小麦水通道蛋白TaTIP1-4DL基因的克隆及表达分析

Cloning and Expression Analysis of Aquaporin TaTIP1-4DL Gene in Wheat

TIPs家族是一类位于液泡膜上的膜内在蛋白,负责调节细胞膨压,可以响应植物逆境胁迫。为进一步探究小麦中TIPs基因的表达模式和生物学功能,利用同源克隆的方法从小麦旗叶中扩增出TaTIP1-4DL基因,其编码区序列全长771 bp,编码257个氨基酸。生物信息学分析显示:该蛋白分子质量为26.3 ku,理论等电点为6.82,为疏水稳定性蛋白;三级结构为保守的沙漏结构,包含6个长α螺旋和2个短α螺旋,符合水通道蛋白的经典模型;氨基酸比对和进化树分析表明,小麦中的TaTIP1-4DL蛋白与山羊草、二穗短柄草中的同源关系较近,并包含6个跨膜区和2个保守的NPA基序;启动子顺式作用元件中包含与逆境相关的响应元件。组织特异性表达分析显示,TaTIP1-4DL基因在小麦不同组织器官中均有表达,且叶片中的表达量最高,茎中最少。胁迫表达分析表明,干旱、脱落酸、高盐对叶片和籽粒中TaTIP1-4DL基因的表达有不同程度的诱导,表达水平普遍呈现先上调后下降的趋势,其中干旱对叶片中该基因的诱导水平最高,干旱处理6 h后,叶片中的表达量达到胁迫前的14倍。

The TIPs family is a type of membrane intrinsic protein located on the tonoplast,which is responsible for regulating cell turgor and responds to external stress.To further explore the expression pattern and biological function of TIPs gene in wheat,the TaTIP1-4DL gene was amplified from flag leaves of wheat by homologous cloning.The coding region was 771 bp in length and encoded 257 amino acids.Bioinformatics analysis showed that the protein had a molecular weight of 26.3 ku and a theoretical isoelectric point of 6.82,belonging to a hydrophobic stability protein.The tertiary structure was a conservative hourglass structure containing six long alpha helices and two short alpha helices,corresponding to the classic model of aquaporin.The amino acid alignment and phylogenetic tree analysis showed that the TaTIP1-4DL protein of wheat was closely related to the homology of Aegilops Linn.and Brachypodium distachyou(L.)Beauv.The promoter cis-acting element contained the response element related to stress.Tissue-specific expression analysis showed that TaTIP1-4DL gene was expressed in different tissues and organs of wheat,and the highest expression level was in leaves and the least in stems.The stress expression analysis showed that the expression level of TaTIP1-4DL gene in leaves and seeds was induced in different degrees by drought,abscisic acid and high salt,and the expression level was generally up-regulated and then decreased.Among them,drought induced the highest level of the gene in leaves.After 6 hours of drought treatment,the expression of the gene in leaves reached 14 times of that before stress.