福清山羊与努比亚黑山羊发情期卵巢组织RNA-Seq分析

doi:10.3864/j.issn.0578-1752.2019.12.014

摘要/Abstract

摘要：

【目的】研究福清山羊与努比亚黑山羊发情期卵巢组织转录组差异表达水平,一方面为山羊繁殖性状形成的分子机制提供理论依据,另一方面为利用努比亚黑山羊杂交改良福清山羊提供可加快遗传进展的分子标记。【方法】利用转录组测序方法对福清山羊和努比亚黑山羊发情期卵巢组织进行研究,筛选品种间的差异表达基因（differentially expressed genes,DEGs）,并对DEGs功能进行注释和若干基因荧光定量PCR（quantitative real-time PCR,qRT-PCR）验证;同时通过与参考基因组比对,分析和筛选测序样品中存在的SNP/InDel。【结果】 6个样品共得到46.68Gb Clean Data,DESeq分析发现了福清山羊和努比亚黑山羊发情期卵巢组织的DEGs149个（包含25个新转录本）,其中表达上调53个,表达下调96个;初步认为输卵管素基因（oviductin,OVN）、类固醇合成快速调节蛋白基因(steroidogenic acute regulatory protein,STAR)、早期生长应答1基因(early growth response 1,EGR1)可作为福清山羊繁殖性能的候选基因。149个DEGs中的108个基因能被GO（gene ontology）数据库注释,30个DEGs能够被COG（Cluster of Orthologous Groups of proteins）数据库注释,91个DEGs能够被KEGG（kyoto encyclopediaof genes and genomes）数据库注释。KEGG通路分析表明DEGs共富集到21条信号通路中,6条通路显著富集。经过进一步的与参考基因组序列比对分析,共发掘1 506个新转录本。经qRT-PCR验证,所选基因（转录本）表达变化模式与转录组测序结果一致,表明测序结果可靠。【结论】在转录组水平上筛选出了福清山羊和努比亚黑山羊发情期卵巢组织的149个DEGs,发掘了1 506个新转录本,初步揭示了OVN、STAR和EGR1在山羊繁殖过程中可能发挥重要作用,为进一步探索山羊繁殖性状相关机理提供参考依据。

关键词: 福清山羊, 努比亚黑山羊, 卵巢, 发情期, 转录组

Abstract:

【Objective】The aim of this study was to analyze the differentially expressed genes of ovary tissue during follicular stage in Fuqing goat and Nubian black goat.【Method】Transcriptome sequencing of ovary tissue during the follicular stage in Fuqing goat and Nubian black goat were performed by using the Illumina HiSeq ^TM 2500 platform with 3 biological replicates per goat breed, and verified by quantitative real-time PCR (qRT-PCR). Differentially expressed genes (DEGs) were selected and enriched based on GO, COG and KEGG database. 【Result】A total of 46.68 Gb clean data were obtained in six samples. 149 DEGs were found between Fuqing goat and Nubian black goat, including 53 DEGs up-regulated genes and 96 DEGs down-regulated genes. According to published papers, the genes, including OVN, STAR and EGR1, were considered as candidate genes of fecundity in goats. 108, 30 and 91 DEGs were enriched by GO, COG and KEGG database, respectively. KEGG pathway analysis showed that DEGs annotated to 21 metabolic pathways, and 6 pathways were enriched significantly, such as autoimmune thyroid disease, and allograft rejection, and phagosome and viral myocarditis associated with reproduction traits in goat. 1506 new genes or transcripts were found by BLAST, including 25 DEGs between two breeds. Verified by qRT-PCR, the pattern of selected genes was consistent with the results of transcriptome sequencing, which showed the sequencing results were reliable. 【Conclusion】149 DEGs and 1506 new genes of ovary tissue during follicular stage in Fuqing goat and Nubian black goat were screened by transcriptonal analysis. DEGs between Fuqing goat and Nubian black goat, including OVN, STAR and EGR1, maybe play an important role in goat reproduction traits.

Key words: Fuqing goat, Nubian black goat, ovary tissue, follicular stage, transcriptome

李文杨,刘远,吴贤锋,高承芳,黄勤楼. 福清山羊与努比亚黑山羊发情期卵巢组织RNA-Seq分析[J]. 中国农业科学, 2019, 52(12): 2171-2182.

LI WenYang,LIU Yuan,WU XianFeng,GAO ChengFang,HUANG QinLou. Transcriptome Analysis of Differentially Gene Expression Associated with Ovary Tissue During the Follicular Stage in Fuqing Goat and Nubian Black Goat[J]. Scientia Agricultura Sinica, 2019, 52(12): 2171-2182.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.12.014

https://www.chinaagrisci.com/CN/Y2019/V52/I12/2171

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参考文献 36

[1]	薛慧良, 周忠孝 . 绵羊繁殖性状候选基因的研究进展. 动物科学与动物医学, 2003,20(3):32-35.
	XUE H L, ZHOU X Z . Advances in candidate genes for reproductive traits in sheep. Animal Science and Veterinary Medicine, 2003, 20(3):32-35. (in Chinese)
[2]	刘远, 林碧芬, 林仕欣, 李文杨, 张晓佩, 高承芳, 董晓宁 . 浅谈福建地方山羊品种高繁殖力资源的开发利用. 中国草食动物科学, 2010,30(6):57-59.
	LIU Y, LIN B F, LIN S X, LI W Y, ZHANG X P, GAO C F, DONG X N . Exploitation and utilization of high fertility resources of Fujian local goat breeds. China Herbivore Science, 2010,30(6):57-59. (in Chinese)
[3]	LING Y H, XIANG H, LI Y S, LIU Y, ZHANG Y H, ZHANG Z J, DING J P, ZHANG X R . Exploring differentially expressed genes in the ovaries of uniparous and multiparous goats using the RNA-Seq (quantification) method. Gene, 2004,550(1):148-153.
[4]	VALERIO C, CLAUDIA A, ITALIA D F, ALFREDO C . Uncovering the complexity of transcriptomes with RNA-Seq. Journal of Biomedicine and Biotechnology, 2010,2010:1-19.
[5]	WANG Z, GERSTEIN M, SNYDER M . RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics, 2009,10(1):57-63. doi: 10.1038/nrg2484
[6]	李丽, 缪中伟, 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. 中国农业科学, 2017,50(18):3608-3619.
	LI L, MIAO Z W, XIN Q W, ZHU ZM, ZHANG L L, ZHUANG X D, ZHENG N Z . Transcriptome analysis of differentially gene expression associated with testis tissue in mule duck and Muscovy duck. Scientia Agricultura Sinica, 2017,50(18):3608-3619. (in Chinese)
[7]	字向东, 罗斌, 夏威, 郑玉才, 熊显荣, 李键, 钟金城, 朱江江, 张正帆 . 基于RNA-Seq技术的牦牛体外受精胚胎发育转录组分析. 中国农业科学, 2018,51(8):1577-1589.
	ZI X D, LUO B, XIA W, ZHENG Y C, XIONG X R, LI J, ZHONG J C, ZHU J J, ZHANG Z F . Transcripomic analysis of IVF embryonic development in the yak( Bos grunniens) Via RNA-Seq. Scientia Agricultura Sinica, 2018, 51(8):1577-1589. (in Chinese)
[8]	罗庆苗, 秦晓玉, 苗向阳 . 利用数字基因表达谱对小尾寒羊高繁性状相关基因的筛选研究. 中国农学通报, 2012,28(23):18-25.
	LUO Q M, QIN X Y, MIAO X Y . The study on related genes of high fecundity using digital gene expression tag profiling in small-tail- han-sheep. Chinese Agricultural Science Bulletin, 2012, 28(23):18-25. (in Chinese)
[9]	DI R, HE J N, SONG S H, TIAN D M, LIU Q Y, LIANG X J, MA Q, SUN M, WANG J D, ZGAO W M, CAO G L, WANG J X, YANG Z M, GE Y, CHU M X . Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season. BMC Genomics, 2014, 15:899.
[10]	FENG X, KI F Z, WANG F, ZHANG G M, PANG J, REN C F, ZHANG T T, YANG H, WANG Z Y, ZHANG Y L . Genome-wide differential expression profiling of mRNAs and IncRNAs associated with prolificacy in Hu sheep. Bioscience Reports, 2018,38, 1-14.
[11]	付绍印, 何小龙, 王标, 李虎山, 储明星, 刘永斌, 张文广 . 不同产羔数绵羊性腺轴比较转录组研究. 中国畜牧兽医, 2017,44(2):488-496.
	FU S Y, HE X L, WANG B, LI H S, CHU M X, LIU Y B, ZHANG W G . Comparative Transcriptome Research of Gonadal Axis for Different Lambing Number of Sheep. China Animal Husbandry and Veterinary Medicine, 2017, 44(2):488-496. (in Chinese)
[12]	李文杨, 刘远, 林仕欣, 张晓佩, 高承芳, 林碧芬, 董晓宁 . BMPR-IB, BMP15, GDF9基因作为福建省4个主要山羊品种多胎性能候选基因的研究. 浙江农业学报, 2012,24(6):975-979.
	LI W Y, LIU Y, LIN S X, ZHANG X P, GAO C F, LIN B F, DONG X N . Studies on BMPR-IB, BMP15, and GDF9 as candidate genes of fecundity in four breeds of goats in Fujian. Acta Agriculture Zhejiang, 2012,24(6):975-979. (in Chinese)
[13]	陈其新, 张建红, 宋彦军, 董济福 . 我国主要肉羊品种肉用性能的初步评价. 中国草食动物科学(全国养羊生产与学术研讨会议论文集), 2012(s1):357-362.
	CHEN Q X, ZHANG J H, SONG Y J, DONG J F . Preliminary evaluation of meat performance of main mutton sheep and goats in China.China Herbivore Science, 2012(s1):357-362. (in Chinese)
[14]	洪开祥, 余荣权, 徐科礼, 陈太金, 许永强, 王清颖 . 努比亚羊与贵州黑山羊杂交性能研究. 中国草食动物科学(全国养羊生产与学术研讨会议论文集), 2014(s1):154-156.
	HONG X K, YU R Q, XU K L, CHEN T J, XU Y Q, WANG Q Y . Study on hybrid performance of Nubian goat and Guizhou black goat.China Herbivore Science, 2012(s1):357-362. (in Chinese)
[15]	KIM D, PERTEA G, TRAPNELL C, PIMENTEL H, KELLEY R, SALZBERG S L . TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biology, 2013,14:R36. doi: 10.1186/gb-2013-14-4-r36
[16]	DONG Y, XIE M, JIANG Y, XIAO N Q, DU X Y, ZHANG W G, TOSSER K G, WANG J H, YANG S, LIANG J, CHEN W B, CHEN J, ZENG P, HOU Y, BIAN C, PAN S K, LI Y X, LIU X, WANG W L, SERVIN B, SAYRE B, ZHU B, SWEENEY D, MOORE R, NIE W H, SHEN Y Y, ZHAO R P, ZHANG G J, LI J Q, FARAUT T, WOMACK J, ZHANG Y P, KIJAS J, COCKETT N, XU X, ZHANG S H, WANG J, WANG W . Sequencing and automated whole-genome optical mapping of the genome of a domestic goat ( Capra hircus). Nature Biotechnology, 2013,31(2):135-141.
[17]	JIANG H, WONG W H . Statistical inferences for isoform expression in RNA-Seq. Bioinformatics, 2009,25(8):1026-1032. doi: 10.1093/bioinformatics/btp113
[18]	FLOREA L, SONG L, SALZBERG S L . Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues. F1000 Research, 2013,2:188. doi: 10.12688/f1000research
[19]	SCHULZE S K, KANWAR R, GOLZENLEUCHTER M, THRNEAU T M, BEYTLER A S . SERE: Single-parameter quality control and sample comparison for RNA-Seq. BMC Genomics, 2012,13(1):524. doi: 10.1186/1471-2164-13-524
[20]	WANG L K, FENG Z X, WANG X, WANG X W, ZHANG X G . DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics, 2010,26, 136-138. doi: 10.1093/bioinformatics/btp612
[21]	ALEXA A, RAHNENFUHRER J . TopGO: enrichment analysis for gene ontology. R package version 2. 8, 2010.
[22]	TATUSOV R L, GALPERIN M Y, NATALE D A . The COG database: a tool for genome scale analysis of protein functions and evolution. Nucleic Acids Research, 2000, 28(1):33-36.
[23]	KANEHISA M, GOTO S, KAWASHIMA S, OKUNO Y, HATTORI M . The KEGG resource for deciphering the genome. Nucleic Acids Research, 2004,32:277-280. doi: 10.1093/nar/gkh063
[24]	MCKENNA A, HANNA M, BANKS E, SIVACHENKO A, CIBULSKIS K, KERNYTSKY A, GARIMELLA K, ALTSHULER D, GABRIEL S, DALY M, DEPRISTO M A . The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research, 2010,20(9):1297-1303. doi: 10.1101/gr.107524.110
[25]	CINGOLANI P, PLATTS A, WANG L L, COON M, NGUYEN T, WANG L, LAND S J, LU X, RUDEN D M . A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly. 2012,6(2):80-92.
[26]	梁素芸, 周正奎, 侯水生 . 基于测序技术的畜禽基因组学研究进展. 遗传, 2017,39(4):276-292.
	LIANG S Y, ZHOU Z K, HOU S S . The research progress of farm animal genomics based on sequencing technologies. Hereditas (Beijing), 2017, 39(4):276-292. (in Chinese)
[27]	兰道亮, 熊显荣, 位艳丽, 徐通, 钟金城, 字向东, 王永, 李健 . 基于RNA-Seq高通量测序技术得牦牛卵巢转录组研究: 进一步完善牦牛基因结构及挖掘与繁殖相关新基因. 中国科学(生命科学), 2014,44(3):307-317.
	LAN D L, XIONNG X R, WEI Y L, XU T, ZHONG J C, ZI X D, WANG Y, LI J . RNA-Seq analysis of yak ovary: improving yak gene structure information and mining reproduction-related genes. Science China-Life Sciences , 2014,44(3):307-317. (in Chinese)
[28]	王旭平, 杨胜林, 陆曼, 柳诚刚, 谭斌, 杨汝才, 周美迪, 王达娜, 李潇蒙 . 番鸭攻击行为的转录组学研究. 农业生物技术学报, 2018,26(4):687-697.
	WANG X P, YANG S L, LU M, LIU C G, TAN B, YANG R C, ZHOU M D, WANG D N, LI X M . Transcriptomics analysis of the aggressive behavior of Muscovy Duck ( Cairna moschata). Journal of Agricultural Biotechnology, 2018,26(4):687-697. (in Chinese)
[29]	BUHI W C . Characterization and biological roles of oviduct-specific, oestrogen-dependent glycoprotein. Reproduction, 2002,123(3):355-362. doi: 10.1530/rep.0.1230355
[30]	张译夫, 潘阳阳, 崔燕, 余四九, 张倩, 樊江峰, 杨琨, 杨融 . 牦牛输卵管蛋白(Ovn)的cDNA克隆及其在发情周期中的差异性表达. 农业生物技术学报, 2015,23(9):1217-1225.
	ZHANG Y F, PAN Y Y, CUI Y, YU S J, ZHANG Q, PAN J F, YANG K , YANG R. cDNA cloning of Yak ( Bos grunniens) Oviductin (Ovn) and its different expression during the estrous cycle. Journal of Agricultural Biotechnology, 2015,23(9):1217-1225. (in Chinese)
[31]	VERHAGE H G, MAVROGIANIS P A , O'DAY B, SCHMIDT A, RIAS E B, DONNELLY K M, BOOMSMA R A, THIBODEAUX J K, FAZLEABAS A T, JAFFE R C. Characteristics of an oviductal glycoprotein and its potential role in the fertilization process. Biology of Reproduction, 1998, 58(5):1098-1101.
[32]	SKIBOLA C F, BRACCI P M, HALPERIN E, NIETERS A, HUBBARD A, PAYNTER R A, SKIBOLA D R, AGANA L, BECKER N, TRESSLER P, FORREST M . Polymorphisms in the estrogen receptor 1 and Vitamin C and Matrix metalloproteinase gene families are associated with susceptibility to lymphoma. PLoS One, 2008,7(3):e2816.
[33]	SAYASITH K, BROWN K A, LUSSIER J G, DORÉ M, SIROIS J . Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation. Journal of Molecular Endocrinology, 2006, 37:239-250.
[34]	吴阳升, 林嘉鹏, 汪立芹, 陈莹, 黄俊成 . 绵羊小卵泡与中卵泡转录组差异特征分析. 江苏农业学报, 2016,32(4):832-843.
	WU Y S, LIN J P, WANG L Q, CHEN Y, HUANG J C . Transcriptome profiling of ovine follicles during growth from small to middle antral sizes. Jiangsu Journal of Agriculture Science, 2016,32(4):832-843. (in Chinese)
[35]	MIAO X, QIN Q L . Genome-wide transcriptome analysis of mRNAs and microRNAs in Dorset and Small Tail Han sheep to explore the regulation of fecundity. Molecular and Cellular Endocrinology, 2015,402(C):32. doi: 10.1016/j.mce.2014.12.023
[36]	段新崇, 魏彦辉, 李阳, 李相运, 周荣艳, 锡建中 . 小尾寒羊间情期和发情期microRNAs差异表达分析. 畜牧兽医学报, 2016,47(7):1324-1332. doi: 10.11843/j.issn.0366-6964.2016.07.004
	DUAN X C, WEI Y H, LI Y, LI X Y, ZHOU R Y, XI J Z . Analyzing the differential expression of microRNAs in estrus and diestrus of small tailed Han sheep. Acta Veterinaria et Zootechnica Sinica, 2016,47(7):1324-1332. (in Chinese) doi: 10.11843/j.issn.0366-6964.2016.07.004

基因名称 Gene	上游引物 Forward primer（5′-3′）	下游引物 Reverse primer（5′-3′）	产物长度 Product size（bp）
18S rRNA	GTAACCCGTTGAACCCCATT	CCATCCAATCGGTAGTAGCG	151
New-Gene 4708	CTCCAAATCGGGCAACACAG	TCCACAGTAATAATCGGCCTCG	77
New-Gene 1125	ACAACAGTGTCAGCATCCGT	TGTTGGAGTCCCGTTATGGC	136
CREB3L4	GGGCTCTTCGAAACAGCAGA	AGTCCCAGCTCCAGGAAAGA	104
THSD7B	AGTTACAAGGCGCATCACCA	TTTCGGCATGATGCAGGACT	159
CRABP1	GTGCAGGAGCTTACCCACTT	CGGGTCCAGTAGGTTTTGGG	93
CTHRC1	TTGGTGCTGGACTAGTGGAC	ATGCGAGACACCGAATTCCA	100

样品 Sample	总Reads Total reads	可定位的Reads Mapped reads	唯一定位Reads Uniq mapped reads	多点定位Reads Multiple mapped reads
F-1	48567844	41489885/85.43%	39687502/81.72%	1802383/3.71%
F-2	49910620	42262711/84.68%	40565566/81.28%	1697145/3.40%
F-3	49890394	42216928/84.62%	40925490/82.03%	1791438/3.59%
N-4	53482074	46603840/87.14%	45110457/84.35%	1293383/2.42%
N-5	53379622	46077636/86.32%	44696951/83.73%	1380685/2.59%
N-6	53967784	46261607/85.72%	45017922/83.42%	1243685/2.30%