小拟南芥转运蛋白基因及NHX2基因的克隆与表达
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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Ap NHX2基因,Ap NHX2开放阅读框1626 bp,编码541个氨基酸。Ap NHX2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Ap NHX2与拟南芥At NHX2亲缘关系最近。实时荧光定量PCR分析显示,Ap NHX2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∶Ap NHX2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. Ap NHX2 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 Ap NHX2 protein has twelve transmembrane domains with structure features of a typical transmembrane protein.Phylogenetic analysis showed that Ap NHX2 is closely related to the Arabidopsis thaliana At NHX2.In addition,quantitative real-time analysis showed that Ap NHX2 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 ∶Ap NHX2 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.
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. Ap NHX2 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 Ap NHX2 protein has twelve transmembrane domains with structure features of a typical transmembrane protein.Phylogenetic analysis showed that Ap NHX2 is closely related to the Arabidopsis thaliana At NHX2.In addition,quantitative real-time analysis showed that Ap NHX2 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 ∶Ap NHX2 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.
引文
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[25]林军,张亮,黄先忠.新疆小拟南芥NAC转录因子ApNAC055的克隆及表达分析[J].石河子大学学报(自然科学版),2017,35(6):747-753.Lin J,Zhang L,Huang X Z.Molecular cloning and expression analysis of Ap NAC055 from Arabidopsis pumila[J].Journal of Shihezi University(Natural Science),2017,35(6):747-753.
[26]黄先忠,张鹏,吕新华,等.新疆小拟南芥Ap CBF1基因的克隆及其过量表达转基因的研究[J].石河子大学学报(自然科学版),2009,27(3):265-268.Huang X Z,Zhang P,Lu X H,et al.Molecular cloning and overexpression of Ap CBF1 from Olimarabidopsis pumila in Xinjiang[J].Journal of Shihezi University(Natural Science),2009,27(3):265-268.
[27]赵云霞,郭丹丽,魏艳玲,等.新疆无苞芥Na+/H+逆向转运蛋白基因Op NHX1的克隆、表达分析与功能验证[J].生物技术通报,2014,(7):74-80.Zhao Y X,Guo D L,Wei Y L,et al.Cloning,expressing and functional analysis of Na+/H+antiporter gene Op N-HX1 from Olimarabidopsis pumila in Xinjiang[J].Biotechnology Bulletin,2014,(7):74-80.
[28]魏艳玲,贾跃腾,黄先忠,等.新疆无苞芥Op NAC083基因的克隆与表达分析[J].石河子大学学报(自然科学版),2015,33(2):222-229.Wei Y L,Jia Y T,Huang X Z,et al.Molecular cloning and expression analyes of Op NAC083 from Olimarabidopsis pumila[J].Journal of Shihezi University(Natural Science),2015,33(2):222-229.
[29]郑丽洁,林军,黄先忠.小拟南芥双键还原酶基因Op D-BR的克隆及表达分析[J].石河子大学学报(自然科学版),2016,34(2):251-258.Zheng L J,Lin J,Huang X Z.Molecular cloning and expression analysis of Op DBR from Olimarabidopsis pumila[J].Journal of Shihezi University(Natural Science),2016,34(2):251-258.
[30]Holmes G D,Hall N E,Gendall A R,et al.Using transcriptomics to identify differential gene expression in response to salinity among Australian Phragmites australis clones[J].Frontiers in Plant Science,2016,7:432.
[31]李红婷,魏露阳,李中虎,等.滨麦叶片转录组分析[J].麦类作物学报,2018,38(9):1084-1093.Li H T,Wei L Y,Li Z H,et al.Transcriptome analysis of Leymus mollis leaf[J].Journal of Triticeae Crops,2018,38(9):1084-1093.
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