CN102676578B - Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer - Google Patents
Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer Download PDFInfo
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Abstract
The invention provides a method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer in the field of biotechnology. The method includes the steps: cloning genes of artemisinic aldehyde delta 11(13) DBR2 from the sweet wormwood, constructing a plant expression vector containing the DBR2 genes, using agrobacterium tumefaciens for mediation to transfer the DBR2 genes into the sweet wormwood so that plants regenerate, using PCR (polymerase chain reaction) to detect integration conditions of the exogenous targeted DBR2 genes, determining the artemisinin content in the transgenic sweet wormwood by means of HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector), and screening the transgenic sweet wormwood plants with the improved artemisinin content. The artemisinin content in the obtained transgenic sweet wormwood can be remarkably increased and is 2.83 times maximally of that of a non-transgenic control plant, and accordingly the method for increasing the artemisinin content in the sweet wormwood lays a foundation for large-scale artemisinin production by the aid of the transgenic sweet wormwood.
Description
Technical field
What the present invention relates to is a kind of method of raising artemislnin content of biological technical field, particularly a kind ofly turns the method that DBR2 gene improves content of artemisinin in sweet wormwood.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Artemisinin (artemisinin) is a kind of sesquiterpene lactones compound containing peroxide bridge structure be separated from its over-ground part, be the medicine of the most effectively treating malaria of generally acknowledging in the world at present, particularly for encephalic malaria and anti-chloroquine malaria, there is quick-acting and feature that is low toxicity.At present, the method for the most effectively treating malaria of world health organisation recommendations is exactly Artemisinin conjoint therapy (ACTs).In addition, along with progressively going deep into Artemisinin pharmacological research, scientist finds that Artemisinin and derivative thereof also have anti-inflammatory, schistosomicide, antitumor and immunoregulatory function.Visible Artemisinin is a kind of natural drug of great potential.
The main source of current Artemisinin extracts from the over-ground part of sweet wormwood plant, but the content of Artemisinin in Artemisia annuna is very low, in different planting environment and varieties of plant, its average content is at the 0.01-1% of sweet wormwood leaf dry weight, the large-scale commercial of this medicine is produced and is restricted.Due to Artemisinin complex structure, synthetic difficulty is large, and yield poorly, cost is high, does not have feasibility.The method of someone trial tissue culture and cell engineering produces Artemisinin, however Artemisinin in callus, content is lower than 0.1% of dry weight, the highest in bud also only have 0.16% of dry weight, and great majority research does not detect Artemisinin in root.Therefore utilize tissue culture and cell engineering not high to the feasibility of producing Artemisinin yet.
Through finding prior art literature search, Waleerat Banyai etc. are in " Plant Cell Tissue and OrganCulture " (plant cell tissue's organ culture), within 2010,103 volume 255-265 pages have delivered the paper being entitled as " Overexpressionof farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth ofArtemisia annua L. " (" overexpression farnesyl pyrophosphate synthase gene can affect the content of Artemisinin in Artemisia annuna and the growth of sweet wormwood "), report by crossing most representation farnesyl pyrophosphate synthase (farnesyl pyrophosphatesynthase, FPS), the content of render transgenic Artemisinin in Artemisia annuna improves 2.5-3.6 doubly, but still only have about 1.3%.But, plant genetic engineering is improve the content of Artemisinin in Artemisia annuna to provide a feasible method.
Sweet wormwood aldehyde Δ 11 (13) reductase enzyme (artemisinic aldehyde Δ 11 (13) double bondreductase in prior art, DBR2) being a key enzyme in artemisinin synthesis approach, is the important target spot of Artemisinin metabolic engineering.Adopt genetic engineering means, transform sweet wormwood with key gene DBR2, the biosynthetic speed limit bottleneck of Artemisinin will be broken, obtain the sweet wormwood plant of Artemisinin high yield, for large-scale production Artemisinin provides a new way.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, provide a kind of and turn the method that DBR2 gene improves content of artemisinin in sweet wormwood.The key gene clone that the present invention relates to, vector construction, genetic transformation, Molecular Detection, Artemisinin extraction and assay are for the present invention, establishing the stable method improving content of artemisinin in sweet wormwood, establishing solid basis for utilizing sweet wormwood scale operation Artemisinin.
The present invention is achieved by the following technical solutions:
Turn the method that DBR2 gene improves content of artemisinin in sweet wormwood, comprise following concrete steps:
(1) gene clone method is adopted to obtain sweet wormwood key gene DBR2;
(2) described DBR2 gene is linked to expression regulation sequence, builds the plant expression vector containing DBR2 gene;
(3) by the described plant expression vector transform Agrobacterium tumefaciens containing DBR2 gene, the Agrobacterium tumefaciens strain containing described DBR2 gene plant expression vector is obtained;
(4) utilize the described Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector to transform sweet wormwood, obtain the transgene abrotanum plant of the integration external source goal gene DBR2 detected through PCR;
(5) carry out HPLC-ELSD mensuration to artemislnin content in described transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves.
Preferably, in described step (1), described gene clone method comprises the following steps: extract sweet wormwood genome total serum IgE, obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL reverse transcription, DNA sequence dna according to SEQ ID NO.1, design amplifies upstream primer and the downstream primer of complete encoder block, described upstream primer is the DNA sequence dna shown in SEQ ID NO.2, described downstream primer is the DNA sequence dna shown in SEQ ID NO.3, and restriction endonuclease sites is introduced respectively so that construction of expression vector on described upstream and downstream primer, with the first described chain cDNA for template, check order after pcr amplification.
Preferably, in described step (2), described structure comprises the following steps containing the plant expression vector of DBR2 gene: first build intermediate carrier pMDT18-DBR2, intermediate carrier pMDT18-DBR2 and expression vector FSN is cut again with XmaI and SacI enzyme, reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extract plasmid and do PCR detection and digestion verification.
Preferred further, described structure intermediate carrier pMDT18-DBR2 comprises following concrete steps: by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme.
Preferably, in described step (4), described conversion comprises the following steps: the preculture of explant; The Dual culture of Agrobacterium and explant; The screening of resistance regeneration plant.
Preferred further, described preculture comprises the following steps: seeds of southernwood 75% alcohol immersion 1min, 20min is soaked again with 20%NaClO, aseptic water washing 3-4 time, blots surface-moisture with aseptic thieving paper, is inoculated in the MS solid medium without hormone, 25 DEG C of illumination cultivation, can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming.
Preferred further, described Dual culture comprises the following steps: forwarded to by described leaf explant in Dual culture substratum, drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast.
Preferred further, described screening comprises the following steps: be transferred in germination screening culture medium the described Dual culture sweet wormwood explant of 3 days in 25 DEG C of illumination cultivation, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, described well-grown resistance Multiple Buds is cut proceed to root media is cultured to and take root, Kan resistance regeneration sweet wormwood plant can be obtained.
Preferably, in described step (4), described PCR detects and comprises the following steps: the primer of design and synthesis DBR2 gene; Carry out DNA cloning; Viewed under ultraviolet radiation, if object band is positive, then this strain is described transgene abrotanum plant.
Preferably, in described step (5), described HPLC-ELSD measures and comprises following condition: chromatographic column used is C-18 reverse phase silica gel post, moving phase select volume ratio be 70: 30 methyl alcohol: water, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 20 μ L, light scattering detector drift tube temperature 40 DEG C, scale-up factor is 7, nebulizer gas pressure 5bar.
The method turning DBR2 gene raising content of artemisinin in sweet wormwood of the present invention; adopt gene engineering method; key gene DBR2 is imported in sweet wormwood plant; obtain the transgene abrotanum strain that artemislnin content significantly improves; the content turning DBR2 gene Artemisinin in Artemisia annuna can reach the 22.6mg/g of dry weight; be 2.83 times of non-transformed common sweet wormwood (8mg/g dry weight), this invention provides high yield for the large-scale production for Artemisinin, to stablize source new drugs significant.
Accompanying drawing explanation
Fig. 1 is the content detection result figure of Artemisinin in sweet wormwood plant of the present invention.
Embodiment
Below embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
embodiment
Step one, the clone of sweet wormwood DBR2 gene
(1) extraction of sweet wormwood genome total serum IgE
Get 100-200mg sweet wormwood young leaflet tablet, after liquid nitrogen flash freezer, grind with mortar rapidly, add and fill 1mL TRlzol (TRlzol Reagents, GIBCOBRL, USA) 1.5mL Eppendorf pipe in, fully after vibration, 5min is put in ambient temperatare, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature places 2-3min, 12,000rmp centrifugal 15min at 4 DEG C; Supernatant liquor (about 600 μ L) is sucked in clean 1.5mL Eppendorf pipe, adds isopyknic Virahol, put upside down mixing, after ambient temperatare puts 10min, 12,000rmp centrifugal 10min at 4 DEG C; Abandon supernatant, add 1mL 75% ethanol purge, after vibration, 7,500rmp centrifugal 5min at 4 DEG C; Be dissolved in appropriate (30-40 μ L) RNAase-free water after drying at room temperature 10-15min; By denaturing formaldehyde gel electrophoresis qualification total serum IgE quality, then on spectrophotometer, measure rna content.
(2) clone of sweet wormwood DBR2 gene
Obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, according to the encoding sequence (DNA sequence dna shown in SEQ ID NO.1) of described sweet wormwood DBR2 gene, design amplifies upstream primer (DNA sequence dna shown in SEQ ID NO.2) and the downstream primer (DNA sequence dna shown in SEQ ID NO.3) of complete encoder block, and on upstream and downstream primer, introduce restriction endonuclease sites (this is determined by the carrier selected) respectively, so that construction of expression vector.With the first described chain cDNA for template, check order after pcr amplification.Determined dna sequence has been checked order by Shanghai Ying Jun biotechnology Services Co., Ltd.Sequencing result shows, the sequence of cloning is consistent with the encoding sequence (DNA sequence dna shown in SEQ ID NO.1) of the sweet wormwood DBR2 gene reported in GenBank.
The present embodiment adopts gene clone method from sweet wormwood, obtain the correct Artemisinin biosynthesizing key gene DBR2 of sequence, for providing an important key gene by turning DBR2 gene raising content of artemisinin in sweet wormwood.
Step 2, containing the structure of the plant binary expression vector of DBR2 gene
(1) structure of intermediate carrier pMDT18-DBR2
Select pMDT18 carrier (Takara, Dalian) for primary element, build intermediate carrier pMDT18-DBR2.Particularly, by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme, being checked order by Shanghai Ying Jun biotechnology Services Co., Ltd confirms the exactness of gene.
(2) containing the structure of the plant expression vector of DBR2 gene
For expression vector, above-mentioned DBR2 gene is connected into the restriction enzyme site position of its correspondence with described FSN (FSN expression vector obtains for transforming on pCAMBIA2300 expression vector and preserves).Particularly, XmaI and SacI double digestion intermediate carrier pMDT18-dbr2 and expression vector FSN.Reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extract plasmid and do PCR detection and digestion verification.
Artemisinin biosynthetic pathway key gene DBR2 is connected to expression regulation sequence by the present embodiment operably, and form the plant expression vector containing DBR2 gene, this expression vector can be used for the content being improved Artemisinin in Artemisia annuna by metabolic engineering strategies.
Step 3, containing the acquisition of DBR2 gene double base plant expression vector agrobacterium tumefaciens engineering bacteria
The above-mentioned plant binary expression vector containing DBR2 gene is proceeded to agrobacterium tumefaciens (as EHA105, for there is the biomaterial of public sale in market, can buy from Australian CAMBIA company, strain number is Gambar 1), and performing PCR of going forward side by side is verified.
Step 4, Agrobacterium tumefaciens mediated DBR2 gene transformation sweet wormwood
(1) preculture of explant
Seeds of southernwood 75% alcohol immersion 1min, 20min is soaked again with 20%NaClO, aseptic water washing 3-4 time, surface-moisture is blotted with aseptic thieving paper, be inoculated in MS (the Murashige and Skoog without hormone, 1962), in solid medium, 25 DEG C, 16h/8h (light/dark) illumination cultivation, can obtain sweet wormwood aseptic seedling.Grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming.
(2) Dual culture of Agrobacterium and explant
By described leaf explant, forward in Dual culture substratum (1/2MS+AS 100 μm of ol/L), drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted, 28 DEG C of light culture 3 days with bacterium liquid.With drip the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene leaf explant for contrast.
(3) screening of resistance regeneration plant
The described Dual culture sweet wormwood explant of 3 days is transferred in germination screening culture medium (MS+6-BA 0.5mg/L+NAA 0.05mg/L+Kan 50mg/L+Cb 500mg/L) in 25 DEG C, 16h/8h (light/dark) illumination cultivation, every two weeks succeeding transfer culture once, can obtain Kan resistance Multiple Buds after 2-3 subculture.Well-grown resistance Multiple Buds is cut proceed to root media (1/2MS+Cb 125mg/L) is cultured to and take root, thus obtain Kan resistance regeneration sweet wormwood plant.
Step 5, the PCR of transgene abrotanum plant detects
Forward primer is designed respectively and reverse primer detects goal gene according to goal gene place expression cassette p35s-dbr2-nos sequence p35s and dbr2.Result shows, the PCR special primer designed by utilization, can amplify the specific DNA fragment of 458bp.And with non-transformed sweet wormwood genomic dna for template time, do not amplify any fragment.
The present embodiment is by described plant expression vector transform Agrobacterium tumefaciens, obtain the Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector for transforming sweet wormwood, Agrobacterium tumefaciens strain constructed by utilization transforms sweet wormwood, obtains the transgene abrotanum plant detected through PCR.The acquisition of transgene abrotanum plant is screen the sweet wormwood strain obtaining higher artemislnin content to provide direct material.
Step 6, utilizes HPLC-ELSD to measure artemislnin content in transgene abrotanum
(1) preparation of HPLC-ELSD condition and system suitability and standardized solution
HPLC: adopt water alliance 2695 system, chromatographic column is C-18 reverse phase silica gel post (Symmetry ShieldTM C18,5 μm, 250 × 4.6mm, Waters), moving phase is methyl alcohol: water, methyl alcohol: the volume ratio of water is 70: 30, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical plate number calculates by Artemisinin peak and is not less than 2000.
ELSD: adopt water alliance 2420 system, light scattering detector drift tube temperature 40 DEG C, scale-up factor (gain) is 7, nebulizer gas pressure 5bar;
Precision takes Artemisinin standard substance (Sigma company) 2.0mg 1mL methyl alcohol and dissolves completely, obtains 2mg/mL Artemisinin standard solution, be stored in-20 DEG C for subsequent use.
In the present embodiment, moving phase is methyl alcohol: water, and when ratio is 70%: 30%, the retention time of Artemisinin is 5.1min, and peak type is good.Theoretical plate number calculates by Artemisinin and is not less than 2000.
(2) making of typical curve
By described 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 parameters, carry out regression analysis respectively with peak area (Y) to standard substance content (X, μ g).By research, in the present embodiment, Artemisinin presents good log-log linear relationship within the scope of 4-20 μ g.The log-log equation of linear regression of Qinghaosu is Y=5.404e+0000X+1.858e+0000, R
2=0.999184.
(3) preparation of sample and the mensuration of artemislnin content
The leaching process of Artemisinin is based on the method reported in Van Nieuwerburgh et al. (2006): the sweet wormwood blade (1-2g fresh weight) taking a morsel fresh, be immersed in 50ml test tube in 10ml chloroform and swayed 1 minute, leach liquor is poured in new test tube and make chloroform volatilize completely, get 3ml dehydrated alcohol and fully dissolve extract, filter after filter filters through 0.22 μm and detect for HPLC.Meanwhile, 60 DEG C of baking ovens are put in the blade collection after chloroform extraction dries, weigh (dry weight calculating sweet wormwood blade);
HPLC-ELSD is adopted to measure artemislnin content, sample feeding volume is 20 μ L, the artemislnin content (mg) in sample is gone out according to peak area substitution linear regression Equation for Calculating, again divided by artemisia leaf dry weight (g) of sample, thus calculate the content of Artemisinin in sweet wormwood plant.
Turn DBR2 gene in an embodiment and significantly improve content of artemisinin in sweet wormwood.The content turning DBR2 gene blue or green senior middle school Artemisinin can reach the 22.6mg/g (as shown in Figure 1) of dry weight, is 2.83 times of non-transformed common sweet wormwood (8mg/g dry weight).
The present embodiment adopts HPLC-ELSD method to determine artemislnin content in transgene abrotanum, adopts the metabolic engineering strategies transforming DBR2 gene to obtain the sweet wormwood plant of Artemisinin high yield, for large-scale production Artemisinin provides a kind of Perfected process.
Claims (7)
1. turn the method that DBR2 gene improves content of artemisinin in sweet wormwood, it is characterized in that, comprise following concrete steps:
(1) gene clone method is adopted to obtain sweet wormwood key gene DBR2;
(2) described DBR2 gene is linked to expression regulation sequence, builds the plant expression vector containing DBR2 gene;
(3) by the described plant expression vector transform Agrobacterium tumefaciens containing DBR2 gene, the Agrobacterium tumefaciens strain containing described DBR2 gene plant expression vector is obtained;
(4) utilize the described Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector to transform sweet wormwood, obtain the transgene abrotanum plant of the integration external source goal gene DBR2 detected through PCR;
(5) carry out HPLC-ELSD mensuration to artemislnin content in described transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves;
In described step (1), described gene clone method comprises the following steps: extract sweet wormwood genome total serum IgE, obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL reverse transcription, DNA sequence dna according to SEQ IDNO.1, design amplifies upstream primer and the downstream primer of complete encoder block, described upstream primer is the DNA sequence dna shown in SEQ ID NO.2, described downstream primer is the DNA sequence dna shown in SEQ ID NO.3, and restriction endonuclease sites is introduced respectively so that construction of expression vector on described upstream and downstream primer, with the first described chain cDNA for template, check order after pcr amplification,
In described step (2), described structure comprises the following steps containing the plant expression vector of DBR2 gene: first build intermediate carrier pMDT18-DBR2, intermediate carrier pMDT18-DBR2 and expression vector FSN is cut again with XmaI and SacI enzyme, reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extracts plasmid and does PCR detection and digestion verification; Described structure intermediate carrier pMDT18-DBR2 comprises following concrete steps: by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme.
2. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, it is characterized in that, in described step (4), described conversion comprises the following steps: the preculture of explant; The Dual culture of Agrobacterium and explant; The screening of resistance regeneration plant.
3. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described preculture comprises the following steps: seeds of southernwood 75% alcohol immersion 1min, then soaks 20min with 20%NaClO, aseptic water washing 3-4 time, surface-moisture is blotted with aseptic thieving paper, be inoculated in the MS solid medium without hormone, 25 DEG C of illumination cultivation, can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming.
4. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described Dual culture comprises the following steps: forwarded to by described leaf explant in Dual culture substratum, drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast.
5. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described screening comprises the following steps: be transferred in germination screening culture medium the described Dual culture sweet wormwood explant of 3 days in 25 DEG C of illumination cultivation, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, described well-grown resistance Multiple Buds is cut proceed to root media is cultured to and take root, Kan resistance regeneration sweet wormwood plant can be obtained.
6. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, is characterized in that, in described step (4), described PCR detects and comprises the following steps: the primer of design and synthesis DBR2 gene; Carry out DNA cloning; Viewed under ultraviolet radiation, if object band is positive, then this strain is described transgene abrotanum plant.
7. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, it is characterized in that, in described step (5), described HPLC-ELSD measures and comprises following condition: chromatographic column used is C-18 reverse phase silica gel post, and moving phase selects volume ratio to be the methyl alcohol of 70:30: water, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 20 μ L, light scattering detector drift tube temperature 40 DEG C, scale-up factor is 7, nebulizer gas pressure 5bar.
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| CN105296536A (en) * | 2015-11-12 | 2016-02-03 | 上海交通大学 | Transgenic sweet wormwood plant and cultivation method thereof |
| CN105695507B (en) * | 2016-04-27 | 2020-02-07 | 上海交通大学 | Method for improving artemisinin content in sweet wormwood herb by transferring ICS1 gene |
| CN106755060B (en) * | 2016-11-17 | 2020-05-26 | 上海交通大学 | Method for improving artemisinin content by co-transferring FPS and DBR2 genes and prepared sweet wormwood herb |
| CN107142287B (en) * | 2017-05-12 | 2020-12-08 | 南京林业大学 | Artemisinic aldehyde double-bond reductase DBR1 and application of recombinant bacterium thereof in preparation of dihydro-beta-ionone |
| CN110628809A (en) * | 2019-07-24 | 2019-12-31 | 中国人民解放军第二军医大学 | Method for improving artemisinin content in sweet wormwood herb by using AaTGA6 gene |
| CN110438148A (en) * | 2019-07-24 | 2019-11-12 | 中国人民解放军第二军医大学 | A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene |
| CN114231655B (en) * | 2021-12-29 | 2023-06-23 | 华智生物技术有限公司 | SNP locus linked with artemisinin content and application thereof |
| CN115918546B (en) * | 2022-09-06 | 2024-05-07 | 上海交通大学 | Transgenic sweet wormwood and method for improving content of artemisinin in sweet wormwood |
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