平欧杂种榛钙调蛋白基因ChaCaM的克隆与表达分析

Cloning and Expression Analysis of Calmodulin Gene in Ping'ou Hybrid Hazelnut(Corylus heterophylla × C. avellana)

以平欧杂种榛(Corylus heterophylla×C.avellana)品种‘达维’为材料,基于转录组测序数据,利用RACE技术克隆钙调蛋白基因,通过生物信息学进行序列预测分析,并利用实时荧光定量PCR技术分析其在不同组织和自交不亲和授粉后不同时间雌蕊中的表达模式。克隆获得一个钙调蛋白基因序列,命名为ChaCaM(Gen Bank登录号:KY211037),其开放阅读框为450 bp,编码149个氨基酸,分子量16.8476 kD。生物信息学分析表明,ChaCaM为酸性稳定蛋白,属于疏水性蛋白,无跨膜域和信号肽存在,主要定位于细胞质基质中,存在糖基化和磷酸化位点。序列分析表明,ChaCaM包含两个EFh结构域,与其他物种的CaM高度相似。系统进化树表明,ChaCaM与烟草、可可和萝卜等的CaM亲缘关系极近,聚类在同一分支下。基因表达分析表明,ChaCaM在平欧杂种榛不同组织中的表达具有差异,且在自交不亲和授粉后的雌蕊中具有明显的表达量变化。ChaCaM具有典型的钙调蛋白基因特征,主要定位于细胞质基质中;表现为组成型表达,推测其参与了榛属植物的自交不亲和反应。

This study cloned calmodulin gene of Ping＇ou hybrid hazelnut（Corylus heterophylla × C. avellana）,and further analyzed sequence features and expression characteristics,which is helpful to understand the role of calmodulin gene in the self-incompatibility reaction in Corylus. Based on the RNA-seq data,the calmodulin gene was cloned using RACE technique in the main cultivar‘Dawei＇of Ping＇ou hybrid hazelnut. The bioinformatic characteristics of the ChaCaM were analyzed using online service. The expression profiles of ChaCaM in various tissues and the pistils at different time after self-incompatibility pollination were investigated by using quantitative real-time PCR（qRT-PCR）. ChaCaM was submitted to Gen Bank with accession number KY211037. This gene contains a 450 bp open reading frame（ORF）that encodes 149 amino acid residues. The molecular weight of ChaCaM was 16.8476 k D. Bioinformatics analysis showed that ChaCaM was an acidic stable hydrophobic protein,without transmembrane domain and signal peptide present. ChaCaM was mainly located in the cytoplasmic matrix,and it contained several phosphorylation sites and glycosylation sites. Sequence analysis showed that ChaCaM contained two EFh domains,which were similar to Ca M from other species. Phylogenetic analysis showed that ChaCaM was closely related to CaM in tobacco,cocoa and radish,and affiliated to the same branch with CaM in other species. Gene expression analysis showed that ChaCaM presented tissue specific expression,especially were significantly changed in the pistils after self-incompatibility pollination. ChaCaM gene has typical characteristics of calmodulin gene,and is mainly located in the cytoplasmic matrix. It belongs to constitutive expression,which may participate in self-incompatibility regulation in Corylus.