MHC-B单倍型鸡MD抗性相关miRNA的鉴定及功能靶基因研究

MD genetic resistance is significant different between species or strains of chicken, and its mechanism has not been clarified, although a large number of studies have shown that MHC is closely related with MD resistance or susceptibility. Recent studies showed that host miRNA involved in host-pathogen interaction and may play a key regulatory role in host genetic resistance. Therefore, we used MHC-B haplotype strains of SPF chicken, which have the same genetic background, but a unique MHC haplotype, is a good model for the mechanism study of MD genetic resistance. The MD-resistant chickens (B21 haplotype) and susceptible chickens (B19 haplotype) will be used, before and after MDV infection, miRNA differential expression profiles in the spleen tissues of three key stages（5,10,21dpi）of MDV infection between the resistance chicken and susceptible chicken will be constructed by deep sequencing technology. We will screen and identify differential expression host miRNAs with MD resistance based on bioinformatics and expression profile analysis, and apply the luciferase reporter gene experiment, gene silencing and over-expression technology to verify the functional target genes (MHC) of miRNA. And then, analyze the inhibition role of miRNAs on MDV replication. Our results will provide the basis for the molecular mechanism of chicken MD genetic resistance difference, and applied miRNA to the disease-resistant breeding of chicken as well as effective prevention and control of MD.

不同品种（系）鸡对马立克氏病（MD）具有不同的遗传抗性。尽管研究表明MHC与MD抗性密切相关，但至今其分子调控机制仍未阐明。近年研究发现宿主miRNA具有抗病毒免疫应答功能很可能在其中发挥着关键作用。因此，为从宿主miRNA调控角度探索MHC相关的MD遗传抗性机制，本项目将以自主培育的B21（抗性）和B19（易感）单倍型鸡为模型，利用高通量测序技术构建B21和B19鸡在MDV病毒感染三个关键阶段（5、10和21dpi）脾脏组织miRNA的差异表达谱，鉴定与MHC-B单倍型鸡MD抗性相关的miRNA；应用双荧光素酶报告基因、miRNA过表达和抑制等实验，验证miRNA作用的功能靶基因（MHC-B区域）；进一步分析宿主miRNA抑制MDV病毒复制的作用。研究结果将不仅为解析MD遗传抗性复杂的分子调控机理奠定基础，并可为将miRNA 作为分子靶标应用于抗病育种和MD有效防控探索新的思路。

不同品种（系）鸡对马立克氏病（MD）具有不同的遗传抗性。尽管研究表明MHC与MD抗性密切相关，但至今其分子调控机制仍未阐明。近年研究发现宿主miRNA具有抗病毒免疫应答功能很可能在其中发挥着关键作用。因此，为从宿主miRNA调控角度探索MHC相关的MD遗传抗性机制，本项目以自主培育的B21（抗性）和B19（易感）MHC-B单倍型SPF鸡为模型，利用高通量测序技术成功构建了B21和B19鸡在MDV病毒感染三个关键阶段脾脏组织miRNA的差异表达谱，鉴定出3个可能与MHC-B单倍型鸡MD抗性相关的miRNAs（miR-7b、miR-21-5p和miR-200b-3p）；应用双荧光素酶报告基因、miRNA过表达和抑制等实验，验证其调控的功能靶基因分别为VDAC1、SATB1和ZEB1；进一步分析了miR-7b、miR-21-5p和miR-200b-3p抑制MDCC-MSB1细胞的增殖和迁移以及miR-7b 抑制MDV病毒复制相关基因的作用。本项目研究结果将不仅为解析MD遗传抗性复杂的分子调控机理奠定基础，并可为将miRNA 作为分子靶标应用于抗病育种和MD有效防控探索新的思路。