广西麻鸡卵巢组织中差异lncRNA筛选及ceRNA网络构建

doi:10.16431/j.cnki.1671-7236.2024.07.002

摘要/Abstract

摘要： 【目的】筛选广西麻鸡卵巢中差异表达的长链非编码RNA(long non-coding RNA,lncRNA)、信使RNA(messenger RNA,mRNA)以及微RNA(microRNAs,miRNA),并进一步筛选出与鸡产蛋性状相关的lncRNA,为后期研究lncRNA对鸡产蛋性状的调控提供理论支持。【方法】根据广西麻鸡从开产到41周龄的产蛋情况筛选出高产和低产的广西麻鸡各3只,并以其卵巢组织为材料,进行转录组测序,使用edgeR和DEseq2软件从测序得到的数据中筛选出差异表达的lncRNA、mRNA以及miRNA,利用miRanda软件和lncRNA的顺式作用分别预测miRNA和lncRNA的靶基因,对差异lncRNA的靶基因进行GO功能和KEGG通路富集分析后,利用Cytoscape(v 3.8.2)软件构建竞争性内源RNA(ceRNA)网络图。利用实时荧光定量PCR验证转录组测序结果。【结果】本研究共获得广西麻鸡卵巢组织中438个差异表达的lncRNAs、18个差异表达的miRNAs以及301个差异表达的mRNAs。对上述差异表达的lncRNA的靶基因进行GO功能和KEGG通路富集分析后发现,其显著富集于激素分泌调节、中胚层形成、中胚层发育以及中胚层形态发生等与产蛋有关的生物过程,以及MARK、甘露糖O-聚糖的生物合成、卵母细胞减数分裂、孕激素介导的卵母细胞成熟以及促性腺激素释放激素等信号通路。通过GO功能和KEGG通路富集分析获得6个与产蛋性状相关的lncRNAs,分别是TCONS_00091814、TCONS_00051866、XR_006939520.1、XR_005839807.1、TCONS_00088588、TCONS_00037324。根据测序结果成功构建出包含9个miRNAs、60个lncRNAs和3个mRNAs的ceRNA网络图,且网络中所有的基因均在低、高产组中差异表达。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】通过RNA测序技术从广西麻鸡卵巢中筛选到了一系列差异表达的lncRNA,并筛选出了6个可能与产蛋性状相关联的lncRNAs,为进一步提高鸡的产蛋性能提供了理论支持。

关键词: 广西麻鸡; lncRNA; 差异表达; 产蛋性状; ceRNA

Abstract: 【Objective】 This study was aimed to screen differentially expressed long non-coding RNA (lncRNA),messenger RNA (mRNA) and microRNA (miRNA) of ovary in Guangxi Ma chicken,and further select lncRNA related to egg production traits,providing theoretical support for the study of lncRNA regulation of egg production traits in chickens.【Method】 Based on the egg production of Guangxi Ma chickens from start of production to 41 weeks of age,three high-yielding and three low yielding Guangxi Ma chickens were selected,and their ovarian tissues were used as materials.RNA sequencing technology was used to sequence them.Differentially expressed lncRNA,mRNA,and miRNA were screened from the sequencing data using edgER and DESeq2 software.the target genes of miRNA and lncRNA were predicted by miRanda software and cis-acting of lncRNA,respectively.GO function and KEGG pathway enrichment analysis were performed on the target genes of differentially expressed lncRNA,and a ceRNA network diagram was constructed using Cytoscape(v 3.8.2) software The transcriptome sequencing results were verified by Real-time quantitative PCR.【Result】 RNA sequencing technology was used to obtain 438 differentially expressed lncRNAs,18 differentially expressed miRNAs,and 301 differentially expressed mRNAs in the ovaries of Guangxi Ma chicken.GO function and KEGG pathway enrichment analysis of the target genes of the differentially expressed lncRNAs showed significant enrichment in bioprocesses related to hormone secretion regulation,mesoderm formation,mesoderm development,and mesoderm morphogenesis related to egg production,as well as signaling pathways such as MARK,biosynthesis of glycosaminoglycans,oocyte meiosis,progesterone-mediated oocyte maturation,and gonadotropin-releasing hormone.Through GO function and KEGG pathway enrichment analysis,six lncRNAs related to egg production traits were obtained,including TCONS_00091814,TCONS_00051866,XR_006939520.1,XR_005839807.1,TCONS_00088588 and TCONS_00037324.A ceRNA network map containing 9 miRNAs with 60 lncRNAs and 3 mRNAs was successfully constructed based on the sequencing results,and all the genes in the network were differentially expressed in the low and high yield groups.Real-time quantitative PCR results of the selected differentially expressed genes were consistent with the gene expression levels from transcriptome sequencing,indicating that the transcriptome sequencing results were reliable.【Conclusion】 A series of differentially expressed lncRNAs were screened from ovary in Guangxi Ma chicken using RNA sequencing technology,and six lncRNAs possibly associated with egg production traits were identified,providing theoretical support for further enhancing egg production performance in chickens.

Key words: Guangxi Ma chicken; lncRNA; differential expression; egg production traits; ceRNA

中图分类号:

S831.2

张珂, 张敏, 李添宝, 张杰, 卢晟盛, 江明生. 广西麻鸡卵巢组织中差异lncRNA筛选及ceRNA网络构建[J]. 中国畜牧兽医, 2024, 51(7): 2739-2750.

ZHANG Ke, ZHANG Min, LI Tianbao, ZHANG Jie, LU Shengsheng, JIANG Mingsheng. Screening of Differential lncRNAs and Construction of ceRNA Network in Ovary Tissue of Guangxi Ma Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2739-2750.

参考文献

[1] 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 Partridge chickens[J].China Animal Husbandry&Veterinary Medicine,2017,44(11):3201-3207.(in Chinese)
[2] GUTTMAN M,RINN J.Modular regulatory principles of large non-coding RNAs[J].Nature,2012,482(7385):339-346.
[3] MILITELLO G,RABIUL M H,PONOMAREVA Y,et al.A novel long non-coding RNA myolinc regulates myogenesis through TDP-43 and Filip1[J]. Journal of Molecular Cell Biology,2018,10(2):102-117.
[4] ZHANG B B,LI M X,WANG H N,et al.An integrative analysis of lncRNAs and mRNAs highlights the potential roles of lncRNAs in the process of follicle selection in Taihang chickens[J].Theriogenology,2023,195:122-130.
[5] LI Q,LI J,LI C,et al.Transcriptome identification and characterization of long non-coding RNAs in the ovary of hens at four stages[J].Animal Biotechnology,2023,34(4):1342-1353.
[6] WEI Q,XUE H,SUN C,et al.Gga-miR-146b-3p promotes apoptosis and attenuate autophagy by targeting AKT1 in chicken granulosa cells[J].Theriogenology,2022,190:52-64.
[7] WEI Q,LI J,HE H,et al.miR-23b-3p inhibits chicken granulosa cell proliferation and steroid hormone synthesis via targeting GDF9[J].Theriogenology,2022,177:84-93.
[8] CHEN Q,DUAN J,WU H,et al.Expression dynamics of gonadotropin-releasing hormone-I and its mutual regulation with luteinizing hormone in chicken ovary and follicles[J].General and Comparative Endocrinology,2019,270:96-102.
[9] HU S,DUGGAVATHI R,ZADWORNY D.Regulatory mechanisms underlying the expression of prolactin receptor in chicken granulosa cells[J].PLoS One,2017,12(1):e0170409.
[10] ZHOU S,LI T,ZHANG M,et al.Epigenetic modification cooperates with Zeb1 transcription factor to regulate Bmp4 to promote chicken PGCs formation[J].Gene,2021,794:145760.
[11] 刘凌斌.产蛋与就巢期蛋鸡卵巢转录组分析及发育相关候选基因筛选[D].成都:四川农业大学,2018. LIU L B.Transcriptome analysis of layers ovaries between egg-laying and broody periods and identifition for candidate genes associated with ovarian development[D].Chengdu:Sichuan Agricultural University,2018.(in Chinese)
[12] JIANG J,CHEN C,CHENG S,et al.Long noncoding RNA lncPGCR mediated by TCF7L2 regulates primordial germ cell formation in chickens[J].Animals (Basel),2021,11(2):292.
[13] LI G,YANG X,LI J,et al.Genome-wide analysis of lncRNA and mRNA expression in the uterus of laying hens[J].Genes (Basel),2023,14(3):639.
[14] 刘广政,丁颖,邹艺琛,等.lncRNA PGCT8正向调控鸡原始生殖细胞形成[J].中国家禽,2023,45(1):16-22. LIU G Z,DING Y,ZOU Y C,et al.lncRNA PGCT8 positively regulates the formation of chicken primordial germ cell[J].China Poultry,2023,45(1):16-22.(in Chinese)
[15] 陈婷,卢晟盛,杨丽丽,等.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 T,LU S S,YANG L L,et al.Based on transcriptome sequencing to explore the relationship between growth and development genes of Guangxi Ma chickens[J].China Animal Husbandry&Veterinary Medicine,2022,49(10):3901-3908.(in Chinese)
[16] WU X,ZHOU X,XIONG L,et al.Transcriptome analysis reveals the potential role of long non-coding RNAs in mammary gland of yak during lactation and dry period[J].Frontiers in Cell and Developmental Biology,2020,8:579708.
[17] 张琼文.隆林山羊肌肉发育关键miRNA-mRNA筛选及miR-495-3p功能研究[D].南宁:广西大学,2021. ZHANG Q W.Screening of key miRNA-mRNA for muscle development in Longlin goat and study on the function of miR-495-3p[D].Nanning:Guangxi University,2021.(in Chinese)
[18] 陈秋月.GnRH1基因在鸡卵巢卵泡中的表达及调控作用分析[D].泰安:山东农业大学,2017. CHEN Q Y.The analysis of expression and regulation of GnRH1 gene in chicken ovarian follicles[D].Tai'an:Shangdong Agricultural University,2017.(in Chinese)
[19] SHIMASAKI S,ZACHOW R J,LI D,et al.A functional bone morphogenetic protein system in the ovary[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(13):7282-7287.
[20] 李婷婷.Bmp4基因对鸡PGCs形成及分化的作用研究[D].扬州:扬州大学,2020. LI T T.Study about the effect of Bmp4 gene on the formation and[D].Yangzhou:Yangzhou University,2020.(in Chinese)
[21] LIU S,ZHANG E,YANG M,et al.Overexpression of Wnt11 promotes chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in synergism with TGF-β[J].Molecular and Cellular Biochemistry,2014,390(1-2):123-131.
[22] 赵静.BPA通过干扰Wnt11介导的非经典Wnt信号通路影响青春期小鼠卵巢发育[D].阜阳:阜阳师范大学,2021. ZHAO J.BPA affects ovarian development through interfering with non-canonical Wnt signalingpathway mediated by Wnt1l in adolescent mice[D].Fuyang:Fuyang Normal University,2021.(in Chinese)
[23] 周祖阳.基于ceRNA研究WNT11基因对山羊卵泡颗粒细胞增殖的影响[D].邯郸:河北工程大学,2022. ZHOU Z Y.The effect of WNT11 on the proliferation of goat granulosa cells based on ceRNA[D].Handan:Hebei University of Engineering,2022.(in Chinese)
[24] 陈宇,白俊艳,王亮,等.鹌鹑GNAS基因的多态性与产蛋性能和蛋品质的关联分析[J].黑龙江畜牧兽医,2021,9:139-142. CHEN Y,BAI J Y,WANG L,et al.Association analysis of GNAS gene polymorphism with laying performance and egg quality in quails[J].Heilongjiang Animal Science and Veterinary Medicine,2021,9:139-142.(in Chinese)
[25] SIKORA K M,MAGEE D A,BERKOWICZ E W,et al.DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits[J].BMC Genetics,2011,12:4.
[26] ADHIKARI D,ZHENG W,SHEN Y,et al.Cdk1,but not Cdk2,is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes[J].Human Molecular Genetics,2012,21(11):2476-2484.
[27] FIRMANI L D,ULIASZ T F,MEHLMANN L M.The switch from cAMP-independent to cAMP-dependent arrest of meiotic prophase is associated with coordinated GPR3 and CDK1 expression in mouse oocytes[J].Developmental Biology,2018,434(1):196-205.
[28] KWONG W Y,ADAMIAK S J,GWYNN A,et al.Endogenous folates and single-carbon metabolism in the ovarian follicle,oocyte and pre-implantation embryo[J].Reproduction,2010,139(4):705-715.
[29] 范雪娇.四氢生物蝶呤对小鼠成纤维细胞糖代谢的作用机制[D].泉州:华侨大学,2014. FAN X J.Regulation of glucose metabolism by tetrahydrobiopterin in murine fibroblasts[D].Quanzhou:Huaqiao University,2014.(in Chinese)
[30] THÖNY B,AUERBACH G,BLAU N.Tetrahydrobiopterin biosynthesis,regeneration and functions[J].Biochemical Journal,2000,347(1):1-16.