鸡NLRC5基因的转录调控及其对早期抗逆性的影响

To explore the solution of addressing the issue of drug residues and dysfunction of chicken immune system in the practical production, Langshan breed with high disease-resistance performance and Anka breed with low disease-resistance are used as experimental materials in this study,according to the early development characteristics of chicken. After predicting the promoter structure and transcription factor binding sites of NLRC5 gene via bioinformatics methods in chicken cloned from Langshan and Anka breed,respectively; a series of variants of promoter of NLRC5 gene are used to construct the recombinants of luciferase reporter gene in order to determine the activity of different variants of promoter and to identify the motif of different transcription factor binding sites. Subsequently, EMSA (electrophoretic mobility shift assay) and mass spectrometry method will be carried out to enrich the transcription factor complex which bind the artificial synthesised probes of binding motif to identify the subunit of transcription factor complex. Those transcription factor expressed by prokaryotic expression system are used to produce monoclonal antibody to carry out CHIP experiment to determine the differential activity of transcription factor binding to the motif between two chicken breeds. The alternative splicing variant will be identified by constructing cDNA library and gene clone. Finally Q-PCR，Northern blot and Western blot technologies are used to analyse the difference expression patterns of alternative splicing variants and NLRC5 related transcription factors. This experimental primarily reveals the regulation mechanism of transcriptional expression of NLRC5 which provides technique support to disease resistance breeding in poultry.

为探求解决当下养殖实际中鸡免疫功能低下和"药残"问题的新途径，本项目以高/低抗性鸡品系（狼山鸡与安卡鸡）半同胞家系为素材，参照家禽早期生长发育特点，运用生物信息学方法对已克隆的鸡NLRC5基因启动子结构、转录因子结合位点进行分析；通过制备一系列启动子突变体，构建荧光素酶报告基因重组体，根据荧光素酶报告基因表达量比较高低抗性家系间启动子调控区差异；并通过凝胶阻滞实验和质谱方法来鉴定与调控元件结合的转录调控因子，然后生产相应的单抗，利用CHIP方法验证高低抗性品系该基因的关键转录因子结合位点的差异。同时应用Q-PCR、Northern blot和Western blot技术检测高抗和低抗性鸡群中该基因转录本mRNA和亚型蛋白表达差异，初步阐明NLRC5的可变剪接和调控区差异对鸡早期抗性的调控作用，为禽类的抗病育种提供技术支撑。

为探求解决当下养殖实际中鸡免疫功能低下和“药残”问题的新途径，本项目对鸡NLRC5基因的转录调控及其在早期抗性的作用进行研究，获得了鸡NLRC5基因序列并了解其启动子遗传特性，鉴定出鸡NLRC5基因调控区的关键转录调控基序、顺式元件和相应的转录调控因子 (NF-κB、STAT1)；探明狼山鸡高、低抗性品种（品系）NLRC5基因调控区的转录活性和关键转录因子表达差异，初步解析了NLRC5基因对鸡早期抗性的调控机理。主要结果如下：①克隆了狼山鸡NLRC5基因CDS及UTR序列，提交至GenBank（JQ044414）。②在狼山鸡、罗斯鸡与安卡鸡的脾脏、盲肠、胸肌、肝脏等组织及两个细胞系（HD11、DF-1）中未发现NLRC5基因的可变剪接体。③通过缺失载体和荧光素酶活性检测，发现NLRC5启动子区存在2个核心区域，分别是-4372到-3756和-2925到-2265，且NLRC5启动子的活性依赖转录起始位点下游区域。④利用TRANSFAC和JASPAR分析发现NLRC5启动子-4372到-2065不包含典型RNA聚合酶Ⅱ结合元件，第一个核心区域内存在顺式元件YY1，第二个核心区域存在NFKB，2个核心区域均含STAT家族的结合位点。⑤转录因子结合实验进一步确认NLRC5第二个核心区域含顺式元件STAT1、CEBP，不含IRF、PPAR。并且STAT1在NLRC5启动子活性调控中具有重要作用。⑥利用MALDI-TOF-MS检测对27个鸡种NLRC5基因全序列中SNP位点进行大规模筛选，发现37个SNP位点，我国地方鸡种SNP位点数普遍高于现代商品肉鸡。在狼山鸡NLRC5启动子区域，在-2470处存在A/G单点突变，并且-2470突变显著降低启动子活性，安卡鸡中未发现影响活性的SNP。⑦利用单碱基突变技术进行-2470处的定点突变，双荧光素酶系统检测进一步说明该位点的突变影响启动子活性，可能受反式作用元件NF-κB、STAT1的调控。⑧克隆自然群体的不同单倍型，根据基因型筛选结果在狼山鸡中构建高低抗性品系AA、GG型群体各80只，1日龄狼山鸡半同胞攻毒结果发现AA型鸡各项抗性指标均优于GG。NLRC5及相关基因的组织和细胞表达研究发现，转录调控因子NF-κB 与NLRC5有显著的线性共表达关系，表明NLRC5确实参与先天免疫且通过抑制NF-κB信号通路从而影响早期抗逆性。