CN106148357A - A kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence and application - Google Patents
A kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence and application Download PDFInfo
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- CN106148357A CN106148357A CN201610727791.7A CN201610727791A CN106148357A CN 106148357 A CN106148357 A CN 106148357A CN 201610727791 A CN201610727791 A CN 201610727791A CN 106148357 A CN106148357 A CN 106148357A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Abstract
The invention discloses a kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence, this coded sequence is designated as AaGSW2, and its nucleotide sequence is as shown in SEQ ID NO:1, and its aminoacid sequence is as shown in SEQ ID NO:2.Coding WRKY class transcription factor AaGSW2 of the present invention, by regulating and controlling the expression of artemisinin synthesis key gene, thus improves the content of arteannuin.In the blade of the common Herba Artemisiae Annuae of non-transgenic, artemislnin content is 9mg/g DW, and the blade artemislnin content of the transgene abrotanum of overexpression AaGSW2 gene expression brings up to 21mg/g DW.This invention for providing high yield for the large-scale production of arteannuin, to stablize source new drugs significant.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence and answer
With.
Background technology
Herba Artemisiae Annuae (Artemisia annua L.) is the annual herb plant of Compositae artemisia.Arteannuin
(artemisinin) it is a kind of sesquiterpene lactones compound containing peroxide bridge structure separated from its aerial parts, is current
The medicine of the maximally effective treatment malaria generally acknowledged in the world, has quick-acting and low especially for encephalic malaria and anti-chlorine quinoline malaria
The feature of poison.At present, the method for the maximally effective treatment malaria of world health organisation recommendations is exactly arteannuin conjoint therapy
(ACTs).It addition, along with progressively going deep into arteannuin pharmacological research, it is anti-that scientist finds that arteannuin and derivant thereof also have
Inflammation, schistosomicide, antitumor and immunoregulatory function.Visible arteannuin is the natural drug of a kind of great potential.
The main source of arteannuin is to extract from the aerial parts of Herba Artemisiae Annuae plant at present, but the content of Artemisinin in Artemisia annuna
The lowest, in different planting environments and varieties of plant, its average content is at the 0.01-1% of Herba Artemisiae Annuae leaf dry weight so that this medicine
The large-scale commercial of thing produces and is restricted.Owing to arteannuin structure is complicated, synthetic difficulty is big, yields poorly, cost
High.Although there being people successfully to utilize culture propagation to produce artemisinin precursors material artelinic acid at present, also need artelinic acid artificial chemistry
Semi-synthetic to arteannuin, this research is still in the laboratory research and development primary stage.Also have tried to by tissue culture and cell engineering
Method produce arteannuin, but arteannuin in callus, content is less than the 0.1% of dry weight, the highest in bud also only have
The 0.16% of dry weight, and great majority research is not detected by arteannuin in root.Come hence with tissue culture and cell engineering
The feasibility producing arteannuin is the highest.
Summary of the invention
It is an object of the invention to overcome deficiency of the prior art, it is provided that a kind of Herba Artemisiae Annuae AaGSW2 albumen coded sequence,
This gene code WRKY class transcription factor (AaGSW2), it participates in the density of regulation and control Herba Artemisiae Annuae glandular hair；Utilize transgenic technology by green grass or young crops
Artemisia AaGSW2 transcription factor overexpression vector converts Herba Artemisiae Annuae with the trichome density of Effective Regulation Herba Artemisiae Annuae epidermis, thus can improve Herba Artemisiae Annuae
The content of element.Artemislnin content brings up to 21mg/g DW (Fig. 2) from the 9mg/g DW of non-transgenic Herba Artemisiae Annuae, this invention for for
The large-scale production of arteannuin provides high yield, to stablize source new drugs significant.
The present invention is to be realized by following technical scheme:
The invention provides a kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence, described coded sequence is designated as AaGSW2, institute
State the nucleotide sequence of AaGSW2 as shown in SEQ ID NO:1；Its aminoacid sequence is as shown in SEQ ID NO:2.
Present invention also offers a peptide species, its aminoacid sequence is as shown in SEQ ID NO:2.
Present invention also offers a kind of recombinant expression carrier, described recombinant expression carrier comprises as shown in SEQ ID NO:1
Nucleotide sequence.
Present invention also offers a kind of recombinant expressed transformant, described recombinant expressed transformant comprises such as SEQ ID NO:1
Shown nucleotide sequence.
Present invention also offers Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2 answering in improving artemislnin content
With.
Further, described application comprises the following steps:
Step 1, being connected on plant expression regulation sequence by Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2, structure contains
The plant expression vector of described Herba Artemisiae Annuae WRKY class transcription factor coded sequence；
Step 2, the described plant expression vector in step 1 is proceeded to Agrobacterium, described Agrobacterium is proceeded to Herba Artemisiae Annuae；
Step 3, pass through antibiotic-screening, it is thus achieved that turning containing described Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2
Change cell, regeneration of transgenic plant.
Further, in step 2 above, freeze-thaw method is used to proceed to.
Present invention also offers a kind of method improving content of artemisinin in sweet wormwood, described method comprises the steps:
Step 1, to Herba Artemisiae Annuae cultigen WRKY class Transcription factor analysis, from Herba Artemisiae Annuae cDNA library clone obtain Herba Artemisiae Annuae WRKY
Class transcription factor AaGSW2；
Step 2, AaGSW2 gene is operatively connectable to expression regulation sequence, is formed containing described AaGSW2 gene
Plant overexpression vector；
Step 3, by containing described AaGSW2 gene plant overexpression vector convert Agrobacterium tumefaciems, it is thus achieved that plant described in having
The Agrobacterium tumefaciens strain of thing overexpression vector；
Step 4, utilize described Agrobacterium tumefaciens strain to convert Herba Artemisiae Annuae, obtain resistance Seedling through hygromycin selection, then examine through PCR
Survey and be transgene abrotanum Seedling for positive plant；
Step 5, to obtain transgene abrotanum carry out trichome density analysis, it is thus achieved that the transgenic that trichome density significantly improves
Herba Artemisiae Annuae, and then obtain the Herba Artemisiae Annuae plant that artemislnin content improves.
Further, described Agrobacterium tumefaciems is EHA105.
Further, in step 4, the described transgene abrotanum plant through PCR detection refers to, separately designs synthesis
The detection primer of AaGSW2 gene, carries out DNA cloning, and it is blue or green that the positive strain of viewed under ultraviolet radiation to purpose band is transgenic
Artemisia plant, described trichome density detection refers under fluorescence microscope, and Herba Artemisiae Annuae secreting type glandular hair shows bright at a particular wavelength
Bright fluorescence, adds up its quantity and density after taking pictures again.
Wherein, artemislnin content in the transgene abrotanum obtained is carried out HPLC-ELSD mensuration.
The present invention clones AaGSW2 gene from Herba Artemisiae Annuae, builds the plant overexpression vector containing AaGSW2 gene, uses crown gall
Agriculture bacillus mediated, use leaf disk method that AaGSW2 gene overexpression vector is converted Herba Artemisiae Annuae；PCR detects external source genes of interest AaGSW2
Integration, by fluorescence microscope and statistics trichome density, it is thus achieved that transgenic that trichome density significantly improves is blue or green
Artemisia；HPLC ELSD detector (HPLC-ELSD) measures content of artemisinin in sweet wormwood, shows the green grass or young crops obtained
Artemislnin content in the transgene abrotanum that Artemisia epidermal gland gross density significantly improves also significantly improves.
In the present invention, can be selected for various carrier known in the art, such as commercially available carrier, including plasmid, cosmid etc..?
When producing the Herba Artemisiae Annuae AaGSW2 protein polypeptide of the present invention, can be operably coupled to Herba Artemisiae Annuae AaGSW2 albumen coded sequence express
Regulating and controlling sequence, thus form Herba Artemisiae Annuae AaGSW2 protein expression vector.
As used herein, " being operably coupled to " refers to that some part of such a situation, i.e. linear DNA molecule can shadow
Ring the activity of same other parts of linear DNA molecule.Such as, if signal peptide DNA as precursor expression and participates in dividing of polypeptide
Secrete, then signal peptide (secretion targeting sequencing) DNA is operably coupled to polypeptid DNA exactly；If promoter controls turning of sequence
Record, then it is to be operably coupled to coded sequence；If ribosome binding site is placed in when can make its position translated, that
It is to be operably coupled to coded sequence.Typically, " being operably coupled to " means adjacent, for secretion targeting sequencing then
Mean in reading frame adjacent.
Below with reference to accompanying drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technique effect.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 shows in preferred embodiment of the present invention the result of AaGSW2 expression in quantitative PCR detection Herba Artemisiae Annuae；
Fig. 2 shows the result using HPLC detection artemislnin content in preferred embodiment of the present invention；*, (T examines P < 0.01
Test).
Detailed description of the invention
The detailed description of the invention provided the present invention below in conjunction with the accompanying drawings elaborates.
Below embodiments of the invention are elaborated: the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following enforcement
Example.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook equimolecular gram
It is grand: the condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989),
Or according to the condition proposed by manufacturer.
Embodiment 1, the clone of Herba Artemisiae Annuae AaGSW2 gene
1. the extraction of Herba Artemisiae Annuae genome total serum IgE
Taking Herba Artemisiae Annuae leaf tissue, be placed in liquid nitrogen grinding, addition fills the 1.5mL Eppendorf (EP) of lysate and is centrifuged
After Guan Zhong, fully vibration, according to the description extracted total RNA of TIANGEN test kit.Total serum IgE is identified with denaturing formaldehyde gel electrophoresis
Quality, then measures rna content on spectrophotometer.
2. the clone of Herba Artemisiae Annuae AaGSW2 gene
With the total serum IgE that extracted as template, under the effect of PowerScript reverse transcription, synthesize cDNA；According to
The sequential design gene-specific primer (SEQ ID NO:3 and SEQ ID NO:4) of AaGSW2 gene, by PCR from total cDNA
Middle amplification AaGSW2 gene, and check order.
Pass through above-mentioned steps, it is thus achieved that the complete encoding sequence (SEQ ID NO:1) of this transcription factor deriving in Herba Artemisiae Annuae
Going out its albumen coded sequence (SEQ ID NO:2), wherein, start codon is ATG, and termination codon is TAG.
Embodiment 2, the structure of plant binary interference expression vector containing AaGSW2 gene
The impact grown for research AaGSW2 gene pairs Herba Artemisiae Annuae secreted glandular hair, builds the overexpression of AaGSW2 overexpression
Carrier PHB-AaGSW2.The structure of expression vector for convenience, introduces the restriction enzyme site of BamH1, reversely draws in forward primer
Introducing the restriction enzyme site of Sac1 in thing, primer is as shown in table 1；
The PCR primer of table 1 PHB-AaGSW2 vector construction
Herba Artemisiae Annuae AaGSW2 gene is operatively connectable to expression regulation sequence by the present embodiment, and this carrier can be used for passing through
Developmental regulation strategy regulates and controls the content of Artemisinin in Artemisia annuna.
Embodiment 3, Agrobacterium tumefaciens mediated AaGSW2 interference carrier genetic transformation Herba Artemisiae Annuae obtain transgene abrotanum plant
1. the acquisition of the Agrobacterium tumefaciems engineering bacteria containing AaGSW2 overexpression vector
By in embodiment 2 containing AaGSW2 plant binary overexpression vector use freeze-thaw method proceed to Agrobacterium tumefaciems (as
EHA105, has the biomaterial of public offering for market, can buy from CAMBIA company of Australia, and strain number is
Gambar 1), performing PCR of going forward side by side is verified.Result shows, the plant binary interference expression vector containing AaGSW2 is the most successfully building up to root
In cancer agrobacterium strains.
The most Agrobacterium tumefaciens mediated AaGSW2 gene transformation Herba Artemisiae Annuae
2.1. the preculture of outer implant
Seeds of southernwood is by 75% soak with ethanol 1min, then soaks 20min, aseptic water washing 3-4 time with 20%NaClO, uses
Surface moisture is blotted in aseptic absorbent paper, is inoculated in MS (Murashige and Skoog, the 1962) solid medium without hormone
In, 25 DEG C, 16h/8h (light/dark) illumination cultivation, Herba Artemisiae Annuae aseptic seedling can be obtained.After Seedling length to about 5cm, clip without
Vaccine leaf explant is used for converting.
2.2. the co-culturing of Agrobacterium and outer implant
By described leaf explant, forwarding to co-culture in culture medium (1/2MS+AS 100 μm ol/L), dropping is containing activation
The 1/2MS suspension of the good described Agrobacterium tumefaciems engineering bacteria containing AaGSW2 plant binary overexpression vector, makes outer implant and bacterium
Liquid is fully contacted, 28 DEG C of light culture 3d.Hang at the 1/2MS fluid medium of the Agrobacterium tumefaciems without genes of interest with dropping
The leaf explant of liquid is comparison.
2.3. the screening of resistance regeneration plant
Outer for the described Herba Artemisiae Annuae co-culturing 3d implant is transferred to germination screening culture medium (MS+6-BA 0.5mg/L+NAA
0.05mg/L+Hyg 50mg/L+Cb 500mg/L) in 25 DEG C, 16h/8h illumination cultivation, successive transfer culture is once every two weeks, warp
Hyg resistance Multiple Buds can be obtained after crossing 2-3 subculture.Well-grown resistance Multiple Buds is cut and proceeds to root media
(1/2MS+Cb 125mg/L) upper cultivation is to taking root, thus obtains Hyg resistance regeneration Herba Artemisiae Annuae plant.
3. the PCR detection of transgene abrotanum plant
35S promoter region and AaGSW2 according to expression cassette upstream, genes of interest place separately design forward primer design
With reverse primer, genes of interest is detected.Result shows, the PCR special primer designed by utilization, can amplify specific DNA
Fragment.And during with non-transformed Herba Artemisiae Annuae genomic DNA for template, do not amplify any fragment.
Described plant expression vector is converted Agrobacterium tumefaciems by the present embodiment, it is thus achieved that for convert Herba Artemisiae Annuae containing AaGSW2
The Agrobacterium tumefaciens strain of plant binary overexpression vector, the Agrobacterium tumefaciens strain constructed by utilization converts Herba Artemisiae Annuae, it is thus achieved that warp
The transgene abrotanum plant of PCR detection.Obtaining as screening the Herba Artemisiae Annuae strain obtaining higher artemislnin content of transgene abrotanum plant
System provides direct material.
Embodiment 4, HPLC-ELSD is utilized to measure artemislnin content in transgene abrotanum
1.HPLC-ELSD condition and system suitability and the preparation of standard solution
HPLC: using water alliance 2695 system, chromatographic column is C-18 reverse phase silica gel post
(SymmetryShieldTM C18,5 μm, 250 × 4.6mm, Waters), flowing is methanol mutually: water, methanol: the volume ratio of water
For 70:30, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical cam curve presses Herba Artemisiae Annuae
Element peak calculates and is not less than 2000.
ELSD: use water alliance 2420 system, evaporative light scattering detector drift tube temperature 40 DEG C, amplify
Coefficient (gain) is 7, nebulizer gas pressure 5bar；
Precision weighs arteannuin standard substance (Sigma company) 2.0mg 1mL methanol and is completely dissolved, and obtains 2mg/mL Herba Artemisiae Annuae
Element standard solution, be stored in-20 DEG C standby.
The present invention flow mutually for methanol (methanol): water, when ratio is 70%:30%, the retention time of arteannuin
For 5.1min, peak type is good.Theoretical cam curve is calculated by arteannuin and is not less than 2000.
2. the making of standard curve
Described reference substance solution difference sample introduction 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under corresponding chromatographic condition is recorded collection of illustrative plates
And chromatographic parameter, with peak area (Y), standard substance content (X, μ g) is carried out regression analysis respectively.By research, blue or green in the present invention
Artemisin presents good log-log linear relationship in 4-20 μ g range.The log-log equation of linear regression of Qinghaosu
For: Y=1.28e+000X+4.71e+000, R=0.979546.
3. the preparation of sample and the mensuration of artemislnin content
Upper Herba Artemisiae Annuae plant, neutralizes bottom and takes Herba Artemisiae Annuae blade fresh for 2g altogether, dry to constant weight in 45 DEG C of baking ovens.Then
From the branch dried, strike inferior lobe, clay into power.Weigh about 0.1g dry powder in 2mL Eppendorf pipe, add 2mL ethanol,
By 40W ultrasonic Treatment 30min, 5000rpm is centrifuged 10min, takes supernatant with 0.22 μm membrane filtration, i.e. can be used for HPLC-
ELSD measures the content of arteannuin.
Using HPLC-ELSD to measure artemislnin content, sample feeding volume is 20 μ l, substitutes into linear regression according to peak area
Equation for Calculating goes out the artemislnin content (mg) in sample, then artemisia leaf dry weight (g) divided by sample, thus calculates Herba Artemisiae Annuae plant
The content of middle arteannuin.
The transfer-gen plant turning AaGSW2 overexpression vector in the present invention has been remarkably improved content of artemisinin in sweet wormwood.
When non-transformed common Herba Artemisiae Annuae content is 9mg/g DW, the same time turns the content of AaGSW2 overexpression vector Artemisinin in Artemisia annuna and puts down
All reaching 21mg/g DW, its content is 2.3 times of non-transformed Herba Artemisiae Annuae content, as in figure 2 it is shown, turn in AaGSW2 overexpression Herba Artemisiae Annuae,
Using HPLC to detect arteannuin, result is three meansigma methodss repeated, and error line represents standard deviation.Statistical analysis t-test examines
Test (*, P < 0.01).
The present embodiment uses HPLC-ELSD method to determine artemislnin content in transgene abrotanum, uses conversion AaGSW2 to surpass
Expression vector metabolic engineering strategies finds that the expression of AaGSW2 gene has obvious incidence relation, for profit with the content of arteannuin
The content carrying out process LAN research and then raising Artemisinin in Artemisia annuna with this gene provides strong experimental evidence.
The Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2 of the present invention is capable of improving artemislnin content in plant,
Described coded sequence is connected on plant expression regulation carrier, builds the plant expression vector containing described coded sequence；To express
Carrier proceeds to Agrobacterium, and Agrobacterium is proceeded to Herba Artemisiae Annuae；Pass through antibiotic-screening, it is thus achieved that the conversion containing described coded sequence is thin
Born of the same parents, regeneration of transgenic plant；In the transgene abrotanum that the present invention obtains, the content of arteannuin is significantly regulated and controled, non-transformed general
When logical Herba Artemisiae Annuae content is 9mg/g DW, the same time turns the content average out to 21mg/g of AaGSW2 overexpression vector Artemisinin in Artemisia annuna
DW, its content is 2.3 times of non-transformed Herba Artemisiae Annuae content.The invention provides one regulation and control content of artemisinin in sweet wormwood transcribe because of
Sub-coded sequence, for utilizing this coded sequence large-scale production arteannuin to lay a solid foundation.
The preferred embodiment of the present invention described in detail above.Should be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations according to the design of the present invention.Therefore, all technical staff in the art
The most on the basis of existing technology by the available technology of logical analysis, reasoning, or a limited experiment
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. a Herba Artemisiae Annuae WRKY class transcription factor coded sequence, it is characterised in that described coded sequence is designated as AaGSW2, described
The nucleotide sequence of AaGSW2 is as shown in SEQ ID NO:1.
2. a Herba Artemisiae Annuae WRKY class transcription factor coded sequence, it is characterised in that the aminoacid sequence of described AaGSW2 coding is such as
Shown in SEQ ID NO:2.
3. a peptide species, it is characterised in that the aminoacid sequence of described polypeptide is as shown in SEQ ID NO:2.
4. a recombinant expression carrier, it is characterised in that described recombinant expression carrier comprises the nucleoside as shown in SEQ ID NO:1
Acid sequence.
5. a recombinant expressed transformant, it is characterised in that described recombinant expressed transformant comprises as shown in SEQ ID NO:1
Nucleotide sequence.
Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2 the most according to claim 1 and 2 is improving artemislnin content
In application.
Application the most according to claim 6, it is characterised in that described application comprises the following steps:
Step 1, Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2 is connected on plant expression regulation sequence, builds containing described
The plant expression vector of Herba Artemisiae Annuae WRKY class transcription factor coded sequence；
Step 2, the described plant expression vector in step 1 is proceeded to Agrobacterium, described Agrobacterium is proceeded to Herba Artemisiae Annuae；
Step 3, pass through antibiotic-screening, it is thus achieved that the conversion containing described Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW2 is thin
Born of the same parents, regeneration of transgenic plant.
Application the most according to claim 7, it is characterised in that in described step 2, uses freeze-thaw method to proceed to.
9. the method improving content of artemisinin in sweet wormwood, it is characterised in that described method comprises the steps: step 1, right
Herba Artemisiae Annuae cultigen H-ZIP IV class Transcription factor analysis, from Herba Artemisiae Annuae cDNA library, clone obtains Herba Artemisiae Annuae WRKY class transcription factor
AaGSW2；
Step 2, AaGSW2 gene is operatively connectable to expression regulation sequence, forms the plant containing described AaGSW2 gene
Overexpression vector；
Step 3, by containing described AaGSW2 gene plant overexpression vector convert Agrobacterium tumefaciems, it is thus achieved that have described plant surpass
The Agrobacterium tumefaciens strain of expression vector；
Step 4, utilize described Agrobacterium tumefaciens strain convert Herba Artemisiae Annuae, obtain resistance Seedling through hygromycin selection, then be detected as through PCR
Positive plant is transgene abrotanum Seedling；
Step 5, the transgene abrotanum obtained is carried out trichome density analysis, it is thus achieved that transgenic that trichome density significantly improves is blue or green
Artemisia, and then obtain the Herba Artemisiae Annuae plant that artemislnin content improves.
Method the most according to claim 9, it is characterised in that described Agrobacterium tumefaciems is EHA105.
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| CN108315335A (en) * | 2018-04-13 | 2018-07-24 | 南京农业大学 | The drought-induced transcription factor PbrWRKY53 of pears and its application in terms of improving plant drought ability |
| CN109735550A (en) * | 2019-01-18 | 2019-05-10 | 上海交通大学 | A kind of nucleotide sequence and its application in raising plant secretion type trichome density |
| WO2020147113A1 (en) * | 2019-01-18 | 2020-07-23 | 上海交通大学 | Nucleotide sequence and application thereof in increasing secretion-type glandular trichome density of plant |
| CN112375767A (en) * | 2020-12-04 | 2021-02-19 | 衡阳师范学院 | Artemisia apiacea WRKY transcription factor AaWRKY4 gene and application |
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| CN108315335A (en) * | 2018-04-13 | 2018-07-24 | 南京农业大学 | The drought-induced transcription factor PbrWRKY53 of pears and its application in terms of improving plant drought ability |
| CN108315335B (en) * | 2018-04-13 | 2020-07-07 | 南京农业大学 | Pear drought-induced transcription factor PbrWRKY53 and application thereof in improving drought resistance of plants |
| CN109735550A (en) * | 2019-01-18 | 2019-05-10 | 上海交通大学 | A kind of nucleotide sequence and its application in raising plant secretion type trichome density |
| WO2020147113A1 (en) * | 2019-01-18 | 2020-07-23 | 上海交通大学 | Nucleotide sequence and application thereof in increasing secretion-type glandular trichome density of plant |
| CN109735550B (en) * | 2019-01-18 | 2022-11-11 | 上海交通大学 | Nucleotide sequence and application thereof in improving density of plant secretory glandular hairs |
| CN112375767A (en) * | 2020-12-04 | 2021-02-19 | 衡阳师范学院 | Artemisia apiacea WRKY transcription factor AaWRKY4 gene and application |
| CN112375767B (en) * | 2020-12-04 | 2022-12-02 | 衡阳师范学院 | Artemisia apiacea WRKY transcription factor AaWRKY4 gene and application thereof |
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