茶树小分子热激蛋白基因CsHSP22.4、CsHSP27.4、CsHSP17.5和CsHSP25.2的克隆与表达分析

Cloning and Expression Analysis of Small Heat Shock Protein Genes CsHSP22.4,CsHSP27.4,CsHSP17.5 and CsHSP25.2 in Camellia sinensis

以‘龙井长叶’茶树为材料,通过RT-PCR技术克隆获得了4个小分子热激蛋白基因,其开放阅读框长度分别为600、732、462和657 bp,编码199、243、153和218个氨基酸,根据其编码蛋白分子量分别命名为:CsHSP22.4、CsHSP27.4、CsHSP17.5和CsHSP25.2。蛋白结构分析显示,CsHSP22.4、CsHSP27.4、CsHSP17.5和CsHSP25.2蛋白均包含1个由CRⅠ、CRⅡ和β6-loop组成的ACD结构域,属于典型的小分子热激蛋白家族成员。亚细胞定位预测和进化树分析表明,CsHSP22.4主要定位于线粒体中,属于MⅠ亚族;CsHSP17.5主要定位于细胞质内,属于CⅠ亚族;而CsHSP27.4和CsHSP25.2则主要定位于叶绿体中,属于CP亚族。实时荧光定量PCR结果表明,CsHSP22.4和CsHSP27.4在叶中表达量最高,CsHSP17.5和CsHSP25.2在茎中表达量最高,具有一定的组织特异性。另外,CsHSP22.4、CsHSP27.4、CsHSP17.5和CsHSP25.2均受高温和干旱胁迫的诱导,其中在高温胁迫下表达量呈爆发性增加,表明其均参与了茶树对高温和干旱胁迫响应的过程,且对高温胁迫具有更高的敏感性。

Four small heat shock protein（s HSP） genes were cloned from Camallia sinensis‘Longjing Changye＇using RT-PCR technology. The open reading frames of them were 600,732,462 and 657 bp,which encoded 199,243,153 and 218 amino acids,respectively. According to the molecular weight of encoded protein,they were named as CsHSP22.4,CsHSP27.4,CsHSP17.5 and CsHSP25.2. Protein structure analysis showed that the four CsHSPs contained a ACD domain consisting of CRⅠ,CRⅡ and β6-loop,typically belonging to the s HSP family member. Subcellular localization prediction and phylogenetic analysis showed that CsHSP22.4 and CsHSP17.5 were mainly localized in the mitochondria and cytoplasm,belonging to the MⅠ and CⅠ subfamily,respectively. CsHSP27.4 and CsHSP25.2 were mainly located in the chloroplast,which belonged to the CP subfamily. q RT-PCR test results indicated that the expression of CsHSP22.4 and CsHSP27.4 was higher in leaf than in other tissues,while the expression of CsHSP17.5 and CsHSP25.2 was higher in stem. The expression of CsHSP22.4,CsHSP27.4,CsHSP17.5 and CsHSP25.2 was induced by heat and drought stress,and the expression level increased explosively under heat stress,implying that they were involved in response to heat and drought stress in Camallia sinensis,although they were more sensitive to heat stress.