拟南芥CaM5的不同剪接产物对其蛋白质结合活性的影响

Effects of Different Alternative Splicing of Arabidopsis CaM5 on Its Protein-binding Activity

摘要:

钙离子(Ca²⁺)/钙调素(calmodulin, CaM)信号参与植物生长发育和对外界刺激响应的调节。拟南芥(Arabidopsis thaliana) CaM家族共有7个基因，编码的蛋白质氨基酸序列具有高度保守性。该研究通过酵母双杂交及生物信息学分析等方法，对CaM5的2个剪接体CaM5.1和CaM5.3进行蛋白结合活性分析。结果表明，拟南芥钙调素结合蛋白(calmodulin-binding protein, CaMBP) IQM3 (IQ motif-containing protein 3)能在酵母中与除CaM5.3外的CaM家族的成员结合。生物信息学分析表明, CaM5.3比CaM5.1和其他CaM亚型成员多出1个由35个氨基酸残基组成的C-末端序列(C-terminal domain, CTD)，可能影响CaM5.3与IQM3结合。在CaM5.1的C-末端添加CML10的CTD，将重组蛋白与IQM3进行结合，证实CaM5.3的CTD是影响其与IQM3结合的关键序列。因此，拟南芥CaM5的不同剪接方式影响其蛋白结合活性，为研究钙调素及钙调素结合蛋白的结合活性提供参考。

Abstract:

Calcium (Ca²⁺)/calmodulin (Cam) signals are involved in the regulation of plant growth and development and response to external stimuli. The Arabidopsis thaliana contains seven genes in CaM family, which encoding proteins with highly conserved amino acid sequence. The protein binding activities of CaM5 splicosomes CaM5.1 and CaM 5.3 were analyzed by yeast two-hybrid and bioinformatics analysis. The results showed that the Arabidopsis calmodulin-binding protein (CaMBP) IQM3 (IQ motif-containing protein 3) could bind to members of CaM family except CaM5.3 in yeast. The bioinformatic analysis revealed that CaM5.3 had one more C-terminal domain (CTD) consisting of 35 amino acid residues than CaM5.1 and other CaM subtypes, which might affect the binding of CaM5.3 to IQM3. CTD of CML10 was added to the C-terminal of CaM5.1, the recombinant protein was binded to IQM3, confirming that CTD of CaM5.3 was the key sequence affecting its binding to IQM3. Therefore, different splicing modes of CaM5 in A. thaliana affect its protein-binding activity, which providing reference for studying the binding activities of calmodulin and calmodulin-binding proteins.