产蛋间隔前期鸽卵泡转录组比较分析揭示卵泡发育相关基因

doi:10.11843/j.issn.0366-6964.2024.08.023

摘要/Abstract

摘要：

旨在对产蛋间隔前期母鸽卵泡颗粒细胞(GC)层进行高通量测序，筛选出与鸽卵泡发育和选择相关的关键基因。本研究选择了80对12月龄产蛋规律、体重相近的白羽王鸽为研究对象，在产蛋间隔第1天(LI1)和第3天(LI3)分别选择3只母鸽，采集F1和SF1卵泡GC层，并利用RNA-seq进行转录组分析，筛选L1F1/L1SF1、L3F1/L3SF1、L1F1/L3F1和L1SF1/L3SF1组，4组间差异表达基因(DEGs)，对DEGs进行GO功能注释和KEGG通路富集分析，通过STRING数据库进行蛋白质互作(PPI)网络分析并使用Cytoscape软件进行可视化处理筛选关键基因，各组随机选择5个DEGs进行RT-qPCR验证转录组的可靠性。在4组比对中分别获得77、2 736、5 698和3 864个DEGs。GO分析显示，4组DEGs都主要富集的GO条目为细胞过程、细胞解剖实体和连接。KEGG通路富集分析显示，L1F1/L1SF1组和L3F1/L3SF1组DEGs均显著富集在类固醇激素生物合成和卵巢类固醇合成等信号通路，L1F1/L3F1组和L1SF1/L3SF1组DEGs均显著富集在糖胺聚糖降解、突触囊泡周期和氧化磷酸化等信号通路。进一步地，进行PPI网络分析并筛选与卵泡发育和选择密切相关的DEGs。各组随机选择5个DEGs进行RT-qPCR验证，表达趋势与测序结果一致。本研究利用RNA-seq技术筛选到与鸽卵泡发育和选择相关的关键基因——EEF2、DDX5、PLK2、IGF2R、LHCGR、HSD3B1、CYP19A1和StAR，为进一步探究鸽卵泡发育和选择的分子调控机制提供了理论依据。

关键词: 鸽, 产蛋间隔, 卵泡发育, 转录组

Abstract:

High throughput sequencing was performed on granulosa cell(GC)layer at early stage of laying interval(LI)to screen key genes involved in pigeon follicle development and selection. Eighty pairs of 12-month-old White King pigeons, with similar laying patterns and body weights were selected in this study. Three female pigeons were selected at first day (LI1) and third day (LI3) of LI, respectively, the GC layer of F1 and SF1 follicles were collected, followed by transcriptome analysis using RNA-seq. Differentially expressed genes (DEGs) among 4 groups: L1F1/L1SF1 group, L3F1/L3SF1 group, L1F1/L3F1 group, and L1SF1/L3SF1 group were analyzed. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs were performed, protein-protein interaction (PPI) network analysis using the STRING database and visualization using Cytoscape software were conducted to selected key genes. Five DEGs from each group were randomly selected for RT-qPCR to verify the reliability of transcriptome. The 77, 2 736, 5 698 and 3 864 DEGs were obtained in the 4 groups, respectively. GO analysis results showed that DEGs in all groups were mainly enriched in cellular processes, cellular anatomical entity and binding. KEGG analysis result showed that DEGs in L1F1/L1SF1 and L3F1/L3SF1 groups were enriched in the steroid hormone biosynthesis and ovarian steroidogenesis signaling pathways. DEGs in L1F1/L3F1 and L1SF1/L3SF1 groups were enriched in the glycosaminoglycan degradation, synaptic vesicle cycle and oxidative phosphorylation signaling pathways. In addition, DEGs closely associated with follicle development and selection were screened by PPI.Five DEGs were randomly selected from each group for RT-qPCR verification, and the expression trends of these DEGs were consistent with the sequencing results. RNA-seq technology was applied to identify key genes associated with pigeon follicle development and selection, which including EEF2, DDX5, PLK2, IGF2R, LHCGR, HSD3B1, CYP19A1 and StAR. These results will provide the theoretical basis for further exploring the molecular mechanisms of follicle development and selection in pigeon.

Key words: pigeon, laying interval, follicular development, transcriptome

中图分类号:

S836.2

陈静, 吴薛蓓, 苗冬枝, 张弛, 郭振玉, 王莹. 产蛋间隔前期鸽卵泡转录组比较分析揭示卵泡发育相关基因[J]. 畜牧兽医学报, 2024, 55(8): 3503-3515.

Jing CHEN, Xuebei WU, Dongzhi MIAO, Chi ZHANG, Zhenyu GUO, Ying WANG. Comparative Analysis of Transcriptome of Pigeon Follicles at Early Stage of Laying Interval Reveals Genes Related to Follicular Development[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3503-3515.

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基因 Gene	引物序列(5′→3′) Primer sequence	目的片段长度/bp Products length
CYP19A1	F: TTGCAAGACCTACCAAGGGC R: GCACAGTATCGGGTACGAGG	185
B2M	F: GCTGAGGCACTGAGAGGAA R: GCTCCTTCGTTGTCACCAG	125
IGF2R	F: TTACACCACCACCATCCACTT R: TAACGGCAGCAGATTGAACAG	199
HSD17B3	F: TGTGTCCTGCGGTTCATCC R: TTCCTCCACCTCTTCTTCAGATAA	101
StAR	F: GCCCTCCAGAAATCGCTCAG R: CGGAATACCTTGCCCACAT	119
DDX5	F: AAGACCAATCTCAGGCGGTG R: AGTCTTCAGCCAGTTGCCTC	172
LHGCR	F: TAACGTATCCCAGCCACTGC R: TGGTGCTTTCGCACACTTTG	105
PLK2	F: TCGCCAAGTGTTATGAGATGAC R: TGCTACTCTGCTGTGAGGAAT	80
EEF2	F: GACAGTGAAGATAAGGACAAGGAG R: AGAAGGCAGGTGAATGGTGAT	111
KRR1	F: CTTGGACCCTTTAGTGGGCT R: TCCGCTTGAACTTGGGCAAA	169
HSD3B1	F: GGGTAAGCTGTCTGGTGAC R: TCACATCTCGGATGTCCCCT	179
FST	F: TGCCAGCGATAACACAACTTAC R: TCTTCTTCTTCCTCTTCATCCTCT	141
GAPDH	F: CTCTACTCATGGCCACTTCCG R: ACAACGTATTCAGCACCAGC	138

样本 Sample	质控后序列/条 Clean reads	Q20/%	Q30/%	比对率/% Mapped reads rate
L1F1-1	42 566 840	96.92	92.22	84.97
L1F1-2	42 566 850	96.93	92.25	83.80
L1F1-3	42 585 948	96.97	92.30	84.06
L1SF1-1	42 815 642	97.09	92.57	84.90
L1SF1-2	42 815 678	97.08	92.58	83.78
L1SF1-3	42 316 180	97.09	92.59	84.08
L3F1-1	42 239 724	96.94	92.27	84.99
L3F1-2	42 153 656	96.97	92.34	82.67
L3F1-3	43 454 914	96.96	92.34	84.01
L3SF1-1	42 260 902	97.03	92.45	83.69
L3SF1-2	42 368 216	97.04	92.52	83.54
L3SF1-3	42 368 343	97.03	92.48	83.24

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