Rybp基因对秦川牛成肌细胞分化作用的研究

发布时间：2018-03-03 21:34

本文选题：牛　切入点：Rybp基因　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着人们生活水平不断提高,牛肉作为一种高蛋白低胆固醇的健康食品越来越受到人们的青睐,市场牛肉的需求量越来越大,供给不足,牛肉价格持续上涨。如何提高牛肉产量和改善牛肉品质成为广大育种工作者关注的热点,而对肉牛肌肉组织生长发育调控机制的研究将从分子水平揭示影响肉牛产肉性能和牛肉品质形成的机理,为肉牛的分子育种工作奠定基础。Ring和YY1结合蛋白(Ring and YY1 binding protein,Rybp)已在小鼠上被证实对肌生成具有抑制作用,而在牛上还未见相关报道,仍需要进一步研究。本研究以Rybp基因为目标基因,利用实时定量PCR技术,检测了该基因在秦川牛16种组织、5个不同发育阶段肌肉、脂肪组织以及不同分化阶段牛成肌细胞中表达水平,利用腺病毒介导该基因在成肌细胞中过量表达,探究Rybp基因对牛成肌细胞分化的作用,通过转录组测序,筛选和鉴定过量表达Rybp基因后的差异表达基因,初步揭示了Rybp基因在牛成肌细胞分化过程中的功能及其对一些重要的成肌细胞分化相关基因的影响。获得的主要研究结果如下:1.秦川牛Rybp基因表达特性分析结果表明,Rybp基因在成年秦川牛睾丸组织中表达量最高,而在背最长肌、里脊、心脏等组织中的表达量相对较低;随着秦川牛年龄的增加,Rybp基因在肌肉组织中的表达水平逐渐升高,在脂肪组织中的表达水平显著下降;在不同分化阶段成肌细胞中,随着成肌细胞分化进程的推进,Rybp基因表达水平逐渐升高,而当分化减速后,Rybp基因表达水平逐渐降低。2.克隆得到秦川牛Rybp基因的CDS区序列,其长度为687bp,编码229个氨基酸残基,然后成功构建并获得了高滴度牛Rybp基因腺病毒过表达载体,病毒滴度为1×109pfu/mL。3.在牛成肌细胞中过表达Rybp基因,可显著抑制肌管的形成,这种差异从过表达后2d开始出现,到6d达到最显著。将过表达Rybp基因6d后的成肌细胞与对照组细胞进行转录组测序,结果共筛选出1311个差异表达基因,其中有707个基因表达水平上调,604个基因表达水平下调,许多参与肌肉发育相关的功能基因表达水平均下调,且差异表达基因显著富集到32条信号通路,包括Wnt等与肌肉发育相关通路。
[Abstract]:With the improvement of people's living standard, beef, as a kind of healthy food with high protein and low cholesterol, is getting more and more popular. Beef prices continue to rise. How to increase beef production and improve beef quality has become the focus of attention of the majority of breeders. The study on the mechanism of the growth and development of beef muscle will reveal the mechanism that affects the performance of beef and the formation of beef quality at the molecular level. To lay the foundation for the molecular breeding of beef cattle. Ring and YY1 binding protein Ring and YY1 binding protein Rybp have been proved to inhibit myogenesis in mice, but have not been reported in cattle. In this study, Rybp gene was used as the target gene and real-time quantitative PCR technique was used to detect the expression of the gene in 16 tissues of Qinchuan cattle and 5 muscles in different developmental stages. The expression levels of adipose tissue and bovine myoblasts at different differentiation stages were investigated by using adenovirus mediated overexpression of the gene in the myoblast, and the effect of Rybp gene on the differentiation of bovine myoblast was investigated by transcriptome sequencing. Screening and identification of differentially expressed genes after overexpression of Rybp gene, The function of Rybp gene in the process of bovine myoblast differentiation and its effect on some important genes related to myoblast differentiation were preliminarily revealed. The main results obtained are as follows: 1. Analysis of the expression characteristics of Rybp gene in Qinchuan cattle. The results showed that the expression of RyBP gene was the highest in the testis of Qinchuan cattle. The expression of RyBP gene in muscle tissues of longissimus dorsi, ridge and heart was relatively low, and the expression level of RyBP gene in muscle tissue increased gradually with the increase of age of Qinchuan cattle, and decreased significantly in adipose tissue. In the myoblasts of different differentiation stages, the expression level of RyBP gene increased with the process of myoblast differentiation, but decreased gradually after the differentiation decelerated. The sequence of CDS region of Rybp gene of Qinchuan cattle was cloned. Its length was 687 BP, encoding 229 amino acid residues, and then the overexpression vector of high titer bovine Rybp gene adenovirus was constructed and obtained. The viral titer was 1 脳 10 9 pfup / mL 路3.The overexpression of Rybp gene in bovine myoblasts could significantly inhibit the formation of myotubes. The difference appeared from the second day after overexpression and reached the most significant at 6 days. After 6 days of overexpression of Rybp gene, 1311 differentially expressed genes were sequenced from myoblasts and control cells. 707 genes were up-regulated and 604 genes were down-regulated. Many functional genes involved in muscle development were down-regulated, and differentially expressed genes were enriched to 32 signal pathways. Including Wnt and other related pathways related to muscle development.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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