Expression system and its method of use, applications

Abstract

This application provides an expression system and a method of using the same, and its applications. The expression system comprises a polynucleotide that enhances the production of mogrosides, the polynucleotide comprising at least one of several protein coding nucleotides that enhance the production of mogrosides, the protein comprising an enzyme and / or functional protein in the mogroside synthesis pathway, and being at least one selected from SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO.

Description

Technical Field

[0001] This application claims the priority of a patent application with the application number 202410467492.9, filed with the China National Intellectual Property Administration on April 17, 2024, and the invention title of "Expression System and Its Usage Method and Applications". All the contents of this application are incorporated herein by reference.

[0002] This application relates to the fields of biotechnology and plant genetic engineering technology, and specifically, to an expression system and its usage method and applications.

Background Art

[0003] With the improvement of people's living standards, metabolic diseases such as diabetes and obesity related to high-carbohydrate diets have become a major threat to human health. Therefore, artificial sweeteners with high sweetness and low calories are widely used in the food field instead of sucrose. However, in recent years, with the popularization of artificial sweeteners, their safety has been questioned. According to research reports, the intake of artificial sweeteners can cause a craving for and dependence on carbohydrates, stimulate appetite through the impairment of calorie compensation, and may lead to an increase in intake, weight gain, and glucose intolerance. In 1983, mogrosides, which are healthy natural non-saccharide sweet components, were discovered in the fruit of Siraitia grosvenorii. Mogrosides have a sweetness approximately 300 times that of sucrose and are regarded as one of the natural high-sweetness and low-calorie sweeteners. Furthermore, they have various pharmacological activities such as anti-tumor, anti-cancer, anti-diabetic, antioxidant, anti-hypoglycemic, and anti-inflammatory effects. In the 1990s, mogrosides were approved by the US Food and Drug Administration (FDA) as food sweetening additives and alternative sugars for patients with diabetes and obesity. Due to these characteristics, the global demand for mogrosides has increased significantly, and they have good application prospects in the food, beverage, and pharmaceutical industries. However, with the increasing demand for mogrosides, a market situation of supply shortage and price increase has occurred. Therefore, improving the production capacity of mogroside synthesis has great market value.

[0004] Monk fruit (Luo Han Guo) is the fruit of a perennial vine belonging to the Cucurbitaceae family, mainly produced in Guangxi Province, and is a unique economic and medicinal plant in China. As the demand for mogrosides increases, the demand for monk fruit itself will also increase. However, there are many varieties of monk fruit currently in circulation and application, and the overall quality of the seeds is not sufficient, resulting in a low mogroside content in monk fruit. Furthermore, the mogroside content varies greatly depending on the variety and origin. Since monk fruit is a major source of mogrosides, increasing the production of mogrosides from monk fruit or other plants is the most direct and effective way to solve the market shortage and high price of mogrosides.

[0005] Therefore, in order to meet the high market demand for mogrosides, there is an urgent need in this field to develop methods to increase the production of mogrosides from monk fruit. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Taking into consideration the problems of the prior art, this application provides an expression system, a method of use thereof, and applications in order to alleviate or solve at least one of the problems described in the background art to at least a certain extent. [Means for solving the problem]

[0007] In one aspect of this application, the application provides an expression system. The expression system comprises a polynucleotide that enhances the production of mogrosides, the polynucleotide comprising at least one of a plurality of protein-coding nucleotides that enhance the production of mogrosides. The protein comprises an enzyme and / or functional protein in the mogroside synthesis pathway, the protein being at least one selected from SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO.

[0008] In some embodiments, the polynucleotide comprises a protein-coding nucleotide that enhances the production of mogrosides, and the protein is (a) at least CDS and / or FTO, (b) At least CDS, EPH1, and CYP87D18, Includes at least one selected from.

[0009] In some embodiments, 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.

[0010] Furthermore, the polynucleotide includes a protein-coding nucleotide that enhances mogroside production, and the protein includes at least SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, and UGT94.

[0011] Selectively, the SQE includes SQE1, SQE2, or a combination thereof, and the EPH includes EPH1, EPH3, or a combination thereof.

[0012] Furthermore, the polynucleotides include sequence numbers 1-9 (SEQ ID NOs: 1-9).

[0013] Furthermore, the polynucleotide comprises at least one of the coding nucleotides DREB1c, CDS, UGT720, UGT94, and FTO.

[0014] Furthermore, the polynucleotide further comprises at least one of a 5'UTR, a 3'UTR, a transcription termination region, and a polyadenylation region, where the 5'UTR is located between a promoter sequence that functions as a translation reader sequence and a coding nucleotide.

[0015] Furthermore, the expression system further includes an expression cassette, the expression cassette including an expression control sequence, where the expression control sequence is operably ligated to the polynucleotide.

[0016] Furthermore, the expression control sequence includes a promoter. The promoter is at least one selected from a constitutive promoter, an inducible promoter, and a tissue-specific promoter. Here, the tissue-specific promoter includes a fruit-specific promoter.

[0017] Furthermore, the expression cassette is located within a plant transformation vector, which comprises at least one of a bacterial Ti plasmid, a Ri plasmid, a Ti-derived plasmid, and a Ri-derived plasmid. Here, the bacteria include Agrobacterium.

[0018] Furthermore, the plant transformation vector has at least one of the selectable marker and the screenable marker.

[0019] Furthermore, the selectable marker includes at least one of antibiotic resistance markers and herbicide resistance markers. The screenable marker includes at least one of a fluorescent protein gene, a β-glucuronidase gene, an amylase gene, a luciferase gene, a Xyle gene, and a β-lactamase gene.

[0020] In another aspect of this application, this application provides the use of the expression system described above for improving the production of mogrosides in monk fruit.

[0021] Furthermore, the Siraitia grosvenorii includes the whole or a part of the Siraitia grosvenorii. The variety of the Siraitia grosvenorii includes at least one selected from Qingpi fruit (Chongpi Guo), Changtan fruit (Changtang Guo), Lajiang fruit (Lajiang Guo), Donggua fruit (Donggua Guo), Chashan fruit (Chashan Guo), and Hongmao fruit (Hongmao Guo).

[0022] In another aspect of the present application, the present application provides a method for improving the production amount of mogrosides in plants. The method includes the step of using the above expression system.

[0023] Furthermore, the method includes the step of applying the expression system to a plant, wherein the polynucleotide is expressed in the plant and codon optimization has been performed.

[0024] Furthermore, the method includes the steps of introducing the expression system into plant cells by transformation treatment to generate transformed plant cells, and culturing the transformed plant cells to generate transformed plants, wherein the plant includes Siraitia grosvenorii.

[0025] Furthermore, the variety of the Siraitia grosvenorii includes at least one selected from Qingpi fruit, Changtan fruit, Lajiang fruit, Donggua fruit, Chashan fruit, and Hongmao fruit.

[0026] Furthermore, the transformation treatment includes at least one of protoplast transfection, introduction by pollen tube pathway, Agrobacterium-mediated transformation, and transformation by particle gun.

[0027] Furthermore, the transformation treatment includes Agrobacterium-mediated transformation, and the Agrobacterium includes a plant transformation vector.

[0028] Furthermore, in the transformed plant, the content of mogrosides changes. The mogrosides include at least one selected from mogroside I, mogroside II, mogroside III, mogroside IV, cyanamide I, and mogroside V.

[0029] In other respects, this application provides a plant or part of a plant produced by a method for improving the production of mogrosides in the above-mentioned plant.

[0030] This application has at least one of the following beneficial effects. 1. The expression system described in this application can be stably expressed in plants and can further improve the mogroside content in plants. 2. The method for improving the production of mogrosides in plants according to this application makes it possible to prepare a healthy natural sweetener (mogroside) economically and in high yield as a substitute for high-calorie or artificial sweeteners on the market. 3. The method for improving the production of mogrosides in plants according to this application makes it possible to obtain plants with high mogroside production, and by cultivating these plants industrially, the production of mogrosides can be significantly increased. This reduces input to technology, equipment, and cultivation conditions, thereby lowering production costs.

[0031] The above summary is provided for illustrative purposes only and is not intended to limit it in any way. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will readily become apparent by referring to the drawings and the detailed description below. [Brief explanation of the drawing]

[0032] In the drawings, unless otherwise specified, the same reference numerals throughout multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. These drawings depict only some embodiments disclosed in this application and should not be considered to limit the scope of this application. [Figure 1] This is a schematic diagram of the mogroside biosynthesis pathway in plants. [Figure 2] This is the expression system constructed in Example 1 of this application. [Figure 3] This figure shows the PCR detection results for each coding nucleotide of the expression system in Example 1 of this application. [Figure 4] This describes the process of obtaining a transformed monk fruit in Example 2 of this application. [Figure 5] This figure shows the PCR detection results of each coding nucleotide in transformed monk fruit in Example 2 of this application. [Figure 6] This is the result of qPCR detection of the relative nucleotide expression levels in transformed monk fruit in Example 2 of this application. [Figure 7] This figure shows the detection results for the mogroside V content in transformed monk fruit in Example 2 of this application. [Figure 8] This is the expression system constructed in Example 3 of this application. [Figure 9] This figure shows the detection results for the mogroside V content in transformed monk fruit in Example 3 of this application. [Modes for carrying out the invention]

[0033] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this application, the technical proposal of this application will be described in more detail below. Several exemplary embodiments are briefly described below. As those skilled in the art will recognize, the embodiments described can be amended in various different ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered illustrative and not limiting in nature.

[0034] In this application, unless otherwise defined, the meaning of all technical and scientific terms is the same as that of a person of the ordinary skill in the art (such as biology, chemistry, medicine, biochemistry, pharmacy, food science, and nutritional science).

[0035] In this specification, “contains” and “includes” should be interpreted as inclusive, not exclusive. “Consists of” and its variations should be interpreted as exclusive, not inclusive.

[0036] In this specification, unless otherwise explicitly indicated, the singular forms “a / an” and “the” refer to multiple objects.

[0037] Mogroside Mogrosides are cucurbitan-type triterpene saponin compounds and natural sweeteners. More than 60 types of mogrosides have been identified and can be classified into mogroside I, mogroside II, mogroside III, mogroside IV, mogroside V, and other mogrosides based on the number of glycosyl groups they contain. The central structure of mogrosides is a cucurbitan-type tetracyclic triterpene structure, and different types of mogrosides are formed by the combination of this structure with a different number of glycosyl groups and other groups (e.g., halogen groups, hydroxyl groups, carboxyl groups, amino groups, nitro groups, ester groups, aldehyde groups, carbonyl groups, carbon-carbon unsaturated bonds). In this application, mogroside refers to cucurbitan-type triterpene saponins containing glycosyl groups. Common mogrosides include mogroside V (88901-36-4), 11-oxomogroside V (126105-11-1), isomogroside V (1126032-65-2), 11-epimogloside 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), and siamenside I (126105-12-2).

[0038] Luo Hanguo This includes the whole or part of the monk fruit plant, such as tissues, cells, protoplasts, cultures, callus, cell masses, grafted individuals, seedlings, clones, micropropagations, cuttings, embryos, pollen, ovules, flowers, leaves, fruits, seeds, vegetatively propagated plants, roots, stems, root tips, etc., or derivatives thereof. Their genetic makeup should be identical or similar to that of the plant from which they were obtained. Furthermore, it includes all stages of development (e.g., seedlings, cuttings before or after rooting, mature or immature plants or leaves).

[0039] contrast or Comparison of Luo Han Guo A “control” or “control monk fruit” refers to a monk fruit that serves as a baseline for comparing changes in the expression type of monk fruit. Controls may include (a) wild-type or naturally occurring monk fruit with the same genotype as the subject, (b) monk fruit with the same genotype as the subject but transformed with an unrelated construct vector (e.g., a construct vector containing a marker gene), (c) monk fruit that has not been transformed in the offspring of the subject, and (d) the subject itself, but in which the introduced expression system is not expressed.

[0040] Expression Cassette An "expression cassette" refers to a polynucleotide encoding a target polypeptide or its variant or fragment, operably linked to at least one expression regulatory sequence. This expression cassette includes a promoter, a coding region, and a termination region in the 5'-3' direction. The regulatory and / or coding regions may be derived from a host cell or a different organism. The expression cassette may include other coding sequences (e.g., genes) for co-conversion with the organism. Alternatively, multiple expression cassettes may be provided with other coding sequences (e.g., genes). Such an expression cassette has multiple restriction sites and / or recombination sites for inserting the target coding polynucleotide or its variant or fragment so that it is subject to transcriptional regulation by a regulatory region such as a promoter.

[0041] Connected to be functional "Operationally linked" refers to the ability for two or more elements to be functionally linked. For example, an operational link between a polynucleotide of interest and a promoter is a link that enables the expression of the polynucleotide of interest. Operationally linked elements can be adjacent or distant. When used to link two protein-coding regions, an operational link means that the coding regions are in the same reading frame.

[0042] Expression regulatory sequences A “regulatory expression sequence” refers to a nucleic acid fragment that affects the expression level of the polypeptide encoded by the expression cassette. Regulatory expression regions include promoters, transcriptional regulatory regions, and termination regions. The termination region may be a natural product of the transcription initiation region, a natural or heterologous polynucleotide or its variant or fragment, or derived from a plant host or other source.

[0043] promoter A "promoter" refers to a nucleic acid sequence that can initiate the transcription of nucleic acids within a cell. Promoters may originate from plants, microorganisms (e.g., bacteria, viruses), or animals, or they may be artificially synthesized or modified nucleic acid sequences.

[0044] SQE "SQE" refers to squalene epoxidase, which can catalyze the epoxidation of squalene to produce 2,3-oxidesqualene and 2,3;22,23-bisoxidesqualene.

[0045] CDS "CDS" refers to cucurbitadienol synthase, which can catalyze 2,3;22,23-bisoxidesqualene to produce the cyclic 24,25-cucurbitadienol.

[0046] EPH "EPH" refers to epoxide hydrolase, which can catalyze 24,25-epoxycucurbitadienol to produce 24,25-dihydroxycucurbitadienol.

[0047] DREB1c "DREB1c" refers to the transcription factor dehydration response element-binding protein 1c.

[0048] CYP87D18 "CYP87D18" refers to cytochrome P450 oxidase, which can oxidize 24,25-dihydroxycubitadienol to produce mogol (11,24,25-trihydroxycubitadienol).

[0049] UGT720 "UGT720" refers to a glucuronosyltransferase that can catalyze the conversion of mogrol to mogroside I, and further catalyze the conversion of mogroside I to mogroside II.

[0050] UGT94 "UGT94" refers to a glucuronosyltransferase that can catalyze mogroside II to mogroside III, mogroside III to mogroside IV or siamenside I, and further catalyze mogroside IV or siamenside I to mogroside V.

[0051] FTO "FTO" refers to an α-ketoglutarate-dependent hydroxylase belonging to the AlkB family, which also has the function of demethylating RNA.

[0052] In one aspect of this application, the application provides an expression system comprising a polynucleotide that improves the production of mogrosides. The polynucleotide comprises at least one of a plurality of protein-coding nucleotides that improve the production of mogrosides. Here, the protein comprises an enzyme and / or functional protein in the mogroside synthesis pathway, specifically 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 an enzyme in the mogroside synthesis pathway and / or one or more nucleotides encoding a functional protein. The expression system has at least one beneficial effect from improving the mogroside content in monk fruit, preparing mogrosides economically and in high yield, and reducing production costs.

[0053] Optimizing the mogroside synthesis pathway in plants is a direct and effective way to increase the production of mogrosides in plants. The mogroside biosynthesis pathway in plants is divided into two steps: the synthesis of mogrol and the glycosylation of mogrol. Mogrol synthesis involves acetyl-CoA producing squalene via the mevalonate pathway, followed by the sequential catalytic action of squalene by squalene epoxidase (SQE), cucurbitadienol synthase (CDS), and epoxide hydrolase (EPH) to produce 24,25-dihydroxycucurbitadienol, and then hydroxylation completed by the catalytic action of CYP87D18, a cytochrome P450 superfamily member, to produce mogrol. In the glycosylation of mogrol, mogrol undergoes sequential glycosylation. First, mogol is sequentially converted to mogroside I and mogroside II by the glucuronosyltransferase UGT720. Mogroside II is then glycosylated by the glucuronosyltransferase UGT94 to produce mogroside III, mogroside IV, and siamenside I, ultimately resulting in mogroside V. Specifically, Figure 1 shows a schematic diagram of the mogroside biosynthesis pathway in plants. Furthermore, improving monk fruit varieties and increasing monk fruit yield can indirectly increase mogroside production. DREB1c or FTO have been shown to significantly improve the yield of rice, wheat, or potatoes. Therefore, DREB1c and FTO also have the potential to improve the yield and quality of monk fruit, thereby indirectly increasing mogroside production.

[0054] According to the embodiments of this application, the polynucleotides used in this application can encode proteins that improve the production of mogrosides, and preferably encode enzymes and / or functional proteins in the mogroside synthesis pathway. As mentioned above, enzymes in the mogroside synthesis pathway include squalene epoxidase (SQE), cucurbitadienol synthase (CDS), epoxide hydrolase (EPH), cytochrome P450 oxidase (CYP87D18), glucuronosyltransferase (UGT720), and glucuronosyltransferase (UGT94). Furthermore, the types of functional proteins referred to herein are not particularly limited. Specifically, functional proteins preferably include proteins that promote plant growth or improve biological productivity. In specific embodiments, functional proteins may be selected from dehydration response element-binding protein (DREB1c), MYB transcription factors, α-ketoglutarate-dependent hydroxylase (FTO) belonging to the AlkB family, or a combination thereof. In other words, the polynucleotide of this application preferably contains at least one of the coding nucleotides of the protein sequences SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. It is even more preferable that it contains at least one of the coding nucleotides of the protein sequences DREB1c, CDS, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide contains at least one coding nucleotide of the CDS protein sequence. For example, in some embodiments, the polynucleotide contains at least one coding nucleotide of the FTO protein sequence.

[0055] In specific examples, SQE includes SQE1, SQE2, or a combination thereof, and EPH includes EPH1, EPH3, or a combination thereof.

[0056] For example, in some embodiments, the polynucleotide comprises one of the coding nucleotides of the protein sequences SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, or FTO. In some embodiments, the polynucleotide comprises the coding nucleotide of the protein sequence SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, or FTO. In some embodiments, the polynucleotide comprises the coding nucleotide of the EPH protein sequence. In some embodiments, the polynucleotide comprises the coding nucleotide of the CDS protein sequence. In some embodiments, the polynucleotide comprises the coding nucleotide of the CYP87D18 protein sequence. In some embodiments, the polynucleotide comprises the coding nucleotide of the FTO protein sequence. In some embodiments, the polynucleotide comprises the coding nucleotide of the protein sequence DREB1c, UGT720, or UGT94.

[0057] For example, in some embodiments, the polynucleotide comprises at least two of the protein sequence coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1 and CYP87D18. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of CDS and FTO.

[0058] For example, in some embodiments, the polynucleotide comprises at least three of the protein sequence coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of CDS, EPH1, and CYP87D18. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, CYP87D18, and FTO.

[0059] For example, in some embodiments, the polynucleotide comprises at least four of the protein sequence coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, CYP87D18, CDS, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, CYP87D18, and CDS, and the protein sequence coding nucleotides of SQE, DREB1c, UGT720, or UGT94. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, EPH3, CYP87D18, and CDS.

[0060] For example, in some embodiments, the polynucleotide comprises at least five of the protein sequence coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, EPH3, CYP87D18, and CDS, and the protein sequence coding nucleotide of SQE or DREB1c.

[0061] For example, in some embodiments, the polynucleotide comprises at least six of the protein sequence coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, EPH3, SQE1, SQE2, CYP87D18, and CDS.

[0062] For example, in some embodiments, the polynucleotide comprises at least seven of the protein sequence coding nucleotides of SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. For example, in some embodiments, the polynucleotide comprises at least the protein sequence coding nucleotides of EPH1, EPH3, SQE1, SQE2, CYP87D18, and CDS, and the protein sequence coding nucleotide of UGT720 or UGT94.

[0063] For example, in some embodiments, the polynucleotides include the coding nucleotides for the protein sequences SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO.

[0064] Furthermore, the source of the protein sequences SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO is not particularly limited, and each may independently originate from animals, plants, or microorganisms. However, in order to better express them in plants, the above protein sequences are preferably derived from plants, and the type of plant is not particularly limited. For example, they can be selected from the Asteraceae, Rosaceae, Caryophyllaceae, Poaceae, Solanaceae, Fabaceae, Vitaceae, Moraceae, Cucurbitaceae, or combinations thereof, and specifically, they may be selected from monk fruit, cucumber, watermelon, bitter melon, loofah, winter melon, pumpkin, zucchini, or combinations thereof.

[0065] According to embodiments of this application, the polynucleotide may further include, in addition to the coding nucleotide of the protein sequence described above, at least one of a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), a transcription termination region, and a polyadenylation region, so that the encoded nucleic acid can be stably expressed. In specific embodiments, the polynucleotide preferably includes a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), a transcription termination region, and a polyadenylation region. Here, the 5'UTR is located between the promoter sequence, which functions as a 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.

[0066] According to embodiments of this application, the expression system further comprises an expression cassette, the expression cassette comprising an expression regulatory sequence. This expression regulatory sequence refers to a nucleotide fragment that affects the expression level of the polypeptide encoded by the expression cassette, and the expression regulatory sequence is operably ligated to a polynucleotide to enable better expression of the polynucleotide. In specific embodiments, the expression regulatory sequence comprises a promoter, a transcription regulatory region, and a termination region sequentially ligated in the 5'-3' direction to express the coding sequence of a transcription regulatory region under the control of the promoter. Specifically, the sequence after the expression regulatory sequence has been operably ligated to the polynucleotide is promoter-polynucleotide-termination region in the 5'-3' direction. In specific embodiments, the transcription regulatory region may be driven by different types of promoters. That is, the type of promoter in the expression regulatory sequence is not particularly limited. For example, the promoter may be a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In specific embodiments, this promoter preferably refers to a promoter that functions in plant cells. Here, constitutive promoters include, but are not limited to, the core promoter of the rsyn7 promoter, the core promoter of the mosaic virus promoter, the plant ubiquitin protein promoter, the plant actin promoter, and promoters modified from these for weak / strong expression. Furthermore, since many tissue-specific promoters are expressed in fruits, it is preferable to include fruit-specific promoters. Specific examples of tissue-specific promoters include, but are not limited to, those known in this field, such as the polygalacturonase gene promoter (CkPGA), CFSP-1, HyPRP, E8S, 2A12, E4, PG promoter, or combinations thereof.

[0067] According to embodiments of this application, the expression cassette may further include other coding sequences in addition to the expression control sequence. Alternatively, multiple expression cassettes may be provided with other coding sequences. In specific examples, the expression cassette has multiple restriction sites and / or recombination sites for inserting a target coding polynucleotide or its variant or fragment so that it is subject to transcriptional control by a regulatory region such as a promoter.

[0068] According to embodiments of this application, the expression cassette may be located within a plant transformation vector so as to be introduced into a plant via the plant transformation vector. Specifically, a plant transformation vector refers to a DNA molecule that delivers polynucleotides to plant cells, and multiple foreign genes or expression cassettes may be present within a single transformation vector. Specifically, the type of plant transformation vector is not particularly limited, as long as it can incorporate and insert a polynucleotide sequence into plant genomic DNA. More specifically, a plant transformation vector includes, but is not limited to, at least one of a bacterial Ti plasmid, Ri plasmid, Ti-derived plasmid, and Ri-derived plasmid. In a preferred example, the bacteria include Agrobacterium. In a specific embodiment, the plant transformation vector in this application is a binary expression vector capable of transforming or transfecting plant cells, and is specifically a plant expression vector. 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 this application may constitute a plant transformation vector, and genetic material (e.g., polynucleotide or expression cassette) may be introduced into the plant body. Transformation methods include, but are not limited to, electroporation, particle gun, Agrobacterium-mediated transformation, and protoplast transfection.

[0069] According to embodiments of this application, a plant transformation vector may have at least one of a selectable marker and a screenable marker for selecting plants that have successfully undergone transformation. Specifically, the types of selectable markers are not particularly limited. For example, they include, but are not limited to, antibiotic resistance markers and herbicide resistance markers. The types of screenable markers are also not particularly limited. For example, they include, but are not limited to, fluorescent protein genes, β-glucuronidase genes, amylase genes, luciferase genes, Xyle genes, and β-lactamase genes.

[0070] Specifically, this application discloses an expression system having polynucleotides and an expression cassette related to the improvement of mogroside production. After transforming a plant with this expression system, a transformed plant capable of expressing the expression system as a polypeptide is obtained by sorting and selection based on the types of selectable markers and / or screenable markers on the plant transformation vector. In the transformed plant, the production of at least one of mogrosides, such as mogroside IV, siamenside I, and mogroside V, is significantly improved. In other words, by utilizing the expression system of this application, a healthy natural sweetener can be prepared economically and in high yield as an alternative to high-calorie or artificial sweeteners on the market.

[0071] In another aspect of this application, this application provides the use of the expression system described above for improving the production of mogrosides in monk fruit.

[0072] According to the embodiments of this application, the varieties of monk fruit in this application are not particularly limited, but it is preferable that they include at least one of the following: Qingpiguo, Changtanguo, Lajiangguo, Dongguaguo, Chashanguo, and Hongmaoguo. Furthermore, monk fruit as used in this application includes the whole or a part of monk fruit, specifically including the whole or a part of any growth stage, for example, the whole monk fruit or a part of any growth stage (e.g., tissue, cells, protoplasts, cultures, callus, cell masses, grafted individuals, seedlings, clones, micropropagations, cuttings, embryos, pollen, ovules, flowers, leaves, fruits, seeds, vegetatively propagated plants, roots, stems, root tips, or derivatives thereof). The genetic composition of the above-mentioned monk fruit parts should be the same as or similar to that of the monk fruit from which they were obtained.

[0073] In another aspect of this application, the application provides a method for improving the production of mogrosides in plants. This method includes the step of using the expression system described above. Specifically, the application provides a method for improving the production of mogrosides in plants using means of biotechnology and genetic engineering. That is, another object of this application is to provide a method for obtaining transgenic plants with high mogroside production in order to produce transgenic plants with high mogroside production.

[0074] The types and composition of polynucleotides, expression cassettes, and plant transformation vectors in the expression system have been described in detail above, so they will not be repeated here.

[0075] According to embodiments of this application, the method optionally includes a step of preparing an expression system. Specifically, this step includes first selecting a coding nucleotide to be expressed, cloning the coding nucleotide sequence, splicing the obtained coding nucleotide sequence to obtain a polynucleotide, constructing an expression cassette, and recombining the polynucleotide and expression cassette into a plant transformation vector to obtain an expression system. That is, the expression system of this application is a modified plant transformation vector having the coding nucleotide of a protein that improves mogroside production. In specific embodiments, cloning the coding nucleotide sequence includes using the cDNA of the selected protein's source species as a template, amplifying it by PCR to obtain the corresponding coding nucleotide, then ligating the obtained coding nucleotide fragment into a transformation vector (e.g., a T vector), and subsequently transforming and expressing it in competent E. coli cells, and using the coding nucleotide sequence for which the sequencing result is correct in subsequent operations. Splicing the obtained coding nucleotide sequence includes fusing the obtained single coding nucleotide into a single long polynucleotide sequence by fusion PCR. The order of linking is not particularly limited and can be arranged selectively, but in specific embodiments, it is preferable to link two adjacent coding nucleotides with a spacer sequence, the P2A peptide, so that each protein can function independently after expression. Methods for recombining polynucleotides and expression cassettes into plant transformation vectors include, but are not limited to, homologous recombination.

[0076] According to embodiments of this application, the method includes the step of applying the above expression system to a plant, where the polynucleotide is expressed in the plant and codon-optimized in order to obtain a plant with high mogroside production. The plant includes monk fruit. The varieties of monk fruit include at least one of the following: Qingpiguo, Changtanguo, Lajiangguo, Dongguaguo, Chashanguo, and Hongmaoguo. Specifically, the method includes introducing the expression system into plant cells by a transformation treatment to generate transformed plant cells, and then culturing the obtained transformed plant cells to obtain a transformed plant. In the obtained transformed plant, the production of at least one of the following mogrosides, such as mogroside I, mogroside II, mogroside III, mogroside IV, siamenside I, and mogroside V, is improved. That is, the method of this application increases the synthesis of mogrosides in plants and further improves the production of mogrosides by introducing an expression system that can promote the synthesis of mogrosides into the plant. In a specific example, the mogroside content in the fresh fruit of transgenic plants obtained by this method exceeds 100 mg / kg. Furthermore, by continuously propagating the transgenic plants obtained by this method, it is possible to obtain transgenic plants with high mogroside production.

[0077] According to embodiments of this application, the method further comprises: introducing the above-mentioned expression system or expression cassette into plant cells by transformation treatment to obtain transformed cells; culturing the transformed cells and performing sorting and selection based on the types of selectable markers and / or screenable markers on the plant transformation vector to obtain positive transformed cells capable of encoding the expression system or expression cassette as a polypeptide; and growing these in a plant cell culture. This plant cell culture can further be cultured as a transformed plant, i.e., a transformant. In specific embodiments, the transformed cells may be "stable transformed" or "transient transformed." Specifically, "stable transformed" refers to the fusion of polynucleotides with nucleotides in plant chromosomes or plastids (chloroplasts, chromoplasts, leucoplasts, etc.) and inheritance by offspring. "Transient transformed" refers to the temporary entry of polynucleotides into the plant but without binding to the plant genome and not being inherited by offspring. Stable transformed cells may be grown into transformed plants for breeding. This makes it possible to obtain stable lines with high mogroside production.

[0078] According to the embodiments of this application, the method of transformation is not particularly limited. Specifically, transformation may be performed by at least one of the following: protoplast transfection, pollen tube pathway transfer, agrobacterium-mediated transformation, and gene gun transformation. In a preferred example, the transformation process includes agrobacterium-mediated transformation. This method is easy to operate, has high transformation efficiency, and makes it easy to obtain stable transformed plants. More specifically, it is preferable that the agrobacterium used in the transformation process is an agrobacterium containing a plant transformation vector. In a specific embodiment, agrobacterium-mediated transformation allows for efficient transformation of plants with a plant transformation vector. The plant transformation vector preferably comprises a Ti plasmid or a Ri plasmid, and the plant transformation vector is replicable in Agrobacterium and Escherichia coli, contains an insertion site for the incorporation of a polynucleotide or expression cassette, contains an expression regulatory sequence, contains a selectable marker, and contains a marker on which the transformation vector can be screened.

[0079] In the aforementioned aspects of this application, a plant or part of a plant is produced. For example, the produced monk fruit includes the whole or a part of a monk fruit, specifically the whole or a part of any growth stage, for example, the whole monk fruit or a part of any growth stage (e.g., tissue, cells, protoplasts, cultures, callus, cell masses, grafted individuals, seedlings, clones, micropropagations, cuttings, embryos, pollen, ovules, flowers, leaves, fruits, seeds, vegetatively propagated plants, roots, stems, root tips, or derivatives thereof). The genetic composition of the above monk fruit parts should be identical or similar to that of the monk fruit from which they were obtained.

[0080] Examples The methods proposed in this application will be described in detail below with specific examples. The following examples are illustrative of this application and do not limit the scope of this application in any way. Furthermore, the methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0081] Example 1 In this embodiment, the expression system of this application was constructed by molecular biological methods. Specifically, this includes selecting the coding nucleotide to be expressed, cloning the coding nucleotide sequence, splicing the coding nucleotide sequence to obtain polynucleotides, constructing an expression cassette, and recombining the polynucleotides and expression cassette into a plant transformation vector to obtain the expression system.

[0082] 1. Selection of coding nucleotides to be expressed In this example, the coding nucleotide sequences for mogroside synthases SQE1, SQE2, EPH1, EPH3, CDS, CYP87D18, UGT720, UGT94, and the transcription factor DREB1c, which increases plant yield, were selected as the nucleotide sequences to be expressed. Here, the coding nucleotide sequences for mogroside synthases SQE1, SQE2, EPH1, EPH3, CDS, CYP87D18, UGT720, and UGT94 are derived from monk fruit, and the coding nucleotide sequence for the transcription factor DREB1c is derived from rice.

[0083] The coding nucleotide sequences are shown in the sequence listing. Specifically, the coding nucleotide sequence for SQE1 from monk fruit is shown in Sequence ID No. 1. The coding nucleotide sequence for SQE2 from monk fruit is shown in Sequence ID No. 2. The coding nucleotide sequence for EPH3 from monk fruit is shown in Sequence ID No. 3. The coding nucleotide sequence for CDS from monk fruit is shown in Sequence ID No. 4. The coding nucleotide sequence for UGT720 from monk fruit is shown in Sequence ID No. 5. The coding nucleotide sequence for UGT94 from monk fruit is shown in Sequence ID No. 6. The coding nucleotide sequence for EPH1 from monk fruit is shown in Sequence ID No. 7. The coding nucleotide sequence for CYP87D18 from monk fruit is shown in Sequence ID No. 8. The coding nucleotide sequence for DREB1c from rice is shown in Sequence ID No. 9.

[0084] 2. Cloning of coding nucleotide sequences Using high-fidelity PCR enzymes, the coding nucleotide sequences for SQE1, SQE2, EPH1, EPH3, CDS, CYP87D18, UGT720, and UGT94 were amplified using monk fruit cDNA as a template, and the coding nucleotide sequence for DREB1c was amplified using rice cDNA as a template. The amplified sequences were ligated into individual T vectors, and then transformed into E. coli for replication and growth. The primer sequences used for amplification of each coding nucleotide sequence are shown in Table 1 below (Note: In Table 1, F indicates a forward primer and R indicates a reverse primer).

[0085] [Table 1]

[0086] 3. Splicing of coding nucleotide sequences The cloned nucleotides were fused to two long polynucleotide sequences, UGT720-UGT94-CYP87D18 and CDS-EPH3-SQE1-SQE2-EPH1-DREB1c, using fusion PCR. To allow each enzyme to function independently after expression, different coding nucleotides were linked with a spacer sequence, the P2A peptide.

[0087] 4. Construction of Expression Cassettes An expression cassette with the structure 35S::UGT720-UGT94-CYP87D18::NOS-polyA was constructed by recombining a constitutive promoter (35S promoter) before the fused UGT720-UGT94-CYP87D18 sequence structure, and then recombining the transcription termination site NOS-polyA after this sequence structure. An expression cassette with the structure UBI::CDS-EPH3-SQE1-SQE2-EPH1-DREB1c::NOS-polyA was constructed by recombining a constitutive promoter (maize UBI promoter) before the fused CDS-EPH3-SQE1-SQE2-EPH1-DREB1c sequence structure, and then incorporating the transcription termination site NOS-polyA after this sequence structure. A spacer sequence AAA was directly ligated between the promoter and the coding nucleotide to improve translation efficiency.

[0088] 5. Recombination of polynucleotides and expression cassettes into plant transformation vectors Using a homologous recombination kit, the constructed 35S::UGT720-UGT94-CYP87D18::NOS-polyA was recombined into the PmeI site of the plant transformation vector, and the constructed UBI::CDS-EPH3-SQE1-SQE2-EPH1-DREB1c::NOS-polyA was recombined into the HindIII and SacI sites of the plant transformation vector. Subsequently, the resulting recombinant expression vectors were transformed into Escherichia coli, and replication and propagation were carried out. The structural diagrams of the constructed recombinant expression vectors are shown in Figure 2.

[0089] 6. The transformed E. coli described above were cultured in antibiotic-containing medium, and plasmids were extracted for PCR identification and sequencing analysis. The primer sequences used for PCR identification and sequencing analysis are shown in Table 1 above. Specifically, as shown in Figure 3, the PCR detection results showed that each gene had a single amplification band in the electrophoresis, and no nonspecific amplification bands were observed. Subsequently, each amplification band in Figure 3 was recovered individually, and sequencing analysis was performed on the recovered sequences to confirm that the sequences of each gene were correctly aligned. In other words, from the above results, it can be seen that in this embodiment, all nucleotides were accurately cloned into the plasmid vector, and the obtained plasmid is an expression system.

[0090] Example 2 In this example, transgenic monk fruit (referring to transgenic monk fruit plants) was prepared by the method described in this application. Specifically, the process includes the preparation of monk fruit explants, preparation of Agrobacterium, transformation and co-culturing of the explants, screening and cultivation, differentiation and rooting, and identification of the transgenic monk fruit. Figure 4 shows images of each growth stage of the transgenic monk fruit obtained in this example. The specific preparation steps are described below.

[0091] 1. Preparation of explants for monk fruit Female monk fruit leaves were selected as the material for the explant. Healthy first and / or second leaves were cut from the top of monk fruit seedlings, first washed with 75% ethanol for 1 minute, then sterilized with a 2% sodium hypochlorite solution containing 0.1% Triton X-100 for 15 minutes, and then washed five times with sterile water (the first four times for 1 minute each, and the last time for 5 minutes). The washed leaves were cut into 0.5 × 0.5 cm leaf discs on sterile filter paper moistened with sterile water, and these leaf discs were used as the prepared explant.

[0092] 2. Preparation for Agrobacterium Agrobacterium GV3101 was selected as the Agrobacterium for infection. The constructed expression system (i.e., the plasmid in Example 1) was introduced into competent Agrobacterium cells by liquid nitrogen freeze-thaw cycle. The specific procedure is as follows:

[0093] Competent Agrobacterium cells were removed from -80°C, slightly thawed at palm temperature, and immediately placed on ice. Next, 100 ng of the validated expression system was added to the competent cells, gently mixed, and left on ice for 20 minutes. Subsequently, the cells were placed in liquid nitrogen for 1 minute, followed by a 37°C water bath for 2 minutes. Then, 600 μL of LB medium was added, and the cells were cultured in a 28°C shaker for 1-3 hours. Finally, an appropriate amount of bacterial suspension was spread onto antibiotic-containing solid LB medium plates and inverted at 28°C. Colonies appeared after 2 days, and positive colonies containing the expression system were identified by PCR targeting these colonies.

[0094] 3. Transformation and co-culture of explants First, the explants of the monk fruit were placed on a pre-culture medium and cultured in the dark for one day. Simultaneously with the start of the pre-culture of the explants, identified positive colonies were picked and cultured overnight with shaking in 5 mL of antibiotic-containing LB medium. The next day, once the bacterial suspension was sufficiently turbid, 1-2 mL of it was added to 50 mL of antibiotic-containing LB medium and cultured with shaking at 28°C until the OD600 was approximately 0.5. Then, the mixture was centrifuged at 3200 g and 4°C for 10 minutes to recover the Agrobacterium, and the supernatant medium was removed. Next, infection medium (liquid MS medium containing 20 mg / L acetosyringone) was added to resuspend the Agrobacterium, and the OD600 was adjusted to 0.1. Finally, the pre-cultured monk fruit explants and the resuspended Agrobacterium were mixed and left in a shaker (28°C, 80 rpm) for 15 minutes. The explants of the monk fruit were removed and placed on sterile filter paper. After waiting for excess culture medium to be absorbed from the explants, the monk fruit explants were transferred to a co-culture medium and co-cultured in a 22°C incubator in the dark for 4 days.

[0095] 4. Screening and Culture After co-culture, the leaf fragments were carefully transferred from the culture medium to empty sterile culture bottles and washed five times with sterile water (the first four times for 2 minutes each, and the last time for 5 minutes). The washed leaf fragments were then transferred to sterile filter paper to absorb the moisture. Next, the leaf fragments were transferred to screening medium (approximately 10 fragments per petri dish). The petri dishes were placed in a light incubator and screening culture was performed (28°C, 16 hours of illumination, 26°C, 8 hours in darkness). After 2-3 weeks of screening culture, the leaf fragments gradually turned yellow, and then some of the leaf fragments turned green again. The re-greened leaf fragments were removed and transferred to new screening medium for a second screening.

[0096] 5. Differentiation and Rooting After approximately four weeks of secondary screening, the resistant leaf fragments gradually grew larger and differentiated into small buds (bud points) visible to the naked eye. The leaf fragments with bud points were transferred to a new screening medium. After a certain period of culture, the bud points gradually grew and formed rootless buds. The buds that had grown to over 1 cm were separated from the original parent plant and inserted into rooting medium. The culture bottles were placed in a light incubator and rooting culture was performed (28°C, 12 hours of illumination, 24°C, 12 hours of darkness). After approximately 3-4 weeks, roots began to differentiate sequentially.

[0097] 6. Identification of transformed monk fruit Using wild-type monk fruit as a control, genomic DNA was extracted from the leaves of transformed monk fruit that had completed rooting, and PCR identification was performed using this as a template. Genomic DNA from the control monk fruit was used as a negative control. The specific procedure is as follows:

[0098] First, small leaf fragments of transformed monk fruit and control monk fruit were placed in 1.5 mL centrifuge tubes, frozen with liquid nitrogen, and then quickly ground into a powder using a grinding pestle. Next, 500 μL of genomic DNA extract (Tris-HCl, 100 mM; EDTA, 50 mM; NaHSO4, 0.38%; SDS, 1.25%; NaOH, 8.3 mM; pH=8.0) was added, and the mixture was vigorously shaken in a vertex mixer before being allowed to stand at room temperature for 5 minutes. After standing, an equal volume of DNA extract was added, the tubes were inverted and mixed, and the mixture was centrifuged at 12,000 rpm at room temperature for 5 minutes. 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, the tubes were inverted and mixed, and the mixture was centrifuged again at 12,000 rpm at room temperature for 5 minutes. The supernatant was discarded, the precipitate was washed twice with 600 μL of 75% ethanol, and finally the precipitate was dried and dissolved in 100 μL of sterile distilled water. Finally, the dissolved genomic DNA was used as a template, and primers (the primer sequences used are as shown in Table 1 above) were designed for the expression cassette of the expression system, and a PCR reaction was performed. As a result, as shown in Figure 5, it is clear that the coding genes of each introduced protein were successfully detected in the transformed monk fruit obtained in this example. In other words, it was confirmed that each coding nucleotide in the expression system was incorporated into the monk fruit plant.

[0099] Next, after PCR identification, RNA was extracted from the leaves of the monk fruit, and after completing reverse transcription, qPCR identification was performed using the obtained cDNA as a template. The primer sequences used for qPCR identification are shown in Table 2 below (Note: In Table 2, F refers to the forward primer and R refers to the reverse primer). As shown in Figure 6, the detection results clearly show that the expression levels of each coding nucleotide in the expression system of the transformed monk fruit obtained in this example are significantly higher than those of the control monk fruit.

[0100] [Table 2]

[0101] 7. Measurement of mogrosides Transgenic monk fruit was cultivated under the same conditions as control monk fruit and harvested at a similar level of maturity. Its mogroside content was then measured. Specifically, the mogroside V content of transgenic monk fruit and control monk fruit was measured and analyzed using liquid chromatography. As shown in Figure 7, the results show that transgenic monk fruit has a significantly higher mogroside V content compared to control monk fruit.

[0102] Example 3 In this example, the coding nucleotide sequence of mogroside synthase CDS (SEQ ID NO: 4) was selected as the nucleotide sequence to be expressed, and a recombinant expression vector was constructed using the same method as in Example 1. Its structure is shown in Figure 8.

[0103] The recombinant expression vector constructed in this example was used to transform monk fruit in the same manner as in Example 2. The mogroside V content of the transformed monk fruit and the control monk fruit was measured and analyzed using liquid chromatography. As shown in Figure 9, the transformed monk fruit had a significantly higher mogroside V content compared to the control monk fruit.

[0104] Furthermore, in other embodiments of this application, following the method and sequence of Example 1, the following protein FTO, FTO and CDS, and three sets of coding nucleotide sequences—CDS, EPH1 and CYP87D18—were selected as nucleotide sequences to be expressed, and after constructing recombinant expression vectors, transformed monk fruit was produced. Here, the coding nucleotide sequence of FTO is shown as SEQ ID NO:44. Detection revealed that all of these transformed monk fruit had significantly higher mogroside V content compared to control monk fruit.

[0105] Overall, the expression system, its usage, and applications proposed in this application enable high-yield production of mogrosides and can be applied to the large-scale, low-cost, and scalable production of mogrosides. After transforming plants with the expression system in this application, transformed plants capable of expressing the expression system as polypeptides can be screened and selected. These transformed plants include mogrosides such as mogroside IV, siamenside I, and mogroside V, which show significantly improved yields. According to this application, healthy natural sweeteners can be prepared economically and in high yield as an alternative to high-calorie or artificial sweeteners available on the market.

[0106] Having described the basic principles, main features, and advantages of this application, it will be apparent to those skilled in the art that this application is not limited to the details of the exemplary embodiments described above, and that it can be realized in other specific forms without departing from the spirit or basic features of this application. Therefore, from any viewpoint, the embodiments should be considered illustrative and non-limiting.

[0107] Furthermore, although this specification is described according to embodiments, each embodiment does not contain only a single, independent technical solution. This method of description in the specification is for clarity, and those skilled in the art should understand that they can view the specification as a whole and combine the technical solutions of each embodiment as appropriate to form other embodiments that are understandable to those skilled in the art.

Claims

1. An expression system, The expression system includes a polynucleotide that improves the production of mogrosides, The polynucleotide comprises at least one of several protein-coding nucleotides that improve the production of mogrosides. The protein comprises an enzyme and / or functional protein in the mogroside synthesis pathway, and is at least one selected from SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, UGT94, and FTO. Expression system.

2. The aforementioned polynucleotide includes a protein-coding nucleotide that improves the production of mogrosides. The aforementioned protein is (a) at least CDS and / or FTO, (b) comprising at least one selected from CDS, EPH1, and CYP87D18, The expression system according to claim 1.

3. The coding nucleotide of the aforementioned CDS is shown in SEQ ID NO: 4, The coding nucleotide of the aforementioned FTO is shown in SEQ ID NO: 44, The coding nucleotide of EPH1 is shown in SEQ ID NO: 7, The coding nucleotide of CYP87D18 is shown in SEQ ID NO:

8. The expression system according to claim 2.

4. The aforementioned polynucleotide includes a protein-coding nucleotide that improves the production of mogrosides. The protein comprises at least SQE, EPH, DREB1c, CDS, CYP87D18, UGT720, and UGT94. Selectively, the SQE includes SQE1, SQE2, or a combination thereof, and the EPH includes EPH1, EPH3, or a combination thereof. The expression system according to claim 1.

5. The aforementioned polynucleotides include SEQ ID NOs: 1 to 9. The expression system according to claim 4.

6. The polynucleotide further comprises at least one of a 5'UTR, a 3'UTR, a transcription termination region, and a polyadenylation region. The aforementioned 5'UTR is located between the promoter sequence, which functions as a translation reader sequence, and the coding nucleotide. The expression system according to any one of claims 1 to 5.

7. The expression system further comprises an expression cassette, The expression cassette includes an expression control sequence, The expression control sequence is operably ligated to the polynucleotide. The expression system according to any one of claims 1 to 5.

8. The expression control sequence includes a promoter, The promoter is at least one selected from a constitutive promoter, an inducible promoter, and a tissue-specific promoter. The tissue-specific promoter includes a fruit-specific promoter. The expression system according to claim 7.

9. The expression cassette is located within the plant transformation vector. The plant transformation vector comprises at least one of the following: a bacterial Ti plasmid, a Ri plasmid, a Ti-derived plasmid, and a Ri-derived plasmid. The aforementioned bacteria include Agrobacterium, The expression system according to claim 7.

10. The plant transformation vector has at least one of the selectable marker and the screenable marker, The selectable marker includes at least one of the antibiotic resistance marker and the herbicide resistance marker. The aforementioned screenable marker includes at least one of the following: a fluorescent protein gene, a β-glucuronidase gene, an amylase gene, a luciferase gene, a Xyle gene, and a β-lactamase gene. The expression system according to claim 9.

11. Uses of the expression system according to any one of claims 1 to 10 for improving the production of mogrosides in monk fruit.

12. The aforementioned monk fruit includes the whole or a part of the monk fruit. The aforementioned varieties of monk fruit include at least one selected from Qingpiguo, Changtanguo, Lajiangguo, Winter Melon Guo, Chashanguo, and Hongmaoguo. The use described in claim 11.

13. A method for improving the production of mogrosides in plants, The method described above includes a step of using the expression system described in any one of claims 1 to 10. method.

14. The method includes the step of applying the expression system to a plant, The polynucleotides in the expression system are expressed in plants and undergo codon optimization. The method according to claim 13.

15. The aforementioned method, The process involves introducing the expression system into plant cells through a transformation treatment to generate transformed plant cells, The process further includes the step of culturing the transformed plant cells to produce a transformed plant, The aforementioned plants include monk fruit, The method according to claim 13.

16. The aforementioned varieties of monk fruit include at least one selected from Qingpiguo, Changtanguo, Lajiangguo, Winter Melon Guo, Chashanguo, and Hongmaoguo. The method according to claim 15.

17. The transformation process includes at least one of the following: protoplast transfection, introduction via pollen tube pathway, Agrobacterium-mediated transformation, and transformation by particle gun. The method according to claim 15.

18. The transformation process includes Agrobacterium-mediated transformation. The aforementioned Agrobacterium contains a plant transformation vector. The method according to claim 15.

19. In the aforementioned transformed plants, the mogroside content changes, The mogroside includes at least one selected from mogroside I, mogroside II, mogroside III, mogroside IV, siamenside I, and mogroside V. The method according to claim 15.

20. A plant or part of a plant produced by a method for improving the production of mogrosides in a plant according to any one of claims 13 to 19.

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