木薯醇腈酶cDNA的克隆及其表达载体的构建

Cloning ofα-Hydroxynitrile Lyase Gene from Cassava and Construction of its Expression Vector

目的克隆木薯醇腈酶(HNL)cDNA构建表达载体,以实现木薯醇睛酶基因的高效表达。方法运用RT-PCR从木薯幼叶组织中扩增出HNL全长cDNA序列,将其克隆至质粒pBluescript SK中进行序列分析,再利用PCR将其再克隆到酵母表达质粒pPIC3.5K上。结果经测序分析表明,克隆的cDNA片段和文献报道的4个木薯HNL cDNA相比,核苷酸同源性最高为99.1%,氨基酸同源性最高为98.8%。结论经双酶切鉴定的结果表明重组表达载体的表达载体构建成功。

Objective To study the cloning of the cDNA of α - HNL and the expression vector construction to realize highly efficient expression in NL gene. Methods The long - length sequence of cDNA of α - HNL was amplified by RT - PCR, then it was cloned into pBluescript SK and analylizd in its sequence. Finall, the cDNA was cloned into an expression vector pPIC3.5K by PCR. Results The sequencing result of the cDNA showed that the sequence encoded for the HNL was not fully consistent with those published. The full sequences analysis demonstrated that the highest homology of cDNA sequence is about 99.1% and the highest homology of amino acid sequence is about 98.8% to those four reported HNL genes from cassava. Conculsion Degestion detection of pPIC3.5K- HNL showed that construction of recombination expression vector was successful.