本文選題：豬　切入點：胚胎期　出處：《中國農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

【摘要】：肌肉生長是豬生產(chǎn)中的重要經(jīng)濟性狀,是一個受多基因調(diào)控的數(shù)量性狀,快速生長型的豬種深受養(yǎng)殖者的青睞,因此對豬肉產(chǎn)量和肉品質(zhì)的研究一直是我國畜牧學者研究的重點。本研究選擇具有慢生長、體型偏小的藏豬(TP);慢生長、體型中等的烏金豬(WJ);快生長、體型正常的大約克豬(LW)作為試驗對象,利用轉(zhuǎn)錄組RNA-seq和蛋白質(zhì)組iTRAQ技術(shù)對豬胚胎60日齡的背最長肌進行分析,鑒定與豬生長性狀相關(guān)的差異基因,并利用PRM技術(shù)對差異基因進行蛋白水平的驗證,旨在通過胚胎時期肌肉生長發(fā)育的差異闡明對出生后豬生長性狀的影響。三個品種豬胚胎60日齡的背最長肌組織RNA-seq得到38.7-50.1G clean reads,檢測到19626個基因在背最長肌組織表達,其中三個品種共表達陽性基因14660個,通過計算所有基因的FPKM值后,以|log2FoldChange|1和90.01作為差異表達基因(DEGs)的篩選標準,我們分別在TP vs.LW 組得到 5115 個 DEGs,TP vs.WJ 組 4274 個 DEGs,WJ vs.LW 組 260 個 DEGs,結(jié)合藏豬、烏金豬、大約克豬出生后的生長特點,確定了 TP vs.LW組和TP vs.WJ組交集的3626個基因作為本研究中重點關(guān)注基因,并初步篩選出15個可能與豬肌肉生長發(fā)育相關(guān)的基因。利用iTRAQ蛋白測序技術(shù)進行蛋白表達水平差異蛋白的篩選,共得到12230個肽段及對應的2474個蛋白。以FoldChange1.5或0.67作為差異表達蛋白(DEPs)的篩選標準,在TP vs.LW組得到 939 個 DEPs,TP vs.WJ 組 981 個 DEPs,WJ vs.LW 組 119 個 DEPs。其中 TP 與 LW 和WJ共同的735個DEPs作為本研究中重點關(guān)注的蛋白,初步篩選了 SYNE2、TRMT10C、GTPBP8、EDEM3、ATP8、PHF1、RIF1、APM1、PDLIM3、GPR84、NR0B1、GPT2、TPTE2 等 12 個基因可能與豬肌肉生長發(fā)育性狀有關(guān),這些基因主要參與細胞的增殖與分化、Ca2+代謝通路、肌纖維和肌原纖維的形成、氨基酸代謝、氧化磷酸化等通路。通過RNA-seq和iTRAQ數(shù)據(jù)的聯(lián)合分析,我們發(fā)現(xiàn)RNA-seq分析中得到的差異基因與iTRAQ分析中得到的差異蛋白之間呈正相關(guān)(Person相關(guān)系數(shù)0.7-0.71)。mRNA水平和蛋白質(zhì)水平表達量趨勢一致的基因有209個。這些基因主要與鈣離子通路、細胞增殖和凋亡、肌原纖維的發(fā)育、肌肉細胞分化、肌內(nèi)膠原含量、成纖維細胞生長的調(diào)節(jié)有關(guān)。其中LDB3、PDLIM5、NR1D2、ROCK2、NDRG2、POSTN、FSCN1、COL3A1、CRYAB、ANKRD2、MAPK12、LOC100627626和LOC100523824等13個基因,可以作為產(chǎn)前成肌細胞增殖和肌肉形成有關(guān)的重要候選基因。采用通過PRM技術(shù)驗證的24個差異基因中,其表達趨勢完全與RNA-seq、iTRAQ—致,進一步證明了數(shù)據(jù)的可靠性、可信性。本研究整合RNA-seq和iTRAQ數(shù)據(jù)進行分析,并利用PRM技術(shù)進行驗證,篩選出了影響豬肌肉生長發(fā)育的差異基因和差異蛋白。MYL1、DOLIM5、CKM、AC4DM、TCAP、PGAM2、LYGM、PGM1、MTFP和CKMT2等10個差異表達基因?qū)ωi骨骼肌的生長發(fā)育以及出生后的產(chǎn)肉性能具有抑制作用。MYL4、COL3A 和STMN1等3個差異表達基因和差異表達蛋白能促進骨骼肌的快速生長和發(fā)育。為下一步深入理解肌肉發(fā)育的分子機理提供新的切入點,為出生后豬生長性狀候選基因的篩選與鑒定提供了寶貴數(shù)據(jù)。
[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.

【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S828

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本文編號：1588414