CN106086037B - A kind of sweet wormwood bZIP class transcription factor coded sequence and cloning process and application - Google Patents
A kind of sweet wormwood bZIP class transcription factor coded sequence and cloning process and application Download PDFInfo
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Abstract
The invention discloses a kind of clone of sweet wormwood AabZIP9 gene coded sequence and its applications.Specifically include the clone of Gene A abZIP9, the building of plant expression vector containing the gene, the peculiar gene promoter of gene pairs qinghaosu biosynthesis pathway activation.The nucleotide sequence of above-mentioned sweet wormwood AabZIP9 gene is as shown in SEQ ID NO.1, and encoded amino acid sequence is as shown in SEQ ID NO.2.The invention also discloses AabZIP9 genes to have the characteristic that qinghaosu biosynthesis pathway specific gene ADS and ALDH1 can be activated to express.AabZIP9 gene can be applied to sweet wormwood quality-improving in the present invention, and the content of Artemisinin in Artemisia annuna can be improved.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of sweet wormwood bZIP class transcription factor coded sequence
AabZIP9 and its cloning process and application.
Background technique
Metabolism is divided into nascent metabolism and secondary metabolism in plant, comes into being metabolite (such as carbohydrate, lipid and nucleic acid)
It is present in all plants, is necessary to plant maintains cell activities, and Secondary metabolites refer to plant
In small molecular organic compounds necessary to a major class non-plant growth and development, synthesis has kind, histoorgan with distribution
With growth and development specificity.For example, the specific medicament ingredient qinghaosu for the treatment of malaria is only in plant ginghao (Artemisia
Annua L.) plant surface secreting type glandular hairs in synthesize and store.In recent years, gradually deep with studying medicinal herb components
Enter, it is found that many medium-height grass the effective elements of the medicines are the tanshinone in Secondary metabolites, such as Radix Salviae Miltiorrhizae, in catharanthus roseus
Vinca alkaloids etc..
Its content in natural plants of most of Secondary metabolites is extremely low, and uses chemically synthesized method,
Process flow is complicated, cost is too high, and there are many more the biosynthesis pathway of Secondary metabolites is unintelligible, Wu Fashi
Existing chemistry is fully synthetic.Therefore, researcher starts to explore other methods for improving Secondary metabolites content.It considers
Secondary metabolites route of synthesis is complicated, and the gene dosage for participating in reaction is more, and is developed, many factors such as environment
It influences, it is hard to work when being modified with to individual gene in approach.And transcription factor can usually regulate and control some Plant Secondary Materials
The expression of multiple enzyme genes on metabolite biosynthesis pathway, to regulate and control the biosynthesis amount of the secondary metabolite.
Currently, discovery has been reported in sweet wormwood, transcription factor can be with the peculiar gene of Effective Regulation artemisinin synthesis approach
Expression, to regulate and control the biosynthesis of qinghaosu, such as AaORA1 transcription factor, AabZIP1 transcription factor, AaMYC2 transcription
Factor etc..Therefore, clone can regulate and control the transcription factor of the peculiar gene expression of qinghaosu biosynthesis pathway, for improveing and mentioning
The content of high Artemisinin in Artemisia annuna has great importance.
Summary of the invention
The purpose of the present invention is to provide a kind of sweet wormwood AabZIP9 gene and the methods for obtaining its nucleotide sequence, also mention
The albumen coded sequence of the gene has been supplied, and has provided the method that the biology of gene function quickly determines.Gene coding
One bZIP class transcription factor, determines its partial biological function.It will be containing the Agrobacterium for having purpose AabZIP9 gene
With the Agrobacterium of the report carrier containing qinghaosu biosynthesis pathway specific gene promoter, infected by tobacco injection instantaneous
The method of expression finds that the gene can activate qinghaosu biosynthesis pathway specific gene to open through double luciferase reporter analyses
The expression of mover provides effective candidate gene for the improvement of qinghaosu metabolic engineering.
The present invention is achieved by the following technical solutions:
The present invention provides a kind of sweet wormwood bZIP class transcription factor coded sequence, which is denoted as AabZIP9,
The nucleotide sequence of AabZIP9 is as shown in SEQ ID NO.1.
The present invention also provides a kind of sweet wormwood bZIP class transcription factor coded sequence, the amino acid sequences of AabZIP9 coding
As shown in SEQ ID NO.2.
The present invention also provides a kind of polypeptides, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.2.
The present invention also provides a kind of recombinant expression carrier, which includes as shown in SEQ ID NO.1
The open reading frame sequence of nucleotide sequence, the open reading frame sequence is as shown in SEQ ID NO.3.Its construction method includes
Following steps:
Step 1 expands AabZIP9 gene open reading frame sequence according to SEQ ID NO.1 sequence design, and amplimer is such as
Under:
Forward primer P3:5 '-CACCATGGCCGCAAACCCTGTCTGG-3 '
Reverse primer P4:5 '-AACGTTACGATGAGAATCCAAAGG-3 '；
Step 2 will connect pENTR-TOPO carrier after pcr amplification product recovery purifying；
Step 3 will connect pENTR-TOPO carrier and pEearlygate104 plant expression vector into AabZIP9 gene
The pEearlygate104-AabZIP9 plant expression vector containing target gene is constructed in such a way that LR reacts.
The present invention also provides a kind of recombinant expression transformants, which includes such as SEQ ID NO.1 institute
The open reading frame sequence of the nucleotide sequence shown, the open reading frame sequence is as shown in SEQ ID NO.3.
Further, the host strain of above-mentioned recombinant expression transformants is Agrobacterium.
The present invention also provides above-mentioned sweet wormwood bZIP class transcription factor coded sequence AabZIP9 in improving artemislnin content
Application.
The present invention also provides the cloning process of above-mentioned sweet wormwood bZIP class transcription factor coded sequence AabZIP9, the clones
Method the following steps are included:
The extraction and purification of step 1, total serum IgE is extracted and is purified using various general plant total RNA extraction reagent boxes and obtained
Obtain sweet wormwood blade total serum IgE；
Sweet wormwood blade total serum IgE is inverted to cDNA with reverse transcriptase by step 2；
Step 3, using above-mentioned cDNA as template, by design gene specific primer, using PCR method expand, obtain PCR
Product, gene specific primer are as follows:
Forward primer P1:5 '-TTTCAAACTGTGGTGGAATGGC-3 '
Reverse primer P2:5 '-CAATAAGAGGCTTAGGAGAGGG-3；
Step 4, the sequencing of PCR product recovery purifying, obtain the nucleotide sequence as shown in SEQ ID NO.1.
The present invention also provides a kind of method of the biological function of the amino acid sequence of quickly detection AabZIP9 coding,
Method includes the following steps:
Step 1 expands AabZIP9 gene open reading frame sequence according to SEQ ID NO.1 sequence design, and amplimer is such as
Under:
Forward primer P3:5 '-CACCATGGCCGCAAACCCTGTCTGG-3 '
Reverse primer P4:5 '-AACGTTACGATGAGAATCCAAAGG-3 '；
Step 2 will connect pENTR-TOPO carrier after pcr amplification product recovery purifying；
Step 3 will connect pENTR-TOPO carrier and pEearlygate104 plant expression vector into AabZIP9 gene
The pEearlygate104-AabZIP9 plant expression vector containing target gene is constructed in such a way that LR reacts；
Step 4, by the promoter ProADS of the peculiar ADS gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProADS of building plant；
Step 5, by the promoter ProCYP71AV1 of the peculiar CYP71AV1 gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProCYP71AV1 of building plant；
Step 6, by the promoter ProDBR2 of the peculiar DBR2 gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProDBR2 of building plant；
Step 7, by the promoter ProALDH1 of the peculiar ALDH1 gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProALDH1 of building plant；
Step 8, by the double fluorescein examining report carriers of pEearlygate104-AabZIP9 plant expression vector and plant
PGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and
PGreenII0800-ProALDH1 is transformed into Agrobacterium respectively, obtains the Agrobacterium tumefaciens attachment comprising purpose carrier；
Step 9, by the Agrobacterium tumefaciens attachment comprising pEearlygate104-AabZIP9 plant expression vector and comprising
After the Agrobacterium tumefaciens attachment mixing of the double fluorescein examining report carriers of plant, growth 5 is injected into such a way that injection is infected
In the tobacco leaf in week；
Step 10 takes the tobacco leaf cultivated after injecting 2 days, with grind into powder after liquid nitrogen flash freezer；
Step 11, using Promega-Dual-Luciferase detection kit fluorescence intensity, determine AabZIP9
The activation of gene and the peculiar gene promoter of artemisinin synthesis approach.
Further, in above-mentioned steps 8, host strain is Agrobacterium GV3101 bacterial strain；In above-mentioned steps 9, include
The Agrobacterium tumefaciens attachment of pEearlygate104-AabZIP9 plant expression vector and include the double fluorescein examining reports of plant
The Agrobacterium tumefaciens attachment mixed proportion of carrier is to mix by 3:1 concentration.
In the present invention, various carriers known in the art can be selected in clone's AabZIP9 gene, such as commercially available carrier, packet
Include plasmid, clay etc..AabZIP9 plant expression vector is constructed in the present invention, and various carriers known in the art also can be selected,
Such as commercially available pCAMBIA serial carrier；The double luciferase reporter carriers of building promoter in the present invention, also can be selected this field
The various other carriers known, such as carrier of commercially available Promega company；Agrobacterium involved in the present invention is Agrobacterium tumefaciems
(Agrobacterium tumefaciens) bacterial strain GV3101, the bacterial strain can open purchases from the market.
Below with reference to attached drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technical effect.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is shown in a preferred embodiment, and tobacco instantaneous conversion AabZIP9 gene significantly improves ADS and ALDH1
The expression activity of this 2 gene promoters.
Specific embodiment
Elaborate below to the embodiment of the present invention: the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as the molecules gram such as Sambrook
It is grand: condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989),
Or according to the normal condition proposed by manufacturer.
The clone of embodiment 1,1 sweet wormwood AabZIP9 gene of embodiment
1, sweet wormwood is cultivated in the controlled environment chamber, and growth conditions is photoperiod 18h/6h (bright/dark), and 25 DEG C；
2, the extraction of sweet wormwood blade total serum IgE.100 milligrams or so tender sweet wormwood leaf tissue materials are taken, is placed in liquid nitrogen and fills
It point is ground to powdered, according to the method for plant total RNA extraction reagent box (Tiangeng is biochemical, Beijing) specification, it is total to extract blade
RNA.3 μ L are taken to carry out the quality that agarose gel electrophoresis identifies total serum IgE the plant total serum IgE of acquisition, then in NanoDrop
The concentration of total serum IgE is measured on (Thermo Fisher, the U.S.) spectrophotometer.
3, the clone of gene.Using the total serum IgE of extraction as template (500ng), according to reverse transcription reagent box PrimeScript
The method of 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian) specification, reverse transcription produce the first chain cDNA；
By the special primer of design, PCR amplification is carried out by template of cDNA, specific primer sequences are as follows:
Forward primer P1:5 '-TTTCAAACTGTGGTGGAATGGC-3 '
Reverse primer P2:5 '-CAATAAGAGGCTTAGGAGAGGG-3
It is 50 μ L, reaction system that PCR, which reacts total volume, are as follows: 5 μ L 10 × KOD buffers, 5 μ L dNTPs, 4 μ L MgSO4,
1 μ L forward primer, 1 μ L reverse primer, 1 μ L cDNA template, 1 μ L KOD enzyme, ddH2O polishing to 50 μ L.
PCR amplification condition, 95 DEG C of 3min of initial denaturation, 35 circulations: 95 DEG C, 30sec；54 DEG C, 30sec；68 DEG C,
100sec, last 68 DEG C of extensions 5min.
PCR product is recovered after purification, it connects flat ends vector pLB (Tiangeng biochemistry Co., Ltd product) and is sequenced,
Obtain pLB-AabZIP9 plasmid vector.
Through the above steps, the sequence of AabZIP9 gene in sweet wormwood is obtained as shown in SEQ ID NO.1, and is derived
Its albumen coded sequence is as shown in SEQ ID NO.2.
The building of embodiment 2, plant expression vector comprising AabZIP9 gene
1, the building of intermediate vector pENTR-TOPO-AabZIP9.
Amplification AabZIP9 gene open reading frame sequence is designed according to SEQ ID NO.1 sequence information, amplimer is such as
Under:
Forward primer P3:5 '-CACCATGGCCGCAAACCCTGTCTGG-3 '
Reverse primer P4:5 '-AACGTTACGATGAGAATCCAAAGG-3 '
PENTR/D-TOPO carrier is purchased in Invitrogen company, is carried for the introduction of the said firm Gateway clone technology
Body adds tetra- bases of CACC according to the requirement of the product description before forward primer ATG base.With pLB-AabZIP9 matter
Grain is template, carries out PCR amplification with flat end high fidelity enzyme KOD, passes through Gateway clone technology after PCR product recovery purifying
Method be connected to pENTR-TOPO carrier, specific method is said according to Invitrogen company pENTR/D-TOPO Cloning Kit
Bright book carries out.
2, comprising the plant expression vector construction of target gene.According to Invitrogen company LR Clonase II
Intermediate vector pENTR-TOPO-AabZIP9 and pEearlygate104 plant expression vector will be carried out LR by Enzyme kit
Reaction, reaction system are prepared by kit specification, are placed in after 25 DEG C of metal baths react 3 hours and are converted bacillus coli DH 5 alpha sense
By state, positive colony PCR verifying is carried out, final acquisition includes the pEearlygate104-AabZIP9 plant table of target gene
Up to carrier.
The building of the double luciferase reporter carriers of 3. artemisinin synthesis approach specific gene promoter of embodiment
1, the promoter of PCR amplification artemisinin synthesis approach specific gene.According to sweet wormwood ADS gene in ncbi database
Promoter (GenBank:DQ448297.1) sequence information designs ADS gene promoter amplifying specific primer, positive and negative special primer
Contain Kpn I and Pst I restriction enzyme site respectively, primer sequence is as follows:
ProADS F 5’-ggtaccACCGGGGACCTCTAGAGATC-3’,
ProADS R 5’-ctgcagGATTTTACAAACTTTGAA-3’。
Likewise, according to promoter (GenBank:FJ870128.1) sequence of sweet wormwood CYP71AV1 gene in ncbi database
Column information designs the CYP71AV1 promoter special primer respectively containing Kpn I and Pst I restriction enzyme site, and primer sequence is as follows:
ProCYP F 5’-ggtaccATGGGTCAATTTCGGGTTG-3’,
ProCYP R 5'-ctgcagTGCTTTTAGTATACTCTTC-3'；
According to promoter (GenBank:KC118524.1) sequence information of sweet wormwood DBR2 gene in ncbi database, design
DBR2 promoter special primer respectively containing Kpn I and Pst I restriction enzyme site, primer sequence are as follows:
ProDBR2 F 5’-ggtaccAAGATGAGATAGGGAACTAAC-3’,
ProDBR2 R 5'-ctgcagTATTGAGTTTGATGTTGACC-3'；
According to promoter (GenBank:KC118522.1) sequence information of sweet wormwood ALDH1 gene in ncbi database, if
The ALDH1 promoter special primer respectively containing Kpn I and Pst I restriction enzyme site is counted, primer sequence is as follows:
ProALDH1 F 5’-ggtaccATGAACCATTAGAAGGGAAGG-3’,
ProALDH1 R 5'-ctgcagCTTTGTTTTTTATGAAA-3'；
Using sweet wormwood genomic DNA as template, above-mentioned 4 promoter fragments of PCR amplification, recovery purifying.
2. promoter PCR product is connected into double luciferase reporter carriers.By the above-mentioned PCR product bis- enzymes of Kpn I and Pst I
It cuts, recycles endonuclease bamhi, 4 digestion segments after the recovery are connected into recycling after Kpn I and Pst I double digestion
PGreenII0800-LUC carrier segments, the double fluorescein examining report carrier pGreenII0800-ProADS of building plant,
PGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH1.
4. tobacco instantaneous conversion of embodiment detects gene and promoter activation
1, the acquisition of Agrobacterium tumefaciens attachment, by pEearlygate104 empty carrier, pEearlygate104-AabZIP9
Expression vector and the double luciferase reporter assay carriers of 4 promoters are transformed into Agrobacterium tumefaciems GV3101 bacterial strain by freeze-thaw method,
Acquisition includes the Agrobacterium tumefaciens attachment of empty carrier, include the Agrobacterium tumefaciens attachment of AabZIP9 gene and 4 include
The Agrobacterium tumefaciens attachment of promoter.
2, the expansion culture of Agrobacterium tumefaciens attachment and processing.Above-mentioned 6 Agrobacterium tumefaciens attachments are being included into 50mg/L
Expand in the LB culture solution of rifampin+20mg/L gentamicin+50mg/L three kinds of antibiotic of kanamycins and cultivates (5mL), 28 DEG C,
220 revs/min, overnight incubation.Second day measurement bacterial concentration, when Agrobacterium bacterial concentration reach OD600 value 2OD~
The rear stopping culture of 2.5OD or so.Agrobacterium is collected in centrifugation, then uses 10mM concentration MgCl2Solution resuspension thallus, adjustment weight
Suspension bacteria liquid OD value is 0.6.Acetosyringone, concentration 200mM are added into resuspension bacterium solution.By above-mentioned resuspension agriculture bar
Bacterium bacterium solution stands 3 hours.
3, infestation method instantaneous conversion tobacco is injected.By after above-mentioned stewing process include empty carrier Agrobacterium engineering bacteria
Strain is mixed by 3:1 concentration ratio respectively with the Agrobacterium tumefaciens attachment that 4 include promoter, as a control group；To include
The Agrobacterium tumefaciens attachment of target gene AabZIP9 expression vector also divides with the Agrobacterium tumefaciens attachment that 4 include promoter
Not An 3:1 concentration ratio mixing, as experimental group.By the syringe of 1ml, mixed Agrobacterium bacterium night is injected into growth
In 5 weeks or so tobacco leafs, then goes under light within dark culturing 1 day and cultivate.
4, Dual-Luciferase is detected.The round punch for being 1.0cm with diameter takes the tobacco cultivated after injecting 2 days
Blade, with grind into powder after liquid nitrogen flash freezer；It is strong using Promega Dual-Luciferase detection kit detection fluorescence
Degree is operated by the method for Promega company specification.
AabZIP9 gene can significantly activate this 2 weights of ADS and ALDH1 in qinghaosu biosynthesis pathway in the present invention
The expression for the structural gene promoter wanted, compared with the control group, AabZIP9 can significantly activate the ability to express of this ADS promoter,
Improve its active 5.4 times or so to control group；Also there is certain activation capability to ALDH1 promoter, improves to the 1.6 of control group
Times or so.And to the promoter of CYP71AV1 and DBR2 without significant activation capability.It is through the invention further to utilize the gene
The content of overexpression and then raising Artemisinin in Artemisia annuna provides strong experimental evidence in sweet wormwood.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (3)
1. a kind of sweet wormwood bZIP class transcription factor coded sequence is improving the application in artemislnin content, which is characterized in that described
Coded sequence is denoted as AabZIP9, and the nucleotide sequence of the AabZIP9 is as shown in SEQ ID NO.1.
2. a kind of improve Artemisinin in Artemisia annuna route of synthesis gene promoter using sweet wormwood bZIP class transcription factor coded sequence
The method of expression activity, which is characterized in that the coded sequence is denoted as AabZIP9, and the nucleotide sequence of the AabZIP9 is such as
Shown in SEQ ID NO.1；It the described method comprises the following steps:
The extraction and purification of step 1, total serum IgE is extracted using various general plant total RNA extraction reagent boxes and purifies acquisition blueness
Mugwort piece total serum IgE；
The sweet wormwood blade total serum IgE is inverted to cDNA with reverse transcriptase by step 2；
Step 3, using the cDNA as template, by design gene specific primer, using PCR method expand, obtain PCR produce
Object, the gene specific primer are as follows:
Forward primer P1:5 '-TTTCAAACTGTGGTGGAATGGC-3 '
Reverse primer P2:5 '-CAATAAGAGGCTTAGGAGAGGG-3；
Step 4, the sequencing of PCR product recovery purifying, obtain the nucleotide sequence as shown in SEQ ID NO.1；
Step 5 expands AabZIP9 gene open reading frame sequence according to SEQ ID NO.1 sequence design, and amplimer is as follows:
Forward primer P3:5 '-CACCATGGCCGCAAACCCTGTCTGG-3 '
Reverse primer P4:5 '-AACGTTACGATGAGAATCCAAAGG-3 '；
Step 6 will connect pENTR-TOPO carrier after pcr amplification product recovery purifying；
Step 7 passes through the pENTR-TOPO carrier and pEearlygate104 plant expression vector that connect into AabZIP9 gene
The mode of LR reaction constructs the pEearlygate104-AabZIP9 plant expression vector containing target gene；
Step 8 connects the promoter ProADS of the peculiar ADS gene of Artemisinin in Artemisia annuna route of synthesis into pGreenII0800-
LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProADS of building plant；
Step 9, by the promoter ProCYP71AV1 of the peculiar CYP71AV1 gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProCYP71AV1 of building plant；
Step 10, by the promoter ProDBR2 of the peculiar DBR2 gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProDBR2 of building plant；
Step 11, by the promoter ProALDH1 of the peculiar ALDH1 gene of Artemisinin in Artemisia annuna route of synthesis connect into
PGreenII0800-LUC carrier, the double fluorescein examining report carrier pGreenII0800-ProALDH1 of building plant；
Step 12, by the double fluorescein examining reports of the pEearlygate104-AabZIP9 plant expression vector and the plant
Carrier pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and
PGreenII0800-ProALDH1 is transformed into Agrobacterium respectively, obtains the Agrobacterium tumefaciens attachment comprising purpose carrier；
Step 13, by the Agrobacterium tumefaciens attachment comprising the pEearlygate104-AabZIP9 plant expression vector and comprising
After the Agrobacterium tumefaciens attachment mixing of the double fluorescein examining report carriers of the plant, life is injected into such a way that injection is infected
In long 5 weeks tobacco leafs；
Step 14 takes the tobacco leaf cultivated after injecting 2 days, with grind into powder after liquid nitrogen flash freezer；
Step 15, using Promega-Dual-Luciferase detection kit fluorescence intensity, determine AabZIP9 gene
With the activation of the peculiar gene promoter of artemisinin synthesis approach.
3. according to the method described in claim 2, it is characterized in that, the Agrobacterium is Agrobacterium in the step 12
GV3101 bacterial strain；In the step 13, the Agrobacterium work comprising the pEearlygate104-AabZIP9 plant expression vector
Journey bacterial strain and Agrobacterium tumefaciens attachment mixed proportion comprising the double fluorescein examining report carriers of the plant are to mix by 3:1 concentration.
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| CN108530525B (en) * | 2018-04-27 | 2021-03-30 | 上海交通大学 | Artemisia apiacea bZIP transcription factor AaABF3 and application thereof |
| CN110819636B (en) * | 2019-11-08 | 2021-09-17 | 浙江中医药大学 | SmbZIP1 gene and application thereof in increasing content of salvianolic acid in salvia miltiorrhiza |
| CN112646813B (en) * | 2020-12-24 | 2022-11-18 | 上海交通大学 | Isolated nucleic acid molecules and uses thereof |
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2016
- 2016-08-17 CN CN201610682249.4A patent/CN106086037B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| A Basic Leucine Zipper Transcription Factor,AabZIP1, Connects Abscisic Acid Signaling with Artemisinin Biosynthesis in Artemisia annua;Fangyuan Zhang等;《Molecular Plant》;20150131;第165页右栏第1段和Supplement Data 1、摘要、第164页右栏第1-2段、第172页右栏第2-3段、第172页右栏第5段至第173页左栏第1段 * |
| Sequence：EY053814.1;ENA;《ENA》;20071111;序列 * |
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