云南半细毛羊精子冷冻前后转录组表达差异分析

doi:10.16431/j.cnki.1671-7236.2022.03.019

摘要/Abstract

摘要： 【目的】本研究旨在对云南半细毛羊精子冷冻前后差异表达的基因与精子冷冻损伤的关系进行研究。【方法】采用电刺激法采集3只云南半细毛羊精液，各分成2份，一份直接用于提取精子RNA，另一份经过冷冻保存7 d后再进行精子RNA提取。利用获得的精子RNA，采用单细胞转录组测序技术分别检测新鲜精子和冻融精子的mRNA转录组，构建mRNA转录组文库，用Hiseq2500测序仪进行测序，对测序结果做进一步过滤处理，然后对差异表达基因进行GO功能注释和KEGG通路分析。【结果】 6份精液样本共产生623 399 754条原始测序序列，过滤处理后得到514 313 802条（82.50%）高质量的Clean reads。生物信息学分析结果表明，冷冻前后差异表达显著的基因共1 213个。其中，解冻后表达量下调的有1 208个，上调的有5个。通过GO功能注释和KEGG通路富集分析，差异表达基因分别归类于56个GO分类，参与40个信号通路。其中与精子功能密切相关的主要包括精子细胞过程、代谢过程、应激反应、生殖、质膜和氧化还原反应；精子冷冻前后差异表达基因参与的信号通路主要有炎症、细胞因子-细胞因子受体相互作用和细胞凋亡等通路。【结论】云南半细毛羊精子的冷冻损伤可能与获得的差异表达基因参与的生物过程有关。此外，获得的差异表达基因也可作为分子标记物应用于绵羊冻精品质的评价。

关键词: 云南半细毛羊; 精子; 冷冻保存; 转录组; 差异表达基因

Abstract: 【Objective】 The purpose of this study was to study the relationship between differentially expressed gene and sperm freezing injury of Yunnan Semi-fine Wool sheep before and after sperm freezing.【Method】 The semen of three Yunnan Semi-fine Wool sheep were collected by electrical stimulation and divided into two parts.One was directly used to extract sperm RNA, and the other was cryopreserved for 7 days before sperm RNA extraction.Using the obtained sperm RNA, the mRNA transcriptomes of fresh and frozen-thawed sperm were examined by single-cell whole transcriptome sequencing, and mRNA transcriptome libraries were constructed and sequenced with Hiseq2500 sequencer, and the sequencing results were further filtered, and the differentially expressed genes were analyzed by GO function annotation and KEGG enrichment analysis.【Result】 6 semen samples had a total of 623 399 754 original sequencing sequences.After filtration, 514 313 802 (82.50%) high-quality Clean reads were obtained.The bioinformatics analysis results showed that there were 1 213 genes with significant difference before and after the freezing and thawing process.Among them, the abundance of 1 208 genes were down-regulated, and 5 genes were up-regulated after freezing and thawing.Through GO annotation and KEGG enrichment analysis, the differential genes were classified into 56 GO terms and were participate in 40 signal pathways.Their biological functions were closely related to sperm functions mainly including cellular process, metabolic process, response to stimulation, reproductive process, reproduction, membrane and antioxidant.Before and after sperm freezing, the main genes involved in inflammation, cytokine-cytokine receptor interaction and apoptosis signal pathway.【Conclusion】 The results showed that the cryoinjuries of sperm of Yunnan Semi-fine Wool sheep might be related to the biological process that the obtained differentially expressed genes were involved.In addition, the obtained differential genes might also be used as potential molecular markers to evaluate the quality of sheep frozen semen.

Key words: Yunnan Semi-fine Wool sheep; sperm; cryopreservation; transcriptome; differential expressed gene

中图分类号:

S826.8

梁家充, 吕春荣, 洪琼花, 吴国权, 权国波. 云南半细毛羊精子冷冻前后转录组表达差异分析[J]. 中国畜牧兽医, 2022, 49(3): 973-981.

LIANG Jiachong, LYU Chunrong, HONG Qionghua, WU Guoquan, QUAN Guobo. Analysis of Sperm Differential Transcriptome Expression Before and After Freezing in Yunnan Semi-fine Wool Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 973-981.

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