甘蔗钙依赖蛋白激酶基因克隆与序列分析

Cloning and sequence analysis of calcium-dependent protein kinase gene in sugarcane

【目的】克隆甘蔗钙依赖蛋白激酶基因(ScCDPK1),为其功能解析和育种利用打下基础。【方法】根据甘蔗cDNA文库中的CDPK基因序列信息设计引物,利用RT-PCR克隆ScCDPK1基因,并采用在线生物信息学分析软件对其推导蛋白的理化性质、跨膜结构、信号肽及保守结构域等进行预测分析。【结果】克隆获得的ScCDPK1基因完整编码区大小1650 bp,编码549个氨基酸,相对分子量61.971 kD,等电点(pI)6.78,不稳定指数35.63。同源性和遗传进化树分析结果显示,ScCDPK1基因与小麦TaCPK7基因亲缘关系最近,同源性达88%;ScCDPK1蛋白与其他已知CDPK蛋白一致,具有CDPKs家族特有的4个保守区域(可变区、蛋白激酶区、自抑制区和钙离子结合区),其二级结构主要由α螺旋(42.80%)和无规则卷曲(32.97%)构成;蛋白功能分类预测结果显示,ScCDPK1蛋白是一种阳离子通道蛋白,可能参与植物胁迫应答、信号转导和翻译调控等生物反应。【结论】ScCDPK1基因编码的蛋白与其他已知CDPK蛋白一致,具有CDPKs家族特有的4个保守区域,是一种阳离子通道蛋白。

[Objective]The calcium-dependent protein kinase gene in sugarcane（ScCDPKl ） was cloned to provide re- ferences for revealing its biological functions and further utilization in breeding. [Method ]The specific primers were designed according to known CDPK gene sequence from sugarcane cDNA library. ScCDPK1 gene was cloned using RT-PCR. The physical and chemical properties, transmembrane domain, signal peptide, and conserved domains were analyzed using online bioinformatics algorithms. [Result]The full coding sequence of ScCDPK1 gene was cloned, which included 1650 bp in length and encoded 549 amino acid residues. Its relative molecular weight was 61.971 kD, isoelectric point（pI） 6.78 and instability index 35.63. Homology and phylogenetic tree analysis showed that ScCDPK1 of sugarcane shared the closest ge- netic relationship with TaCPK7 of wheat and had 88% similarity. Like other known CDPK proteins, ScCDPKI protein con- tained four conserved domains（variable domain, protein kinase domain, self-inhibition domain and calcium binding domain）. Its protein secondary structure mainly consisted of alpha helix （42.80%） and random coil （32.97%）. Protein functional analysis showed that ScCDPK1 protein was a kind of cation channel protein which play important roles in many biology processes such as plant stress response, signal transduction and translation. [Conclusion]Like other known CDPK proteins, the protein encoded by ScCDPK1 gene contains four conserved domains which is peculiar to CDPKs family. And it is a cation channel protein.