蓖麻类钙调蛋白基因RcCML42克隆与表达载体构建

Cloning and Expression Vector Construction of Castor Calmodulin-like Proteins Gene RcCML42

为了探究类钙调蛋白基因家族成员在蓖麻体内的作用及表达模式,在蓖麻体内挖掘家族基因CML42,并对其进行克隆与表达分析。以哲蓖三号植株为试验材料,克隆获得RcCML42完整编码区序列(CDS),利用生物信息学方法对RcCML42基因序列进行分析,包括编码氨基酸、蛋白分子量及等电点、跨膜结构域、氨基酸序列一致性等内容。通过荧光定量PCR(qRT-PCR)技术,检测RcCML42基因在蓖麻植株根、茎、叶位置不同时间点的特异性表达情况。结果显示,RcCML42 CDS长度为597 bp,编码198个氨基酸,含有3个EF-hand钙结合结构域。蛋白分子量为22.27 ku,等电点为4.55,不存在跨膜结构域。氨基酸序列一致性分析表明,RcCML42与巴西橡胶树一致性最高,达86.84%。qRT-PCR结果显示,RcCML42基因在蓖麻根、茎、叶组织中均有表达,在经NaCl处理后,RcCML42在根中的表达量呈现先下降后上升的趋势。RcCML42在茎中12 h高度表达,在叶中为8 h高度表达。且在茎中的表达量显著高于其在根、叶中的表达。CML是植物体内最重要的一类钙感受器,通过与钙调素结合蛋白结合进而对植物细胞进行调控。当植物受到环境中NaCl侵害时,CML可以参加体内钙离子的调节,进而减轻盐胁迫对植物体的损害。综上,推测RcCML42在盐胁迫条件下的蓖麻信号转导中起重要作用。

In order to explore the role and expression patterns of members of the Calmodulin-like proteins gene family in castor,the family gene CML42 was extracted from castor,and its cloning and expression were analyzed.Zhebi No.3 plant was used as the material to clone and obtain the complete coding region sequence(CDS)of RcCML42,bioinformatics method was used to analyze RcCML42 gene sequence,included amino acid coding,protein molecular weight and isoelectric,transmembrane domain,amino acid sequence consistency and so on.Using qRT-PCR to analyze the changes of expression of castor RcCML42 gene in root,stem and leaf tissues at different time points.The results showed that RcCML42 CDS was 597 bp in length,encoded 198 amino acids,contained three EF-hand calcium-binding domains,the protein molecular weight was 22.27 ku,the isoelectric point was 4.55,and there was no transmembrane domain.Analysis of amino acid sequence identity showed that RcCML42 had the highest identity with Hevea brasiliensis,reaching 86.84%.The results of qRT-PCR analysis showed that the expression of RcCML42 in the roots after NaCl treatment showed a trend of first decreasing and then rising.The expression level of RcCML42 was highly expressed at 12 h in the stem and at 8 h in the leaf.And expression in stems was significantly higher than its expression in roots and leaves CML is one of the most important calcium receptors in plants.It regulates plant cells by binding with calmodulin binding protein.When plants are affected by NaCl in the environment,CML can participate in the regulation of calcium ions in the body,thus reducing the damage to plants caused by salt stress.In summary,RcCML42 plays an important role in the signal transduction of castor under salt stress.