基于RNA-seq技术对皖南花猪和大约克猪背最长肌差异基因的筛选

发布时间：2018-06-23 03:48

本文选题：皖南花猪 + 大约克猪　；参考：《安徽农业大学》2015年硕士论文

【摘要】：中外猪种在生长速度和肉质性状方面存在很大差异,国外猪种如大约克猪具有生长速度快、饲料利用率高、瘦肉率高的优点,缺点是肉质差,适应性差;中国地方品种猪如皖南花猪具有适应性强、耐粗饲、肉质好的优点,缺点是生长速度慢、瘦肉率低。本试验以皖南花猪和大约克猪的背最长肌为研究对象,探索不同品种猪肉质性状产生差异的分子机制。采用高通量转录组测序(RNA-seq)技术筛选出皖南花猪和大约克猪背最长肌组织差异表达基因;利用DAVID数据库对差异基因进行GO功能注释和pathway通路分析;利用荧光定量PCR方法对筛选到的12个差异基因进行验证。本研究的主要结果如下:(1)RNA-seq测序结果显示,皖南花猪3个样本得到的总Reads数分别为65 584126,64 327 738和59 244 502,其中Clean reads达到94.4%、94.3%和94.2%。大约克猪3个样本得到的总reads数分别为57 969 134,68 254 168和72 298 142,其中Clean reads达到93.8%、93.7%和93.8%。表明测序饱和度良好,测序数据真实可靠。(2)对测序数据进行分析,筛选到347个差异基因,其中94个为上调基因,253个为下调基因。(3)GO功能富集得到分类条目有487个。其中细胞组分相关的差异基因数量为62个,所占比例为12.7%;分子功能相关的差异基因数量为116个,所占比例为23.9%;生物过程相关的差异基因数量为309个,所占比例为63.4%。GO分析表明在皖南花猪和大约克猪背最长肌中上调表达的差异基因主要显著性富集在葡萄糖代谢过程、糖酵解、肌肉收缩过程以及肌肉器官发育过程等;在皖南花猪和大约克猪背最长肌中下调表达的差异基因主要显著性富集在肌肉收缩、肌原纤维蛋白、细胞骨架蛋白结合、脂肪酸代谢过程、脂质代谢过程、线粒体基质、ATP代谢过程等。(4)KEGG数据库注释的差异表达基因有6620个,Pathway显著性富集分析发现这些基因参与了146条生物学代谢通路,其中有22条存在显著性富集(P0.01)。参与脂肪代谢途径的差异基因数目最多(37个),主要包括脂肪酸代谢、PPAR信号通路、丙酸代谢、脂肪酸生物合成、脂肪细胞因子信号转导通路、脂肪酸伸长率和不饱和脂肪酸生物合成等信号通路。(5)从筛选出的与肉质性状有关的差异基因中随机挑选12个差异表达基因,其中上调基因为SLC37A4、GPI、AK1;下调基因为FABP3、CAST、AMPD3、CSRP3、PDK4、PFKL、PRKG1、CPT1B、以及ACTN2。通过qRT-PCR试验进行验证,结果表明qRT-PCR试验验证的差异基因其表达水平与RNA-seq测序得到的差异基因的表达水平相一致,表明RNA-seq测序的结果准确,可靠有效。综上,本研究获得了猪背最长肌组织基因表达谱,筛选出了一些与肉质性状和生长性状有关的候选基因,为后续进一步研究中外猪种肉质性状差异的分子机理和挖掘与肉质性状相关的候选基因等提供了理论依据。
[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.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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