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

Abstract:

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.