半番鸭与番鸭精巢组织差异表达转录组测序分析

doi:10.3864/j.issn.0578-1752.2017.18.016

摘要/Abstract

摘要： 【目的】研究半番鸭与番鸭精巢组织转录组差异表达基因，为进一步阐明半番鸭不育的遗传机制奠定理论基础。【方法】利用转录组测序方法对半番鸭和番鸭的精巢组织进行研究，筛选其差异表达基因，并对其功能进行注释和荧光定量PCR(quantitative real-time PCR, QRT-PCR)验证。【结果】测序共获得43.84Gb Clean Data，组装后共获得193 535条Unigene。DESeq分析发现3 597个基因在两个鸭品种间差异表达，其中上调基因1 194个和下调基因2 403个，包括与生殖功能相关的基因，如成纤维细胞生长因子（fibroblast growth factor, FGF）、蛋白激酶A（protein kinaseA, PKA）、丝裂原活化蛋白激酶7（mitogen-activated protein kinase 7-like, partial, BMK）、生长因子受体结合蛋白2（growth factor receptor-bound protein 2, GRB2）、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)和肿瘤坏死因子受体超家族成员6（tumor necrosis factor receptor superfamily member 6, FAS）等。GO (gene Ontology)分析发现382个差异基因获得功能注释，其中97个基因涉及发育繁殖生物学过程。KEGG (kyoto Encyclopedia of Genes and Genomes)通路分析表明差异表达基因共富集到50 条信号通路中，其中17个通路显著富集，包括丝裂原活化蛋白激酶信号转导通路 (mitogen-activated protein kinase signaling pathway, MAPK)、甘油酯代谢（glycerolipid metabolism）以及钙信号途径 (calcium signaling pathway)信号通路等，与生殖功能密切相关的有促性腺激素释放激素信号通路（gonadotropin releasing hormone (GnRH) signaling pathway）和MAPK信号转导通路。经QRT-PCR验证，差异基因表达变化模式与转录组测序结果一致，测序结果可靠。【结论】在转录组水平上筛选出半番鸭和番鸭精巢组织差异表达基因，揭示了GnRH和MAPK信号通路在鸭的生殖活动中发挥了重要作用，为进一步探索半番鸭生殖系统的分化机理提供可靠的参考依据。

关键词: 半番鸭, 番鸭, 精巢组织, 差异表达基因, 转录组

Abstract: 【Objective】 The purpose of this study is to analyze the transcriptome differential gene expression of mule duck and muscovy duck testis, results of the study will lay a theoretical foundation for the further elucidation of the mechanisms of genetic sterility of mule duck. 【Method】 Transcriptome sequencing of testis from mule duck and muscovy duck was performed using the Illumina HiSeq 2500 platform with 2 biological replicates per duck breed, and verified by quantitative real-time PCR (QRT-PCR). 【Result】 After removing sequencing adaptors and the low-quality reads, a total of 43.84 Gb clean reads were obtained, the Q30 base percentage at 91.36% and above, and clean reads were assembled into 193 535 Unigene. The r2 Differential expression analysis showed that 3 597 differentially expressed genes were found between two duck breeds, including 1 194 up regulated genes and 2 403 down-regulated genes. Several genes were related with reproductive function, such as fibroblast growth factor, FGF, protein kinase A (PKA), mitogen-activated protein kinase 7-like, partial (BMK),growth factor receptor-bound protein 2 (GRB2), c-Jun N-terminal kinase (JNK )and tumor necrosis factor receptor superfamily member 6(FAS),and so on. With Gene Ontology (GO) annotation, 382 differentially expressed genes were identified including 97 related annotation genes involving development and reproduction biological process. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differentially expression genes annotated to 50 metabolic pathway, and 17 pathways were enriched significantly, such as calcium signaling pathway, glycerolipid metabolism and MAPK signaling pathway, and gonadotropin releasing hormone (GnRH) signaling pathway and MAPK signaling pathway associated with testis physiology and reproduction activities. Verified by QRT-PCR, the pattern of differential gene expression was consistent with the results of transcriptome sequencing, which showed the sequencing results were reliable. 【Conclusion】 Differentially expressed genes of mule duck and muscovy duck testis were screened by transcriptional analysis, revealed that the GnRH and MAPK signaling pathway play an important role in duck reproductive activities, which provide reliable reference for exploring the differentiation mechanism of reproductive system in mule duck.

Key words: mule duck, muscovy duck, testis tissue, differentially expressed genes, transcriptome

李丽，缪中纬，辛清武，朱志明, 章琳俐，庄晓东，郑嫩珠. 半番鸭与番鸭精巢组织差异表达转录组测序分析[J]. 中国农业科学, 2017, 50(18): 3608-3619.

LI Li, MIAO ZhongWei, XIN QingWu, ZHU ZhiMing, ZHANG LinLi, ZHUANG XiaoDong, ZHENG NenZhu. Transcriptome Analysis of Differential Gene Expression Associated with Testis Tissue in Mule Duck and Muscovy Duck[J]. Scientia Agricultura Sinica, 2017, 50(18): 3608-3619.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.18.016

https://www.chinaagrisci.com/CN/Y2017/V50/I18/3608

参考文献

[1] 檀俊秩, 陈晖, 宋建捷, 刘玉涛. 半番鸭繁殖性状的研究. 福建农业学报, 1998, 13(2): 41-45.

TAN J Z, CHEN H, SONG J J, LIU Y T. Studies on reproductive character of mule duck. Journal of Fujian Agricultural Sciences, 1998, 13(2):41-45. (in Chinese)

[2] 刘军须, 蔡月花, 张敬各, 张福英. 雄性不育大鼠近交系MIJ的建立及其遗传特征观察. 动物学杂志, 2008, 43(5) :37-44.

LIU J X, CAI Y H, ZHANG J G, ZHANG F Y. Establishment and genetic characteristics of rat inbred strain MIJ with spontaneous male infertility. Journal of Animal Science, 2008, 43(5):37-44. (in Chinese)

[3] 张庆波, 李齐发, 李家璜, 李新福, 刘振山, 潘增祥, 宋大伟, 谢庄. 牛精子发生相关新基因b-DAZL的克隆、生物信息学分析与组织表达研究. 自然科学进展, 2008, 18(5):493-504.

ZHANG Q B, LI Q F, LI J H, LI X F, LIU Z S, PAN Z X, SONG D W, XIE Z. Cloning, bioinformatics analysis and tissue expression of a novel b-DAZL gene related to spermatogenesis in cattle. Progress in Natural Science, 2008, 18(5):493-504. (in Chinese)

[4] LI S J, WANG C, YU W H, ZHAO S H, GONG Y Z. Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks. PloS One, 2012, 7: e36592.

[5] LI Q H, WANG N, DU Z, HUX X, CHENL, FEI J, WANGY Y, LI. N. Gastrocnemius transcriptome analysis reveals domestication induced gene expression changes between wild and domestic chickens. Genomics, 2012, 100: 314-319.

[6] 陈黎, 黄学涛, 田勇, 陶争荣, 卢立志. 利用转录组测序筛选与鸭青壳性状形成相关的基因. 农业生物技术学报, 2016, 24(7): 1064-1072.

CHEN L, HUANG X T, TIAN Y, TAO Z R, LU L Z. Identifying genes associated with blue eggshell in ducks (Anasplatyrhynchos domesticus) by transcriptome analysis. Journal of Agricultural Biotechnology, 2016, 24(7): 1064-1072. (in Chinese)

[7] BAUERSACHS S, WOLF E. Transcriptome analyses of bovine, porcine and equine endometrium during the pre-implantation phase[J]. Animal reproduction science. 2012,134:84-94.

[8] FIEDLER T J, HUDDER A, MCKAY S J, SHIVKUMAR S, CAPO T R, SCHMALE M C, WALSH P J. The transcriptome of the early life history stages of the california sea hare aplysia californica, comparative biochemistry and physiology Part D. Genomics & Proteomics, 2010, 5:165-170.

[9] 张伟. 中华绒螯蟹精巢组织文库构建和基因的克隆与序列分析[D]. 上海: 华东师范大学, 2012.

ZHANG W. Construction of cDNA library of Chinese mitten crab, eriocheir sinensis ：East China Normal University, 2012. (in Chinese)and cloeing and molecular characterization of gene[D]. Shanghai

[10] 钟志君. 猪睾丸和卵巢组织转录组的差异分析[D]. 雅安：四川农业大学, 2012 .

ZHONG Z J. Repertoire of porcine microRNA in adult ovary and testis by deep sequencing[D]. Sichuan: Sichuan Agricultural University, 2012 (in Chinese)

[11] 张升利, 付成东, 梁拥军, 李文通, 孙砚胜, 史东杰, 张欣. 长尾草金鱼成熟期雌雄性腺RNA-Seq转录组分析. 水产科学, 2014, 33(12): 750-753.

ZHANG S L, FU C D, LIANG Y J, LI W T, SUN Y S, SHI D J, ZHANG X. The RNA-Seq transcriptome analysis in male gonads of Long-tailed goldfish rassius auratus. Fisheries Science, 2014, 33(12): 750-753. (in Chinese)

[12] Xu Q, Zhao W M, Chen Y, Tong Y Y, Rong G H, Huang Z Y, Zhang Y, Chang G B, Wu X S, Chen G H. Transcriptome profiling of the goose (Anser cygnoides) ovaries identify laying and broodiness phenotypes. PloS One, 2013, 8:e55496.

[13] 朱志明, 陈红萍, 林如龙, 缪中纬, 辛清武, 李丽, 张丹青, 郑嫩珠. 山麻鸭开产期和产蛋高峰期卵巢组织转录组分析. 中国农业科学, 2016, 49(5):998-1007.

ZHU Z M, CHEN H P, LIN R L, MIAO Z W, XIN Q W, LI L, ZHANG D Q, ZHENG N Z. Transcriptome analysis of ovary tissue in early laying period and egg laying peak period of shanma Ducks. Scientia Agricultura Sinica, 2016, 49(5):998-1007. (in Chinese)

[14] SCHULZE S K, KANWAR R, GÖLZENLEUCHTER M, THERNEAU T M, BEUTLER A S. SERE: Single-parameter quality control and sample comparison for RNA-Seq. [BMC genomics Italic], 2012, 13(1): 524.

[15] XIANG L X, HE D, DONG W R, ZHANG Y W, SHAO J Z. Deep sequencing-based transcriptome profiling analysis of bacteria challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish. BMC Genomics, 2010, 11: 472.

[16] WANG B, GUO G W, WANG C, LIN Y, WANG X M, ZHAO M M, GUO Y, HE M H, ZHANG Y, PAN L. Survey of the transcriptome of aspergillus oryzae via massively parallel mRNA sequencing. Nucleic Acids Research, 2010, 38(15): 5075-5087.

[17] LI M Y, TAN H W, WANG F, JIANG Q, XU Z S, TIAN C, XIONG A S. De novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley (Petroselinum crispum). PLoS One, 2014, 9(9): e108977.

[18] ZHU S Y, TANG S M, TANG Q M, LIU T M. Genome-wide transcriptional changes of ramie (Boehmeria nivea L. Gaud) in response to root-lesion nematode infection. Gene, 2014, 552: 67-74.

[19] CONG F, LIU X L, HAN Z X, SHAO Y H, KONG X P, LIU S W. Transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection. Genomics, 2013, 14: 743-756.

[20] FRASER B A, WEADICK C J, JANOWITZ I, RODD F H, HUGHES K A. Sequencing and characterization of the guppy (Poecilia reticulata) transcriptome. BMC Genomics, 2011, 12:202.

[21] LI G Y, XIE P, LI H Y, HAO L, XIONG Q, QIU T.Involment of p53, Bax, and Bcl-2 pathway in microcystins-induced apoptosis in rat testis. Environmental Toxicology, 2011, 26(2):111-117.

[22] 郑炜, 赵宗胜, 李青峰, 班谦, 李洪涛, 梁耀伟. Solexa测序技术分析鸡与鹌鹑属间杂交雌性和雄性胚胎的差异microRNAs. 中国兽医学报, 2014, 34(1):116-122.

ZHENG W, ZHAO Z S, LI Q F, BAN Q, LI H T, LIANG H T. Analysis of difference microRNAs in female and male embryos of chicken-quail hybrid by solexa sequencing. Chinese Journal of Veterinary Science, 2014, 34(1):116-122. (in Chinese)

[23] DRUMMOND A E. TGFβ signaling in the development of ovarian function. Cell and Tissue Research, 2005, 322(l): 107-115.

[24] KONRAD L, KEILANI M M, LAIBLE L, NOTTELMANN U, HOFMANN R. Effects of TGF-betas and a specific antagonist on apoptosis of immature rat male germ cells in vitro. Apoptosis, 2006, 11(5): 739-748.
[25] SUN Q Y, BREITBART H, SCHATTEN H. Role of the MAPK cascade in mammalian germ cells. Reproduction, Fertility, and Development, 1999, 11(8):443-450.

[26] HARRIS V K, COTICCHIA C M, KAGAN B L, AHMAD S, WELLSTEIN A, RIEGE A T, HARRIS V K, COTICCHIA C M. Induction of the angiogenic modulator fibroblast growth factor- binding protein by epidermal growth factor is mediated through both MEK/ERK and p38 signal transduction path ways. Journal Biology Chemistry, 2000, 275(15): 10802-10811.

[27] ALMOG T, NAOR Z. Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions. Molecular and Cellular Endocrinology, 2008, 282:39-44.

[28] JOHNSON C, JIA Y, WANG C, LUE Y H, SWERDLOFF R S, ZHANG X S, HU Z Y, LI Y C, LIU Y X, AMIYA P. Sinha Hikim. Role of Caspase 2 in apoptotic signaling in primate and murine germ cells. Biology of Reproduction, 2008, 79 (5): 806.

[29] SHOW M D, HILL C M, ANWAY M D, WRIGHT W W, ZIRKIN B R. Phosphorylation of mitogen-activated protein kinase 8 (MAPK8) is associated with germ cell apoptosis and redistribution of the Bcl2- Modifying Factor (BMF). Journal of Andrology, 2008，29(3): 338-344.

[30] VALVE E, PENTTILA T L, PARANKO J, HA¨RKO¨NEN P. FGF-8 is expressed during specific phases of rodent oocyte and spermatogonium development. Biochemical and Biophysical Research Communications, 1997, 232(1) :173-177.

[31] DVORAK P, DVORAKOV D, HAMPL A. Fibroblast growth factor signaling in embryonic and cancer stem cells. FEBS Letters , 2006, 580: 2869-2874.

[32] MIALON A, SANKINEN M, SO¨DERSTRO¨M H, JUNTTILA T T, HOLMSTRO¨M T, KOIVUSALO R, PAPAGEORGIOU A C, JOHNSON R S, HIETANEN S, ELENIUS K, WESTERMARCK J. DNA topoisomerase I is a cofactor for c-jun in the regulation of epidermal growth factor receptor expression and cancer cell proliferation. Molecular and Cellular Biology, 2005, 12(25): 5040-5051.

[33] 陈丽莉,肖亚梅,刘文彬,赵如榕,刘姣,刘筠,李万程.ERK/JNK在黄鳝雌、雄发育阶段生殖腺中的表达和定位.动物学报，2007, 53(2): 325-331.

CHEN L L, XIAO Y M, LIU W B, ZHAO R R, LIU J, LI W C. Expression and location of ERK/JNK in ovary and spermary in rice field eels monopterus albus. Journal of Animal Science, 2007, 53(2):325-331. (in Chinese)

[34] SHEIKH A Q, TAGHIAN T, HEMINGWAY B, CHO H, KOGAN A B, NARMONEVA1 D A. Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field. Journal of the Royal Society Interface, 2013, 10(78): 1-13.

[35] Vien H Y Lee, Leo T O Lee, Billy K C Chow. Gonadotropin- releasing hormone: regulation of the GnRH gene. FEBS Journal, 2008, 275: 5458-5478.

[36] BELOV A A, MOHAMMADI M. Grb2, a double-edged sword of receptor tyrosine kinase signaling. Science Signaling, 2012, 5(249): 49.

[37] HAINES E, MINOO P, FENG Z Q, RESALATPANAH N, NIE X M, CAMPIGLIO M, ALVAREZ L, COCOLAKIS E, RIDHA M, FULVIO M D, GOMEZ-CAMBRONERO J, LEBRUN J J, ALI S. Tyrosine phosphorylation of Grb2:Role in prolactin /epidermal growth factor cross talk in mammary epithelial cell growth and differentiation. Molecular and Cellular Biology, 2009, 29(10):2505-2520.

[38] YUAN Y Y, CHEN W Y, SHI Q X, MAO L Z,YU S Q, FANG X, ROLDAN E R S. Zona pellucida induces activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa. Biology of Reproduction, 2003, 68(3): 904-913.

[39] SANTI C M, SANTOS T, HERNÁNDEZ-CRUZ A, DARSZON A. Properties of a novel pH-dependent Ca²⁺ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function. The Journal of General Physiology, 1998,112: 33-53.