CN102604987A - Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer - Google Patents
Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer Download PDFInfo
- Publication number
- CN102604987A CN102604987A CN2012100135067A CN201210013506A CN102604987A CN 102604987 A CN102604987 A CN 102604987A CN 2012100135067 A CN2012100135067 A CN 2012100135067A CN 201210013506 A CN201210013506 A CN 201210013506A CN 102604987 A CN102604987 A CN 102604987A
- Authority
- CN
- China
- Prior art keywords
- dxr
- gene
- sweet wormwood
- plant
- improves
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for improving an artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer. The method comprises the following steps of: cloning DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene in the Artemisia annua L., so as to establish a plant expression carrier containing the DXR gene; transferring the DXR gene into Artemisia annua L. through agrobacterium tumefaciens-mediated transformation to grow a plant; detecting the integration condition of the exogenous target gene DXR by a PCR (Polymerase Chain Reaction); detecting the artemisinin content in the transgenic Artemisia annua L. through high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD); and screening the transgenic Artemisia annua L. plant with improved artemisinin content. The artemisinin content of the transgenic Artemisia annua L. obtained by the invention is obviously improved and high up to 2.4 times that of the non-transferred plant. Therefore, the invention provides the method for improving the artemisinin content in the Artemisia annua L. and lays a foundation for producing artemisinin by using the transgenic Artemisia annua L. in a large scale.
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 of method of changeing artemislnin content in the DXR 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 the 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; Waleerat Banyai etc. are in " Plant Cell Tissue and Organ Culture " (plant cell tissue's organ culture); Volume 255-265 page or leaf had been delivered the paper that is entitled as " Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L. " (" overexpression farnesyl pyrophosphate synthase gene can influence the growth of the content and the sweet wormwood of Artemisinin in the sweet wormwood ") in 2010 103; Report is through overexpression farnesyl pyrophosphate synthase (farnesyl pyrophosphate synthase; FPS); The content of Artemisinin has improved 2.5-3.6 doubly in the render transgenic sweet wormwood, but still has only about 1.3%.But, plant genetic engineering provides a feasible method for the content that improves Artemisinin in the sweet wormwood.
(1-deoxy-D-xylulose-5-phosphate reductoisomerase DXR) is a key enzyme in the Artemisinin route of synthesis to 1-deoxy-D-xylulose in the prior art-5-phosphoric acid reduction isomerase, is the important target spot of Artemisinin metabolic engineering.Adopt genetic engineering means, transform sweet wormwood, 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 key gene DXR.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of DXR of commentaries on classics gene to improve the method for artemislnin content in the sweet wormwood.The key gene clone who the present invention relates to, vector construction, genetic transformation, Molecular Detection, Artemisinin extract and assay is used for the present invention; Set up the method for artemislnin content in the stable raising sweet wormwood, established solid basis for utilizing sweet wormwood scale operation Artemisinin.
The present invention realizes through following technical scheme:
1, a kind of method of changeing artemislnin content in the DXR gene raising sweet wormwood, this method comprises the steps:
(1) adopt gene clone method to obtain sweet wormwood key gene DXR;
(2) be linked to expression regulation sequence to said DXR gene, make up the plant expression vector that contains the DXR gene;
(3) the said plant expression vector that contains the DXR gene is transformed agrobacterium tumefaciens, obtain to contain the agrobacterium tumefaciens bacterial strain of said DXR gene plant expression vector;
(4) utilize the agrobacterium tumefaciens bacterial strain of the said DXR of containing gene plant expression vector to transform sweet wormwood, obtain transgene abrotanum plant through the integration DXR gene of PCR detection;
(5) artemislnin content in the said transgene abrotanum is carried out HPLC-ELSD and measure, screening obtains the transgene abrotanum plant that artemislnin content improves.
Preferably, in said step (1), said gene clone method may further comprise the steps: extract the total RNA of sweet wormwood genome; The total RNA of sweet wormwood genome that is obtained is obtained the first chain cDNA through ThermoScript II XL reverse transcription; According to the dna sequence dna shown in the SEQ ID NO:1, design amplifies the upstream primer and the downstream primer of complete encoder block, and said upstream primer is the dna sequence dna shown in the SEQ ID NO:2; Said downstream primer is the dna sequence dna shown in the SEQ ID NO:3; And on said upstream and downstream primer, introduce restriction endonuclease sites respectively so that construction of expression vector is a template with the described first chain cDNA, behind pcr amplification, check order.
Preferably; In said step (2), the plant expression vector that said structure contains the DXR gene may further comprise the steps: make up earlier intermediate carrier pMDT18-dxr, again with XmaI and SacI enzyme hit a carrier pMDT18-dxr and expression vector FSN; Reclaim DXR 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.
Further preferred, said structure intermediate carrier pMDT18-dxr may further comprise the steps: through introducing the full length gene of XmaI and SacI restriction enzyme site before and after the high-fidelity enzymatic amplification DXR gene respectively, be connected on the pMDT18 carrier through ligase enzyme.
Preferably, in said step (4), said conversion may further comprise the steps: the preparatory cultivation of explant; The common cultivation of Agrobacterium and said explant; The screening of resistance regeneration plant.
Further preferred, 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 time 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 about 5cm after, clip aseptic seedling leaf explant is used for transforming.
Further preferred; 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 engineering bacteria of the good said DXR of the containing gene plant binary expression vector of activation; Said explant is fully contacted with bacterium liquid, 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.
Further preferred; 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 subculture, 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 the Kan resistance sweet wormwood plant that regenerates.
Preferably, in said step (4), said PCR detects and may further comprise the steps: the primer of the synthetic DXR gene of design; Carry out DNA cloning; Ultraviolet ray is observed down, if the purpose band is positive, then this strain system is said transgene abrotanum plant.
Preferably, in said step (5), said HPLC-ELSD measures and comprises following condition: used chromatographic column is a C-18 reverse phase silica gel post; It is 70: 30 methyl alcohol that moving phase is selected volume ratio for use: water, 30 ℃ of column temperatures, flow velocity 1.0mL/min; Sample size 20 μ L; 40 ℃ of light scattering detector drift tube temperatures, scale-up factor are 7, nebulizer gas pressure 5bar.
Commentaries on classics DXR gene of the present invention improves the method for artemislnin content in the sweet wormwood; Adopt gene engineering method; Key gene DXR is imported in the sweet wormwood plant, obtained the transgene abrotanum strain system that artemislnin content significantly improves, the content that changes Artemisinin in the DXR gene sweet wormwood can reach the 19.2mg/g of dry weight; Be 2.4 times of the common sweet wormwood of non-conversion (8mg/g dry weight), this invention for for the large-scale production of Artemisinin high yield is provided, to stablize source new drugs significant.
Description of drawings
Fig. 1 is the content detection figure as a result of Artemisinin in the sweet wormwood plant of the present invention.
Embodiment
Elaborate in the face of embodiments of the invention down: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme 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
Step 1, the clone of sweet wormwood DXR gene
(1) 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 1mL 75% ethanol and clean, after the vibration, descend 7, the centrifugal 5min of 500rmp in 4 ℃; Be dissolved in behind the 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.
(2) clone of sweet wormwood DXR gene
The total RNA of sweet wormwood genome that is obtained is obtained the first chain cDNA through ThermoScript II XL (AMV) reverse transcription; Encoding sequence (dna sequence dna shown in the SEQ ID NO:1) according to said sweet wormwood DXR gene; Design amplifies the upstream primer (dna sequence dna shown in the SEQ ID NO:2) and the downstream primer (dna sequence dna shown in the SEQ ID NO:3) 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 English fine horse biotechnology Services Co., Ltd.Sequencing result shows, the encoding sequence (dna sequence dna shown in the SEQ ID NO:1) of the sweet wormwood DXR gene of being reported among the sequence of being cloned and the GenBank is consistent.
Present embodiment adopts gene clone method from sweet wormwood, to obtain the correct Artemisinin biosynthesizing key gene DXR of sequence, for artemislnin content provides an important key gene in the DXR gene raising sweet wormwood through changeing.
Step 2 contains the structure of the plant binary expression vector of DXR gene
(1) structure of intermediate carrier pMDT18-dxr
(Takara Dalian) is primary element, makes up intermediate carrier pMDT18-dxr to select the pMDT18 carrier for use.Particularly,, be connected on the pMDT18 carrier, confirm the exactness of gene by the order-checking of Shanghai English fine horse biotechnology Services Co., Ltd through ligase enzyme through introducing the full length gene of XmaI and SacI restriction enzyme site before and after the high-fidelity enzymatic amplification DXR gene respectively.
(2) contain the structure of the plant expression vector of DXR gene
With described FSN (the FSN expression vector is transformed on the pCAMBIA2300 expression vector and obtained and preservation) is expression vector, and above-mentioned DXR gene is connected into its corresponding restriction enzyme site position.Particularly, XmaI and SacI double digestion intermediate carrier pMDT18-dxr and expression vector FSN.Reclaim DXR 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 Artemisinin biosynthetic pathway key gene DXR operability be connected in expression regulation sequence, form the plant expression vector that contains the DXR gene, this expression vector can be used for improving through the metabolic engineering strategy content of Artemisinin in the sweet wormwood.
Step 3 contains the acquisition of DXR gene double base plant expression vector agrobacterium tumefaciens engineering bacteria
Change the plant binary expression vector of the above-mentioned DXR of containing gene 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 Gambar 1), the performing PCR of going forward side by side checking.
Step 4, Agrobacterium tumefaciens mediated DXR 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 time 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
Above-mentioned leaf explant is forwarded in the common culture medium (1/2MS+AS 100 μ mol/L); Dropping contains the 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria of the good said DXR of the containing gene plant binary expression vector of activation; Above-mentioned explant is fully contacted, 28 ℃ of dark cultivations 3 days with bacterium liquid.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+Kan 50mg/L+Cb 500mg/L) to be gone up in 25 ℃, 16h/8h (light/dark) 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.
Step 5, the PCR of transgene abrotanum plant detects
According to goal gene place expression cassette p35s-dxr-nos sequence p35s with dxr 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 576bp.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 DXR 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.
Step 6 utilizes HPLC-ELSD to measure artemislnin content in the transgene abrotanum
(1) preparation of HPLC-ELSD condition and system suitability and standardized solution
HPLC: adopt water alliance 2695 systems, chromatographic column is C-18 reverse phase silica gel post (Symmetry Shield TM C18,5 μ m; 250 * 4.6mm, Waters), moving phase is methyl alcohol: water; Methyl alcohol: the volume ratio of 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 alliance 2420 systems, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor (gain) is 7, nebulizer gas pressure 5bar;
Precision takes by weighing Artemisinin 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 methyl alcohol among the present invention: water, ratio are 70%: 30% o'clock, and the RT of Artemisinin is 5.1min, and the peak type is good.Theoretical plate number is calculated by Artemisinin and is not less than 2000.
(2) 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 scope.The log-log equation of linear regression of Artemisinin reference substance is Y=5.404e+0000X+1.858e+0000, R
2=0.999184.
(3) mensuration of the preparation of sample and 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 filter filtration, 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 DXR gene in the present invention and significantly improve artemislnin content in the sweet wormwood.The content that changes the blue or green senior middle school of DXR gene Artemisinin can reach the 19.2mg/g (as shown in Figure 1) of dry weight, is 2.4 times of the common sweet wormwood of non-conversion (8mg/g dry weight).
Present embodiment adopts the HPLC-ELSD method to measure artemislnin content in the transgene abrotanum, and the metabolic engineering strategy of employing conversion DXR gene has obtained the sweet wormwood plant of Artemisinin high yield, for the large-scale production Artemisinin provides a kind of Perfected process.
Claims (10)
1. one kind is changeed the method that the DXR gene improves artemislnin content in the sweet wormwood, it is characterized in that this method comprises the steps:
(1) adopt gene clone method to obtain sweet wormwood key gene DXR;
(2) be linked to expression regulation sequence to said DXR gene, make up the plant expression vector that contains the DXR gene;
(3) the said plant expression vector that contains the DXR gene is transformed agrobacterium tumefaciens, obtain to contain the agrobacterium tumefaciens bacterial strain of said DXR gene plant expression vector;
(4) utilize the agrobacterium tumefaciens bacterial strain of the said DXR of containing gene plant expression vector to transform sweet wormwood, obtain transgene abrotanum plant through the integration DXR gene of PCR detection;
(5) artemislnin content in the said transgene abrotanum is carried out HPLC-ELSD and measure, screening obtains the transgene abrotanum plant that artemislnin content improves.
2. commentaries on classics DXR gene according to claim 1 improves the method for artemislnin content in the sweet wormwood; It is characterized in that in said step (1), said gene clone method may further comprise the steps: extract the total RNA of sweet wormwood genome; The total RNA of sweet wormwood genome that is obtained is obtained the first chain cDNA through ThermoScript II XL reverse transcription; According to the dna sequence dna shown in the SEQ ID NO:1, design amplifies the upstream primer and the downstream primer of complete encoder block, and said upstream primer is the dna sequence dna shown in the SEQ ID NO:2; Said downstream primer is the dna sequence dna shown in the SEQ ID NO:3; And on said upstream and downstream primer, introduce restriction endonuclease sites respectively so that construction of expression vector is a template with the described first chain cDNA, behind pcr amplification, check order.
3. commentaries on classics DXR gene according to claim 1 improves the method for artemislnin content in the sweet wormwood, it is characterized in that, in said step (2); The plant expression vector that said structure contains the DXR gene may further comprise the steps: make up intermediate carrier pMDT18-dxr earlier; With XmaI and SacI enzyme hit a carrier pMDT18-dxr and expression vector FSN, reclaim DXR gene fragment and the big fragment of FSN carrier again, connect conversion; The picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
4. commentaries on classics DXR gene according to claim 3 improves the method for artemislnin content in the sweet wormwood; It is characterized in that; Said structure intermediate carrier pMDT18-dxr may further comprise the steps: through introducing the full length gene of XmaI and SacI restriction enzyme site before and after the high-fidelity enzymatic amplification DXR gene respectively, be connected on the pMDT18 carrier through ligase enzyme.
5. commentaries on classics DXR 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 said explant; The screening of resistance regeneration plant.
6. commentaries on classics DXR gene according to claim 5 improves the method for artemislnin content in the sweet wormwood, and it is characterized in that said preparatory cultivation may further comprise the steps: seeds of southernwood is with 75% alcohol immersion 1min; Soak 20min with 20%NaClO again, aseptic water washing 3-4 time blots surface-moisture with aseptic thieving paper; Be inoculated in the MS solid medium of no hormone, 25 ℃ of 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.
7. commentaries on classics DXR gene according to claim 5 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 engineering bacteria of the good said DXR of the containing gene plant binary expression vector of activation; Said explant is fully contacted with bacterium liquid, 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.
8. commentaries on classics DXR gene according to claim 5 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 obtaining the Kan resistance bud of growing thickly behind 2-3 the subculture; 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.
9. commentaries on classics DXR 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 DXR gene of design; Carry out DNA cloning; Ultraviolet ray is observed down, if the purpose band is positive, then this strain system is said transgene abrotanum plant.
10. commentaries on classics DXR 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: used chromatographic column is a C-18 reverse phase silica gel post, and it is 70: 30 methyl alcohol that moving phase is selected volume ratio for use: water, 30 ℃ of column temperatures; Flow velocity 1.0mL/min, sample size 20 μ L, 40 ℃ of light scattering detector drift tube temperatures; Scale-up factor is 7, nebulizer gas pressure 5bar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100135067A CN102604987A (en) | 2012-01-17 | 2012-01-17 | Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100135067A CN102604987A (en) | 2012-01-17 | 2012-01-17 | Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102604987A true CN102604987A (en) | 2012-07-25 |
Family
ID=46522755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100135067A Pending CN102604987A (en) | 2012-01-17 | 2012-01-17 | Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102604987A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531752A (en) * | 2014-12-12 | 2015-04-22 | 重庆医药高等专科学校 | Method for cultivating sweet wormwood herb with high artemisinin content by co-transforming genes Anti-Dxr and Anti-Fpps |
| CN105695507A (en) * | 2016-04-27 | 2016-06-22 | 上海交通大学 | Method for increasing content of artemisinin in artemisia annua through ICS1 gene transferring |
| CN105713921A (en) * | 2016-04-11 | 2016-06-29 | 上海交通大学 | Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes |
| CN105861544A (en) * | 2016-04-11 | 2016-08-17 | 上海交通大学 | Method for improving content of artemisinin in sweet wormwood herb through transferring F3H gene |
| CN106349352A (en) * | 2016-10-27 | 2017-01-25 | 上海交通大学 | Artemisia apiacea translocator AaPDR3 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182544A (en) * | 2007-11-15 | 2008-05-21 | 上海交通大学 | Method for enhancing arteannuin content in southernwood by transforming ads gene |
| CN102242145A (en) * | 2011-04-20 | 2011-11-16 | 上海交通大学 | Method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene |
-
2012
- 2012-01-17 CN CN2012100135067A patent/CN102604987A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182544A (en) * | 2007-11-15 | 2008-05-21 | 上海交通大学 | Method for enhancing arteannuin content in southernwood by transforming ads gene |
| CN102242145A (en) * | 2011-04-20 | 2011-11-16 | 上海交通大学 | Method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene |
Non-Patent Citations (3)
| Title |
|---|
| PUI KWAN FUNG ET AL: "Computational Analysis of the Evolution of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase, an Important Enzyme in Plant Terpene Biosynthesis", 《CHEMISTRY & BIODIVERSITY》 * |
| 景福远: "利用基因工程技术提高青蒿中青蒿素含量的研究", 《中国优秀硕士学位论文全文数据库-农业科技辑》 * |
| 杨瑞仪等: "青蒿素生物合成基因的转录谱分析", 《广州中医药大学学报》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531752A (en) * | 2014-12-12 | 2015-04-22 | 重庆医药高等专科学校 | Method for cultivating sweet wormwood herb with high artemisinin content by co-transforming genes Anti-Dxr and Anti-Fpps |
| CN104531752B (en) * | 2014-12-12 | 2017-08-08 | 重庆医药高等专科学校 | The method that cotransformation Gene A nti Dxr and Anti Fpps cultivate Gaoqing punt-pole cellulose content sweet wormwood |
| CN105713921A (en) * | 2016-04-11 | 2016-06-29 | 上海交通大学 | Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes |
| CN105861544A (en) * | 2016-04-11 | 2016-08-17 | 上海交通大学 | Method for improving content of artemisinin in sweet wormwood herb through transferring F3H gene |
| CN105713921B (en) * | 2016-04-11 | 2019-02-19 | 上海交通大学 | Turn the method that CHI gene improves content of artemisinin in sweet wormwood |
| CN105695507A (en) * | 2016-04-27 | 2016-06-22 | 上海交通大学 | Method for increasing content of artemisinin in artemisia annua through ICS1 gene transferring |
| 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 |
| CN106349352A (en) * | 2016-10-27 | 2017-01-25 | 上海交通大学 | Artemisia apiacea translocator AaPDR3 and application thereof |
| CN106349352B (en) * | 2016-10-27 | 2019-09-24 | 上海交通大学 | Sweet wormwood transport protein AaPDR3 and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101182544B (en) | Method for enhancing arteannuin content in southernwood by transforming ads gene | |
| CN102676578A (en) | Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer | |
| CN102776225A (en) | Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene | |
| Shi et al. | Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots | |
| CN102643838A (en) | Method for improving content of artemisinin in artemisia apiacea by tran-ALDH1 gene | |
| CN103088027B (en) | PDR transport protein gene promoter for controlling ginsenoside accumulation, and its application | |
| Benyammi et al. | Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots | |
| Sharifi et al. | The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L. | |
| CN102242145A (en) | Method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene | |
| CN102604987A (en) | Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer | |
| CN112375767B (en) | Artemisia apiacea WRKY transcription factor AaWRKY4 gene and application thereof | |
| CN101182543B (en) | Method for enhancing arteannuin content in southernwood using gene cyp71av1 and cpr co-transformation | |
| CN101182545B (en) | Method for enhancing arteannuin content in southernwood using gene hmgr and fps co-transformation | |
| CN102776212A (en) | Production method of high-artemisinin-content transgene sweet wormwood plants | |
| Beigmohamadi et al. | Potential of the genetically transformed root cultures of Plumbago europaea for biomass and plumbagin production | |
| CN101182546B (en) | Method for enhancing arteannuin content in southernwood by RNA interference | |
| Marwani et al. | Development of hairy root culture of Andrographis paniculata for in vitro adrographollide production | |
| CN101597620B (en) | Method for improving the content of artemisia annua patchouli calcohol by utilizing pts gene and RNA interferon ads gene | |
| CN102558325A (en) | Preparation method of artemisinin AaORA protein, encoding gene and transgenic artemisinin plant | |
| CN102499037B (en) | Method for rapid propagation of genetically modified sweet wormwood by hydroponics | |
| CN105296536A (en) | Transgenic sweet wormwood plant and cultivation method thereof | |
| CN101560519B (en) | Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene | |
| CN104531753B (en) | The method that cotransformation Sps, Hmgr and Dxs gene cultivate bud Gaoqing punt-pole cellulose content sweet wormwood | |
| CN101831456B (en) | Method for increasing content of camptothecin in camplotheca acuminata calluses by adopting camplotheca acuminata transgene aoc | |
| CN105936914A (en) | Asparagus chalcone synthetase gene, encoded protein and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120725 |