SAGP基因克隆及反义表达载体的构建

为了研究不同启动子驱动的反义SAGP抑制内源ADP-Glc PPase小亚基编码基因表达的效果,创造适于外源基因表达的突变体,利用RT-PCR方法,由马铃薯块茎cDNA克隆获得ADP-Glc PPase小亚基编码基因(SAGP)的1 821 bp cDNA。核酸数据库检索和序列比对分析表明,已克隆的SAGP基因cDNA推测编码607个氨基酸组成的多肽,其中第312和411的天冬酰氨均突变为丝氨酸。以pCambia为基础,分别构建了CaMV35S和GBSSI块茎启动子驱动的小亚基cDNA反义结构植物表达载体。

Cloning of SAGP gene and constructing of antisense transformation vectors

To compare effect of expression repression antisense SAGP driven by two promoters and subsequently to create potato mutation of tuber starch deficiency,a full length of cDAN encoding small subunit of ADP-Glc PPase(SAGP) was cloned and sequenced through RT-PCR with tuber of potato cultivar Zheng 1011.The sequence BLAST and alignment showed that SAGP of the 1 821 bp cDNA encodes a deduced peptide composed of 607 amino acids,and two mutations,N312S and N411S,occurred on the pepetide.With known vector pCambia for plant transformation,two antisense recombinant vectors,pS-SAGP and pG-SAGP in which SAGP were driven by promoters of 35S and GBSSI respectively,were constructed.