CN106086038B - Artemisia apiacea WRKY transcription factor coding sequence, cloning method and application - Google Patents

Abstract

The invention discloses clone of a sweet wormwood WRKY gene coding sequence and application thereof. Specifically comprises cloning of a gene AaWRKY2, construction of a plant expression vector containing the gene, and activation of the gene on a specific gene promoter in an artemisinin biosynthesis pathway. The nucleotide sequence of the sweet wormwood AaWRKY2 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The invention also discloses the characteristic that the AaWRKY2 gene has the characteristic of activating the expression of the 2 gene promoters of ADS and DBR2 with specificity in the artemisinin biosynthesis pathway. The AaWRKY2 gene can be applied to quality improvement of the sweet wormwood through over-expression and the like, and the content of artemisinin in the sweet wormwood can be increased.

Description

Artemisia apiacea WRKY transcription factor coding sequence, cloning method and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a sweet wormwood WRKY transcription factor coding sequence AaWRKY2 and application thereof.

Background

Metabolism in plants is divided into primary metabolism and secondary metabolism, wherein primary metabolites (such as saccharides, lipids and nucleic acids) exist in all plants and are necessary for the plants to maintain cell life activities, and plant secondary metabolites refer to a large group of small-molecule organic compounds which are not necessary for plant growth and development in plants, and the synthesis and distribution of the small-molecule organic compounds have species, tissue organ and production and development specificity. For example, artemisinin, a specific pharmaceutical ingredient for the treatment of malaria, is synthesized and stored only in the secretory glandular hairs on the surface of the plant Artemisia annua L. In recent years, with the research of Chinese herbal medicine ingredients, it is found that the effective ingredients of many Chinese herbal medicines are plant secondary metabolites, such as tanshinone in salvia miltiorrhiza, vinca alkaloid in vinca and the like.

Most plant secondary metabolites have extremely low content in natural plants, but the chemical synthesis method has complex process flow and high cost, and the biosynthesis routes of many plant secondary metabolites are unclear, so that the chemical total synthesis cannot be realized. Therefore, researchers have begun exploring other ways to increase the level of secondary metabolites in plants. Considering that the synthesis path of plant secondary metabolites is complex, the number of genes involved in reaction is large, and the genes are influenced by multiple factors such as development and environment, the modification of a single gene on the path sometimes cannot be effective. The transcription factor can regulate the expression of multiple enzyme genes in the biosynthesis pathway of a plant secondary metabolite, thereby regulating the biosynthesis amount of the secondary metabolite.

At present, it has been reported in artemisia apiacea that transcription factors can effectively regulate and control the expression of specific genes of an artemisinin synthesis pathway, so as to regulate and control the biosynthesis of artemisinin, such as AaORA1 transcription factors, AaWRKY2 transcription factors, AaMYC2 transcription factors and the like. Therefore, the cloning of the transcription factor capable of regulating and controlling the expression of the specific gene of the artemisinin biosynthesis pathway has important significance for improving and increasing the content of artemisinin in the artemisia apiacea.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the present invention provides the following technical solutions to increase the content of artemisinin in artemisia apiacea:

the invention provides a sweet wormwood WRKY transcription factor coding sequence, which is marked as AaWRKY2, and the nucleotide sequence of AaWRKY2 is shown as SEQ ID No. 1.

The invention also provides a southernwood WRKY transcription factor coding sequence, and the amino acid sequence coded by AaWRKY2 is shown in SEQ ID NO. 2.

The invention also provides a polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.

The invention also provides a recombinant expression vector, which comprises an open reading frame sequence of the nucleotide sequence shown as SEQ ID NO.1, wherein the open reading frame sequence is shown as SEQ ID NO. 3. The construction method comprises the following steps:

step 1, designing and amplifying AaWRKY2 gene open reading frame sequence according to SEQ ID NO.1 sequence, wherein amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3', respectively;

step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR/D-TOPO vector;

and 3, constructing a pEarlygate104-AaWRKY2 plant expression vector containing the target gene by using the pENTR/D-TOPO vector connected with the AaWRKY2 gene and a pEarlygate104 plant expression vector in an LR reaction mode.

The invention also provides a recombinant expression transformant, which comprises an open reading frame sequence of the nucleotide sequence shown as SEQ ID NO.1, and the open reading frame sequence is shown as SEQ ID NO. 3.

Further, the host strain of the recombinant expression transformant is agrobacterium.

The invention also provides application of the sweet wormwood WRKY transcription factor coding sequence AaWRKY2 in improving the content of artemisinin.

The invention also provides a cloning method of the sweet wormwood WRKY transcription factor coding sequence AaWRKY2, which comprises the following steps:

step 1, extracting and purifying total RNA, namely extracting and purifying by adopting various general plant total RNA extraction kits to obtain total RNA of artemisia apiacea leaves;

step 2, reversing total RNA of the artemisia apiacea leaves into cDNA by using reverse transcriptase;

and 3, using the cDNA as a template, designing a gene specific primer, and amplifying by adopting a PCR method to obtain a PCR product, wherein the gene specific primer is as follows:

forward primer P1: 5'-AAGATGGAATATGGAAGAGG-3'

Reverse primer P2: 5' -ATCAAACAAAGAGTCCACAGAC-3;

and 4, recovering, purifying and sequencing the PCR product to obtain the nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides a method for rapidly detecting the biological function of the amino acid sequence encoded by the AaWRKY2, which comprises the following steps:

step 1, designing and amplifying AaWRKY2 gene open reading frame sequence according to SEQ ID NO.1 sequence, wherein amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3', respectively;

step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR/D-TOPO vector;

step 3, constructing a pEarlygate104-AaWRKY2 plant expression vector containing a target gene by connecting the pENTR/D-TOPO vector connected with the AaWRKY2 gene with a pEarlygate104 plant expression vector in an LR reaction mode;

step 4, connecting a promoter ProADS of a special ADS gene in an artemisinin synthesis approach in artemisia apiacea into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII 0800-ProADS;

step 5, connecting a promoter ProCYP71AV1 of a specific CYP71AV1 gene in an artemisinin synthesis way in the sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProCYP71AV 1;

step 6, connecting a promoter ProDBR2 of a specific DBR2 gene in an artemisinin synthesis way in sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProDBR 2;

step 7, connecting a promoter ProALDH1 of a specific ALDH1 gene in an artemisinin synthesis way in sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProALDH 1;

step 8, respectively transforming a pEarlygate104-AaWRKY2 plant expression vector and a plant double-fluorescein detection report vector pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH1 into agrobacterium to obtain an agrobacterium engineering strain containing a target vector;

step 9, mixing an agrobacterium engineering strain containing a pEarlygate104-AaWRKY2 plant expression vector and an agrobacterium engineering strain containing a plant double-fluorescein detection report vector, and injecting the mixture into tobacco leaves growing for 5 weeks in an injection infection mode;

step 10, taking tobacco leaves cultured for 2 days after injection, quickly freezing the tobacco leaves by using liquid nitrogen, and grinding the tobacco leaves into powder;

and step 11, detecting the fluorescence intensity by adopting a Promega-Dual-Luciferase detection kit, and determining the activation effect of the AaWRKY2 gene and the specific gene promoter of the artemisinin synthesis pathway.

Further, in the step 8, the host bacterium is agrobacterium GV3101 strain; in the step 9, the mixing ratio of the agrobacterium engineering strain containing the pEarlygate104-AaWRKY2 plant expression vector to the agrobacterium engineering strain containing the plant double-fluorescein detection report vector is 3:1, mixing.

In the present invention, the AaWRKY2 gene may be cloned using various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, and the like. In the invention, the AaWRKY2 plant expression vector is constructed, and various vectors known in the field, such as a commercial pCAMBIA series vector, can be selected; the promoter bifluorin reporter vector constructed in the present invention can be selected from various other vectors known in the art, such as commercially available vectors from Promega corporation; the Agrobacterium involved in the present invention is Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain GV3101, which is publicly available on the market.

The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows that in a preferred embodiment, the tobacco transient transformation AaWRKY2 gene significantly improves the expression activity of promoters of 2 genes, namely ADS and DBR 2.

Detailed Description

The following examples illustrate the invention in detail: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1, example 1 cloning of AaWRKY2 Gene of Artemisia annua

1. Culturing herba Artemisiae Annuae in artificial climate chamber with photoperiod of 18h/6h (light/dark) and growth condition of 25 deg.C;

2. and (4) extracting total RNA of the artemisia apiacea leaves. 100 mg of young sweet wormwood leaf tissue material is taken and put in liquid nitrogen to be fully ground into powder, and the total RNA of the leaves is extracted according to the method of the instruction of a plant total RNA extraction kit (Tiangen Biochemical, Beijing). mu.L of the obtained plant total RNA was subjected to agarose gel electrophoresis to identify the quality of the total RNA, and then the concentration of the total RNA was determined on a NanoDrop (Thermo Fisher, USA) spectrophotometer.

3. Cloning of the gene. Reverse transcription is carried out by taking the extracted total RNA as a template (500ng) according to the method of the instruction of a reverse transcription Kit PrimeScript1st Strand cDNA Synthesis Kit (TaKaRa, Dalian) to produce first Strand cDNA; through the designed specific primer, the cDNA is taken as a template for PCR amplification, and the specific primer sequence is as follows:

forward primer P1: 5'-AAGATGGAATATGGAAGAGG-3'

Reverse primer P2: 5' -ATCAAACAAAGAGTCCACAGAC-3

The total volume of the PCR reaction is 50 mu L, and the reaction system is as follows: mu.L of 10 XKOD buffer, 5. mu.L of dNTPs, 4. mu.L of MgSO4, 1. mu.L of forward primer, 1. mu.L of reverse primer, 1. mu.L of cDNA template, 1. mu.L of KOD enzyme, and ddH2O to 50. mu.L.

PCR amplification conditions, pre-denaturation 95 ℃ for 3min, 35 cycles: 95 ℃ for 30 sec; 54 ℃ for 30 sec; extension at 68 ℃ for 100sec, and finally at 68 ℃ for 5 min.

After the PCR product was recovered and purified, the blunt-ended vector pLB (a product of Tiangen Biochemical Co., Ltd.) was ligated and sequenced to obtain a pLB-AaWRKY2 plasmid vector.

Through the steps, the sequence of the AaWRKY2 gene in the artemisia apiacea is shown as SEQ ID NO.1, and the protein coding sequence is deduced to be shown as SEQ ID NO. 2.

Example 2 construction of plant expression vector containing AaWRKY2 Gene

1. Construction of intermediate vector pENTR/D-TOPO-AaWRKY 2.

The sequence of the open reading frame of the AaWRKY2 gene is designed and amplified according to the sequence information of SEQ ID NO.1, and amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3'

pENTR/D-TOPO vector was purchased from Invitrogen, a portal vector for the Gateway cloning technology of this company, and four bases of CACC were added before the bases of the forward primer ATG, as required by the product instructions. pLB-AaWRKY2 plasmid is used as a template, PCR amplification is carried out by using blunt-end high-fidelity enzyme KOD, a PCR product is recovered and purified and then is connected to a pENTR/D-TOPO vector by a method of Gateway cloning technology, and the specific method is carried out according to the instruction of pENTR/D-TOPO cloning kit of Invitrogen company.

2. The intermediate vector pENTR/D-TOPO-AaWRKY2 and the pEarlygate104 plant expression vector are subjected to LR reaction according to an LR ClonaseIIenzyme kit of Invitrogen, a reaction system is prepared according to a kit specification, the reaction system is placed at 25 ℃ in a metal bath for reaction for 3 hours, then Escherichia coli DH5 α competence is transformed, positive cloning PCR verification is carried out, and the pEarlygate104-AaWRKY2 plant expression vector containing the target gene is finally obtained.

Example 3 construction of artemisinin Synthesis pathway specific Gene promoter Bifluorescein reporter vectors

1. PCR amplifies the promoter of artemisinin synthesizing path specific gene. According to the sequence information of a promoter (GenBank: DQ448297.1) of an ADS gene of artemisia apiacea in an NCBI database, designing an ADS gene promoter amplification specific primer, wherein the forward and reverse specific primers respectively contain Kpn I enzyme cutting sites and Pst I enzyme cutting sites, and the primer sequences are as follows:

ProADS F 5’-ggtaccACCGGGGACCTCTAGAGATC-3’,

ProADS R 5’-ctgcagGATTTTACAAACTTTGAA-3’。

similarly, based on the sequence information of the promoter (GenBank: FJ870128.1) of the Artemisia annua CYP71AV1 gene in NCBI database, CYP71AV1 promoter specific primers containing Kpn I and Pst I restriction enzyme sites are designed, and the primer sequences are as follows:

ProCYP F 5’-ggtaccATGGGTCAATTTCGGGTTG-3’,

ProCYP R 5’-ctgcagTGCTTTTAGTATACTCTTC-3’；

according to the sequence information of a promoter (GenBank: KC118524.1) of an artemisia apiacea DBR2 gene in an NCBI database, specific primers of a DBR2 promoter respectively containing Kpn I enzyme cutting sites and Pst I enzyme cutting sites are designed, and the sequences of the primers are as follows:

ProDBR2 F 5’-ggtaccAAGATGAGATAGGGAACTAAC-3’,

ProDBR2 R 5’-ctgcagTATTGAGTTTGATGTTGACC-3’；

according to the sequence information of the promoter (GenBank: KC118522.1) of the Artemisia apiacea ALDH1 gene in NCBI database, ALDH1 promoter specific primers respectively containing Kpn I and Pst I enzyme cutting sites are designed, and the primer sequences are as follows:

ProALDH1 F 5’-ggtaccATGAACCATTAGAAGGGAAGG-3’,

ProALDH1 R 5’-ctgcagCTTTGTTTTTTATGAAA-3’；

and (3) performing PCR amplification on the 4 promoter fragments by using the genomic DNA of the artemisia apiacea as a template, and recovering and purifying.

2. The PCR product of the promoter is connected into a double-fluorescein report vector. And carrying out double enzyme digestion on the PCR product by using Kpn I and Pst I, recovering enzyme digestion fragments, connecting the fragments obtained after 4 enzyme digestion recoveries into pGreenII0800-LUC vector fragments recovered after double enzyme digestion by using Kpn I and Pst I, and constructing plant double-fluorescein detection report vectors pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH 1.

Example 4 detection of Gene and promoter activation by transient transformation of tobacco

1. The method comprises the steps of obtaining an agrobacterium engineering strain, and transforming a pEarlygate104 empty vector, a pEarlygate104-AaWRKY2 expression vector and 4 promoter double-fluorescence detection report vectors into an agrobacterium tumefaciens GV3101 strain through a freeze-thaw method to obtain the agrobacterium engineering strain containing the empty vector, the agrobacterium engineering strain containing the AaWRKY2 gene and 4 agrobacterium engineering strains containing the promoters.

2. And (3) carrying out enlarged culture and treatment on the agrobacterium engineering strain. The 6 Agrobacterium engineering strains were grown up in LB medium containing three antibiotics 50mg/L rifampicin +20mg/L gentamicin +50mg/L kanamycin (5mL), at 28 ℃ at 220 rpm overnight. And measuring the concentration of the bacterial liquid on the second day, and stopping culturing when the concentration of the agrobacterium liquid reaches an OD600 value of about 2-2.5 OD. The Agrobacterium was collected by centrifugation and then treated with 10mM MgCl₂The solution was resuspended in bacteria, and the OD of the resuspended bacterial solution was adjusted to 0.6. Acetosyringone was added to the resuspension broth at a concentration of 200 mM. And standing the re-suspended agrobacterium liquid for 3 hours.

3. The injection infection method is used for instantaneously transforming the tobacco. Mixing the agrobacterium engineering strain containing the empty vector after standing treatment and 4 agrobacterium engineering strains containing promoters according to the concentration ratio of 3:1 respectively to be used as a control group; the agrobacterium engineering strain containing the expression vector of the target gene AaWRKY2 and 4 agrobacterium engineering strains containing promoters are mixed according to the concentration ratio of 3:1 respectively to be used as an experimental group. The mixed agrobacterium was injected into tobacco leaves growing for about 5 weeks by a 1ml syringe, cultured in the dark for 1st, and then cultured in the light.

4. Dual-Luciferase assay. Taking tobacco leaves cultured for 2 days after injection by using a circular puncher with the diameter of 1.0cm, quickly freezing the tobacco leaves by using liquid nitrogen, and grinding the tobacco leaves into powder; the fluorescence intensity was measured using the Promega Dual-Luciferase assay kit, according to the protocol of Promega corporation.

The AaWRKY2 gene can obviously activate the expression of 2 important structural gene promoters including ADS and DBR2 in an artemisinin biosynthesis pathway, compared with a control group, the AaWRKY2 can obviously activate and improve the expression activity of the ADS promoter to about 2.2 times of that of the control group, can improve the expression activity of the DBR2 promoter to about 2.5 times of that of the control group, and has no obvious activation capability on the CYP71AV1 promoter and the ALDH1 promoter. The invention provides powerful experimental evidence for further utilizing the over-expression of the gene in the sweet wormwood herb to further improve the content of the artemisinin in the sweet wormwood herb.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (8)

1. A sweet wormwood WRKY transcription factor is characterized by being named as AaWRKY2, and the nucleotide sequence of AaWRKY2 is shown as SEQ ID No. 1.

2. The Artemisia annua WRKY transcription factor as claimed in claim 1, wherein the amino acid sequence of AaWRKY2 is shown as SEQ ID NO. 2.

3. A recombinant expression vector is characterized in that the recombinant expression vector comprises an open reading frame sequence of a nucleotide sequence shown as SEQ ID NO.1, and the open reading frame sequence is shown as SEQ ID NO. 3.

4. The use of the Artemisia annua WRKY transcription factor as claimed in claim 1 or 2 for increasing artemisinin content.

5. The method for cloning the Artemisia annua WRKY transcription factor as claimed in claim 1, wherein the cloning method comprises the following steps:

step 1, extracting and purifying total RNA, namely extracting and purifying by adopting various general plant total RNA extraction kits to obtain total RNA of artemisia apiacea leaves;

step 2, reverse transcriptase is used for inverting the total RNA of the artemisia apiacea leaves into cDNA;

and 3, using the cDNA as a template, designing a gene specific primer, and amplifying by adopting a PCR method to obtain a PCR product, wherein the gene specific primer is as follows:

forward primer P1: 5'-AAGATGGAATATGGAAGAGG-3'

Reverse primer P2: 5' -ATCAAACAAAGAGTCCACAGAC-3;

and 4, recovering, purifying and sequencing the PCR product to obtain the nucleotide sequence shown as SEQ ID NO. 1.

6. The method of constructing a recombinant expression vector according to claim 3, comprising the steps of:

step 1, designing and amplifying AaWRKY2 gene open reading frame sequence according to SEQ ID NO.1 sequence, wherein amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3', respectively;

step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR/D-TOPO vector;

and 3, constructing a pEarlygate104-AaWRKY2 plant expression vector containing the target gene by using the pENTR/D-TOPO vector connected with the AaWRKY2 gene and a pEarlygate104 plant expression vector in an LR reaction mode.

7. A method for rapidly detecting the biological function of the AaWRKY2 transcription factor of claim 2, comprising the steps of:

step 1, designing and amplifying AaWRKY2 gene open reading frame sequence according to SEQ ID NO.1 sequence, wherein amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3', respectively;

step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR/D-TOPO vector;

step 3, constructing a pEarlygate104-AaWRKY2 plant expression vector containing a target gene by connecting the pENTR/D-TOPO vector connected with the AaWRKY2 gene with a pEarlygate104 plant expression vector in an LR reaction mode;

step 4, connecting a promoter ProADS of a special ADS gene in an artemisinin synthesis approach in artemisia apiacea into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII 0800-ProADS;

step 5, connecting a promoter ProCYP71AV1 of a specific CYP71AV1 gene in an artemisinin synthesis way in the sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProCYP71AV 1;

step 6, connecting a promoter ProDBR2 of a specific DBR2 gene in an artemisinin synthesis way in sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProDBR 2;

step 7, connecting a promoter ProALDH1 of a specific ALDH1 gene in an artemisinin synthesis way in sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProALDH 1;

step 8, respectively transforming the pEarlygate104-AaWRKY2 plant expression vector and the plant double-fluorescein detection report vector pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH1 into agrobacterium to obtain agrobacterium engineering strains containing target vectors;

step 9, mixing the agrobacterium engineering strain containing the pEarlygate104-AaWRKY2 plant expression vector and the agrobacterium engineering strain containing the plant double-fluorescein detection report vector, and injecting the mixture into tobacco leaves growing for 5 weeks in an injection infection mode;

step 10, taking the tobacco leaves which are cultured for 2 days after injection, quickly freezing the tobacco leaves by using liquid nitrogen, and grinding the tobacco leaves into powder;

and step 11, detecting the fluorescence intensity by adopting a Promega-Dual-Luciferase detection kit, and determining the activation effect of the AaWRKY2 gene and the specific gene promoter of the artemisinin synthesis pathway.

8. The method according to claim 7, wherein in step 8, the Agrobacterium is the GV3101 strain; in the step 9, the mixing ratio of the agrobacterium engineering strain containing the pEarlygate104-AaWRKY2 plant expression vector to the agrobacterium engineering strain containing the plant double-fluorescein detection report vector is 3:1, mixing.

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