海甘蓝硫甙合成关键酶S-GT基因两种表达载体的构建

Cloning and Expression Vector Construction for Genes of Thiohydroximate S-glucosyltransferase in Crambe abyssinica

Thiohydroximate S-glucosylt ransferase(S-GT)是硫甙生物合成的一个关键酶。根据发表的甘蓝型油菜S-GT基因cDNA序列设计引物,以海甘蓝总DNA为模板进行PCR扩增,获得S-GT基因全长,构建了海甘蓝S-GT基因的植物双元表达载体pCambia2300sn-Ca SGT。用PCR的方法对CaSGT基因的外显子进行了拼接,将得到的序列正向插入到了大肠杆菌表达载体pET-32b(+)中,得到重组质粒pET-32b(+)-CaS-GT,为进一步研究CaSGT的生物学特性打下基础。

Thiohydroximate S-glucosyltransferase （S-GT） plays a key role in the process of glucosinolate （GS） biosynthesis. Full-length CaGST gene was amplified from Crarnbe abyssinica genomic DNA according to the cDNA sequence of S-GT gene in Brassica napus, and then the sequence was cloned into plasmid pCambia2300sn and located between 35S promoter and NOS terminater, Meanwhile, we assembled the two extrons of CaSGTgene by PCR strategy and cloned the sequence into pET-32b （＋） to get the recombinant expression vector PET-32b （＋）-CaSGT. tion