基于RNA-Seq技术筛选影响猪肌纤维性状的候选基因
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摘要
旨在探究影响肌纤维性状的候选基因和信号通路。本研究以马身猪和大白猪为研究对象,采用HE染色法分析6月龄马身猪和大白猪背最长肌肌纤维直径和密度,利用RNA-Seq分析马身猪和大白猪背最长肌组织中基因表达情况,并对差异表达基因进行GO和KEGG富集分析,再通过qRT-PCR验证RNA-Seq结果的准确性。结果显示,马身猪背最长肌肌纤维直径极显著低于大白猪(P<0.01),而肌纤维密度极显著高于大白猪(P<0.01)。转录组测序结果显示,在6月龄马身猪和大白猪背最长肌中,差异倍数在2倍以上的基因共105个,其中,马身猪相对于大白猪上调的基因有55个,下调的基因有50个。GO富集分析表明,差异表达基因主要富集在与线粒体相关的细胞组分和骨骼肌分化有关的生物学过程中;KEGG分析结果显示,这些差异表达基因主要参与到与氧化磷酸化有关的信号途径。qRT-PCR和RNA-Seq对6个DEGs表达情况的检测结果表明它们的表达趋势相同,说明RNA-Seq结果准确可靠。结合差异基因表达丰度、GO和KEGG富集分析结果,本研究发现,MYL3、MYH3、MYH6基因通过影响肌纤维的组成而影响肌纤维特性,ND6基因通过参与线粒体氧化磷酸化过程影响肌纤维类型,MICU2基因通过维持线粒体Ca~(2+)浓度的稳态影响细胞功能,编码转录因子的EGR1和FOS基因通过调节细胞增殖、分化和凋亡等过程影响肌肉生长发育。本研究初步揭示了造成马身猪和大白猪肌纤维性状差异的主要原因,为猪肉品质的改善提供了相应的理论依据。
The aim of this study was to explore the candidate genes and signal pathways affecting muscle fiber characteristics. In this study, muscle fiber diameter and density of longissimus dorsi of Mashen and Large White pigs aged 6 months were measured by HE staining, and the gene expressions in longissimus dorsi of the 2 pig breeds at 6 months old was detected by using RNA-Seq. GO and KEGG enrichment analysis were used to study the function of differentially expressed genes(DEGs). The accuracy of RNA-Seq results was verified by qRT-PCR. The results showed that the fiber diameter of longissimus dorsi of Mashen pig was significantly shorter than that of Large White pig(P<0.01), and the fiber density of Mashen pig was greater than that of Large White pig(P<0.01). A total of 105 DEGs at least 2-fold change were identified, including 55 up-regulated genes and 50 down-regulated genes in longissimus dorsi of Mashen pig compared to Large White pig. GO enrichment analysis showed that the DEGs were mainly enriched in the cellular component related to mitochondrion and skeletal muscle differentiation biological processes, and KEGG analysis showed that these DEGs were mainly involved in the signaling pathways related to oxidative phosphorylation. The expression trends of 6 selected DEGs detected by qRT-PCR and RNA-Seq were the same, which indicated that the results of RNA-Seq were accurate and reliable. Based on the results of expression abundance, GO and KEGG analysis of DEGs, the genes of MYL3, MYH3 and MYH6 were found to affect muscle fiber characteristic by influencing the composition of muscle fiber. ND6 gene was related to muscle fiber type by involving in oxidative phosphorylation pathway in mitochondria. MICU2 gene was involved in keeping homeostasis of Ca~(2+) concentration in mitochondria and further affected cellular function. The genes of EGR1 and FOS coding transcription factors might affect muscle growth and development by regulating cell proliferation, differentiation and apoptosis. This study preliminarily explored the reason causing significantly different muscle fiber characteristics between Mashen and Large White pigs, and provided a theoretical basis for improving meat quality in pig production.
The aim of this study was to explore the candidate genes and signal pathways affecting muscle fiber characteristics. In this study, muscle fiber diameter and density of longissimus dorsi of Mashen and Large White pigs aged 6 months were measured by HE staining, and the gene expressions in longissimus dorsi of the 2 pig breeds at 6 months old was detected by using RNA-Seq. GO and KEGG enrichment analysis were used to study the function of differentially expressed genes(DEGs). The accuracy of RNA-Seq results was verified by qRT-PCR. The results showed that the fiber diameter of longissimus dorsi of Mashen pig was significantly shorter than that of Large White pig(P<0.01), and the fiber density of Mashen pig was greater than that of Large White pig(P<0.01). A total of 105 DEGs at least 2-fold change were identified, including 55 up-regulated genes and 50 down-regulated genes in longissimus dorsi of Mashen pig compared to Large White pig. GO enrichment analysis showed that the DEGs were mainly enriched in the cellular component related to mitochondrion and skeletal muscle differentiation biological processes, and KEGG analysis showed that these DEGs were mainly involved in the signaling pathways related to oxidative phosphorylation. The expression trends of 6 selected DEGs detected by qRT-PCR and RNA-Seq were the same, which indicated that the results of RNA-Seq were accurate and reliable. Based on the results of expression abundance, GO and KEGG analysis of DEGs, the genes of MYL3, MYH3 and MYH6 were found to affect muscle fiber characteristic by influencing the composition of muscle fiber. ND6 gene was related to muscle fiber type by involving in oxidative phosphorylation pathway in mitochondria. MICU2 gene was involved in keeping homeostasis of Ca~(2+) concentration in mitochondria and further affected cellular function. The genes of EGR1 and FOS coding transcription factors might affect muscle growth and development by regulating cell proliferation, differentiation and apoptosis. This study preliminarily explored the reason causing significantly different muscle fiber characteristics between Mashen and Large White pigs, and provided a theoretical basis for improving meat quality in pig production.
引文
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[22] YUAN Y,WANG W X,LI H Z,et al.Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma[J].BMC Cancer,2015,15:346.
[23] ZHANG C L,WANG J M,WANG G Z,et al.Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes[J].Gene,2016,577(2):209-214.
[24] GRABAREK Z.Structural basis for diversity of the EF-hand calcium-binding proteins[J].J Mol Biol,2006,359(3):509-525.
[25] KONING M,WERKER P M N,VAN LUYN M J,et al.Hypoxia promotes proliferation of human myogenic satellite cells:a potential benefactor in tissue engineering of skeletal muscle[J].Tissue Eng Part A,2011,17(13-14):1747-1758.
[26] TAJSHARGHI H,OLDFORS A.Myosinopathies:pathology and mechanisms[J].Acta Neuropathol,2013,125(1):3-18.
[27] 张莺莺,昝林森,王洪宝.利用基因芯片技术筛选秦川牛公牛与阉牛肌肉组织差异表达基因[J].遗传,2010,32(11):1166-1174.ZHANG Y Y,ZAN L S,WANG H B.Genome array on differentially expressed genes of muscle tissue in intact male and castrated Qinchuan cattle[J].Hereditas (Beijing),2010,32(11):1166-1174.(in Chinese)
[28] XIONG X W,LIU X X,ZHOU L S,et al.Genome-wide association analysis reveals genetic loci and candidate genes for meat quality traits in Chinese Laiwu pigs[J].Mamm Genome,2015,26(3-4):181-190.
[29] 胡帅帅,王文洲,闫晓荣,等.新西兰白兔中Myh6基因的表达及与肌纤维性状的相关性[J].华南农业大学学报,2017,38(2):12-17.HU S S,WANG W Z,YAN X R,et al.Myh6 gene expression and its correlation with muscle fiber traits of New Zealand white rabbits[J].Journal of South China Agricultural University,2017,38(2):12-17.(in Chinese)
[30] KIRICHOK Y,KRAPIVINSKY G,CLAPHAM D E.The mitochondrial calcium uniporter is a highly selective ion channel[J].Nature,2004,427(6972):360-364.
[31] DE LA FUENTE S,MATESANZ-ISABEL J,FONTERIZ R I,et al.Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells[J].Biochem J,2014,458(1):33-40.
[32] PATRON M,CHECCHETTO V,RAFFAELLO A,et al.MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity[J].Mol Cell,2014,53(5):726-737.
[33] WAGNER E.Functions of AP1 (Fos/Jun) in bone development[J].Ann Rheum Dis,2002,61 (Suppl 2):ii40-ii42.
[34] SHAULIAN E,KARIN M.AP-1 as a regulator of cell life and death[J].Nat Cell Biol,2002,4(5):E131-E136.
[35] 崔亚凤,严云勤,李树峰,等.Egr1对小鼠成肌细胞C2C12分化的影响[J].中国细胞生物学学报,2018,40(1):25-32.CUI Y F,YAN Y Q,LI S F,et al.The effect of Egr1 on C2C12 differentiation[J].Chinese Journal of Cell Biology,2018,40(1):25-32.(in Chinese)
[36] ISHIKAWA H,SHOZU M,OKADA M,et al.Early growth response gene-1 plays a pivotal role in down-regulation of a cohort of genes in uterine leiomyoma[J].J Mol Endocrinol,2007,39(5):333-341.
[2] JOO S T,KIM G D,HWANG Y H,et al.Control of fresh meat quality through manipulation of muscle fiber characteristics[J].Meat Sci,2013,95(4):828-836.
[3] LEFAUCHEUR L,GERRARD D.Muscle fiber plasticity in farm mammals[J].J Anim Sci,2000,77(Suppl_E):1-19.
[4] MALTIN C A,WARKUP C C,MATTHEWS K R,et al.Pig muscle fibre characteristics as a source of variation in eating quality[J].Meat Sci,1997,47(3-4):237-248.
[5] OZAWA S,MITSUHASHI T,MITSUMOTO M,et al.The characteristics of muscle fiber types of longissimus thoracis muscle and their influences on the quantity and quality of meat from Japanese Black steers[J].Meat Sci,2000,54(1):65-70.
[6] CHOI Y M,NAM K W,CHOE J H,et al.Growth,carcass,fiber type,and meat quality characteristics in Large White pigs with different live weights[J].Livest Sci,2013,155(1):123-129.
[7] KARLSSON A H,KLONT R E,FERNANDEZ X.Skeletal muscle fibres as factors for pork quality[J].Livest Prod Sci,1999,60(2-3):255-269.
[8] YANG Y L,LIANG G M,NIU G L,et al.Comparative analysis of DNA methylome and transcriptome of skeletal muscle in lean-,obese-,and mini-type pigs[J].Sci Rep,2017,7:39883.
[9] RYU Y C,KIM B C.The relationship between muscle fiber characteristics,postmortem metabolic rate,and meat quality of pig longissimus dorsi muscle[J].Meat Sci,2005,71(2):351-357.
[10] XU Y J,QIAN H,FENG X T,et al.Differential proteome and transcriptome analysis of porcine skeletal muscle during development[J].J Proteomics,2012,75(7):2093-2108.
[11] LI Y,XU Z Y,LI H Y,et al.Differential transcriptional analysis between red and white skeletal muscle of Chinese Meishan pigs[J].Int J Biol Sci,2010,6(4):350-360.
[12] AYUSO M,FERNáNDEZ ,et al.Comparative analysis of muscle transcriptome between pig genotypes identifies genes and regulatory mechanisms associated to growth,fatness and metabolism[J].PLoS One,2015,10(12):e0145162.
[13] ZHAO Y Y,GAO P F,LI W,et al.Study on the developmental expression of Lbx1 gene in Longissimus dorsi of Mashen and Large White pigs[J].Ital J Anim Sci,2015,14(1):109-113.
[14] 贾静敏.马身猪和大白猪肌球蛋白重链(MyHC)的发育性变化及肌纤维组织学观察[D].太谷:山西农业大学,2012.JIA J M.Developmental changes of myosin heavy chain (MyHC) in horses and large white pigs and histological observation of muscle fibers[D].Taigu:Shanxi Agricultural University,2012.(in Chinese)
[15] GIL M,DELDAY M I,GISPERT M,et al.Relationships between biochemical characteristics and meat quality of Longissimus thoracis and Semimembranosus muscles in five porcine lines[J].Meat Sci,2008,80(3):927-933.
[16] CHOI Y M,KIM B C.Muscle fiber characteristics,myofibrillar protein isoforms,and meat quality[J].Livest Sci,2009,122(2-3):105-118.
[17] KIM J M,LEE S H,RYU Y C.Comparisons of meat quality and muscle fibre characteristics on multiple pig breeds and sexes using principal component analysis[J].Anim Prod Sci,2018,58(11):2091-2099.
[18] SCHIAFFINO S,REGGIANI C.Fiber types in mammalian skeletal muscles[J].Physiol Rev,2011,91(4):1447-1531.
[19] CHOI Y M,RYU Y C,KIM B C.Influence of myosin heavy- and light chain isoforms on early postmortem glycolytic rate and pork quality[J].Meat Sci,2007,76(2):281-288.
[20] 束婧婷,章明,宋迟,等.基于表达谱芯片挖掘清远麻鸡和科宝肉鸡比目鱼肌差异表达基因[J].畜牧兽医学报,2016,47(1):25-33.SHU J T,ZHANG M,SONG C,et al.Identification of differentially expressed genes for myofiber characteristics in soleus muscles between Qingyuan partridge chickens and Cobb broilers[J].Acta Veterinaria et Zootechnica Sinica,2016,47(1):25-33.(in Chinese)
[21] 孟宪然,杜琛,王静,等.基于RNA-Seq识别山羊肉品质候选基因[J].畜牧兽医学报,2015,46(8):1300-1307.MENG X R,DU C,WANG J,et al.RNA-Seq approach for identifying candidate genes of meat quality in goats[J].Acta Veterinaria et Zootechnica Sinica,2015,46(8):1300-1307.(in Chinese)
[22] YUAN Y,WANG W X,LI H Z,et al.Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma[J].BMC Cancer,2015,15:346.
[23] ZHANG C L,WANG J M,WANG G Z,et al.Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes[J].Gene,2016,577(2):209-214.
[24] GRABAREK Z.Structural basis for diversity of the EF-hand calcium-binding proteins[J].J Mol Biol,2006,359(3):509-525.
[25] KONING M,WERKER P M N,VAN LUYN M J,et al.Hypoxia promotes proliferation of human myogenic satellite cells:a potential benefactor in tissue engineering of skeletal muscle[J].Tissue Eng Part A,2011,17(13-14):1747-1758.
[26] TAJSHARGHI H,OLDFORS A.Myosinopathies:pathology and mechanisms[J].Acta Neuropathol,2013,125(1):3-18.
[27] 张莺莺,昝林森,王洪宝.利用基因芯片技术筛选秦川牛公牛与阉牛肌肉组织差异表达基因[J].遗传,2010,32(11):1166-1174.ZHANG Y Y,ZAN L S,WANG H B.Genome array on differentially expressed genes of muscle tissue in intact male and castrated Qinchuan cattle[J].Hereditas (Beijing),2010,32(11):1166-1174.(in Chinese)
[28] XIONG X W,LIU X X,ZHOU L S,et al.Genome-wide association analysis reveals genetic loci and candidate genes for meat quality traits in Chinese Laiwu pigs[J].Mamm Genome,2015,26(3-4):181-190.
[29] 胡帅帅,王文洲,闫晓荣,等.新西兰白兔中Myh6基因的表达及与肌纤维性状的相关性[J].华南农业大学学报,2017,38(2):12-17.HU S S,WANG W Z,YAN X R,et al.Myh6 gene expression and its correlation with muscle fiber traits of New Zealand white rabbits[J].Journal of South China Agricultural University,2017,38(2):12-17.(in Chinese)
[30] KIRICHOK Y,KRAPIVINSKY G,CLAPHAM D E.The mitochondrial calcium uniporter is a highly selective ion channel[J].Nature,2004,427(6972):360-364.
[31] DE LA FUENTE S,MATESANZ-ISABEL J,FONTERIZ R I,et al.Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells[J].Biochem J,2014,458(1):33-40.
[32] PATRON M,CHECCHETTO V,RAFFAELLO A,et al.MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity[J].Mol Cell,2014,53(5):726-737.
[33] WAGNER E.Functions of AP1 (Fos/Jun) in bone development[J].Ann Rheum Dis,2002,61 (Suppl 2):ii40-ii42.
[34] SHAULIAN E,KARIN M.AP-1 as a regulator of cell life and death[J].Nat Cell Biol,2002,4(5):E131-E136.
[35] 崔亚凤,严云勤,李树峰,等.Egr1对小鼠成肌细胞C2C12分化的影响[J].中国细胞生物学学报,2018,40(1):25-32.CUI Y F,YAN Y Q,LI S F,et al.The effect of Egr1 on C2C12 differentiation[J].Chinese Journal of Cell Biology,2018,40(1):25-32.(in Chinese)
[36] ISHIKAWA H,SHOZU M,OKADA M,et al.Early growth response gene-1 plays a pivotal role in down-regulation of a cohort of genes in uterine leiomyoma[J].J Mol Endocrinol,2007,39(5):333-341.
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