肉鸡健康及促生长相关肠道功能菌的靶向筛选与机制探究

The characters of poultry are not only influenced by genetic and nutritional factors, also associate with the gut microbes. We have already screened some strains which can improve the immunity and growth characters of poultry, but the relationship between some good characters of Chinese native broiler breeds and their gut microbes is not very clear. At the same while, which role did plenty of uncultured functional microbes played in poultry healthy is still unknown. Therefore, we intend to choose white feather broiler and Lueyang black-bone silky slightly (which famous as its high value of meat quality and disease resistance), compare their health and growth characters under same feeding conditions and select individuals with remarkable differences to collect caecum microbes. Use Metagenomic approaches to find functional genes on immune and growth promoting, then using the microfluidic technology to targeted screening and culturing the beneficial microbes for further Investigating on how they affect intestinal development, immune mature, body fat deposition, muscle development etc. The implementation of this project could provide theoretical basis and technical support to finding new strains which can promote the healthy and growth of the poultry.

家禽的性状不仅受到遗传和营养因素的调控，还受肠道微生物的影响。目前我们已经分离到一些可提高家禽免疫及促进生长的有益菌株，但一些地方品种鸡在生长及抗病方面的优势与其肠道微生物的关系尚不明确。同时，还存在大量未知有益微生物有待筛选，它们在家禽生长和健康上发挥的作用也不清楚。因此，本项目拟选用引进商业化品种（白羽肉鸡）和具有较高肉用价值且抗逆性强的陕西地方品种肉鸡（略阳乌鸡）作为研究对象，对比其在相同饲养条件下健康状况及生长状况的差异，选取具有明显表型差异的个体采集肠道微生物，通过宏基因组测序分析其功能基因，寻找其中对免疫和生长有促进作用的功能菌，结合微流控技术对功能菌进行靶向筛选和培养，并进一步验证其在家禽肠道发育、免疫成熟、体脂沉积及肌肉发育等方面发挥的作用及机制。该项目的实施，为寻找可促进家禽健康生长的优良新菌种及其在生产上的应用提供理论基础和技术支持。

益生菌在调控家禽肠道微生态中发挥重要作用，是良好的抗生素替代品之一。本项目以寻找可应用于促进家禽健康生长的优良新菌种为目标，利用耐酸、耐胆盐、粘附力、温度耐受、免疫特性、毒力基因表达、溶血能力、生物膜形成等检测方法，筛选出对免疫和生长有促进作用的功能益生菌，并进一步通过高通量测序分析、基因无痕敲除、超表达、qPCR、Western Blot、免疫荧光染色、ELISA等技术验证其在家禽肠道发育、免疫成熟、生产性能等方面发挥的作用及机制。结果如下：①从320株乳酸菌中获得了有望用于饲料添加的37株候选益生菌菌株，并进一步靶向筛选出5株具有提高肉鸡免疫力和生产性能的功能益生菌。②乳酸菌胞外多糖的表达量与其免疫调节活性呈正相关，胞外多糖表达量高的植物乳杆菌LPZ01能够显著降低肉鸡盲肠中沙门氏菌的定植并有效减缓沙门氏菌引起的肠道炎症。③植物乳杆菌LPZ01能够通过调控miRNAs的表达影响细胞炎症因子IFN-γ、LITAF和IL-6的水平（其中miR-193a-5p直接靶向抑制上皮细胞中免疫相关靶基因TRAF3的mRNA和蛋白水平的表达），进而参与宿主免疫应答反应的调节。④双组份系统LiaRS和BsrRS能够调控屎肠球菌对胆盐的耐受，其中BsrXRS具有自调控特性，能够调控下游27个靶基因，涉及细胞膜流动性、脂肪酸生物合成、细胞壁稳态、DNA错配修复等多个细胞功能来抵抗胆盐的毒性。⑤两个胆盐水解酶BSH1和BSH2对甘氨酸和牛磺酸结合型胆盐都具有较强的水解能力，能够帮助屎肠球菌耐受该类胆盐。⑥在高海拔低氧地区增加鸡舍中的氧浓度，配合饲喂乳酸杆菌JM113，能有效增强肉鸡的免疫应答，改善其生产性能和显著降低死亡率，JM113菌株具有胞外多糖产生相关的基因簇和转录因子cpsA，cpsA能够调控该乳酸菌的生长、胞外多糖的产生、生物膜的形成和自凝集，进而提高乳酸菌在肠道中的定植与生存。.本研究结果为提高肉鸡免疫力和生产性能的益生菌饲料添加剂提供了优势候选菌株，也为研究益生菌调控家禽肠道免疫功能的机理研究奠定了理论基础。