小桐子磷酸葡萄糖变位酶cPGM与pPGM基因的克隆及原核表达分析

Cloning and Prokaryotic Expression of Phosphoglucomutase Genes cPGM and pPGM from Jatropha curcas

为了探究磷酸葡萄糖变位酶(PGM)在植物蔗糖与淀粉代谢中的重要作用,基于同源序列比对的方法,在小桐子基因组中鉴定到1个细胞质型PGM基因(命名为JccPGM)与1个叶绿体型PGM基因(命名为JcpPGM),利用qRT-PCR方法检测JccPGM与JcpPGM基因在小桐子不同器官与低温条件下的表达特性,同时,构建了pGEX-4T-1-JccPGM与pGEX-4T-1-JcpPGM原核表达载体并在大肠杆菌BL21(DE3)中进行了表达分析。结果表明:两者分别编码582,637 aa的蛋白质。聚类分析表明,小桐子pPGM在其N端包含叶绿体定位信号肽,而cPGM较pPGM多4段肽链序列-^(108)VGVDGS^(113)-、-^(183)SGPE^(186)-、-^(283)GKSNSE^(288)-、-^(470)SLGEVN^(475)-。qRT-PCR表达分析显示,小桐子cPGM与pPGM基因都在叶片中高表达,而在根与种子中表达量较低。通过BL21(DE3)诱导表达,分别得到90.7,97.0 ku的蛋白条带,与理论融合蛋白的分子量一致。综上所述,本研究为开展小桐子cPGM与pPGM基因表达蛋白的功能分析以及其在蔗糖与淀粉积累、逆境应答中的机制研究奠定了基础。

In order to explore the function of phosphoglucomutase(PGM)in the metabolism of sucrose and starch in plants.Based on homologous sequence alignment,a cytoplasmic PGM gene(named JccPGM)and a chloroplastic PGM gene(named JcpPGM)were identified from the Jatropha curcas genome.The expression levels of JccPGM and JcpPGM in different organs and under chilling stress were detected by qRT-PCR method.The prokaryotic expression recombinant vectors of pGEX-4T-1-JccPGM and pGEX-4T-1-JcpPGM were constructed,and then fusion proteins were induced in Escherichia coli BL21(DE3)strains.The results showed that JccPGM and JcpPGM encoded proteins of 582,637 aa,respectively.Cluster analysis showed that pPGM contained a chloroplast localization signal peptide at the N-terminal,whereas cPGM had four more peptide sequences of-^(108)VGVDGS^(113)-,-^(183)SGPE^(186)-,-^(283)GKSNSE^(288)-,-^(470)SLGEVN^(475)-than pPGM.qRT-PCR analysis revealed that cPGM and pPGM expressed specifically in different organs,abundantly in leaves,but scarcely in roots and seeds.Escherichia coli BL21(DE3)induction and SDS-PAGE analysis indicated that the fusion protein molecular weights were 90.7,97.0 ku,respectively,which was consistent with the predicted weights.In conclusion,this study lays a foundation for further studies on the protein functions of cPGM and pPGM and the mechanisms underlying sucrose and starch accumulation and stress responses in J.curcas.