基于胚胎肌肉组织转录组和蛋白质组数据鉴定猪生长性状相关基因

发布时间：2018-03-09 11:52

本文选题：猪　切入点：胚胎期　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：肌肉生长是猪生产中的重要经济性状,是一个受多基因调控的数量性状,快速生长型的猪种深受养殖者的青睐,因此对猪肉产量和肉品质的研究一直是我国畜牧学者研究的重点。本研究选择具有慢生长、体型偏小的藏猪(TP);慢生长、体型中等的乌金猪(WJ);快生长、体型正常的大约克猪(LW)作为试验对象,利用转录组RNA-seq和蛋白质组iTRAQ技术对猪胚胎60日龄的背最长肌进行分析,鉴定与猪生长性状相关的差异基因,并利用PRM技术对差异基因进行蛋白水平的验证,旨在通过胚胎时期肌肉生长发育的差异阐明对出生后猪生长性状的影响。三个品种猪胚胎60日龄的背最长肌组织RNA-seq得到38.7-50.1G clean reads,检测到19626个基因在背最长肌组织表达,其中三个品种共表达阳性基因14660个,通过计算所有基因的FPKM值后,以|log2FoldChange|1和90.01作为差异表达基因(DEGs)的筛选标准,我们分别在TP vs.LW 组得到 5115 个 DEGs,TP vs.WJ 组 4274 个 DEGs,WJ vs.LW 组 260 个 DEGs,结合藏猪、乌金猪、大约克猪出生后的生长特点,确定了 TP vs.LW组和TP vs.WJ组交集的3626个基因作为本研究中重点关注基因,并初步筛选出15个可能与猪肌肉生长发育相关的基因。利用iTRAQ蛋白测序技术进行蛋白表达水平差异蛋白的筛选,共得到12230个肽段及对应的2474个蛋白。以FoldChange1.5或0.67作为差异表达蛋白(DEPs)的筛选标准,在TP vs.LW组得到 939 个 DEPs,TP vs.WJ 组 981 个 DEPs,WJ vs.LW 组 119 个 DEPs。其中 TP 与 LW 和WJ共同的735个DEPs作为本研究中重点关注的蛋白,初步筛选了 SYNE2、TRMT10C、GTPBP8、EDEM3、ATP8、PHF1、RIF1、APM1、PDLIM3、GPR84、NR0B1、GPT2、TPTE2 等 12 个基因可能与猪肌肉生长发育性状有关,这些基因主要参与细胞的增殖与分化、Ca2+代谢通路、肌纤维和肌原纤维的形成、氨基酸代谢、氧化磷酸化等通路。通过RNA-seq和iTRAQ数据的联合分析,我们发现RNA-seq分析中得到的差异基因与iTRAQ分析中得到的差异蛋白之间呈正相关(Person相关系数0.7-0.71)。mRNA水平和蛋白质水平表达量趋势一致的基因有209个。这些基因主要与钙离子通路、细胞增殖和凋亡、肌原纤维的发育、肌肉细胞分化、肌内胶原含量、成纤维细胞生长的调节有关。其中LDB3、PDLIM5、NR1D2、ROCK2、NDRG2、POSTN、FSCN1、COL3A1、CRYAB、ANKRD2、MAPK12、LOC100627626和LOC100523824等13个基因,可以作为产前成肌细胞增殖和肌肉形成有关的重要候选基因。采用通过PRM技术验证的24个差异基因中,其表达趋势完全与RNA-seq、iTRAQ—致,进一步证明了数据的可靠性、可信性。本研究整合RNA-seq和iTRAQ数据进行分析,并利用PRM技术进行验证,筛选出了影响猪肌肉生长发育的差异基因和差异蛋白。MYL1、DOLIM5、CKM、AC4DM、TCAP、PGAM2、LYGM、PGM1、MTFP和CKMT2等10个差异表达基因对猪骨骼肌的生长发育以及出生后的产肉性能具有抑制作用。MYL4、COL3A 和STMN1等3个差异表达基因和差异表达蛋白能促进骨骼肌的快速生长和发育。为下一步深入理解肌肉发育的分子机理提供新的切入点,为出生后猪生长性状候选基因的筛选与鉴定提供了宝贵数据。
[Abstract]:Muscle growth is an important economic traits in pig production, is a quantitative trait controlled by many genes, pig farmers by the rapid growth of the favor, so the study on meat production and meat quality of animal husbandry has been the focus of scholars in China. This study has slow growth, smaller Tibet pig (TP); slow growth, medium-sized Wujin pigs (WJ); fast growth, the normal size of Yorkshire pigs (LW) as the test object, using RNA-seq and iTRAQ transcriptome proteome technology on pig embryos at 60 days of age the longissimus muscle were analyzed, and identification of genes related to growth traits in pigs the verification and protein level on different genes by using PRM technology, through the difference of embryonic muscle growth to clarify the influence of growth traits of pigs. After the birth of three varieties of pig embryos at 60 days of age the longissimus muscle tissue RNA-seq 38.7-50 .1G clean reads, detected the expression of 19626 genes in longissimus muscle tissue, of which three species were positive expression of 14660 genes, all genes by calculating the value of FPKM, |log2FoldChange|1 and 90.01 differentially expressed genes (DEGs) as the selection criteria, we obtained 5115 DEGs in TP vs.LW group, TP vs.WJ group 4274 DEGs vs.LW group, WJ 260 DEGs, the combination of Tibetan pigs, Wujin pigs, growth characteristics of Yorkshire pigs after birth, to determine the TP vs.LW group and TP vs.WJ group as the intersection of 3626 genes in this study focus on the gene, and initial screening genes related to the growth and development of 15 possible with pigs muscle. Screening of protein expression level of proteins by iTRAQ protein sequencing technology, there are 2474 protein 12230 peptides and corresponding to FoldChange1.5 or 0.67. As the differentially expressed protein (DEPs) screening standard in TP Group vs.LW had 939 DEPs, 981 DEPs TP vs.WJ group, WJ group vs.LW 119 DEPs. TP and LW WJ and 735 DEPs as a common focus in the study of protein, preliminary screening of SYNE2, TRMT10C, GTPBP8, EDEM3, ATP8, PHF1, RIF1, APM1, PDLIM3, GPR84, NR0B1 GPT2, TPTE2, etc. 12 genes and porcine muscle growth traits, proliferation and differentiation of these genes mainly involved in cell metabolism, Ca2+ pathway, amino acid metabolism, muscle fibers and myofibrils, oxidative phosphorylation pathway. Through the combined analysis of RNA-seq and iTRAQ data, we found a positive correlation between the difference the analysis of iTRAQ gene and the difference obtained in RNA-seq analysis of protein (Person correlation coefficient 0.7-0.71).MRNA and protein level expression trend of 209 genes. These genes with calcium ion pathway, cell proliferation And apoptosis, myofibril development, muscle cell differentiation, collagen content in muscle and fibroblast growth regulation. The LDB3, PDLIM5, NR1D2, ROCK2, NDRG2, POSTN, FSCN1, COL3A1, CRYAB, ANKRD2, MAPK12, LOC100627626 and LOC100523824 13 genes can be used as prenatal myoblast proliferation and muscle the formation of important candidate genes related with 24 genes. Through the verification of the PRM technology, its expression trend fully with RNA-seq, iTRAQ, further proved the reliability of the data, the credibility of the integration of RNA-seq and iTRAQ. The research data were analyzed and verified using PRM technology, selected the differences in the effects of porcine skeletal muscle growth the development of gene and proteins.MYL1, DOLIM5, CKM, AC4DM, TCAP, PGAM2, LYGM, PGM1, MTFP and CKMT2 10 expression of porcine skeletal muscle growth genes and meat production performance after birth can inhibit The role of.MYL4 gene and expression of COL3A and STMN1 3 differentially expressed protein can promote the rapid growth and development of skeletal muscle. Offer a new perspective to understand the molecular mechanism of muscle development in the next step, provides valuable data for the screening and identification of postnatal porcine growth traits.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S828

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