鸭胚胎发育中后期胸肌发育阻滞的RNA-seq分析

doi:10.3864/j.issn.0578-1752.2018.22.015

摘要/Abstract

摘要：

【目的】选择两个中国地方品种高邮鸭和金定鸭,对鸭胚胎发育中后期胸肌进行转录组分析研究,旨在探明胸肌发育阻滞的分子变化机制,为鸭骨骼肌调控机理研究打下基础。【方法】在21胚龄和27胚龄两个时间点,分别解剖高邮鸭、金定鸭各3只,采集胸大肌,提取总RNA构建文库,利用Illumina的HiseqTM2000进行高通量测序,并利用生物信息学方法进行差异表达基因挖掘、基因功能注释等分析,探讨21胚龄和27胚龄两个时间点之间胸肌发育阻滞的分子机制。【结果】高邮鸭、金定鸭21胚龄和27胚龄胸大肌组织RNA-seq质量Q20均在94%以上,Q30均在89%以上,测序得到的结果可靠,可用于后续分析。RNA水平相关性检查和基因mRNA表达量聚类图结果都表明,21胚龄（27胚龄）高邮鸭和金定鸭之间表达模式的相关性高于高邮鸭（金定鸭）21胚龄和27胚龄之间的表达模式。不同品种内时间点之间的差异基因数量（高邮鸭6 128个,金定鸭6 452个）远多于同一时间点不同品种间的差异基因数量（21胚龄522个,27胚龄299个）。qRT-PCR验证试验结果与RNA-seq分析结果相关性较强。通过GO和KEGG富集分析发现,高邮鸭、金定鸭胸肌在21胚龄到27胚龄阶段,能量代谢相关基因（主要为辅酶Q相关基因、ATP酶合成相关基因和细胞色素C相关基因）均显著上调,DNA复制和细胞周期相关基因（主要为微型染色体维持蛋白（MCM）相关基因、复制因子C（RFC）相关基因）均显著下调。相关基因表达的变化可能与此阶段成肌细胞增殖速度减慢,逐渐退出细胞周期开始准备下一阶段融合成多核肌管并形成肌纤维有关。对肌肉生长发育相关的关键基因分析发现,促进肌肉生长的IGF1和诱导成肌细胞末端分化的MyoG显著下调,促进肌纤维分化融合的MUSTN1基因、诱导肌祖细胞向成肌细胞转化的MyoD1基因显著上调。【结论】鸭胚胎中后期胸肌发育过程中大量基因差异表达。其中能量代谢相关基因的上调和DNA复制、细胞周期相关基因的下调,以及肌肉发育相关基因MUSTN1显著上调,IGF1、MyoG等显著下调,可能与鸭胚胎中后期胸肌发育阻滞现象密切相关。

关键词: 鸭, 胸肌发育, 转录组测序, 差异表达基因

Abstract:

【Objective】 In order to find the molecular varying mechanism involving the development arrest of pectoralis, Chinese native breeds, including Gaoyou Duck (GYD) and Jinding Duck (JDD), were selected for RNA-seq study using the pectoralis samples during the semi-late embryonic period.【Method】3 ducks of GYD and JDD , respectively, in the 21th embryonic day (ed21) and ed27 were selected to collect pectoralis major muscle and to extract total RNA to build cDNA library for RNA-seq with HiseqTM2000 of Illumina. At last, bioinformatics methods were used to extract differentially expressed genes (DEGs) between different breeds and time points, and to analyze the gene function annotation for studying molecular mechanism of pectoralis development retardation during ed21 and ed27. 【Result】 The results showed that the base ratios with quality value higher than 20 in reads (Q20) were more than 94%, and the base ratios of Q30 were more than 89%, which indicated a reliable sequencing result for the following analysis. The RNA level correlation inspection and mRNA expression level cluster graph both manifest that the correlation of mRNA expression patterns of GYD and JDD at ed21 or ed27 were higher than that of GYD (JDD) during ed21 and ed27. The numbers of DEGs between ed21 and ed27 (6128 DEGs for GYD and 6452 DEGs for JDD) were both apparently more than the numbers of DEGs between GYD and JDD in ed21 (522 DEGs) and ed27 (299 DEGs). qRT-PCR results of selected genes showed a strong correlation with RNA-seq results. GO and KEGG enrichment analysis showed the results that the genes involved in energy metabolism (mainly was coenzyme Q related genes, ATP enzymic synthesis related genes, and cytochrome C related genes) up regulated and DNA replication and cell cycle related-genes (mainly was minichromosome maintenance complex related genes and replication factor C related genes) down regulated significantly. This varies of related genes expression might relate to the slow myoblast proliferation and gradually exit the cell cycle to prepare for the next stage of fusing to multi-nuclei myotube and form myofiber. In the key genes involving in muscle growth and development, IGF1 (a gene promoting muscle growth) and MyoG (inducing terminal differentiation of myoblast) both down regulated dramatically. However, MUSTN1 gene, accelerating muscle fibre into differentiation and fusing stage, and MyoD gene, promoting muscle progenitor cell differentiated to myoblast, were expressed in a higher level in ed27 than in ed21. 【Conclusion】 A lot of genes differentially expressed between ed21 and ed27 in pectoralis muscle of duck, among of which the up-regulation of energy metabolism related genes, the down-regulation of DNA replicate and cell cycle related genes, and up-regulated MUSTN1, down-regulated IGF1 and MyoG, might closed relate to arrest phenomenon of pectoralis development during the semi-late stage of duck embryos.

Key words: duck, pectoralis muscle development, RNA-seq, differentially expressed genes

刘宏祥,徐文娟,朱春红,陶志云,宋卫涛,章双杰,李慧芳. 鸭胚胎发育中后期胸肌发育阻滞的RNA-seq分析[J]. 中国农业科学, 2018, 51(22): 4373-4386.

LIU HongXiang,XU WenJuan,ZHU ChunHong,TAO ZhiYun,SONG WeiTao,ZHANG ShuangJie,LI HuiFang. RNA-seq Analysis on Development Arrest of Duck Pectoralis Muscle During Semi-Late Embryonic Period[J]. Scientia Agricultura Sinica, 2018, 51(22): 4373-4386.

图/表 14

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图4

高邮鸭及金定鸭21胚龄和27胚龄mRNA差异表达基因GO功能分类图 B1：细胞成分运动（cellular component movement）,B2：基于微管的过程（microtubule-based process）,B3：基于微管的运动（microtubule-based movement）,B4：细胞周期（cell cycle）,B5：分解代谢过程的正向调控（positive regulation of catabolic process）,B6：自噬调控（regulation of autophagy）,B7：自噬正调控（positive regulation of autophagy）,B8：细胞膜组织（cellular membrane organization）,B9：（evasion or tolerance of host defenses by virus）,B10：细胞分解过程的正向调控（positive regulation of cellular catabolic process）,B11：宿主防御逃逸（avoidance of host defenses）,B12：宿主防御回避或耐受（evasion or tolerance of host defenses）,B13：其它共生互作生物的防御逃逸avoidance of defenses of other organism involved in symbiotic interaction）,B14：其他共生互作生物的防御回避或耐受（evasion or tolerance of defenses of other organism involved in symbiotic interaction）,B15：其他共生互作生物的防御响应（response to defenses of other organism involved in symbiotic interaction）,B16：对宿主防御的响应（response to host defenses）,B17：对宿主的响应（response to host） C1：线粒体（mitochondrion）,C2：核糖体（ribosome）,C3：核糖核蛋白复合物（ribonucleoprotein complex）,C4：细胞外基质（extracellular matrix）,C5：大分子复合物（macromolecular complex）,C6：非膜结合细胞器（non-membrane-bounded organelle）,C7：胞内非膜结合细胞器（intracellular non-membrane-bounded organelle） M1：核糖体结构成分（structural constituent of ribosome）,M2：肌动活性（motor activity）,M3：微管肌动活性（microtubule motor activity）,M4：结构分子活性（structural molecule activity）,M5：微管结合（microtubule binding）,M6：细胞支架蛋白结合（cytoskeletal protein binding）,M7：DNA依赖的ATP酶活性（DNA-dependent ATPase activity）,M8：微管蛋白结合（tubulin binding）,M9：焦磷酸酶活性（pyrophosphatase activity）,M10：氧化还原酶活性（oxidoreductase activity）,M11：作用于酸酐的水解酶活性（hydrolase activity, acting on acid anhydrides）,M12：作用于含磷酸酐的水解酶活性（hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides）,M13：核苷三磷酸酶活性（nucleoside-triphosphatase activity）"

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表9

GO与KEGG联合分析后的共有基因"

基因名 Gene name	基因描述 Gene description	Ensembl No.	上/下调 Up or down	基因名 Gene name	基因描述 Gene description	Ensembl No.	上/下调 Up or down
ATP5F1	ATP合成酶：H+转运线粒体Fo复合物亚基5F1 ATP synthase, H+ transporting, mitochondrial Fo complex subunit 5F1	ENSAPLG00000013428	上调 Up	ANAPC2	（细胞分裂）后期启动复合物亚基2 Anaphase promoting complex subunit 2	ENSAPLG00000001836	下调Down
ATP5H	ATP合成酶：H+转运线粒体Fo复合物亚基5H ATP synthase, H+ transporting, mitochondrial Fo complex subunit 5H	ENSAPLG00000006404	上调 Up	DNA2	DNA复制解螺旋/核酸酶2 DNA replication helicase/nuclease 2	ENSAPLG00000015576	下调Down
ATP5J	ATP合成酶：H+转运线粒体Fo复合物亚基5J ATP synthase, H+ transporting, mitochondrial Fo complex subunit 5J	ENSAPLG00000009600	上调 Up	FEN1	Flap结构特异性核酸内切酶1 Flap structure-specific endonuclease 1	ENSAPLG00000001725	下调Down
COX5A	细胞色素c氧化酶亚基5A Cytochrome c oxidase subunit 5A	ENSAPLG00000014886	上调 Up	MAD1L1	MAD1有丝分裂阻滞缺陷类似物1 MAD1 mitotic arrest deficient like 1	ENSAPLG00000015788	下调Down
COX7A2L	细胞色素c氧化酶亚基类7A2 Cytochrome c oxidase subunit 7A2 like	ENSAPLG00000012898	上调 Up	MCM2	微型染色体维持复合物组分2 Minichromosome maintenance complex component 2	ENSAPLG00000004520	下调Down
LOC101800937	线粒体中细胞色素c氧化酶亚基7B Cytochrome c oxidase subunit 7B, mitochondrial	ENSAPLG00000007286	上调 Up	MCM3	微型染色体维持复合物组分3 Minichromosome maintenance complex component 3	ENSAPLG00000013058	下调Down
NDUFA5	NADH：泛醌氧化还原酶亚基A5 NADH: Ubiquinone oxidoreductase subunit A5	ENSAPLG00000014770	上调 Up	MCM4	微型染色体维持复合物组分4 Minichromosome maintenance complex component 4	ENSAPLG00000003491	下调 Down
NDUFA7	NADH：泛醌氧化还原酶亚基A7 NADH: Ubiquinone oxidoreductase subunit A7	ENSAPLG00000011378	上调 Up	MCM5	微型染色体维持复合物组分5 Minichromosome maintenance complex component 5	ENSAPLG00000007931	下调 Down
NDUFB1	NADH：泛醌氧化还原酶亚基B1 NADH: Ubiquinone oxidoreductase subunit B1	ENSAPLG00000006000	上调 Up	ORC5	起点识别复合物亚基5 Origin recognition complex subunit 5	ENSAPLG00000002905	下调 Down
NDUFB3	NADH：泛醌氧化还原酶亚基B3 NADH: Ubiquinone oxidoreductase subunit B3	ENSAPLG00000015508	上调 Up	POLE	DNA聚合酶ε催化亚基 DNA polymerase epsilon, catalytic subunit	ENSAPLG00000009950	下调 Down
NDUFB4	NADH：泛醌氧化还原酶亚基B4 NADH: Ubiquinone oxidoreductase subunit B4	ENSAPLG00000007537	上调 Up	RB1	RB转录共阻遏因子1 RB transcriptional corepressor 1	ENSAPLG00000007734	下调 Down
NDUFB8	NADH：泛醌氧化还原酶亚基B8 NADH: Ubiquinone oxidoreductase subunit B8	ENSAPLG00000015152	上调 Up	RFC1	复制因子C亚基1 Replication factor C subunit 1	ENSAPLG00000010594	下调 Down
NDUFC2	NADH：泛醌氧化还原酶亚基C2 NADH: Ubiquinone oxidoreductase subunit C2	ENSAPLG00000006009	上调 Up	RFC2	复制因子C亚基2 Replication factor C subunit 2	ENSAPLG00000009389	下调 Down
NDUFS6	NADH：泛醌氧化还原酶亚基S6 NADH: Ubiquinone oxidoreductase subunit S6	ENSAPLG00000014333	上调 Up	TFDP1	转录因子Dp-1 Transcription factor Dp-1	ENSAPLG00000014438	下调 Down
SDHD	琥珀酸盐脱氢酶复合物亚基D Succinate dehydrogenase complex subunit D	ENSAPLG00000005936	上调 Up	YWHAZ	酪氨酸3-单氧酶/色氨酸5-单氧酶活化蛋白ζ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta	ENSAPLG00000002635	下调 Down
UQCRB	泛醌-细胞色素c还原酶结合蛋白 Ubiquinol-cytochrome c reductase binding protein	ENSAPLG00000012746	上调 Up

表9

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基因 Gene	上游引物 Up-primer	下游引物 Lower-primer	产物长度 Product length (bp)	退火温度 Temperature (℃)
F16P1	CCCTAAAGGAAAGCTGAGAC	CTCGGGCACTATATCCAGTA	115	60
COL1A1	GCCAACGAAATCGAGATCAG	CGTCTTTGTCGTCTTGTACT	126	60
WIPI1	GAAATCCCAGATGTTTACATCG	TCCGTCCCTTTCTTGAAGT	113	60
ARMC3	TTGGCATTGGCTGTAGTTTAG	TACTCTTGAGGAGGAAGCAGAA	115	60
CEA20	TCTCCGAGCCAGAAATCC	TCTTCCACCAGTACACGTC	105	60
GLIS3	ATGACGCAGAGACAAACT	TTGTGTTGTACGTTCTTCTGAG	188	60
SRSF4	CTCTTACTCCAGAAGCCGA	GATCTACTCTTGGAGCGAC	105	60
LAMC3	GAGGCCCAGAAGAAGATCA	ACAACCTGTGCCCTCTTA	131	60
TNNI1	CTGCACGAGAAGGTTGAG	GCAGGTCAAGCACTTTGAT	109	60
TNNT2	TCTCCAACATGCTGCATT	CTGAGGTGGTCGATGTTC	130	60
TNNC1	ATGCTGCTTTAACTGGAATG	CACCACAGGGTGGAAATC	179	60
MYOZ1	CAGAAGATTCAGTCTCACAAGT	AGGTCTTTATCCAAGCCAC	103	60
MYBPH	TCATGGGCAACACCTACTC	GGATCTTCTCTGGCTGGTAA	129	60
COEA1	CTTCCTGCCAGCAATTAC	AGCCAAACACTTCCATCATA	134	60
KCRS	GACCAGTGCATCCAAACC	TCTCAGCAAACACCTCGTAG	103	60
DEP1A	ATCTGTTGTTTGTTGCTTCC	TGTATCATCAAAGAGCGTGTG	134	60
ACTB*	TGAGAGTAGCCCCTGAGGAGCAC	TAACACCATCACCAGACTCCATCAC	198	60
GAPDH*	CTTCGGAATAGGGAGGAGAC	CGGAGATGATGACACGCTTA	131	60

品种 Breeds	样品 Sample	总碱基数（Gbp）Total nucleotides	Q20（%）	Q30（%）	GC（%）
高邮鸭 GY	21胚龄 21 ed	5.45	94.42	89.55	51.47
高邮鸭 GY	27胚龄 27 ed	6.25	94.34	89.46	51.89
金定鸭 JD	21胚龄 21 ed	6.16	94.28	89.37	50.11
金定鸭 JD	27胚龄 27 ed	5.79	94.33	89.37	51.42

项目 Statistical content	品种 Breeds	21胚龄 21 ed		27胚龄 27 ed
项目 Statistical content	品种 Breeds	数量 Number	百分比 Percentage	数量 Number	百分比 Percentage
总读段数 Total reads	高邮鸭/GY	43570695	-	50059412	-
总读段数 Total reads	金定鸭/JD	49264929	-	46361619	-
总比对数 Total mapped	高邮鸭/GY	26927881	61.80	29480876	58.89
总比对数 Total mapped	金定鸭/JD	31722102	64.39	27119296	58.50
多次比对数 Multiple mapped	高邮鸭/GY	560376	1.29	668497	1.33
多次比对数 Multiple mapped	金定鸭/JD	636927	1.29	603064	1.30
唯一比对数 Uniquely mapped	高邮鸭/GY	26367505	60.51	28812378	57.56
唯一比对数 Uniquely mapped	金定鸭/JD	31085175	63.10	26516232	57.19

品种 Breeds	RPKM值 RPKM value	0-1	1-3	3-15	15-60	> 60	总计 Total
高邮鸭 GY	21胚龄 21 ed	5197（27.22%）	2114（11.07%）	5245（27.47%）	4711（24.67%）	1828（9.57%）	19095
高邮鸭 GY	27胚龄 27 ed	5757（30.15%）	2186（11.45%）	5053（26.46%）	4310（22.57%）	1789（9.37%）	19095
金定鸭 JD	21胚龄 21 ed	5144（26.93%）	2152（11.27%）	5254（27.51%）	4744（24.84%）	1802（9.44%）	19095
金定鸭 JD	27胚龄 27 ed	5884（30.81%）	2166（11.34%）	4894（25.63%）	4356（22.81%）	1796（9.40%）	19095

基因名 Gene symbol	相关系数 Correlation coefficient	描述 Description
F16P1	0.81	果糖-1,6-二磷酸酶1 Fructose-1,6-bisphosphatase 1
COL1A1	0.79	胶原蛋白α-1（I）链 Collagen alpha-1(I) chain
WIPI1	0.87	WD重复区域磷酸肌醇互作蛋白1 WD repeat domain phosphoinositide-interacting protein 1
ARMC3	0.88	Armadillo重复蛋白3 Armadillo repeat-containing protein 3
CEA20	0.89	癌胚抗原相关细胞粘附因子20 Carcinoembryonic antigen-related cell adhesion molecule 20
GLIS3	0.75	锌指蛋白GLIS3 Zinc finger protein GLIS3
SRSF4	0.79	富含丝氨酸/精氨酸剪切因子 Serine/arginine-rich splicing factor 4
LAMC3	0.82	层连蛋白亚基γ3 Laminin subunit gamma-3
TNNI1	0.96	慢肌中肌钙蛋白1 Troponin 1, slow skeletal muscle
TNNT2	0.81	心肌型肌钙蛋白2 Troponin T2, Cardiac Type
TNNC1	0.70	慢肌和心肌中肌钙蛋白C Troponin C, slow skeletal and cardiac muscles
MYOZ1	0.76	Myozenin蛋白1 Myozenin-1
MYBPH	0.72	Myosin结合蛋白H Myosin-binding protein H
COEA1	0.87	胶原蛋白α-1（XIV）链 Collagen alpha-1(XIV) chain
KCRS	0.82	线粒体中S型肌酸激酶 Creatine kinase S-type, mitochondrial
DEP1A	0.75	含DEP区域蛋白1A DEP domain-containing protein 1A