斑马鱼PKR剪接异构体克隆、鉴定及转录表达分析

Cloning,identification and transcription analysis of PKR transcript variant from Zebrafish

目的:克隆斑马鱼PKR(ZPKR)及其剪接异构体(ZPKRV)并对其进行鉴定和转录表达分析。方法:采用基因克隆法克隆ZPKR及ZPKRV;利用生物信息学方法分析其结构差异,设计定量分析引物;使用病毒双链RNA类似物(Poly I:C)刺激,提取斑马鱼组织总RNA并反转录合成c DNA,通过q PCR检测ZPKR及ZPKRV转录表达的差异,推测ZPKR及ZPKRV的功能。结果:首次在斑马鱼组织中克隆到ZPKRV;生物信息分析显示,ZPKRV仅仅在ZPKR的双链RNA结合结构域和激酶区之间的链接区缺失了28个氨基酸残基,并且缺失的序列包括链接区的碱性区;Poly I:C刺激后的转录表达显示,在刺激后的12 h,ZPKR表达上调到第一个峰值,24 h又下调,48 h再上调;而ZPKRV仅在刺激后24 h表达上调到最高,然后下调。结论:ZPKRV碱性区的缺失,可能会导致其单链RNA结合功能的丧失,进而促进蛋白的翻译表达;ZPKR和ZPKRV同时表达,可能通过显性的负效应,降低PKR在抗病毒免疫反应中对蛋白翻译的抑制作用,促进抗病毒免疫细胞因子的翻译表达。

Objective:To clone the Zebrafish PKR(ZPKR)and ZPKR transcript variant(ZPKRV),then analyze its transcriptional expression.Methods:ZPKR and ZPKRV were cloned by gene cloning method.Bioinformatics approaches were used to analyze the structural differences and design primers for quantification it.Furthermore,Zebrafish were stimulated with Poly I:C and the total RNA were extracted for reverse transcription.Then fluorescence qPCR was utilized to detect the difference in the transcriptional expression of ZPKR and ZPKRV,and to speculate on their function.Results:It was the first time that the ZPKRV was cloned in Zebrafish tissue.Bioinformatics analysis revealed that compared with ZPKR,ZPKRV was only deleted 28 amino acid residues in the linker between dsRBD and KD,including the conserved basic region in the linker.After Poly I:C stimulation,the transcriptional expression of ZPKR was up-regulated to the first peak at 12 h and then down-regulated at 24 h,again it was up-regulated at 48 h.However,ZPKRV was only up-regulated at 24 h,and it was down-regulated subsequently.Conclusion:The deletion of the basic region in the Zebrafish PKRV may result in the loss of its single-stranded RNA binding function,which in turn promotes protein translation.ZPKR and ZPKRV coexisted in Zebrafish may reduce the inhibitory effect of PKR on protein translation in the antiviral immune response and enhance the translation of antiviral immune cytokines through dominant-negative effect.