陆地棉水通道蛋白GhNIP6.1基因的克隆及表达分析  

Cloning of Aquaporin Gene GhNIP6.1 from the Upland Cotton and Its Expression in Different Cotton Tissues

作　　者：巩元勇[1] 郭书巧[1] 束红梅[1] 倪万潮[1] 

机构地区：[1]江苏省农业科学院经济作物研究所/农业部长江下游棉花和油菜重点实验室,江苏南京210014

出　　处：《棉花学报》2013年第1期1-8,共8页Cotton Science

基　　金：国家转基因重大专项(2011ZX08005-001);国家自然科学基金(31101452);江苏省农业科学院博士后科研资助计划(012066511108);江苏省自然科学基金(BK2010465)

摘　　要：根据EST拼接的序列设计引物,利用RT-PCR技术,从陆地棉品种苏棉18中克隆获得了一个水通道蛋白基因GhNIP6.1。该基因开放阅读框包含903个核苷酸,编码300个氨基酸,分子量为30.97kD,理论等电点为8.99。GhNIP6.1蛋白的生物信息学分析表明:GhNIP6.1含有6个跨膜区,由5个环相连,其中环A、C和E在细胞膜外,环B、D和蛋白的N、C末端都位于细胞膜内,N末端79个氨基酸,C末端18个氨基酸,具有MIP家族典型的保守氨基酸序列;该蛋白的三级结构同拟南芥的AtNIP6.1非常相似,亚细胞定位也同AtNIP6.1一致,可能位于质膜中。进化分析发现,GhNIP6.1蛋白同拟南芥的AtNIP6.1蛋白相似性最高。进一步扩增棉花核基因组获得了2021bp的DNA序列,它包含5个外显子和4个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则。半定量分析表明,该基因在根、茎和叶中都有表达,其中真叶含量较高,子叶中没有表达。Primers were designed according to the assembled ESTs of Gossypium hirsutum, and an aquaporin gene GhNIP6.1 was obtained from upland cotton Sumian 18 in this experiment by RT-PCR. The gene contains an ORF comprising of 903 nucleotides which encodes 300 amino acids with a predicted molecular mass of 30.97 kD and theoretical isoelectric point of 8.99. Bioinformatics analysis indicates the GhNIP6.1 exhibited a typical structure with six trans-membrane domains which linked by five loops, loop A, C and E were at outside of membrane, loop B, D, N terminal and C terminal were at inside of membrane. N terminal has 79 amino acids and C terminal has 18 amino acids. The amino acid sequence of GhNIP6.1 has the conserved peptides of MIP family. The result of predicted protein 3 D structure of GhNIP6.1 showed that it was highly similitude with that of AtNIP6.1. The subcellular localization of GhNIP6.1 maybe localize at plasma membrane, which is consistent with AtNIP6.1. Phylogenic analysis displays GhNIP6.1 has the highest similarity with Arabidopsis thaliana AtNIP6.1 protein. A genomic DNA sequence with 2021 bp clone containing an aquaporin was isolated from the upland cotton in this experiment, and the GhNIP6.1 gene contains eight introns and seven exons. All exon/intron junctions of the gene contain the gt/ag consensus splicing site. Semi-quantitative analysis of GhNIP6.1 expression showed that the gene expressed extensively in cotton roots, stems, leaves, except cotyledon, and leaf is the most highly expressed tissue.

关 键 词：陆地棉 水通道蛋白 GhNIP6.1 表达序列标签 

分 类 号：S562[农业科学—作物学]