三角帆蚌活化蛋白激酶C受体1基因(HcRACK1)的克隆及表达分析

Molecular cloning and expression analysis of receptor for activated C kinase 1(HcRACK1) from Hyriopsis cumingii

为了探究RACK1基因在三角帆蚌免疫系统中的调控机制,采用RACE技术克隆得到三角帆蚌RACK1基因(命名为HcRACK1)的c DNA全序列,并对该序列进行分析。结果显示,HcRACK1基因全长为1249 bp,其中开放阅读框957 bp,编码318个氨基酸。蛋白质结构域分析显示HcRACK1含有RACK1家族特有的7个WD40结构域。运用荧光定量PCR技术分析HcRACK1在正常组织中及受到嗜水气单胞菌侵染和重金属镉暴露后相关组织表达量的变化。结果显示,HcRACK1在各个组织中均有表达,其中闭壳肌中表达量最高;嗜水气单胞菌侵染后,HcRACK1在血淋巴中的表达量逐渐上升,24 h时达到峰值,随后下降;暴露在100μg/L浓度的重金属镉中,HcRACK1在肝胰腺中的表达量逐渐上升,在第3天达到峰值;血淋巴在第2天达到峰值,随后下降;鳃中HcRACK1的表达量无明显变化。上述结果表明,Hc RACK1与细菌及镉引起的机体的氧化应激反应有关,并能参与到机体的免疫反应中。

In the present study, full length c DNA sequence of receptor for activated C kinase 1 gene of Hyriopsis cumingii（HcRACK1） was cloned by using 3’RACE and 5’RACE techniques, and the sequence and structural analysis of the HcRACK1 gene was conducted by using bioinformatics method. The results showed that the fulllength c DNA of HcRACK1 consisted of 1249 bp in length, containing an open reading frame（ORF） of 957 bp encoding 318 amino acids. Analysis of protein domain features showed that the deduced polypeptide contained seven WD40 domains characteristic of RACK1 protein family. The tissue distribution of HcRACK1 in unchallenged H. cumingii and temporal expression pattern of HcRACK1 challenged with bacteria and exposed to100 μg/L cadmium were analyzed by quantitative real-time PCR（q RT-PCR）. The transcript was detected in all tissues tested, and the expression level was the highest in adductor muscle. After challenge with bacteria,expression level of HcRACK1 in haemocytes was gradually increased and until 24 h post challenge reached the peak. After exposure to cadmium, its expression level in digestive gland gradually increased and reached the peak on 3 d post exposure, and in haemocytes on 2 d post exposure, and then increased over time. It showed no obvious change in gill. These results suggested that HcRACK1 was related to the oxidative stress response caused by bacterial challenge and cadmium exposure, and it also has a potential link with organism’s immune response.