CN101182543B - Method for enhancing arteannuin content in southernwood using gene cyp71av1 and cpr co-transformation - Google Patents
Method for enhancing arteannuin content in southernwood using gene cyp71av1 and cpr co-transformation Download PDFInfo
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- CN101182543B CN101182543B CN2007101704223A CN200710170422A CN101182543B CN 101182543 B CN101182543 B CN 101182543B CN 2007101704223 A CN2007101704223 A CN 2007101704223A CN 200710170422 A CN200710170422 A CN 200710170422A CN 101182543 B CN101182543 B CN 101182543B
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Abstract
The invention is a method for adopting the cotransformation of genes cyp71av1 and cpr to increase the arteannuin content in southernwood in the gene engineering technology field. The invention clones the gene cyp71av1 of the CYP71AV1 and the gene cpr of the CPR from the southernwood to construct the plant expression vector which contains DNA molecules and agrobacterium tumefaciems is used for mediation; the genes of cyp71av1 and cpr are admitted into the southernwood for regenerating plant; a PCR is used for detecting the integration situation of the foreign object genes of the cyp71av1 and the cpr and a high-efficiency liquid phase chromatography-evaporative light scattering detector is used for detecting the arteannuin content in southern wood and the transgenic southernwood plant with increased arteannuin content is selected. The invention obtains the transgenic southernwood with obviously increased arteannuin content and the highest content is 1.82 times the content of the non-transgenic plant.
Description
Technical field
What the present invention relates to is a kind of method of gene engineering technology field, particularly a kind of method that adopts two key gene cyp71av1 and cpr cotransformation to improve artemislnin content in the sweet wormwood.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Artemisinin is from the isolating a kind of sesquiterpene lactones compound that contains the peroxide bridge structure of its over-ground part, is the medicine of present the most effective treatment malaria of generally acknowledging in the world, particularly has quick-acting and characteristics low toxicity for encephalic malaria and anti-chloroquine malaria.At present, the method for the most effective treatment 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 derivative thereof also have anti-inflammatory, schistosomicide, antitumor and immunoregulatory function.As seen 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 very low (0.01%-1%) in the sweet wormwood makes the large-scale commercial applications production of this medicine be restricted.Because the Artemisinin complex structure, the synthetic difficulty is big, yields poorly, and the cost height 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, Dahua Chen etc. are at " Plant Science " (" plant science ", 2000 155 phase 179-185 pages or leaves) delivered the paper that is entitled as " Expression of a chimericfarnesyl diphosphate synthase gene in Artemisia annua L.transgenic plantsvia Agrobacterium tumefaciens-mediated transformation " (" by agrobacterium mediation converted representation farnesyl pyrophosphate synthase gene in the sweet wormwood plant "), report is by overexpression farnesyl pyrophosphate synthase (farnesyl diphosphate synthase, FPS), the content of Artemisinin has improved 2-3 doubly in the render transgenic sweet wormwood, but still has only about 1%.But, metabolic engineering provides a feasible method for the content that improves Artemisinin in the sweet wormwood.
AMORPHADIENE oxydase (amorpha-4 in the prior art, 11-diene C-12 oxidase, CYP71AV1) be a key enzyme in the Artemisinin route of synthesis, and cytochrome P450 reductase (cytochrome P450 reductase CPR) is the oxidasic redox companion body of AMORPHADIENE.Adopt genetic engineering means,, will break Artemisinin biosynthesizing speed limit bottleneck, thereby obtain the sweet wormwood plant of Artemisinin high yield, for the large-scale production Artemisinin provides a new way with key gene cyp71av1 and companion body cpr cotransformation sweet wormwood thereof.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of method that adopts gene cyp71av1 and cpr cotransformation to improve artemislnin content in the sweet wormwood is provided.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 that improves artemislnin content in the sweet wormwood, established solid basis for utilizing transgene abrotanum scale operation Artemisinin.
The present invention is achieved by the following technical solutions: the present invention clones cyp71av1 and cpr gene from sweet wormwood, structure contains the plant expression vector of described dna molecular, with Agrobacterium tumefaciens mediated, cyp71av1 and cpr gene are imported the sweet wormwood and the plant that regenerates simultaneously; PCR detects the integration situation of external source goal gene cyp71av1 and cpr, and high performance liquid chromatography-light scattering detector (HPLC-ELSD) is measured artemislnin content in the sweet wormwood, and screening obtains the transgene abrotanum plant that artemislnin content improves.
The present invention includes following concrete steps:
(1) adopt gene clone method to obtain sweet wormwood key gene cyp71av1 and cpr;
(2) cyp71av1 and cpr gene operability be connected in expression regulation sequence, form the plant expression vector that contains cyp71av1 and cpr gene;
(3) plant expression vector that will contain cyp71av1 and cpr gene transforms the agrobacterium tumefaciens (biomaterial that public sale is arranged for market, can buy from many companies such as Australian CAMBIA company), obtain to be used to transform the agrobacterium tumefaciens bacterial strain that contains cyp71av1 and cpr gene plant expression vector of sweet wormwood;
(4) utilize constructed agrobacterium tumefaciens bacterial strain to transform sweet wormwood, obtain the transgene abrotanum plant that detects through PCR;
(5) artemislnin content in the transgene abrotanum that obtains is carried out HPLC-ELSD and measure, screening obtains the transgene abrotanum plant that artemislnin content significantly improves.
The described transgene abrotanum plant that detects through PCR is meant that the detection primer of synthetic cyp71av1 of design and cpr gene carries out DNA cloning respectively, and the positive plant that the purpose band is observed in ultraviolet ray down is the transgene abrotanum plant.
Described HPLC-ELSD measures artemislnin content in the sweet wormwood, method is as follows: chromatographic column C-18 reverse phase silica gel post, 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, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor (gain) is 7, nebulizer gas pressure 5bar.
Cyp71av1 of the present invention and cpr cotransformation improve the method for artemislnin content in the sweet wormwood; adopt gene engineering method; key gene cyp71av1 and cpr are imported in the sweet wormwood plant; obtained the transgene abrotanum plant that artemislnin content significantly improves; the content of Artemisinin can reach 18.2mg/g DW in corotation cyp71av1 and the cpr gene sweet wormwood; be 1.82 times of the common sweet wormwood of non-conversion (10mg/g DW), this invention for provide high yield for the Artemisinin large-scale production, to stablize source new drugs significant.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided 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, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of sweet wormwood cyp71av1 and cpr gene
1. the extraction of the total RNA of sweet wormwood genome
Sweet wormwood (the higher sweet wormwood kind of sun artemislnin content at Chongqing tenth of the twelve Earthly Branches is originated in the employing) young leaflet tablet that takes a morsel behind liquid nitrogen flash freezer, grinds with mortar rapidly, add and fill 1mL TRIzol (TRIzol Reagents, GIBCOBRL is in 1.5mL Eppendorf pipe USA), fully after the vibration, under room temperature, place 5min, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature is placed 2-3min, in 4 ℃, 12, the centrifugal 15min of 000g; Supernatant liquor (about 600 μ L) is sucked in the clean 1.5mL Eppendorf pipe, adds isopyknic Virahol, put upside down mixing, place 10min under the room temperature after, in 4 ℃, 12, the centrifugal 10min of 000g; Abandon supernatant, add 1mL 75% ethanol and clean, after the vibration, in 4 ℃, 7, the centrifugal 5min of 500g; Be dissolved in behind the drying at room temperature 15-20min in an amount of (30-50 μ L) RNAase-free water; Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
2. the clone of sweet wormwood cyp71av1 and cpr gene
The total RNA of sweet wormwood genome that is obtained is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, according to the encoding sequence (sequence 1 in the sequence table) of described sweet wormwood cyp71av1 gene and the encoding sequence (sequence 2 in the sequence table) of cpr gene, design amplifies the upstream and downstream primer of complete encoder block respectively, 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, checks order behind pcr amplification.Determined dna sequence adopts 3730 automatic sequencers to finish by Shanghai English fine horse biotechnology Services Co., Ltd.Sequencing result shows, the sweet wormwood cyp71av1 gene of being reported among the sequence of being cloned and the GenBank (sequence 1 in the sequence table) and the encoding sequence of cpr gene (sequence 2 in the sequence table) are consistent.
Present embodiment adopts gene clone method to obtain sequence correct Artemisinin biosynthesizing key gene cyp71av1 and cpr from sweet wormwood, and artemislnin content provides two important key genes in the sweet wormwood in order to improve by two key gene cotransformation strategies.
Embodiment 2
The structure that contains the plant binary expression vector of cyp71av1 and cpr gene
1. the structure of intermediate carrier pMD18-T::p35S-gfp*gus-nos
Selecting pMD18-T and pCAMBIA1304 for use is primary element, makes up intermediate carrier pMD18-T::p35S-gfp*gus-nos.Particularly, go up a pair of primer of sequences Design of p35S-gfp*gus-nos according to pCAMBIA1304, and on the upstream and downstream primer, introduce restriction endonuclease sites respectively, so that construction of expression vector.With the pCAMBIA1304 plasmid is masterplate, and pcr amplification gfp*gus fusion gene expression cassette is connected to the pMD18-T carrier, transformation and selection, and the order-checking of picking mono-clonal is confirmed errorless.
2. the structure of intermediate carrier pMD18-T::p35S-cyp71av1-nos and pMD18-T::p35S-cpr-nos
Based on described pMD18-T::p35S-gfp*gus-nos, replace gfp*gus fusion gene on it respectively with cyp71av1 among the embodiment 1 and cpr gene.Particularly, SpeI/BstEII double digestion pMD18-T::cyp71av1, pMD18-T::cpr and pMD18-T::p35S-gfp*gus-nos, reclaim cyp71av1, the big fragment of cpr and pMD18-T::p35S-gfp*gus-nos, cyp71av1 is connected transformed into escherichia coli with big fragment respectively with cpr, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
3. the structure of plant binary expression vector pCAMBIA2300::gfp*gus
Based on described pMD18-T::p35S-gfp*gus-nos, the expression cassette that will contain the gfp*gus fusion gene places plant binary expression vector pCAMBIA2300, is built into plant binary expression vector pCAMBIA2300::gfp*gus.Concrete operations are as follows: with SmaI and PstI double digestion pCAMBIA2300, reclaim big fragment, with SwaI and PstI double digestion pMD18-T::p35S-gfp*gus-nos, reclaim the expression cassette of gfp*gus.The gfp*gus expression cassette is connected with the big fragment of pCAMBIA2300, transformed into escherichia coli, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
4. the structure of plant binary expression vector pCAMBIA2300::gfp*gus-cyp71av1
Based on described pMD18-T::p35S-cyp71av1-nos, to contain cyp71av1 expression of gene box and insert among the plant binary expression vector pCAMBIA2300::gfp*gus, be built into plant binary expression vector pCAMBIA2300::gfp*gus-cyp71av1.Concrete operations are as follows: with SmaI and PstI double digestion pCAMBIA2300::gfp*gus, reclaim big fragment, with SwaI and PstI double digestion pMD18-T::p35S-cyp71av1-nos, reclaim cyp71av1 expression of gene box.Cyp71av1 expression of gene box is connected with the big fragment of pCAMBIA2300::gfp*gus, transformed into escherichia coli, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
5. the structure of plant binary expression vector pCAMBIA2300::gfp*gus-cyp71av1-cpr
Based on described pMD18-T::p35S-cpr-nos, to contain cpr expression of gene box and insert among the plant binary expression vector pCAMBIA2300::gfp*gus-cyp71av1, be built into plant binary expression vector pCAMBIA2300::gfp*gus-cyp71ay1-cpr.Concrete operations are as follows: with SmaI and PstI double digestion pCAMBIA2300::gfp*gus-cyp71av1, reclaim big fragment, with SwaI and PstI double digestion pMD18-T::p35S-cpr-nos, reclaim cpr expression of gene box.Cpr expression of gene box is connected with the big fragment of pCAMBIA2300::gfp*gus-cyp71av1, transformed into escherichia coli, 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 cyp71av1 and cpr operability be connected in expression regulation sequence, formation contains the plant expression vector of cyp71av1 and cpr gene, and this expression vector can be used for improving by the metabolic engineering strategy content of Artemisinin in the sweet wormwood.
Embodiment 3
Agrobacterium tumefaciens mediated cyp71av1 and cpr gene genetic transform sweet wormwood and obtain the transgene abrotanum plant
1. contain the acquisition of cyp71av1 and cpr gene double base plant expression vector agrobacterium tumefaciens engineering bacteria
Change the plant binary expression vector that contains cyp71av1 and cpr gene among the embodiment 2 over to agrobacterium tumefaciens (as EHA105, the biomaterial that public sale is arranged for market, can buy from Australian CAMBIA company, strain number is Gambar1), the performing PCR of going forward side by side checking.The result shows, contains cyp71av1 and cpr gene plant binary expression vector successfully is building up to the agrobacterium tumefaciens bacterial strain.
2. Agrobacterium tumefaciens mediated cyp71av1 and cpr gene transformation sweet wormwood
2.1. the pre-cultivation of explant
Seeds of southernwood is with 75% alcohol immersion 1min, soak 20min with 20%NaClO again, aseptic water washing 3-4 time, blot 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.
2.2. the common cultivation of Agrobacterium and explant
With described leaf explant, forward in the common culture medium (1/2 MS+AS, 100 μ mol/L), dropping contains the 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria of good described cyp71av1 of containing of activation and cpr gene plant binary expression vector, explant is fully contacted, 28 ℃ of dark 3d that cultivate with bacterium liquid.Is contrast with dropping at the leaf explant of the 1/2MS of the agrobacterium tumefaciens that does not have goal gene liquid nutrient medium suspension.
2.3. the screening of resistance regeneration plant
The described sweet wormwood explant of cultivating 3d altogether 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 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. the PCR of transgene abrotanum plant detects
According to the sequences Design forward primer of CaMV 35S, design reverse primer respectively according to the sequence of cyp71av1 and cpr gene goal gene is detected.The result shows, can amplify the specific DNA fragment of 580bp and 2584bp respectively with CaMV 35S forward primer and cyp71av1 reverse primer and CaMV 35S forward primer and cpr reverse primer.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 cyp71av1 and cpr 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.
Embodiment 4
Utilize HPLC-ELSD to measure artemislnin content in the transgene abrotanum
1.HPLC-ELSD the preparation of condition and system suitability and standardized solution
HPLC: adopt water alliance 2695 systems, chromatographic column is C-18 reverse phase silica gel post (SymmetryShieldTM C18,5 μ m, 250 * 4.6mm, Waters), 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 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 5 bar;
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 ℃ standby.
Moving phase is methyl alcohol among the present invention: water, and methyl alcohol: the volume ratio of water is 70: 30 o'clock, and the retention time 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. the making of typical curve
With 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 parameter carry out regression analysis with peak area (Y) to standard substance content (X, μ g) respectively.By 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=1.28e+000X+4.71e+000, R=0.979546.
3. the 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 dehydrated alcohol and fully dissolve extract, be used for HPLC and detect.Simultaneously, 60 degree 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 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.
Corotation cyp71av1 and cpr gene have significantly improved artemislnin content in the sweet wormwood in the present invention.The content of Artemisinin can reach 18.2mg/g DW in corotation cyp71av1 and the cpr gene sweet wormwood, is 1.82 times of the common sweet wormwood of non-conversion (10mg/g DW).
Present embodiment adopts the HPLC-ELSD method to measure artemislnin content in the transgene abrotanum, and the metabolic engineering strategy of employing cotransformation cyp71av1 and cpr gene has obtained the sweet wormwood plant of Artemisinin high yield, for the large-scale production Artemisinin provides a kind of Perfected process.
The sequence table that the present invention relates to:
＜110〉Shanghai Communications University
＜120〉adopt gene cyp71av1 and cpr cotransformation to improve the method for artemislnin content in the sweet wormwood
<160>2
<170>PatentIn?version?3.4
<210>1
<211>1488
<212>DNA
＜213〉sweet wormwood (Artemisia annua)
<400>1
atgaagagta?tactaaaagc?aatggcactc?tcactgacca?cttccattgc?tcttgcaacg 60
atccttttgt?tcgtttacaa?gttcgctact?cgttccaaat?ccaccaaaaa?aagccttcct 120
gagccatggc?ggcttcccat?tattggtcac?atgcatcact?tgattggtac?aacgccacat 180
cgtggggtta?gggatttagc?cagaaagtat?ggatctttga?tgcatttaca?gcttggtgaa 240
gttccaacaa?tcgtggtgtc?atctccgaaa?tgggctaaag?agattttgac?aacgtacgac 300
attacctttg?ctaacaggcc?cgagacttta?actggtgaga?ttgttttata?tcacaatacg 360
gatgttgttc?ttgcacctta?tggtgaatac?tggaggcaat?tacgtaaaat?ttgcacattg 420
gagcttttga?gtgttaagaa?agtaaagtca?tttcagtcac?ttcgtgaaga?ggagtgttgg 480
aatttggttc?aagagattaa?agcttcaggt?tcagggagac?cggttaacct?ttcagagaat 540
gttttcaagt?tgattgcaac?gatacttagt?agagccgcat?ttgggaaagg?gatcaaggac 600
cagaaagagt?taacggagat?tgtgaaagag?atactgaggc?aaactggtgg?ttttgatgtg 660
gcagatatct?ttccttcaaa?gaaatttctt?catcatcttt?cgggcaagag?agctcggtta 720
actagccttc?gcaaaaagat?cgataattta?atcgataacc?ttgtagctga?gcatactgtt 780
aacacctcca?gtaaaactaa?cgagacactc?ctcgatgttc?ttttaaggct?caaagacagt 840
gctgaattcc?cattaacatc?tgataacatt?aaagccatca?ttttggatat?gtttggagca 900
ggcacagaca?cttcctcatc?cacaatcgaa?tgggcgattt?cggaactcat?aaagtgtccg 960
aaagcaatgg?agaaagtaca?agcggaattg?aggaaagcat?tgaacggaaa?agaaaagatc 1020
catgaggaag?acattcaaga?actaagctac?ttgaacatgg?taatcaaaga?aacattgagg 1080
ttgcaccctc?cactaccctt?ggttctgcca?agagagtgcc?gccaaccagt?caatttggct 1140
ggatacaaca?tacccaataa?gaccaaactt?attgtcaacg?tctttgcgat?aaatagggac 1200
cctgaatatt?ggaaagacgc?tgaagctttc?atccctgaac?gatttgaaaa?tagttctgca 1260
actgtcatgg?gtgcagaata?cgagtatctt?ccgtttggag?ctgggagaag?gatgtgtcct 1320
ggagccgcac?ttggtttagc?taacgtgcag?ctcccgctcg?ctaatatact?atatcatttc 1380
aactggaaac?tccccaatgg?tgtgagctat?gaccagatcg?acatgaccga?gagctctgga 1440
gccacgatgc?aaagaaagac?tgagttgtta?ctcgttccaa?gtttctag 1488
<210>2
<211>2115
<212>DNA
＜213〉sweet wormwood (Artemisia annua)
<400>2
atgcaatcaa?caacttccgt?taagttatct?cccttcgatc?taatgacggc?gttacttaac 60
ggcaaggtat?cgttcgacac?atcaaacaca?tcggatacga?atattccgtt?agcggtgttt 120
atggagaatc?gtgagctttt?gatgatttta?actacttcgg?ttgcggtgtt?gatcggatgc 180
gttgtggtgc?ttgtgtggag?acggtcgtcg?tcggcggcga?agaaagcggc?ggagtcgccg 240
gtgattgttg?tgccgaagaa?agtgacggag?gatgaggttg?atgacggacg?gaagaaagtt 300
actgtgtttt?ttggaactca?gactggtact?gctgaaggtt?ttgctaaggc?gcttgttgaa 360
gaagctaaag?cgcgatatga?aaaggcggtg?tttaaagtga?ttgatttgga?tgattatgct 420
gctgaagatg?atgagtatga?ggagaagtta?aagaaagaat?ctcttgcttt?tttcttttta 480
gctacgtatg?gagatggtga?gccgacagat?aatgctgcta?gattctataa?atggtttacc 540
gagggtgaag?agaaaggtga?atggcttgac?aagcttcaat?acgcagtgtt?tggacttggt 600
aacagacagt?atgagcattt?caacaagatt?gcgaaggtgg?tcgatgaaaa?acttgtggag 660
cagggtgcaa?agcgccttgt?tcctgttggc?atgggagacg?atgatcaatg?tatcgaagac 720
gacttcactg?catggaaaga?gttggtgtgg?cctgagttgg?atcaattact?tcgtgatgag 780
gatgatacat?ctgttgccac?tccatacaca?gctgctgttg?gagaataccg?tgttgtgttc 840
catgacaaac?cagagacata?tgatcaggat?caactgacaa?atggccatgc?tgttcatgat 900
gctcaacatc?catgcagatc?caatgtcgct?gtcaaaaagg?agctccattc?ccctctatct 960
gaccggtctt?gcactcattt?ggaatttgat?atctctaata?ctggattatc?gtatgaaact 1020
ggggaccatg?ttggagtcta?cgttgagaat?ctaagtgaag?ttgtggacga?agctgaaaaa 1080
ttaataggtt?taccgccgca?cacttatttc?tcagtacata?ctgataacga?agacgggaca 1140
ccacttggtg?gagcctcttt?gccacctcct?ttccctccat?gcactttaag?aaaagcattg 1200
gcttcctatg?ccgatgtttt?gagctctcct?aaaaagtcag?ctttgcttgc?tttagctgct 1260
catgctactg?attctactga?agctgataga?ctgaaatttt?ttgcgtctcc?tgctggaaag 1320
gatgaatatg?ctcagtggat?agttgcaagc?cacagaagtc?tccttgaggt?catggaggcc 1380
ttcccatcag?ctaagcctcc?gcttggtgtt?ttttttgcat?ctgtcgcccc?acgtttgcag 1440
ccgagatact?attccatttc?ttcttcccca?aagtttgcgc?caaataggat?tcatgtaact 1500
tgtgcattag?tgtatgagca?aacaccatca?ggccgcgttc?acaagggagt?ctgttcaaca 1560
tggatgaaga?atgccgtgcc?tatgacagaa?agccaggatt?gcagttgggc?cccaatttat 1620
gttagaacat?ccaatttcag?acttccttct?gatcctaagg?tcccagttat?catgattggc 1680
ccaggcactg?gattggctcc?atttagaggt?ttccttcagg?aaaggttagc?tcagaaggaa 1740
gctgggactg?agctcggaac?agccatctta?ttcttcggat?gcaggaatcg?caaagtggat 1800
ttcatatatg?aggacgagct?taataatttc?gtggagacgg?gggctctttc?cgagcttgtt 1860
acggccttct?ctcgtgaagg?tgccactaag?gagtacgtgc?aacacaagat?gactcagaag 1920
gcttcggata?tctggaattt?actctctgag?ggagcatatt?tgtatgtttg?cggtgatgcc 1980
aaaggcatgg?ccaaagatgt?acatcggact?ctgcacacta?ttgtgcaaga?acagggatct 2040
ctagactcct?caaaggcgga?gctctacgtg?aagaatctac?aaatggcagg?aagatatctc 2100
cgtgatgtat?ggtaa 2115
Claims (4)
1. method that adopts gene cyp71av1 and cpr cotransformation to improve artemislnin content in the sweet wormwood, it is characterized in that, clone AMORPHADIENE oxidase C YP71AV1 gene cyp71av1 and cytochrome P450 reductase CPR gene cpr from sweet wormwood, structure contains the plant expression vector of described dna molecular, with Agrobacterium tumefaciens mediated, cyp71av1 and cpr gene are imported the sweet wormwood and the plant that regenerates simultaneously, PCR detects the integration situation of external source goal gene cyp71av1 and cpr, high performance liquid chromatography-light scattering detector is measured artemislnin content in the sweet wormwood, and screening obtains the transgene abrotanum plant that artemislnin content improves.
2. employing gene cyp71av1 according to claim 1 and cpr cotransformation improve the method for artemislnin content in the sweet wormwood, it is characterized in that, comprise following concrete steps:
(1) adopt gene clone method to obtain sweet wormwood enzyme gene cyp71av1 and cpr;
(2) cyp71av1 and cpr gene operability be connected in expression regulation sequence, form the plant expression vector that contains cyp71av1 and cpr gene;
(3) plant expression vector that will contain cyp71av1 and cpr gene transforms agrobacterium tumefaciens, obtains to be used to transform the agrobacterium tumefaciens bacterial strain that contains cyp71av1 and cpr gene plant expression vector of sweet wormwood;
(4) utilize constructed agrobacterium tumefaciens bacterial strain to transform sweet wormwood, obtain the transgene abrotanum plant that detects through PCR;
(5) artemislnin content in the transgene abrotanum that obtains is carried out HPLC-ELSD and measure, obtain the transgene abrotanum plant that artemislnin content improves, screening obtains the transgene abrotanum plant that artemislnin content improves.
3. employing gene cyp71av1 according to claim 2 and cpr cotransformation improve the method for artemislnin content in the sweet wormwood, it is characterized in that, the described transgene abrotanum plant that detects through PCR is meant, the detection primer of cyp71av1 and cpr gene is synthesized in design respectively, carry out DNA cloning, the positive plant that the purpose band is observed in ultraviolet ray down is the transgene abrotanum plant.
4. employing gene cyp71av1 according to claim 2 and cpr cotransformation improve the method for artemislnin content in the sweet wormwood, it is characterized in that, described high performance liquid chromatography and light scattering detector are measured artemislnin content in the sweet wormwood plant, method is as follows: chromatographic column C-18 reverse phase silica gel post, 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, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor are 7, nebulizer gas pressure 5bar.
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| CN101475935B (en) * | 2009-01-08 | 2010-12-08 | 上海交通大学 | Cyp7lav1 gene promoter |
| CN101665796B (en) * | 2009-09-04 | 2012-04-25 | 上海柏泰来生物技术有限公司 | Artemisia apiacea4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase coding sequence |
| CN102776212A (en) * | 2011-12-20 | 2012-11-14 | 上海交通大学 | Production method of high-artemisinin-content transgene sweet wormwood plants |
| CN102643838A (en) * | 2012-01-17 | 2012-08-22 | 上海交通大学 | Method for improving content of artemisinin in artemisia apiacea by tran-ALDH1 gene |
| CN102776225A (en) * | 2012-07-18 | 2012-11-14 | 上海交通大学 | Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene |
| CN105296536A (en) * | 2015-11-12 | 2016-02-03 | 上海交通大学 | Transgenic sweet wormwood plant and cultivation method thereof |
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