百脉根LjIPT3启动子的克隆及表达载体的构建

目的]为深入研究LjIPT3的表达调控和功能奠定基础。方法]利用TAIL-PCR技术克隆百脉根IPT基因LjIPT3起始密码子的上游序列,利用生物信息学手段对其序列特征和潜在的调控元件进行分析,并构建启动子表达载体。结果]利用TAIL-PCR技术成功克隆了LjIPT3基因5′端上游调控序列。将目的片段连接到pMD18-T载体上,获长度为1 910 bp序列,其3′端有部分序列与LjIPT3的5′端相同,证明克隆的片段为LjIPT3起始密码子ATG的上游区域。该序列含有1个TATA-box和3个CAAT-box,还包含光响应元件、MYB结合位点、不同的激素反应元件以及各种胁迫元件,说明该基因的表达可能受多种因素的调控。成功构建了驱动报告基因GUS的植物表达载体。结论]该研究为研究植物细胞分裂素的合成代谢和植物生长发育的调控奠定了基础。

Cloning of LjIPT3 Promoter in Lotus japonicus and Its Expression Vector Construction

Objective] The research aimed to lay the foundation for deep study on the expression regulation and functions of LjIPT3.Method] The upstream sequence of start codon in IPT gene(LjIPT3) of Lotus japonicus were cloned by using TAIL-PCR technology.Its sequence characteristics and the potential regulatory elements were analyzed by bioinformatics means and the promoter expression vector was constructed.Result] The upstream regulatory sequence at 5′ end of LjIPT3gene was successfully cloned by using TAIL-PCR technology.The target fragment was connected with pMD18-T vector and the sequence with the length of 1 910 bp was obtained.Some sequence at 3′ end was the same with the 5′ end of LjIPT3,which proved that the cloned fragment was the upstream region of the start codon in LjIPT3gene.There were one TATA-box and 3CAAT-box in this sequence.It also contained photoresponse,MYB binding sites,different hormone responsive element and all kinds of stress elements.These indicted that the expression of this gene was probably regulated by many kinds of factors.The plant expression vector for driving reporter gene GUS was successfully constructed.Conclusion] This research laid the foundation for studying the anabolism of phytocytomine and the regulation of plant growth and development.