奶牛乳脂性状关键候选基因及其突变位点功能验证研究

奶牛遗传改良对奶业发展至关重要。基因组选择技术已切实加快奶牛群体遗传进展。未来基因编辑技术的应用也是加快奶牛遗传改良的重要手段，前提是目标性状关键功能基因或突变的作用被验证。牛奶中乳脂含量越来越被关注，培育特定乳脂含量奶牛新品系也值得被重视。大量乳脂性状候选基因被鉴定，但基本没有深入系统的验证；因此，本项目将针对前期鉴定到的乳脂性状候选基因，IGF1R、FGFR4、PKLR和FADS2，利用遗传效应分析来确认它们与乳脂量、乳脂率和24种脂肪酸的关联，评价其育种价值；针对具备效应的基因和SNP，在细胞水平上利用基因干扰、过表达、双荧光素酶活性、凝胶阻滞和细胞功能检测等技术对其作用进行验证；进一步以CRISPR/Cas9基因编辑小鼠为模型，验证关键基因和突变位点对乳脂性状形成的作用；旨在解析关键基因和突变对乳脂性状形成的作用机理，并提出可用于奶牛基因组选择芯片开发和基因编辑奶牛的重要分子信息。

Genetic improvement of dairy cattle is very important to the development of dairy industry. The genomic selection has effectively accelerated the genetic progress of dairy cattle population. In the future, the application of gene editing is also an important means to speed up the genetic improvement of dairy cattle, but the premise is that the function of key functional genes or mutations of target traits is verified. The content of milk fat is more and more concerned by people. It is also worthy of attention to cultivate new dairy lines with specific milk fat content. A large number of candidate genes for milk fat traits have been identified, but there is no in-depth systematic verification. Therefore, in this project, we will analyze the genetic effects of the previously identified candidate genes for milk fat traits, IGF1R, FGFR4, PKLR, and FADS2, to confirm their associations with milk fat content, milk fat percentage and 24 fatty acids, and evaluate their breeding values. For the genes and SNPs with effect, we will verify their effects on the formation of milk fat traits at the cellular level by using RNA interference, over expression, dual-luciferase activity detection, electrophoretic mobility shift assay and cell function detection. Further, CRISPR / cas9 gene editing mice will be used as the model to verify the effect of key gene and mutation on the formation of milk fat traits. The purpose of this study is to reveal the mechanism of the key candidate gene/mutation on the formation of milk fat traits and to propose important molecular information for the development of genomic selection chips and gene editing of dairy cattle.