棉花GhDHAR3基因克隆、功能序列分析及烟草的遗传转化  

Cloning and Sequence Analysis of a Cotton DHAR3 cDNA and Genetic Transformation of Tobacco

作　　者：王芳[1] 王斐[1] 孙辉[1] 李鸿彬[1,2] 

机构地区：[1]石河子大学生命科学学院,新疆石河子832003 [2]石河子大学农业生物技术重点实验室,新疆石河子832003

出　　处：《西北农业学报》2011年第5期88-93,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(30860031);农业部转基因专项(2009ZX08005-027B);兵团博士基金(2008JC01)

摘　　要：对棉花EST数据库进行同源搜索、比对和序列拼接,经RT-PCR从陆地棉纤维组织中扩增得到脱氢抗坏血酸还原酶基因GhDHAR3的cDNA,其开放阅读框为792bp,编码由263个氨基酸组成的蛋白质。同源性比对分析显示GhDHAR3蛋白具有较高的保守性,进化树分析表明其与拟南芥AtDHAR3亲缘关系较近。利用软件进行蛋白质序列分析,GhDHAR3蛋白具有GST-N家族和GST-C-DHAR典型的结构域,分别属于硫氧还蛋白超家族和GST-C末端超家族。将GhDHAR3基因构建到植物真核表达载体pCAMBIA2300中,利用农杆菌通过叶盘法转化烟草,经PCR分子鉴定,获得了含GhDHAR3转基因烟草植株。首次克隆棉花DHAR基因,通过结构域分析其可能的作用并成功转化烟草。Ascorbate acid plays important role in multiple physiological processes such as plant cell development and stress responses.Here,a cotton cDNA,enconding a 792 bp open reading frame and dehydroascorbate reductase3（GhDHAR3） protein of 263 amino acid residues,was cloned by RT-PCR method from developing cotton fibers.Homology blast and phylogenetic tree analyses showed that GhDHAR3 contained conserved functional domain and belonged to the arabidopsis AtDHAR3 protein family.Motif search results indicated that GhDHAR3 protein contained typical GST N terminal family and GST C terminal subfamily motif belonging to the thioredoxin（TRX）-like superfamily and GST C-terminal alpha helical domain family respectively.Signal peptide was not found in GhDHAR3 protein sequence.Plant over-expression vector,constructed by enzyme digestion of GhDHAR3 and pCAMBIA2300 and successive ligation,was transformed of tobacco.Transgenic tobacco plants were obtained successfully after molecular identification.

关 键 词：棉花 脱氢抗坏血酸还原酶 植物表达载体构建 遗传转化 

分 类 号：S562[农业科学—作物学]