小麦叶绿体psbA基因的克隆及表达

CLONING AND EXPRESSION OF WHEAT CHLOROPLAST psbA GENE

用ＰＣＲ直接从小麦叶绿体基因组扩增到完整的ｐｓｂＡ基因，并构建了ｐｓｂＡ基因的克隆ｐＴＤ１Ⅱ。为研究ｐｓｂＡ基因在Ｅ·ｃｏｌｉ中的表达，将完整的ｐｓｂＡ基因正向插入高表达载体ｐＫＫ２２３－３中构建表达克隆ｐＫＴＤ１。含有重组表达质粒的转化细胞经ＩＰＴＧ诱导后提取总蛋白，经ＳＤＳ－ＰＡＧＥ分析，小麦叶绿体ｐｓｂＡ基因能在Ｅ·ｃｏｌｉ中表达３２ｋＤ的Ｄ１蛋白，表达的Ｄ１蛋白占细菌总蛋白的３％以上。

A PCR clone pTD1,Ⅱ containing complete psbA gene for chloroplast D1 protein was obtained from wheat ctDNA. To study expression of the psbA gene in E. coli, the gene was inserted in an expression vector PKK223-3 to construct the expression plasmid pKTD1.After induction with IPTG the transformed E. coli cells harboring this plasmid can express the 32kD protein. The expression level of the protein was 3% of the total bacterial proteins.