CN106047895B - Artemisia apiacea bZIP transcription factor coding sequence, cloning method and application - Google Patents
Artemisia apiacea bZIP transcription factor coding sequence, cloning method and application Download PDFInfo
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Abstract
The invention discloses a clone of a sweet wormwood AabZIP6 gene coding sequence and application thereof. Specifically comprises cloning of a gene AabZIP6, construction of a plant expression vector containing the gene, and activation of the gene on a specific gene promoter of an artemisinin biosynthesis pathway. The nucleotide sequence of the southernwood AabZIP6 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 AabZIP6 gene can activate the expression of the artemisinin biosynthesis pathway specific genes ADS and ALDH 1. The AabZIP6 gene can be applied to the quality improvement of the sweet wormwood and can improve the content of artemisinin in the sweet wormwood.
Description
Technical Field
the invention relates to the technical field of genetic engineering, in particular to an artemisia apiacea bZIP transcription factor coding sequence AabZIP6, a cloning method 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.
Currently, it has been reported in artemisia apiacea that transcription factors can effectively regulate the expression of specific genes of an artemisinin synthesis pathway, thereby regulating the biosynthesis of artemisinin, such as AaORA1 transcription factors, AabZIP1 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
The invention aims to provide a sweet wormwood AabZIP6 gene and a method for obtaining a nucleotide sequence thereof, and also provides a protein coding sequence of the gene and a method for quickly determining the biological function of the gene. The gene codes a bZIP transcription factor, and partial biological functions of the bZIP transcription factor are determined. Through a tobacco injection infection transient expression method and a bifluorescent reporter analysis, the gene can activate the expression of the artemisinin biosynthesis pathway specific gene promoter, and an effective candidate gene is provided for the improvement of artemisinin metabolic engineering.
The invention is realized by the following technical scheme:
the invention provides a sweet wormwood bZIP transcription factor coding sequence, which is marked as AabZIP6, and the nucleotide sequence of AabZIP6 is shown as SEQ ID NO. 1.
The invention also provides a sweet wormwood bZIP transcription factor coding sequence, and the amino acid sequence coded by AabZIP6 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 an AabZIP6 gene open reading frame sequence according to the sequence of SEQ ID NO.1, wherein amplification primers are as follows:
Forward primer P3: 5'-CACCATGTCGAGGCCACCTCGACTTCC-3'
reverse primer P4: 5'-GTTAATATGAAGCTTGCCCATGTC-3', respectively;
Step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR-TOPO vector;
And 3, constructing a pEearlygate104-AabZIP6 plant expression vector containing the target gene by using the pENTR-TOPO vector connected with the AabZIP6 gene and a pEearlygate104 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 artemisia apiacea bZIP transcription factor coding sequence AabZIP6 in improving the artemisinin content.
The invention also provides a cloning method of the sweet wormwood bZIP transcription factor coding sequence AabZIP6, 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'-GTAGGAATTTGGTCAACTATGTCG-3'
Reverse primer P2: 5'-GGGACGGATGGTATCTAAATGC-3', respectively;
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 coded by the AabZIP6, which comprises the following steps:
step 1, designing and amplifying an AabZIP6 gene open reading frame sequence according to the sequence of SEQ ID NO.1, wherein amplification primers are as follows:
Forward primer P3: 5'-CACCATGTCGAGGCCACCTCGACTTCC-3'
Reverse primer P4: 5'-GTTAATATGAAGCTTGCCCATGTC-3', respectively;
step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR-TOPO vector;
step 3, constructing a pEearlygate104-AabZIP6 plant expression vector containing a target gene by connecting the pENTR-TOPO vector connected with the AabZIP6 gene with a pEearlygate104 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 pEearlygate104-AabZIP6 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 pEearlygate104-AabZIP6 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 AabZIP6 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 pEearlygate104-AabZIP6 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 AabZIP6 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 AabZIP6 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 AabZIP6 gene significantly improves the expression activity of promoters of 2 genes, namely ADS and ALDH 1.
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 AabZIP6 Gene in 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. Using the extracted total RNA as a template (500ng), and carrying out reverse transcription to produce first-Strand cDNA according to the method of a reverse transcription Kit PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian connection); 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'-GTAGGAATTTGGTCAACTATGTCG-3'
Reverse primer P2: 5'-GGGACGGATGGTATCTAAATGC-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-AabZIP6 plasmid vector.
through the steps, the sequence of the AabZIP6 gene in the artemisia apiacea is shown in SEQ ID NO.1, and the protein coding sequence is deduced to be shown in SEQ ID NO. 2.
Example 2 construction of plant expression vector containing AabZIP6 Gene
1. Construction of intermediate vector pENTR-TOPO-AabZIP 6.
the AabZIP6 gene open reading frame sequence is designed and amplified according to the SEQ ID NO.1 sequence information, and the amplification primers are as follows:
Forward primer P3: 5'-CACCATGTCGAGGCCACCTCGACTTCC-3'
Reverse primer P4: 5'-GTTAATATGAAGCTTGCCCATGTC-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-AabZIP6 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-TOPO vector by a method of Gateway cloning technology, and the specific method is carried out according to the Specification of pENTR/D-TOPO cloning kit of Invitrogen company.
2. And constructing a plant expression vector containing the target gene. According to an LR clone II Enzyme kit of Invitrogen company, an LR clone II Enzyme kit is used for LR reaction between an intermediate vector pENTR-TOPO-AabZIP6 and a pEearlygate104 plant expression vector, a reaction system is prepared according to a kit instruction, the reaction system is placed at 25 ℃ in a metal bath for reaction for 3 hours, then escherichia coli DH5 alpha competence is transformed, positive clone PCR verification is carried out, and the pEearlygate104-AabZIP6 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. And (3) obtaining an agrobacterium engineering strain, namely transforming a pEearlygate104 empty vector, a pEearlygate104-AabZIP6 expression vector and 4 promoter double-fluorescence detection report vectors into the agrobacterium tumefaciens GV3101 strain by a freeze-thaw method to obtain the agrobacterium engineering strain containing the empty vector, the agrobacterium engineering strain containing the AabZIP6 gene and 4 agrobacterium engineering strains containing the promoters.
2. The 6 agrobacterium engineering strains are subjected to amplification culture (5mL) in LB culture solution containing three antibiotics of 50mg/L rifampicin, 20mg/L gentamicin and 50mg/L kanamycin at 28 ℃ for 220 r/min for overnight culture, the concentration of the bacteria liquid is measured on the second day, the culture is stopped when the concentration of the agrobacterium liquid reaches an OD600 value of about 2 OD-2.5 OD, the agrobacterium is centrifuged, the agrobacterium is collected, the bacteria is resuspended in 10mM MgCl 2 solution, the OD value of the resuspended bacteria liquid is adjusted to 0.6, acetosyringone is added into the resuspended bacteria liquid, the concentration of the acetosyringone is 200 mM., and the resuspended agrobacterium liquid is kept still 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 AabZIP6 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 AabZIP6 gene can obviously activate the expression of 2 important structural gene promoters, namely ADS and ALDH1, in an artemisinin biosynthesis pathway, compared with a control group, the AabZIP6 can obviously activate the expression capability of the ADS promoter, and the activity of the AabZIP6 gene is improved to about 3.4 times that of the control group; the promoter of ALDH1 has certain activation capability, and is improved to about 2.6 times of that of a control group. But has no obvious activation capability on the promoters of CYP71AV1 and DBR 2. 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 (3)
1. the application of the artemisia apiacea bZIP transcription factor coding sequence in improving the artemisinin content is characterized in that the coding sequence is marked as AabZIP6, and the nucleotide sequence of AabZIP6 is shown as SEQ ID No. 1.
2. a method for improving the expression activity of an artemisinin synthesis pathway gene promoter in artemisia apiacea by utilizing a nucleotide sequence of an artemisia apiacea bZIP transcription factor coding sequence is characterized in that the coding sequence is marked as AabZIP6, and the nucleotide sequence of AabZIP6 is shown as SEQ ID No. 1;
the 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'-GTAGGAATTTGGTCAACTATGTCG-3'
Reverse primer P2: 5' -GGGACGGATGGTATCTAAATGC-3;
Step 4, recovering, purifying and sequencing the PCR product to obtain a nucleotide sequence shown as SEQ ID NO. 1;
Step 5, designing and amplifying an AabZIP6 gene open reading frame sequence according to the sequence of SEQ ID NO.1, wherein amplification primers are as follows:
forward primer P3: 5'-CACCATGTCGAGGCCACCTCGACTTCC-3'
reverse primer P4: 5'-GTTAATATGAAGCTTGCCCATGTC-3', respectively;
Step 6, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR-TOPO vector;
Step 7, constructing a pEearlygate104-AabZIP6 plant expression vector containing a target gene by connecting the pENTR-TOPO vector connected with the AabZIP6 gene with a pEearlygate104 plant expression vector in an LR reaction mode;
Step 8, connecting a promoter ProADS of a special ADS gene in an artemisinin synthesis pathway in artemisia apiacea into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII 0800-ProADS;
Step 9, 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 10, 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 11, 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 12, respectively transforming the pEearlygate104-AabZIP6 plant expression vector and the 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 13, mixing the agrobacterium engineering strain containing the pEearlygate104-AabZIP6 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 14, 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 15, detecting the fluorescence intensity by adopting a Promega-Dual-Luciferase detection kit, and determining the activation effect of the AabZIP6 gene and the specific gene promoter of the artemisinin synthesis pathway.
3. The method according to claim 2, wherein in the step 12, the agrobacterium is agrobacterium GV3101 strain; in the step 13, the mixing ratio of the agrobacterium engineering strain containing the pEearlygate104-AabZIP6 plant expression vector to the agrobacterium engineering strain containing the plant double-fluorescein detection report vector is 3:1, mixing.
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