基因组可变剪切相关SNPs的功能鉴定及其在奶牛乳腺炎易感性中的作用

Mastitis resistance or susceptibility is a complex trait that is comprehensively affected by the genetic component, physiological and environmental factors. However, the molecular mechanism underlying the formation of resistance is poorly understood. Genetic variations, such as single nucleotide polymorphisms (SNPs) through their effects on gene expression by modulating alternative splicing patterns, influence many aspects of the human and animal phenotypes, including disease susceptibility. Our previous study has proved that alternative splicing is an important mechanism regulating immune-related gene differential expression and is associated with mastitis resistance in dairy cow. Many studies have also demonstrated that variants in genes related to immune response that could be associated with mastitis. MicroRNAs (miRNAs) are small endogenous RNAs of approximately 22 nucleotides that play important roles in posttranscriptional regulation of gene expression. Recent evidences show that the alternative splicing can negatively modulate biogenesis of pre-miRNAs located across the gene exon-intron splicing sites. However, what is not known is whether genetic splicing-related mutations are the molecular basis linking with the alternative splicing mechanism and mastitis resistance phenotypes relevent. Taken together, we hypothesized that genetic splicing-related SNPs would disturb the gene splicing patterns and result in the aberrant transcripts of mastitis-associated genes and subsequently play a bona fide biological role on bovine resistance or susceptibility to mastitis, so far as to, SNPs would potentially influence mature microRNA expression by regulating splicing of gene overlapping with pre-miRNAs, which maybe one of the new mechanisms for the mastitis resistance formation. To confirm our hypotheses, therefore, we will be seeking to investigate the splicing patterns of transcriptome and genomic variations using the RNA-seq and genotyping by sequencing methods; to identify the genomic splicing-related SNPs via integrated bioinformatics combinating with our previous miRNAs profile and proteome data from cow's mammary gland tissues; to determinate whether the SNPs would generate the aberrant transcripts using the minigene transcriptional system; to assess the genetic effects of splicing-related SNPs on mastitis resistance using the experimental and reference cattle populations. It is anticipated that we would expound partly the molecular basis of mastitis resistance or susceptibility formation and find a potential novel molecular mechanism, as well as exploit a set of splicing-related functional SNPs that would be further applied in the molecular breeding program in dairy cattle.

乳腺炎抗性是一个复杂性状，受遗传、环境等综合因素影响。抗性形成的分子遗传机制尚不明了。遗传变异可调控基因可变剪切，影响人类、动物疾病表型。我们提出“单核苷酸多态（SNPs）突变影响乳腺炎抗性相关基因的剪切模式，产生异常转录本及差异表达，导致奶牛个体乳腺炎抗性/易感性差异,并表现病原体特异性”的假设。本项目拟应用RNA测序、基因分型测序等研究健康和不同病原体感染引起的乳腺炎奶牛乳腺和嗜中性粒细胞转录组与可变剪切模式差异；利用生物信息学、Minigene剪切系统等鉴定和验证可导致基因产生异常转录本，或者通过影响基因剪切调控microRNAs表达的SNPs；参考群体验证SNPs遗传效应；整合转录组、牛QTLdb、及前期的蛋白质组和microRNA谱结果，揭示乳腺炎抗性相关基因的调控网络，部分阐明乳腺炎抗性形成的分子基础和新机制；重点挖掘基因组剪切相关SNPs分子标记，以应用于奶牛乳腺炎抗病育种。

乳腺炎抗性是一个复杂性状，受遗传、环境等综合因素影响。遗传变异可调控基因可变剪切，影响人类、动物疾病表型。利用转录组测序研究了健康和乳腺炎奶牛嗜中性粒细胞中基因可变剪切的模式差异，检测到831个基因存在可变剪切事件。通过RT-PCR以及克隆测序技术在奶牛乳腺组织和嗜中性粒细胞中检测到CCL5、LGR4、SLAMF7、IL6R基因存在新的可变剪切体，并对这些基因不同转录本进行表达水平分析。在CCL5和IL6R基因发生剪切的区域发现存在两个SNP，这两个SNP位点位于外显子剪接增强子(ESE)基序区域。构建mini-基因剪切系统，进行细胞转染和测定，验证这些SNP是否是引起基因产生可变剪切的原因。同时将这些SNP不同基因型与奶牛体细胞评分进行关联分析，验证它们的遗传效应。对SLAMF7-不同剪切体蛋白进行表达、定位和抗菌活性分析，纯化的蛋白SLAMF7对革兰氏阳性细菌金黄色葡萄球菌可能具有较弱的抗菌活性。鉴定到位于牛bta-miR-2899和SCS相关QTL中的SNP(rs109462250)，将该SNP与SCS进行关联分析，表明AA基因型是乳腺炎抗性基因型。研究结果分析了乳腺炎抗性相关基因的调控网络，解析了剪切相关SNPs与乳腺炎抗性的关系，揭示了奶牛乳腺炎抗性形成的分子基础。