基于转录组测序挖掘广西麻鸡生长发育相关基因

doi:10.16431/j.cnki.1671-7236.2022.10.022

摘要/Abstract

摘要： 【目的】通过对1和60日龄广西麻鸡腿肌进行转录组测序(RNA-Seq),筛选出广西麻鸡生长发育关键基因。【方法】选择1和60日龄健康母鸡各3只,分别采集腿肌组织,利用Illumina Novaseq^TM 6000平台进行mRNA转录组测序,利用DESeq2软件进行差异表达基因的分析,并对差异表达基因进行GO功能和KEGG通路富集分析,进一步筛选出生长发育相关基因,并用实时荧光定量PCR验证转录组测序结果。【结果】转录组测序结果显示,6个样本分别获得34 296 198~41 647 642条clean reads。与1日龄腿肌相比,60日龄腿肌共获得2 304个差异表达基因,其中998个基因上调,1 306个基因下调。GO功能富集分析发现,共获得富集条目572条,其中生物过程381条,细胞组分70条,分子功能121条。KEGG通路富集分析发现,共获得34个显著富集的信号通路,其中心肌收缩、MAPK信号通路、紧密连接、肌动蛋白细胞骨架信号通路、心肌细胞中的肾上腺素能信号传导等与生长发育相关。通过GO功能和KEGG通路富集分析获得5个生长发育相关基因,分别为肌球蛋白重链10(MYH10)、肌球蛋白链15(MYH15)、成纤维细胞生长因子10(FGF10)、成纤维细胞生长因子16(FGF16)、肌肉生长抑制素(GDF8)基因,其中MYH10、MYH15、FGF10为上调差异表达基因,FGF16和GDF8为下调差异表达基因。所选差异表达基因实时荧光定量PCR结果与转录组测序的基因表达水平一致,说明转录组测序结果可靠。【结论】本试验通过对广西麻鸡1和60日龄2个不同生长阶段进行转录组测序分析,获得MYH10、MYH15、FGF10、FGF16、GDF8共5个与生长发育相关的关键基因,为后续进一步探讨广西麻鸡生长发育的分子调控机制提供参考。

关键词: 广西麻鸡; 腿肌; 转录组测序; 差异表达基因

Abstract: 【Objective】 The purpose of this study was to screen out the key genes for growth and development of Guangxi Ma chickens through transcriptome sequencing (RNA-Seq) of leg muscles at 1- and 60-day-old.【Method】 In this study,three healthy 1- and 60-day-old hens were selected,and leg muscle tissues were collected respectively.Illumina Novaseq^TM 6000 platform was used for mRNA transcriptome sequencing.DESeq2 software was used to analyze the differentially expressed genes,and GO function and KEGG pathway enrichment analysis of the differentially expressed genes were performed.The genes related to birth growth and development were further screened,and the transcriptome sequencing results were verified by Real-time quantitative PCR.【Result】 Transcriptome sequencing reaults showed that 34 296 198-41 647 642 clean reads were obtained from 6 samples.Compared with 1-day-old leg muscle,a total of 2 304 differentially expressed genes were obtained at 60-day-old leg muscle,of which 998 genes were up-regulated and 1 306 genes were down-regulated.Go function enrichment analysis found that 572 enrichment items were obtained,including 381 biological processes,70 cell components and 121 molecular functions.The enrichment analysis of KEGG pathway found that a total of 34 signal pathways were obtained.Myocardial contraction,MAPK signal pathway,tight junction,actin cytoskeleton signal pathway and adrenergic signal transduction in cardiomyocytes were related to growth and development.Through GO function and KEGG pathway enrichment analysis,5 growth and development related genes were obtained,which were myosin heavy chain 10 (MYH10),myosin heavy chain 15 (MYH15),fibroblast growth factor 10 (FGF10),fibroblast growth factor 16 (FGF16) and myostatin (GDF8) genes, respectively.MYH10,MYH15 and FGF10 were up-regulated differentially expressed genes,FGF16 and GDF8 were down-regulated differentially expressed genes.The results of Real-time quantitative PCR of the selected differentially expressed genes were consistent with the gene expression levels of transcriptome sequencing,indicating that the transcriptome sequencing results were reliable.【Conclusion】 By analyzing the transcriptome data of 1- and 60 day-old Guangxi Ma chickens,5 genes related to growth and development were obtained,including MYH10,MYH15,FGF10,FGF16 and GDF8,which provided a reference for further exploring the regulation mechanism of growth and development of Guangxi Ma chickens.

Key words: Guangxi Ma chickens; leg muscle; RNA-Seq; differentially expressed genes

中图分类号:

S831.2

陈婷, 卢晟盛, 杨丽丽, 李添宝, 张敏, 江明生. 基于转录组测序挖掘广西麻鸡生长发育相关基因[J]. 中国畜牧兽医, 2022, 49(10): 3901-3908.

CHEN Ting, LU Shengsheng, YANG Lili, LI Tianbao, ZHANG Min, JIANG Mingsheng. Based on Transcriptome Sequencing to Explore the Relationship Between Growth and Development Genes of Guangxi Ma Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3901-3908.

参考文献

[1] 杜文娅,李淑霞,邹乐勤,等.广西麻鸡个体产蛋规律研究[J].广西畜牧兽医,2020,36(1):9-11. DU W Y,LI S X,ZOU L Q,et al.Study on individual laying law of Guangxi Ma chicken[J]. Guangxi Journal of Animal Husbandry & Veterinary Medicine,2020,36(1):9-11.(in Chinese)
[2] 杜文娅,邹乐勤,杨祝良,等.广西5个品种地方鸡肌肉氨基酸分析和营养评价[J].中国畜牧杂志,2021,57(9):109-113. DU W Y,ZOU L Q,YANG Z L,et al.Amino acid analysis and nutritional evaluation of muscle of five local chicken breeds in Guangxi[J].Chinese Journal of Animal Science,2021,57(9):109-113.(in Chinese)
[3] 董亚宁,王巧巧,李倩倩,等.肌纤维发育相关基因在不同地方鸡种早期生长发育中的表达分析[J].畜牧与兽医,2021,53(10):14-21. DONG Y N,WANG Q Q,LI Q Q,et al.Expression analysis of muscle fiber development related genes in early growth and development of chicken breeds in different places[J].Animal Husbandry & Veterinary Medicine,2021,53(10):14-21.(in Chinese)
[4] 许家明,张正芬,华国洪,等.广西麻鸡屠宰性能和肉品质研究[J].中国畜牧兽医,2017,44(11):3201-3207. XU J M,ZHANG Z F,HUA G H,et al.Study on slaughter performance and meat quality of Guangxi Ma chicken[J].China Animal Husbandry & Veterinary Medicine, 2017,44(11):3201-3207.(in Chinese)
[5] 张秀梅,康丽,李显耀,等.汶上芦花鸡和济宁百日鸡母鸡的肌肉营养成分及风味的比较[J].中国畜牧兽医,2015,42(1):109-117. ZHANG X M,KANG L,LI X Y,et al.Comparison of muscle nutrients and flavor between Wenshang Luhua chicken and Jining Bairi chicken[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):109-117.(in Chinese)
[6] 虎红红,母童,马正旭,等.基于RNA-Seq技术对静原鸡不同部位肉质相关差异基因的筛选[J].基因组学与应用生物学,2021,40(Z1):2038-2046. HU H H,MU T,MA Z X,et al.Screening of meat quality related genes in different parts of Jingyuan chicken based on RNA-Seq technology[J]. Genomics and Applied Biology,2021,40(Z1):2038-2046.(in Chinese)
[7] XING S,LIU R,ZHAO G,et al.RNA-Seq analysis reveals Hub genes involved in chicken intramuscular fat and abdominal fat deposition during development[J].Frontiers in Genetics,2020,11:1009.
[8] LIU L,LIU X,CUI H,et al.Transcriptional insights into key genes and pathways controlling muscle lipid metabolism in broiler chickens[J]. BMC Genomics,2019,20(1):863.
[9] XINF K,CHEN Y,WANG L,et al.Epididymal mRNA and miRNA transcriptome analyses reveal important genes and miRNAs related to sperm motility in roosters[J]. Poultry Science,2021,101(1):101558.
[10] 张剑,曹婧,耿爱莲,等.不同日龄北京油鸡腿肌中长链脂肪酸变化规律研究[J].中国家禽,2021,43(7):10-17. ZHANG J,CAO J,GENG A L,et al.Study on the changes of long chain fatty acids in leg muscle of Beijing Oil chicken at different ages[J]. China Poultry,2021,43(7):10-17.(in Chinese)
[11] 任宇,丁玥竹,农燕鸿,等.云南独龙鸡和白来航鸡卵巢组织转录组水平的比较分析[J].畜牧兽医学报,2022,53(1):43-52. REN Y,DING Y Z,NONG Y H,et al.Comparative analysis of transcriptome levels in ovarian tissues of Yunnan Dulong chicken and bailaihang chicken[J].Chinese Journal of Animal and Veterinary Sciences, 2022,53(1):43-52.(in Chinese)
[12] 李春晖,何程实,吴建鑫,等.调控鸡胚肝脏组织抗氧化功能差异基因的筛选[J].合肥工业大学学报,2021,44(10):1430-1436. LI C H,HE C S,WU J X,et al Screening of genes regulating the antioxidant function of chicken embryo liver[J]. Journal of Hefei University of Technology, 2021,44(10):1430-1436.(in Chinese)
[13] YANG S,WANG Y,WANG L,et al.RNA-Seq reveals differentially expressed genes affecting polyunsaturated fatty acids percentage in the Huangshan Black chicken population[J].PLoS One,2018,13(4):e0195132.
[14] HE M,WU P,CHEN F,et al.Transcriptome analysis of leg muscles in fast and slow growth Bian chickens[J].Animal Biotechnology,2020,31(4):295-305.
[15] YIN X,WU Y,ZHANG S,el al.Transcriptomic profile of leg muscle during early growth and development in Haiyang Yellow chicken[J].Archives Animal Breeding,2021,64(2):405-416.
[16] OTANIO T,FURUSE M.Tight junction structure and function revisited[J]. Trends in Cell Biology,2020,30(10):805-817.
[17] WANG J,GARERI C,ROCKMAN H A.G-protein-coupled receptors in heart disease[J].Circulation Research,2018,123(6):716-735.
[18] ZHANG Z,DU H,YANG C,et al.Comparative transcriptome analysis reveals regulators mediating breast muscle growth and development in three chicken breeds[J]. Animal Biotechnology,2019 30(3):233-241.
[19] ROSSI A C,MAMMUCARI C,ARGENTINI C,et al.Two novel/ancient myosins in mammalian skeletal muscles:MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles[J].The Journal of Physiology, 2010,588(2):353-364.
[20] RIVETTI S,CHEN C,BELLUSCI S.FGF10/FGFR2b signaling in mammary gland fevelopment,homeostasis,and cancer[J].Frontiers in Cell and Developmental Biology,2020,8:415.
[21] ALVAREZ Y,ALONSO M T,VENDRELL V,et al.Requirements for FGF3 and FGF10 during inner ear formation[J].Development,2003,130(25):6329-6338.
[22] FANTETTI K N,FEKETE D M.Members of the BMP,Shh,and FGF morphogen families promote chicken statoacoustic ganglion neurite outgrowth and neuron survival in vitro[J].Developmental Neurobiology,2021 72(9):1213-1228.
[23] AHSAN M,LI X,LUNDBERG A E,et al.Identification of candidate genes and mutations in QTL regions for chicken growth using bioinformatic analysis of NGS and SNP-chip data[J]. Frontiers in Genetics,2016,4:226.
[24] HOTTA Y,SASAKI S,KONISHI M,et al.FGF16 is required for cardiomyocyte proliferation in the mouse embryonic heart[J].Developmental Dynamics,2008,237(10):2947-2954.
[25] MCFARLAND D C,VELLEMAN S G,PESALL J E,el al.The role of myostatin in chicken (Gallus domesticus) myogenic satellite cell proliferation and differentiation[J].General and Comparative Endocrinology,2007,151(3):351-357.
[26] KUBOTA K,SATO F,ARAMAKI S,et al.Ubiquitous expression of myostatin in chicken embryonic tissues:Its high expression in testis and ovary[J]. Comparative Biochemistry and Physiology, 2007,148(3):550-555.
[27] SUN Y,LIU W Z,LIU T,et al.Signaling pathway of MAPK/ERK in cell proliferation,differentiation,migration,senescence and apoptosis[J].Journal of Receptor and Signal Transduction Research,2015,35(6):600-604.
[28] HARTSOCK A,NELSON W J.Adherens and tight junctions:Structure,function and connections to the actin cytoskeleton[J].Biochimica et Biophysica Acta (BBA) - Biomembranes,2008,1778(3):660-669.
[29] GONZLEZ-MARISCAL L,LECHUGA S,GARAY E.Role of tight junctions in cell proliferation and cancer[J].Progress in Histochemistry and Cytochemistry,2007,42(1):1-57.