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