灵芝三萜合成关键元件GLCYP450及其还原酶GLNADPH的基因克隆及共表达载体构建

Construction of the coexpression vector containing key element GLCYP450 involved in Ganoderma triterpene biosynthesis and its reductase gene GLNADPH

细胞色素P450是灵芝三萜生物合成途径中的关键元件,其催化反应过程中需要有NADPH/NADH为其传递电子。本课题组根据已获得的赤芝(Ganoderma lucidum(Leyss.Ex Fr.)Karst.)基因组数据中的CYP450和NADPH转录本序列,利用RT-PCR方法获得灵芝CYP450(GLCYP450)和NADPH(GLNADPH)基因的全长cDNA序列,并在序列两端引入相应的限制性内切酶位点,通过酶切及连接反应,将GLCYP450和GLNADPH重组到酵母表达载体pESC-URA中,成功构建了酵母表达载体质粒pESC-GLNADPH-GLCYP450,为通过合成生物学手段研究灵芝三萜生物合成奠定基础。

Cytochrome P450 （CYP450） is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G. lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5＇ and 3＇ ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.