香格里拉水韭磷酸烯醇式丙酮酸羧化酶基因(PEPC)的克隆及其表达载体构建

Cloning of the phosphoenolpyruvate carboxylase gene(PEPC)from Isoetes shangrilaensis X.Liu and construction of its expression vector

以中国特有植物香格里拉水韭(Isoetes shangrilaensis X.Liu)为材料,通过转录组测序数据分析筛选出磷酸烯醇式丙酮酸羧化酶基因(IsPEPC),根据该基因序列,从香格里拉水韭cDNA中克隆获得磷酸烯醇式丙酮酸羧化酶(PEPCase)的编码基因IsPEPC,并将此基因插入pCAMBIA-2300-N-eGFP及pMD质粒载体上,再采用农杆菌介导的花序浸染法将2个重组载体分开转入野生型拟南芥(Arabidopsis thaliana(L.)Heynh.)中。结果显示:IsPEPC基因蛋白编码序列长度为2928 bp,编码975个氨基酸;同源性检索分析结果表明,该蛋白与其近源物种江南卷柏(Selaginella moellendorffii Hieron.)的PEPC基因蛋白序列同源性为79.8%。对转基因的T1代拟南芥通过抗性筛选并在gDNA水平上阳性鉴定,初步鉴定得到pC2300-N-eGFP-IsPEPC转基因株系26个和pMD-IsPEPC转基因株系32个。

Using Chinese Isoetes shangrilaensis X.Liu,the phosphoenolpyruvate carboxylase gene(IsPEPC)sequence information was screened by transcriptome sequencing analysis,and the IsPEPC gene encoding the phosphoenolpyruvate carboxylase(PEPCase)was cloned from cDNA based on the gene sequence.The IsPEPC gene was inserted into the pCAMBIA-2300-N-eGFP and pMD plasmid vectors,after which the recombinant vectors were transferred into wild-type Arabidopsis thaliana(L.)Heynh.by agrobacterium-mediated inflorescence infiltration.Results showed that the protein-coding sequence of the IsPEPC gene was 2928 bp in length and encoded 975 amino acids.Homologous searching showed that the protein was related to the PEPC protein sequence of the source species Selaginella moellendorffii Hieron.,with 79.8%sequence homology.Transgenic T1 A.thaliana was screened for resistance and positively identified at the gDNA level.We initially identified 26 pC2300-N-eGFP-IsPEPC transgenic lines and 32 pMD-IsPEPC transgenic lines.