Construction method of tandem gene, expression frame, expression vector and rice germplasm of synthetic ginsenoside Ro

Abstract

This invention provides a tandem gene, expression cassette, expression vector, and rice germplasm construction method for synthesizing ginsenoside Ro, belonging to the field of genetic engineering technology. This invention provides a tandem gene for synthesizing ginsenoside Ro, which involves tandemly connecting four key genes from *Saccharomyces cerevisiae*, one gene from *Licoricea javanica*, and five genes from ginseng, and constructing gene expression units for each, thereby forming a tandem expression cassette. This invention connects the tandem expression cassette with a plant expression vector to construct a recombinant expression vector, transforms the recombinant expression vector into *Agrobacterium*, and successfully transforms the recombinant expression vector into the rice genome using an *Agrobacterium*-mediated genetic transformation method. The rice can express the recombinant expression vector correctly and efficiently in the grains, laying a solid technical foundation for the large-scale synthesis of ginsenoside Ro.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for constructing a tandem gene, expression cassette, expression vector, and rice germplasm for synthesizing ginsenoside Ro. Background Technology

[0002] Ginsenoside Ro is an oleanolic acid-type pentacyclic triterpenoid saponin. As an important natural functional active substance in ginseng, it has significant advantages in terms of its natural source and lack of toxic side effects, and possesses broad pharmacological application value. Studies have shown that ginsenoside Ro can exert a potent anti-inflammatory effect by regulating the level of inflammatory factors in the body, effectively alleviating tissue inflammatory responses; at the same time, it can regulate the activity of immune cells, enhance the body's immune function, and improve the body's resistance to pathogens; in terms of cardiovascular protection, it can improve vascular endothelial function, inhibit platelet aggregation, and help maintain cardiovascular homeostasis; in addition, its hepatoprotective effect is particularly prominent, which can reduce chemical or pathological liver damage, protect the integrity of hepatocytes, and also has auxiliary physiological functions such as anti-oxidation and metabolic regulation. Currently, the main method for obtaining ginsenoside Ro is direct extraction from ginseng plants. However, ginseng has a long growth cycle of 4-6 years, with stringent requirements for soil and climate conditions. Furthermore, ginsenoside Ro is present in extremely low concentrations within ginseng, accounting for only a small percentage of total ginsenosides. This results in a cumbersome extraction process with low yields. Additionally, impurities are easily left behind during extraction, further increasing purification costs. This traditional extraction method not only hinders large-scale production but also fails to meet the growing market demand in the pharmaceutical and health product sectors. Therefore, developing efficient and sustainable new methods for obtaining ginsenoside Ro is urgently needed. Summary of the Invention

[0003] This invention provides a method for constructing a tandem gene, expression cassette, expression vector, and rice germplasm for synthesizing ginsenoside Ro. This method enables the synthesis of ginsenoside Ro from rice, thereby producing high-value-added rice and providing an efficient and sustainable source crop for the production of ginsenoside Ro.

[0004] This invention provides a tandem gene for synthesizing ginsenoside Ro, comprising the encoding genes of the following enzymes connected in sequence: 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, licorice β-amyrin synthase 1, ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18; The 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxygenase mentioned above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ); The β-amyrin synthase 1 from licorice root (Glycyrrhiza glabra) is derived from licorice (Glycyrrhiza glabra). Glycyrrhiza glabra ); The ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18 are derived from ginseng ( Panax ginseng ).

[0005] In one specific embodiment of the present invention, the amino acid sequences of each enzyme in the tandem gene are shown in SEQ ID No. 1 to SEQ ID No. 10 in sequence.

[0006] In one specific embodiment of the present invention, the nucleotide sequences of the encoding genes of each enzyme in the tandem gene are shown in SEQ ID No. 11 to SEQ ID No. 20 in sequence.

[0007] This invention also provides an expression cassette for synthesizing ginsenoside Ro, comprising expression units of the encoding genes of the following enzymes connected in sequence: 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, licorice β-amyrin synthase 1, ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18; The 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxygenase mentioned above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ); The β-amyrin synthase 1 from licorice root (Glycyrrhiza glabra) is derived from licorice (Glycyrrhiza glabra). Glycyrrhiza glabra ); The ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18 are derived from ginseng ( Panax ginseng ); Each enzyme's encoding gene expression unit includes a rice endosperm-specific globular protein promoter and terminator.

[0008] In one specific embodiment of the present invention, the rice endosperm-specific globulin promoter is... GluB-1 The promoter, with a nucleotide sequence as shown in SEQ ID No. 21; the rice endosperm-specific globulin terminator is... GluB-1 Terminator, nucleotide sequence as shown in SEQ ID No. 22.

[0009] The present invention also provides a recombinant expression vector comprising at least one of the above-described tandem genes or the above-described expression cassette.

[0010] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0011] The present invention also provides a method for constructing rice expressing ginsenoside Ro, comprising the following steps: connecting the above expression frame with a plant expression vector to construct a recombinant expression vector; The recombinant expression vector was transformed into the target rice to construct rice expressing ginsenoside Ro.

[0012] The present invention also provides rice expressing ginsenoside Ro constructed using the above-described construction method.

[0013] The present invention also provides a method for producing ginsenoside Ro, comprising extracting ginsenoside Ro from the grains or tissues of the rice described above.

[0014] Beneficial effects: This invention constructs a complete pathway for the synthesis of heterologous ginsenoside Ro using genes from different species, and uses genes from brewer's yeast ( Saccharomyces cerevisiae The four key genes Sc HMG1 (3-Hydroxy-3-methylglutaryl-CoA reductase) ScERG20 (Farnesy pyrophosphate synthase) ScERG9 (Squalene synthase) and ScERG1 (Squalene epoxidase), derived from licorice ( Glycyrrhiza glabra A gene GgbAS1 (Glycyrrhiza glabra β-amyrin synthase 1), and ginseng-derived ( Panax ginseng 5 genes PgOAS1 (Ginseng Oleanolic Acid Synthase 1) PgCPR2 (Ginsenoside P450 reductase 2) PgCSyGT1 (Ginseng cellulose synthase type glycosyltransferase 1) PgUGT8 (Ginseng UDP-glucosyltransferase 8) and PgUGT18 (and ginseng UDP-glucosyltransferase 18) were tandemly linked and connected to the rice endosperm-specific globulin promoter and terminator to construct gene expression units, thereby forming tandem expression frames.

[0015] This invention connects the tandem expression cascade with a plant expression vector to construct a recombinant expression vector. The recombinant expression vector is then transformed into Agrobacterium and successfully transferred into the rice genome using Agrobacterium-mediated genetic transformation. Examples demonstrate that the average content of ginsenoside Ro in transgenic rice seeds reached 0.17 g / g dry weight. Furthermore, the content of oleanolic acid, a key intermediate in transgenic rice, was detected, showing an average of 2.36 ng / g dry weight. This fully demonstrates that this optimized gene combination can be efficiently expressed in rice endosperm, laying a solid technical foundation for the large-scale synthesis of ginsenoside Ro.

[0016] This invention utilizes rice seeds as a "molecular farm," which, compared to microbial fermentation systems with potential allergens and complex downstream purification processes, as well as natural extraction methods for ginseng that are costly and dependent on long growth cycles, offers advantages such as high safety, low production costs, and convenient storage and transportation. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the ginsenoside Ro synthesis pathway constructed in this invention; Figure 2 Diagram of the carrier structure for synthesizing ginsenoside Ro; Figure 3 Electrophoresis image of RT-PCR detection of exogenous genes in transgenic rice plants; Figure 4 UPLC-MS / MS chromatogram of ginsenoside R0 in rice seeds; Figure 5 The UPLC-MS / MS chromatogram of oleanolic acid in rice seeds. Detailed Implementation

[0018] This invention provides a tandem gene for synthesizing ginsenoside Ro, comprising the encoding genes of the following enzymes connected in sequence: 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, licorice β-amyrin synthase 1, ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18; The 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxygenase mentioned above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ); The β-amyrin synthase 1 from licorice root (Glycyrrhiza glabra) is derived from licorice (Glycyrrhiza glabra). Glycyrrhiza glabra ); The ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18 are derived from ginseng ( Panax ginseng ).

[0019] In the tandem genes described in this invention, each gene is merely the coding sequence of its corresponding gene, and it has been confirmed that the tandem genes can be expressed in crops. In one embodiment, it is required to express the tandem genes in rice; therefore, codon optimization was performed to obtain a biosynthetic ginsenoside Ro-related gene that can be stably expressed in rice. In this invention, the optimization of the coding region gene structure follows the following principles: (1) Optimize gene codons according to the codon preference of rice to improve gene translation efficiency.

[0020] (2) Eliminate the recognition sites of commonly used restriction endonucleases inside the gene to facilitate the construction of expression cassettes.

[0021] (3) Eliminate inverted repeat sequences, stem-loop structures and transcription termination signals to balance GC / AT within the gene and improve RNA stability.

[0022] (4) Make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein.

[0023] (5) Optimize the mRNA secondary structure free energy to improve gene expression efficiency.

[0024] This invention begins with 3-hydroxy-3-methylglutaryl-CoA reductase derived from Saccharomyces cerevisiae, whose encoding gene is Sc. HMG1 It is the rate-limiting enzyme in the mevalonate (MVA) pathway, catalyzing the conversion of HMG-CoA to mevalonate (MVA). The Sc described in this invention... HMG1 After codon optimization, it is named Sc HMG1S Its amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 11.

[0025] This invention is derived from farnesyl pyrophosphate (FPP) synthase in Saccharomyces cerevisiae, the encoding gene of which is: ScERG20 It is the core node of the MVA pathway, catalyzing a two-step continuous condensation: the first step condenses IPP (isopentene pyrophosphate) and DMAPP (dimethylallyl pyrophosphate) into GPP (geranyl pyrophosphate, C 10 The second step involves condensing GPP and IPP into FPP (farnesyl pyrophosphate, C100000). 15 The present invention describes... ScERG20 After codon optimization, it was named ScERG20S,Its amino acid sequence is shown in SEQ ID No. 2, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 12.

[0026] This invention is derived from squalene synthase in Saccharomyces cerevisiae, the encoding gene of which is: ScERG9 It is a key branch point in sterol synthesis in the MVA pathway and the first step in the flow of FPP to ergosterol. It can catalyze the condensation of two FPP molecules to generate squalene (C). 30 The present invention describes... ScERG9 The name after codon optimization is ScERG9S, Its amino acid sequence is shown in SEQ ID No. 3, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 13.

[0027] This invention is derived from squalene epoxidase in Saccharomyces cerevisiae, the encoding gene of which is: ScERG1 It is the key rate-limiting enzyme in ergosterol synthesis, catalyzing the synthesis of squalene (C... 30 The 2,3-position of the epoxidation of ) yields 2,3-epoxysqualene. This invention describes... ScERG1 The name after codon optimization is ScERG1S Its amino acid sequence is shown in SEQ ID No. 4, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 14.

[0028] This invention is derived from licorice ( Glycyrrhiza glabra The gene encoding β-amyrin synthase 1 from licorice is: GgbAS1 It is a core enzyme in triterpenoid synthesis and a key starting point for the synthesis of glycyrrhizin and oleanolic acid triterpenoids. Using 2,3-epoxysqualene as the sole substrate, it catalyzes the production of β-amyrin (a pentacyclic triterpenoid with an oleanolic skeleton). The present invention... GgbAS1 The name after codon optimization is GgbAS1S Its amino acid sequence is shown in SEQ ID No. 5, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 15.

[0029] This invention is derived from ginseng ( Panax ginseng The gene encoding ginsenoside synthase 1 is: PgOAS1 It is the core rate-limiting enzyme in the synthesis of oleanolic acid-type ginsenosides (such as ginsenoside Ro), catalyzing the production of β-amyrin and oleanolic acid from 2,3-epoxysqualene. The present invention... PgOAS1 The name after codon optimization is PgOAS1S Its amino acid sequence is shown in SEQ ID No. 6, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 16.

[0030] This invention relates to ginseng cytochrome P450 reductase 2 derived from ginseng, the encoding gene of which is: PgCPR2 It is the electron transport core for ginsenoside synthesis, providing electrons to all ginseng CYP450 oxidases (such as CYP716A and CYP72A), and is an essential co-expression element for ginsenoside synthesis in yeast. The present invention describes... PgCPR2 The name after codon optimization is PgCPR2S Its amino acid sequence is shown in SEQ ID No. 7, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 17.

[0031] This invention relates to ginseng cellulose synthase-type glycosyltransferase 1 derived from ginseng, the encoding gene of which is: PgCSyGT1 This is a key C3-position glucuronyl transferase in the synthesis of oleanolic acid-type ginsenosides, specifically catalyzing the glucuronidation of oleanolic acid C3-OH, which is the first glycosylation step in the formation of ginsenoside Ro; using oleanolic acid as a substrate and UDP-glucuronic acid (UDP-GlcA) as a sugar donor. The present invention... PgCSyGT1 The name after codon optimization is PgCSyGT1S Its amino acid sequence is shown in SEQ ID No. 8, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 18.

[0032] This invention relates to ginseng UDP-glucosyltransferase 8 derived from ginseng, whose encoding gene is: PgUGT8 Using UDP-glucose (UDP-Glc) as a sugar donor, responsible for adding glucose at the C28-COOH site, is an essential step in the synthesis of ginsenoside Ro. The present invention... PgUGT8 The name after codon optimization is PgUGT8S Its amino acid sequence is shown in SEQ ID No. 9, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 19.

[0033] This invention relates to ginseng UDP-glucosyltransferase 18 derived from ginseng, whose encoding gene is: PgUGT18 Using UDP-glucose (UDP-Glc) as a sugar donor, it specifically adds glucose to the outer side of C3-O-glucuronic acid (GlcA) and is a key enzyme in the final step of ginsenoside Ro synthesis. Its encoding gene is [gene information missing]. PgUGT18 The present invention describes PgCSyGT1 The name after codon optimization is PgUGT18S Its amino acid sequence is shown in SEQ ID No. 10, and the nucleotide sequence encoding the gene is shown in SEQ ID No. 20.

[0034] In this invention, in addition to the sequences shown above, other homologous sequences may also be used. Homologous enzymes or mutant enzymes with the same amino acid sequence homology and function are all within the scope of protection of this invention. Homologous genes or mutated genes encoding genes are also included, as long as the encoded protein has a function similar to the corresponding enzyme. This invention does not specifically limit the type of variation or mutation; it can be the deletion, addition, alteration, extension, or modification of one or more amino acids / nucleotides.

[0035] This invention also provides an expression cassette for synthesizing ginsenoside Ro, comprising expression units of the encoding genes of the following enzymes connected in sequence: 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, licorice β-amyrin synthase 1, ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18; The 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxygenase mentioned above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ); The β-amyrin synthase 1 from licorice root (Glycyrrhiza glabra) is derived from licorice (Glycyrrhiza glabra). Glycyrrhiza glabra ); The ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18 are derived from ginseng ( Panax ginseng ); Each enzyme-encoded gene expression unit includes a rice endosperm-specific globulin promoter and a terminator.

[0036] This invention uses each gene as an expression unit, and 10 expression units are cascaded according to the gene connection order to obtain a complete expression frame. The expression unit of this invention includes a rice endosperm-specific globulin promoter, a coding gene, and a terminator connected in sequence, wherein the rice endosperm-specific globulin promoter is... GluB-1 The promoter sequence is shown in SEQ ID No. 21; the rice endosperm-specific globulin terminator is... GluB-1 Terminator, sequence as shown in SEQ ID No. 22.

[0037] The present invention also provides a recombinant expression vector comprising at least one of the above-described tandem genes or the above-described expression cassette.

[0038] The recombinant expression vector of the present invention contains the above-mentioned Sc HMG1S , ScERG20S ,ScERG9S , ScERG1S , GgbAS1S , PgOAS1S , PgCPR2S , PgCSyGT1S , PgUGT8S and PgUGT18S At least one gene in the gene sequence is present, requiring the construction of multiple recombinant expression vectors to satisfy the expression of the aforementioned 10 genes. The recombinant expression vector of this invention may also contain at least one expression unit from the aforementioned 10 gene expression units, requiring the construction of multiple recombinant expression vectors to satisfy the expression of the aforementioned 10 genes. The recombinant expression vector of this invention may also contain the aforementioned expression frame. The recombinant expression vector uses a plant expression vector as the base vector. In one embodiment, the pCAMBIA1301 expression vector (GenBank accession number AF234297.1) is used as the base vector, and the complete expression frame is ligated into the pCAMBIA1301 expression vector to construct the recombinant expression vector.

[0039] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0040] The recombinant host cell described in this invention can be an intermediate host, such as Agrobacterium EHA105, that delivers the recombinant expression vector to the genome of the target plant; or it can be a plant cell containing the recombinant expression vector and expressing ginsenoside Ro.

[0041] The present invention also provides a method for constructing rice expressing ginsenoside Ro, comprising the following steps: connecting the above expression frame with a plant expression vector to construct a recombinant expression vector; The recombinant expression vector was transformed into the target rice to construct rice expressing ginsenoside Ro.

[0042] In one embodiment of the present invention, the recombinant expression vector is transformed into Agrobacterium EHA105, and the recombinant expression vector is transferred into a rice host using an Agrobacterium-mediated genetic transformation method, thereby constructing rice that can express ginsenoside Ro.

[0043] The present invention also provides rice expressing ginsenoside Ro constructed using the above-described construction method.

[0044] The transgenic rice plants described in this invention can amplify the above 10 genes, and they are expressed to different degrees in the transgenic rice endosperm, indicating that the above 10 exogenous genes have been successfully integrated and effectively transcribed in the rice endosperm. Ginsenoside Ro and oleanolic acid can be detected in rice grains.

[0045] The present invention also provides a method for producing ginsenoside Ro, comprising extracting ginsenoside Ro from the grains or tissues of the rice described above.

[0046] The present invention does not specifically limit the extraction method of ginsenoside Ro. It can be extracted using conventional extraction methods in the field. For example, in the examples, refer to the paper published by Yao Ruijiao et al., Study on extraction and purification process of total saponins from stems and leaves of Panax notoginseng [J].

[0047] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for constructing a tandem gene, expression cassette, expression vector, and rice germplasm for synthesizing ginsenoside Ro, provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products. This invention relates to molecular biology experiments; unless otherwise noted, all references are made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1994, Science Press). The rice seeds (Xiushui 134) used were preserved by the Agricultural Synthetic Biology Research Center of the Institute of Biotechnology, Shanghai Academy of Agricultural Sciences. Unless otherwise specified, all reagents used in this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd. or Shanghai Sinopharm Group Co., Ltd.

[0049] Example 1: Optimization and Synthesis of Ginsenoside Ro Synthesis Gene Using the four key genes Sc of brewer's yeast HMG1 , ScERG20 , ScERG9 and ScERG1 One gene of licorice GgbAS1 The 5 genes of ginseng PgOAS1 , PgCPR2 , PgCSyGT1 , PgUGT8 and PgUGT18 Using the original sequence as a template, sequence optimization was performed, and the optimized gene nucleotide sequences are shown in SEQ ID No. 11~SEQ ID No. 20, respectively. The whole gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0050] Example 2 Construction of Multi-Gene Plant Transformation Vector The 10 optimized genes from Example 1 were ligated to rice endosperm-specific promoters to construct ten gene expression cassettes. The ten gene expression cassettes were then sequentially ligated using the ClonExpress MultiS multi-fragment one-step seamless rapid cloning kit (Novizan) (ScHMG1S-ScERG20S-ScERG9S-ScERG1S-GgbAS1S-PgOAS1S-PgCPR2S-PgCSyGT1S-PgUGT8S-PgUGT18S) to form a complete sequence containing a multi-gene expression cassette. EcoRI and HindIII restriction sites were introduced at both ends of the complete sequence. The complete sequence was analyzed by Sangon Biotech (Shanghai) Co., Ltd. Finally, the correctly sequenced complete synthetic fragment was digested with EcoRI and HindIII and ligated into the same digestion vector pCAMBIA1301 to obtain a multi-gene plant transformation vector containing ten genes: pCAMBIA1301-ScHMG1S-ScERG20S-ScERG9S-ScERG1S-GgbAS1S-PgOAS1S-PgCPR2S-PgCSyGT1S-PgUGT8S-PgUGT18S. Figure 1 and Figure 2 ).

[0051] Example 3 Agrobacterium-mediated genetic transformation of rice 1) Preparation of Agrobacterium The recombinant vector pCAMBIA1301-ScHMG1S-ScERG20S-ScERG9S-ScERG1S-GgbAS1S-PgOAS1S-PgCPR2S-PgCSyGT1S-PgUGT8S-PgUGT18S containing the multi-gene tandem expression cassette from Example 2 was transformed into Agrobacterium EHA105 via electroporation. The target Agrobacterium was then recultured at an OD concentration. 600 A suspension of Agrobacterium-mediated transformation of rice was prepared with a concentration of 0.3-0.5.

[0052] 2) Rice genetic transformation Genetic transformation was performed according to conventional rice genetic transformation methods, referring to the paper published by Zhou Lei et al., "Optimization Study of Agrobacterium-mediated Rice Genetic Transformation Method" [J]. Xiushui 134 was selected as the host. Seeds were sterilized with 0.1% mercuric chloride and placed on induction medium. After induction culture, pale yellow callus tissue was generated. The callus tissue was co-cultured with Agrobacterium for 2-3 days and then placed on selection medium for dark culture for 14 days. After differentiation, rice seedlings were obtained, followed by rooting, seedling strengthening, and transplanting to obtain mature rice.

[0053] Example 4: Molecular Identification of Transgenic Rice DNA was extracted from the rice cultured in Example 3 and detected using the primer pairs described in Table 1: PCR reaction system (50μL): 10×PCR buffer 5.0μL; dNTPs 4μL (2.5 mmol / L); template 1μL (20ng~50ng); primer R 1μL; primer F 1μL; Taq enzyme 0.2μL; add sterile water to a final volume of 50μL.

[0054] Reaction program: 94℃ for 5 min; 94℃ for 20 s, 56℃ for 30 s, 72℃ for 30 s, 32 cycles; 72℃ for 10 min.

[0055] Table 1 PCR amplification primers

[0056] PCR results showed that transgenic plants could amplify a fragment of the same size as the target band, while the wild-type control did not show this band. Figure 3 ).

[0057] Example 5: Construction of a rice molecular farm for producing ginsenoside Ro The rice plants that tested positive in Example 4 were planted in the farmland according to the following steps: 1) Select suitable land, plow, fertilize, irrigate, etc., to ensure that the soil fertility and moisture meet the needs of rice growth.

[0058] 2) Sow the selected rice seeds in the field. There are two sowing methods: direct sowing and seedbed sowing. Direct sowing involves scattering the seeds directly in the field, while seedbed sowing involves planting the seedlings in a seedbed first, and then transplanting them into the field after they have grown to a certain height.

[0059] 3) After sowing, the field needs to be managed regularly, including weeding, irrigation, fertilization, and pest and disease control.

[0060] 4) After the rice matures, it is harvested. Harvesting can be done manually or by machine.

[0061] 5) After harvesting, the rice needs to be threshed, which separates the rice grains from the husks for easier storage and retrieval.

[0062] Example 6: UPLC-MS / MS Targeted Metabolomics Detection of Ginsenoside Ro Content and Key Intermediate Substance Content The transgenic rice plants (T1 generation) obtained in Example 5 were planted until maturity, and their seeds were harvested. Then, the contents of ginsenoside Ro and oleanolic acid in the seeds were detected by UPLC-MS / MS. The specific steps are as follows: 1) Metabolite extraction Five seeds were randomly selected, and the seeds were dehulled and crushed. 0.5 g of the powder was placed in a 10 mL centrifuge tube, and 5 mL of 70% methanol aqueous solution was added. The mixture was vortexed for 3 min. Then, ultrasonic extraction was performed for 35 min, with vortexing for 30 s every 10 min during the ultrasonic process. The mixture was then centrifuged at 8000 rpm for 10 min, and all the supernatant was transferred to a new centrifuge tube. 5 mL of 70% methanol aqueous solution was added to the residue, and the ultrasonic and centrifugation steps were repeated. The supernatants from both extractions were combined. Finally, the resulting liquid was concentrated to 1 mL by nitrogen blowing and then processed.

[0063] 2) Detection of ginsenoside Ro Detection was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and the target compounds were separated by chromatographic separation using a liquid chromatography column. Liquid chromatograph: Agilent Xevo TQ's 1260 HPLC-MS / MS. Column: Agilent ZORBAX Eclipse Plus C18 (3.5µm, 2.1m). 150mm).

[0064] The parameters are shown in Tables 2 and 3: Table 2 Parameters of Liquid Chromatography

[0065] Table 3 Liquid Phase Gradient Elution Program

[0066] MS parameters Mass spectrometry analysis was performed using a Waters triple quadrupole mass spectrometer equipped with an ESI ion source in dynamic multiple reaction monitoring (MRM) mode. The ion source parameters are shown in Table 4. Table 4 Ion source parameters

[0067] MRM channels are shown in Table 5: Table 5. Ion pairs monitored by MRM channels

[0068] The results of ginsenoside RO detection are as follows: Table 6. Ginsenoside Ro content

[0069] The results showed that ginsenoside Ro could be detected in all different strains. Figure 4 The average content reached 0.17 mg / g.

[0070] 3) Oleanolic acid detection Detection was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and the target compounds were separated by chromatographic separation using a liquid chromatography column. Liquid chromatograph: Agilent Xevo TQ's 1260 HPLC-MS / MS. Column: Agilent ZORBAX Eclipse Plus C18 (3.5µm, 2.1m). 150mm).

[0071] The parameters are shown in Tables 7 and 8: Table 7 Parameters of Liquid Chromatography

[0072] Table 8 Liquid Phase Gradient Elution Procedure

[0073] MS parameters Mass spectrometry analysis was performed using a Waters triple quadrupole mass spectrometer equipped with an ESI ion source in dynamic multiple reaction monitoring (MRM) mode. The ion source parameters are shown in Table 9. Table 9 Ion source parameters

[0074] MRM channels are shown in Table 10: Table 10 MRM channel monitoring of ion pairs

[0075] The results of the oleanolic acid test are as follows: Table 11 Oleanolic acid content

[0076] The results showed that oleanolic acid could be detected in all different strains. Figure 4 The average content reached 2.36 ng / g.

[0077] In summary, this invention successfully synthesizes ginsenoside Ro in rice by introducing and optimizing a complete set of ginsenoside Ro synthesis genes, providing a novel and efficient technical solution for the low-cost and safe production of high-value functional active substance ginsenoside Ro using plant systems.

[0078] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tandem gene for synthesizing ginsenoside Ro, characterized in that, The gene encoding the following enzymes is linked in sequence: 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, licorice β-amyrin synthase 1, ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18; The 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxygenase mentioned above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ); The β-amyrin synthase 1 from licorice root (Glycyrrhiza glabra) is derived from licorice (Glycyrrhiza glabra). Glycyrrhiza glabra ); The ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18 are derived from ginseng ( Panax ginseng ).

2. The tandem gene according to claim 1, characterized in that, The amino acid sequences of each enzyme in the tandem gene are shown in SEQ ID No. 1 to SEQ ID No. 10, respectively.

3. The tandem gene according to claim 1 or 2, characterized in that, The nucleotide sequences of the encoding genes of each enzyme in the tandem gene are shown in SEQ ID No. 11 to SEQ ID No. 20, respectively.

4. An expression cassette for synthesizing ginsenoside Ro, characterized in that, The expression units include the gene encoding the following enzymes linked in sequence: 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, licorice β-amyrin synthase 1, ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18; The 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxygenase mentioned above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ); The β-amyrin synthase 1 from licorice root (Glycyrrhiza glabra) is derived from licorice (Glycyrrhiza glabra). Glycyrrhiza glabra ); The ginseng oleanolic acid synthase 1, ginseng cytochrome P450 reductase 2, ginseng cellulose synthase-type glycosyltransferase 1, ginseng UDP-glucosyltransferase 8, and ginseng UDP-glucosyltransferase 18 are derived from ginseng ( Panax ginseng ); Each enzyme's encoding gene expression unit includes a rice endosperm-specific globular protein promoter and terminator.

5. The expression box according to claim 4, characterized in that, The rice endosperm-specific globular protein promoter is GluB-1 The promoter, with its nucleotide sequence shown in SEQ ID No. 21; The rice endosperm-specific globulin terminator is: GluB-1 Terminator, nucleotide sequence as shown in SEQ ID No.

22.

6. A recombinant expression vector comprising at least one gene of the tandem gene of any one of claims 1 to 3 or the expression cassette of claim 4 or 5.

7. A recombinant host cell comprising the recombinant expression vector of claim 6.

8. A method for constructing rice expressing ginsenoside Ro, characterized in that, The process includes the following steps: connecting the expression frame described in claim 4 or 5 to a plant expression vector to construct a recombinant expression vector; The recombinant expression vector was transformed into the target rice to construct rice expressing ginsenoside Ro.

9. Rice expressing ginsenoside Ro, constructed using the method described in claim 8.

10. A method for producing ginsenoside Ro, characterized in that, This includes extracting ginsenoside Ro from the grains or tissues of the rice described in claim 9.

Images