蓖麻细胞色素P450基因片段的克隆及RNAi表达载体的构建

CLONING OF CYTOCHROME PARTIAL P450 GENE FROM CASTOR(Ricinus Communis L.) AND CONSTRUCTION OF RNAi VECTOR

采用PCR方法克隆了蓖麻细胞色素P450基因的正反2个片段。正向片段长为525个碱基,反向片段长为421个碱基。经测序分析,得到的正向基因片段和反向基因片段与GenBank上的蓖麻细胞色素P450基因(XM_002525863.1)的碱基同源性分别为98.85%和99.51%。利用载体pHANNIBAL和限制性内切酶将正反2个片段连接成具有发夹结构的中间载体pHAN-P450S-P450A,然后将中间载体和表达载体pBI-121连接,构建了P450基因的RNAi植物表达载体,为利用RNA干扰抑制P450基因表达的研究奠定了基础。

Sense and anti-sense fragments of castor cytochrome P450 were cloned by PCR.A 525bp sense fragment and a 421bp anti-sense fragment were obtained,which had a homology of 98.85% and 99.51% with the original sequence,respectively.An intermediate vector pHAN-P450S-P450A with hairpin structure was constructed,which was subsequently digested with restriction enzyme,and was ligated into the pBI-121 vector,producing the final plant expression vector.This result provided a foundation for further research of P450 gene expression by RNA interference technology.