蓖麻Na^+/H^+逆向转运蛋白基因NhaD克隆与表达载体构建

Cloning and Expression Vector Construction of Na^+/H^+ Antiporter in Castor(Ricinus communis)

为发掘耐盐相关基因,以蓖麻叶片为材料,设计特异性引物,克隆蓖麻Na^+/H^+逆向转运蛋白基因(NhaD),并对该序列进行生物信息学分析。结果表明,NhaD全长1 743 bp,可编码580个氨基酸。将其命名为Rc NhaD。其编码蛋白的分子量为61.821 kD,等电点(pI)值6.74。利用DNAMAN软件对该序列与其它植物氨基酸序列进行同源性比对,结果显示与木薯等植物的同源性在85%以上,该蛋白含有ArsB蛋白的功能域,属于ArsB/NhaD转运蛋白家族成员。对其二级结构分析显示该蛋白为疏水性蛋白,该蛋白具有11个跨膜区域,是典型的跨膜蛋白。根据Rc NhaD全长设计带有KpnⅠ与XbaⅠ酶切位点的引物扩增出序列全长,用KpnⅠ与XbaⅠ进行双酶切后与表达载体pCG-1301连接,成功构建了Rc NhaD的表达载体。本研究为进一步研究该基因功能提供指导。

In order to discover salt-tolerant genes of castor,the halophyte Castor(Ricinus communis L.)leaves were used as experimental material.The specific primers were designed to clone the Na^+/H^+antiporter(NhaD)gene,and its sequence was analyzed by the approaches of bioinformatics.The full-length cDNA of this gene is 1 743 bp and encode 580 amino acids,which was named as RcNhaD.The predicted molecular weight is 61.821 kD and the isoelectric point(p I)is 6.74.The homology of the amino acid sequence of RcNhaD was compared with that of other plants by the DNAMAN software.The results showed that the homology of RcNhaD is over 85%similarities as the cassava etc.The protein of RcNhaD contains the functional domain of the ArsB protein and belongs to the member of the ArsB/NhaD transporter family.Secondary structure prediction of castor RcNhaD protein has a hydrophobic protein that includes 11 transmembrane regions,it is obvious that RcNhaD is a typical transmembrane protein.Based on the full length Rc NhaD sequence,the specific primer with digestion sites of KpnⅠand XbaⅠwas designed for amplification full-length sequence;the amplified RcNhaD with digestion of double enzyme KpnⅠand XbaⅠwas connected to the expression vector pCG1301 connection to successfully construct RcNhaD expression vector.This study provides guidance for further study on the function of this gene.