非洲菊f3h基因的克隆及反义表达载体的构建

Molecular Cloning and Construction of Antisense Expression Vector of Flavanone 3-hydroxylase Gene from Gerbera jamesonii

黄烷酮3-羟化酶(F3H)是植物花青素合成代谢中早期重要关键酶之一,对花色起着重要的调控作用.根据其它物种的f3h基因设计简并引物,通过RT-PCR技术,从非洲菊中克隆到500bp的片段,经BlastN比对,证实该片段为非洲菊f3h基因保守片段.将其反向插入到高效表达载体pCAMBIA1304+CaMV35S启动子下游,通过PCR鉴定和双酶切鉴定表明成功构建高效反义表达载体pCAMBIA1304+-antif3h,并转化到根癌农杆菌LBA4404形成工程菌株,作为今后研究非洲菊花色调控机制以及培育新品种的有力工具.

Flavanone 3-hydroxylase （F3H） is one of the key enzymes in the early stage of anthocyanin biosnythesis and plays an important regulation role for floral colour. In the present study, degenerate primers were designed based on f3h genes of other plants, and a 500 bp conservative fragment was obtained from Gerbera jarnesonii by RT-PC, and then Blast N in on-line NCBI confirmed that it was a conservative fragment of the G. jamesonii f3h gene. The PCR product of the antisense f3h gene was cloned into the downstream of CaMV 35S promoter of the efficient expression vector pCAMBIA1304^＋ plasmid. PCR and double restriction enzyme identification showed that an antisense expression vector, pCAMBIA1304^＋-antif3h, was established successfully, and the vector was transformed into Agrobacteriurn turnefaciens LBA4404, which can be used as a tool to research floral colour regulation mechanism and breed new species.