• ISSN 1008-505X
  • CN 11-3996/S
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. 小麦TaCLC-e-3AL基因功能分析及互作蛋白鉴定[J]. 植物营养与肥料学报. DOI: 10.11674/zwyf.2023492
引用本文: 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. 小麦TaCLC-e-3AL基因功能分析及互作蛋白鉴定[J]. 植物营养与肥料学报. DOI: 10.11674/zwyf.2023492
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. Functional analysis of TaCLC-e-3AL and the identification of interacting proteins in wheat (Triticum aestivum L.)[J]. Journal of Plant Nutrition and Fertilizers. DOI: 10.11674/zwyf.2023492
Citation: 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. Functional analysis of TaCLC-e-3AL and the identification of interacting proteins in wheat (Triticum aestivum L.)[J]. Journal of Plant Nutrition and Fertilizers. DOI: 10.11674/zwyf.2023492

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.)

  • 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,利用双杂交酵母筛选与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该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NO3,并能够维持植株体内NO3/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NO3,并维持NO3/Cl的比值,表明TaCLC-e-3AL可能调控Cl和NO3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。

     

    Abstract:
    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 screening.
    Results The TaCLC-e-3AL gene was cloned from wheat and bioinformatics analysis indicated that its encoded protein contained 11 transmembrane domains, which had 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 could store more NO3 and maintain NO3/Cl homeostasis, and not have significant changes in root length or fresh weight. 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 chloroplasts. The overexpression of TaCLC-e-3AL in Arabidopsis thaliana has led to the raised NO3N storage but normal NO3/Cl homeostasis in plants under low nitrogen stress, thereby, TaCLC-e-3AL is thought regulate the synergistic transport of Cl and NO3. Through interaction with aquaporin, voltage-dependent anion channel and other proteins, TaCLC-e-3AL participates in response to drought stress, photosynthesis, and ion stress in wheat.

     

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