海甘蓝硫甙合成关键酶S-GT基因克隆及种子特异性hpRNAi载体构建

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

根据甘蓝型油菜S-GT(thiohydroximate S-glucosyltransferase)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。克隆的海甘蓝S-GT序列与甘蓝型油菜序列相比,除74 bp的内含子部分外有92个碱基的差别,相似性高达93.4%。分析显示该序列均有完整的开放阅读框,并表明所克隆的海甘蓝S-GT序列编码465个氨基酸,在第10个位点上比甘蓝型油菜序列少一个丙氨酸(A),总共有23个氨基酸不同,相似性为95.06%。根据获得的基因序列设计引物扩增出同一基因序列相同但是带有不同酶切位点的两个片段,将两个片段反向插入到已构建的带有种子特异表达载体内含子的两端,成功构建了海甘蓝S-GT基因的种子特异性hpRNAi载体,为特异性降低海甘蓝的种子硫甙奠定了基础。

Thiohydroximate S-glucosyltransferase （S-GT） plays a key role in the process of glucosinolate （GS） biosynthesis. In this research, PCR primers were designed according to the eDNA sequence of S-GT gene in Brassica napus and full length of the S-GT genes in Crarnbe abyssinica were obtained by using the genomic DNA as PCR templates. Sequence alignment revealed that CaSGT gene we cloned had an intron of 74 bp and coded a protein of 465 amino acids and shared a similarity of 93.4% at the DNA sequence level and a similarity of 95.06% at the amino acid level with the B. napus S-GT gene sequence published （AF304430）. Two small fragments with identical sequences but different restriction sites were amplified from the cloned S-GT sequence and ligated in opposite orientations into the seed-specific vector 2300-nap-intron to produce hpRNAi vectors to silence the internal S-GT activities in seeds of C. abyssinica Without lowering the glucosinolate level in the vegetative organs.