水稻多逆境响应基因OsMsr13的克隆与功能分析

Cloning and Functional Analysis of a Multiple-stress Responsive Gene,OsMsr13,in Rice

为了深入研究作物耐逆境胁迫的分子机制和发现新的水稻耐逆基因,采用Affymetrix基因芯片技术与水稻表达芯片(含51,279个转录本),分析了培矮64S不同生长发育时期、不同组织器官全基因组在低温、干旱、高温逆境胁迫下的表达水平变化,筛选出一批耐多逆境候选功能基因。OsMsr13是其中一个受低温、高温与干旱诱导,在各生长发育时期与组织器官均显著上调的基因。根据序列生物信息学分析,OsMsr13含有7个外显子和6个内含子c,DNA全长序列为1603bp,完整的ORF为1317bp,编码一个含438个氨基酸残基的蛋白质;在OsMsr13启动子区域发现多个与逆境应答相关的顺式作用元件;数据库查找和蛋白质多序列的比对表明OsMsr13的预测蛋白属于钙调素结合蛋白家族。为了进一步研究OsMsr13基因的功能,通过RT-PCR方法扩增到包含OsMsr13完整ORF的cDNA克隆,构建了含有OsMsr13重组基因的过量表达载体pCAM-JIT-OsMsr13,并经农杆菌介导法将重组基因导入到水稻中。转OsMsr13株系苗期的抗逆性试验表明OsMsr13过量表达能显著地提高水稻的耐寒性,但在抗旱和耐高温方面,转基因植株和野生型9311没有观察到多大差别。

To understand mechanisms underlying stress tolerance and discover novel stress-tolerance genes in rice（Oryza sativa L.）,GeneChip Rice Genome Array（Affymetrix） was used to investigate the global genome expression profiles of the indica cultivar Pei＇ai 64S subjected to cold,drought or heat stresses.A large number of genes were highly up-and down-regulated under the stresses.OsMsr13（Oryza sativa L.Multiple Stresses Responsive Gene 13） was one of the genes highly induced in different plant organs at different development stages by different stresses.The sequence analysis showed that OsMsr13 contains seven exons and six introns and encodes a protein of 438 amino acid residues.Several cis-elements related to stress responses were identified in the promoter region of OsMsr13.GenBank sequence database searching and multiple sequence alignments showed that OsMsr13 may belong to the calmodulin-bind superfamily.To study its function,the cDNA of gene OsMsr13 was cloned through RT-PCR amplification and ligated into the over-expression vector pCAM-JIT（recombined from pCAMBIA1300 and pJIT163）.The constructed vector was delivered into rice by the Agrobacterium-mediated transformation method.The obtained result showed that over expression of OsMsr13 in rice enhanced cold tolerance of rice plants,but showed no effect on heat and drought tolerance.