草原红牛和荷斯坦牛肌肉组织差异表达基因筛选及肉质性状相关基因分析

发布时间：2018-01-26 03:57

本文关键词： 抑制性消减杂交基因芯片荧光定量PCR 肉质性状中国草原红牛中国荷斯坦牛　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：日渐提升的生活水平要求,对国内牛肉市场上的牛肉品质提出了更多的要求,民众的生活中逐渐出现了越来越多的优质或是高档的牛肉产品,这也要求研发人员需要深入进行牛肉的品质研究。通常,我们所说的肉质包含了肉的营养成分、适口性和观感等多方面的内容。在以往,肉品的外观和风味,是消费者所最关心的,也是通过这几个指标对肉质进行评价。随着生物信息学迅速发展,动物遗传育种与繁殖学已经有常规的育种向分子育种转变,与此同时基因差异表达调控机制成为改良遗传性状以及基因功能鉴定的研究热点。本文利用抑制性消减杂交(suppression subtractive hybridization SSH)方法构建了中国草原红牛与中国荷斯坦牛背最长肌差异表达消减cDNA文库,共获得859个差异表达克隆,对全文库进行了测序并与GenBank数据库进行比对分析。结果共获得789个新ESTs,84个未知功能蛋白基因,356个已知功能蛋白基因。在已知功能蛋白基因中,表达上调的基因有123个,表达下调的基因有233个,对筛选出的阳性克隆进行功能分类,功能注释结果显示,筛选出的差异基因的功能主要在细胞位置、分子功能和生物过程这三大类中。对于SSH消减文库中所筛选出的克隆,我们采用基因芯片技术对克隆是否存在假阳性进行验证并进一步筛选出差异基因,采用Sam法筛选差异基因,共1296个基因片段,Ratio=2/5情况下,筛选出32个差异表达基因,其中24个基因表达上调,8个基因表达下调;Ratio=4/5情况下有43个差异表达基因,其中18个基因表达上调,25个基因表达下调,结果与SSH文库结果进行比对,基因差异表达情况一致,证明SSH文库质量高,假阳性率低。参照国家标准对草原红牛和普通杂种肉牛的脂肪酸进行测定,前者脂肪酸含量高于后者。挑选出文库筛选出的与脂肪代谢功能相关的差异表达基因GLTPD1和PPARγ,采用荧光定量PCR技术测定两个基因在草原红牛和普通杂种肉牛中的表达量,两者在草原红牛中的表达量高于普通杂种肉牛。将脂肪酸和荧光定量PCR测定结果进行关联性分析,结果说明GLTPD1和PPARγ可以作为研究肉质性状的候选基因。
[Abstract]:The rising living standard demands more and more demands on beef quality in domestic beef market, and more and more high-quality or high-grade beef products appear gradually in the people's life. This also requires researchers to conduct in-depth research on beef quality. Generally speaking, meat quality includes nutrition, palatability and perception of meat. In the past, the appearance and flavor of meat. With the rapid development of bioinformatics, there has been a shift from conventional breeding to molecular breeding in animal genetics and reproduction. At the same time, the regulation mechanism of gene differential expression has become a hot topic in the identification of genetic traits and gene function. Suppression subtractive hybridization SSH). Methods A subtractive cDNA library was constructed for the differential expression of longissimus dorsi muscle between Chinese steppe red cattle and Chinese Holstein cattle. A total of 859 differentially expressed clones were obtained. The whole library was sequenced and compared with the GenBank database. A total of 789 new ests and 84 unknown functional protein genes were obtained. Among 356 known functional protein genes, 123 genes were up-regulated and 233 genes were down-regulated. The results of functional annotation showed that the functions of the differentially screened genes were mainly in the three categories of cell location, molecular function and biological process. The clones screened in the subtractive library of SSH were selected. We used gene chip technology to verify the existence of false positive clones and further screening of differential genes, using Sam method to screen differential genes, a total of 1296 gene fragments. In the case of Ratio=2/5, 32 differentially expressed genes were screened, among which 24 genes were up-regulated and 8 genes were down-regulated. There were 43 differentially expressed genes in Ratio=4/5, of which 18 genes were up-regulated and 25 genes were down-regulated. The results were compared with those of SSH library. The difference of gene expression showed that the quality of SSH library was high and the false positive rate was low. The fatty acids of steppe red cattle and common hybrid beef cattle were determined with reference to the national standard. The fatty acid content of the former was higher than that of the latter. The differentially expressed genes GLTPD1 and PPAR 纬 related to fat metabolism function were selected from the library. Fluorescence quantitative PCR was used to detect the expression of two genes in steppe red cattle and common hybrid beef cattle. The results of fatty acid analysis and fluorescence quantitative PCR analysis were used to analyze the correlation between the results of fatty acid and fluorescence quantitative PCR. The results showed that GLTPD1 and PPAR 纬 could be used as candidate genes for studying meat quality traits.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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