小麦<i>TaCLC-e-3AL</i>基因功能分析及互作蛋白鉴定

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

doi:10.11674/zwyf.2023492

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 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 NO₃⁻ and maintained stable NO₃⁻/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 chloroplasts. The overexpression of TaCLC-e-3AL in Arabidopsis thaliana could raise NO₃⁻-N storage and thereby keep the normal NO₃⁻/Cl⁻ homeostasis in plants under low nitrogen stress, so the TaCLC-e-3AL is thought regulating 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.

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.)