利用miRNA测序技术鉴定调控种公鸡精子发生的候选基因

doi:10.7668/hbnxb.20193391

摘要/Abstract

摘要：

精子发生是一个复杂的过程,目前家禽的精子发生机制尚未彻底阐明。因此,鉴定调控种公鸡精子发生的候选基因,对于探究家禽的精子发生机制具有重要意义。对8只海兰褐公鸡的睾丸与附睾样本进行miRNA测序,筛选差异表达miRNA并对其进行靶基因预测和功能分析。结果表明,在睾丸和附睾样本中共检测到718个miRNA,其中109个miRNA差异表达。这些差异表达的miRNA中有40个在睾丸组织中显著上调,69个在附睾组织中显著上调。对差异表达miRNA进行靶基因预测并取交集,共得到11 077个靶基因,其中在睾丸组织中上调差异表达miRNA的靶基因有4 784个,在附睾组织中上调差异表达miRNA的靶基因有6 293个。对差异表达miRNA的靶基因进行功能分析,GO富集分析结果表明,睾丸和附睾中靶基因主要富集于细胞过程的负调节、细胞生物合成过程的调节、生物合成过程的调节等生物学过程。KEGG分析结果表明,与精子发生相关的靶基因显著富集于MAPK信号通路。本研究发现miR-31、miR-499、miR-375和miR-34c可能是调控鸡精子发生的候选基因。

关键词: 鸡, 睾丸, 附睾, miRNA, 精子发生

Abstract:

Spermatogenesis is a complex process.At present,the mechanism of spermatogenesis in poultry has not been fully clarified.Therefore,Identification of candidate genes regulating spermatogenesis in breeder cocks is of great significance to explore the mechanism of spermatogenesis in poultry.miRNA sequencing was performed on testis and epididymis samples of 8 Hailan brown cocks,the differentially expressed miRNAs were screened and their target genes were predicted and analyzed.A total of 718 miRNAs were detected in testis and epididymis samples,of which 109 miRNAs were differentially expressed,among these differentially expressed miRNAs,40 were significantly up-regulated in testicular tissue and 69 in epididymis.The target genes of differentially expressed miRNAs were predicted and intersected.A total of 11 077 target genes were obtained,among them,4 784 target genes up-regulated differentially expressed miRNA in testis and 6 293 target genes up-regulated differentially expressed miRNA in epididymis.Functional analysis of target genes differentially expressing miRNA,Go enrichment analysis showed that the target genes in testis and epididymis were mainly concentrated in biological processes such as negative regulation of cell process,regulation of cell biosynthesis process,regulation of biosynthesis process and so on.KEGG analysis showed that the target genes related to spermatogenesis were significantly enriched in MAPK signaling pathway.It found that miR-31,miR-499,mir-375 and miR-34c might be candidate genes to regulate chicken spermatogenesis.

Key words: Chicken, Testis, Epididymis, miRNA, Spermatogenesis

丛百林, 薛吕帆, 盖凯, 赵延辉, 陈鹏, 陈余, 王梁, 吕学泽, 郭勇, 盛熙晖. 利用miRNA测序技术鉴定调控种公鸡精子发生的候选基因[J]. 华北农学报, 2022, 37(S1): 333-338. doi: 10.7668/hbnxb.20193391.

CONG Bailin, XUE Lüfan, GAI Kai, ZHAO Yanhui, CHEN Peng, CHEN Yu, WANG Liang, LÜ Xueze, GUO Yong, SHENG Xihui. Identification of Candidate Genes Regulating Spermatogenesis in Breeder Cocks by miRNA Sequencing[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(S1): 333-338. doi: 10.7668/hbnxb.20193391.

http://www.hbnxb.net/CN/Y2022/V37/IS1/333

图/表 8

图1 样本间表达水平相关性分析 G.睾丸;F.附睾。

Fig.1 Correlation analysis of expression levels among samples G.Testis;F.Epididymis.

表1 睾丸与附睾中表达量最高的10个miRNA

Tab.1 Ten miRNAs with the highest expression in testis and epididymis

miRNA	睾丸中表达量 (平均值) Expression in testis (average value)	miRNA	附睾中表达量 (平均值) Expression in epididymis (average value)
gga-mir-21	99 010.250	gga-mir-21	1 107 495.250
gga-mir-19b	88 695.375	gga-mir-26a	945 307.625
gga-mir-29a	87 730.000	gga-mir-34b	623 894.500
gga-mir-29c	87 001.625	gga-mir-101	524 502.125
gga-mir-101	85 919.250	gga-mir-125b	466 637.375
gga-mir-26a	82 653.000	gga-mir-34c	330 340.625
gga-mir-19a	69 895.625	gga-mir-29a	302 676.000
gga-mir-125b	4 8597.875	gga-mir-29c	300 093.250
gga-let-7a	33 634.000	gga-mir-142	191 980.125
gga-let-7j	32 795.875	gga-mir-199	179 682.750

图2 鸡睾丸与附睾样本中的差异表达miRNA 红色.上调;绿色.下调;蓝色.无显著差异;FC.表达量差值;FDR.错误发现率。

Fig.2 Differentially expressed miRNAs in chicken testis and epididymis Red.Up regulation;Green.Down;Blue.No significant difference;FC.Expression difference;FDR.Error discovery rate.

表2 睾丸与附睾中差异倍数最高的10个miRNA

Tab.2 The 10 miRNAs with the highest multiple differences between testis and epididymis

miRNA	Log₂(FC)	P-value	差异类型 Different type
gga-mir-146c	6.902 743 454	9.08E-37	上调
gga-mir-203a	6.309 846 537	7.50E-34	上调
gga-mir-216c	6.565 912 100	1.91E-31	上调
gga-mir-31	5.788 709 614	3.41E-29	上调
gga-mir-499	5.535 957 062	4.64E-28	上调
gga-mir-375	-8.685 414 283	1.94E-50	下调
gga-mir-34c	-6.647 415 315	1.78E-41	下调
gga-mir-34b	-6.060 662 629	1.42E-37	下调
gga-mir-429	-7.167 204 066	3.12E-35	下调
gga-mir-200b	-6.557 815 014	8.41E-35	下调

图3 睾丸组织上调差异表达miRNA靶基因GO富集分析 1.细胞过程的负调节;2.神经系统发育;3.细胞发育;4.细胞分化;5.细胞大分子生物合成过程的调控;6.细胞发育过程;7.大分子生物合成过程的调控;8.生物合成过程的调节;9.神经元投射;10.轴突;11.质膜结合细胞投影;12.细胞投影;13.躯体发育室;14.转录抑制复合物;15.突触;16.细胞连接;17.转录辅抑制活性;18.蛋白激酶活性;19.转录调节活性;20.跨膜受体蛋白激酶活性;21.DNA结合;22.序列特异性DNA结合;23.转录调控区序列特异性DNA结合;24.调节区核酸结合。

Fig.3 Enrichment analysis of up-regulated differentially expressed miRNA target gene go in testicular tissue 1.Negative regulation of cellular process;2.Nervous system development;3.Cell development;4.Cell differentiation;5.Regulation of cellular macromolecule biosynthetic;6.Cellular developmental process;7.Regulation of macromolecule biosynthetic;8.Regulation of biosynthetic process;9.Neuron projection;10.Axon;11.Plasma membrane bounded cell projection;12.Cell projection;13.Somatodendritic compartment;14.Transcription repressor complex;15.Synapse;16.Cell junction;17.Transcription corepressor activity;18.Protein kinase activity;19.Transcription regulator activity;20.Transmembrane receptor protein kinase activity;21.DNA binding;22.Sequence-specific DNA binding;23.Transcription regulatory region sequence-specific DNA binding;24.Regulatory region nucleic acid binding.

图4 睾丸组织上调差异表达miRNA靶基因KEGG通路分析 1.MAPK信号通路;2.肌动蛋白骨架的调节;3.黏着斑;4.细胞衰老;5.自噬动物;6.心肌细胞的肾上腺素能信号传导;7.Fox信号通路;8.TGF-β信号通路;9.ErbB信号通路;10.粘附连接。

Fig.4 Up regulation of differentially expressed miRNA target gene KEGG pathway in testis 1.MAPK signaling pathway;2.Regulation of actin cytoskeleton;3.Focal adhesion;4.Cellular senescence;5.Autophagy-animal;6.Adrenergic signaling in cardiomyocytes;7.Fox signaling pathway;8.TGF-beta signaling pathway;9.ErbB signaling pathway;10.Adheres junction.

图5 睾丸组织下调差异表达miRNA靶基因GO富集分析 1.RNA代谢过程的调控;2.含核碱基化合物代谢过程的调控;3.神经系统发育;4.大分子生物合成过程的调控;5.细胞生物合成过程的调节;6.生物合成过程的调节;7.核酸模板转录调控;8.RNA生物合成过程的调控;9.轴突;10.转录调节活性;11.序列特异性DNA结合;12.DNA结合;13.核酸结合;14.DNA结合转录因子活性;15.转录调控区序列特异性DNA结合;16.调节区核酸结合;17.序列特异性双链DNA结合。

Fig.5 Down regulation and enrichment of differentially expressed miRNA target gene GO in testicular tissue 1.Regulation of RNA metabolic process;2.Regulation of nucleobase-containing compound metabolic process;3.Nervous system development;4.Regulation of macromolecule biosynthetic process;5.Regulation of cellular biosynthetic process;6.Regulation of biosynthetic process;7.Regulation of nuclei acid-templated transcription;8.Regulation of RNA biosynthetic process;9.Axon;10.Transcription regulator activity;11.Sequence-specific DNA binding;12.DNA binding;13.Nucleic acid binding;14.DNA-binding transcription factor activity;15.Transcription regulatory region sequence-specific DNA binding;16.Regulatory region nucleic acid binding;17.Sequence-specific double-stranded DNA binding.

图6 睾丸组织下调差异表达miRNA靶基因KEGG通路分析 1.MAPK信号通路;2.内吞作用;3.黏着斑;4.肌动蛋白细胞骨架的调节;5.Wnt信号通路;6.紧密连接;7.细胞衰老;8.TGF-β信号通路;9.黏附连接;10.Notch信号通路。

Fig.6 Down regulation of differentially expressed miRNA target gene KEGG pathway in testis 1.MAPK signaling pathway;2.Endocytosis;3.Focal adhesion;4.Regulation of actin cytoskeleton;5.Wnt signaling pathway;6.Tight junction;7.Cellular senescence;8.TGF-beta signaling pathway;9.Adheres junction;10.Notch signaling pathway.

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