蓖麻液泡膜质子泵RcVP1基因克隆与表达分析

Cloning and expression analysis of vacuolar membrane proton pump RcVP1 gene from castor(Ricinus communis L.)

为挖掘耐盐基因,培育耐盐蓖麻新种质,本研究设计特异性引物克隆蓖麻液泡膜H+-PPase基因,将其命名为Rc VP1,该基因开放阅读框为2 304 bp,编码767个氨基酸,其编码蛋白质的分子量和等电点分别为8.04×104和4.94。Rc VP1含有保守的H+-PPase结构域,包括CS1、CS2、CS3高度保守区,有13个跨膜结构域。多重序列比对结果表明Rc VP1氨基酸序列与毛白杨的相似性最高,达95.00%,与Ⅰ型H+-PPase拟南芥AVP1的相似性高达88.53%,与Ⅱ型H+-PPase拟南芥AVP2的相似性只有36.41%,属于Ⅰ型液泡膜H+-PPase跨膜蛋白质。半定量PCR分析结果表明,该基因在蓖麻叶片、茎中受盐胁迫诱导上调表达,在种子中抑制表达。

In order to explore the salt-tolerant genes and cultivate a new ger^nplasm of salt-tolerant castor,the specific primers were designed to clone the H+-PPase gene,named RcVPl.The open reading frame(ORF)of RcVPl gene was2304bp and encoded767amino acids.The molecular weight and isoelectric point of the encoded protein were8.04x104and4.94,respectively.RcVP1contained three conserved H+-PPase domains(C S1,C S2,C S3),and it had13transmembrane domains.Multiple alignment results showed that the amino acid sequence of RcVP1had95.00%and88.53%similarities with that of Populus tomentosa H+-PPase and Arabidopsis thaliana type I H+-PPase AVP1,respectively.However,it only had36.41%similarities with that of Arabidopsis thaliana type n H+-PPase AVP2.Therefore RcVPl belonged to type I vacuolar membrane proton pump transmembrane protein.Semi-quantitative PCR analysis results displayed that RcVPl gene up-regulated in the leaves and stems,down-regulated in the seeds under salt stress.