马铃薯和拟南芥GAPC酶基因的克隆及分析

Cloning and analysis of the cytosolic glyceraldehyde 3-phosphate dehydrogenase( GAPC) gene from Solanum tuberosum and Arabidopsis thaliana

胞质三磷酸甘油醛脱氢酶(GAPC)是糖酵解中的关键酶,近年的研究表明其对盐、低温、高温、氧化、高渗、低磷等多种逆境胁迫均有响应。本实验采用RT-PCR,分别从马铃薯和拟南芥中克隆GAPC的编码区序列(CDS),并作生物信息学比较,同时构建2个基因的植物表达载体。结果表明,StGAPC和AtGAPC2的CDS长度均为1017bp,相似性84%,编码338个氨基酸,相似性为92%。蛋白分子量分别为36.65和36.91 kDa,理论等电点为6.34和6.67,均属稳定蛋白;三级结构预测表明两者和水稻(2e5rC)GAPC蛋白结构相似,具有GAPC保守功能域。多种植物GAPC基因的序列进化分析表明同科属植物的同源性较高可归于同一分支,与现有的分类系统相对应。以pBI121为基础载体,构建了由CaMV35S启动GAPC基因的植物表达载体,导入农杆菌EHA105,经PCR检测确定获得阳性克隆。本研究为获得转GAPC基因的材料并进一步研究GAPC在逆境中的功能奠定基础。

Cytosolic glyceraldehyde 3-phosphate dehydrogenase （GAPC） takes part in glycolysis and is a classical housekeeping enzyme in both prokaryotes and eukaryotes.Increasing evidence indicates that GAPC is involved in various plant abiotic and biotic stress responses such as salt,low phosphate,reactive oxygen species （ROS） and heat in phytophthora infestans.The AtGAPC2 gene has been found in responses to low phosphate and osmotic stress in Arabidopsis.Analysis of sequences and protein structure of GAPC could help in studies of the function and mechanisms of GAPC acting in plant stress resistance.The fragments encoding GAPC were obtained from Solanum tuberosum （StGAPC） and Arabidopsis thialiana （AtGAPC2） seedlings by reverse transcription polymerase chain reaction （RT-PCR）.The CDS lengths of StGAPC and AtGAPC2 were 1017 bp,encoding a protein subunit of 338 amino acids.Bioinformatics analysis demonstrated that the StGAPC and AtGAPC2 had 84％ similarity in nucleotide sequence and 92％ similarity in amino acid sequence.Molecular weights of the proteins were 36.65 and 36.91 kDa,the PI （theoretical isoelectric point） were 6.34 and 6.67,and both of them had stable protein structures.The 3D structures of StGAPC and AtGAPC2 were predicted by homology comparative modeling in the Swiss-Model,and showed that the 3D structures were highly similar to Oryza sativa GAPDH C chain （2e5rC）.Predicted proteins contained conserved GAPC domains which included an NAD binding domain constructed by βαβαβ Rossmann coil and a catalyzed domain in the saddle super secondary structure composed of seven β foldings.CDS sequences of GAPC showed that the same family of plants has a high homology attributed to the same branch in the evolutionary tree and this evolutionary relationship corresponded to the existing classification system.pBI121 was constructed as a base vector and the gene expression vector in the plant was driven by a CaMV35S promoter.The recombinant vector was introduced into Agrobacterium strain EHA105 and positive clones were determined by PCR.This research is a basis to obtain transgen ic materials and for further study on the function of GAPC.