桑树钙调素蛋白基因MCaM-3的克隆及序列分析与诱导表达

Cloning,Sequence Analysis and Induced Expression of a Calmodulin Gene MCaM-3 From Mulberry

钙调素蛋白(calmodulin CaM)作为真核生物细胞内的多功能Ca2+结合蛋白,在调节植物的生长发育、环境适应性和抗逆性方面具有重要作用。利用桑树表达序列标签(expressed sequence tags,ESTs)克隆了一个编码桑树CaM基因的全长cDNA序列,命名为MCaM-3(GenBank登录号:GQ303247)。序列分析表明,MCaM-3全长951bp,存在143 bp的5′端非翻译序列(5′-UTR)和358 bp的3′端非翻译序列(3′-UTR),其开放读码框(ORF)长450bp,编码149个氨基酸,预测蛋白质分子质量为16.85 kD,等电点为3.95。用在线软件SMART分析显示,MCaM-3蛋白含有4个Ca2+结合基序(EFh)结构域,可以结合Ca2+。同源分析表明,CaM基因在桑树与拟南芥、杨树、大豆、小麦、水稻、玉米各物种间具有很高的保守性。基于桑树和其它19个物种CaM基因的系统进化分析表明,桑树与蓖麻、烟草、大黄、樱桃的亲缘关系较近。半定量RT-PCR检测表明,与正常生长环境相比,MCaM-3基因mRNA的转录水平在低温、干旱、盐胁迫条件下均显著提高,初步推测MCaM-3基因与桑树的抗逆性有一定关联。

Calmodulin（CaM） is a multifunctional Ca^2＋-binding protein in eukaryotic cells.It plays important roles in regulating the growth and development,environment adaptation and stress tolerance in plants.A full-length cDNA sequence coding for CaM in mulberry,designated MCaM-3（GenBank accession number GQ303247）,was cloned based on expressed sequence tags from mulberry tree.Sequence analysis showed that the MCaM-3 is 951 bp long and contains a 143 bp 5′-UTR（untranslated region） and a 358 bp 3′-UTR.Its open reading frame（ORF） is of 450 bp,encoding 149 amino acids with a predicted molecular weight of 16.85 kD and an isoelectric point of 3.95.Online SMART analysis showed that the protein of MCaM-3 has four Ca^2＋-binding functional domains,being able to bind calcium ion.Homology analysis revealed that,CaM genes are highly conservative in mulberry and other species including Arabidopsis,poplar,soybean,wheat,rice and maize.Phylogenetic analysis based on MCaM-3 and CaM genes with other 19 species showed that mulberry shows closer relationships with castor oil plant,tobacco,rhubarb and cherry.The results of semi-quantitative RT-PCR analysis showed that the transcriptional level of MCaM-3 mRNA increased significantly under the conditions of low temperature,drought and salt stress respectively compared to the normal growth environment,suggesting its involvement in general response to abiotic stresses in mulberry.