甜菜树贝壳杉烯合酶基因的克隆与原核表达

Cloning and Expression of the Ent-Kaurene Synthase Gene from Yunnanopilia longistaminata(W.Z.Li,C.Y.Wu et D.Z.Li)(Opiliaceae)

本文作者研究了赤霉素合成途径中的关键酶-贝壳杉烯合酶,对其功能的研究可以为以后优化品种提供基因层面的基础。首先通过RACE-PCR技术,从甜菜树叶片中克隆得到贝壳杉烯合酶基因(Ent-kaurene synthase gene,Yl KS)的全长c DNA,共2 512 bp(Gen Bank登录号为KP872698),其ORF长度为2 232 bp,共编码743个氨基酸,预测蛋白质相对分子质量大小为84 987,蛋白质等电点为5.264,表明该蛋白质呈酸性。将该基因构建到表达载体pET32a中,得到重组质粒pET-32a-Yl KS。通过将p GG/An2、p IRS和重组质粒pET-32a-Yl KS 3个质粒共转入菌株BL21(DE3)中,得到重组菌株,并进行发酵。通过SDS-PAGE分析发现,重组蛋白成功得到了表达。最后通过对发酵产物进行萃取,并使用GC-MS进行检测,确定了该基因所编码的酶确实是贝壳杉烯合酶。

Yunnanopilia longistaminata is a wild woody vegetable in China,and its tender stems and leaves are edible. So far,studies of Y. longistaminata have mostly been focused on tissue culture,physiology and biochemistry,while less concentrated on the gene level. Gibberellin（GA） can enhance vegetative growth,such as the bolting,and the growth of stems and leaves. Using molecular biology methods,we studied the ent-kaurene synthase,a key enzyme in the synthesis pathway of gibberellins. Ent-kaurene synthase gene（YlKS） was cloned from the leaf of Y. longistaminata through RACE-PCR. The full gene length was 2 512 bp（GenBank accession number KP872698）. Results showed that the ORF length of YlKS was 2 232 bp and the molecular weight of its encoded protein（YlKS） was 84 987. The theoretical isoelectric point of YlKS was 5.264,which suggested that YlKS protein was acidic. The gene was cloned into the vector pET32a to get the plasmid pET-32a-YlKS. Plasmids pIRS,pGG/An2 and pET-32a-YlKS were co-transformed into BL21（DE3） strain to obtain a new recombinant strain,and then the recombinant strain was used for fermentation. The SDS-PAGE analysis showed that the recombinant protein was expressed successfully. Finally,we used n-hexane to extract the fermentation products and the GC-MS analysis confirmed that the gene in this study was Ent-kaurene synthase encoding gene.