马铃薯水通道蛋白基因cDNA克隆、序列分析及表达  

Cloning,Sequence Analysis and Expression of cDNA Encoding Plasma Aquaporins StPIP1 of Solanum tuberosum

作　　者：吴旺泽[1] 彭晓莉[2] 王晓明[3] 王蒂[1] 

机构地区：[1]甘肃农业大学农学院 [2]兰州大学生命科学学院,兰州730000 [3]仲恺农业技术学院农业与园林学院,广州510225

出　　处：《农业生物技术学报》2007年第4期677-683,共7页Journal of Agricultural Biotechnology

基　　金：国家基础研究发展规划(973)项目(No.2006CB708200);甘肃省农业厅项目(No.GNSW-2006-01)资助

摘　　要：以干旱胁迫处理的马铃薯(Solanum tuberosum L.)普通栽培种Gannongshu No.2叶片为材料,提取总RNA,采用RT-PCR方法,扩增出编码288aa序列的水通道蛋白基因StPIP1(Solanum tuberosum L.plasma membrane intrinsic protein gene)cDNA,GenBank登录号为DQ999080。用生物软件对StPIP1分析表明,StPIP1含有6个跨膜区,具有MIP家族的特征信号序列SGXHXNPAVT,还具有质膜水通道蛋白家族的特征信号序列GGGANXXXXGY和TGI/TNPARSL/FGAAI/VI/VF/YN。聚类分析表明和其它14个物种PIP1类水通道蛋白同源性在92%以上,属于质膜水通道蛋白PIP1类。三级结构预测表明和菠菜(Spinacia oleracea)(2B5F)水通道蛋白结构相似,单体由5个短环相连的6个亲水的跨膜螺旋,N-末端、C-末端以及B、D环位于细胞内,A环、C环及E环在细胞外。构建以rd29A启动子和CaMV35S启动子驱动的StPIP1和GFP(绿色荧光蛋白)融合基因表达载体,用基因枪转化洋葱(Allium cepa)表皮进行瞬时表达,结果表明StPIP1表达受干旱胁迫的调节。Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins that facilitate water transport across biological membranes.The gene encoding the Solanum tuberosum L. aquaporin （StPIPI） cDNA was cloned from leaf ofS. tuberosum cv. Gannongshu No.2 under PEG6000 stress by RT-PCR. The StPIPI cDNA was 867 bp in length, encoded a protein of 288 amino acids with a predicted molecular mass of 30. 9 kD（GenBank Accessin No.DQ 999080）. StPIP1 exhibited a typical structure with six membrane-spanning domains and an internal symmetry showing two highly conserved Asn-Pro-Ala （NPA） motifs, and possessing the MIP family signal consensus sequence SGXHXNPAVT and the higher plant PIP highly conservative sequence GGGANXXXXGY and TGI/TNPARSL/FGAAI/VI/VF/YN.The StPIPI amino acids showed 92%-97% identity to other 14 plant species PIPI subfamily, so StPIPI should be a plasma membrane intrinsic proteins of PIP1 subfamily. The protein 3D structure was predicted by homology comparatvie modeling in Swiss-Model, the results from Swiss-Model showed that the 3D structure of StPIP1 was highly similitude with Spinacia oleracea （2B5F）. Two plant expression vectors that schleped StPIP1 and GFP （green fluorencent protein） fusion genes were constructed, in which StPIP1 and GFP fusion genes were regulated by rd29A and CaMV35S promoters, respectively. Particle bombardment procedures were used to transfer StPIP1 and GFP fusion genes to onion （Allium cepa）epidermis cells, and transient expression was presented. ,The results showed that expression of StPIP1 was regulated by drought stress.

关 键 词：马铃薯 水通道蛋白 基因克隆 序列分析 蛋白质结构预测 瞬时表达 

分 类 号：S188[农业科学—农业基础科学]