转录组测序筛选牛卵泡发育偏差和优势卵泡选择相关基因

doi:10.11843/j.issn.0366-6964.2024.11.023

摘要/Abstract

摘要：

旨在筛选牛卵泡发育偏差和优势卵泡(dominant follicle, DF)选择相关基因。本研究选取6头10月龄健康海福特青年母牛，同期发情后平均分为两组，第一组采集第一个卵泡发育波出现偏差前第一大卵泡(the largest follicle at predeviation, PDF1)和第二大卵泡(the second largest follicle at predeviation, PDF2)，第二组采集第一个卵泡发育波出现偏差后第一大卵泡(the largest follicle at onset of deviation, ODF1)和第二大卵泡(the second largest follicle at onset of deviation, ODF2)，提取卵泡颗粒细胞(granule cells, GCs) RNA进行转录组测序，测序结果对照参考基因组，PDF1-VS-PDF2组筛选影响卵泡发育偏差的差异表达基因(differentially expressed genes, DEGs)，ODF1-VS-ODF2组、ODF1-VS-PDF1组和ODF1-VS-PDF2组筛选影响优势卵泡选择的DEGs并进行GO和KEGG富集分析、PPI分析筛选关键基因，通过RT-qPCR和Western blotting验证筛选基因的准确性。结果显示，PDF1-VS-PDF2组发现220个DEGs，179个上调，41个下调，GO和KEGG分析显示PI3K-Akt信号通路、TGF-β信号通路与卵泡发育相关，PPI分析显示MYC、BRCA1、EZH2、ARID1A、SMARCA4为中枢基因；ODF1-VS-ODF2组发现184个DEGs，93个上调，91个下调，GO和KEGG分析显示PI3K-Akt信号通路、TGF-β信号通路、mTOR信号通路、TNF信号通路和Jak-STAT信号通路与卵泡发育相关，PPI分析显示POLR2A、FOS、HIF1A、KIT、SOCS3为中枢基因；ODF1-VS-PDF1组和ODF1-VS-PDF2组共发现837个DEGs，360个上调，477个下调，GO和KEGG分析显示mTOR信号通路、TNF信号通路、TGF-β信号通路、PI3K-Akt信号通路和孕激素(progestin, P₄)介导的卵母细胞成熟通路与卵泡发育相关，PPI分析显示HIF1A、RPS9、COL1A2、PIK3R1、COL4A1、ITSN1、GNB1、RPL3、ESPL1、CUL7为中枢基因。RT-qPCR结果表明BRCA1、ARID1A、EZH2在PDF1表达量高于PDF2，POLR2A在ODF1表达量高于ODF2。Western blotting结果表明BRCA1和EZH2在PDF1表达量高于PDF2。本研究筛选出MYC、BRCA1、EZH2、ARID1A、SMARCA4可能在牛卵泡发育偏差发挥作用，POLR2A、FOS、HIF1A、KIT、SOCS3、RPS9、COL1A2、PIK3R1、COL4A1、ITSN1、GNB1、RPL3、ESPL1和CUL7可能在牛DF选择过程发挥作用，试验证实筛选的基因翻译为蛋白质发挥作用。研究结果为探索牛卵泡发育偏差和优势卵泡选择基因调控理论奠定基础。

关键词: 牛, 转录组, 卵泡发育, 基因

Abstract:

The study aimed to screen genes related to the deviation of follicular development and dominant follicle selection in cattle. In this study, 6 healthy 10-month-old Hayford young cows were divided into two groups on average after synchronization. The first group collected the first and second largest follicles before the deviation of the first follicle development wave. The second group collected the first and second largest follicles after the deviation of the first follicle development wave. RNA of follicular granulosa cells was extracted for transcriptome sequencing, and the sequencing results were compared with the reference genome. The PDF1-VS-PDF2 group was screened for differentially expressed genes affecting follicular development. ODF1-VS-ODF2 group, ODF1-VS-PDF1 group and ODF1-VS-PDF2 group screened DEGs that affected dominant follicle selection, and performed GO and KEGG enrichment analysis and PPI analysis to screen key genes. The accuracy of the screened genes was verified by RT-qPCR and Western blotting. The results showed that 220 DEGs were found in the PDF1-VS-PDF2 group, 179 of which were up-regulated and 41 were down-regulated. GO and KEGG analysis showed that PI3K-Akt signaling pathway and TGF-β signaling pathway were related to follicular development. PPI analysis showed that MYC, BRCA1, EZH2, ARID1A and SMARCA4 were hub genes. A total of 184 DEGs were found in ODF1-VS-ODF2 group, 93 were up-regulated and 91 were down-regulated. GO and KEGG analysis showed that TGF-β signaling pathway, PI3K-Akt signaling pathway, Jak-STAT signaling pathway, TNF signaling pathway, mTOR signaling pathway and cell adhesion molecules were related to follicular development. PPI analysis showed that POLR2A, FOS, HIF1A, KIT and SOCS3 were hub genes. A total of 837 DEGs were found in ODF1-VS-PDF1 group and ODF1-VS-PDF2 group, of which 360 were up-regulated and 477 were down-regulated. GO and KEGG analysis showed that mTOR signaling pathway, TNF signaling pathway, TGF-β signaling pathway, PI3K-Akt signaling pathway, progestin (P₄)-mediated oocyte maturation pathway were related to follicular development. PPI analysis showed that HIF1A, RPS9, COL1A2, PIK3R1, COL4A1, ITSN1, GNB1, RPL3, ESPL1, CUL7 were the hub genes. RT-qPCR results showed that the expression of BRCA1, ARID1A and EZH2 in PDF1 was higher than that in PDF2, and the expression of POLR2A in ODF1 was higher than that in ODF2. Western blotting results showed that the expression of BRCA1 and EZH2 in PDF1 was higher than that in PDF2. In this study, MYC, BRCA1, EZH2, ARID1A and SMARCA4 may play a role in the deviation of bovine follicle development. POLR2A, FOS, HIF1A, KIT, SOCS3, RPS9, COL1A2, PIK3R1, COL4A1, ITSN1, GNB1, RPL3, ESPL1 and CUL7 may play a role in the selection process of bovine DF. The results of this study laid a foundation for exploring the theory of regulation of bovine follicle development deviation and dominant follicle selection gene.

Key words: bovine, transcriptome, follicular development, genes

中图分类号:

S823.2

周宏泰, 闫俊蓉, 李鹏飞. 转录组测序筛选牛卵泡发育偏差和优势卵泡选择相关基因[J]. 畜牧兽医学报, 2024, 55(11): 5059-5071.

Hongtai ZHOU, Junrong YAN, Pengfei LI. Transcriptome Sequencing was Used to Screen Genes Related to Follicular Development Bias and Dominant Follicle Selection in Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5059-5071.

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基因 Gene	上游引物(5'→3') Forward primer	下游引物(5'→3') Reverse primer
BRCA1	F：GCGTCCTTCCAAACAAGTGTATCAG	R：CCAAGCCAGTTTCCCTTTCTTCATC
ARID1A	F：ACAGCCGAATCTCATGCCTTCC	R：AGCGGAGAACTGATTGCCATAGG
POLR2A	F：TCAACTGTATCCGCACCCATAGC	R：GCCATTCTCCACCACCACCTTAG
EZH2	F：AGGAGGAGAAGAAGGACGAGACC	R：GTGCCGATGAGGACCCTGAAC
β-actin	F：GCGTTACACCCTTTTTCTTGACA	R：TCACCTTCACCGTTCCAGTT

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