黑龙江林蛙热休克蛋白hsp70家族基因的鉴定及其在感染下的表达分析

Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection

摘要:

热休克蛋白(heat shock protein, HSPs)是一类广泛存在于各类生物体中高度保守的应激蛋白，当生物体受到环境中物理、化学和生物等因素的刺激时，机体出于自我保护会迅速产生应激反应，合成该类蛋白，从而减少外界刺激对机体的损伤，提高自身抵抗能力，HSP70是该家族的重要成员。为探究HSP70在两栖类抗感染中的作用，以黑龙江林蛙(Rana amurensis)作为研究对象，基于同源克隆的方法，采用反转录PCR(reverse transcription PCR, RT-PCR)技术克隆Ra-hsp70家族基因；采用生物信息学方法对Ra-hsp70家族基因进行序列特征、立体结构、亲缘关系的分析；再利用实时荧光定量PCR (real-time quantitative PCR, qRT-PCR)技术对Ra-hsp70家族基因在细菌感染下的表达谱进行分析；应用免疫组织化学技术对HSP70蛋白进行表达和定位分析。结果表明，获得的Ra-HSP70家族的4个成员HSP70、HSPA5、HSPA8和HSPA13均包含有3段保守的HSP70家族标签序列，属于典型的HSP70家族成员；4个成员在系统发育树中分布在4个不同的分支，且含有相同亚细胞定位基序的成员分布在同一分支；感染嗜水气单胞菌(Aeromonas hydrophila, Ah)后，Ra-hsp70家族基因的4个成员的相对表达量均出现显著上调(P<0.01)，但是在不同组织中到达峰值的时间不同。免疫组织化学法检测到HSP70蛋白在肝脏、肾脏、皮肤和胃组织的胞质中均有不同程度表达。Ra-hsp70家族基因的4个成员能够不同程度地响应细菌感染，推测参与了机体对抗致病菌侵染的生物学过程，并在其中发挥不同的生物学作用，为两栖类HSP70基因的功能研究提供了理论基础。

Abstract:

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P<0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.