present application is transformed into plants, the transformed plants that can express the polypeptides of the expression system are screened and selected, and the transformed plants contain Momordica grosvenori with significantly improved yield, such as Momordica grosvenori IV, siamenoside I and Momordica grosvenori V. The present application can economically and simultaneously produce healthy natural sweeteners in high yield to replace high-calorie or artificially synthesized sweeteners on the market.

[0139] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be realized in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting.

[0140] In addition, it should be understood that although the present specification is described in terms of embodiments, each embodiment does not contain only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. An expression system wherein, The expression system comprises polynucleotides for increasing mogroside production, the polynucleotides comprising at least one of the coding nucleotides of the proteins for increasing mogroside production; The proteins comprise enzymes and / or functional proteins of the mogroside synthesis pathway, selected from at least one of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94 and FTO.

2. The expression system of claim 1, wherein, The polynucleotides comprise coding nucleotides of the proteins for increasing mogroside production, the proteins comprising at least one of the following: (a) at least CDS and / or FTO; (b) at least CDS, EPH1 and CYP87D18.

3. The expression system of claim 2, wherein, The coding nucleotide of the CDS is shown in SEQ ID NO: 4, the coding nucleotide of the FTO is shown in SEQ ID NO: 44, the coding nucleotide of the EPH1 is shown in SEQ ID NO: 7, and the coding nucleotide of the CYP87D18 is shown in SEQ ID NO:

8.

4. The expression system of claim 1, wherein, The polynucleotides comprise coding nucleotides of the proteins for increasing mogroside production, the proteins comprising at least SQE, EPH, DREB1c, CDS, CYP87D18, UGT720 and UGT94; Optionally, the SQE comprises SQE1, SQE2 or a combination thereof, and the EPH comprises EPH1, EPH3 or a combination thereof.

5. The expression system of claim 4, wherein, The polynucleotides comprise SEQ ID NOs: 1-9.

6. The expression system according to any one of claims 1 to 5, wherein, The polynucleotides further comprise at least one of 5' UTR, 3' UTR, transcription termination region and polyadenylation region; The 5' UTR is located between the promoter sequence initiating the function of the translation leading sequence and the coding nucleotide.

7. The expression system according to any one of claims 1 to 5, wherein, The expression system further comprises an expression cassette, and the expression cassette comprises a control expression sequence; The control expression sequence is operably linked to the polynucleotides.

8. The expression system of claim 7, wherein, The control expression sequence comprises a promoter; The promoter is selected from at least one of constitutive promoter, inducible promoter and tissue-specific promoter; The tissue-specific promoter comprises a fruit-specific promoter.

9. The expression system of claim 7, wherein, The expression cassette is located in a plant transformation vector, the plant transformation vector comprising at least one of Ti plasmid, Ri plasmid, Ti-derived plasmid and Ri-derived plasmid of bacteria; The bacteria comprise Agrobacterium.

10. The expression system of claim 9, wherein, The plant transformation vector has at least one of selectable marker and screenable marker; The selectable marker comprises at least one of antibiotic resistance marker and herbicide resistance marker; The screenable marker comprises at least one of fluorescent protein gene, beta-glucuronidase gene, amylase gene, luciferase gene, Xyle gene and beta-lactamase gene.

11. Use of the expression system of any one of claims 1-10 for increasing mogroside production in Momordica grosvenori.

12. The use according to claim 11, wherein, The Momordica grosvenori comprises the whole or part of Momordica grosvenori; The variety of the Momordica grosvenori comprises at least one of Qingpiguo, Changtan guo, Lajiangguo, Dongguaguo, Chashanguo and Hongmaoguo.

13. A method of increasing the production of mogroside in a plant, wherein, comprising the step of using the expression system of any one of claims 1-10.

14. The method of claim 13, wherein, The method comprises the step of applying the expression system to a plant, wherein the polynucleotide in the expression system is expressed in the plant and is codon-optimized.

15. The method of claim 13, wherein, The method further comprises: introducing the expression system into a plant cell by a transformation process to produce a transformed plant cell; culturing the transformed plant cell to produce a transformed plant, The plant comprises a Siraitia grosvenorii.

16. The method of claim 15, wherein, The Siraitia grosvenorii comprises at least one of Qingpi Guo, Changtan Guo, Lajiang Guo, Dongguaguo, Chashan Guo, and Hongmao Guo.

17. The method of claim 15, wherein, The transformation process comprises at least one of protoplast transfection, pollen tube pathway introduction, Agrobacterium-mediated transformation, and biolistic transformation.

18. The method of claim 15, wherein, The transformation process comprises Agrobacterium-mediated transformation, and the Agrobacterium comprises a plant transformation vector.

19. The method of claim 15, wherein, The transformed plant has an altered content of mogrosides; The mogrosides comprise at least one of mogroside I, mogroside II, mogroside III, mogroside IV, siamenoside I, and mogroside V.

20. A plant or plant part produced by the method of increasing the production of mogrosides in a plant of claims 13-19.

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