4个基因在副猪嗜血杆菌感染仔猪脑组织中DNA甲基化与mRNA表达联合验证

Integrated Validation for DNA Methylation and mRNA Expression of 4 Genes in Brain of Glaessrerlla parasuis-infected Piglets

【目的】验证候选基因在副猪嗜血杆菌(Glaesserella parasuis,GPS)感染仔猪脑组织中DNA甲基化与mRNA表达之间的联合调控关系,为揭示副猪嗜血杆菌引起仔猪脑膜炎的表观致病机理提供理论依据。【方法】选取6头28日龄断奶仔猪,随机均分为对照组和GPS组,GPS组仔猪腹腔注射1 mL 2×109 CFU/mL副猪嗜血杆菌SH0165菌液,对照组仔猪腹腔注射等量生理盐水,7 d后采集仔猪脑组织提取DNA和RNA。采用实时荧光定量PCR检测4个候选基因(LYPD1、PITPNM1、SYP、ACVR1B)的mRNA表达量,并利用重亚硫酸盐测序(bisulfite sequencing PCR,BSP)、甲基化特异性PCR(methylation-specific PCR,MSP)检测副猪嗜血杆菌感染前后4个基因在仔猪脑组织中的DNA甲基化变化。【结果】本研究成功将MSP方法应用于基因的DNA甲基化测序研究,使其不局限于定点检测甲基化位点。实时荧光定量PCR结果显示,与对照组相比,GPS组仔猪LYPD 1、PITPNM 1、SYP、ACVR 1 B基因mRNA表达均显著或极显著下调(P<0.05;P<0.01)。DNA甲基化测序结果显示,除ACVR 1 B基因外,各基因DNA甲基化均上调。【结论】除ACVR 1 B基因外,LYPD 1、PITPNM 1、SYP 3个基因DNA甲基化与mRNA表达水平呈反向关联调控,副猪嗜血杆菌感染后仔猪脑组织DNA甲基化变化对4个候选基因的表达具有不同的调控模式。

【Objective】The aim of this study was to verify the joint regulatory relationship between DNA methylation and mRNA expression of candidate genes in the brain tissue of piglets infected by Glaesserella parasuis(GPS),in order to provide a theoretical basis for revealing the apparent pathogenic mechanism of meningitis caused by GPS in piglets.【Method】Six 28-day-old weaned piglets were randomly divided into control and GPS groups.The piglets in GPS group were intraperitoneally injected with 1 mL of 2×109 CFU/mL GPS SH0165 solution,and the piglets in control group were intraperitoneally injected with the same amount of normal saline,and the brain of the piglets were collected 7 days later to extract DNA and RNA.The mRNA expression of 4 candidate genes(LYPD1,PITPNM1,SYP and ACVR1B)were detected by Real-time quantitative PCR,and bisulfite sequencing PCR(BSP)and methylation-specific PCR(MSP)were used to detect the DNA methylation changes of 4 genes in brain tissues of piglets before and after GPS infection.【Result】This study successfully applied the MSP method to DNA methylation sequencing of genes,making it not limited to fixed-point detection of methylation sites.The results of Real-time quantitative PCR showed that compared with control group,the mRNA expression of LYPD 1,PITPNM 1,SYP and ACVR 1 B genes in GPS group were significantly or extremely significantly down-regulated(P<0.05 or P<0.01).The results of DNA methylation sequencing showed that the DNA methylation level of 4 genes were up-regulated except for ACVR 1 B gene.【Conclusion】Except for ACVR 1 B gene,the DNA methylation of LYPD 1,PITPNM 1 and SYP genes was inversely associated with mRNA expression,and the DNA methylation changes in brain of piglet after GPS infection had different regulatory patterns for the expression of 4 candidate genes.