CN114107334A

CN114107334A - A mulberry resveratrol synthase gene and a method for using the same to enhance the drought tolerance of mulberry and increase the content of resveratrol in mulberry white bark

Info

Publication number: CN114107334A
Application number: CN202111323717.6A
Authority: CN
Inventors: 盖英萍; 冀宪领; 张利琴; 刘朝阳; 辛有超
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-03-01
Anticipated expiration: 2041-11-10
Also published as: CN114107334B

Abstract

The invention discloses a mulberry resveratrol synthase gene and a method for using the gene to enhance the drought tolerance of mulberry and increase the content of resveratrol in mulberry white bark. The invention utilizes a resveratrol synthase gene derived from mulberry tree, named as MulSTS, transforms seedlings of mulberry mulberry through transgenic technology mediated by Agrobacterium rhizogenes, obtains hairy roots of mulberry tree transgenic with MulSTS gene, and utilizes the obtained transgenic hairy roots. The root is the rootstock, and the grafted mulberry seedlings are obtained by grafting. The drought tolerance of the grafted mulberry seedlings obtained by this method was significantly improved, and the content of resveratrol in the root phloem was significantly higher than that of the control mulberry grafted seedlings. The invention provides a method for cultivating drought-resistant mulberry varieties, improves the drought tolerance of the mulberry tree, widens the planting range of the mulberry tree, and improves the medicinal quality of the root bark (mulberry white bark) of the mulberry tree at the same time. It provides a reference for the improvement of drought tolerance and medicinal value, and has broad application value.

Description

Mulberry resveratrol synthase gene and method for enhancing drought tolerance of mulberry and improving resveratrol content in white mulberry root-bark by using same

Technical Field

The invention relates to the technical field of biology and agriculture, in particular to a mulberry resveratrol synthase gene and a method for enhancing drought resistance of mulberry and improving resveratrol content in white mulberry root-bark by using the same.

Background

The mulberry is a material base for silkworm industry production, can be used for picking leaves and breeding silkworms, and also has high edible, medicinal and feeding values. Cortex mori is the dry root bark (phloem) of mulberry, has been recorded in Shen nong's herbal Jing as a medicine for purging lung-heat, relieving asthma, inducing diuresis and relieving edema. The modern medical research results also show that the cortex mori radicis has the efficacies of reducing blood sugar, resisting inflammation, resisting oxidation, resisting tumors, resisting depression, protecting heart and nerves, regulating immunocompetence and the like, has high medicinal value and wide application, and the quality standard thereof is collected in the pharmacopoeia of the people's republic of China in the calendar and is one of the traditional Chinese medicinal materials commonly used in clinic. The resveratrol is used as a main drug effect component of the white mulberry root-bark, has various physiological activities, and the content of the resveratrol is one of main indexes for measuring the quality of the white mulberry root-bark. In plants, resveratrol is synthesized by using 4-coumaroyl-coenzyme A and malonyl-coenzyme A as substrates under the catalytic action of resveratrol synthase, and the resveratrol synthase is a key enzyme for synthesizing resveratrol. Resveratrol is also present in a few species such as grapes, peanuts, giant knotweed, sorghum and the like, besides being present in mulberry tissues in a high content. Although many plants contain substrates required for resveratrol synthesis, resveratrol cannot be synthesized or can be synthesized only in a trace amount due to the absence of resveratrol synthase or extremely low expression level of resveratrol synthase. Therefore, the resveratrol synthase gene is integrated into the genome of a species which does not contain the resveratrol synthase or has extremely low resveratrol synthase expression quantity by using a genetic engineering means for expression, the content of the resveratrol in plant tissues can be increased or the plant which can not synthesize the resveratrol can synthesize the resveratrol, so that the nutritional value and the health-care quality of agricultural and forestry crops are improved. At present, patent applications related to plant resveratrol synthase genes and applications exist at home and abroad, wherein the patent applications comprise a mulberry resveratrol synthase gene invented by the name of Simmondsia chinensis and the like (Chinese patent CN 104975032A), and a resveratrol synthase gene derived from a mulberry is disclosed, and the resveratrol content in transgenic arabidopsis thaliana can be increased by expressing the gene in arabidopsis thaliana. The invention discloses the cloning of mulberry resveratrol synthetase gene and the construction of plant expression vector (Chinese patent CN 103898130A) invented by the Wangbang and the like, discloses the cloning of resveratrol synthetase gene from mulberry and the construction technology of plant expression vector thereof, and the expression of the gene in tobacco improves the content of resveratrol in the tobacco. "Mulberry resveratrol synthetase, coding gene and recombinant vector and application thereof" invented by Cao Hao Tao et al (Chinese patent CN 109266663A) discloses a mulberry resveratrol synthetase, coding gene and application of recombinant vector, the gene is expressed in yeast, can use p-coumaric acid as substrate to convert and synthesize resveratrol.

Resveratrol synthase belongs to a member of plant type III polyketide synthase family (PKS), exists in a plant body in the form of a gene family, and has high homology with chalcone synthase which is another member of the gene family. There is currently no uniform specification for the nomenclature of PKS family members, and many PKS members, although named resveratrol synthase genes, do not necessarily have resveratrol synthase function. The functions of most PKS family members in the mulberry are not identified, and reports that the resveratrol synthetase gene is over-expressed in the mulberry and the content of resveratrol in the phloem of the root system of the mulberry is improved are not found at present.

Drought is one of the major abiotic stress factors affecting the growth, development, yield, quality and planting range of agricultural and forestry crops. According to investigation, arid land and semi-arid land in China account for about 45% of the total land area in China, and the arid land becomes an important factor influencing the sustainable and healthy development of agriculture and forestry in China. The mulberry is not only used as a feed tree for silkworms, but also an important ecological tree for water and soil conservation, wind prevention and sand fixation and saline-alkali soil improvement. The sericulture industry is currently moving from economically developed regions in south east to inland arid and semiarid regions. Therefore, the drought tolerance of the mulberry is improved, the cultivation area is expanded, and the method has important significance for realizing the economic and ecological values of the mulberry. At present, the mulberry in production mostly adopts grafting to breed nursery stocks, stock used for grafting is mainly derived from seedling mulberry nursery stocks, stress resistance screening and quality improvement are not carried out, the obtained mulberry grafting nursery stocks have poor stress resistance, and reports that drought-resistant mulberry stock is obtained by using a transgenic technology and then the mulberry nursery stocks are bred by a grafting method, so that the drought resistance of the mulberry is enhanced are not found. Besides pharmacological activity, resveratrol in plants is an important plant protection agent and antitoxin and participates in the stress response process of plants to pathogenic bacteria, ultraviolet radiation, mechanical damage, hormone and the like. Research reports have been reported that the disease resistance of plants is enhanced by transferring resveratrol synthase genes into plants without the resveratrol synthase genes. However, the main objective of the previous studies on the biosynthesis of resveratrol is mainly genetic improvement of plant disease resistance, and no research report on the enhancement of the plant resistance to abiotic stress such as drought by transferring resveratrol synthase gene is found. Particularly for the mulberry, an efficient genetic transformation system cannot be established at present, the transgenic mulberry is difficult to obtain, and the research report of improving the drought resistance of the mulberry by a transgenic technology is not seen so far.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a mulberry resveratrol synthase gene and a method for enhancing drought resistance of mulberry and improving the content of resveratrol in white mulberry root-bark by using the same. The resveratrol synthase gene is over-expressed in the mulberry, so that the drought tolerance of the mulberry can be enhanced, and the planting area of the mulberry is widened; the content of resveratrol in the phloem of the root system of the mulberry can be increased, the medicinal quality of the mulberry is improved, a reference is provided for improving the drought tolerance and the medicinal quality of other agricultural and forestry plants, and the application value is wide.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a resveratrol synthase gene selected from at least one of nucleic acid molecules represented by the following (1) to (3):

(1) nucleic acid molecule with sequence shown as SEQ ID NO. 1;

(2) nucleic acid molecule with sequence shown as SEQ ID NO. 2;

(3) a nucleic acid molecule which has 98 percent or more of sequence homology with the nucleotide sequence shown in SEQ ID NO.2 and is functionally equivalent to the nucleotide sequence shown in SEQ ID NO. 2.

In a second aspect of the invention, a plant expression vector containing the resveratrol synthase gene is provided.

Furthermore, the invention also provides a recombinant bacterium containing the plant expression vector.

In a third aspect of the invention, the resveratrol synthase gene, the plant expression vector or the recombinant bacterium is applied to improving the drought resistance of plants and/or improving the content of resveratrol.

Preferably, the plant is mulberry.

In a fourth aspect of the invention, the resveratrol synthase gene, the plant expression vector or the recombinant strain are applied to cultivation of drought-tolerant mulberry varieties.

In the fifth aspect of the invention, the resveratrol synthase gene, the plant expression vector or the recombinant bacterium are applied to the production of the white mulberry root-bark with high resveratrol content.

The sixth aspect of the invention provides a method for enhancing drought tolerance of mulberry and improving resveratrol content in phloem of root system by using transgenic resveratrol synthase gene rootstock, which comprises the following steps:

(1) placing the resveratrol synthase gene sequence under a CaMV35S strong promoter to construct a plant expression vector;

(2) transferring the plant expression vector constructed in the step (1) into agrobacterium rhizogenes K599 competent cells to obtain a transformant;

(3) transforming the mulberry by using the transformant in the step (2) to obtain hairy root transgenic mulberry;

(4) a mulberry grafted seedling with enhanced drought resistance and resveratrol content in root bark is obtained by taking a hairy root transgenic mulberry as a stock and grafting.

Preferably, in the step (1), the specific construction method of the plant expression vector comprises:

using mulberry leaf cDNA as a template, performing PCR reaction by using primers shown in SEQ ID NO.5 and SEQ ID NO.6, connecting a PCR product with a pMD19-T Simple cloning vector, transforming escherichia coli DH5 alpha competent cells by using the connecting product, screening positive clones, and performing sequence determination; and (3) carrying out enzyme digestion on the recombinant plasmid containing the mulberry resveratrol synthase gene fragment through sequence determination by using Bam HI and Kpn I, recovering the mulberry resveratrol synthase gene fragment, and connecting the recovered mulberry resveratrol synthase gene fragment with a pROKII expression vector digested by the same restriction enzyme, so as to construct a plant expression vector.

The invention has the beneficial effects that:

(1) the invention provides an endogenous gene in a mulberry for the first time, and the gene can enhance the drought tolerance of the mulberry and improve the content of resveratrol in the phloem of the root system of the mulberry.

(2) The drought-resistant mulberry stock is obtained by utilizing a transgenic technology, and the mulberry seedling with stronger drought resistance and higher medicinal quality is obtained by grafting the mulberry stock.

(3) The invention transforms the seedling mulberry by agrobacterium rhizogenes mediated transgenic technology to obtain the hairy root of the mulberry with the resveratrol synthase gene, uses the obtained transgenic hairy root as a stock to obtain the mulberry by grafting, obviously enhances the drought resistance of the obtained mulberry and obviously improves the resveratrol content in the phloem of the root system. The invention provides a method for cultivating drought-resistant mulberry seedlings, enhances the drought resistance of mulberry trees, widens the planting range of the mulberry trees, improves the medicinal quality of the mulberry trees, reduces the risk of transmission and genetic drift of transgenic mulberry trees through pollen and fruits, can obviously reduce the worry about the safety of transgenic organisms, provides reference for the improvement of the drought resistance and the medicinal quality of other forest trees, and has wide application value.

Drawings

FIG. 1: a schematic diagram for constructing a plant overexpression vector of a mulberry resveratrol synthase gene.

FIG. 2: and (3) identifying transgenic mulberry hairy roots. A: and (3) PCR identification result of transgenic mulberry hairy roots. B: and detecting the expression abundance of the MulSTS in hairy roots of the transgenic mulberry. M: DNA molecule Marker; WT: wild mulberry root system; OE 1-3: transgenic mulberry hairy roots.

FIG. 3: and (4) measuring the content of resveratrol in phloem of mulberry root system. WT is the wild mulberry root phloem, OE1-3 is the root phloem of different MulSTS gene-transferred strains.

FIG. 4: effect of drought stress on growth rate of mulberry.

FIG. 5: impact of drought stress on mulberry biomass.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, many PKS family members are designated resveratrol synthase genes, but some genes do not have resveratrol synthase function. The functions of most of the PKS family members in mulberry are not identified at present, and for mulberry, an efficient genetic transformation system cannot be established at present, it is difficult to obtain transgenic mulberry, and it may be more difficult to apply the gene isolated from mulberry to mulberry for overexpression than to apply the gene to other plants. Therefore, no research report for improving the drought resistance of the mulberry by a transgenic technology is found so far.

Based on the above, the invention carries out intensive research on the resveratrol synthase gene in the mulberry, obtains the resveratrol synthase gene derived from the mulberry (Morus multicaulis Perr), is named as MulSTS, and the RNA sequence of the resveratrol synthase gene is shown as SEQ ID NO. 1; the sequence of the coding gene of MulSTS is shown in SEQ ID NO. 2. Researches find that the drought resistance of mulberry grafted seedlings obtained by taking a mulberry root system transformed with a MulSTS gene as a stock is obviously enhanced, the planting range of the mulberry is widened, the resveratrol content in mulberry root bark is obviously increased, and the medicinal quality of the mulberry root bark is improved, so that the invention is provided.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier.

Example 1: cloning method of MulSTS gene of mulberry

(1) Extracting total RNA of mulberry leaves by using a nucleic acid extraction kit, synthesizing cDNA by using a reverse transcription kit, wherein the reaction system is as follows:

5X PrimeScript RT Master Mix 2.0μL

RNA(1μg/μL) 1.0μL

dH₂O(RNase-free) up to 10.0μL

adding the above reagents, mixing, reverse transcription reacting in 35 deg.C water bath for 20min, rapidly ice-cooling for 5min, inactivating at 85 deg.C for 1min to obtain the final product, namely mulberry cDNA, and storing at-20 deg.C.

(2) A pair of specific primers MulSTS-5 and MulSTS-3 is designed, and the specific sequences are as follows:

MulSTS-5：5′-ATGGCACCGAACAACGTGCC-3′；(SEQ ID NO.3)

MulSTS-3：5′-CTATGCAATAATGGGGACAC-3′；(SEQ ID NO.4)

PCR amplification is carried out by taking mulberry leaf cDNA as a template, and the system is as follows:

the reaction procedure was as follows:

after the reaction is finished, detecting the PCR product by using 1% agarose gel electrophoresis, recovering the target fragment for sequencing, and obtaining the mulberry MulSTS gene (the sequence is shown as SEQ ID NO. 2) with correct sequencing.

Example 2: construction of Mulberry MulSTS gene plant expression vector

(1) According to the nucleotide sequence of the separated mulberry MulSTS gene, primers MulSTS-F and MulSTS-R with enzyme cutting sites are designed, and the sequences are specifically as follows:

MulSTS-F：5′-GGATCCATGGCACCGAACAACGTG-3′；(SEQ ID NO.5)

MulSTS-R：5′-GGTACCCTATGCAATAATGGGGAC-3′；(SEQ ID NO.6)

using the cDNA of mulberry leaf as a template, PCR reaction was performed in the same manner as in example 1.

(2) Connecting the PCR product with a pMD19-T Simple cloning vector, transforming Escherichia coli DH5 alpha competent cells by the connecting product, screening positive clones by ampicillin (50mg/L) resistance, extracting recombinant plasmids after PCR identification of bacterial liquid, and performing sequence determination after enzyme digestion identification.

(3) The recombinant plasmid containing Mulberry MulSTS gene fragment after sequence determination is cut by Bam HI and Sac I, and the recovered MulSTS gene fragment is connected with pROKII expression vector cut by the same restriction enzyme. And transforming the connecting product into escherichia coli DH5 alpha competent cells, screening positive clones for kanamycin (50mg/L) resistance, and performing bacteria liquid PCR identification and plasmid DNA restriction enzyme digestion identification on the selected positive clones. Thus, a plant expression vector of MulSTS gene of mulberry was constructed (FIG. 1).

Example 3: obtaining transgenic mulberry with hairy root

(1) And transforming the constructed plant expression vector of the MulSTS gene into an agrobacterium rhizogenes K599 competent cell by a freeze thawing method, and screening positive clones for kanamycin (50mg/L) resistance. Selecting agrobacterium (agrobacterium single colony carrying recombinant plasmid) and inoculating the agrobacterium in LB liquid medium (tryptone L0g/L, yeast powder 5g/L, sodium chloride L0g/L, kanamycin 50mg/L), culturing at 28 ℃ and 250r/min under oscillation for about 48h to the late logarithmic growth phase; diluting the bacterial liquid by 10 times with MS culture solution for later use.

(2) And (3) sowing the plump 12 # Guisangyou seeds on a seedbed, covering a small arched shed for protection and culture, and when two true leaves grow out of the nursery stock, fixing the seedling and keeping the seedling distance to be 5 cm. Injecting the Agrobacterium rhizogenes suspension into the upper embryonic axis of the seedling, burying the injection part with moist fine sand, culturing under weak light for 3d, then culturing under normal light for about 20d, gradually removing the arched shed, and continuing culturing for about 30 d.

3. Taking out the seedling, screening out the seedling with hairy root at the injection part, cutting off the original root system, cutting 2 leaves on the overground part, transplanting to the field for culture, and culturing with the seedling distance of 7-10 cm.

Example 4: PCR identification of hairy root transgenic mulberry

(1) DNA of hairy root transgenic mulberry plants and wild type plant roots is extracted by a CTAB method.

(2) Designing a specific primer 35S-5 according to the 35S promoter sequence, which comprises the following steps:

35S-5：5′-GACGCACAATCCCACTATCC-3′；(SEQ ID NO.7)

respectively taking the extracted DNA of the hairy roots and wild mulberry roots of the transgenic mulberry as templates and 35S-5(SEQ ID NO.7) and MulSTS-3(SEQ ID NO.4) as primers to carry out PCR amplification, wherein the reaction system is as follows:

the reaction procedure was as follows:

after the reaction, the PCR product was detected by electrophoresis on a 1% agarose gel. As can be seen from the results of agarose gel electrophoresis (FIG. 2A), a band of about 1200bp can be amplified in the DNA of the root system of the transgenic plant (the band is obtained by amplifying the 35S promoter and the MulSTS sequence), and the DNA of the non-transgenic plant has no 35S promoter sequence, so that the band cannot be amplified in the DNA of the wild-type plant, which indicates that the MulSTS gene started by the 35S promoter is successfully introduced into the hairy root genome of the mulberry.

Example 5: expression abundance of MulSTS gene in hairy root of transgenic mulberry and resveratrol content detection

(1) The test adopts a fluorescent quantitative PCR method to detect the expression level of the MulSTS gene in the root system of the transgenic mulberry.

(2) Total RNA of wild type and transgenic mulberry root systems is extracted by a Trizol method, genomic DNA is removed by DNase I, and the total RNA is reversely transcribed into cDNA.

(3) The mulberry ACTIN-1 is used as an internal reference gene, reverse transcription products of wild type and transgenic mulberry RNA are used as templates, and primers of fluorescent quantitative PCR are as follows:

MulACTIN-F：5′-CACTGAGGCTCCTTTGAACCC-3′(SEQ ID NO.8)；

MulACTIN-R：5′-AGGTCGAGACGGAGAATAGCATG-3′(SEQ ID NO.9)。

reaction System and reaction step reference

Premix Ex TaqTM II instruction carries out reaction, and whether the MbSTS7 gene can be successfully expressed in the root system of the transgenic mulberry and the expression abundance of the gene is analyzed.

The results are shown in FIG. 2B. As can be seen from fig. 2B: the MulSTS is highly expressed in the hairy root system of the transgenic mulberry.

(4) Taking the hairy roots of MulSTS gene-transferred mulberry and phloem of wild type roots, cutting into 2mm multiplied by 2mm, drying at 80 ℃, weighing 4g of dried sample, adding 100mL of 80% ethanol, carrying out reflux extraction for 4h, filtering, fixing the volume to 100mL, and determining the content of resveratrol by using a high performance liquid chromatography technology.

The results are shown in FIG. 3. As can be seen from fig. 3: the resveratrol content in the hairy root of the transgenic mulberry is obviously higher than that in the wild type.

Example 6: drought resistance analysis of mulberry obtained by grafting by taking transgenic mulberry hairy roots as rootstocks

(1) The hairy roots of the transgenic mulberry are cultured for one year and then are taken out before the mulberry sprouts in the spring of the next year, the mulberry is taken as a stock, the mulberry branch of Nongsang No. 14 is selected as a scion, and a seedling (marked as a transgenic genome) is bred by grafting. Meanwhile, the root of a wild Guisangyou 12 mulberry is taken as a stock, and a mulberry seedling bred by grafting with a Nongsang No. 14 mulberry branch as a scion is taken as a contrast (marked as a wild group).

(2) Respectively planting the grafted survival transgenic group and wild type group mulberry in flowerpots, respectively selecting 60 mulberry trees with basically consistent growth vigor of the transgenic group and the wild type group to carry out a drought tolerance test when the seedlings grow to about 30cm, normally watering one half of 30 pots in each group, keeping 75% of field water capacity (contrast treatment), carrying out drought treatment on the other half of the 30 pots, keeping 30% of field water capacity, treating for 7d, and measuring the plant height and the total dry weight (total biomass) of the overground part and the underground part. The results are shown in FIGS. 4 and 5. The results show that the mulberry seedlings grafted by taking the transgenic mulberry hairy roots as the rootstocks have higher growth amount and biomass under drought stress than the control mulberry seedlings, show stronger drought resistance and have good application prospect.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

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Claims

1. A resveratrol synthase gene, characterized in that, it is selected from at least one of the nucleic acid molecules shown in the following (1)-(3):

(1) a nucleic acid molecule whose sequence is shown in SEQ ID NO.1;

(2) a nucleic acid molecule whose sequence is shown in SEQ ID NO.2;

(3) A nucleic acid molecule that has 98% or more sequence homology with the nucleotide sequence shown in SEQ ID NO. 2 and is functionally equivalent to the nucleotide sequence shown in SEQ ID NO. 2.

2. A plant expression vector containing the resveratrol synthase gene of claim 1.

3. The recombinant bacteria containing the plant expression vector of claim 2.

4. The application of the resveratrol synthase gene of claim 1, the plant expression vector of claim 2 or the recombinant bacteria of claim 3 in improving the salt tolerance of plants.

5. The application according to claim 4, wherein the plant is a mulberry tree.

6. The application of the gene of claim 1, the plant expression vector of claim 2 or the recombinant bacteria of claim 3 in improving plant drought tolerance and/or increasing the content of resveratrol.

7. The application according to claim 6, wherein the plant is a mulberry tree.

8. The application of the resveratrol synthase gene of claim 1, the plant expression vector of claim 2 or the recombinant bacteria of claim 3 in the production of Morus alba with high resveratrol content.

9. a method utilizing trans-resveratrol synthase gene stock to enhance mulberry drought tolerance and improve resveratrol content in root phloem, is characterized in that, comprises the following steps:

(1) the resveratrol synthase gene sequence described in claim 1 is placed under the strong promoter of CaMV35S, and a plant expression vector is constructed;

(2) transferring the plant expression vector constructed in step (1) into Agrobacterium rhizogenes K599 competent cells to obtain transformants;

(3) utilize the transformant of step (2) to transform mulberry to obtain hairy root transgenic mulberry;

(4) Using hairy root transgenic mulberry as rootstock, grafted mulberry seedlings with enhanced drought tolerance and increased resveratrol content in root bark were obtained by grafting.

10. method according to claim 9, is characterized in that, in step (1), the concrete construction method of described plant expression vector is:

Using mulberry leaf cDNA as a template, using the primers shown in SEQ ID NO.5 and SEQ ID NO.6 to carry out PCR reaction, the PCR product was ligated with pMD19-T Simple cloning vector, and the ligated product was transformed into E. coli DH5α competent cells, and screened The positive clone was cloned and sequenced; the recombinant plasmid containing the mulberry resveratrol synthase gene fragment after sequence determination was digested with BamHI and Kpn I, and the mulberry resveratrol synthase gene fragment was recovered with the same restriction. The pROKII expression vector digested by Dicer is connected to construct a plant expression vector.