卵形鲳鲹卵巢不同发育时期的转录组测序分析

Transcriptome analysis of ovaries at the different developmental stages of Trachinotus ovatus

  • 摘要: 【目的】鉴定筛选出与卵形鲳鲹卵巢发育相关的候选基因及信号通路,为揭示其卵巢性成熟过程的分子机制打下基础。【方法】挑选卵巢发育处于І期和Ш期的雌性卵形鲳鲹,分别构建卵形鲳鲹卵巢І期和Ш期的cDNA文库,采用Illumina HiSeqTM 2500进行转录组测序,经过滤、质量控制及拼接组装后获得的Unigenes在七大数据库(Nr、Nt、Pfam、KOG/COG、Swiss-Prot、KEGG和GO)中进行比对;通过FPKM及DEGseq筛选出差异表达基因,以GOseq和KOBAS对差异表达基因分别进行功能注释及信号通路富集分析,并采用MISA和GATK3进行SSR鉴定及SNP分析。【结果】卵形鲳鲹卵巢组织转录组测序获得的325156432条Raw reads,经过滤筛选得到317206752条Clean reads,拼接组装后得到59554条Unigenes;69.65%的Unigenes在Nr、Nt、Pfam、KOG/COG、Swiss-Prot、KEGG和GO等七大数据库中注释成功,其中有24599条Unigenes被注释到GO数据库,15997条Unigenes被注释到KEGG数据库。在卵形鲳鲹卵巢组织的2个发育时期共鉴定获得56115个基因,经差异表达分析后获得17737个差异基因,其中8169个基因在卵巢Ш期上调表达、9568个基因在卵巢Ш期下调表达。GO功能注释分析发现,卵形鲳鲹卵巢差异表达基因主要注释在细胞过程、氮化合物代谢过程、初级代谢过程、核、核部分、离子结合及水解酶活性等条目上;而KEGG信号通路富集分析结果显示,17737个差异表达基因显著富集在318条代谢途径上,其中前20条KEGG信号通路包括2-氧代羧酸代谢、PI3K-Akt信号通路、甲状腺激素信号通路、磷脂酶D信号通路、Fc εRI信号通路和细胞周期等。卵形鲳鲹卵巢转录组(59554条Unigenes)中共存在30133个SSRs和82490个SNPs。【结论】GnRHRFSHRFSHβCYP11ASIRT3PEG3等差异表达基因及PI3K-Akt信号通路和VEGF信号通路等与卵形鲳鲹卵巢的发育密切相关,共同调节卵巢的发育与成熟,在卵巢性成熟过程中发挥重要作用。

     

    Abstract: 【Objective】The candidate genes and pathways related to the ovarian development of Trachinotus ovatus were identified,so as to lay the foundation of revealing the molecular mechanism on ovarian sexual maturation.【Method】 The stage І and stage Ш ovarian tissue of T. ovatus were selected to construct the cDNA library for stage І and stage Ш,respectively. Transcriptome sequencing was performed by Illumina HiSeqTM2005. After filtering,quality control,and assembly,the unigenes obtained were mapped to the seven databases(Nr,Nt,Pfam,KOG/COG,Swiss-Prot,KEGG and GO). The differentially expressed genes(DEGs)were identified by FPKM and DEGseq. The GOseq and KOBAS were used for functional annotation and signal pathway enrichment analysis of DEGs,respectively. And the simple sequence repeat(SSR)identification and single nucleotide polymorphisms(SNP)analysis were performed by MISA and gatk3.【Result】The results showed that a total of 325156432 raw reads were generated from ovarian tissues. After filtering,317206752 clean reads were selected,and then were assembled into 59554 unigenes,69.65% of which were successfully annotated in Nr,Nt,Pfam,KOG/COG,Swiss-Prot,KEGG and GO databases. 24599 and 15997 unigenes were annotated to the GO database and KEGG database,respectively. The analysis showed that a total of 17737 DEGs were found in the ovaries of T. ovatus,among which 8169 DEGs were up-regulated at stage Ш ovary,and 9568 DEGs were down-regulated in stage Ш ovary. The GO functional annotation analysis demonstrated that,cellular process,nitrogen compound metabolic process,primary metabolic process,nucleus,nuclear part,ion binding and hydrolase activity. The results of KEGG signaling pathway enrichment analysis showed that 17737 DEGs were significantly enriched on 318 metabolic pathways. The top 20 KEGG signaling pathway including 2-Oxocarboxylic acid metabolism,PI3K-Akt signaling pathway,thyroid hormone signaling pathway,phospholipase D signaling pathway,Fc epsilon RI signaling pathway and cell cycle. Finally,a total of 30133 SSRs and 82490 SNPs were obtained from the 59554 unigenes of ovarian transcriptome of T. ovatus.【Conclusion】Six DEGs,including GnRHR,FSHR,FSHβ,CYP11A,SIRT3 and PEG3,as well as two KEGG pathways such as PI3K-Akt signaling pathway and VEGF signaling pathway are closely involved in the ovarian development of T. ovatus,which co-regulates ovarian development and maturation.

     

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