转录组测序筛选牛卵泡发育相关基因及其表达差异分析

doi:10.3864/j.issn.0578-1752.2018.15.015

摘要/Abstract

摘要： 【目的】通过不同生理状态牛卵泡高通量测序筛选与卵泡发育相关的基因。【方法】母牛同期发情后，B超声波连续监测并适时采集第一卵泡波出现偏差前的最大卵泡（the largest follicle at predeviation，PDF1）和第二大卵泡（the second largest follicle at predeviation，PDF2），构建RNA文库后Illumina平台测序，经数据库比对，设定参数筛选高表达基因、差异表达基因并进行GO分析，Genecards基因功能查询进一步筛选与卵泡发育直接相关的调控基因，qRT-PCR对筛选的基因进行表达量验证分析。【结果】两个转录本中共获得263个差异表达基因；GO功能聚类分析共分为三大类90组：其中生物学过程占64.4%，细胞组分占17.8%，分子功能占17.8%；获得一些重要的功能富集通路，如调控信号转导和细胞因子应答的生物途径；基因表达量分析筛选出10个高表达的上调和下调基因，获得参与雌激素合成和胎儿性别发育的CYP17A1、参与类固醇激素合成的LOC785462、细胞发育过程中调节细胞凋亡的DACH1以及调节ERK和MEK1/2信号通路的MAP3K3。Genecards功能查询共获得6个基因与卵泡发育关系较为密切，其中上调基因分别为PRSS35, PTGRF, ARID4B, GPR116；下调基因为APOA1和CPXM1；qRT-PCR结果显示，PRSS35和ARID4B在DF中的表达量显著高于SF（P＜0.05），CPXM1在SF中的表达量极显著高于DF（P＜0.01），PTGRF, GPR116和APOA1在DF和SF中的表达量不存在显著差异（P＞0.05），但其表达变化趋势与高通量测序结果相一致。【结论】转录组测序结果真实可靠，PRSS35和ARID4B在卵泡发育过程中发挥正调控作用，CPXM1在卵泡发育过程中发挥负调控作用，获得的牛卵泡发育相关基因和重要调节途径，对后期深入探讨卵泡发育调控机理的研究具有重要意义。

关键词: 牛, 卵泡, 转录组, PDF1, PDF2, 发育

Abstract: 【Objective】 The study focused on screening some genes involved in follicular development through high- throughput sequencing in bovine follicles at different physiological states. 【Method】Cows were selected for estrus synchronization, and the largest follicle and second largest follicle at predeviation during the first follicle wave were gathered by B-type ultrasonography. Thereafter, the RNA libraries were constructed and RNA sequencing was performed by Illumina platform. Compared with the database, parameters were set to screen high-expressed genes and differentially expressed genes, and then GO analysis was conducted. Further screened regulatory genes directly related to follicular development by Genecards, and qRT-PCR was performed to validate expression of screened genes associated with follicular development. 【Result】 Results showed that 263 differentially expressed genes were obtained from the two transcripts, which could be assigned into 90 groups under three categories by GO clustering analysis (biological processes, 64.4%; cell component, 17.8%; molecular function, 17.8%). Some important functional enrichment pathways were obtained, such as regulating signal transduction and cytokine response biological pathways; 10 high-expressed up-regulated and down-regulated genes were selected by gene expression analysis, CYP17A1 involved in estrogen synthesis and fetal gender development, LOC785462 participated in steroid hormone synthesis, DACH1 regulated cell apoptosis in process of cell development, and MAP3K3 regulated ERK and MEK1/2 signaling pathways. Six genes were found closely associate with follicular development by Genecards: up-regulated genes included PRSS35, PTGRF, ARID4B and GPR116, down-regulated genes included APOA1 and CPXM1. qRT-PCR results showed that expression of PRSS35 and ARID4B were significantly higher in DF than in SF (P＜0.05), and CPXM1 expression was significantly higher in SF than in DF (P＜0.01). There was no significant difference in the expression of PTGRF, GPR116 and APOA1 between DF and SF (P＞0.05), however, the expression variation trend was consistent with high-throughput sequencing results. 【Conclusion】The transcriptome sequencing results were accurate, PRSS35 and ARID4B played positive roles and CPXM1 played a negative role in regulating follicular development, genes related to bovine follicular development and important regulated pathways, which were of great significance to further study the regulation mechanism of follicular development.

Key words: bovine, follicle, transcript, PDF1, PDF2, development

李鹏飞，孟金柱，景炅婕，毕锡麟，王锴，朱芷葳，吕丽华. 转录组测序筛选牛卵泡发育相关基因及其表达差异分析[J]. 中国农业科学, 2018, 51(15): 3000-3008.

LI PengFei, MENG JinZhu, JING JiongJie, BI XiLin, WANG Kai, ZHU ZhiWei, Lü LiHua. Follicular Development Related Genes Screening and Differential Expressed Analysis by Transcriptome Sequencing in Bovine Ovary[J]. Scientia Agricultura Sinica, 2018, 51(15): 3000-3008.

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