hbFGF基因的克隆及其植物双元表达载体的构建

目的]为利用植物基因工程技术获取hbFGF基因奠定基础。方法]采用RT-PCR方法克隆hbFGF基因,将其插入双元表达载体pCambia1301中,构建植物表达载体pCambia1301-hbfgf,并将该表达载体导入发根农杆菌菌株C58C1。结果]1%琼脂糖凝胶电泳结果显示,hbFGF基因被成功连接到克隆载体pMD18-T上,且重组质粒pMD18-T-hbfgf的DNA测序结果与GenBank中登录的hbFGFcDNA序列完全一致;双元表达载体重组质粒pCambia1301-hbfgf经BamHI消化后进行1%琼脂糖凝胶电泳,结果显示,电泳图谱中出现1200、10000bp2个片段,说明hbFGF基因已被成功克隆到植物表达载体pCambia1301中;农杆菌转化子PCR产物电泳图谱中483bp处出现条带,说明pCambia1301-hbfgf已被转入农杆菌中。结论]该研究成功获得了可直接用于遗传改良的工程菌pCambia1301-hbfgf-C58C1。

Cloning of hbFGF Gene and Construction of Its Binary Plant Expression Vector

Objective]The aim was to lay a foundation for obtaining hbFGF gene by utilizing plant genetic engineering technique.Method]The hbFGF gene was cloned by using RT-PCR method,it was inserted into binary expression vector pCambia1301 for constructing plant expression vector pCambia1301-hbfgf and introducing this expression vector into Agrobacterium rhizogenes Strain C58C1.Result]The results from 1% agarose gen electrophoresis (AGE) showed that hbFGF gene was connected to clone vector pMD18-T successfully and the DNA sequencing results of recombinant plasmid pMD18-T-hbfgf accorded with the cDNA sequence of hbFGF logged in GenBank completely.The recombinant plasmid pCambia1301-hbfgf of binary expression vector was treated with BamHI digestion before 1% AGE,the results was that there were 2 fragments of 1 200 and 10 000 bp appeared in electrophoregram,indicating that hbFGF gene had been cloned into plant expression vector pCambia1301 successfully.There was bands appeared at 483 bp in electrophoregram of PCR products of agrobacterium transformer,indicating that pCambia1301-hbfgf had been introduced into agrobacterium.Conclusion]In this research,the engineering strain pCambia1301-hbfgf-C58C1,which could be used for genetic improvement directly,was obtained.