小拟南芥液泡膜H^+-PPase基因OpVP1的克隆、序列分析及表达

Cloning,Sequence Analysis and Expression Analysis of Vacuolar H^+-PPase Gene OpVP1 in Olimarabidopsis pumila

植物液泡膜质子转运无机焦磷酸酶(V-PPase)酸化植物液泡并为液泡的次级转运系统提供能量,在植物耐盐性起着重要的作用。为了克隆小拟南芥液泡膜H+-PPase基因,采用RT-PCR结合RACE的方法从小拟南芥叶片的cDNA中克隆了1个液泡膜H+-PPase基因,命名为OpVP1。OpVP1基因的cDNA全长为2698bp,开放阅读框(ORF)为2313bp,编码770个氨基酸。OpVP1蛋白与琴叶拟南芥、拟南芥相似性最高,分别为98.6%、98.4%。系统进化分析表明OpVP1基因属于Ⅰ型液泡膜质子焦磷酸酶基因。OpVP1蛋白的分子量是80745.9Da,等电点pI为5.13,含有14个跨膜螺旋结构。三维结构分析表明OpVP1蛋白是由2个单体组成的二聚体蛋白。qRT-PCR表明OpVP1基因在角果中表达高于根、茎、叶和花中的表达。

The H＋-translocating inorganic pyrophosphatase（H＋-PPase） can acidify vacuoles in plants and provide energy for secondary transit system of vacuoles,which plays an important role in salt tolerance of plants.To clone the H＋-PPase gene of Olimarabidopsis pumila,we identified a homolog gene named OpVP1 by RT-PCR and RACE using the leaf cDNA as PCR template.The full-length cDNA of OpVP1 was 2 698 bp,the open reading of which was 2 313 bp in length and encoded a putative 770 amino acids.The deduced amino acid sequence of OpVP1 shared highest similarity with Arabidopsis lyrata and Arabidopsis thaliana,with an identity of 98.6%,98.4%,respectively.The phylogenetic analysis indicated that OpVP1 belonged to classⅠtype vacuolar H＋-inorganic pyrophosphatase gene.The molecular weight of OpVP1 protein was 80745.9 Da and the isoelectric point was 5.13.Computional analysis showed OpVP1 was a typical membrane protein with 14 transmembrane domains.Three-dimensional structural analysis showed OpVP1 protein was a dimeric protein,which was composed of two monomers.qRT-PCR analysis showed that the expression level of OpVP1 was higher in silique than in root,stem,leaf and flower.