论文部分内容阅读
液泡膜H+-ATPase在植物应对非生物胁迫中起着十分重要的作用，对其关键亚基基因的克隆及分析，有助于阐释V-ATPase在逆境下的调节及响应机制。本研究利用RT-PCR技术从棉花中克隆了一个液泡膜H+-ATPase亚基A基因，命名为GhV HA-A。该基因ORF为1872 bp，编码623个氨基酸，预测的蛋白质理论分子量为68.41 kD，等电点为5.17。通过氨基酸序列比对和系统进化树分析发现，GhVHA-A与可可V-ATPase A亚基的同源性最高，达到97.9%，在进化上亲缘关系较近；且在编码蛋白的氨基酸序列上有两个与ATP或GTP的磷酸基团相互作用的保守结构域“GAFGCGKT”和“PSVNWLISYS”，说明A亚基是相对保守的。本研究构建了植物表达载体pCAMBIA 1304-V HA-A ，并转入到根癌农杆菌(GV3101)中，为后续研究棉花V-ATPase A亚基基因在干旱胁迫应答中的功能奠定试验基础。“,”V-H+-ATPase is c ommo nly found in plants and plays a vital role in abiotic stress tolerance. Cloning and expression analysis of the gene (s) encoding key V-ATPase subunit (s), and helps to elucidate V-H+-ATPase reg-ulation and response under diverse stresses. RT-PCR method was used to clone a V-ATPase subunit A gene, a gene coding for GhV HA-A was isolated from cotton (Gossypium hirsutum L.). The open reading frame was 1 872 bp, encoding 623 amino acid residues with a predicted molecular mass of 68.41 kD and a basic isoelectric point of 5.17. Homology analysis and phylogenetic tree analysis founded that GhVHA-A shares 97.9%amino acid identity with the V-ATPase subunit A from The ob roma c ac ao. Two conserved domains, GAFGCGKT and PSVNWLISYS, interacted with the phosphate groups ofthe coding region, indicating the highly conserved vacuolar H+-ATPase subunit A. In this study, the plant expression vector pCAMBIA1304-V HA-A was constructed and transformated into Agrobacterium (GV3101). It might lay a foundation of experiment for studing the response of cotton V-ATPase subunit A gene to drought stress.