番茄脯氨酸脱氢酶基因的克隆与RNAi植物表达载体的构建

摘要

[Objective]The full length cDNA of Proline dehydrogenase (ProDH) gene was cloned.The RNA interference plant expression vector of ProDH genes was constructed and was transformed into Agrobacterium tumefaciens.[Methods]Apair of specific primers were designed according to database information of ProDH gene in GenBan,and the cDNA was cloned.The forward and reverse fragments were selected and amplified in base of analysis of gene sequences.The RNA interference plant expression vector of ProDH genes (PBI121-PDHi) was constructed,which was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method.[Results] Sequencing results showed that the full length of the cloned fragment was 2 001 bp,and the CDS was 1 491 bp which encoded 380 amino acids.The cloned fragment shared a homology of 100％with the reported sequence of ProDH gene.[Conclusion] The transformation was confirmed by PCR amplification with specific,which would provide a foundation for further functional study.%[目的]克隆了脯氨酸脱氢酶ProDH基因的全长cDNA,构建了ProDH基因的RNA干扰植物表达载体,并转化到农杆菌.[方法]以“耐运2000番茄”幼苗为试材,根据GenBan中公布的番茄脯氨酸脱氢酶ProDH基因的序列信息设计了一对特异性引物,克隆了该基因的全长cDNA,分析基因序列选择正反向片段并扩增,并通过酶切、连接的方法构建了ProDH基因的RNA干扰植物表达载体PBI121-PDHi利用冻融法将表达载体转化到农杆菌EHA105中.[结果]所克隆到的ProDH基因片段长2 001 bp,其中CDS为1 491 bp,编码380个氨基酸.测序结果与公布序列同源性100％,因此可以用来构建干扰载体;通过酶切与测序鉴定,证明表达载体构建成功.[结论]经特异性引物扩增检测,证明表达载体已转入农杆菌,为进一步的研究该基因奠定了基础.