高山离子芥磷脂酶Dα编码区基因序列克隆与表达载体构建

The coding sequence clone and expression vector construction of PLDα gene fromChorispora Bungeana

磷脂酶D(PLD)是重要的细胞磷脂代谢酶,在植物的生长及对不良环境胁迫的抵御反应中起重要作用.本试验以高山离子芥(Chorispora Bungeana)为材料,取幼叶分离mRNA,反转录合成cDNA,PCR扩增加XbaΙ和SacΙ酶切位点的高山离子芥磷脂酶Dα(PLDα)编码区序列、测序.结果表明插入片段为PLDα目的基因片段,全长约1 600bp,blast比对发现该序列与Genbank中报道的拟南芥PLDα基因相比同源率为94.6%.回收纯化PCR产物,克隆至pTG19-T载体双酶切后将目的基因片段定向克隆至pBI-121载体,构建高山离子芥PLDα基因编码区片段的表达载体pBI121-PLDα,双酶切及PCR鉴定结果显示表达载体构建成功,为下一步进行抗逆转基因作物选育奠定了基础.

Phospholipase D（PLD） is a major kind of phospholipid hydrolase in plant , and play important roles in response to environmental stress . According to coding sequence of PLDα gene from Chorispora Bungeana and the sequence of pBI-121 vector , a pair of specific primers add XbaΙand SacΙrestriction sites to the ends of PLDα gene is designed . The purified PCR product is linked to pTG19-T vector and the sequencing results show that the full-length of the inserted gene fragment is about 1 600 bp and showing 94.6% homology to the sequence of A rabidopsis thaliana PLDα. The recombinant plasmid is identified by restriction analysis and PCR , sequence analysis indicates that the pBI121-PLDα expression vector is constructed successfully .