新疆无苞芥类钙调素蛋白基因OpCML13的克隆与分析
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国家自然科学基金(31060149);石河子大学高层次人才启动项目(RCZX200902);新世纪优秀人才支持计划(NCET-12-1072)


Clone and Expression Analysis of Calmodulin-Like Protein Gene OpCML13 from Olimarabidopsis pumila of Xinjiang
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    摘要:

    通过对无苞芥幼苗cDNA文库的随机克隆测序分析,获得1条与拟南芥编码类钙调素13(calmodulin-like13,CML13)高度相似的EST(GenBank登录号为JZ152285)。利用RT-PCR技术从无苞芥cDNA中克隆了该基因,命名为OpCML13,该基因开放阅读框(ORF)为447 bp,编码148个氨基酸。生物信息学分析表明,OpCML13蛋白的二级结构含有4个Ca2+结合基序(EF-hands)结构,是一个亲水蛋白,无信号肽,无跨膜区域,为非跨膜蛋白;同源建模发现该蛋白包含8个α-螺旋结构和10个β-转角。系统进化树分析表明,OpCML13编码产物与拟南芥AtCML13和AtCML14进化关系较近,属于同一进化分支。半定量RT-PCR结果显示,OpCML13基因在无苞芥的不同组织中都有表达,在根中表达量最高;高盐胁迫处理6~24 h,OpCML13基因的表达明显增强,随后逐渐恢复正常表达水平;低温、干旱和ABA处理6 h后基因表达明显上调,并且一直保持较高的表达水平。研究表明,OpCML13基因在无苞芥逆境胁迫中可能起着重要作用。

    Abstract:

    An expressed sequence of tags (EST) which was highly similar to the calmodulin-like13 (CML13) gene of Arabidopsis was obtained,through sequencing of clones randomly selected from previously constructed cDNA library of Olimarabidopsis pumila leaves.This gene was cloned from leaf cDNA of O.pumila by RT-PCR technique,which was designated as OpCML13.Bioinformatics analysis showed that the open reading frame (ORF) of OpCML13 gene contained 447 bp encoding a peptide of 148 amino acids residues.The structural analysis of OpCML13 showed that it included four EF-hands motifs,and it is a hydrophilic protein,and no signal peptide and no transmembrane domain were found in OpCML13 protein indicating it is not a transmembrane protein.The tertiary structural analysis of OpCML13 through Swiss-model showed that it contained eight α-helixes and 10-β-turns.Phylogenetic tree revealed that OpCML13 showed closer genetic kinship with AtCML13 and AtCML14 of A.thaliana indicating that they belonged to the same evolutionary branch.Expression analysis by RT-PCR indicated that OpCML13 displayed a much broader expression range in different tissues in O.pumila,with a maximum expression in root.Under stress,expression of OpCML13 was induced with different kinetics and magnitude.The expression of OpCML13 was up-regulated as early as 6~24 h of salt treatment before rapidly returning normal level.OpCML13 mRNA significantly increased after cold,drought and ABA treatment for 6 h and remained elevated for at least 6 h.Our research indicated that OpCML13 played an important role in adversity stress.

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赵云霞,魏艳玲,黄先忠.新疆无苞芥类钙调素蛋白基因OpCML13的克隆与分析[J].西北植物学报,2014,34(3):431-437

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  • 在线发布日期: 2014-04-10
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