本文選題：皖南花豬 + 大約克豬�。� 參考：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：中外豬種在生長速度和肉質(zhì)性狀方面存在很大差異,國外豬種如大約克豬具有生長速度快、飼料利用率高、瘦肉率高的優(yōu)點,缺點是肉質(zhì)差,適應(yīng)性差;中國地方品種豬如皖南花豬具有適應(yīng)性強、耐粗飼、肉質(zhì)好的優(yōu)點,缺點是生長速度慢、瘦肉率低。本試驗以皖南花豬和大約克豬的背最長肌為研究對象,探索不同品種豬肉質(zhì)性狀產(chǎn)生差異的分子機制。采用高通量轉(zhuǎn)錄組測序(RNA-seq)技術(shù)篩選出皖南花豬和大約克豬背最長肌組織差異表達(dá)基因;利用DAVID數(shù)據(jù)庫對差異基因進行GO功能注釋和pathway通路分析;利用熒光定量PCR方法對篩選到的12個差異基因進行驗證。本研究的主要結(jié)果如下:(1)RNA-seq測序結(jié)果顯示,皖南花豬3個樣本得到的總Reads數(shù)分別為65 584126,64 327 738和59 244 502,其中Clean reads達(dá)到94.4%、94.3%和94.2%。大約克豬3個樣本得到的總reads數(shù)分別為57 969 134,68 254 168和72 298 142,其中Clean reads達(dá)到93.8%、93.7%和93.8%。表明測序飽和度良好,測序數(shù)據(jù)真實可靠。(2)對測序數(shù)據(jù)進行分析,篩選到347個差異基因,其中94個為上調(diào)基因,253個為下調(diào)基因。(3)GO功能富集得到分類條目有487個。其中細(xì)胞組分相關(guān)的差異基因數(shù)量為62個,所占比例為12.7%;分子功能相關(guān)的差異基因數(shù)量為116個,所占比例為23.9%;生物過程相關(guān)的差異基因數(shù)量為309個,所占比例為63.4%。GO分析表明在皖南花豬和大約克豬背最長肌中上調(diào)表達(dá)的差異基因主要顯著性富集在葡萄糖代謝過程、糖酵解、肌肉收縮過程以及肌肉器官發(fā)育過程等;在皖南花豬和大約克豬背最長肌中下調(diào)表達(dá)的差異基因主要顯著性富集在肌肉收縮、肌原纖維蛋白、細(xì)胞骨架蛋白結(jié)合、脂肪酸代謝過程、脂質(zhì)代謝過程、線粒體基質(zhì)、ATP代謝過程等。(4)KEGG數(shù)據(jù)庫注釋的差異表達(dá)基因有6620個,Pathway顯著性富集分析發(fā)現(xiàn)這些基因參與了146條生物學(xué)代謝通路,其中有22條存在顯著性富集(P0.01)。參與脂肪代謝途徑的差異基因數(shù)目最多(37個),主要包括脂肪酸代謝、PPAR信號通路、丙酸代謝、脂肪酸生物合成、脂肪細(xì)胞因子信號轉(zhuǎn)導(dǎo)通路、脂肪酸伸長率和不飽和脂肪酸生物合成等信號通路。(5)從篩選出的與肉質(zhì)性狀有關(guān)的差異基因中隨機挑選12個差異表達(dá)基因,其中上調(diào)基因為SLC37A4、GPI、AK1;下調(diào)基因為FABP3、CAST、AMPD3、CSRP3、PDK4、PFKL、PRKG1、CPT1B、以及ACTN2。通過qRT-PCR試驗進行驗證,結(jié)果表明qRT-PCR試驗驗證的差異基因其表達(dá)水平與RNA-seq測序得到的差異基因的表達(dá)水平相一致,表明RNA-seq測序的結(jié)果準(zhǔn)確,可靠有效。綜上,本研究獲得了豬背最長肌組織基因表達(dá)譜,篩選出了一些與肉質(zhì)性狀和生長性狀有關(guān)的候選基因,為后續(xù)進一步研究中外豬種肉質(zhì)性狀差異的分子機理和挖掘與肉質(zhì)性狀相關(guān)的候選基因等提供了理論依據(jù)。
[Abstract]:There are great differences in growth rate and meat quality between Chinese and foreign pig breeds. Foreign pig breeds such as Yorkshire pig have the advantages of fast growth rate, high feed utilization rate and high lean meat rate, but the shortcomings are poor meat quality and poor adaptability. Chinese local breeds such as Wannanhua pig have the advantages of strong adaptability, high tolerance to coarse feed and good meat quality, but the disadvantages are slow growth rate and low lean meat rate. In this study, the longissimus dorsi muscle of Wannanhua pig and Yorkshire pig was used as the research object to explore the molecular mechanism of the difference in quality traits of different breeds of pork. High-throughput transcriptome sequencing (RNA-seq) technique was used to screen differentially expressed genes in longissimus dorsi and longissimus muscularis of Wannanhua pig and Yorkshire pig respectively. Go functional annotation and pathway analysis were used to analyze the differential genes. The 12 differentially screened genes were verified by fluorescence quantitative PCR. The main results of this study were as follows: (1) the results of RNA-seq sequencing showed that the total read numbers of the three samples were 65,584126,64327,738 and 59,244,502, respectively. The total reads numbers of the three samples were 57969 13468254 168 and 72 298 142, respectively. The Clean reads reached 93.893 7% and 93.8% respectively. The results showed that the saturation of sequencing was good and the sequence data were reliable. (2) 347 differentially expressed genes were screened by analyzing the sequencing data, of which 254 genes were up-regulated and 253 genes were down-regulated. (3) 487 items were obtained by functional enrichment of go. Among them, the number of differentially expressed genes related to cellular components was 62, the proportion was 12.7, the number of differentially related genes related to molecular function was 116, the proportion was 23.9. the number of differentially related genes related to biological processes was 309. The result of 63.4. go analysis showed that the up-regulated genes in longissimus dorsi muscle were mainly concentrated in glucose metabolism, glycolysis, muscle contraction and muscle organ development. The down-regulated genes were mainly concentrated in muscle contraction, myofibrillar protein, cytoskeleton protein binding, fatty acid metabolism and lipid metabolism. Mitochondrial matrix ATP metabolism and so on. (4) 6620 differentially expressed genes in KEGG database were found to be involved in 146 biological metabolic pathways, 22 of which were significantly enriched (P0.01). The number of differentially expressed genes involved in fat metabolism pathway is the highest (37), including fatty acid metabolism PPAR signaling pathway, propionic acid metabolism, fatty acid biosynthesis, adipocyte factor signal transduction pathway, and the main genes involved in fat metabolism pathway. Fatty acid elongation and unsaturated fatty acid biosynthesis. (5) Twelve differentially expressed genes were randomly selected from selected differentially expressed genes related to meat quality traits, including SLC37A4 GPIK1, FABP3CASTAMPD3, CSRP3PDK4PFKKL1, CPT1B, and ACTN2. The results of qRT-PCR test showed that the expression level of differentially expressed genes was the same as that of RNA-seq sequencing. The results showed that the results of RNA-seq sequencing were accurate and effective. In conclusion, we obtained the gene expression profile of pig's longissimus dorsi muscle, and screened out some candidate genes related to meat quality and growth traits. It provides a theoretical basis for further study on the molecular mechanism of meat quality traits difference between Chinese and foreign pigs and the mining of candidate genes related to meat quality traits.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828

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