CN107475265A - Nucleotide sequence, carrier and the method for improving content of artemisinin in sweet wormwood - Google Patents
Nucleotide sequence, carrier and the method for improving content of artemisinin in sweet wormwood Download PDFInfo
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Abstract
The present invention relates to genetic engineering field, in particular to nucleotide sequence, carrier and the method for improving content of artemisinin in sweet wormwood.A kind of nucleotide sequence, it is formed by connecting successively by the CRY1 genes in the promoter pCYP and sweet wormwood in sweet wormwood.The sequence can effectively facilitate the synthesis of Artemisinin in Artemisia annuna, improve the content of qinghaosu.Present invention also offers the carrier containing described nucleotide sequence, and good basis is provided for being transferred to for nucleotide sequence.Present invention also offers the method for improving content of artemisinin in sweet wormwood, above-mentioned nucleotide sequence or above-mentioned carrier are transferred in sweet wormwood, PCR detects the integration of external source target gene, the content of qinghaosu in high performance liquid chromatography and EISD measure transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves, and is laid the foundation to mass produce qinghaosu using transgene abrotanum.
Description
Technical field
The present invention relates to genetic engineering field, in particular to nucleotide sequence, carrier and improves Artemisinin in Artemisia annuna
The method of content.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.In its secondary metabolite
Qinghaosu is important anti-malaria medicaments, particularly more efficient to encephalic malaria and anti-chlorine quinoline malaria.Meanwhile in artemisia annua essential oil
Also it is rich in the monoterpene and sequiterpene of a large amount of Various Functions, such as 1,8- eucalyptol, carypohyllene, artemisia ketone etc..At present, qinghaosu
Conjoint therapy (ACTs) is the method for the maximally effective treatment malaria of world health organisation recommendations.With to qinghaosu pharmacological research
Progressively deeply, scientist has found that qinghaosu and its derivative also have anti-inflammatory, antitumor, anticancer and immunoregulatory work(
Energy.
But content of the qinghaosu in plant ginghao is very low, large-scale commercial production is restricted, can not be complete
The market demand in the full up foot whole world.Because qinghaosu is complicated, artificial synthesized difficulty is big, yields poorly, and cost is high, without can
Row.Qinghaosu is produced by yeast engineering, early investment cost is big, and limits throughput is unable to meet demand.Prior art table
Bright plant genetic engineering provides a feasible method to improve the content of Artemisinin in Artemisia annuna.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of nucleotide sequence, and the sequence can effectively facilitate the conjunction of Artemisinin in Artemisia annuna
Into improving the content of Artemisinin in Artemisia annuna.
The second object of the present invention is to provide the carrier containing described nucleotide sequence, and offer is transferred to for nucleotide sequence
Good basis.
The third object of the present invention is to provide a kind of method for improving content of artemisinin in sweet wormwood, by being transferred to target core
Acid sequence, screening obtain the transgene abrotanum plant for carrying the nucleotide sequence, and the artemislnin content in the plant improves, to utilize
Transgene abrotanum large-scale production qinghaosu lays the foundation.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of nucleotide sequence, it is formed by connecting successively by the CRY1 genes in the promoter pCYP and sweet wormwood in sweet wormwood.
Energy source of the light as photosynthesis of plant, be regulation development of plants and morphogenetic most important environment because
Son.Light can adjust gene expression and metabolism in plant.Research shows that sweet wormwood is abiotic in light, salt stress and heavy metal etc.
Qinghaosu yield significantly improves under conditions.Illumination effect terpene synthesizes, and the arabidopsis after light processing can accumulate more
Sequiterpene.Light can adjust qinghaosu metabolic pathway key gene ADS and CYP71AV1 gene expression amount.
Plant by phytochrome (absorb 600-750nm red/far-red light), cryptochrome (absorbing 320-500nm blue lights),
The light receptors such as UV-B photosensory cells (absorbing 280~320nm) respond different light quality, light intensity and photoperiod, wherein cryptochrome
Mediate the reaction of a variety of photoinductions.Found through experiment, arabidopsis cryptochrome CRY1 genes are overexpressed in sweet wormwood can improve green grass or young crops
Artemisin content.On this basis, the present inventor clones sweet wormwood cryptochrome CRY1 genes, and connects with the promoter pCYP in sweet wormwood
Connect, convert sweet wormwood, probe into metabolic regulation of the blue light receptor to qinghaosu approach, the positive strain for screening to obtain is qinghaosu high yield
Sweet wormwood plant, this provides a new way for large-scale production qinghaosu.
Further, the DNA sequence dna of the promoter pCYP is as shown in SEQ ID NO.1, the DNA sequences of the CRY1 genes
Row are as shown in SEQ ID NO.2.
Further, the amino acid sequence of the CRY1 gene codes is as shown in SEQ ID NO.12.
Further, the DNA sequence dna of the promoter pCYP is prepared by the following method：
The total serum IgE reverse transcription of sweet wormwood is cDNA, then with sense primer as shown in SEQ ID NO.3 and anti-sense primer such as
Enter performing PCR amplification shown in SEQ ID NO.4, obtain the DNA sequence dna of the promoter pCYP；
The DNA sequence dna of the CRY1 genes is prepared by the following method：
The total serum IgE reverse transcription of sweet wormwood is cDNA, then with sense primer as shown in SEQ ID NO.5 and anti-sense primer such as
Enter performing PCR amplification shown in SEQ ID NO.6, obtain the DNA sequence dna of the CRY1 genes.
Present invention also offers the carrier containing the nucleotide sequence.
The carrier is prepared by the following method：
The DNA sequence dna of the promoter pCYP is connected on cloning vector by ligase, is then introduced restriction enzyme site and is entered
Performing PCR expands, and obtained product carries out digestion, and expression vector carries out corresponding digestion, product connection, obtains first vector；
The DNA sequence dna of the CRY1 genes is connected on cloning vector by ligase, is then introduced restriction enzyme site and is carried out
PCR is expanded, and obtained product carries out digestion, and the first vector carries out corresponding digestion, product connection, produces the carrier；
Wherein, the promoter pCYP and the restriction enzyme site that the CRY1 genes introduce are identical in the part that both connect,
Remaining is different, and all exists in the multiple cloning sites of the expression vector.
Preferably, the expression vector is pCAMBIA2300；
The cloning vector is pLB carriers.
Preferably, the restriction enzyme site that the DNA sequence dna of the promoter pCYP introduces is BamH I and Sac I；
The restriction enzyme site that the DNA sequence dna of the CRY1 genes introduces is Hind III and BamH I.
Further, the DNA sequence dna of the promoter pCYP introduces the upstream and downstream used in the PCR amplifications that restriction enzyme site is carried out
Primer is as shown in SEQ ID NO.7 and SEQ ID NO.8；
The DNA sequence dna of the CRY1 genes introduces the upstream and downstream primer such as SEQ used in the PCR amplifications that restriction enzyme site is carried out
Shown in ID NO.9 and SEQ ID NO.10.
Present invention also offers a kind of method of high content of artemisinin in sweet wormwood, comprise the following steps：
Above-mentioned nucleotide sequence or above-mentioned carrier are transferred in sweet wormwood, screening carries the green grass or young crops of above-mentioned nucleotide sequence
Wormwood artemisia；
The sweet wormwood is planted, extracted to obtain qinghaosu.
Further, the nucleotide sequence, which is transferred to the method for sweet wormwood, includes electroporation, microinjection and particle gun；
The carrier is transferred in sweet wormwood by Agrobacterium tumefaciens mediated transgenic method.
Further, the sweet wormwood being transferred to is the blade of tender seedling.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention provides a kind of nucleotide sequence, and the sequence can effectively facilitate the synthesis of Artemisinin in Artemisia annuna, improve sweet wormwood
The content of middle qinghaosu.
(2) carrier provided by the invention containing described nucleotide sequence, good base is provided for being transferred to for nucleotide sequence
Plinth.
(3) a kind of method for improving content of artemisinin in sweet wormwood provided by the invention, by being transferred to target nucleic acid sequence, sieve
Select and screen the transgene abrotanum plant that acquisition carries the nucleotide sequence, the artemislnin content in the plant improves, and turns base to utilize
Because sweet wormwood large-scale production qinghaosu lays the foundation.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is the artemislnin content testing result for carrying the sweet wormwood and non-transformed common sweet wormwood that turn pCYP-CRY1 genes
Column diagram.
Embodiment
It is an object of the invention to overcome deficiency of the prior art, there is provided one kind turns pCYP-CRY1 genes and improves sweet wormwood
The method of middle artemislnin content.Gene cloning of the present invention, vector construction, genetic transformation, Molecular Detection, qinghaosu extraction
And assay is used for the present invention, the stable method for improving content of artemisinin in sweet wormwood is established, to be given birth on a large scale using sweet wormwood
Production qinghaosu is laid a good foundation.
Generally, the present invention is to provide it is a kind of turn pCYP-CRY1 genes improve content of artemisinin in sweet wormwood method,
Comprise the following steps：
(1) the promoter pCYP of CYP genes is obtained using gene clone method；
(2) CRY1 genes are obtained using gene clone method；
(3) pCYP the and CRY1 genes are connected to expression regulation sequence, build the plant expression of the gene containing pCYP-CRY1
Carrier；
(4) plant expression vector of the gene containing pCYP-CRY1 is converted into Agrobacterium tumefaciems, acquisition contains the pCYP-
The Agrobacterium tumefaciens strain of CRY1 gene plant expression vectors, then converts sweet wormwood；
(5) screening obtains the transgene abrotanum plant for carrying external source target gene pCYP-CRY1.
In step (1), the DNA sequence dna of pCYP genes is as shown in SEQ ID NO.1, the amino acid sequence of the pCYP genes
As shown in SEQ ID NO.13.
PCYP genes are prepared by the following method：
Sweet wormwood genome total serum IgE is extracted, the sweet wormwood genome total serum IgE obtained is obtained the by reverse transcriptase XL reverse transcriptions
One chain cDNA, according to the DNA sequence dna shown in SEQ ID NO.1, design amplifies the sense primer and anti-sense primer of promoter, institute
Sense primer is stated as the DNA sequence dna shown in SEQ ID NO.3, the anti-sense primer is the DNA sequence dna shown in SEQ ID NO.4,
Using the first described chain cDNA as template, using the sense primer and the anti-sense primer as primer pair, connected after PCR is expanded
Intermediate carrier PLB-pCYP is obtained on to PLB carriers to be sequenced.
In step (2), the DNA sequence dna of CRY1 genes is as shown in SEQ ID NO.2, and the amino acid sequence of CRY1 genes is such as
Shown in SEQ ID NO.14.
CRY1 genes are prepared by the following method：
Sweet wormwood genome total serum IgE is extracted, the sweet wormwood genome total serum IgE obtained is obtained the by reverse transcriptase XL reverse transcriptions
One chain cDNA, according to the DNA sequence dna shown in SEQ ID NO.2, design amplifies the sense primer of complete encoder block and downstream is drawn
Thing, the sense primer are the DNA sequence dna shown in SEQ ID NO.5, and the anti-sense primer is the DNA shown in SEQ ID NO.6
Sequence, using the first described chain cDNA as template, using the sense primer and the anti-sense primer as primer pair, expanded through PCR
After be connected on PLB carriers obtain intermediate carrier PLB-CRY1 be sequenced.
In step (3), the plant expression vector of the gene containing pCYP-CRY1 is built：
After PLB-pCYP sequencings are correct, by introducing Hind III and BamH I respectively before and after high-fidelity enzymatic amplification pCYP genes
The full length gene of restriction enzyme site obtains target gene fragment, i.e., using PLB-pCYP as template, its DNA sequence dna of sense primer such as SEQ
Shown in ID NO.7 and its DNA sequence dna of anti-sense primer enters performing PCR amplification as shown in SEQ ID NO.8 using high-fidelity enzyme, and recovery is drawn
Enter the pCYP genetic fragments of restriction enzyme site.The Hind III and digestion expression vector pCAMBIA2300 of BamH I is used again, is reclaimed
PCAMBIA2300 carrier large fragments, both products connection of recovery, conversion, picking monoclonal, extraction plasmid do PCR detections and
Digestion verification.
After PLB-CRY1 sequencings are correct, by introducing BamH I and the enzymes of Sac I respectively before and after high-fidelity enzymatic amplification CRY1 genes
The full length gene of enzyme site obtains target gene fragment, i.e., using PLB-CRY1 as template, its DNA sequence dna of sense primer such as SEQ ID
Shown in NO.9 and its DNA sequence dna of anti-sense primer enters performing PCR amplification as shown in SEQ ID NO.10 using high-fidelity enzyme, and recovery introduces
The CRY1 genetic fragments of restriction enzyme site.Expression vector pCAMBIA2300 of the digestion with PCYP, recovery pCAMBIA2300 carriers are big
Fragment, by seamless clone technology by CRY1 genetic recombination to the expression vector pCAMBIA2300 with pCYP on, convert and picking
Monoclonal, extraction plasmid do PCR detections and digestion verification.
In step (4), pCYP-CRY1 genetic transformation, comprise the following steps：The preculture of explant；Agrobacterium and explant
The co-cultivation of body；The screening of resistance regeneration plant；
Wherein, the preculture comprises the following steps：Seeds of southernwood soaks 1min with 75% ethanol, then uses 20%NaClO
20min is soaked, aseptic water washing 3-4 times, surface moisture is blotted with sterile blotting paper, is inoculated in the MS solid mediums of no hormone
In, 25 DEG C of illumination cultivations, you can obtain sweet wormwood aseptic seedling, after seedling length to 5cm or so, clip tests for sterility explant is used for
Conversion.
The co-cultivation comprises the following steps：The blade explant is gone to and co-cultured in culture medium, is added dropwise containing activation
The 1/2MS suspensions of the Agrobacterium tumefaciems engineering bacteria of the good binary expression vector of gene plant containing pCYP-CRY1, make explant
Fully contact with bacterium solution, 28 DEG C of light cultures 3 days, trained with the 1/2MS liquid being added dropwise in the Agrobacterium tumefaciems without target gene
The blade explant for supporting base suspension is control.
In step (5), screening comprises the following steps：The sweet wormwood explant for co-culturing 3 days is transferred to germination screening
In 25 DEG C of illumination cultivations on base, squamous subculture once, can obtain kanamycins after 2-3 squamous subculture every two weeks
(Kan) resistance Multiple Buds, well-grown Kan resistances Multiple Buds is cut to be transferred on root media and cultivated to taking root,
Kan resistances regeneration sweet wormwood plant can be obtained.
Further, performing PCR detection is also entered to the Kan resistances regeneration sweet wormwood plant of acquisition, PCR detections comprise the following steps：
Expression cassette Pcambia-pCYP-CRY1 sequences pCYP and CRY1 according to where target gene separately design forward primer and reversely drawn
Thing；Carry out DNA cloning；Viewed under ultraviolet radiation, if purpose band is the positive, the strain is the transgene abrotanum plant；
The DNA sequence dna of the forward primer is as shown in SEQ ID NO.11, the DNA sequence dna such as SEQ ID of the reverse primer
Shown in NO.10.
Further, in addition to artemislnin content in the transgene abrotanum high performance liquid chromatography and evaporative light are carried out
Scatter detector measure (HPLC-ELSD) measure, screening obtain the transgene abrotanum plant that artemislnin content improves.
Further, the HPLC-ELSD measure includes following condition：Chromatographic column used is C-18 reverse phase silica gel posts, stream
Dynamic is mutually 70 from volume ratio:30 first alcohol and water, 30 DEG C, flow velocity 1.0mL/min of column temperature, the μ L of sample size 20, evaporative light-scattering
40 DEG C of detector drift tube temperature, amplification coefficient 7, nebulizer gas pressure 5bar.
The method for turning pCYP-CRY1 genes and improving content of artemisinin in sweet wormwood of the present invention, will using gene engineering method
The over-express vector of the promoter pCYP driving enzyme, namely chalcone isomerase CRY1 genes of cytochrome P 450 monooxygenases gene imports
In sweet wormwood plant, the transgene abrotanum strain that artemislnin content significantly improves is obtained, turns sweet wormwood in pCYP-CRY1 gene sweet wormwoods
The content of element can reach the 17.52mg/g of dry weight, be 1.90 times of non-transformed common sweet wormwood (9.21mg/g dry weights), the hair
The bright large-scale production for qinghaosu and offer high yield, stable source new drugs are significant.
The technology contents of the present invention are described further with reference to the accompanying drawings and examples.Following examples will help
The present invention is further understood in those skilled in the art, but the invention is not limited in any way.Do not noted in the following example
The experimental method of bright actual conditions, generally cloned according to normal condition, such as Sambrook equimoleculars：Laboratory manual is shown in New
York:Condition described in Cold Spring Harbor Laboratory Press versions in 1989, or according to manufacturer
Proposed condition.
Agrobacterium tumefaciems EHA105 of the present invention exists《Huang Yali, Jiang Xiliang, field Yunlong, Guo Ping, Zhu Changxiong；Root
The research of the agriculture bacillus mediated trichoderma harzianum genetic transformation of cancer, Chinese biological engineering magazine, 2008,28 (3)：38-43》Document
Disclosed in.Agrobacterium tumefaciems EHA105 can be obtained by disclosing commercially available commercial channel, such as can be public from Australian CAMBIA
Department buys, strain number Gambar1.
Embodiment 1
Step 1, the clone of sweet wormwood pCYP and CRY1 gene
(1) extraction of sweet wormwood genome total serum IgE
The extraction of sweet wormwood genome total serum IgE uses the RNA kits of TIANGEN companies.Take 50-100mg sweet wormwood young tender leafs
Piece rapid grind into powder in liquid nitrogen, 550 μ L lysates RL (having added mercaptoethanol) are added, the acutely concussion that is vortexed mixes.
9800rpm centrifuges 5min, draws 450 μ L and is transferred on Filter column CS, and CS is placed in collecting pipe, and 12000rpm is centrifuged 5 minutes, small
The heart is drawn in the centrifuge tube of the μ L to RNase-free of supernatant 400 in collecting pipe, is slowly added to the anhydrous second of 0.5 times of supernatant volume
Alcohol, mix, be transferred in adsorption column CR3,12000rpm centrifugation 1min, abandon waste liquid, CR3 is put back in collecting pipe.Added into CR3
350 μ L protein liquid removals RW1,12000rpm centrifugation 1min, abandon waste liquid, CR3 are put back in collecting pipe.80 μ L are added to CR3 centers
DNase1 working solutions (the μ L RDD solution of 10 μ L DNase1 storing liquids+70, soft mix), be stored at room temperature 15 minutes.To CR3
It is middle to add 350 μ L protein liquid removals RW1,12000rpm centrifugation 1min, waste liquid is abandoned, CR3 is put back in collecting pipe.Added into CR3
500 μ L rinsing liquids RW (addition ethanol), are stored at room temperature 2 minutes, 12000rpm centrifugation 1min, outwell the waste liquid in collecting pipe, will
CR3 is put back in collecting pipe, is repeated once.12000rpm centrifuges 5min.CR3 is placed in room temperature and places 5min, CR3 is put into new
In RNase-free, 70 μ LRNase-free dd H are vacantly added dropwise to the middle part of adsorbed film2O, 2min is stored at room temperature,
12000rpm centrifuges 2min, obtains RNA solution.10 μ L are taken to be put in RNase-free tubules, for running glue and surveying concentration；It is remaining
60 μ L be put in RNase-free centrifuge tubes -80 DEG C of preservations.
(2) clone of sweet wormwood pCYP and CRY1 genes
The sweet wormwood genome total serum IgE obtained is obtained into the first chain cDNA by reverse transcriptase XL (AMV) reverse transcription, according to green grass or young crops
The coded sequence (DNA sequence dna shown in SEQ ID NO.1) of wormwood artemisia pCYP genes, design amplify the sense primer (SEQ of promoter
DNA sequence dna shown in ID NO.3) and anti-sense primer (DNA sequence dna shown in SEQ ID NO.4), with the first described chain cDNA
For template, acquisition intermediate carrier PLB-pCYP on PLB carriers is connected to by ligase after PCR is expanded, is sequenced, DNA
Sequencing is sequenced by Shanghai Sani bio tech ltd and completed.Sequencing result shows, sequence (the SEQ ID cloned
DNA sequence dna shown in NO.1) with the sequence of sweet wormwood pCYP genes of report there is nonsense point mutation.
According to the coded sequence (DNA sequence dna shown in SEQ ID NO.2) of the sweet wormwood CRY1 genes, design has amplified
The sense primer (DNA sequence dna shown in SEQ ID NO.5) and anti-sense primer (the DNA sequences shown in SEQ ID NO.6 of whole encoder block
Row), using the first described chain cDNA as template, it is connected to after PCR is expanded by ligase on PLB carriers and obtains intermediate carrier
PLB-CRY1, it is sequenced, determined dna sequence is sequenced by Shanghai Sani bio tech ltd and completed.Sequencing result shows,
The sequence (DNA sequence dna shown in SEQ ID NO.2) cloned and the code sequence for the sweet wormwood CRY1 genes reported in GenBank
Nonsense point mutation is shown, but they are completely the same on amino acid sequence.
The correct sweet wormwood cytochrome P 450 monooxygenases gene of sequence is obtained from sweet wormwood using gene clone method
Promoter pCYP and enzyme, namely chalcone isomerase CRY1 genes, for by turn pCYP-CRY1 genes improve content of artemisinin in sweet wormwood
Basis is provided.
Step 2, the structure of the plant expressing vector of the gene containing pCYP-CRY1
(1) pCYP and CRY1 target gene fragment obtains
Using PLB-pCYP as template, design introduces the sense primer of the restriction enzyme sites of Hind III (shown in SEQ ID NO.7
DNA sequence dna) and with the anti-sense primers of BamH I (DNA sequence dna shown in SEQ ID NO.8) be primer pair, carried out using high-fidelity enzyme
PCR is expanded.
Using PLB-CRY1 as template, design introduces the sense primer (DNA shown in SEQ ID NO.9 of the restriction enzyme sites of BamH I
Sequence) and anti-sense primer (DNA sequence dna shown in SEQ ID NO.10) with Sac I be primer pair, carried out using high-fidelity enzyme
PCR is expanded.
(2) structure of the plant expression vector of the gene containing pCYP and CRY1
Using pCAMBIA2300 as expression vector, above-mentioned pCYP promoters are connected into its corresponding restriction enzyme site position, then will
CRY1 genes are connected into its corresponding restriction enzyme site position.Specifically, with Hind III and the double digestion expression vectors of BamH I
PCAMBIA2300, the pCYP genetic fragments of the corresponding restriction enzyme site of introducing of recovery and pCAMBIA2300 carriers large fragment are connected
Connect, convert, picking monoclonal, extraction plasmid does PCR detections and digestion verification.After sequencing is correct, first vector is obtained.
Obtained by the full length gene for introducing BamH I and the restriction enzyme sites of Sac I before and after high-fidelity enzymatic amplification CRY1 genes respectively
Target gene fragment；Digestion first vector, pCAMBIA2300 carrier large fragments are reclaimed, phase is introduced into by seamless clone technology
The CRY1 genetic recombination of restriction enzyme site is answered to the expression vector pCAMBIA2300 with pCYP, is converted and picking monoclonal, extraction
Plasmid does PCR detections and digestion verification.
The present embodiment gives birth to the promoter pCYP of cytochrome P 450 monooxygenases gene (CYP71AV1) and sweet wormwood flavones
Thing route of synthesis key gene CRY1 is operatively connectable to expression regulation sequence, forms the plant of the gene containing pCYP-CRY1
Thing expression vector, the expression vector can be used for the content that Artemisinin in Artemisia annuna is improved by metabolic engineering strategies.
Step 3, the acquisition of the double base plant expression vector Agrobacterium tumefaciems of gene containing pCYP-CRY1
It (such as EHA105, is market that the plant binary expression vector of the above-mentioned gene containing pCYP-CRY1 is transferred into Agrobacterium tumefaciems
Have the biomaterial of public offering, can be bought from Australian CAMBIA companies, strain number is Gambar 1), and carry out
PCR is verified.
Step 4, Agrobacterium tumefaciens mediated pCYP-CRY1 genetic transformation sweet wormwood
(1) preculture of explant
75% ethanol of seeds of southernwood soaks 1min, then soaks 20min with 20%NaClO, aseptic water washing 3-4 times, uses
Sterile blotting paper blots surface moisture, is inoculated in MS (Murashige and Skoog, 1962) solid medium of no hormone
In, 25 DEG C, 16h/8h (light dark) illumination cultivation, you can obtain sweet wormwood aseptic seedling.After seedling length to 5cm or so, clip aseptic seedling
Blade explant is used to convert.
(2) co-cultivation of Agrobacterium and explant
By the blade explant, the co-cultivation culture medium (μ of 1/2MS+AS 100 for adding acetosyringone (AS) are gone to
Mol/L in), the 1/2MS of the Agrobacterium tumefaciems engineering bacteria containing the pCYP-CRY1 gene plant binary expression vectors activated is added dropwise
Suspension, explant is set fully to be contacted with bacterium solution, 28 DEG C of light cultures 3 days.To be added dropwise in the Agrobacterium tumefaciems without target gene
1/2MS fluid nutrient medium suspensions blade explant for control.
(3) screening of resistance regeneration plant
By it is described co-culture the sweet wormwood explant of 3 days be transferred to addition 6-benzyl aminopurine (6-BA), methyl α-naphthyl acetate (NAA),
Germination screening base (the MS+6-BA0.5mg/L+NAA 0.05mg/L+Kan of kanamycins (Kan), carbenicillin (cb)
50mg/L+cb 500mg/L) in 25 DEG C, 16h/8h (light dark) illumination cultivation, every two weeks squamous subculture once, by 2-3 times
Kan resistance Multiple Buds can be obtained after squamous subculture, well-grown resistance Multiple Buds are cut and are transferred to root media (1/
2MS+cb 125mg/L) on cultivate to taking root, you can obtain Kan resistances regeneration sweet wormwood plant.
Step 5, the PCR detections of transgene abrotanum plant
Expression cassette pCAMBIA2300 sequences pCYP and CRY1 according to where target gene separately design forward primer (such as SEQ
DNA sequence dna shown in ID NO.11) and reverse primer (DNA sequence dna as shown in SEQ ID NO.10) target gene is examined
Survey.As a result show, using designed PCR special primers, specific DNA fragment can be amplified, and with non-transformed sweet wormwood genome
When DNA is template, any fragment is not amplified.
Plant expression vector is converted Agrobacterium tumefaciems by the present embodiment, obtains the base containing pCYP-CRY1 for converting sweet wormwood
Because of the Agrobacterium tumefaciens strain of plant expression vector, sweet wormwood is converted using constructed Agrobacterium tumefaciens strain, obtains and is examined through PCR
The transgene abrotanum plant of survey.The sweet wormwood strain that the acquisition of transgene abrotanum plant obtains higher artemislnin content for screening provides
Direct material.
Step 6, artemislnin content in transgene abrotanum is determined using HPLC-ELSD
(1) preparation of HPLC-ELSD conditions and system suitability and standard liquid
HPLC：Using the systems of water alliance 2695, chromatographic column is C-18 reverse phase silica gel posts (Symmetry
Shiled TM C18,5 μm, 250 × 4.6mm, Waters), mobile phase is first alcohol and water, methanol：The volume ratio of water is 70:30,
30 DEG C, flow velocity 1.0mL/min of column temperature, the μ L of sample size 10, sensitivity (AUFS=1.0), theoretical cam curve are calculated by qinghaosu peak
Not less than 2000.
ELSD：Using the systems of water alliance 2420,40 DEG C of EISD drift tube temperature, amplification
Coefficient (gain) is 7, nebulizer gas pressure 5bar；
Precision weighs qinghaosu standard items (Sigma companies) 2.0mg and is completely dissolved with 1mL methanol, obtains 2mg/mL sweet wormwoods
Plain standard solution, be stored in -20 DEG C it is standby.
Mobile phase is first alcohol and water in the present embodiment, volume ratio 70%：30%, the retention time of qinghaosu is
5.1min, peak type are good.Theoretical cam curve is calculated by qinghaosu is not less than 2000.
(2) making of standard curve
The reference substance solution is distinguished to μ L of sample introduction 2,4 μ L, 6 μ L, 8 μ L, 10 μ L record collection of illustrative plates under corresponding chromatographic condition
And chromatographic parameter, regression analysis is carried out to standard items content (X, μ g) with peak area (Y) respectively.By studying, in the present embodiment
Good log-log linear relationships are presented in qinghaosu in 4-20 μ g ranges.(3) measure of the preparation of sample and artemislnin content
Take fresh sweet wormwood blade to be ground after 55 DEG C of oven for drying, take 0.1g samples to add 1mL first in 2mL EP pipes
Supernatant is drawn after alcohol ultrasound 30min, 12000rpm centrifugation 10min to manage in new EP；Precipitation adds 1mL methanol and continues ultrasound
Supernatant is drawn after 30min, 12000rpm centrifugation 10min, filter 1mL after supernatant twice is mixed manages in new EP.
Artemislnin content is determined using HPLC-ELSD, sample feeding volume is 20 μ L, and linear regression is substituted into according to peak area
Equation calculates the artemislnin content (mg) in sample, then divided by sample sweet wormwood leaf dry weight (g), so as to calculate sweet wormwood plant
The content of middle qinghaosu.
The present embodiment transfer pCYP-CRY1 genes significantly improve content of artemisinin in sweet wormwood.As shown in figure 1, turn pCYP-
The content of CRY1 gene Artemisinin in Artemisia annuna can reach the 17.52mg/g of dry weight, be non-transformed common sweet wormwood (9.21mg/g
Dry weight) 1.90 times.CK represents non-transformed common sweet wormwood in figure；PCYP-CRY1-41 to pCYP-CRY1-69 represents to screen respectively
The different transgenic lines arrived.
The present embodiment determines artemislnin content in transgene abrotanum using HPLC-ELSD methods, using conversion CYP-CRY1
The metabolic engineering strategies of gene obtain the sweet wormwood plant of qinghaosu high yield, and a kind of ideal is provided for large-scale production qinghaosu
Method.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Sequence table
<110>Guangzhou nm crystallite bio tech ltd of Shanghai Communications University
<120>Nucleotide sequence, carrier and the method for improving content of artemisinin in sweet wormwood
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1150
<212> DNA
<213>Sweet wormwood (Artemisia annua L.)
<400> 1
aatgggtcaa tttcgggttg aggttttctg acctgccaac ctgacaaccc gaacccgcca 60
atccgaaccc gacccgaatt gccaccccta aatacatgta agcatttgga ttgcaataaa 120
cactacaaaa ctcatagatc cgtatattta caaaactaag aaaaaagaca aagctaatct 180
aaacaataca aaactctaaa agccgattga cacaactaca aaacatgacg cacaagcaat 240
acaaaaatta ggggtgttca agcaacacta caaaacatca tcactactga catctttatg 300
tgttttaacc cactcataag tattatacta atacatgtta caagcctctc atgtattata 360
caactgacat cttgttttta tcatcattta tgtaatggcc ctgtgacaat aaacaaaatg 420
atagccatat aatccacttg cgtcgttggc ttttcaagtt gataccatct aacattctaa 480
cacacgtata gcaaacataa tgaagtaaaa tacacaaaat caaattagta tattggtcaa 540
ataaggatag caatggtcca tttttgacaa actcagtagc ataacatgtt ttatatctaa 600
tagctttttc cagtttataa attcatatct tttacgtgtc aatttcttga ctttaacact 660
tatataagtt ataatcttca ttttttggca acaactatta tatttttagg tcatgtttaa 720
ttttaatttt gacattaagt ttacataact ttaatactat aatacactac tttcagtttg 780
gatgaacacc ttgatttcca ttataacatc tctgacgtca acttttgaat ctaattaagg 840
tcactcacaa actaaaactt tcggcatccc cttattttgt tgaatgtaac caaaccacgt 900
aagttttcct ttcttggtca atgcataaat gtaaaagatg gcatgttata ttaactaaac 960
aattaatacg aaataatttt tcataataaa actggacata aatgtcaaga gtggactact 1020
gcacattcaa tgagggtgtc attgaaattc caaatggggt agcacatatt caattccgca 1080
tcaaccccat acaacaatat aaacaacctt aattaacatt tgacaccatg aagagtatac 1140
taaaagcatg 1150
<210> 2
<211> 2013
<212> DNA
<213>Sweet wormwood (Artemisia annua L.)
<400> 2
atgtcaggag gtgttagtat agtgtggttt aggagagatc taagggtaga agataatcca 60
gctttggctg caggtgtgag ggcaggtcag gtgatagctg tgtatatatg ggcaccagaa 120
gaagaaggtc attattatcc tggaagggta tcaagatggt ggttaaaaca aagtttagct 180
ttattggatt catctttgaa aaaccttggt acttctttgg ttactaaaag gtctactgat 240
agtgttgcat cacttcttga ggttgttaag tcaactggtg ctactcaggt cctatttaat 300
cacttatatg atcctttgtc tctcgttagg gatcatcgaa tgaaggaggt tttaaccaat 360
aatggcatag cagtgcgttc cttcaacgca gatttgctat atgaaccatg ggaaattctt 420
gatgataagg gccgcccgtt caatacgttt acagaattct gggataggtg ccttagtatg 480
ccttatgatc ctgaatctcc acaactccca ccaaagaaga tcatctcagg tgacttatca 540
aagtgccctt cagagacaat aatatttgag gatgaatcag agaaaggaag caacgctctt 600
ctagcgcgag catggacacc cggttggagc aatgcagata aagcactagc agctttcatg 660
aacgggccat tactagagta ctccagaaac cgtagaaaag ctgatagtgc cacaacctca 720
ttcctatctc cacacctaca ttttggagaa cttagcgtac gaaaagtttt tcatttgctc 780
cgaatcaaac aagtcctttg ggccaatgaa ggtaacactg caggcgaaga aagtgtaaat 840
ttattcctta aatctatagg tcttagggaa tattcaaggt acatgagttt taatcatcca 900
tatagtcacg aaaggcctct tttgggtcat ttgaagtttt tcccttggag aatagatgaa 960
ggttatttta aggcgtggag acaaggtaga acgggttacc cgttggttga tgcagggatg 1020
agagaactgt gggctactgg gtggctgcat gacaggatac gggtcgtggt ttcgagtttc 1080
tttgtgaagg ttttacagtt accctggaga tggggcatga agtatttttg ggatacactt 1140
ttggatgctg atcttgaaag tgatgctttg ggttggcagt atataacggg tactttacca 1200
gatggccgtg agtttgaccg tattgataat ccacagtttg aggggtatac attcgaccca 1260
aatggcgaat acgtgcgtag gtggcttcct gaactcgcta gactaccaac cgagtggatc 1320
caccacccat gggatgcacc agaatacgta cttcaagctg ctggtatcga gcttgggtca 1380
aactaccctc tccccatagt caaactagat gcagcgaaat ctcgattaca agaagcactt 1440
gctcaaatgt ggcaatatga agcttcatct agagccattc ttgaaaacgg attagaagaa 1500
ggtcttggag actcaaccga agctacttcc ttaatagcat tccctcaaga tatggatgag 1560
atggatatgg acagtaatgc catcagagcc acaactcata caaccaccac tatccgtcat 1620
tatgaagatc aaatggttcc aagtatgaca acgtctttgt taagaggcgg tgaagaagaa 1680
gaatcttctt cagatgtcgg aaatcaagca gaagatagca gggctgaagt tcctattaat 1740
caaggcatgg atgatactcg tatgggaaga acagatgata tgcagcaagt gaatgatatc 1800
actgcggcat tgaggagtat ggatgatttg caagccgaat cttctaacac ttctggttca 1860
acgagggaaa gagacggggg tgttgtgcca gtgtggtcac cttcaacttc tagtttcaac 1920
gagacgtttg ttggtgaaga tagtacttat ttgcaaaggc atccgcaatc tcaccagtta 1980
gtcaactgga ggcggctttc ccaaaccggg tga 2013
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 3
aatgggtcaa tttcgggttg ag 22
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 4
catgctttta gtatactctt ca 22
<210> 5
<211> 46
<212> DNA
<213>Artificial sequence ()
<400> 5
atgtcaggag gtgttagtat ag 22
<210> 6
<211> 43
<212> DNA
<213>Artificial sequence ()
<400> 6
tcacccggtt tgggaaagcc gc 22
<210> 7
<211> 28
<212> DNA
<213>Artificial sequence ()
<400> 7
ccaagcttaa tgggtaaatt tcgggttg 28
<210> 8
<211> 28
<212> DNA
<213>Artificial sequence ()
<400> 8
cgggatccca tgcttttagt atactctt 28
<210> 9
<211> 46
<212> DNA
<213>Artificial sequence ()
<400> 9
gaagagtata ctaaaagcag gatccatgtc aggaggtgtt agtata 46
<210> 10
<211> 46
<212> DNA
<213>Artificial sequence ()
<400> 10
gaacgatcgg ggaaattcga gctccccggt ttgggaaagc cgcctc 46
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 11
tcaaccccat acaacaatat aa 22
<210> 12
<211> 670
<212> PRT
<213>Sweet wormwood (Artemisia annua L.)
<400> 12
Met Ser Gly Gly Val Ser Ile Val Trp Phe Arg Arg Asp Leu Arg Val
1 5 10 15
Glu Asp Asn Pro Ala Leu Ala Ala Gly Val Arg Ala Gly Gln Val Ile
20 25 30
Ala Val Tyr Ile Trp Ala Pro Glu Glu Glu Gly His Tyr Tyr Pro Gly
35 40 45
Arg Val Ser Arg Trp Trp Leu Lys Gln Ser Leu Ala Leu Leu Asp Ser
50 55 60
Ser Leu Lys Asn Leu Gly Thr Ser Leu Val Thr Lys Arg Ser Thr Asp
65 70 75 80
Ser Val Ala Ser Leu Leu Glu Val Val Lys Ser Thr Gly Ala Thr Gln
85 90 95
Val Leu Phe Asn His Leu Tyr Asp Pro Leu Ser Leu Val Arg Asp His
100 105 110
Arg Met Lys Glu Val Leu Thr Asn Asn Gly Ile Ala Val Arg Ser Phe
115 120 125
Asn Ala Asp Leu Leu Tyr Glu Pro Trp Glu Ile Leu Asp Asp Lys Gly
130 135 140
Arg Pro Phe Asn Thr Phe Thr Glu Phe Trp Asp Arg Cys Leu Ser Met
145 150 155 160
Pro Tyr Asp Pro Glu Ser Pro Gln Leu Pro Pro Lys Lys Ile Ile Ser
165 170 175
Gly Asp Leu Ser Lys Cys Pro Ser Glu Thr Ile Ile Phe Glu Asp Glu
180 185 190
Ser Glu Lys Gly Ser Asn Ala Leu Leu Ala Arg Ala Trp Thr Pro Gly
195 200 205
Trp Ser Asn Ala Asp Lys Ala Leu Ala Ala Phe Met Asn Gly Pro Leu
210 215 220
Leu Glu Tyr Ser Arg Asn Arg Arg Lys Ala Asp Ser Ala Thr Thr Ser
225 230 235 240
Phe Leu Ser Pro His Leu His Phe Gly Glu Leu Ser Val Arg Lys Val
245 250 255
Phe His Leu Leu Arg Ile Lys Gln Val Leu Trp Ala Asn Glu Gly Asn
260 265 270
Thr Ala Gly Glu Glu Ser Val Asn Leu Phe Leu Lys Ser Ile Gly Leu
275 280 285
Arg Glu Tyr Ser Arg Tyr Met Ser Phe Asn His Pro Tyr Ser His Glu
290 295 300
Arg Pro Leu Leu Gly His Leu Lys Phe Phe Pro Trp Arg Ile Asp Glu
305 310 315 320
Gly Tyr Phe Lys Ala Trp Arg Gln Gly Arg Thr Gly Tyr Pro Leu Val
325 330 335
Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly Trp Leu His Asp Arg
340 345 350
Ile Arg Val Val Val Ser Ser Phe Phe Val Lys Val Leu Gln Leu Pro
355 360 365
Trp Arg Trp Gly Met Lys Tyr Phe Trp Asp Thr Leu Leu Asp Ala Asp
370 375 380
Leu Glu Ser Asp Ala Leu Gly Trp Gln Tyr Ile Thr Gly Thr Leu Pro
385 390 395 400
Asp Gly Arg Glu Phe Asp Arg Ile Asp Asn Pro Gln Phe Glu Gly Tyr
405 410 415
Thr Phe Asp Pro Asn Gly Glu Tyr Val Arg Arg Trp Leu Pro Glu Leu
420 425 430
Ala Arg Leu Pro Thr Glu Trp Ile His His Pro Trp Asp Ala Pro Glu
435 440 445
Tyr Val Leu Gln Ala Ala Gly Ile Glu Leu Gly Ser Asn Tyr Pro Leu
450 455 460
Pro Ile Val Lys Leu Asp Ala Ala Lys Ser Arg Leu Gln Glu Ala Leu
465 470 475 480
Ala Gln Met Trp Gln Tyr Glu Ala Ser Ser Arg Ala Ile Leu Glu Asn
485 490 495
Gly Leu Glu Glu Gly Leu Gly Asp Ser Thr Glu Ala Thr Ser Leu Ile
500 505 510
Ala Phe Pro Gln Asp Met Asp Glu Met Asp Met Asp Ser Asn Ala Ile
515 520 525
Arg Ala Thr Thr His Thr Thr Thr Thr Ile Arg His Tyr Glu Asp Gln
530 535 540
Met Val Pro Ser Met Thr Thr Ser Leu Leu Arg Gly Gly Glu Glu Glu
545 550 555 560
Glu Ser Ser Ser Asp Val Gly Asn Gln Ala Glu Asp Ser Arg Ala Glu
565 570 575
Val Pro Ile Asn Gln Gly Met Asp Asp Thr Arg Met Gly Arg Thr Asp
580 585 590
Asp Met Gln Gln Val Asn Asp Ile Thr Ala Ala Leu Arg Ser Met Asp
595 600 605
Asp Leu Gln Ala Glu Ser Ser Asn Thr Ser Gly Ser Thr Arg Glu Arg
610 615 620
Asp Gly Gly Val Val Pro Val Trp Ser Pro Ser Thr Ser Ser Phe Asn
625 630 635 640
Glu Thr Phe Val Gly Glu Asp Ser Thr Tyr Leu Gln Arg His Pro Gln
645 650 655
Ser His Gln Leu Val Asn Trp Arg Arg Leu Ser Gln Thr Gly
660 665 670
Claims (10)
1. a kind of nucleotide sequence, it is characterised in that connected successively by the promoter pCYP in sweet wormwood with the CRY1 genes in sweet wormwood
Form.
2. nucleotide sequence according to claim 1, it is characterised in that the DNA sequence dna of the promoter pCYP such as SEQ ID
Shown in NO.1, the DNA sequence dna of the CRY1 genes is as shown in SEQ ID NO.2, further, the ammonia of the CRY1 gene codes
Base acid sequence is as shown in SEQ ID NO.12.
3. nucleotide sequence according to claim 1, it is characterised in that the DNA sequence dna of the promoter pCYP passes through following
It is prepared by method：
The total serum IgE reverse transcription of sweet wormwood is cDNA, then with sense primer as shown in SEQ ID NO.3 and anti-sense primer such as SEQ ID
Enter performing PCR amplification shown in NO.4, obtain the DNA sequence dna of the promoter pCYP；
The DNA sequence dna of the CRY1 genes is prepared by the following method：
The total serum IgE reverse transcription of sweet wormwood is cDNA, then with sense primer as shown in SEQ ID NO.5 and anti-sense primer such as SEQ ID
Enter performing PCR amplification shown in NO.6, obtain the DNA sequence dna of the CRY1 genes.
4. the carrier containing the nucleotide sequence described in claim any one of 1-3.
5. carrier according to claim 4, it is characterised in that the carrier is prepared by the following method：
The DNA sequence dna of the promoter pCYP is connected on cloning vector by ligase, is then introduced restriction enzyme site and is entered performing PCR
Amplification, obtained product carry out digestion, and expression vector carries out corresponding digestion, product connection, obtains first vector；
The DNA sequence dna of the CRY1 genes is connected on cloning vector by ligase, is then introduced restriction enzyme site and is entered performing PCR expansion
Increase, obtained product carries out digestion, and the first vector carries out corresponding digestion, product connection, produces the carrier；
Wherein, the promoter pCYP and the restriction enzyme site that the CRY1 genes introduce are identical in the part that both connect, remaining
Difference, and all the expression vector multiple cloning sites exist.
6. carrier according to claim 5, it is characterised in that the expression vector is pCAMBIA2300；
The cloning vector is pLB carriers.
7. carrier according to claim 5, it is characterised in that the digestion position that the DNA sequence dna of the promoter pCYP introduces
Point is BamH I and Sac I；
The restriction enzyme site that the DNA sequence dna of the CRY1 genes introduces is Hind III and BamH I.
8. carrier according to claim 7, it is characterised in that the DNA sequence dna of the promoter pCYP introduces restriction enzyme site
Upstream and downstream primer used in the PCR amplifications of progress is as shown in SEQ ID NO.7 and SEQ ID NO.8；
The DNA sequence dna of the CRY1 genes introduces the upstream and downstream primer such as SEQ ID used in the PCR amplifications that restriction enzyme site is carried out
Shown in NO.9 and SEQ ID NO.10.
A kind of 9. method for improving content of artemisinin in sweet wormwood, it is characterised in that comprise the following steps：
Nucleotide sequence described in claim any one of 1-3 or the carrier described in claim any one of 4-8 are transferred to sweet wormwood
In, screening carries the sweet wormwood of above-mentioned nucleotide sequence；
The sweet wormwood is planted, extracted to obtain qinghaosu.
10. according to the method for claim 9, it is characterised in that the method that the nucleotide sequence is transferred to sweet wormwood is worn including electricity
Kong Fa, microinjection and particle gun；
The carrier is transferred in sweet wormwood by Agrobacterium tumefaciens mediated transgenic method；
Further, the sweet wormwood being transferred to is the blade of tender seedling.
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| CN113755504A (en) * | 2021-09-29 | 2021-12-07 | 西南大学 | Artemisia apiacea mediator AaMED25 gene and application thereof |
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