基于高通量测序的梅花鹿卵巢衰老的转录组分析

doi:10.16431/j.cnki.1671-7236.2023.08.013

摘要/Abstract

摘要： 【目的】获取青年梅花鹿和老年梅花鹿卵巢组织的转录组信息,为深入研究梅花鹿卵巢衰老的潜在机制提供依据。【方法】以4岁龄青年梅花鹿和10岁龄老年梅花鹿卵巢作为试验样本,对样本进行石蜡切片、苏木精-伊红染色和卵泡计数;用Trizol法提取卵巢样本RNA构建转录组文库,应用Illumina HiSeq^TM2000测序平台测序,对测序结果进行差异表达基因、GO功能、KEGG通路和蛋白互作网络分析,并挑选部分差异表达基因进行实时荧光定量PCR验证。通过RIPA裂解液提取卵巢样本蛋白并对组蛋白H3赖氨酸4三甲基化(H3K4me3)蛋白在青年和老年梅花鹿中的表达情况进行Western blotting检测。【结果】组织学分析结果表明,老年梅花鹿的卵巢卵泡数显著低于青年梅花鹿(P＜0.05),而闭锁卵泡数极显著高于青年梅花鹿(P＜0.01)。转录组学分析结果表明,老年和青年梅花鹿卵巢间共有1 477个差异表达基因,其中表达上调基因503个,表达下调基因974个。GO功能和KEGG通路富集结果表明,免疫系统过程、转录、DNA损伤修复、炎症反应、凋亡等生物事件与卵巢衰老有关。实时荧光定量PCR结果显示,共有17个基因在老年和青年梅花鹿卵巢中表达量差异达显著或极显著水平(P＜0.05;P＜0.01),表明RNA-Seq结果可靠。Western blotting结果表明,H3K4me3蛋白在老年和青年梅花鹿卵巢中表达量存在显著差异(P＜0.05)。【结论】青年和老年梅花鹿卵巢在卵泡数方面存在显著差异,通过RNA-Seq筛选出PI3K-Akt信号通路影响DNA损伤修复效率,参与梅花鹿卵巢衰老。H3K4me3在老年梅花鹿卵巢中高表达与转录激活有关。

关键词: 梅花鹿; 卵巢衰老; RNA-Seq; H3K4me3

Abstract: 【Objective】 This study was aimed to obtain the transcriptome information of ovary tissues in young and old sika deer,and provide a basis for further study the potential mechanism of ovarian aging in sika deer.【Method】 The ovaries of 4 years young sika deer and 10 years old sika deer were used as test samples.The samples were processed with paraffin sectioning,hematoxylin-eosin staining and follicle counting.RNA was extracted from the ovarian samples using the Trizol method,and a transcriptome library was constructed.The Illumina HiSeq^TM 2000 sequencing platform was used to sequence,the sequencing results were analyzed for differentially expressed genes,GO function,KEGG pathway and protein interaction network analysis.Selected differentially expressed genes were further analyzed using Real-time quantitative PCR.The expression of trimethylation of lysine 4 on histone H3 protein subunit (H3K4me3) protein in ovaries of young and old sika deer were detected by Western blotting analysis using RIPA lysis solution.【Result】 The histological analysis results showed that the number of ovarian follicles in old sika deer was significantly lower than that in young sika deer (P＜0.05),and the number of atresia follicles in old sika deer was extremely significantly higher than that of young sika deer (P＜0.01).Transcriptome analysis showed that there were 1 477 differentially expressed genes of ovaries between old and young sika deer,of which 503 genes were upregulated and 974 genes were downregulated.GO function and KEGG pathway enrichment analysis showed that the immune system process,transcription,DNA damage repair,inflammatory response,apoptosis and other biological events were related to ovarian aging.Real-time quantitative PCR results showed that there were 17 genes with significant or extremely significant expression of ovaries between old and young sika deer (P＜0.05 or P＜0.01),indicating that RNA-Seq results were reliable.Western blotting analysis results showed that the expression of H3K4me3 protein in ovaries was significantly different between old and young sika deer (P＜0.05).【Conclusion】 The number of follicles in ovaries of young and old sika deer showed a significant difference.The PI3K-Akt signaling pathway had been identified to affect the efficiency of DNA damage repair and participate in ovarian aging of sika deer via RNA-Seq.The high expression of H3K4me3 in ovaries of old sika deer was related to transcription activation.

Key words: sika deer; ovarian aging; RNA-Seq; H3K4me3

中图分类号:

S825

林乐丰, 刁云飞, 范冰峰, 刘理想, 邵静, 赵向远, 许保增. 基于高通量测序的梅花鹿卵巢衰老的转录组分析[J]. 中国畜牧兽医, 2023, 50(8): 3157-3170.

LIN Lefeng, DIAO Yunfei, FAN Bingfeng, LIU Lixiang, SHAO Jing, ZHAO Xiangyuan, XU Baozeng. Transcriptome Analysis of Aging Sika Deer (Cervus nippon) Ovaries Based on High-throughput Sequencing[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3157-3170.

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