奶牛乳脂代谢关键候选基因的鉴定与功能分析

Identification and functional analysis of key candidate genes for milk fat metabolism in dairy cattle

为深入挖掘影响荷斯坦奶牛乳脂代谢的关键候选基因,利用实时荧光定量聚合酶链式反应(qRT-PCR)技术检测课题组前期通过转录组和加权基因共表达网络分析(WGCNA)得到的15个候选差异基因的组织表达谱。确定乳脂代谢关键候选基因后检测其在原代奶牛乳腺上皮细胞(BMECs)中的定位,并进行克隆测序,以及结构和功能分析。结果表明,ENPP 2、PI4K2A、CTSH和PTPRR基因在乳腺组织中的表达水平均高于其他组织,同时PI4K2A和CTSH在乳腺组织和原代BMECs中的表达相对于其他基因处于较高水平。通过qRT-PCR结合前期转录组测序的结果,最终确定PI4K2A为调控奶牛乳脂合成的关键候选基因,其主要在BMECs的细胞质中表达。克隆测序发现,奶牛PI4K2A基因编码区(CDS)全长1440 bp,编码479个氨基酸。结构和功能分析表明,PI4K2A为非分泌型且不稳定的亲水蛋白,含有40个磷酸化位点,翻译后的修饰作用非常丰富。PI4K2A蛋白的二级和三级结构均以无规则卷曲为主,α螺旋次之,且在不同物种中具有较高的保守性。本研究确定PI4K2A为奶牛乳脂代谢的重要候选基因,研究结果可以为奶牛乳脂代谢的分子调控机制研究提供理论依据。

In order to deeply explore the key candidate genes affecting milk fat metabolism in Holstein cows,this study used real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)to examine the tissue expression profiles of 15 candidate differential genes obtained by transcriptome and weighted gene co-expression network analysis(WGCNA)in the previous phase.After the key candidate genes for milk fat metabolism were identified,their localization in primary dairy mammary epithelial cells(BMECs)was examined,and clonal sequencing and functional analysis were performed.Results showed that the expression levels of ENPP2,PI4K2A,CTSH and PTPRR genes were higher in breast tissue than in other tissues,respectively,while the expression of PI4K2A and CTSH were at higher levels in both breast tissue and primary BMECs compared to other genes.The results of qRT-PCR combined with sequencing of the transcriptome finally identified PI4K2A as a key candidate gene regulating milk fat synthesis in dairy cows,mainly expressed in the cytoplasm of BMECs.Clone sequencing study revealed that the CDS(coding sequence)region of the cow PI4K2A gene was 1440 bp in length and encoded 479 amino acids.Structural and functional analysis showed that PI4K2A was a non-secretory and unstable hydrophilic protein containing 40 phosphorylation sites with abundant post-translational modifications.Both the secondary and tertiary structure of PI4K2A protein were predominantly random coil,followed by α-helix,and was highly conserved across species.This study identified PI4K2A as an important candidate gene for milk fat metabolism in dairy cow,providing theoretical basis for the study of the molecular regulation mechanism of milk fat metabolism in dairy cow.