枣树水通道蛋白基因生物信息学分析及原核表达载体的构建

The Biological Information Analysis and Construction of the Recombinant Prokaryotic Expression Plasmid of Ziziphus jujuba Aquaporin(AQP)

利用生物信息学软件对已获得的枣树水通道蛋白基因cDNA序列ZjPIP2(DDBJ/EMBL/GenBank的注册号为:AB530493)进行了同源性及功能位点等多项参数分析,结果表明,此序列为全长846 bp的开放读码框(ORF),编码281氨基酸,分子量为29.87 kD,理论等电点为8.77;具有膜蛋白(MIP)家族典型的保守氨基酸序列HINPAVTFG和2个NPA保守肽段。序列相似性分析表明,该蛋白与已知的其他12种植物膜蛋白中的水通道蛋白具有极高的同源性,属于水通道蛋白的质膜膜内蛋白PIP2类。三级结构预测表明,其与菠菜(Spinacia oleracea)水通道蛋白(1z98A)有相似的三维结构。以克隆载体pSPORT1携带的枣树水通道蛋白基因cDNA序列为模板,PCR扩增后,经BamHⅠ和SalⅠ消化,与pET28a载体进行重组连接,测序结果显示,其原核表达载体构建成功。此结果为研究枣水通道蛋白基因在植物组织的分布、生物学功能以及可能的活性调节方式奠定了基础。

Analyzing the homology and functional sites and many other parameters of the aquaporin＇s eDNA sequences that we have obtained by bioinformatics software, the results show that this sequence is an open reading frame （ORF）whose full-length is 846 bp, encoding 281 amino acids. Its molecular weight is 29.87 kD; theoretical isoelectric point is 8.77; and it has a conserved amino acid sequence ＂HINPAVTFG＂and two ＂NPA＂conservative peptide which are MIP＇s （major intrinsic protein） typical conserved amino acid sequence; similarity analysis shows that the protein has highly homologous with other known aquaporins of the other 12 plants, belonging to the PIP2 （plasma membrane intrinsic protein, PIP ）class. Tertiary structure prediction shows that it has similar three-dimensional structure with the Spinacia oleracea＇s aquaporins （lz98A）. Using the cloning vector pSPORT1 carrying Ziziphus jujube aquaporin gene＇s eDNA sequence as a template to amplify the objective gene, then the PCR products were digested by BamH Ⅰ and So/Ⅰ and connected to recombinant with prokaryotic expression plasmid pET28a, and the sequencing results show that the prokaryotie expression vector was successfully constructed. This data lays solid foundations for further research on the protein, the distribution of plant tissue, the biological function, and the possible ways of active regulation.