CN103740737B - A kind of D-amino acid dehydrogenase gene bll6812S and its preparation method and application - Google Patents
A kind of D-amino acid dehydrogenase gene bll6812S and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of high-efficiency nitrogen-fixing soybean D-amino acid dehydrogenase gene bll6812S and its preparation method and application of raw rhizobium slowly that derives from, is its nucleotide sequence as SEQ? ID? is the amino acid sequence of its coding as SEQ shown in No1? ID? shown in No2. This D-amino acid dehydrogenase gene bll6812S be by derive from high-efficiency nitrogen-fixing soybean slowly the D-amino acid dehydrogenase gene bll6812 of raw rhizobium transform according to plant-preference codon, utilize method for synthesizing gene to be prepared from. Described bll6812S genetic transformation arabidopsis is obtained to transgenic arabidopsis, and this transgenic arabidopsis is obviously better than wild type arabidopsis in the performance of resistance glyphosate, can be applicable in glyphosate resistant crops breeding.
Description
Technical field
The invention belongs to field of crop genetic breeding, be specifically related to a kind of high-efficiency nitrogen-fixing soybean raw rhizobium slowly that derive fromD-amino acid dehydrogenase gene bll6812S and its preparation method and application.
Background technology
Glyphosate (Glyphosate) claims again: glyphosate, agriculture reach (Roundup) etc., because physicochemical property is stablized and has a heightEffect, wide spectrum, low toxicity, low-residual, easily decompose, spoiled soil environment and most plants is had to the advantages such as natural disposition of going out by force not, fromSince within 1971, Monsanto Company succeeds in developing, now become the herbicide of global marketing amount maximum. Therefore, glyphosate also becomesThe first choice of antiweed genetically modified crops research, resistance glyphosate genetically modified crops are turning of current global sown area maximumGene crops.
The toxic action mechanism of glyphosate is the competitive 5-enol pyruvic acid shikimic acid-3-suppressing in shikimic acid pathwayPhosphonic acids synzyme (5-enolpyruvylshikimate-3-phosphatesynthase is called for short EPSPS), causes branched acidBiosynthesis block, the biosynthesis of blocking-up aromatic amino acid and some aromatics, thus upset the normal nitrogen of plantMetabolism and make weeds death. At present, anti-glyphosate plants genetic engineering mainly adopts three kinds of methods. The first, plant cell passes throughThe extraordinary expression of EPSPS produces resistance to the glyphosate of doses. The second, plant cell is lived by the effect of EPSPS geneProperty site change to glyphosate produce resistance, i.e. usually said EPSPS Antiglyphosate gene. Utilize this mechanism of action to make to plantThe conventional gene that thing has a glyphosate resistance is mainly two kinds of CP4-EPSPS and aroA. The 3rd, import coding glyphosate degraded phaseCorrelation gene, as glyphosate oxidoreductase gene (Glyphosateoxidoreductase, GOX), and glyphosate N-acetyl turnsMove enzyme (glyphosateNacetyhransferase) gene, i.e. GAT gene.
Current commercial resistance glyphosate genetically modified crops are all almost About Monsanto Chemicals's product (trade nameRoundupReady), and mainly adopted to express the CP4-EPSPs of the low sensitivity of glyphosate was improved to crop resisting glyphosateProperty, but this resistance mechanism can not be eliminated the systemic glyphosate of plant, and plant is very slow to glyphosate metabolism, makesBecome glyphosate constantly accumulation in plant, this may cause the harm of two aspects: the one, and to causing of breeding plant organInfringement, this infringement shows as poor pollination and blasting so that output and reduces. There are some researches show that first generation resistance glyphosate turnsGene soybean compared with non-transgenic soybean yield reducation 6.7%[Duke, and Powle, PestManag.Sci., 2008,64:319-325]; The 2nd, may cause certain potential hazard to the healthy of eater. Nearest research shows, longThe feed that phase feeding is added with glyphosate can cause mouse fetus and the conceived many functions of female mouse to occur abnormal [DaruichDeng, Environ.Res., 2001,85:226-231]. In addition, glyphosate can disturb mouse cell to produce the enzyme [Wailh of testosteroneDeng, Environ.Health.Persp., 2000,108:769-776.] and human placenta's cell in human estrin synzyme[Environ.Health.Persp. such as Richard, 2005,, 13:716.]. This is also that people query resistance glyphosate transgenosis foodThe one of the main reasons of product security, thereby the residual resistance mechanism of exploitation nothing/low glyphosate is very necessary.
Little at the application report of resistance glyphosate about D-amino acid in existing document and scientific research. Amino acid sequenceHomology analysis shows, D-AAO (D-aminoacidoxidase is called for short DAAO, EC1.4.3.3) and sweet ammoniaAcid oxidase (glycineoxidase is called for short GO, EC1.4.3.19) and monomer sarcosine oxidase (monomericSarcosineoxidase, EC1.5.3.1) belong to the II member of glutathione reductase family [Dym and Eisenberg,ProteinSci., 2001,10:1712-1728]. DAAO and GO can catalytic amino acid oxidase deaminations, produce corresponding α-oneAcid, ammonia/amine and H2O2, but both substrate spectrums are different. DAAO can generate aminomethyl phosphonic acid (AMPA) and glyoxalic acid by catalysis glyphosate,Thereby reduce the toxicity of glyphosate. In recent years, DAAO is applied to and creates glyphosate resistance plant research by people, but does not have at presentAbout D-amino acid dehydrogenase gene being applied to the relevant report of cultivating anti-glyphosate plants.
Summary of the invention
Technical problem to be solved by this invention is to provide one to derive from high-efficiency nitrogen-fixing soybean raw rhizobium slowly(Bradyrhizobiumdiazoefficiens) D-amino acid dehydrogenase gene bll6812S and preparation method thereof and shouldWith. The present invention by derive from high-efficiency nitrogen-fixing soybean slowly the D-amino acid dehydrogenase gene (bll6812) of raw rhizobium be transformed into canThe D-amino acid dehydrogenase gene (bll6812S) of high efficient expression in plant, and by this D-amino acid dehydrogenase geneBll6812S arabidopsis thaliana transformation, and then the ability of raising transgenic arabidopsis resistance glyphosate, this D-amino acid dehydrogenase geneBll6812S is extremely important for the plant variety of cultivating resistance glyphosate, has important theory significance and realistic meaning.
Therefore, one of technical problem to be solved by this invention, is to provide one to derive from high-efficiency nitrogen-fixing soybean raw slowlyThe D-amino acid dehydrogenase gene bll6812S of rhizobium.
Two of technical problem to be solved by this invention, high-efficiency nitrogen-fixing soybean raw rhizobium are slowly provided described in being to provideThe preparation method of D-amino acid dehydrogenase gene bll6812S.
Three of technical problem to be solved by this invention, high-efficiency nitrogen-fixing soybean raw rhizobium are slowly provided described in being to provideThe application of D-amino acid dehydrogenase gene bll6812S in Plant Transformation.
Four of technical problem to be solved by this invention, high-efficiency nitrogen-fixing soybean raw rhizobium are slowly provided described in being to provideD-amino acid dehydrogenase gene bll6812S, improving application in glyphosate resistance of plant performance and turn cultivating resistance glyphosateApplication in gene plant.
In order to achieve the above object, technical scheme of the present invention is as follows:
Derive from a high-efficiency nitrogen-fixing soybean D-amino acid dehydrogenase gene bll6812S for raw rhizobium slowly, its nucleotidesSequence is as shown in SEQIDNo1, and the amino acid sequence of its coding is as shown in SEQIDNo2. Described D-amino acid dehydrogenase baseBecause bll6812S coding reading frame forms 421 amino acid whose protein of coding by 1266bp.
The high-efficiency nitrogen-fixing soybean D-amino acid dehydrogenase gene bll6812S of raw rhizobium slowly that derives from of the present invention,Employing manual method is synthetic, by high-efficiency nitrogen-fixing soybean slowly raw rhizobium D-amino acid dehydrogenase gene (bll6812) according toPlant-preference password is transformed, and utilizes gene synthetic method [Xiongetal., NuclAcidsRes, 2004,32:e98]Carry out chemical synthesis, on the constant basis of the amino acid sequence that keeps D-amino acid dehydrogenase gene bll6812, design primerBll6812S-1～bll6812S-32, the synthetic D-amino acid dehydrogenase gene of the present invention of the performing PCR of going forward side by side amplification(bll6812S). The primer of specific design is as follows:
bll6812S-1：
GAA,GGA,TCCATGCCAGAGGGTCGTCACGTCGCTATCATCGGTGCTGGTGCTGTCGGTGTCATCTCCGCT
bll6812S-2：
CGATCAGAGTGACACGATGACCCTCACGCAGAGCCTCGATAGCGGAGATGACACCGACAG
bll6812S-3：
TCATCGTGTCACTCTGATCGACCCAGGTGAGCCAGGTGGTGAGCAGGCTGCTTCCTACGG
bll6812S-4:
TGGTGGGATGACGGAGTGGGAGGACAGCCAACCAGCGTTACCGTAGGAAGCAGCCTGCTC
bll6812S-5：
CCCACTCCGTCATCCCACCAGCTGAGCCAGGTATCTGGAAGAAGGTTCCAGGTTATCTGA
bll6812S-6:
AGGTAGGACCAACGGATAGCCAGTGGACCCAGTGGGTCCATCAGATAACCTGGAACCTTC
bll6812S-7：
GCTATCCGTTGGTCCTACCTGCCAAAGGCTCTGCCTTGGCTGATCAAGTACCTGCTGTCC
bll6812S-8:
GAGCGAAAGCAGTCTTCTCGACACGAGCCTCAGTCCAACCGGACAGCAGGTACTTGATCA
bll6812S-9：
CGAGAAGACTGCTTTCGCTCTGCGTGACCTGCTGAAGGACGCTCCACTGCTGCACCGTAA
bll6812S-10:
ACGCTCGATCAGCTCTGGGACACCAGCCTCCTCAGCCAGCTTACGGTGCAGCAGTGGAGC
bll6812S-11：
TCCCAGAGCTGATCGAGCGTAACGGTGTCATGCACGCTTTCCCATCTCGTGGTAACTTCG
bll6812S-12:
ACACCGACCTTCTTACGCAGACGCCAACCCAGGTCGTTGTCGAAGTTACCACGAGATGGG
bll6812S-13：
CTGCGTAAGAAGGTCGGTGTCGCTTGGCTGGAGCTGAACGCTGACGAGATGCGTCAGCGT
bll6812S-14:
CGACGACACCGAAGGAGTAACGTGGGTGCAGGTCTGGCTCACGCTGACGCATCTCGTCAG
bll6812S-15：
TTACTCCTTCGGTGTCGTCGTCGAGGAAGCTGGTCGTTGTCGTGACCCAGGTGCTTACGT
bll6812S-16:
CTTAGCACCAGAAGCCAGAGCGTGGTTAGCCAGAGCAGCGACGTAAGCACCTGGGTCACG
bll6812S-17：
CTCTGGCTTCTGGTGCTAAGCTGGTTCGTGCTAAGGCTACTGGTCTGAAGCTGTCTGGTA
bll6812S-18:
GCGATCTCACCAGTCTCAGTGACGACAGCAACCAGCTTGTTACCAGACAGCTTCAGACCA
bll6812S-19：
ACTGAGACTGGTGAGATCGCTTGTGATGCTGCTGTCGTTGCTGCTGGTGCTCGTTCCAAG
bll6812S-20:
TCTCCAGTGGCAGTGGATCACCAACAGAAGCAGTCAGCTGCTTGGAACGAGCACCAGCAG
bll6812S-21：
TGATCCACTGCCACTGGAGACTGAACGTGGTTATCACGTCATGATCGAGAACCCAGAGAC
bll6812S-22：
CATCTTAGCGTCGGAAGCCATGATGGAGGAACGTGGACCAGTCTCTGGGTTCTCGATCAT
bll6812S-23:
TGGCTTCCGACGCTAAGATGGTCGTCAACTGGACTAACAAGGGTCTGCGTGCTGCTGGTA
bll6812S-24:
TTCCAGTTTGGAGCAGCTTCCAGACCAGCAATCTCGACAGTACCAGCAGCACGCAGACCC
bll6812S-25：
GAAGCTGCTCCAAACTGGAAACGTGCTGAGATCCTGCGTGACCACCTGTTCTCCATGTTC
bll6812S-26:
TCTTGATACGAGAAGCTGGGATGTCACGTGGCAGCTTTGGGAACATGGAGAACAGGTGGT
bll6812S-27：
CCCAGCTTCTCGTATCAAGACTTGGTTCGGTCATCGTCCATCCATGCCAGATGGTCTGCC
bll6812S-28:
GTAGACGATGTCACGAGAAGCACGAGCATGACCGATGCATGGCAGACCATCTGGCATGGA
bll6812S-29：
CTTCTCGTGACATCGTCTACGCTTTCGGTCACGGTCACGTTGGTCTGGTCGGTTCTGCTC
bll6812S-30:
TGCTTACCAGACAGCAGCTGAGCGACCAGACGACCAGTACGAGCAGAACCGACCAGACCA
bll6812S-31
CAGCTGCTGTCTGGTAAGCAGCCAGAGATCCCACTGGCTCCATTCTCTCCAACTCGTTTC
bll6812S-32:
GAG,CTCTTACAGGAAACGAGTTGGAGAGAATG
D-amino acid dehydrogenase gene bll6812S of the present invention is proceeded to arabidopsis, and it is sweet to be applied to raising Genes For Plant Tolerance grassThe ability of phosphine, and then cultivate resistance glyphosate genetically modified plants, specifically comprise:
1) structure of recombinant plasmid AH752
After pcr amplification finishes, with 1% Ago-Gel recovery, get 10 μ l recovery products and be directly connected with T/A cloning vector,4 DEG C of connections are spent the night, and obtain DNA and connect product, in Efficient Conversion DH5 α competent cell.
Utilize BamHI and SacI to carry out after double digestion PCR product, reclaim bll6812S fragment, will by T4DNA ligaseThe bll6812S gene reclaiming is connected with the plant expression vector 1301 that contains two 35S promoters, and enzyme is cut qualification and sequencingObtain the recombinant plasmid AH752 that contains genes of interest bll6812S gene. This expression vector also comprises gus reporter gene and bandIntrone kalamycin resistance marker gene.
2) agrobacterium mediation converted arabidopsis
Utilize electric shocking method that above-mentioned recombinant plasmid AH752 is imported in agrobacterium tumefaciens lba4404, Agrobacterium is dipped in colored method and transformsThe Agrobacterium LBA4404 containing bll6812S gene building is transformed into [Cloughetal., Theplant in arabidopsisJournal, 1998,16 (6): 735-743], utilize 50 μ g/mL hygromycin to carry out transformed plant screening, raw on hygromycin plateLong normal seedling is transplanted seedlings, and sowing, carries out positive seedling qualification. Treat that seedling grows to about 3 weeks, get blade extracting RNA, carry outPcr amplification genes of interest, the fragment that acquisition length is 1266bp, proves that genes of interest successfully imports in arabidopsis.
3) Resistance Identification to glyphosate after synthetic bll6812S gene-transformed plant
The transgenic arabidopsis selfing that successfully proceeds to bll6812S gene was isozygotied for 3 generations, obtain the transformant that isozygotys, collectSeed. After planting, 22 DEG C of growths, after 3 weeks, spray 10mM glyphosate 3 days continuously, cultivate non-transgenic arabidopsis (wild simultaneouslyRaw type arabidopsis) experiment in contrast. Observe transgenic arabidopsis and the resistance effect of non-transgenic arabidopsis to glyphosate. 4After it, wild type arabidopsis survival rate is 0, and transgenic arabidopsis strain survival rate can reach 95.3%, and result shows wild typeObvious to the Resistant Difference of glyphosate with transgenosis type arabidopsis, transgenic arabidopsis obviously improves glyphosate resistance.
Beneficial effect of the present invention:
The present invention adopts gene synthetic method to synthesize a kind of high-efficiency nitrogen-fixing soybean D-amino acid of raw rhizobium slowly that derives fromDehydrogenase gene bll6812S, this bll6812S gene can successfully be transformed in arabidopsis, and in arabidopsis high efficient expression,To transgenic arabidopsis; The arabidopsis plant and the wild type arabidopsis plant that proceed to bll6812S gene have on glyphosate resistanceSignificantly difference, transgenic arabidopsis, compared with wild type arabidopsis, has obvious resistance glyphosate ability, shows bll6812SGene proceed to the ability that has improved arabidopsis plant resistance glyphosate.
Brief description of the drawings
Fig. 1 is the structure schematic diagram of the present invention plant recombinant plasmid AH752 of containing genes of interest;
Fig. 2 is the pcr amplification result figure of the positive seedling of transgenic arabidopsis in the embodiment of the present invention 4, and wherein, M is DNAMark, WT is wild type plant, 1,2,3 are respectively three different strains of transgenic arabidopsis.
Fig. 3 turns bll6812S gene arabidopsis and wild type arabidopsis after spraying glyphosate in the embodiment of the present invention 5Resistance glyphosate phenotypic map, wherein: CK is wild type arabidopsis, and 752-4,752-8,752-14 are respectively three of transgenic arabidopsisIndividual strain.
Detailed description of the invention
Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing. It should be noted that described embodiment is only in order to sayBright technical scheme of the present invention and unrestricted, although the present invention is had been described in detail with reference to preferred embodiment, this areaThose of ordinary skill should be appreciated that and can modify or be equal to replacement the technical scheme of invention, and do not depart from the present inventionThe spirit and scope of technical scheme, it all should be encompassed in claim scope of the present invention.
Test material and source thereof that the present invention is used comprise:
Wild type arabidopsis (Colombia's type, EcotypeColumbia), 23 DEG C of phjytotrons are cultivated, 16h illuminationCultivate. High-efficiency nitrogen-fixing soybean slowly raw rhizobium USDA110 bacterial strain is preserved by Academy of Agricultural Sciences, Shanghai City biotechnology research. GreatlyEnterobacteria (Escherichiacoli) DH5 α is studied by Academy of Agricultural Sciences, Shanghai City biotechnology research institute plant genetic engineeringPreserve chamber. Cloning vector pMD-18-SimpleT, all kinds of restriction enzyme, Taq polymerase, ligase, dNTP, 10 × PCRBuffer and DNAmarker are purchased from precious bioengineering Dalian Co., Ltd. All chemical reagent are all from U.S.'s sigma chemistryCompany and Shanghai traditional Chinese medicines chemical reagent are bought. ABIPRIAMBig-DyeTerminatorDNA sequencing kit is purchased from U.S.Application system company of state.
Molecular biology conventional in the present invention operates specifically referring to " molecular cloning " [MolecularCloning.2nded.ColdSpringHarborLaboratoryPress，1989】。
If the present invention's reagent used does not clearly indicate, all purchased from Sigma-aldrich (Sigma-Aldrich）。
Embodiment 1: the synthetic high-efficiency nitrogen-fixing soybean D-amino acid dehydrogenase base of raw rhizobium slowly that derives from of gene synthetic methodBecause of bll6812S
Utilize gene synthetic method [Xiongetal., NuclAcidsRes, 2004,32:e98] Clone Origin in efficientlyFixed nitrogen soybean is the D-amino acid dehydrogenase gene (bll6812) of raw rhizobium USDA110 bacterial strain slowly, is keeping bll6812 geneAmino acid sequence constant basis on, according to plant-preference password again composite coding high-efficiency nitrogen-fixing soybean raw rhizobium slowlyD-amino acid dehydrogenase gene bll6812S, the synthetic primer bll6812S-1～bll6812S-32 of bll6812S gene is as follows:
bll6812S-1：
GAA,GGA,TCCATGCCAGAGGGTCGTCACGTCGCTATCATCGGTGCTGGTGCTGTCGGTGTCATCTCCGCT
bll6812S-2：
CGATCAGAGTGACACGATGACCCTCACGCAGAGCCTCGATAGCGGAGATGACACCGACAG
bll6812S-3：
TCATCGTGTCACTCTGATCGACCCAGGTGAGCCAGGTGGTGAGCAGGCTGCTTCCTACGG
bll6812S-4:
TGGTGGGATGACGGAGTGGGAGGACAGCCAACCAGCGTTACCGTAGGAAGCAGCCTGCTC
bll6812S-5：
CCCACTCCGTCATCCCACCAGCTGAGCCAGGTATCTGGAAGAAGGTTCCAGGTTATCTGA
bll6812S-6:
AGGTAGGACCAACGGATAGCCAGTGGACCCAGTGGGTCCATCAGATAACCTGGAACCTTC
bll6812S-7：
GCTATCCGTTGGTCCTACCTGCCAAAGGCTCTGCCTTGGCTGATCAAGTACCTGCTGTCC
bll6812S-8:
GAGCGAAAGCAGTCTTCTCGACACGAGCCTCAGTCCAACCGGACAGCAGGTACTTGATCA
bll6812S-9：
CGAGAAGACTGCTTTCGCTCTGCGTGACCTGCTGAAGGACGCTCCACTGCTGCACCGTAA
bll6812S-10:
ACGCTCGATCAGCTCTGGGACACCAGCCTCCTCAGCCAGCTTACGGTGCAGCAGTGGAGC
bll6812S-11：
TCCCAGAGCTGATCGAGCGTAACGGTGTCATGCACGCTTTCCCATCTCGTGGTAACTTCG
bll6812S-12:
ACACCGACCTTCTTACGCAGACGCCAACCCAGGTCGTTGTCGAAGTTACCACGAGATGGG
bll6812S-13：
CTGCGTAAGAAGGTCGGTGTCGCTTGGCTGGAGCTGAACGCTGACGAGATGCGTCAGCGT
bll6812S-14:
CGACGACACCGAAGGAGTAACGTGGGTGCAGGTCTGGCTCACGCTGACGCATCTCGTCAG
bll6812S-15：
TTACTCCTTCGGTGTCGTCGTCGAGGAAGCTGGTCGTTGTCGTGACCCAGGTGCTTACGT
bll6812S-16:
CTTAGCACCAGAAGCCAGAGCGTGGTTAGCCAGAGCAGCGACGTAAGCACCTGGGTCACG
bll6812S-17：
CTCTGGCTTCTGGTGCTAAGCTGGTTCGTGCTAAGGCTACTGGTCTGAAGCTGTCTGGTA
bll6812S-18:
GCGATCTCACCAGTCTCAGTGACGACAGCAACCAGCTTGTTACCAGACAGCTTCAGACCA
bll6812S-19：
ACTGAGACTGGTGAGATCGCTTGTGATGCTGCTGTCGTTGCTGCTGGTGCTCGTTCCAAG
bll6812S-20:
TCTCCAGTGGCAGTGGATCACCAACAGAAGCAGTCAGCTGCTTGGAACGAGCACCAGCAG
bll6812S-21：
TGATCCACTGCCACTGGAGACTGAACGTGGTTATCACGTCATGATCGAGAACCCAGAGAC
bll6812S-22：
CATCTTAGCGTCGGAAGCCATGATGGAGGAACGTGGACCAGTCTCTGGGTTCTCGATCAT
bll6812S-23:
TGGCTTCCGACGCTAAGATGGTCGTCAACTGGACTAACAAGGGTCTGCGTGCTGCTGGTA
bll6812S-24:
TTCCAGTTTGGAGCAGCTTCCAGACCAGCAATCTCGACAGTACCAGCAGCACGCAGACCC
bll6812S-25：
GAAGCTGCTCCAAACTGGAAACGTGCTGAGATCCTGCGTGACCACCTGTTCTCCATGTTC
bll6812S-26:
TCTTGATACGAGAAGCTGGGATGTCACGTGGCAGCTTTGGGAACATGGAGAACAGGTGGT
bll6812S-27：
CCCAGCTTCTCGTATCAAGACTTGGTTCGGTCATCGTCCATCCATGCCAGATGGTCTGCC
bll6812S-28:
GTAGACGATGTCACGAGAAGCACGAGCATGACCGATGCATGGCAGACCATCTGGCATGGA
bll6812S-29：
CTTCTCGTGACATCGTCTACGCTTTCGGTCACGGTCACGTTGGTCTGGTCGGTTCTGCTC
bll6812S-30:
TGCTTACCAGACAGCAGCTGAGCGACCAGACGACCAGTACGAGCAGAACCGACCAGACCA
bll6812S-31
CAGCTGCTGTCTGGTAAGCAGCCAGAGATCCCACTGGCTCCATTCTCTCCAACTCGTTTC
bll6812S-32:
GAG,CTCTTACAGGAAACGAGTTGGAGAGAATG
The synthetic design principle of gene comprises: by plant-preference codon, avoid occurring in gene the PolyA tailings such as ATTTASignal, avoids 6 or more continuous A+T sequence, avoids 5 or more G+C sequence, and the ratio 40～60% of G+C is anti-Only introne cutting sequence, reduces the two-layer configuration hairpins of gene inside, avoids 2,3 with CG and TA dual oligonucleotide(CG easily causes and methylates in plant).
Taking bll6812S-1, bll6812S-32 as outside primer, draw as inner side taking bll6812S-2～bll6812S-31Thing, utilizes the PCR bll6812S fragment that increases, and in 50 μ l reaction systems, the primer of inner side is respectively 1.5ng, outsideTwo primers are respectively 30ng, 1 μ lKODFXtaq enzyme (Toyobo company, Japan), 5 μ l10 × PCRbuffer, 4 μ lDNTP, adds sterilized water and is settled to 50 μ l. Amplification condition is: 94 DEG C of preheating 1min; 94 DEG C, 30s, 50 DEG C, 30s, 72 DEG C, 1min.Totally 25 circulations.
After PCR finishes, with 1% Ago-Gel recovery, get 10 μ l recovery products directly and pMD-18-SimpleT cloneCarrier is connected, and 4 DEG C of connections are spent the night, and obtains DNA and connects product, and Efficient Conversion is in DH5 α competent cell.
Extracting plasmid from DH5 α transformant cell, carries out pcr amplification taking extracting plasmid as template, obtained oneThe fragment of 1266bp, shows through order-checking and BLAST comparison gene and the high-efficiency nitrogen-fixing soybean raw rhizobium slowly that PCR method is syntheticThe amino acid sequence of bll6812 gene is in full accord. This gene is the present invention and derives from high-efficiency nitrogen-fixing soybean raw rhizobium slowlyBll6812S gene, its nucleotide sequence is as shown in SEQIDNo1, the amino acid sequence of its coding is as SEQIDNo2 instituteShow.
Embodiment 2: high-efficiency nitrogen-fixing soybean is raw rhizobium D-amino acid dehydrogenase gene bll6812S expression vector slowlyBuild
Carry out double digestion PCR product with BamHI and SacI respectively, with 1% Ago-Gel recovery DNA fragmentation, pass throughT4DNA ligase by the bll6812S genetic fragment of recovery with contain two 35S promoter 1301 plasmids and be connected, enzyme is cut and is identified and orderRow analysis is measured and has been obtained the recombinant plasmid AH752 that contains D-amino acid dehydrogenase gene bll6812S gene, as shown in Figure 1.This expression vector also comprises gus reporter gene and band introne kalamycin resistance marker gene, and carrier as shown in Figure 1.
Embodiment 3: Agrobacterium is cultivated
Recombinant plasmid imports in Agrobacterium through electric shocking method. Picking agrobacterium tumefaciens lba4404 list bacterium is cultivated to 25mLYEBBase (50mg/L rifampin) overnight incubation, gets 5mL bacterium liquid and is transferred to 100mLYEB culture medium (50mg/L rifampin), is cultured toOD600=0.7-0.8, bacterium liquid is placed 10 minutes on ice, the centrifugal 10min of 5000rpm, 4 DEG C, collect thalline, add 100mL asepticDistilled water cleans twice. Add 4mL10% glycerine suspension thalline, forward 50ml centrifuge tube to. The centrifugal 10min of 5500rpm, 4 DEG C. ReceiveCollection thalline, adds 500 μ L10% glycerine suspension thalline, forwards 1.5ml centrifuge tube to, obtains Agrobacterium competent cell. Get on 70 μ LState the Agrobacterium competent cell of purchasing, add the 1 μ L recombinant plasmid AH752 yellow rifle head of decaptitating to mix, forward 0.1cm electricity toHit in cup. Shock parameters: 200 Ω, 1.7KV, 2.5F, adds 800 μ LSOC(referring to " molecular cloning experiment refers to immediately after electric shockSouth " [SambrookandRussell, 2001]) nutrient solution. Cultivate after 1 hour, get 100 μ L and be coated with resistance plate screening transformant,28 DEG C of cultivations, filter out the bacterial strain that successfully imports recombinant plasmid AH752.
Embodiment 4: arabidopsis dips in colored method and transforms
Connect bacterium containing the agrobacterium strains list bacterium colony of object plasmid and contain 28 DEG C of cultivations in corresponding antibiotic LB culture medium at 5mL2 days. 5mL bacterium liquid is forwarded in the liquid LB culture medium of 500mL to 28 DEG C and cultivate 16-24 hour (OD=1.5-2.0), liquid canPreserve 30 days at 4 DEG C. Centrifugal collection thalline under room temperature, centrifugal 10 minutes of 4000g. Outstanding with the fresh sucrose solution of equal-volume 5%Floating. After adding 0.02% Silwet-77 to mix, transfer in beaker, obtain transformed bacteria liquid. After being inverted, soaks wild type arabidopsisEnter 10 seconds in transformed bacteria liquid. Lotus throne and inflorescence all will infect. 300 milliliters of conversions for each bacterial strain, turn 2-3 alms bowl. After 7 days againTransform 1 time. After infecting, transgenic arabidopsis plant bacterium liquid air is done 3-5 second. With preservative film by good transgenic arabidopsis plant circle,Keep flat 16-24 hour. After conversion, be not placed under high temperature and high light. Open preservative film, keep certain humidity, 1 of regrowthSowing after month. Utilize 50 μ g/mL hygromycin to carry out transformed plant screening, the normal seedling of growing on hygromycin plate movesSeedling, sowing, carries out positive seedling qualification.
Treat that seedling grows to about 3 weeks, get blade extracting RNA, reverse transcription is CDNA, with bll6812S-1, bll6812S-32For primer, utilize the PCR bll6812S fragment that increases, in 50 μ l reaction systems, primer 2 μ g, 1 μ lKODFXtaq enzyme(Toyobo company, Japan), 5 μ l10 × PCRbuffer, 4 μ ldNTP, add sterilized water and are settled to 50 μ l. Amplification condition is: 94DEG C preheating 1min; 94 DEG C, 30s, 50 DEG C, 30s, 72 DEG C, 1min. Totally 30 circulations.
Carry out pcr amplification genes of interest, result as shown in Figure 2. As can be known from Fig. 2, in wild type plant, do not detectLength is the fragment of 1266bp, and proceed to, in bll6812S gene plant, to have very bright length be the band of 1266bp, provesThese genetically modified plants are all positive seedlings, and illustration purpose gene successfully imports in arabidopsis.
Embodiment 5: the Resistance Identification to glyphosate after synthetic bll6812S gene-transformed plant
The above-mentioned transgenic arabidopsis selfing that successfully proceeds to bll6812S gene was isozygotied for 3 generations, obtains the transformant that isozygotys,Collect seed. After planting, 22 DEG C of growths, after 3 weeks, spray 10mM glyphosate 3 days continuously, cultivate non-transgenic arabidopsis (simultaneouslyWild type arabidopsis) experiment in contrast. Observe transgenic arabidopsis and the resistance effect of non-transgenic arabidopsis to glyphosate.
Within 4 days, add up afterwards the seedling survival rate of wild type arabidopsis and transgenic arabidopsis, wild type arabidopsis survival rate is 0,And genetically modified three strain survival rates are respectively 90.5%, 92.4% and 95.3%, as shown in Figure 3. Experimental result shows wild typeObvious to the Resistant Difference of glyphosate with transgenosis type arabidopsis, transgenic arabidopsis obviously improves glyphosate resistance, explanationThe synthetic bll6812 gene of the present invention can improve glyphosate resistance of plant performance.
In sum, the present invention is raw slowly by designing 32 primers and utilizing gene synthetic method will come from high-efficiency nitrogen-fixing soybeanThe bll6812 gene of rhizobium carried out again transformation, obtained its nucleotide sequence as shown in SEQIDNo1Bll6812s gene. Also this bll6812s gene is successfully imported in arabidopsis simultaneously, detect the transfer-gen plant that discovery obtainsAll there is very strong resistance glyphosate ability, show that the reforming composite bll6812 gene of the present invention has raising glyphosate resistance of plantAbility, the technical field that can be used for cultivating anti-glyphosate plants.
Finally it should be noted that above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although ginsengAccording to preferred embodiment, the present invention is had been described in detail, those of ordinary skill in the art should be appreciated that can to inventionTechnical scheme is modified or is equal to replacement, and does not depart from the spirit and scope of technical solution of the present invention, and it all should be encompassed inIn claim scope of the present invention.
Claims (2)
1. D-amino acid dehydrogenase gene bll6812S is at the application improving in glyphosate resistance of plant performance, described D-aminoThe nucleotide sequence of acidohydrogenase gene bll6812S as shown in SEQIDNo1, coding amino acid sequence as SEQIDNoShown in 2.
2. D-amino acid dehydrogenase gene bll6812S is in an application of cultivating in anti-glyphosate plants, and described D-amino acid is de-The nucleotide sequence of hydrogenase gene bll6812S as shown in SEQIDNo1, coding amino acid sequence as SEQIDNo2 instituteShow.
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| US6329573B1 (en) * | 1996-07-02 | 2001-12-11 | The Board Of Trustees Of Southern Illinois University | Plants containing the gdhA gene and methods of use thereof |
| CN102264907A (en) * | 2008-10-23 | 2011-11-30 | 巴斯夫植物科学有限公司 | Plants with increased yield (NUE) |
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| US6329573B1 (en) * | 1996-07-02 | 2001-12-11 | The Board Of Trustees Of Southern Illinois University | Plants containing the gdhA gene and methods of use thereof |
| CN102264907A (en) * | 2008-10-23 | 2011-11-30 | 巴斯夫植物科学有限公司 | Plants with increased yield (NUE) |
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| Title |
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| 奇异变形杆菌的L-氨基酸脱氢酶在大肠杆菌中的异源表达和鉴定;Jin-Oh Baek,等;《生物工程学报》;20081225;第24卷(第12期);2129 * |
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