CN102776225A - Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene - Google Patents
Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene Download PDFInfo
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Abstract
The invention discloses a method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene. The method includes the steps of cloning WRKY type transcription factor AaWRKY1 gene in sweet wormwood; constructing a plant expression vector containing the AaWRKY1; introducing the AaWRKY1 gene into the sweet wormwood to regrow a plant under mediation of Agrobacterium Tumefaciens; performing PCR (polymerase chain reaction) to detect integration status of the foreign target gene AaWRKY1; measuring the content of artemisinin in the transgenic sweet wormwood by efficient liquid chromatography and an evaporative light scattering detector, and screening to obtain the plants of sweet wormwood with artemisinin content increased. The content of artemisinin in the transgenic sweet wormwood with the AaWRKY1 gene is increased to 24.59mg/g DW by the method and is 4.44 times of that of the non-transgenic sweet wormwood. The method provides new high-yield and stable drug sources for large-scale production of artemisinin.
Description
Technical field
The present invention relates to a kind of method of utilizing transgenic technology to improve artemislnin content in the sweet wormwood, particularly a kind of method of changeing artemislnin content in the AaWRKY1 gene raising sweet wormwood.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Artemisinin (artemisinin) is from the isolating a kind of sesquiterpene lactones compound that contains the peroxo bridge structure of its over-ground part; Be the medicine of the efficacious therapy malaria of generally acknowledging in the world at present, particularly have quick-acting and characteristics low toxicity for encephalic malaria and anti-chloroquine malaria.At present, the method for the efficacious therapy malaria of world health organisation recommendations is exactly Artemisinin conjoint therapy (ACTs).In addition, along with to the Artemisinin pharmacological research progressively deeply, scientist finds that Artemisinin and verivate thereof also have anti-inflammatory, schistosomicide, antitumor and immunoregulatory function.It is thus clear that Artemisinin is a kind of natural drug that has potentiality.
The main source of Artemisinin is the over-ground part extraction from the sweet wormwood plant at present; Yet the content of Artemisinin is very low in the sweet wormwood; Its average content makes the large-scale commercial applications production of this medicine be restricted at 0.01～1% of sweet wormwood leaf dry weight in different planting environments and the varieties of plant.Because the Artemisinin complex structure, the synthetic difficulty is big, yields poorly, and cost is high, does not have feasibility.Someone attempts producing Artemisinin with the method for tissue culture and cell engineering, yet Artemisinin content in callus is lower than 0.1% of dry weight, and the highest in bud also have only 0.16% of dry weight, and great majority research does not detect Artemisinin in root.Therefore it is not high to utilize tissue culture and cell engineering to produce the feasibility of Artemisinin yet.
Through the prior art literature search is found; Ma etc. 2009 have delivered the paper that is entitled as " Isolation and Characterization of AaWRKY1; an Artemisia annua Transcription Factor that Regulates the Amorpha-4; 11-diene Synthase Gene; a Key Gene of Artemisinin Biosynthesis " (" separation and the clone of the WRKY class transcription factor AaWRKY1 of regulation and control Artemisinin biosynthesizing key enzyme ADS ") in " Plant & Cell Physiology " (plant and stechiology); Report can combine with the promotor of ADS with the preliminary AaWRKY1 that verified of gel retardation assasy through the yeast list is assorted, and through making up the instantaneous commentaries on classics sweet wormwood of overexpression carrier, finds that the expression of the enzyme in the Artemisinin route of synthesis in the sweet wormwood has all increased.This explanation AaWRKY1 has important effect in Artemisinin synthetic.
Sweet wormwood WRKY class transcription factor AaWRKY1 can regulate and control first key enzyme ADS in the Artemisinin route of synthesis in the prior art, is the critical elements of Artemisinin metabolic engineering.Adopt genetic engineering means,, will break the biosynthetic speed limit bottleneck of Artemisinin, obtain the sweet wormwood plant of Artemisinin high yield, for the large-scale production Artemisinin provides a new way with AaWRKY1 gene transformation sweet wormwood.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of AaWRKY1 of commentaries on classics gene to improve the method for artemislnin content in the sweet wormwood.The present invention has set up the method for artemislnin content in the stable raising sweet wormwood, for the large-scale production of Artemisinin provides high yield, stable source new drugs.
The object of the invention is realized through following technical scheme:
A kind of method of changeing artemislnin content in the AaWRKY1 gene raising sweet wormwood comprises the steps:
Step 1 adopts gene clone method to obtain sweet wormwood WRKY class transcription factor AaWRKY1 gene;
Step 2 is linked to expression regulation sequence to said AaWRKY1 gene, makes up the plant expression vector that contains the AaWRKY1 gene;
Step 3 transforms agrobacterium tumefaciens with the said plant expression vector that contains the AaWRKY1 gene, obtains to be used to transform the agrobacterium tumefaciens bacterial strain of containing of sweet wormwood of said AaWRKY1 gene plant expression vector;
Step 4 utilizes the agrobacterium tumefaciens bacterial strain of said structure to transform sweet wormwood, obtains the transgene abrotanum plant through the integration external source goal gene AaWRKY1 gene of PCR detection;
Step 5 is carried out HPLC-ELSD to artemislnin content in the said transgene abrotanum and is measured, and screening obtains the transgene abrotanum plant that artemislnin content improves.
Preferably, in said step 4, said conversion may further comprise the steps: the preparatory cultivation of explant; The common cultivation of Agrobacterium and explant; The screening of resistance regeneration plant.
Preferably, said preparatory cultivation may further comprise the steps: seeds of southernwood soaks 20min with 20%NaClO again with 75% alcohol immersion 1min; Aseptic water washing 3～4 times blots surface-moisture with aseptic thieving paper, is inoculated in the MS solid medium of no hormone; 25 ℃ of illumination cultivation; Can obtain the sweet wormwood aseptic seedling, treat that seedling grows to 5cm after, clip aseptic seedling leaf explant is used for transforming.
Preferably; The said cultivation altogether may further comprise the steps: said leaf explant is forwarded in the common culture medium; Dropping contains the 1/2MS suspension of the agrobacterium tumefaciens of the said AaWRKY1 of the containing gene plant of activatory expression vector; Said explant is fully contacted with said suspension, and 28 ℃ of dark cultivations 3 days are contrast with the leaf explant that drips at the 1/2MS of the agrobacterium tumefaciens that does not have goal gene liquid nutrient medium suspension.
Preferably; Said screening may further comprise the steps: the said sweet wormwood explant of cultivating altogether 3 days is transferred on the germination screening culture medium in 25 ℃ of illumination cultivation; Per two all succeeding transfer culture once; Through can obtaining the Kan resistance bud of growing thickly behind 2～3 subcultures, said well-grown resistance bud of growing thickly is cut to change over to be cultured on the root media and taken root, can obtain Kan resistance regeneration sweet wormwood plant.
Preferably, in said step 4, said PCR detects and may further comprise the steps: the primer of the synthetic AaWRKY1 gene of design; Carry out DNA cloning; The positive strain that the purpose band is observed in ultraviolet ray down is to be the transgene abrotanum plant.
Preferably, in said step 5, said HPLC-ELSD measures the following condition that comprises: chromatographic column C-18 reverse phase silica gel post; Moving phase is methyl alcohol: water, and methyl alcohol: the volume ratio of water is 70:30,30 ℃ of column temperatures; Flow velocity 1.0mL/min, sample size are 20 μ L, 40 ℃ of light scattering detector drift tube temperatures; Scale-up factor is 7, nebulizer gas pressure 5bar.
The present invention clones the AaWRKY1 gene from sweet wormwood, make up the plant expression vector that contains the AaWRKY1 gene, with Agrobacterium tumefaciens mediated, the AaWRKY1 gene is imported the sweet wormwood and the plant that regenerates; PCR detects the integration situation of external source goal gene AaWRKY1, and HPLC-ELSD measures artemislnin content in the sweet wormwood, and screening obtains the transgene abrotanum plant that artemislnin content improves.
The artemislnin content that transgene abrotanum strain provided by the invention is significantly improves; The content that changes Artemisinin in the AaWRKY1 gene sweet wormwood can reach the 24.59mg/g of dry weight; Be 4.44 times of the common sweet wormwood of non-conversion, for the large-scale production of Artemisinin provides high yield, stable source new drugs.
Description of drawings
Fig. 1 is the content detection figure as a result of Artemisinin in the transgene abrotanum strain provided by the invention.
Embodiment
Below in conjunction with specific embodiment the present invention is elaborated.Following examples will help those skilled in the art further to understand the present invention, but not limit the present invention in any form.Should be pointed out that to those skilled in the art, under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement.These all belong to protection scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; For example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of sweet wormwood AaWRKY1 gene
1.1 the extraction of the total RNA of sweet wormwood genome
Get 100-200mg sweet wormwood young leaflet tablet, behind liquid nitrogen flash freezer, grind with mortar rapidly, adding fills 1mL TRlzol (TRlzol Reagents; GIBCOBRL is in 1.5mL Eppendorf pipe USA), fully after the vibration; In room temperature held 5min, add 200 μ L chloroforms, use forced oscillation 15sec; After room temperature is placed 2～3min, descend 12, the centrifugal 15min of 000rmp in 4 ℃; Supernatant (about 600 μ L) is sucked in the clean 1.5mL Eppendorf pipe, add isopyknic Virahol, put upside down mixing, behind the room temperature held 10min, descend 12, the centrifugal 10min of 000rmp in 4 ℃; Abandon supernatant, add lmL75% ethanol and clean, after the vibration, descend 7, the centrifugal 5min of 500rmp in 4 ℃; Be dissolved in behind drying at room temperature 10～15min in an amount of (30～40 μ L) RNAase-free water; Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
1.2 the clone of sweet wormwood AaWRKY1 gene
The total RNA of sweet wormwood genome that is obtained is obtained the first chain cDNA through the ThermoScript II reverse transcription; Encoding sequence (shown in the sequence in the sequence table 1) according to said sweet wormwood AaWRKY1 gene; Design amplifies the upstream and downstream primer of complete encoder block; And on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.With the described first chain cDNA is template, behind pcr amplification, checks order.Determined dna sequence is accomplished by the order-checking of Shanghai invitrogen company.Sequencing result shows; (GenBank is numbered: encoding sequence FJ390842) is at 552 for the sweet wormwood AaWRKY1 gene of being reported among sequence of being cloned (sequence 1 in the sequence table) and the GenBank; 576; 663 have three nonsense point mutation, but they are in full accord on aminoacid sequence.
Present embodiment adopts gene clone method from sweet wormwood, to obtain the WRKY class transcription factor AaWRKY1 of first key enzyme ADS of the correct regulation and control Artemisinin biosynthetic pathway of sequence.
Embodiment 2
The structure that contains the plant binary expression vector of AaWRKY1 gene
2.1 the structure of intermediate carrier pMD18T-AaWRKY1
(Takara Dalian) is primary element, makes up intermediate carrier pMD18T-AaWRKY1 to select the pMD18T-simple carrier for use.Particularly,, be connected on the pMD18T carrier, confirm the exactness of gene by the order-checking of Shanghai invitrogen company through ligase enzyme through introducing the full length gene of KpnI and SacI restriction enzyme site before and after the high-fidelity enzymatic amplification AaWRKY1 gene respectively.
2.2 the structure of plant expression vector FSN::p35S-AaWRKY1-NOS
With described FSN (the FSN expression vector gets by transforming on the pCAMBIA2300 expression vector) is expression vector, and AaWRKY1 gene among the embodiment 1 is connected into its corresponding restriction enzyme site position.Particularly, KpnI and SacI double digestion intermediate carrier pMD18T-AaWRKY1 and expression vector FSN.Reclaim AaWRKY1 gene fragment and the big fragment of FSN carrier, connect conversion, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
Present embodiment with sweet wormwood WRKY class transcription factor AaWRKY1 gene operability be connected in expression regulation sequence, form the plant expression vector that contains the AaWRKY1 gene, this expression vector can be used for improving through the metabolic engineering strategy content of Artemisinin in the sweet wormwood.
Embodiment 3
Agrobacterium tumefaciens mediated AaWRKY1 gene genetic transforms sweet wormwood and obtains the transgene abrotanum plant
3.1 contain the acquisition of AaWRKY1 gene double base plant expression vector agrobacterium tumefaciens engineering bacteria
Change the plant binary expression vector that contains the AaWRKY1 gene among the embodiment 2 over to agrobacterium tumefaciens (like EHA105, for there is the biomaterial of public sale in market, can buy from Australian CAMBIA company, strain number is Gambar1), the performing PCR of going forward side by side checking.
3.2 Agrobacterium tumefaciens mediated AaWRKY1 gene transformation sweet wormwood
(1) the preparatory cultivation of explant
Seeds of southernwood is with 75% alcohol immersion 1min; Soak 20min with 20%NaClO again, aseptic water washing 3～4 times blots surface-moisture with aseptic thieving paper; Be inoculated in MS (the Murashige and Skoog of no hormone; 1962) in the solid medium, 25 ℃, 16h/8h (light/dark) illumination cultivation can obtain the sweet wormwood aseptic seedling.After treating that seedling grows to about 5cm, clip aseptic seedling leaf explant is used for transforming.
(2) the common cultivation of Agrobacterium and explant
With described leaf explant; Forward in the common culture medium (1/2MS+AS100 μ mol/L); Dropping contains the 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria of the good said AaWRKY1 of the containing gene plant binary expression vector of activation, and explant is fully contacted with bacterium liquid, 28 ℃ of dark cultivations 3 days.Leaf explant to drip at the 1/2MS of the agrobacterium tumefaciens that does not have goal gene liquid nutrient medium suspension is contrast.
(3) screening of resistance regeneration plant
The described sweet wormwood explant of cultivating altogether 3 days is transferred to germination screening culture medium (MS+6-BA 0.5mg/L+NAA 0.05mg/L ten Kan 50mg/L+Cb 500mg/L) to be gone up in 25 ℃, 16h/8h illumination cultivation; Per two all succeeding transfer culture once, through obtaining the Kan resistance bud of growing thickly behind 2-3 subculture.Well-grown resistance bud of growing thickly is cut to change over to be cultured on the root media (1/2MS+Cb 125mg/L) and taken root, thereby obtain Kan resistance regeneration sweet wormwood plant.
3.3 the PCR of transgene abrotanum plant detects
According to goal gene place expression cassette p35S-AaWRKY1-NOS sequence p35S with AaWRKY1 designs forward primer respectively and reverse primer detects goal gene.The result shows, utilizes the PCR special primer that is designed, and can amplify the specific DNA fragment of about 1.4kb.And when being template, do not amplify any fragment with non-conversion sweet wormwood genomic dna.
Present embodiment transforms agrobacterium tumefaciens with described plant expression vector; Acquisition is used to transform the agrobacterium tumefaciens bacterial strain that contains AaWRKY1 gene plant expression vector of sweet wormwood; Utilize constructed agrobacterium tumefaciens bacterial strain to transform sweet wormwood, obtain the transgene abrotanum plant that detects through PCR.The acquisition of transgene abrotanum plant provides direct material for the sweet wormwood strain system that screening obtains higher artemislnin content.
Embodiment 4
Utilize HPLC-ELSD to measure artemislnin content in the transgene abrotanum
4.1HPLC-ELSD the preparation of condition and system suitability and standardized solution
HPLC: adopt water alliance2695 system, chromatographic column is C-18 reverse phase silica gel post (Symmetry Shield TM C18,5 μ m; 250 * 4.6mm, Waters), moving phase is that the volume ratio of Jia Chun ﹕ water Jia Chun ﹕ water is 70 ﹕ 30,30 ℃ of column temperatures, flow velocity 1.0mL/min; Sample size 10 μ L, sensitivity (AUFS=1.0), theoretical plate number is calculated by the Artemisinin peak and is not less than 2000.
ELSD: adopt water alliance2420 system, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor (gain) is 7, nebulizer gas pressure 5bar.
Precision takes by weighing blue or green high plain standard substance (Sigma company) 2.0mg and dissolves fully with 1mL methyl alcohol, obtains 2mg/mL Artemisinin standard solution, be stored in-20 ℃ subsequent use.
Moving phase is Jia Chun ﹕ water among the present invention, and when ratio was 70% ﹕ 30%, the RT of Artemisinin was 5.1min, and the peak type is good.Theoretical plate number is calculated by blue or green rhzomorph and is not less than 2000.
4.2 the making of typical curve
With said reference substance solution difference sample introduction 2 μ L under corresponding chromatographic condition, 4 μ L, 6 μ L, 8 μ L, 10 μ L record collection of illustrative plates and chromatographic parameter carry out regression analysis with peak area (Y) to standard substance content (X, μ g) respectively.Through research, Artemisinin presents good log-log linear relationship among the present invention in 4～20 μ g scopes.The log-log equation of linear regression of Artemisinin reference substance is Y=7.286836X+2.033682, R
2=0.997766.
4.3 the preparation of sample and the mensuration of artemislnin content
The leaching process of Artemisinin is based on reported method among the Van Nieuwerburgh et al. (2006): the sweet wormwood blade that takes a morsel fresh (1～2g fresh weight); In the 50mL test tube, it is immersed in and swayed in the 10mL chloroform 1 minute; Leach liquor poured into make chloroform volatilization fully in the new test tube; Get the 3mL absolute ethyl alcohol and fully dissolve extract, after 0.22 μ m filters the filtration of worry head, be used for HPLC and detect.Simultaneously, 60 ℃ of baking ovens are put in the blade collection behind the chloroform extraction dries, weigh (dry weight of calculating the sweet wormwood blade).
Adopt HPLC-ELSD to measure artemislnin content; The sample feeding volume is 20 μ L; Go out the artemislnin content (mg) in the sample according to the linear regression equation calculation of peak area substitution,, thereby calculate the content of Artemisinin in the sweet wormwood plant again divided by the artemisia leaf dry weight (g) of sample.
Change the AaWRKY1 gene in the present invention and significantly improve artemislnin content in the sweet wormwood.The content that changes Artemisinin in the AaWRKY1 gene sweet wormwood can reach the 24.59mg/g of dry weight, is 4.44 times of the common sweet wormwood of non-conversion.
Present embodiment adopts the HPLC-ELSD method to measure artemislnin content in the transgene abrotanum, and the metabolic engineering strategy of employing conversion AaWRKY1 gene has obtained the sweet wormwood plant of Artemisinin high yield, for the large-scale production Artemisinin provides a kind of Perfected process.
Claims (7)
1. one kind is changeed the method that the AaWRKY1 gene improves artemislnin content in the sweet wormwood, it is characterized in that, comprises the steps:
Step 1 adopts gene clone method to obtain sweet wormwood WRKY class transcription factor AaWRKY1 gene;
Step 2 is linked to expression regulation sequence to said AaWRKY1 gene, makes up the plant expression vector that contains the AaWRKY1 gene;
Step 3 transforms agrobacterium tumefaciens with the said plant expression vector that contains the AaWRKY1 gene, obtains to be used to transform the agrobacterium tumefaciens bacterial strain of containing of sweet wormwood of said AaWRKY1 gene plant expression vector;
Step 4 utilizes the agrobacterium tumefaciens bacterial strain of said structure to transform sweet wormwood, obtains the transgene abrotanum plant through the integration external source goal gene AaWRKY1 gene of PCR detection;
Step 5 is carried out HPLC-ELSD to artemislnin content in the said transgene abrotanum and is measured, and screening obtains the transgene abrotanum plant that artemislnin content improves.
2. commentaries on classics AaWRKY1 gene according to claim 1 improves the method for artemislnin content in the sweet wormwood, it is characterized in that in said step 4, said conversion may further comprise the steps: the preparatory cultivation of explant; The common cultivation of Agrobacterium and explant; The screening of resistance regeneration plant.
3. commentaries on classics AaWRKY1 gene according to claim 2 improves the method for artemislnin content in the sweet wormwood, and it is characterized in that said preparatory cultivation may further comprise the steps: it is 75% alcohol immersion 1min that seeds of southernwood uses volume(tric)fraction; Use massfraction to soak 20min again as 20%NaClO; Aseptic water washing 3～4 times blots surface-moisture with aseptic thieving paper, is inoculated in the MS solid medium of no hormone; 25 ℃ of illumination cultivation; Can obtain the sweet wormwood aseptic seedling, treat that seedling grows to 5cm after, clip aseptic seedling leaf explant is used for transforming.
4. commentaries on classics AaWRKY1 gene according to claim 2 improves the method for artemislnin content in the sweet wormwood; It is characterized in that; The said cultivation altogether may further comprise the steps: said leaf explant is forwarded in the common culture medium; Dropping contains the 1/2MS suspension of the agrobacterium tumefaciens of the said AaWRKY1 of the containing gene plant of activatory expression vector; Said explant is fully contacted with said suspension, and 28 ℃ of dark cultivations 3 days are contrast with the leaf explant that drips at the 1/2MS of the agrobacterium tumefaciens that does not have goal gene liquid nutrient medium suspension.
5. commentaries on classics AaWRKY1 gene according to claim 2 improves the method for artemislnin content in the sweet wormwood; It is characterized in that; Said screening may further comprise the steps: the said sweet wormwood explant of cultivating altogether 3 days is transferred on the germination screening culture medium in 25 ℃ of illumination cultivation; Per two all succeeding transfer culture once; Through can obtaining the Kan resistance bud of growing thickly behind 2～3 subcultures, said well-grown resistance bud of growing thickly is cut to change over to be cultured on the root media and taken root, can obtain Kan resistance regeneration sweet wormwood plant.
6. commentaries on classics AaWRKY1 gene according to claim 1 improves the method for artemislnin content in the sweet wormwood, it is characterized in that, in said step 4, said PCR detects and may further comprise the steps: the primer of the synthetic AaWRKY1 gene of design; Carry out DNA cloning; The positive strain that the purpose band is observed in ultraviolet ray down is to be the transgene abrotanum plant.
7. commentaries on classics AaWRKY1 gene according to claim 1 improves the method for artemislnin content in the sweet wormwood, it is characterized in that, in said step 5; Said HPLC-ELSD measures and comprises following condition: chromatographic column C-18 reverse phase silica gel post, moving phase are methyl alcohol: water, and methyl alcohol: the volume ratio of water is 70:30; 30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size are 20 μ L; 40 ℃ of light scattering detector drift tube temperatures, scale-up factor are 7, nebulizer gas pressure 5bar.
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| CN105924510A (en) * | 2016-05-06 | 2016-09-07 | 上海交通大学 | Artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1 and application |
| CN106086038A (en) * | 2016-08-17 | 2016-11-09 | 上海交通大学 | A kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence and cloning process and application |
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| CN104152463A (en) * | 2014-07-31 | 2014-11-19 | 上海交通大学 | Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof |
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