小麦类钙调素TaCML8-A基因的克隆和表达分析

Cloning and Expression Analysis of Calmodulin-like Protein TaCML8-A Gene in Wheat

为挖掘小麦抗逆基因,研究类钙调素基因在植物抗逆反应的分子机制,利用电子克隆结合RT-PCR的方法克隆小麦类钙调素基因TaCML8-A。生物信息学分析显示,该基因的CDS区全长为519 bp,编码172个氨基酸,包含PTZ00184和FRQ1超家族,含有4个EF-hand结构域,其中包含4个钙离子结合位点;编码蛋白质分子质量为18.31 ku,等电点为4.54,属于酸性蛋白质。亚细胞定位结果显示,TaCML8-A蛋白质在细胞核和细胞膜中均有分布。核苷酸序列比对发现,TaCML8-A与水稻OsCML14亲缘关系最近,相似性为79.17%。qRT-PCR结果显示,在盐、渗透和冷胁迫处理中,TaCML8-A基因在小麦地上部分和根中的表达均上升,表明植物可能通过提高TaCML8-A基因的表达来响应盐、渗透和冷胁迫;热激时TaCML8-A基因在根和地上部分分别受到诱导和抑制,从而发挥不同的功能。从小麦中成功克隆出类钙调素基因TaCML8-A,并进行表达分析,初步推测该基因可能参与调控植物的非生物胁迫,其结果可为后续研究其生物学功能提供基础理论支撑。

In order to explore stress resistance genes in wheat and study the molecular mechanism of calmodulin-like genes in plant stress resistance,a calmodulin-like gene TaCML8-A was cloned from wheat by electronic cloning combined with RT-PCR.Bioinformatics analysis showed that the open reading frame length of the gene was 519 bp,encoding 172 amino acid sequences,including PTZ00184 and FRQ1 superfamily,four EF-hand domains,including four calcium binding sites.The molecular weight of the encoded protein was 18.31 ku and the isoelectric point was 4.54.It belonged to acidic protein.Subcellular localization showed that TaCML8-A protein was distributed in the nucleus and cell membrane.Nucleotide sequence alignment showed that TaCML8-A had the closest genetic relationship with rice OsCML14,with a similarity of 79.17%.qRT-PCR results showed that the expression of TaCML8-A gene in shoot and root of wheat increased under salt,osmotic and cold stress,indicating that plants might respond to thoese stresses by increasing the expression of TaCML8-A.During heat shock,TaCML8-A gene was induced and inhibited in root and shoot,respectively,so as to play different functions.The calmodulin-like gene TaCML8-A was successfully cloned from wheat and analyzed for expression.It is preliminarily speculated that the gene might be involved in regulating abiotic stress of plants,so as to provide basic theoretical support for the follow-up study of its biological function.