小麦TaCLC-e-3AL基因功能分析及互作蛋白鉴定  

Functional analysis of TaCLC-e-3AL and the identification of interacting proteins in wheat(Triticum aestivum L.)

作　　者：宋腾钊 毛培钧 杨瑞鹏 李冰冰 刘腾飞 谢玉民 李妍珂 袁梦营 程西永 许海霞 SONG Teng-zhao;MAO Pei-jun;YANG Rui-peng;LI Bing-bing;LIU Teng-fei;XIE Yu-min;LI Yan-ke;YUAN Meng-ying;CHENG Xi-yong;XU Hai-xia(The Shennong Laboratory/College of Agronomy,Henan Agricultural University/National Key Laboratory of Wheat and Maize Crop Science,Zhengzhou,Henan 450046,China)

机构地区：[1]神农种业实验室/河南农业大学农学院/省部共建小麦玉米作物学国家重点实验室,河南郑州450002

出　　处：《植物营养与肥料学报》2024年第5期835-847,共13页Journal of Plant Nutrition and Fertilizers

基　　金：神农种业实验室一流课题项目(SN01-2022-01);河南省重大科技专项(221100110300);河南省重点研发专项(231111110700)。

摘　　要：【目的】分析小麦氯离子通道TaCLC-e-3AL基因功能并鉴定其互作蛋白,以解析TaCLC-e-3AL参与小麦响应低氮胁迫的作用机制。【方法】以拟南芥AtCLC-e氨基酸序列为参考序列,通过BlastP对小麦基因组数据库进行比对,获得TaCLC-e-3AL(TraesCS3A02G253600)、TaCLC-e-3B(TraesCS3B02G285500)和TaCLC-e-3DL(TraesCS3D02G254500)3个基因,分析其基因结构和系统进化关系。将TaCLC-e-3AL-p1300-GFP融合蛋白表达载体转化至小麦原生质体中,分析TaCLC-e-3AL的亚细胞定位特征;采用转基因拟南芥进行异源功能验证,利用酵母双杂交筛选与TaCLC-e-3AL相互作用的蛋白。【结果】生物信息学分析表明,TaCLC-e-3AL编码的蛋白含有11个跨膜结构域,与乌拉尔图小麦TuCLC-e同源性最高。组织表达量预测分析表明,TaCLC-e-3AL基因在小麦的叶片和茎部表达量较高。顺式作用元件分析表明,其可能响应光、激素以及逆境胁迫等信号。亚细胞定位显示,TaCLC-e-3AL蛋白经内质网分选后定位于叶绿体内。过表达TaCLC-e-3AL转基因拟南芥植株在低氮胁迫条件下可以储存更多的NO_(3)^(-),并能够维持植株体内NO_(3)^(-)/Cl^(-)的稳态,不引起植株根长和鲜重的显著变化。酵母双杂交文库筛选显示TaCLC-e-3AL与水通道、叶绿体a/b结合蛋白和电压依赖性阴离子通道3个蛋白互作,表明TaCLC-e-3AL可能与它们协同参与干旱胁迫响应、光合作用、信号传导等生物过程。【结论】小麦氯离子通道蛋白TaCLC-e-3AL位于叶绿体内。TaCLC-e-3AL基因转化至拟南芥中过表达,在低氮胁迫条件下较野生型可以在植株体内储存更多的NO_(3)^(-),并维持NO_(3)^(-)/Cl^(-)的值,表明TaCLC-e-3AL可能调控Cl^(-)和NO_(3)^(-)的协同运输。TaCLC-e-3AL通过与水通道蛋白、叶绿体a/b结合蛋白、电压依赖性阴离子通道蛋白互作,参与小麦的干旱胁迫、光合作用和离子胁迫应答。【Objectives】This study aimed to elucidate the mechanism of TaCLC-e-3AL involvement in response to low nitrogen stress by analyzing the function and identifying the interacted proteins of TaCLC-e-3AL.【Methods】The amino acid sequence of Arabidopsis thaliana AtCLC-e was used as a reference sequence,and three genes,TaCLC-e-3AL(TraesCS3A02G253600),TaCLC-e-3B(TraesCS3B02G285500)and TaCLC-e-3DL(TraesCS3D02G254500),were obtained from wheat genome database by BlastP,and their structures and phylogenetic relationships were then analyzed.The TaCLC-e-3AL-p1300-GFP fusion was transformed into wheat protoplasts to analysis the subcellular localization characteristics of TaCLC-e-3AL.Transgenic Arabidopsis thaliana experiments were employed to determine the function of TaCLC-e-3AL.The interactors of TaCLC-e-3AL were identified by yeast two-hybrid system screening.【Results】The TaCLC-e-3AL gene was cloned from wheat,and bioinformatics analysis indicated that the encoded protein of TaCLC-e-3AL gene contained 11 transmembrane domains,and showed the highest homology with TuCLC-e in Triticum urartu.TaCLC-e-3AL gene was highly expressed in leaves and stems of wheat,and several light,hormone and stress response elements were detected in its promoter region using the method of cis-acting elements analysis.Subcellular localization in protoplasts showed that TaCLC-e-3AL was mainly expressed in chloroplasts.When TaCLC-e-3AL was overexpressed in Arabidopsis thaliana under low nitrogen condition,the transgenic plants stored more NO_(3)^(-)and maintained stable NO_(3)^(-)/Cl−homeostasis,the root length and fresh weight were not impacted significantly.The screening results of the yeast two-hybrid system showed that TaCLC-e-3AL interacted with aquaporin,voltage-dependent anion channel and other proteins,indicating the possible involvement of TaCLC-e-3AL in drought stress response,photosynthesis,signal transduction and other biological processes.【Conclusions】The wheat chloride ion channel protein TaCLC-e-3AL is located in the ch

关 键 词：小麦 氯离子通道蛋白 TaCLC-e-3AL 功能分析 互作蛋白 

分 类 号：S512.1[农业科学—作物学]