新疆雪莲DFR基因的分离及同源遗传转化

Isolation and Homologous Genetic Transformation of DFR in Saussurea involucrata Kar.et Kir.

二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase,DFR)是植物重要的次生代谢产物花青素生物合成途径中的关键酶。运用RT-PCR和RACE技术从新疆雪莲(Saussurea involucrata Kar.et Kir.)中克隆得到DFR基因(GenBank登录号为JN092126)。DFR基因的cDNA全长序列含有1个1 029 bp的开放阅读框(ORF),编码343个氨基酸,该基因推断的蛋白与水母雪莲DFR基因推断的蛋白高度同源,相似性达到92%;以不同物种中DFR氨基酸序列进行比对分析,推断的蛋白含有与NADPH特异结合的结构域。将该基因运用农杆菌介导的叶片转化法进行同源转化,将含有转DFR基因的愈伤组织进行悬浮培养,紫外分光光度法测定愈伤组织的总黄酮含量,结果表明转基因愈伤组织的总黄酮含量明显高于非转基因愈伤组织的含量。该研究为提高新疆雪莲药用化学成分黄酮类物质及实现新疆雪莲花青素的人工生物合成的研究奠定基础,对解决天山雪莲资源匮乏提供参考。

The dihydroflavonol-4-reductase （DFR） is an important enzyme in plant secondary metabolites and catalyzes the key step in the biosynthesis of anthocyanins. DFR gene （GenBank Accession JN092126 ） was cloned from Saussurea involucrata Kar. et Kir. by RT-PCR and RACE. The eDNA sequence of DFR was consis- ted of 1 029 bp open reading frame （ORF） encoding 343 amino acid, the deduced DFR protein has high homolo- gy with S. medusa （92% identity）. Homology analysis showed that deduced DFR protein has the special domain regioselectivity towards NADPH. DFR gene of S. involucrata was under the control of the cauliflower mosaic virus （CaMV） 35S promoter, homologous transformation was conducted using an Agrobacter-ium rihizogenes-mediated transformation system. The results of UV spectrophotometry showed that Sinvolucrata callus after suspension cul- ture had an obviously higher average content of total flavonoids than non-transgenic callus. This study will im- prove the products of medicinal compositions and realize the synthesis of anthocyanins in S. involucrata.