小拟南芥转运蛋白基因及NHX2基因的克隆与表达  

Analysis of transporter genes of Arabidopsis pumila and cloning and expression analysis of NHX2 gene

作　　者：孙琦 黄薇 刘芳 金玉环 肖建旺 黄先忠[1] Sun Qi;Huang Wei;Liu Fang;Jin Yuhuan;Xiao Jianwang;Huang Xianzhong(Special plant genomics laboratory,College of Life Sciences,Shihezi University,Shihezi,Xinjiang 832003,China)

机构地区：[1]石河子大学生命科学学院特色植物基因组学实验室

出　　处：《石河子大学学报（自然科学版）》2019年第2期200-209,共10页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(U1303302)

摘　　要：转运蛋白(transport protein)是膜蛋白的一大类,介导生物膜内外的化学物质及信号的交换。植物体内存在多个与Na +转运相关的蛋白,其中液泡膜Na +/H +逆向转运蛋白(Vacuolar Na^+/H^+ antiporter,NHX)在离子稳态和提高植物耐盐性方面发挥着重要作用。为了深入了解转运蛋白基因在短命植物小拟南芥( Arabidopsis pumila)耐盐方面的作用,本研究首先基于小拟南芥响应高盐胁迫叶片转录组数据筛选出1157个转运蛋白基因,按功能分为Na^+转运蛋白,K^+转运蛋白,Ca^2+转运蛋白,ABC转运蛋白以及糖转运蛋白等,其中功能注释为Na +转运蛋白的基因有24个。K均值(K-means)聚类分析结果显示,1157个转运蛋白基因分布于20个K subcluster,其中在K6、K9、K15子聚类中的基因数量分布较多,分别为172、193、190个。在分布于K6子聚类的Na^+转运蛋白基因中,有一个编码NHX2蛋白的基因经盐胁迫处理后明显上调表达。采用RT-PCR克隆了ApNHX 2基因, ApNHX2开放阅读框1626 bp,编码541个氨基酸。ApNHX2蛋白是一个典型的跨膜转运蛋白,具有12个跨膜结构区。系统进化分析表明ApNHX2与拟南芥AtNHX2亲缘关系最近。实时荧光定量PCR分析显示,ApNHX 2基因在小拟南芥各组织中均有表达,但在花中表达量最高。为进一步研究该基因的功能,构建了过量表达载体35S∶ApNHX2并转化农杆菌GV3101。本研究为进一步阐述转运蛋白基因在小拟南芥响应盐胁迫中的功能机制奠定了基础。Transport proteins are a large class of membrane proteins,which mediate the exchange of chemical substances and signal inside and outside biomembranes.There are several different kinds of transport proteins related to Na^+ transport in plants,of which vacuolar Na^+/H^+ antiporters play an important role in ion homeostasis and salt tolerance.To understand the molecular functions of transporter genes in salt tolerance of ephemeral plant Arabidopsis pumila, 1157 transporter protein-related genes were identified based on the transcriptome data,which were divided into six categories based on their annotated functions:calcium transporter,potassium transporter,sodium transporter,ABC transporter,sugar transporter and other transporters.There are 24 genes annotated as sodium transporter-related genes.K-means clustering analysis showed that 1157 transporter genes were distributed in 20 K subclusters,of which the number of genes in K6,K9 and K15 sub-clusters was bigger,and their number was 172,193 and 190,respectively.A Na^+ antiporter gene NHX2 was categoried into the K6 sub-cluster and its expression was obviously up-regulated in response to salinity. ApNHX2 gene was cloned through RT-PCR,and the length of open reading frame was 1626 bp,encoding 541 amino acid residues.Bioinformatics prediction revealed that ApNHX2 protein has twelve transmembrane domains with structure features of a typical transmembrane protein.Phylogenetic analysis showed that ApNHX2 is closely related to the Arabidopsis thaliana AtNHX2.In addition,quantitative real-time analysis showed that ApNHX2 was expressed in all tissues of Arabidopsis pumila with the highest level in flowers.To further study its function,a plant overexpression vector 35 S ∶ ApNHX2 was constructed and then transformed into Agrobacterium tumerfaciens strain GV3101.This study is helpful to elucidate the functional mechanism of transporter genes in response to salt stress in Arabidopsis pumila.

关 键 词：小拟南芥 NA^+/H^+逆向转运蛋白 基因克隆 组织表达 

分 类 号：Q943.2[生物学—植物学]