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

作　　者：王海波 李芙蓉[1] 杨金翠 高永 郭俊云 WANG Haibo;LI Furong;YANG Jincui;GAO Yong;GUO Junyun(College of Biological Resource and Food Engineering,Qujing Normal University,Qujing 655011,China;Key Laboratory of Yunnan Province Universities of the Diversity and Ecological Adaptive Evolution for Animals and Plants on YunGui Plateau,Qujing Normal University,Qujing 655011,China)

机构地区：[1]曲靖师范学院生物资源与食品工程学院,云南曲靖655011 [2]曲靖师范学院云南省高校云贵高原动植物遗传多样性及生态适应性进化重点实验室,云南曲靖655011

出　　处：《华北农学报》2021年第4期23-30,共8页Acta Agriculturae Boreali-Sinica

基　　金：云南省地方本科高校(部分)基础研究联合专项(202001BA070001-003);云南省大学生创新创业训练计划项目(202010684038);国家自然科学基金(31460179;31760103);国家级大学生创新创业训练计划项目(201810684018)。

摘　　要：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.

关 键 词：小桐子 磷酸葡萄糖变位酶 基因克隆 原核表达 

分 类 号：Q78[生物学—分子生物学] S759.03[农业科学—森林经理学]