| 摘要: |
| 【目的】完善和优化绵羊（Ovis aries）基因组注释信息，解析绵羊睾丸组织在不同繁殖力水平的遗传机理。【方法】以高繁殖力绵羊品种湖羊和低繁殖力绵羊品种藏绵羊（各3只）为研究对象，采集其睾丸组织，提取RNA，构建cDNA 文库，进行RNA-Seq测序，对测序数据进行组装。利用Cuffcompare软件对重新组装的测序数据与绵羊参考基因组进行比对分析，优化已注释基因的结构，并挖掘新转录本，对新转录本进行GO和KEGG通路分析。使用DESeq软件挖掘差异表达新转录本，对其进行GO和KEGG通路分析。随机选取表达显著上调和下调的新转录本各5个，采用实时荧光定量PCR（RT-qPCR）对其表达水平进行验证。【结果】对6只供试羊构建了6个独立的cDNA文库，共获得266 009 566个原始读段，质量控制后得到133 004 783个过滤后读段；6个cDNA文库过滤后读段的GC含量在49.61%～51.14%，符合碱基组成规律，Q30比例≥92.96%。对4 273个已注释基因的结构进行了优化，其中5′非翻译区域（UTR）延伸基因2 027个，3′ UTR延伸基因1 907个，5′和3′ UTR同时延伸基因339个。挖掘到12 178个新转录本，GO功能分析显示，2 416个新转录本注释到生物学过程、细胞组成和分子功能；KEGG富集发现新转录本显著富集在单纯疱疹病毒Ⅰ型感染、过氧物酶体、河马信号通路。与湖羊相比，藏绵羊睾丸组织中有155个新转录本表达差异显著，其中103个表达上调，52个表达下调；GO功能分析显示，差异新转录本富集在细胞过程、膜部分、绑定等条目；KEGG富集发现差异新转录本显著富集在钙信号通路和核因子-κB炎症信号通路等。10个差异表达新转录本的RT-qPCR验证试验结果与RNA-Seq结果趋势一致。【结论】从湖羊和藏绵羊睾丸组织中共挖掘出12 178个新转录本，其中155个差异表达。这些新转录本主要通过参与细胞功能、生物调节和代谢途径,以及通过核因子-κB炎症信号和钙信号通路广泛参与调控生殖细胞的增殖与分化、睾酮的分泌、免疫应答、Ca2+跨膜转运等途径，进而调控绵羊睾丸的发育和精子的发生。 |
| 关键词: 湖羊；藏绵羊；睾丸；新转录本 家畜繁殖 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目 (32160792) |
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| Annotation and functional analysis of new transcripts from testicular tissues of sheep with different fertility levels |
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FU Xiaoyu,LIU Miaomiao,YANG Yanan,et al
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| Abstract: |
| 【Objective】This study improved and optimized the annotation information of sheep genome and analyzed genetic mechanism of testicular tissue of sheep (Ovis aries) at different fecundity levels.【Method】Three Hu sheep (high fertility) and three Tibetan sheep (low fertility) were included in this study.The testis tissues were collected,RNA were extracted,cDNA libraries were constructed,RNA-Seq sequencing was performed,and sequencing data was assembled.The recombined sequencing data were compared with the sheep reference genome using cuffcompare software to optimize the structure of annotated genes,mine new transcripts,and perform GO and KEGG pathway analysis on the new transcripts.Five new transcripts with significant up-regulation and five new transcripts with down-regulated expression were randomly selected and their expression levels were verified by real-time fluorescence quantitative PCR (RT-qPCR). 【Result】Six independent cDNA libraries were constructed,and a total of 266 009 566 raw reads were obtained. After quality control,133 004 783 clean reads were obtained.The GC contents of the six cDNA libraries were 49.61%—51.14%,which was consistent with the rule of base composition,and the Q30 proportions were ≥92.96%.Extensions were achieved with 2 027 transcripts for the 5′ untranslated region (UTR),1 907 transcripts for the 3′ UTR region,and 339 genes for both 5′ and 3′ UTR at the same time.A total of 12 178 new transcripts were mined and GO functional analysis showed that 2 416 new transcripts were annotated to biological processes,cell composition,and molecular functions.KEGG enrichment showed that the new transcripts were significantly enriched in herpes simplex virus type Ⅰ infection,peroxisome,and hippopotamus signaling pathways.Compared with Hu sheep,the expression of 155 new transcripts in the testis of Tibetan sheep was significantly different,of which 103 were up-regulated and 52 were down regulated.The differential new transcripts were enriched in cell process,membrane part,and binding terms,according to GO functional analysis. KEGG enrichment found that the new transcripts were significantly enriched in the calcium signaling pathway and NF-κB inflammatory signaling pathway.The RT-qPCR validation test of 10 differentially expressed new transcripts were consistent with RNA-Seq.【Conclusion】A total of 12 178 new transcripts were extracted from the testis tissues of Hu sheep and Tibetan sheep,of which 155 were differentially expressed.These new transcripts regulated testis development and spermatogenesis by participating in cellular function,biological regulation,and metabolic pathways,as well as calcium and NF-κB signaling pathways involved in the regulation of reproductive cell proliferation and differentiation,testosterone secretion,immune response,and Ca2+ signal transduction,so as to regulate the development of sheep testis and spermatogenesis. |
| Key words: Hu sheep Tibetan sheep testis new transcript livestock breeding |
