CN102399813A - Application of jasmonic acid carboxymethyl transferase gene in cultivation of belladonna with high yield of tropane alkaloids - Google Patents

Abstract

The invention provides a strategy and a method for improving the content of endogenous methyl jasmonate and promoting the biosynthesis of tropane alkaloids in belladonna so as to improve the yield of TAs in belladonna. The method comprises introducing belladonna into coding region of jasmonic acid carboxymethyl transferase gene or nucleotide sequence with the same function, and culturing belladonna with high yield of tropane alkaloids. Relates to cloning of target gene, construction of expression vector and genetic transformation of belladonna. The process is to separate a target gene by using high-fidelity RT-PCR, construct a plant high-efficiency expression vector carrying the target gene, introduce the target gene into belladonna for high-efficiency expression by adopting a transgenic technology, break the rate-limiting reaction in a MeJA biosynthesis pathway in the belladonna, improve the content of endogenous MeJA, promote the biosynthesis of TAs in the belladonna by using the endogenous high-content MeJA, promote the metabolic flow to the direction of a target product, improve the synthesis capacity of the TAs in the belladonna, and further improve the yield of the TAs in the belladonna. The invention not only provides a new method for cultivating belladonna with high TAs yield by utilizing the genetic engineering technology, but also provides a novel high-quality medicine source for the production of TAs including hyoscyamine and scopolamine.

Description

Application of jasmonic acid carboxymethyltransferase gene in cultivating belladonna with high yield of tropane alkaloids

technical field

The invention belongs to the fields of molecular biology, breeding and genetic engineering, and relates to a method for cultivating belladonna ( Atropa belladonna L.) with high yield of tropane alkaloids (tropane alkaloids, TA) by using genetic engineering technology. It specifically involves the strategy of increasing the content of endogenous Jasmonic acid methyl ester (MeJA) in belladonna to promote the synthesis of TA, and the key enzyme gene jasmonic acid carboxymethyltransferase (jasmonic acid methyl ester) in the biosynthetic pathway of methyl jasmonate. acid carboxyl methyltransferase (JMT) target gene cloning, expression vector construction, and specific procedures for obtaining transgenic belladonna with high yield of tropane alkaloids. The present invention also provides the high-yielding transgenic belladonna obtained by genetic engineering and its cultured progeny, regenerated plants, plant tissues or seeds.

Background technique

The plant growth regulator methyl jasmonate (Jasmonic acid methyl ester, MeJA) is a small molecule compound that can induce the excessive accumulation of various secondary metabolites in different plants. MeJA is widespread in the plant kingdom, especially seed plants. MeJA chemistry originates from membrane lipids, and MeJA is finally synthesized after seven enzymatic steps ^[2] . Most of the functional genes in this pathway have been cloned and analyzed functionally, and the rate-limiting steps have been clarified. The most important rate-limiting step of the MeJA synthesis pathway is located at the end of the metabolic pathway, that is, the synthesis of MeJA from jasmonic acid (jasmonic acid or jasmonate, JA) catalyzed by JMT. This enzymatic step is the most important position in the genetic modification of the MeJA synthesis pathway. Previous studies have shown that treating plant cells including periwinkle, yew, and scopolamine with exogenous MeJA can greatly improve the synthesis ability of their secondary metabolites. Plants themselves have the ability to synthesize MeJA, and under normal circumstances, the ability of plants to synthesize MeJA is very low. Based on this, modifying the MeJA biosynthetic pathway of belladonna at the molecular level through genetic engineering, thereby changing the level of MeJA in plant cells, may have an important impact on the secondary metabolic pathway of belladonna TA and develop into a new metabolic engineering strategy .

Contents of the invention

The first object of the present invention is to provide a strategy for genetically improving belladonna by using genetic engineering technology. This strategy is to increase the content of endogenous MeJA in belladonna and promote the synthesis of scopolane alkaloids to achieve high-yield scopolamine. Alkane alkaloids for the purpose of belladonna.

In another aspect of the present invention, the method for improving the content of endogenous MeJA in belladonna belladonna is also provided, the method will be the key enzyme JMT gene in the MeJA biosynthesis pathway or the coding region of the nucleotide sequence with the same function The plant high-efficiency expression vector of DNA molecules, overexpressed in belladonna, increases the content of endogenous MeJA in belladonna, and promotes the synthesis of anisopolane alkaloids, including the cloning of the target gene, the construction of the expression vector, and the acquisition of anisopolamine Specific procedures for transgenic belladonna with high production of alkanes alkaloids.

In another aspect of the present invention, a plant expression vector is also provided, which comprises the coding region of the above-mentioned JMT gene nucleotides, which can be derived from plants with MeJA biosynthesis pathways or encode mutants with the same catalytic function and Artificially synthesized nucleotide sequences. In the example the sequence is from Arabidopsis.

In another aspect of the present invention, a host cell transformed with the above-mentioned plant expression vector is also provided. In the example the host cell is belladonna.

Technical scheme of the present invention is as follows:

The purpose of the present invention is to provide a method for increasing the TA content of belladonna, and achieve the purpose of obtaining transgenic belladonna with high TA content by overexpressing JMT gene in belladonna. It is characterized in that the plant expression vector carrying the JMT coding region of belladonna is used to overexpress JMT in belladonna cells, tissues, organs and plants by any transgenic method, thereby improving the biosynthesis ability of MeJA in belladonna and promoting the biosynthesis of TA. The present invention is achieved like this:

(1) Obtain the JMT gene coding region by gene cloning;

(2) The JMT gene coding region is operably linked to the expression control sequence to form a plant expression vector;

(3) obtaining sterile explants of belladonna;

(4) Using any transgenic method to transfer the JMT gene coding region to belladonna cells, tissues, organs, or plants;

(5) Screen and identify belladonna transformants under specific conditions;

(6) The transgenic belladonna cultivated under suitable conditions to obtain transgenic offspring.

The plant expression vector involved in the present invention comprises the DNA of the JMT gene coding region.

The living body obtained by the above method is the cells, tissues, organs and plants of the transgenic belladonna. Its characteristics are: the JMT gene coding region driven by the CaMV 35S promoter is introduced, the expression level of the JMT gene is greatly improved, the MeJA synthesis ability is greatly improved, the TA synthesis ability is enhanced, and the TA content is also greatly increased.

In the present invention, term " JMT " is jasmonate carboxymethyltransferase gene, comprises the gene of coding jasmonate carboxymethyltransferase or has the plant high-efficiency expression vector of the DNA molecule of the coding region of the nucleotide sequence of the same function , Gene nucleotide coding region. The enzyme encoded by this gene catalyzes the synthesis of MeJA from jasmonic acid (jasmonic acid or jasmonate, JA), and this enzymatic step is the most important position in the genetic modification of the MeJA synthesis pathway. The gene can be derived from a plant with MeJA biosynthesis pathway, or encode a mutant with the same catalytic function and an artificially synthesized nucleotide sequence.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids and the like. In the present invention, the term "living body" refers to cells, tissues, organs and plants of belladonna.

In the present invention, the term "tropane alkaloids" or "TA" includes hyoscyamine and scopolamine.

In the present invention, the term "any transgenic method" includes Agrobacterium tumefaciens Ti plasmid-mediated gene transformation, Agrobacterium rhizogenes Ri plasmid-mediated gene transformation, plant virus vector-mediated gene transformation, such as PEG-mediated gene transformation, lipid Plastid-mediated gene transformation, electric shock method-mediated gene transformation, ultrasonic-mediated gene transformation, microinjection-mediated gene transformation, laser microbeam-mediated gene transformation, particle gun method-mediated gene transformation, pollen tube channel-mediated gene transformation , Germ cell immersion method mediated gene transformation.

In the present invention, the term "screening and identifying belladonna transformants under specific conditions" refers to the use of antibiotics (kanamycin, hygromycin, G418, etc.) or herbicides (grass Aminophosphine, glyphosate) to select transformants of belladonna resistant to antibiotics; PCR, Southern blotting, Northern blotting, and Western blotting can be used to identify belladonna transformants.

In the present invention, the term "transgenic belladonna cultured under suitable conditions to obtain transgenic offspring" refers to the in vitro culture of identified transformants and the detection of the expression level of the JMT gene to screen excellent transformants with high TA yields cultured to obtain transgenic offspring.

In the present invention, we cloned the coding region of the JMT gene from belladonna, constructed a high-efficiency antisense expression vector for plants, and genetically transformed belladonna to break the rate-limiting reaction step in the TA biosynthetic pathway in belladonna, thereby Improve the production capacity of TA, and then screen to obtain high-yielding transgenic belladonna for cultivation, and obtain transgenic offspring.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are generally in accordance with conventional conditions, such as the conditions described in "Molecular Cloning" (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions suggested by the manufacturer. the

Example 1

Cloning of Coding Region of Arabidopsis JMT Gene

Design primers according to the reported JMT nucleotide sequence (GenBank accession number AY008434) and Bgl II and BstE II restriction sites: upstream primer fjmt: 5'-ccagatctATGGAGGTAATGCGAGTTCTTCAC-3', where agatct is the Bgl II site; downstream primer rjmt: 5'-cggttaccTCAACCGGTTCTAACGAGCGAAA G-3', where ggttacc is the BstE II site. Using the cDNA reverse-transcribed from Arabidopsis flower RNA as a template, PCR amplification was performed to extract total RNA from Arabidopsis leaves (RNA extraction kit from Shanghai Huashun Bioengineering Co., Ltd.), and reverse-transcribed into cDNA (TaKaRa RNA PCR Kit), and then carry out PCR amplification. The PCR reaction system is 50ul, including deionized water, 10×PCR buffer 5ul, dNTP 1ul, MgCl ₂ 3ul, each primer 1ul, cDNA template 1ul, Taq enzyme 0.5ul. The PCR conditions were 94°C for 5 minutes, followed by 30 cycles of 94°C for 45 seconds, 64.5°C for 50 seconds, and 72°C for 1min30sec, and finally extended at 72°C for 8 minutes.

All PCR products were subjected to agarose/1×TAE gel electrophoresis and ultraviolet detection, and the target DNA band was recovered. Take 4.5??L of the recovered product, 0.5??L of PMD 18-T vector DNA, and 5??L of Ligation Solution. The total volume is 10??L. After centrifugation and mixing, ligate overnight at 16°C. Transform the overnight reaction mixture into DH5α competent cells, and screen positive white colonies on LB solid plates containing 100mg/L Amp, 20??L 20mg/mLX-gal and 5??L 200mg/mL IPTG. Pick a single white colony and shake and expand it in LB liquid medium containing Amp (100mg/L) for PCR detection (the reaction system and conditions are the same as before), select a tube of bacterial liquid that passed the test, and send a part to sequencing; Expand the culture and extract the recombinant plasmid, and carry out the identification of Bgl II and BstE II double enzyme digestion. The enzyme digestion reaction system is: 24??L deionized water, 5??L 10×H buffer, 20??L recombinant plasmid, 1??L Bgl II. Then add 1??L BstE II, digest at 60°C for 6 h. After the reaction, add 5??L of 10×Loading Buffer to stop the reaction, and then conduct agarose gel electrophoresis detection and photography. And the recombinant plasmids were named pMD 18-T - AtJMT .

Example 2

Construction of Plant Expression Vector Carrying Arabidopsis JMT Gene Coding Region

The pCAMBIA1304 ⁺ transformed in the laboratory was selected as the vector to construct the plant expression vector. The construction process (see Figure 3-4) is as follows: use Bgl II and BstE II to double digest pMD 18-T - AtJMT and pCAMBIA1304 ⁺ respectively; (enzyme digestion system and conditions are the same as above) recover AtJMT small fragments and pCAMBIA1304 ⁺ large fragments; respectively Ligate the AtJMT small fragment with pCAMBIA1304 ⁺ large fragment; connection system and conditions: take 4.5??L AtJMT small fragment, 0.5??L pCAMBIA1304 ⁺ large fragment, 5??LSolutionⅠ, a total of 10??L add 200??L EP tube, mix well, ligate overnight at 16°C, transform all the ligation products into DH5α, screen positive single clones on LB solid plate containing kanamycin, pick positive single clones in LB liquid containing Kan (50mg/L) Expand the culture in the culture medium, detect the target gene by PCR (the reaction system and conditions are the same as before), and extract the plasmid, and use Bgl II and BstE II double enzyme digestion to verify; the recombinant plasmid containing AtJMT will be obtained.

The plasmid can be introduced into Agrobacterium LBA4404 or EHA105 to obtain engineering bacteria, which can be used for transformation of belladonna.

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Example 3

Obtaining sterile explants of belladonna

Method 1: Using explants to establish sterile explants of belladonna

Take belladonna young shoots and young stems, wash them with running water for 1 hour; then soak them in 2% (M/V) NaClO solution for 10 minutes, rinse them with sterile water for 3 times; then use 0.1% (M/V) mercuric chloride (HgCl ₂ ) Soak in the solution for 15 minutes, rinse with sterile water for 6 times; then inoculate in a sterile cluster bud induction medium (the medium is contained in a 150ml Erlenmeyer flask, sterilized at 121 ⁰ C for 20 minutes), the culture The basic formula is: MS basic medium, adding plant growth regulator 1.25 mg/L BA (benzyl adenine), 30 g/L sucrose and 0.6 g/L PVP (polyvinylpyrrolidone) to adjust the pH value of the medium to 5.8, and then Add 5% agar powder. Young shoots of belladonna were cultivated in a light incubator under the following conditions: 25 ⁰ C, 12 hours of light, and light intensity of 55 μmol. m ^-2 .s ^-1 . After 40 days, sterile belladonna explants can be obtained, which can be used for genetic transformation when the leaves grow to a size of 3cm×3cm.

Method 2: Using belladonna seeds to germinate under sterile conditions to obtain sterile explants

Take the mature seeds of belladonna, soak them in 2% (M/V) NaClO solution for 20 minutes, rinse them _three times with sterile water; Rinse 6 times with sterile water; remove its seed coat under aseptic conditions; inoculate belladonna embryos on the seed germination medium (the medium is contained in a 150ml Erlenmeyer flask, sterilized at 121 ⁰ C for 20 minutes), the The medium formula is: MS basic medium, add 30g/L sucrose, adjust the pH value of the medium to 5.8, and then add 5% agar powder. Cultivate the young shoots of belladonna in a light incubator at 25 ⁰ C and in the dark. After the seeds germinated, the culture conditions were changed to: 25 ⁰ C, 12 hours of light, and the light intensity was 25 μmol. m ^-2 .s ^-1 . Leaves can be used for genetic transformation when they grow to a size of 3cm×3cm.

Example 4

Genetic transformation of belladonna with Agrobacterium tumefaciens to obtain transgenic belladonna

1. Agrobacterium tumefaciens LBA4404- AtJMT , EHA105- AtJMT . Take it out from the refrigerator before use, inoculate it into 50ml YEB liquid culture (add kanamycin to reach a final concentration of 100mg/L), shake and culture twice at 28 ⁰ C and 200rpm;

2. When the OD ₆₀₀ of the second activation reaches 0.3, add 100 μmol/mL acetosyringone, continue to culture at 28 ⁰ C with shaking at 200 rpm, and when the OD ₆₀₀ reaches 0.6, centrifuge at 4000 rpm for 10 minutes at room temperature;

3. Discard the supernatant, suspend the bacteria with MS liquid medium (100 μmol/mL acetosyringone), dilute to 5 times the original volume, and culture at 28 ⁰ C, 200 rpm, so that the concentration of the bacteria reaches OD ₆₀₀ =0.3 Left and right; called transformation solution; can be used for genetic transformation of belladonna; steps 1, 2, and 3 are called activation of Agrobacterium tumefaciens;

4. Take sterile belladonna terminal buds, lateral buds, stems and other plant parts, cut the stems into 1 cm pieces, or cut the leaves into about 2 cm ² , use a sterile scalpel to make a "+" shaped wound, put Put it into the above transformation solution, take it out after 10 minutes of infection, blot dry with sterile toilet paper, and insert it into MS solid medium supplemented with 100 μmol/mL acetosyringone for co-cultivation for 2 days, the culture conditions are: 25 ⁰ C, under dark conditions nourish.

5. After co-cultivation, transfer to MS solid medium supplemented with 1.25 mg/L BA (add 250 mg/L cephalosporin to achieve the purpose of sterilization; add 10 mg/L hygromycin as a screening pressure to obtain transgenic belladonna clustered buds), the culture conditions were: 25 ⁰ C, 12 hours of light, and the light intensity was 55 μmol. m ^-2 .s ^-1 . After 40 days, new belladonna cluster shoots were obtained.

6. When the clustered buds induced from belladonna transformed explants grow to about 1 cm, cut out individual clustered buds and inoculate them on MS solid medium without plant growth regulators (add 250 mg/L cephalosporin Bacteria to achieve the purpose of sterilization; add 10 mg/L hygromycin as a screening pressure to obtain transgenic belladonna cluster buds) subculture; the normal growth of belladonna cluster buds on this medium is the transgenic belladonna cluster buds. After that, subculture once every 25 days, and after 5 subcultures, the Agrobacterium can be removed. Then only take root on MS solid medium supplemented with 0.25 mg/L NAA. Transgenic belladonna seedlings can be transplanted one week after hardening.

Example 5

PCR detection of transgenic belladonna

1, the extraction of belladonna genomic DNA, the method is as follows:

1) Take a small amount of belladonna, put it into a 1.5ml Eppendorf tube, and add 500 microliters of extraction buffer.

2) After fully grinding with a small glass rod, put it in a water bath at 60°C for 50 minutes, and mix it by inverting frequently;

3) Centrifuge at room temperature for 10 minutes at 12000rpm;

4) Take the supernatant, add 500ul saturated phenol [Tris-HCl (pH8.0) saturated, absorb the lower layer], mix gently, and stand at 4°C for 5 minutes until the layers are separated;

5) 12000rpm, centrifuge at room temperature for 10min;

6) Aspirate the supernatant (about 250 microliters), add 2 times the volume of absolute ethanol (stored at -20°C), mix well, and let stand at room temperature until the DNA precipitates;

7) Centrifuge at 8000rpm for 5 minutes at 4°C;

8) Wash twice with 75% ethanol, centrifuge slightly, absorb residual ethanol, and place at room temperature to make ethanol evaporate completely.

9) Add 50ul TE (100ug/ml RNaseA, 50mM Tris.Cl, 10mM EDTA, pH 8.0) to dissolve the DNA. 37°C water bath for 1 hour.

10) Add 40ul chloroform/isoamyl alcohol (24:1), mix gently, and let stand for 5 minutes until the layers are separated.

11) Centrifuge at 12000 rpm for 10 minutes at room temperature.

12) Pipette the supernatant (about 35ul) into a new Eppendorf tube and store at -20°C for PCR detection.

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2. PCR detection of transgenic belladonna, the method is as follows:

Since the target gene JMT used does not come from belladonna itself, it can be directly detected by PCR. Since the JMT gene is in the same border with the hygromycin resistance gene on p1304 ⁺ , the hygromycin resistance gene can also be detected in the DNA of the transgenic belladonna to confirm the transformant. Detection primers of JMT gene fjmt: 5'-ccagatctATGGAGGTAATGCGAGTTCTTCAC-3', rjmt: 5'-cggttaccTCAACCGGTTCTA ACGAGCGAAA G-3'. The PCR conditions were 94°C for 5 minutes, followed by 30 cycles of 94°C for 45 seconds, 64.5°C for 50 seconds, and 72°C for 1min30sec, and finally extended at 72°C for 8 minutes. The hygromycin resistance gene (812bp) was detected using primers fhygr (5'-cgatttgtgtacgcccgacagtc-3') and rhygr (5'-CGATGTAGGAGGGCGTG GATATG'-3). The PCR program was: denaturation at 94°C for 5 min→30 cycles (94°C for 50 sec→58°C for 50 sec→72°C for 1 min)→72°C for 6 min.

The positive control was amplified with p1304 ⁺ - AtJMT as a template, and the negative control was amplified with natural leaf DNA of belladonna. PCR products were detected by agarose gel electrophoresis and ultraviolet light.

Example 6

Target gene expression level detection:

Using the transgenic belladonna with positive molecular detection as the material, Northern hybridization was used to detect the expression level of the target gene, that is, AtJMT , and high-expression transgenic lines were screened out. The method of Northern hybridization to detect the expression level of the target gene was as follows:

① Preparation and labeling of probes: The target gene verified by sequencing was used as a template, and the probe was amplified by PCR using the same primers as those used for cloning the target gene. Use Gene Images ^™ Contents CDP-Star™ labeling module (RPN3540) from Amersham Pharmacia Company, and operate according to the instructions attached to the kit. Dilute the template (DNA or RNA) concentration with DD water or TE to 2-25ng/ul; the template is generally from the PCR product, or the product of enzyme digestion, and take 5ul of the recovered product for electrophoresis. If it is clearly visible, there should be no problem with the labeled probe . Denature the DNA template in a boiling water bath for 10 minutes, with a volume of at least 20ul, and immediately ice bath (put it in an ice box). Add the following reagents to the centrifuge tube: 10ul Nucleotide mix; 5ul Primer; 50ng denatured DNA template; 1ul Klenow enzyme (5U/ul); add water to 50ul; M pH=8.0) After that, wash the nylon membrane with the probe in 2×SSC solution at 65°C for 15 minutes, and observe whether the probe is marked under a purple light; store the probe in the dark at -20°C for later use;

②Preparation of agarose-formaldehyde denaturing gel: Add: 37.5 mL of DEPC-treated water; 7.5 mL of 10× MOPS; 0.55 g of RNase-free low-melting point agarose; The volume is 45 mL; melt the gel in a microwave oven, add 10 mL of formaldehyde when it is cooled to 60°C, mix well, pour the gel after cooling to about 45°C, insert a 3 mm comb, and set aside after the gel is cooled;

③Preparation and electrophoresis of RNA denatured samples: extract total RNA from transgenic belladonna leaves; use ethanol precipitation method to concentrate RNA samples to more than 3??g/??L, and calculate the ??L of RNA required for each sample , prepare the corresponding number of RNase-free 500??L Eppendorf tubes, and add after numbering: 30??g total RNA; 7??L formaldehyde; 20??L deionized formamide; 2??L 10× MOPS ; Make up to 40??L of DEPC-treated water; after mixing, denature at 65°C for 10 min, cool on ice, add a small amount of ethidium bromide and 6??L RNase-free sample buffer, and load the sample immediately. Electrophoresis at 1 v.cm-1 in 1× MOPS buffer, transfer to membrane when bromophenol blue migrates to half the distance of the gel;

④Northern gel transfer, fixation and hybridization detection: 1) Take out the gel after electrophoresis, cut off the excess colloid, and cut off the upper left corner as a mark; 2) Wash the gel with DEPC-treated water for a while; 3) Same as Like Southern hybridization, use DEPC-treated 20×SSC to transfer membranes; 4) Similar to Southern hybridization, pre-hybridize, hybridize (same as Southern hybridization, only the hybridization temperature is changed to 65°C) and wash in an RNase-free environment;

⑤Finally, according to the strength of the hybridization signal, the strength of gene expression was distinguished, and the lines with high expression were selected for further analysis.

Example 7

Comparison of hyoscyamine and scopolamine contents in wild-type belladonna and transgenic belladonna

The content of scopolamine and scopolamine was determined according to the method established by Hashimoto et al. (1993). The results were: the contents of scopolamine in the main root, leaf and stem of wild-type belladonna were: 8.1 mg/g (dry weight), 1.4 mg/g (dry weight), 0.3 mg/g (dry weight); The contents of transgenic belladonna taproot, leaves and stems were: 13.4mg/g (dry weight), 2.3 mg/g (dry weight), 1.4 mg/g (dry weight); scopolamine in wild-type belladonna taproot, The contents in leaves and stems were: 0.6 mg/g (dry weight), 0.2 mg/g (dry weight), 0.03 mg/g (dry weight); the contents of scopolamine in the main root, leaves and stems of transgenic belladonna were respectively For: 0.82 mg/g (dry weight), 0.5 1 mg/g (dry weight), 0.06 mg/g (dry weight).

Claims (3)

1. A plant expression vector, which comprises the coding region of the jasmonate carboxymethyltransferase gene nucleotide, which is derived from a plant with the MeJA biosynthetic pathway or encoded with a mutant with the same catalytic function and an artificially synthesized nucleus nucleotide sequence. 2. A method for increasing the content of endogenous MeJA and the content of tropane alkaloids in belladonna belladonna, the method uses the key enzyme jasmonic acid carboxyl methyltransferase (JMT) in the MeJA biosynthesis pathway ) gene or a plant high-efficiency expression vector of a DNA molecule in the coding region of a nucleotide sequence with the same function, overexpressed in belladonna, increases the content of endogenous MeJA in belladonna, and promotes TA biosynthesis. The process includes: (1) Obtain the JMT gene coding region by gene cloning; (2) Connect the JMT gene coding region to the expression control sequence to construct a plant expression vector carrying the JMT gene; (3) obtaining sterile explants of belladonna; (4) Transgenic methods are used to transfer the JMT gene coding region to belladonna cells, tissues, organs, and plants; (5) Screen and identify belladonna transformants under specific conditions; (6) The transgenic belladonna cultured under suitable conditions to obtain transgenic offspring whose genome integrates the coding region of the nucleotide of the jasmonic acid carboxymethyltransferase gene from the plasmid in claim 1 . 3. A host cell transformed with the plant expression vector according to claim 1, wherein the host cell is belladonna.