WANGWen-ling,WANGXian-lei,XiongLi-man,et al.ConstructionandVerificationofDoubleT-DNAPlantExpressionVectoroftheAt2Gene[J].Northern Horticulture,2012,36(12):107-112.
At2基因双T-DNA植物表达载体的构建
- Title:
- ConstructionandVerificationofDoubleT-DNAPlantExpressionVectoroftheAt2Gene
- 文章编号:
- 1001-0009（2012）12-0107-06
- Keywords:
- At2gene; doubleT-DNA; plantexpressionvector; marker-free
- 分类号:
- Q946
- 文献标志码:
- A
- 摘要:
- 以甜瓜品种‘MR-1’为试材，采用PCR技术，扩增出了At2基因并在两端添加BamHI和SacI酶切位点。结果表明：在NCBI上进行BLAST显示其氨基酸序列与GeneBank中已公布的At2基因的同源性为99%；经DNAMAN软件分析，核苷酸序列同源性为99.43%。将其连接在中间表达载体G-pPTN133上，得到替换了GUS基因的中间载体A-pPTN133。A-pPTN133再经ScaI酶切后,插入到经ScaI酶切的pPTN133-中,构建成双T-DNA植物表达载体At2-pPTN133。经酶切和PCR鉴定证明了所构建载体的正确性。
- Abstract:
- Takingmeloncultivars‘MR-1’asmaterial,theenzymaticresistancegenesAt2wasamplifiedbyPCRmethodandtheBamHIenzymesiteandSacIenzymesitewasasoadded.Theresultsshowedthatitsaminoacidsequencehomologywas99%comparedwith“At2”genepublishedinGeneBankafterBLASTinNCBI;thenucleotidesequencehomologywas99.43%afteranalysisbyDNAMAN.TheacquiredgenewasligatedtoplasmidG-pPTN133，andthereforetheGUSgenewasreplacedofvectorA-pPTN133wasconstructed.TheplasmidA-pPTN133wasdigestedbySacIandthenligatedtothevectorpPTN133-whichwasasodigestedbySacI,sotheplantexpressionvectorAt2-pPTN133withdoubleT-DNAwasfinallyobtained.TheresultsshowedthatenzymesdigestionandPCRdemonstratedthecorrectnessofthevector.ThedoubleT-DNAvectorcanbetransformedintomelonmediatedbyAgrobacteriumtumufaciens.
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备注/Memo
第一作者简介：王文玲（1986-）,女，在读硕士,现主要从事植物分子生物学研究工作。E-mail:wangwenling86@163.com。
责任作者：李冠(1949-),男,硕士,教授,现主要从事植物生理生化与分子生物学研究工作。E-mail：guanli6666@126.com。
基金项目：国家自然科学基金资助项目(31060148，31150004);新疆高新技术研究发展计划资助项目（201111120)。
收稿日期：2012-03-27