盘羊及其杂交羊SPLUNC1特性研究及抗肺炎支原体差异表达基因的筛选

The hybridization of wild argali and bashbay sheep is interspecific hybridization. The resistance of Mycoplasma pneumoniae (Mp) infection in the hybrid offspring is weakened, which arosed a lot of death in these mules in early growth. Short palate，lung，and nasal epithelium clone 1 (SPLUNC1) serves as a secretory protein，which play an important role in promoting lung innate defense against Mp infection. In this study, Xinjiang wild argali and bashbay sheep and their hybrid sheep are objects. Mycoplasma pneumonia of sheep is the disease model. Study on SPLUNC1 gene by using techniques such as artificial insemination, gene clone, gene sequencing, gene expression, real-time PCR, cell culture, electron microscope, flow cytometry, artificial infection and ELISA. Comparative genomics in argali, bashbay sheep and their hybrid sheep will be analyzed.Function of SPLUNC1 protein from different species sheep will be compared. Effects of SPLUNC1 in these argali hybrids that reduced Mp infection resistances as well as death will be revealed from genetic structure, gene expression level, protein function, actual disease resistance. Meanwhile, differentially expressed genes of anti-Mp from different species sheep will be screened by Digital Gene Expression Profiling technology. It can also provide a theoretical basis for further research the molecular mechanism of susceptible Mp in the hybrid sheep.

盘羊与巴什拜羊杂交为种间杂交，其杂交后代对肺炎支原体感染抗性减弱，并造成在生长发育早期大量死亡。短腭、肺及鼻咽上皮克隆1（SPLUNC1）为一分泌性蛋白，在促进肺部先天防御肺炎支原体感染过程中发挥了重要作用。本研究以新疆野生盘羊、巴什拜羊及其杂交后代为研究对象，以支原体肺炎为疾病模型，从羊的SPLUNC1基因入手，通过人工授精、基因克隆、测序、基因表达、real-time PCR、细胞培养、电镜观察，流式细胞、人工感染、ELISA等研究手段，进行比较基因组学分析，比较以上不同品种羊SPLUNC1蛋白的功能，以期从基因结构、基因表达水平、蛋白功能、实际抗病等多个角度去揭示SPLUNC1在盘羊杂交后代对肺炎支原体抗性减弱而死亡中所起到的作用。同时运用数字基因表达谱技术，筛选不同品种羊抗肺炎支原体差异表达基因，为进一步研究杂交羊易感支原体肺炎的分子作用机制奠定基础。

野生盘羊与巴什拜羊杂交为种间杂交，其杂交后代羔羊易感绵羊支原体肺炎并造成大量死亡，本项目以SPLUNC1基因为研究对象，针对杂交后代易被Mo感染设计了相关实验内容，取得了以下研究成果：.1．利用RACE技术对上述三种羊的SPLUNC1基因cDNA全长序列进行了扩增及序列分析，证实野生盘羊、巴什拜羊及其杂交后代羊的SPLUNC1 cDNA序列全长分别为1076bp，1063bp和1117bp，其中ORF均为768bp，共编码255个氨基酸，杂交后代羊与巴什拜羊相比其核苷酸序列同源性为99%，氨基酸序列同源性为99.61%。.2.利用真核表达系统在毕赤酵母中成功表达三种羊的SPLUNC1基因，使用Ni-NTA琼脂亲和层析纯化法对重组SPLUNC1蛋白进行纯化。.3.重组SPLUNC1蛋白体外功能研究证实，三种羊的重组SPLUNC1蛋白对Mo可起到明显的抑制作用，均能显著地抑制巴氏杆菌和大肠杆菌的生长,能显著提高Mo感染的外周中性粒细胞NE的活性；巴什拜羊的重组SPLUNC1蛋白体外生物学活性要高于盘羊和杂交羊。.4.重组SPLUNC1蛋白体内功能研究证实，重组SPLUNC1蛋白对感染Mo的盘羊杂交羊的6个相关细胞因子具有很好调节作用，结果非常有利于机体恢复健康。重组SPLUNC1蛋白治疗组羔羊没有死亡，而对照组死亡率33%；说明重组SPLUNC1蛋白对绵羊支原体肺炎的治疗具有明显效果。.5.利用RNA-Seq技术筛选出了11个感染Mo后肺组织差异表达基因，并发现盘羊杂交羊对Mo易感的主要原因是感染Mo后，机体的免疫球蛋白类别转换受阻，形成高IgM综合征，造成IgG、IgA和IgE水平大大降低，导致了免疫缺陷；另外，盘羊杂交羊的B细胞不能完成向浆细胞和记忆B细胞的分化，导致其出现严重的病理损伤。以上结果从分子水平阐述了巴什拜羊和盘羊杂交羊对Mo抗病和易感的分子机理。