鸡毒支原体感染相关miRNAs鉴定及其分子调控机制研究

Chronic respiratory disease（CRD）infected by Mycoplasma gallisepticum（MG）is the most important factor depressing the economic benefit of poultry industry. However, there isn't active prevention measure nowaday. Although miRNA take contribution to the pathopoiesis of most etiological agent, the research of miRNA contribution on the effect of MG infection is still unknown. miRNAs differentially expressed in broiler trachea and lung infected by MG-HS strain will be screened with Illumina extensive sequencing technology, and further identified with real time PCR, Northern blot and hybridization in situ. The potential target genes of miRNA will be forecasted with bioinformatics, and assessed with analytical method of miRNA-mRNA compound and report gene pGL3Luciferase. miRNA contribution will be investigated with antisense oligonucleotide and miRNA overexpresssion technique. Moreover, the function of target genes will be demonstrated with the target gene silencing by RNA interference. According to the above results,The molecular regulating mechanism of miRNAs in broilers to Mycoplasma gallisepticum infection could be illuminated. This research will be beneficial to explain the molecular mechanism of CRD etiopathogenesis，and contribute theory and technological foundation to CRD prevention, treatment and molecular breeding for CRD resistance.

MG引起的鸡慢性呼吸道病是降低养禽业经济效益的重大疾病，迄今未找到理想的防治措施。研究表明，miRNA在病原致病中发挥着重要的作用，而在MG致病中miRNA的研究尚未见报道。本项目以MG-HS感染鸡为对象，拟利用Illumina深度测序技术鉴定感染前后气管中差异表达的miRNAs，并用real time-PCR、Northern blot、原位杂交等技术进行验证；采用生物信息学技术预测及分析差异表达miRNA潜在的靶基因，利用miRNA-mRNA复合物分析及pGL3 Luciferase报告基因载体等方法对靶基因进行鉴定；通过反义寡核苷酸及超表达miRNA技术对特异miRNA进行功能研究；利用逆转录病毒介导超表达和RNAi技术研究靶基因的功能，阐明miRNAs在MG-HS感染中的分子调控机制。项目的完成将诠释MG的致病机理，为分子抗病育种和miRNA疗法提供理论基础和科学方法。

鸡毒支原体（MG）引起的鸡慢性呼吸道疾病是降低养禽业经济效益的重大传染病，迄今未找到理想的防治措施。研究表明，miRNA 在病原致病中发挥着重要的作用。课题运用纯化的GST-pMGA1.2及His-pMGA1.2蛋白成功制备了抗pMGA1.2的多克隆及单克隆抗体；以MG-HS感染与未感染鸡胚为研究对象，利用Illumina深度测序技术发现感染后3rd、10th、11th等3个关键时期肺组织中差异表达的miRNAs分别为45、68及30个，新发现8个miRNAs；3个时期差异表达的miRNAs分别靶向6280、7181及4536个基因；miRNA-GO发现miNRA-8，miRNA-499，miRNA-17等家族在MG感染过程中占主导地位；Pathway-net发现，MAPK通路、Wnt 通路和Calcium通路等在MG感染过程中起主要作用；Gene-net发现，PLCB家族基因、MAPK家族基因及PIK家族基因在MG感染过程扮演中心控制枢纽的角色。在细胞和动物水平，我们对其中7个差异表达的miRNAs进行了鉴定，深入解析了miR-19a及miR-146c抵御MG感染的分子机制。利用荧光素酶报告基因载体及超表达和抑制实验验证了各个miRNAs的靶基因；采用逆转录病毒介导超表达和RNAi技术研究了靶基因的功能。其中miR-19a、miR-146c、miR-130、miR-21、miR-16及miR-101在MG感染后极显著上调。miR-19a通过靶基因ZMYND11激活NF-kB通路，提高前炎性因子表达、加快细胞周期进程、促进细胞增殖来抵御MG感染；miR-146c通过靶基因MMP16提高TLR6、TLR2表达，激活NF-κB通路抵御MG感染；miR-101通过靶基因EZH2抑制细胞的增殖，加剧MG感染； miR-130通过靶向PTEN促进细胞增殖，激活NF-kB抵御MG感染；miR-21通过靶向MAP3K1而抵御MG感染；miR-16通过靶基因PIK3R1和AKT3激活NF-kB通路来抵御MG感染；miR-99a在MG感染后极显著下调，通过靶向SMARCA5促进细胞的增殖来抵御MG感染。本项目的完成阐明了相关miRNA/mRNA及其分子信号通路在MG感染中的致病机理，揭示了动物组织和细胞抗御MG感染的分子机理，为鸡分子抗病育种和miRNA疗法提供理论基础和科学方法。