The invention relates to hematoxyloxymethyltransferase CsOMT50 and its application in 2apos; application of-O-methylisoliquiritigenin synthesis

Abstract

The invention provides hematoxyloxymethyltransferase CsOMT50 and application of the hematoxyloxymethyltransferase CsOMT50 in synthesis of 2 '-O-methylisoliquiritigenin, and belongs to the technical field of genetic engineering. In-vivo verification of tobacco and hematoxylin species and prokaryotic cell expression are carried out on hematoxyloxymethyltransferase CsOMT50, CsOMT50 overexpression in tobacco, hematoxylin or prokaryotic cells is obtained, and the yield of 2 '-O-methylisoliquiritigenin can be increased. The CsOMT50 is subjected to point mutation, the synthesis condition of the 2 '-O-methylisoliquiritigenin can be regulated and controlled, the 275bp amino acid of the amino acid sequence of the CsOMT50 is mutated from H to R, and the yield of the 2'-O-methylisoliquiritigenin can be increased.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a hematoxymethyltransferase CsOMT50 and its application in the synthesis of 2'-O-methylisoglycyrrhizin. Background Technology

[0002] Sappanwood ( Caesalpinia sappan Sappanwood (L.) is a legume belonging to the genus Sappanwood, widely distributed in the pantropical region. It has a medicinal history of over 1800 years in my country. The Chinese Pharmacopoeia lists "promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain" as its core efficacy. Extracts from its dried heartwood have identified several active ingredients, including isoliquiritin and brassinolide. Among them, 2'-O-methylisoglycyrrhizin (4,4'-dihydroxy-2'-methoxychalcone), as a characteristic methylated derivative of isoliquiritin, exhibits significant anti-inflammatory, antioxidant, and potential anti-tumor activities, demonstrating important application value in the field of natural drug development. However, the current acquisition of 2'-O-methylisoglycyrrhizin faces severe bottlenecks: the content of this component in natural Sappanwood is extremely low, and its supply stability and product enrichment efficiency are difficult to meet industrial needs due to the influence of growth cycle, geographical climate, and cultivation conditions; the chemical synthesis route involves multiple methylation modifications, resulting in poor reaction selectivity, numerous byproducts, and insufficient environmental friendliness, thus limiting its practical application. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a hematoxymethyltransferase CsOMT50 and its application in the synthesis of 2'-O-methylisoglycyrrhizin, by regulating the hematoxymethyltransferase... CsOMT50 Overexpression of 2'-methyl isoliquiritigenin or point mutation of the amino acid sequence of hematoxymethyltransferase CsOMT50 can significantly improve the synthesis capacity and yield of 2'-O-methyl isoliquiritigenin.

[0004] The objective of this invention is achieved through the following technical solution: This invention provides a hematoxymethyltransferase CsOMT50, the amino acid sequence of which is shown in SEQ ID NO.11.

[0005] Preferably, the nucleotide sequence encoding CsOMT50 is shown in SEQ ID NO.12.

[0006] This invention provides the application of the hematoxymethyltransferase CsOMT50 described in the above technical solution in the synthesis of 2'-O-methylisoglycyrrhizin.

[0007] Preferably, overexpression of hematoxymethyltransferase CsOMT50 increases the yield of 2'-O-methylisoglycyrrhizin.

[0008] Preferably, the production of 2'-O-methyl isoliquiritigenin can be regulated by a 275bp point mutation in the amino acid sequence of CsOMT50.

[0009] Preferably, the amino acid at the 275th bp of the CsOMT50 amino acid sequence is mutated from H to R to increase the yield of 2'-O-methylisoglycyrrhizin.

[0010] Preferably, the amino acid at the 275th bp of the CsOMT50 amino acid sequence is mutated from H to L to reduce the yield of 2'-O-methylisoglycyrrhizin.

[0011] This invention provides a method for increasing the yield of 2'-O-methylisoglycyrrhizin, comprising: Overexpressing CsOMT50 in cells; Alternatively, the amino acid sequence of CsOMT50 in cells could be mutated from H to R at a 275bp interval.

[0012] This invention provides the application of hematoxymethyltransferase CsOMT50 in breeding for high yield of 2'-O-methyl isoglycyrrhizin.

[0013] This invention provides a method for breeding plants to produce high yields of 2'-O-methyl isoglycyrrhizin, comprising: Overexpress CsOMT50 in plants; or mutate 275 bp of the amino acid sequence of CsOMT50 in plants from H to R; then screen for positive plants.

[0014] This invention provides a hematoxymethyltransferase CsOMT50 and its application in the synthesis of 2'-O-methylisoglycyrrhizin. The invention validates the hematoxymethyltransferase CsOMT50 in vivo in tobacco and sappanwood species, as well as in prokaryotic cells, demonstrating that overexpression of CsOMT50 in tobacco, sappanwood, or prokaryotic cells can increase the yield of 2'-O-methylisoglycyrrhizin. Furthermore, point mutation of CsOMT50 can regulate the synthesis of 2'-O-methylisoglycyrrhizin. Mutating the 275th bp amino acid of CsOMT50 from H to R increases the yield of 2'-O-methylisoglycyrrhizin; mutating the 275th bp amino acid of CsOMT50 from H to L decreases the yield of 2'-O-methylisoglycyrrhizin. This invention provides important gene resources and theoretical support for the study of 2'-O-methyl isoliquiritigenin biosynthesis and the development of 2'-O-methyl isoliquiritigenin-based drugs by exploring the hematoxymethyltransferase CsOMT50. Attached Figure Description

[0015] Figure 1 CsOMT50 tobacco fluorescence image; Figure 2 Liquid chromatography diagram for validating the function of CsOMT50 in transgenic tobacco; CK: injection of isoliquiritigenin substrate only; CsOMT50; injection of CsOMT50 + substrate; Figure 3 This is a secondary mass spectrum of CsOMT50 from genetically modified tobacco. Figure 4 Figure 1 shows the results of 2'-O-methyl isoglycyrrhizin content detection in genetically modified tobacco and wild-type tobacco. Figure 5 Liquid phase diagram for crude protein catalytic analysis of transgenic tobacco CsOMT50; Figure 6 Secondary mass spectra of crude protein catalytic analysis from transgenic tobacco CsOMT50; Figure 7 Figure showing the detection results of 2'-O-methyl isoglycyrrhizin content produced by crude protein catalysis in genetically modified tobacco CsOMT50; Figure 8 Fluorescence image of hairy roots of transgenic sappanwood; Figure 9 The graph shows the relative expression levels of the CsOMT50 gene in transgenic and wild-type sappanwood. Figure 10 Liquid chromatography diagram for functional verification of transgenic sappanwood CsOMT50; CK: wild type; CsOMT50: CsOMT50 bacterial suspension treatment; Figure 11 This is the secondary mass spectrum of transgenic hematoxylin CsOMT50; Figure 12 Figure 1 shows the results of 2'-O-methyl isoglycyrrhizin content detection in genetically modified sappanwood and wild-type tobacco. Figure 13 Liquid phase diagram for crude protein catalytic analysis of transgenic hematoxylin CsOMT50; Figure 14 Secondary mass spectra of crude protein catalytic analysis from transgenic hematoxylin CsOMT50; Figure 15 The results of the production of 2'-O-methyl isoliquiritigenin by crude protein catalysis of transgenic and wild-type sappanwood CsOMT50 are shown in the figure. Figure 16 The image shows the SDS-PAGE analysis results of CsOMT50 protein generated in prokaryotic cells. Figure 17 This is a liquid phase diagram of the product of CsOMT50 catalyzing isoliquiritigenin generated in prokaryotic cells. Figure 18 Secondary mass spectra of CsOMT50 generated within prokaryotic cells for catalytic analysis. Figure 19The content of 2'-O-methyl isoliquiritigen catalyzed by CsOMT50 generated in prokaryotic cells; Figure 20 The image shows the SDS-PAGE analysis results of the CsOMT50H275R protein. Figure 21 The image shows the SDS-PAGE analysis results of the CsOMT50H275L protein. Figure 22 Liquid phase diagram of the product of isoliquiritigenin catalyzed by the CsOMT50 mutant; Figure 23 Secondary mass spectra of CsOMT50H275R catalyst; Figure 24 The content of 2'-O-methyl isoliquiritigenin produced by the CsOMT50 mutant catalysis. Detailed Implementation

[0016] This invention provides a hematoxymethyltransferase CsOMT50, the amino acid sequence of which is shown in SEQ ID NO. 11. As an optional embodiment of this invention, the nucleotide sequence encoding CsOMT50 is shown in SEQ ID NO. 12.

[0017] This invention provides the application of the hematoxymethyltransferase CsOMT50 described in the above-mentioned technical solution in the synthesis of 2'-O-methylisoglycyrrhizin. As an optional embodiment of this invention, overexpression of hematoxymethyltransferase is performed. CsOMT50 To increase the yield of 2'-O-methylisoglycyrrhizin, as an optional embodiment of the present invention, the yield of 2'-O-methylisoglycyrrhizin can be regulated by a point mutation at 275 bp of the amino acid sequence of CsOMT50. Preferably, the yield of 2'-O-methylisoglycyrrhizin is increased by mutating the amino acid at 275 bp of the CsOMT50 sequence from H to R. Alternatively, the yield of 2'-O-methylisoglycyrrhizin is decreased by mutating the amino acid at 275 bp of the CsOMT50 sequence from H to L.

[0018] This invention validates the hematoxymethyltransferase CsOMT50 in vivo and in prokaryotic cells using tobacco and sappanwood species, demonstrating that overexpression of CsOMT50 in tobacco, sappanwood, or prokaryotic cells increases the yield of 2'-O-methylisoglycyrrhizin. Furthermore, point mutation of the CsOMT50 amino acid sequence at 275 bp regulates 2'-O-methylisoglycyrrhizin synthesis. Mutating the 275 bp amino acid from H to R increases 2'-O-methylisoglycyrrhizin yield; mutating it from H to L decreases 2'-O-methylisoglycyrrhizin yield.

[0019] This invention provides a method for increasing the yield of 2'-O-methyl isoglycyrrhizin, comprising: overexpressing CsOMT50 in cells; or, mutating a 275bp amino acid sequence of CsOMT50 in cells from H to R. As an optional embodiment of this invention, the cells include prokaryotic cells and / or plant cells. This invention does not specifically limit the overexpression method; any conventional overexpression method in the art can be used. This invention also does not specifically limit the point mutation method of the amino acid sequence; any conventional point mutation method in the art can be used. This invention can significantly increase the yield of 2'-O-methyl isoglycyrrhizin by overexpressing CsOMT50 in cells. This invention further significantly increases the yield of 2'-O-methyl isoglycyrrhizin by mutating a 275bp amino acid sequence of CsOMT50 from H to R and then overexpressing it in cells.

[0020] This invention provides the application of hematoxymethyltransferase CsOMT50 in breeding for high yield of 2'-O-methyl isoglycyrrhizin.

[0021] This invention provides a method for breeding plants to produce high yields of 2'-O-methyl isoglycyrrhizin, comprising: overexpressing CsOMT50 in plants; or mutating a 275bp amino acid sequence of CsOMT50 in plants from H to R; and then screening for positive plants. This invention does not specifically limit the plant used; as an optional embodiment, the plant may be Nicotiana benthamiana and / or Caesalpinia sappan. The method for overexpressing CsOMT50 is not specifically limited, and any conventional overexpression method in the art can be used. The method for point mutation of the 275bp amino acid sequence of CsOMT50 in the plants described in this invention is not specifically limited, and any conventional point mutation method in the art can be used. As an optional embodiment of the present invention, the primers used for the point mutation can be CsOMT50H275R-F and CsOMT50H275R-R; the nucleotide sequence of CsOMT50H275R-F is shown in SEQ ID NO.7; the nucleotide sequence of CsOMT50H275R-R is shown in SEQ ID NO.8. The present invention does not specifically limit the screening method for the positive plants; any conventional screening method in the art can be used. As an optional embodiment of the present invention, the screening method can be screening for positive strains by qRT-PCR.

[0022] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] Cultivation conditions for *Hematoxylin and argyrophyllin*: photoperiod of 16 h light / 8 h dark, day / night temperature of 25℃, relative humidity of 55%, and light intensity of 64.50 PPFD (photosynthetic photon flux density of 64.50 μmol / (m²)). 2 ·s)).

[0024] Example 1: Process of mining the CsOMT50 gene in Hematoxylin and argyrophyllin By combining genomic, metabolomic, and transcriptomic data, co-expression network analysis and linear correlation analysis were used to identify biosynthetic genes of sappanins-type isoflavones. [The text then abruptly shifts to a different topic:] ...screening... CsOMT50 It is a key gene for high isoflavone levels in Sappanins-type cells.

[0025] CsOMT50 The nucleotide sequence is shown in SEQ ID NO.12, specifically as follows: MGSTSTEIQNLKASSSQTEDEACLSAMYLTTNQVYPAVLNAAIDLDLFEIIANKASPPGAFMSAREIASHLPSQHPDMPDRLERMLRLLASYTLLTCSSRATEDGTAVTVYRLSPSGKYCVTGETGGNLASFTTFLCYKALLEIWMNFKEAVVDPDIDLFKKVHGKTSYEYFGTDPKLNHIFNKA MADVCAVEMRRLLQIYKGFDGISTLVDVGGGNGQNLKLIISNYPSIKGINFDLPQVIEHAPPIPGIEHIGGDMFASVPKGDAIILKAVCHNWSDEKCVEFLRKCHEALPENGKVIIVEFILPESPEPTEASKLVSTLDNLMFITVGGRERTEKQFESLGKRSGFSRFQVACRAFSALGVVEFYK.

[0026] 1. Cloning of the CsOMT50 gene from *Caesalpinia sappan* and in vivo functional verification in tobacco: CsOMT50 was obtained by gene cloning, and a plant overexpression vector was constructed. The CsOMT50 gene was transiently transformed into *Nicotiana benthamiana*. Nicotiana benthamiana In the relevant verification, the following steps are performed.

[0027] 1) Construction of CsOMT50 overexpression vector: The pCAMBIA1380-CsOMT50 plant overexpression vector was commissioned to Beijing Qingke Biotechnology Co., Ltd., and the specific steps are as follows: Using pCAMBIA1380 as the vector, the restriction enzyme sites were XbaI and SalI. The CsOMT50 target gene containing these restriction sites was amplified. The PCR product was detected by 1% agarose gel electrophoresis, and the target band was excised and purified using a gel extraction kit. The pCAMBIA1380 vector and the CsOMT50 target gene were digested with XbaI and SalI, respectively. Both the digested vector and the target gene were then subjected to agarose gel electrophoresis, and the linearized vector and the digested target gene fragment were recovered. The linearized vector and the digested target gene fragment were ligated to obtain pCAMBIA1380-CsOMT50, i.e., the CsOMT50 overexpression vector. The CsOMT50 overexpression vector was then transformed and positive clones were screened. Specifically, the ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing hygromycin, and incubated overnight at 37°C. Single colonies were selected, and colony PCR, plasmid double enzyme digestion, and finally sequencing were performed to confirm that the CsOMT50 sequence was correct and the reading frame was correct.

[0028] 2) CsOMT50 overexpression vector transformed into Agrobacterium The Agrobacterium used for transformation was GV3101 Chemically Competent Cell from Qingke Biotechnology. The CsOMT50 overexpression vector was transformed into competent Agrobacterium cells using the freeze-thaw method. The competent cells were thawed on ice; the recombinant plasmid was added, gently mixed, and the cells were incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min. 700 μL of antibiotic-free LB broth was added, and the cells were incubated at 28°C with shaking at 200 rpm for 2–3 h. The cells were centrifuged at 4°C and 5000 rpm for 5 min, and a portion of the supernatant was discarded, retaining approximately 100 μL of resuspended cells. The cells were then plated onto LB agar plates containing both antibiotics (rifampicin 50 μg / mL + kanamycin 50 μg / mL). The plates were inverted and incubated at 28°C for 2–3 days until single colonies appeared.

[0029] Single colonies were picked and positive clones were verified to confirm that the CsOMT50 overexpression vector was correctly transformed.

[0030] Tobacco transient transformation (1) Take the recombinant Agrobacterium bacterial suspension stored at -80℃ and spread it on Kan and Rif double antibody plates. Grow at 28℃ for two days. After Agrobacterium colonies grow, select a single colony and transfer it to 1 mL of Kan and Rif double antibody LB liquid medium. Incubate at 28℃ and 200 rpm. Continue to transfer the bacterial suspension to 50 mL of Kan and Rif double antibody LB liquid medium and incubate overnight until OD. 600 The bacterial culture was obtained at approximately 0.6~0.8. (2) Preparation of injection buffer for transient expression of tobacco: Injection buffer for transient expression of tobacco: 0.5 M MES, 1 mL; 20 mM Na3PO4, 1 mL; D-glucose, 50 mg; 1 M acetylsylgenone, 1 µL; water to 10 mL. Hematoxylin infection resuspension: 1 M magnesium chloride, 0.5 mL; 0.5 M MES, 1 mL; D-glucose, 50 mg; 100 μmol / L acetylsylgenone, 83 µL; water to 50 mL. The pH was adjusted to 5.7 using KOH. (3) Centrifuge the above bacterial culture at 3000 rpm for 10 min, discard the supernatant, add injection buffer to resuspend the precipitate, and continue centrifuging at 3000 rpm for 10 min. Discard the supernatant and repeat twice. (4) Resuspend the precipitate again with injection buffer. The final OD of Agrobacterium bacterial culture was obtained. 600 Between 0.3 and 0.5. (5) Use a disposable 1 mL syringe to inject Agrobacterium tumefaciens solution into the mesophyll from the back of the tobacco leaf, and wipe the leaves dry of any remaining solution. (6) Incubate the tobacco in the dark for 2 to 3 days after injection.

[0031] 3) qRT-PCR analysis of transgenic tobacco (1) Tobacco RNA extraction Total RNA was extracted from tobacco using the TaKaRa MiniBEST Plant RNA Extraction Kit. The instructions can be found on the TaKaRa website (https: / / www.takarabiomed.com.cn / ). After spotting an appropriate amount of tobacco total RNA sample, electrophoresis was performed at 110 V for about 15 min to check RNA integrity. The electrophoresis buffer needs to be replaced. The operation should be as rapid as possible to minimize RNA degradation. RNA concentration and A230 / A260 and A260 / A280 are parameters for judging the quality of the extracted RNA. They were obtained by measuring 1 μL of total RNA sample with a spectrophotometer. (2) Synthesis of cDNA first strand Tobacco RNA was extracted and the experiment was performed according to the instructions of the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit to obtain cDNA. Genomic DNA was removed. The reaction system is shown in Table 1 below. The mixture was gently mixed and reacted at 42℃ for 2 min. After the reaction, the mixture was placed on ice. The reverse transcription reaction was then performed. The system is shown in Table 2 below. The mixture was gently mixed and reacted at 37℃ for 15 min, then at 85℃ for 5 seconds. After the reaction, it was stored at -20℃. (3) qRT-PCR analysis The qRT-PCR reaction was performed using the NovoStart® Fast SYBR qPCR SuperMi rapid real-time PCR kit. cDNA was used as the template, and the primer sequences are shown in Table 3. The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The qRT-PCR reaction system is shown in Table 4 below, and the reaction procedure is shown in Table 5.

[0032] Table 1. Genomic DNA Removal Reaction System

[0033] Table 2 Reverse transcription reaction system

[0034] Table 3 qRT-PCR primer sequences

[0035] Table 4 qRT-PCR reaction system

[0036] Table 5 qRT-PCR reaction procedure

[0037] Fluorescence phenotypic screening was performed 3 days after tobacco injection. Based on a fluorescent reporter gene (e.g., GFP) linked to the target gene, fluorescence phenotypic screening of transgenic tobacco leaves was conducted using fluorescence microscopy. The results showed that the experimental group exhibited stronger fluorescence than the control group (see details). Figure 1 The successful transformation of the transgenic plants was confirmed. qRT-PCR results also showed that the expression level of the target gene CsOMT50 in the plants was significantly higher than that in the wild-type plants.

[0038] 4) Metabolic detection of transiently transformed tobacco (1) Sample extraction Tobacco leaves showing significant expression of the CsOMT50 gene in qRT-PCR were cut off, wrapped in aluminum foil, and rapidly frozen in liquid nitrogen and stored at -80℃. The samples were then freeze-dried under vacuum and ground into powder using a grinder. 50 mg of sample powder was dissolved in 1 mL of 70% methanol internal standard extract. 500 μL of petroleum ether was added, vortexed for 5 min, allowed to stand for separation, and centrifuged at 12000 rpm for 10 min at 4℃. All supernatant was filtered through a 0.22 μm PTFE membrane and stored at -20℃ for LC-MS / MS analysis. (2) Chromatographic and mass spectrometry acquisition conditions The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC™ AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (Applied Biosystems 6500 QTRAP, https: / / sciex.com.cn / ).

[0039] The main liquid chromatography conditions included: ① Column: Agilent SB-C18 1.8 µm, 2.1 mm × 100 mm; ② Mobile phase: Phase A was ultrapure water (with 0.1% formic acid added), and Phase B was acetonitrile (with 0.1% formic acid added); ③ Elution gradient: 0.00-9.00 min: Phase B ratio increased linearly from 5% to 95%; 9.00-10.00 min: Phase B ratio was 95%; 10.00-11.00 min: Phase B ratio decreased to 5%; 11.00-14.00 min: Phase B ratio was 5%; ④ Flow rate: 0.35 mL / min; Column temperature: 40℃; Injection volume: 2 μL.

[0040] The mass spectrometry conditions mainly included: an electrospray ionization (ESI) source temperature of 500 °C; an ion spray voltage (IS) of 5500 V (positive ion mode) / -4500 V (negative ion mode); and source gas I (GSI), gas II (GSII), and curtain gas (CUR) set to 50, 60, and 25 psi, respectively, with collision-induced ionization parameters set to high. QQQ scans used MRM mode with the collision gas (nitrogen) set to medium. Further optimization of the declustering potential (DP) and collision energy (CE) was performed for each MRM ion pair. A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.

[0041] Liquid chromatography-mass spectrometry (LC-MS) for in vivo verification of CsOMT50 function in transgenic tobacco is shown below. Figure 2 As shown. Figure 2 CK indicates injection of isoliquiritigenin substrate only; CsOMT50 indicates injection of CsOMT50 plus injection of isoliquiritigenin substrate. The concentration of isoliquiritigenin substrate was 1 mg / mL. The isoliquiritigenin substrate was mixed with the bacterial culture during the transient conversion of tobacco and injected at the same time as the bacterial culture injection. The injected tobacco was cultured in the dark for 2-3 days, and metabolite analysis was performed.

[0042] The secondary mass spectrum of CsOMT50 from genetically modified tobacco is shown below. Figure 3 As shown in the figure. The results of 2'-O-methyl isoglycyrrhizin content in genetically modified tobacco and wild-type tobacco are as follows. Figure 4 As shown.

[0043] Using UPLC-MS / MS, no obvious chromatographic peak of 2'-O-methylisoglycyrrhizin was detected in the liquid chromatogram of the control group, and a secondary mass spectrum could not be obtained; the liquid chromatogram of the CsOMT50+ substrate group showed a chromatographic peak at 10.84 min. Figure 2 The secondary mass spectrum of 2'-O-methylisoglycyrrhizin was extracted, with a mass-to-charge ratio (m / z) of 269.04, indicating the presence of 2'-O-methylisoglycyrrhizin. Figure 3 The content of 2'-O-methyl isoliquiritigenin was 103.33 ng / mL. Figure 4 ).

[0044] 5) Instantaneous conversion of tobacco crude protein catalysis (1) Cut tobacco leaves that showed significant gene expression in qRT-PCR results, wrap them in tin foil and freeze them in liquid nitrogen. Weigh about 2 g of frozen leaves and quickly put them back into liquid nitrogen. Grind the tobacco leaves into powder in a mortar that has been pre-cooled in liquid nitrogen. Transfer the powder to a 10 mL centrifuge tube, resuspend it with 1.5~2 mL of lysis extract NB1, and mix it thoroughly with a shaker. Place it on ice for 30 minutes and pre-cool the benchtop centrifuge. Centrifuge at 15,000×g for 1 hour at 4℃. Take the supernatant on ice and filter it with a double-layer Miracloth filter (wet the filter with NB1 before filtering). Take 200 μL of enzyme solution (i.e. the supernatant obtained from filtration) for catalysis experiments.

[0045] Catalytic experiment: The in vitro enzymatic reaction system is shown in Table 6 below. The experimental group is the enzyme solution; CK is the enzyme solution extracted from crude tobacco protein injected with only isoliquiritigenin substrate from wild-type tobacco; CsOMT50 is the enzyme solution extracted from crude tobacco protein injected with both CsOMT50 and isoliquiritigenin substrate. The substrate is isoliquiritigenin (the concentration of both substances is 1 mM). The reaction was carried out at 37℃ and 200 rpm for 30 minutes.

[0046] Table 6 In vitro enzymatic reaction system

[0047] After the reaction was complete, 1 mL of methanol solution was added to terminate the reaction. The reaction solution was centrifuged at high speed for 20 min, and an appropriate amount of the supernatant was added to an Agilent liquid chromatography vial for analysis of the enzyme activity product using ultra-high performance liquid chromatography (UPLC).

[0048] (2) The acquisition conditions and methods for chromatographic mass spectrometry are the same as those in 4 above.

[0049] Liquid chromatography-mass spectra of crude protein catalytic analysis of transgenic tobacco CsOMT50 are shown below. Figure 5 As shown. The secondary mass spectrum of crude protein catalytic analysis of transgenic tobacco CsOMT50 is shown below. Figure 6 As shown. The crude protein catalysis of transgenic tobacco CsOMT50 produces 2'-O-methyl isoglycyrrhizin, with the content as shown in the figure. Figure 7 As shown. Catalysis was performed by extracting crude tobacco protein. Results showed that no obvious 2'-O-methyl isoglycyrrhizin peak was detected in the liquid chromatogram catalyzed by wild-type tobacco crude protein, and a secondary mass spectrum could not be obtained; however, a peak appeared at 10.84 min in the liquid chromatogram catalyzed by the CsOMT50+ substrate group of tobacco crude protein. Figure 5 The secondary mass spectrum of 2'-O-methylisoglycyrrhizin was extracted, with a mass-to-charge ratio (m / z) of 269.04, indicating the presence of 2'-O-methylisoglycyrrhizin. Figure 6The content of 2'-O-methyl isoliquiritigenin was 136.33 ng / mL. Figure 7 The above experiments show that CsOMT50 has the function of catalyzing the synthesis of 2'-O-methylisoglycyrrhizin in tobacco.

[0050] Example 2: Creation of the CsOMT50 transgenic line from Hematoxylin and argyrophyllin 1. The constructed CsOMT50 plant overexpression vector (same as in Example 1) was transformed into the K599 Agrobacterium tumefaciens strain ( Agrobacterium tumefaciens (The method is the same as in Example 1). The bacteria were activated and cultured at 28°C for 48 h on TY solid medium containing Kan. Single colonies were picked and inoculated into TY liquid medium, and cultured at 28°C with shaking at 200 rpm until the logarithmic growth phase (OD600≈0.8). The bacterial suspension was centrifuged at 8000 rpm for 10 min to collect the cells, and resuspended in the infiltration solution until OD600=0.6. Plump *Caesalpinia sappan* seeds were selected, and the seeds were punctured using a 1 mL sterile syringe needle. The punctured seeds were completely immersed in the prepared *Agrobacterium* resuspension. The resuspension was placed in a plant in vivo transformation system, and a negative pressure of 4 kPa was applied for 120 s each time, repeated 3 times. The seeds were then sown in the pre-prepared cultivation substrate to a depth of about 3 cm. The planting pots were placed in a greenhouse or artificial climate chamber, with 16 h of light and 8 h of darkness, and normal cultivation and watering management at 25°C.

[0051] 2. qRT-PCR analysis of transgenic sappanwood 1) The method for extracting hematoxylin RNA is the same as in Example 1.

[0052] 2) The synthesis of the first strand of cDNA is the same as in Example 1.

[0053] 3) qRT-PCR Analysis: The qRT-PCR reaction was performed using the NovoStart® FastSYBR qPCR SuperMi rapid real-time PCR kit. Primer sequences are shown in Table 7 and were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The qRT-PCR reaction system is shown in Table 8, and the reaction procedure is shown in Table 9.

[0054] Table 7 qRT-PCR primer sequences

[0055] Table 8 qRT-PCR reaction system

[0056] Table 9 qRT-PCR reaction procedure

[0057] First, based on the fluorescent reporter gene linked to the target gene, the transgenic hairy roots of *Caesalpinia sappan* were screened for fluorescence phenotype using a Zeiss confocal microscope. The results showed that CsOMT50 exhibited significant fluorescence in the hairy roots of *Caesalpinia sappan*. Figure 8 Secondly, qRT-PCR experiments revealed that the expression level of the CsOMT50 (CsapChr2G050450) gene in the *Caesalpinia sappan* strain was significantly higher than that in the wild type, being 9.02 times higher. Figure 9 ).

[0058] 3. Metabolome of transiently transformed sappanwood 1) Sample extraction method is the same as in Example 1.

[0059] 2) Chromatographic and mass spectrometric acquisition conditions were the same as in Example 1. No substrate injection was involved in the sappanwood treatment. Using UPLC-MS / MS, the control group showed a detected 2'-O-methylisoglycyrrhizin peak, but a secondary mass spectrum could not be obtained; the concentration was 0.07 μg / g. The characteristic ion chromatogram of the CsOMT50 group showed a clear 2'-O-methylisoglycyrrhizin peak, with an elution time of 10.88 min (…). Figure 10 The secondary mass spectrum of 2'-O-methylisoglycyrrhizin was extracted, with a mass-to-charge ratio (m / z) of 269.04, indicating the presence of 2'-O-methylisoglycyrrhizin. Figure 11 2'-O-methylisoglycyrrhizin ( ), with a content of 67.71 μg / g. Figure 12 ).

[0060] 4. Instantaneous Conversion of Crude Protein from Sappanwood Catalysis 1) The crude protein extraction method from Sappanwood was the same as in Example 1. 2) The chromatographic and mass spectrometric acquisition conditions and methods were the same as in Example 1. Catalysis was performed by extracting crude protein from the hairy roots of Sappanwood. The results showed that the wild type detected a chromatographic peak of 2'-O-methylisoglycyrrhizin, with a content of 25.78 ng / mL; the liquid chromatogram of the CsOMT50+ substrate group catalyzed by crude protein from Sappanwood showed a peak at 10.84 min (…). Figure 13 The secondary mass spectrum of 2'-O-methylisoglycyrrhizin was extracted, with a mass-to-charge ratio (m / z) of 269.04, indicating the presence of 2'-O-methylisoglycyrrhizin. Figure 14 ), the concentration of 2'-O-methyl isoliquiritigenin was 101.87 ng / mL. Figure 15 The above experiments showed that wild-type sappanwood contains low levels of 2'-O-methyl isoliquiritin; when transformed with CsOMT50, sappanwood synthesized a higher content of 2'-O-methyl isoliquiritin. Through overexpression of the CsOMT50 gene, the synthesis of 2'-O-methyl isoliquiritin in sappanwood was successfully achieved.

[0061] Example 3: In vitro functional verification of CsOMT50 1. Prokaryotic expression of pET28-CsOMT50: The pET28-CsOMT50 expression vector was developed by Beijing Qingke Biotechnology Co., Ltd., and the specific steps are as follows: Using pET-28a as the vector, the restriction enzyme sites were NcoI and Xhol. The CsOMT50 target gene containing these restriction sites was amplified. The PCR product was detected by 1% agarose gel electrophoresis. The target band was excised, and the CsOMT50 target gene was recovered and purified using a gel extraction kit. The pET-28a vector and the CsOMT50 target gene were digested with NcoI and Xhol, respectively. Both the digested vector and the target gene were then subjected to agarose gel electrophoresis, and the linearized vector and the digested target gene fragment were recovered. The linearized vector and the digested target gene fragment were ligated to obtain pET-28a-CsOMT50, i.e., the CsOMT50 overexpression vector. The CsOMT50 overexpression vector was transformed and positive clones were screened as follows: The ligation product (CsOMT50 overexpression vector) was added to E. coli DH5α competent cells and incubated on ice for 30 min; heat-shocked at 42℃ for 90 s, then immediately incubated on ice for 2 min; 500 μL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ and 200 rpm for 1 h; 100 μL of the bacterial culture was spread on LB agar plates containing kanamycin (final concentration 50 μg / mL) and incubated overnight at 37℃. Single colonies were picked for PCR amplification, and electrophoresis was performed to detect the presence of the target band. The plasmid of positive colonies was extracted, double-digested with NcoI and XhoI, and electrophoresis was performed to observe whether the vector fragment and the target gene fragment were released. Sequencing verification: The plasmid with correct digestion was sent for sequencing to confirm that the CsOMT50 sequence was correct and the reading frame was correct.

[0062] The pET28-CsOMT50 plasmid was transformed into the Transetta(DE3) expression strain using the same transformation method as above. After transformation, a single colony was picked and added to 1 mL of LB liquid medium containing 100 mg / mL Kan, and cultured at 37°C with shaking at 200 rpm until the bacterial culture became turbid. Strains with positive bacterial tests were stored at -80°C with 50% glycerol added. Using the empty Transetta(DE3) vector as a negative control, 500 μL of both the experimental and control groups was added to 10 mL of LB liquid medium containing Kan and cultured at 37°C with shaking at 200 rpm until turbidity was reached. 10 mL of activated bacterial culture was added to 500 mL of LB liquid medium containing Kan and cultured at 37°C with shaking at 200 rpm until the OD600 was between 0.5 and 0.6. IPTG inducer was added to bring the final IPTG concentration in the bacterial culture to 0.5 mM, and the culture was incubated at 16°C with shaking at 200 rpm for 12 h.

[0063] 2. Extraction of target protein (1) Centrifuge the IPTG-induced bacterial culture at 5000 rpm at 4℃ for 10 min, discard the supernatant, and collect the bacterial cells. (2) Resuspend in 40 mL PBS solution, centrifuge at 5000 rpm at 4℃ for 10 min, wash, discard the supernatant, and resuspend in 40 mL PBS solution. (3) Use an ultrasonic cell disruptor, amplitude bar No. 2, 33% power, disrupt for 2 s, pause for 5 s, and continue for 30 min. (4) Centrifuge the disrupted bacterial culture at 14000 rpm at 4℃ for 10 min, transfer the supernatant to a new centrifuge tube, add PBS solution to the precipitate and resuspend, and retain both the supernatant and the resuspended solution for gel electrophoresis. (5) Take 1 mL of His-tag and add 1 mL of PBS solution, add to a gravity column, discard the eluent, repeat 4 times, and for the last time, leave 1 mL of the mixture and add it to the supernatant, and slowly rotate on a shaker at 4℃ for 1 h. (6) Pass the supernatant containing His-tag through a gravity column. The effluent is called flowthrough (sample retention for gel running). The target protein binds to His-tag in the gravity column. Elute the protein with 5 mL of Elution Buffer to obtain the purified protein (sample retention for gel running).

[0064] 3. SDS-PAGE electrophoresis analysis (1) Prepare electrophoresis gel using the Omni-Easy™ One-Step PAGE Gel Rapid Preparation Kit (10%). Refer to the instructions at http: / / www.epizyme.cn / index.php?c=article&id=1225. (2) Take 40 μL of supernatant, precipitate resuspension, flowthrough, and purified protein into a 1.5 mL centrifuge tube, add 10 μL of 5× protein loading buffer to each, boil at 100℃ for 10 min, and then centrifuge at 12000 rpm for 2 min. (3) Take 20 μL of protein sample and load it into a 10% PAGE gel, and centrifuge at 120 V for 70 min. (4) Remove the gel, cut off the stacking gel, put the separating gel into Coomassie Brilliant Blue rapid staining solution, stain on a shaker at 20 rpm for 30 min, and then destain overnight with water. (5) Observe the destaining of the gel the next day.

[0065] SDS-PAGE results of CsOMT50 protein are as follows: Figure 16 As shown. Figure 16 In this context, M stands for two-color pre-stained protein marker; CL: protein supernatant resuspension; FT: protein flow buffer; W1: protein washing buffer; and E1~E3: protein elution buffer.

[0066] The CsOMT50 protein, after prokaryotic expression and centrifugation, was subjected to SDS-PAGE electrophoresis, followed by staining with Coomassie Brilliant Blue rapid staining solution and overnight destaining. The results are as follows: Figure 16 As shown, the size of the CsOMT50 protein is 40.39 kDa, and the size of the His tag on the pET28 vector is 0.8 kDa. Therefore, the size of the recombinant protein after the CsOMT50 protein is fused with the HIS tag is 41.19 kDa.

[0067] 4. Concentration of the target protein (1) Add the purified protein to the upper layer of the ultrafiltration tube, centrifuge at 4000 rpm for 20 min at 4℃, and discard the eluent. (2) Add 10 mL of PBS buffer to the upper layer of the ultrafiltration tube, centrifuge at 4000 rpm for 30 min, discard the eluent, and repeat once. (3) Collect the remaining liquid in the upper layer of the ultrafiltration tube, which is the concentrated protein solution, and store it at -80℃ for subsequent experiments.

[0068] 5. Validation of CsOMT50 catalytic function (1) The in vitro enzymatic reaction system is shown in Table 10 below. The experimental group, i.e., the treatment group corresponding to CsOMT50, is concentrated protein; the positive control group is ChOMT protein, and the negative control, i.e., CK, is boiled protein. The substrate is isoliquiritigenin (concentration of 1 mM). The reaction was carried out at 37℃ and 200 rpm for 30 minutes. (2) After the reaction was completed, 1 mL of methanol solution was added to terminate the reaction. (3) The reaction solution was centrifuged at high speed for 20 min, and an appropriate amount of the upper liquid was added to an Agilent liquid chromatography vial. The enzyme activity product was analyzed by ultra-high performance liquid chromatography (UPLC).

[0069] Table 10 In vitro enzymatic reaction system

[0070] 6. Detection of in vitro enzyme activity products: Qualitative and quantitative determination of samples was performed using UHPLC-Q-TOF / MS. Chromatographic conditions: Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a C-18 column; column temperature 40℃; flow rate 0.4 mL / min; injection volume 2 µL; mobile phase composition A: water + 25 mM ammonium acetate + 0.5% formic acid, B: methanol; gradient elution program as follows: 0–0.5 min, 5% B; 0.5–10 min, B linearly changing from 5% to 100%; 10.0–12.0 min, B maintained at 100%; 12.0–12.1 min, B linearly changing from 100% to 5%; 12.1–16 min, B maintained at 5%; throughout the analysis, samples were placed in an autosampler at 4℃. To avoid the influence of instrument signal fluctuations, continuous analysis of samples was performed in a randomized order. Mass spectrometry conditions: The first and second stage spectra of the samples were acquired using an AB Triple TOF 6600 mass spectrometer. The ESI source conditions after chromatographic separation were as follows: Ion Source Gas1 (Gas1): 60, Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600℃, IonSapary Voltage Floating (ISVF): ±5500 V (positive and negative modes); TOF MS scan m / z range: 60-1000 Da, product ion scan m / z range: 25-1000 Da, TOF MS scan accumulation time: 0.20 s / spectra, product ion scan accumulation time: 0.05 s / spectra; the secondary mass spectrometer was obtained using information dependent acquisition (IDA) in high sensitivity mode, with Declustering potential (DP): ±60 V (positive and negative modes), Collision Energy: 35±15 eV, and IDA settings as follows: Exclude isotopes within 4 Da, Candidate ions to monitor per cycle: 10. Mass spectrometry data were analyzed using MSDIAL (ver. 4.9.221218 Windows x64).

[0071] In vitro catalysis and LC-MS / MS detection revealed that no chromatographic peak of 2'-O-methylisoglycyrrhizin was detected in the negative control (CK: boiled protein), making it impossible to obtain a secondary mass spectrum and accurately quantify the protein; the CsOMT50-catalyzed liquid chromatogram showed a peak at 51.90 min. Figure 17 The secondary mass spectrum of 2'-O-methylisoglycyrrhizin was extracted, with a mass-to-charge ratio (m / z) of 269.04, indicating the presence of 2'-O-methylisoglycyrrhizin. Figure 18 The concentration of 2'-O-methylisoglycyrrhizin was 69.99 ng / mL. Figure 19 ).

[0072] Example 4: Functional Validation of CsOMT50 Mutant 1. Mutation sites were selected using HelixFold3 (https: / / paddlehelix.baidu.com / app / all / helixfold3 / forecast) to predict the binding of CsOMT50 protein to substrate ligands, protein catalytic functional sites, and protein pockets; the 3D model of the substrate ligand was visualized using PyMOL3.1 (https: / / pymol.org / ) to identify the active amino acid sites for CsOMT50 candidate substrate binding and catalytic activity.

[0073] 2. Primer Design: To introduce a single-base site-directed mutation into the plasmid (pET28-CsOMT50 plasmid), only one pair of primers needs to be designed to amplify the plasmid via reverse PCR. The primer sequences used for the CsOMT50 mutation site are shown in Table 11.

[0074] Table 11 Primer sequences used for the CsOMT50 mutation site

[0075] 3. Site-directed mutagenesis 1) Preparation of PCR reaction system Site-directed mutagenesis was performed using the Hieff Mut™ Site-Directed Mutagenesis Kit. After thawing and thoroughly mixing each reaction group, the reaction mixtures were prepared in an ice-water bath to increase amplification specificity. The PCR reaction systems are shown in Table 12.

[0076] Table 12 PCR reaction system

[0077] The PCR reaction program was as follows: pre-denaturation: 95℃ for 30s; 34 cycles (denaturation: 95℃ for 15s; annealing: 58℃ for 15s; extension: 72℃ for 1 min); final extension: 72℃ for 5 min; storage: 4℃.

[0078] 2) The amplified product DpnI was digested to remove the methylated template plasmid. The reaction system is shown in Table 13.

[0079] Table 13 Reaction System

[0080] After gently blowing and mixing, place the above reaction system at a constant temperature of 37°C for 1-2 hours.

[0081] 3) Recombination reaction, the recombination reaction system is shown in Table 14.

[0082] Table 14 Recombination Reaction System

[0083] (1) Place the above system at 50℃ for 20 min; (2) After the reaction is complete, immediately place the reaction tube in an ice-water bath to cool; (3) After that, the reaction product can be directly converted; or it can be stored at -20℃ and thawed and converted when needed.

[0084] 4) E. coli transformation: The E. coli used for transformation was TOP10 Chemically CompetentCell from Qingke Biotechnology.

[0085] (1) Take 10 µL of cooled reaction solution and add it to 100 µL of competent cells. Gently tap the tube wall a few times to mix. Place on ice for 30 min. (2) Heat shock at 42℃ for 45-90 s and incubate in an ice-water bath for 2 min. (3) Add 700 µL of LB medium and shake at 37℃, 200-250 rpm for 1 h. (4) Centrifuge at 5000 rpm for one minute to collect the bacteria. Take about 100 µL of supernatant, gently pipette to resuspend the bacterial block, and spread it on a plate containing 50 mg / mL Kan antibiotic. Invert the plate and incubate overnight at 37℃. (5) Screening of positive clones: Single colonies from the long bacterial plate were picked and placed in LB liquid containing the corresponding antibiotic. The plate was then placed in a shaker at 37°C and shaken gently at 200 r for 5 hours. The turbid bacterial solution was then taken out for PCR verification. The system is shown in Table 15 below. The PCR reaction program was as follows: pre-denaturation: 98°C for 2 min; 33 cycles (denaturation: 98°C for 10 s; annealing: 58°C for 15 s; extension: 72°C for 1 min); final extension: 72°C for 3 min; storage: 4°C. 1 mL of the positive bacterial solution was sent to Qingke Biotechnology Co., Ltd. for sequencing verification.

[0086] Table 15 PCR Validation Reaction System

[0087] (6) Extraction of positive clone plasmids: High purity plasmid DNA small-scale extraction kit (Genesand Biotech) was used to extract positive clone plasmids. The kit instructions can be found on the Genesand Biotech website (https: / / genesand.com / ).

[0088] 5) Prokaryotic expression and purification of the CsOMT50 mutant were performed using the same method as in Example 3.

[0089] 6) Validation of the catalytic function of the CsOMT50 mutant, using the same method as in Example 3.

[0090] 7) Results (1) Expression and purification of CsOMT50 mutant protein The flow-through buffer, after prokaryotic expression and purification, and the purified protein were subjected to SDS-PAGE electrophoresis. Following electrophoresis, the protein was stained with Coomassie Brilliant Blue and destained overnight. The results are as follows: Figure 20 and Figure 21 As shown in the figure. The marker is a two-color pre-stained protein marker; FT: protein flow buffer; W: protein washing buffer; E1~E2: protein elution buffer. Both CsOMT50H275R and CsOMT50H275L proteins are 40.39 kDa, and the His tag on the pET28 vector is 0.8 kDa. Therefore, the recombinant proteins after fusing CsOMT50H275R and CsOMT50H275L proteins with the His tag are both 41.19 kDa. The figure shows a band at 41.19 kDa, indicating that further purification steps can be performed to obtain a purer protein for subsequent research.

[0091] (2) In vitro enzyme activity verification of CsOMT50 mutant In vitro catalysis and LC-MS / MS detection revealed that no chromatographic peak of 2'-O-methyl isoglycyrrhizin was detected in the negative control (CK: boiled protein), and a secondary mass spectrum could not be obtained, making accurate quantification impossible; compared to wild-type CsOMT50, the liquid chromatogram catalyzed by CsOMT50H275R showed a peak at 10.81 min. Figure 22 The secondary mass spectrum of 2'-O-methylisoglycyrrhizin was extracted, with a mass-to-charge ratio (m / z) of 269.04, indicating the presence of 2'-O-methylisoglycyrrhizin. Figure 23 ), the concentration of 2'-O-methyl isoliquiritigenin was 281.15 ng / ml. Figure 24The H275R single-point mutant exhibited a 3.11-fold increase in catalytic activity towards the substrate under the same reaction conditions, significantly surpassing the wild type. The H275L single-point mutant showed decreased catalytic activity under the same reaction conditions. This result functionally demonstrates that His275 is a key "activity gate" site for CsOMT50, thus establishing H275R as the decisive mutation for enhanced CsOMT50 activity.

[0092] 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 hematoxymethyltransferase CsOMT50, characterized in that, The amino acid sequence of CsOMT50 is shown in SEQ ID NO.

11.

2. The hematoxymethyltransferase CsOMT50 according to claim 1, characterized in that, The nucleotide sequence encoding CsOMT50 is shown in SEQ ID NO.

12.

3. The application of the hematoxymethyltransferase CsOMT50 as described in claim 1 or 2 in the synthesis of 2'-O-methylisoglycyrrhizin.

4. The application according to claim 3, characterized in that, Overexpression of hematoxymethyltransferase CsOMT50 increases the yield of 2'-O-methylisoglycyrrhizin.

5. The application according to claim 3, characterized in that, The production of 2'-O-methyl isoliquiritigenin can be regulated by a 275bp point mutation in the amino acid sequence of CsOMT50.

6. The application according to claim 5, characterized in that, The yield of 2'-O-methylisoglycyrrhizin was increased by mutating the amino acid at 275 bp of the CsOMT50 amino acid sequence from H to R.

7. The application according to claim 5, characterized in that, The production of 2'-O-methylisoglycyrrhizin was reduced by mutating the amino acid at 275 bp of the CsOMT50 amino acid sequence from H to L.

8. A method for increasing the yield of 2'-O-methylisoglycyrrhizin, characterized in that, include: Overexpressing CsOMT50 in cells; Alternatively, the amino acid sequence of CsOMT50 in cells could be mutated from H to R at a 275bp interval.

9. Application of hematoxymethyltransferase CsOMT50 in breeding for high yield of 2'-O-methyl isoglycyrrhizin.

10. A method for breeding plants to produce high yields of 2'-O-methyl isoglycyrrhizin, characterized in that, include: Overexpression in plants CsOMT50 ; Alternatively, the amino acid sequence of CsOMT50 in plants could be mutated from H to R at 275 bp. Then, positive plants are selected.

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