甘蓝型油菜CONSTANS基因植物过表达载体构建及转基因植株的获得

Construction of Overexpression Vector of CONSTANS Gene Plant in Brassica napus and Production of Transgenic Plants

甘蓝型油菜CONSTANS目的基因的克隆以NCBI上公布的序列设计全长扩增引物进行扩增。对目的基因片段测序并进行酶切位点分析,设计酶切引物(BamHⅠ,SacⅠ),通过BamHⅠ和SacⅠ双酶切将目的基因片段连接在真核表达载体pBI121上,PCR鉴定结果表明PBI121+CONSTANS过表达载体已成功构建。将过表达载体转化感受态EHA105细胞,通过拟南芥花絮侵染T0代种子,用含卡拉霉素(50 mg/L)的MS固体培养基进行筛选,获得阳性植株。通过PCR鉴定,结果表明PBI121+CONSTANS过表达载体已成功导入拟南芥中,获得转基因阳性苗12株,为下一步基因功能分析做好了准备。

The CONSTANS target gene from Brassica napus was cloned,and the full length amplified primer of it was designed and amplified on the sequence released on NCBI.The target gene fragments were sequenced and the restriction loci were analyzed.The enzyme primers（BamHⅠ,SacⅠ） were designed,and the target gene fragments were connected to the eukaryotic expression vector p BI121 through BamHⅠand SacⅠ double enzyme digestion.PCR identification result indicated that PBI121 ＋CONSTANS overexpression vector was successfully constructed.The overexpression vector was transformed into the competent EHA105 cell,and the seeds of T0 were infected by Arabidopsis thaliana.The positive plants were screened by MS solid medium containing Cara（50 mg/L）.PCR identification showed that PBI121＋CONSTANS overexpression vector was successfully introduced into Arabidopsis thaliana and 12 transgenic positive seedlings were obtained,which did the preparation for the gene function analysis.