本文關(guān)鍵詞： 抑制性消減雜交 基因芯片 熒光定量PCR 肉質(zhì)性狀 中國(guó)草原紅牛 中國(guó)荷斯坦牛　出處：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：日漸提升的生活水平要求,對(duì)國(guó)內(nèi)牛肉市場(chǎng)上的牛肉品質(zhì)提出了更多的要求,民眾的生活中逐漸出現(xiàn)了越來(lái)越多的優(yōu)質(zhì)或是高檔的牛肉產(chǎn)品,這也要求研發(fā)人員需要深入進(jìn)行牛肉的品質(zhì)研究。通常,我們所說(shuō)的肉質(zhì)包含了肉的營(yíng)養(yǎng)成分、適口性和觀感等多方面的內(nèi)容。在以往,肉品的外觀和風(fēng)味,是消費(fèi)者所最關(guān)心的,也是通過(guò)這幾個(gè)指標(biāo)對(duì)肉質(zhì)進(jìn)行評(píng)價(jià)。隨著生物信息學(xué)迅速發(fā)展,動(dòng)物遺傳育種與繁殖學(xué)已經(jīng)有常規(guī)的育種向分子育種轉(zhuǎn)變,與此同時(shí)基因差異表達(dá)調(diào)控機(jī)制成為改良遺傳性狀以及基因功能鑒定的研究熱點(diǎn)。本文利用抑制性消減雜交(suppression subtractive hybridization SSH)方法構(gòu)建了中國(guó)草原紅牛與中國(guó)荷斯坦牛背最長(zhǎng)肌差異表達(dá)消減cDNA文庫(kù),共獲得859個(gè)差異表達(dá)克隆,對(duì)全文庫(kù)進(jìn)行了測(cè)序并與GenBank數(shù)據(jù)庫(kù)進(jìn)行比對(duì)分析。結(jié)果共獲得789個(gè)新ESTs,84個(gè)未知功能蛋白基因,356個(gè)已知功能蛋白基因。在已知功能蛋白基因中,表達(dá)上調(diào)的基因有123個(gè),表達(dá)下調(diào)的基因有233個(gè),對(duì)篩選出的陽(yáng)性克隆進(jìn)行功能分類,功能注釋結(jié)果顯示,篩選出的差異基因的功能主要在細(xì)胞位置、分子功能和生物過(guò)程這三大類中。對(duì)于SSH消減文庫(kù)中所篩選出的克隆,我們采用基因芯片技術(shù)對(duì)克隆是否存在假陽(yáng)性進(jìn)行驗(yàn)證并進(jìn)一步篩選出差異基因,采用Sam法篩選差異基因,共1296個(gè)基因片段,Ratio=2/5情況下,篩選出32個(gè)差異表達(dá)基因,其中24個(gè)基因表達(dá)上調(diào),8個(gè)基因表達(dá)下調(diào);Ratio=4/5情況下有43個(gè)差異表達(dá)基因,其中18個(gè)基因表達(dá)上調(diào),25個(gè)基因表達(dá)下調(diào),結(jié)果與SSH文庫(kù)結(jié)果進(jìn)行比對(duì),基因差異表達(dá)情況一致,證明SSH文庫(kù)質(zhì)量高,假陽(yáng)性率低。參照國(guó)家標(biāo)準(zhǔn)對(duì)草原紅牛和普通雜種肉牛的脂肪酸進(jìn)行測(cè)定,前者脂肪酸含量高于后者。挑選出文庫(kù)篩選出的與脂肪代謝功能相關(guān)的差異表達(dá)基因GLTPD1和PPARγ,采用熒光定量PCR技術(shù)測(cè)定兩個(gè)基因在草原紅牛和普通雜種肉牛中的表達(dá)量,兩者在草原紅牛中的表達(dá)量高于普通雜種肉牛。將脂肪酸和熒光定量PCR測(cè)定結(jié)果進(jìn)行關(guān)聯(lián)性分析,結(jié)果說(shuō)明GLTPD1和PPARγ可以作為研究肉質(zhì)性狀的候選基因。
[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 緯 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 緯 could be used as candidate genes for studying meat quality traits.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823

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本文編號(hào)：1464606