At2基因双T-DNA植物表达载体的构建  

Construction and Verification of Double T-DNA Plant Expression Vector of the At2 Gene

作　　者：王文玲[1] 王贤磊[1] 熊丽曼[1] 高兴旺[1] 李冠[1] 

机构地区：[1]新疆大学生命科学与技术学院,新疆乌鲁木齐830046

出　　处：《北方园艺》2012年第12期107-112,共6页Northern Horticulture

基　　金：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)

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。Taking melon cultivars ’MR-1’ as material,the enzymatic resistance genes At2 was amplified by PCR method and the BamHⅠenzyme site and SacⅠenzyme site was aso added.The results showed that its amino acid sequence homology was 99%compared with ＂At2＂ gene published in GeneBank after BLAST in NCBI;the nucleotide sequence homology was 99.43%after analysis by DNAMAN.The acquired gene was ligated to plasmid G-pPTN133,and therefore the GUS gene was replaced of vector A-pPTN133 was constructed The plasmid A-pPTN133 was digested by SacⅠand then ligated to the vector pPTN133^- which was aso digested by SacⅠ,so the plant expression vector At2-pPTN133 with double T-DNA was finally obtained.The results showed that enzymes digestion and PCR demonstrated the correctness of the vector.The double T-DNA vector can be transformed into melon mediated by Agrobacterium tumufaciens.

关 键 词：At2基因 双T-DNA 植物表达载体 无选择标记 

分 类 号：Q946[生物学—植物学]