大豆GmGST12基因的克隆及表达分析  

Cloning and Expression Analysis of GmGST12 in Soybean

作　　者：韩少怀 李佳佳[1] 张璟曜 张浩[1] 李艳伟[1] 丁先龙[1] 贺亭亭[1] 杨守萍[1] 

机构地区：[1]南京农业大学大豆研究所/国家大豆改良中心/农业部大豆生物学与遗传育种重点实验室/作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《大豆科学》2015年第5期782-788,共7页Soybean Science

基　　金：国家高技术发展研究计划"863计划"(2011AA10A105);国家转基因重大专项(2011ZX08004-005;2013ZX08004-005;2014ZX08004-005);教育部长江学者和创新团队发展计划(PCSIRT13073);江苏省现代作物生产协同创新中心

摘　　要：植物中的活性氧(ROS)作为细胞内或细胞间的信号起重要作用,但其浓度过高可能会导致植物出现雄性不育,而谷胱甘肽S-转移酶类(GSTs)对于解除ROS对细胞的毒害具有重要作用。前期在大豆质核互作雄性不育系NJCMS1A与其保持系NJCMS1B的比较转录组学研究中发现一个差异表达基因Gm GST12。本研究通过同源克隆法从NJCMS1A和NJCMS1B花蕾中克隆了Gm GST12基因,其编码区序列(CDS)全长均为708 bp,且核苷酸序列相同,编码含235个氨基酸的谷胱甘肽S-转移酶;系统发生分析表明,Gm GST12与拟南芥At GSTU9的同源性最高,二者氨基酸序列相似度为52%;组织表达分析表明,Gm GST12在NJCMS1A花蕾中的表达水平极显著地高于NJCMS1B花蕾中的表达水平,而在根、茎和叶中的表达水平差异不显著;亚细胞定位结果显示Gm GST12定位于细胞核和细胞质中;此外,还构建了植物过表达载体p CAMBIA3301-Gm GST12,以用于下一步转基因功能验证研究。以上结果为进一步研究大豆质核互作雄性不育的分子机理提供了基础。The reactive oxygen species （ROS） play an important role in plants which act as intracellular or intercellular sig- nals. But high concentration of ROS might lead to plant male sterility. Glutathione S-transferases（GSTs） are crucial to the de- toxification for the cells. Based on comparative transcriptome analysis between the cytoplasmic-nuclear male sterile line NJC- MS1A and its maintainer NJCMS1B in Glycine max, a differential expression gene of GmGST12 was identified in our previous research. In the present study, GmGST12 gene was cloned from the flower buds of NJCMS1A and NJCMS1B by homology- based cloning method. The coding DNA sequences （CDSs） of GmGST12 from NJCMS1A and NJCMS1B were 708 bp long with the same nucleotide sequences and coded the glutathione S-transferases protein consisted of 235 amino acids. Phylogenet- ic analysis showed that the homology between GmGSTI2 and AtGSTU9 from Arabidopsis thaliana was the highest and the iden- tity of the amino acid sequences was 52% . The tissues expression analysis showed that the mRNA expression level of GmGST12 in flower buds of NJCMS1A was significantly higher than that in flower buds of NJCMS1B, but no difference was found in root, steam and leaf. The result of subcellular localization indicated that GmGST12 was located in the cytoplasm and nucleus. In addition, the plant overexpression vector pCAMBIA3301-GmGST12 was constructed for further transgenic re- search. All the above results provided the foundation for future study on the molecular mechanism of cytoplasmic-nuclear male sterility in soybean.

关 键 词：大豆 质核互作雄性不育 谷胱甘肽S-转移酶 基因克隆 表达分析 

分 类 号：S565.1[农业科学—作物学]