意大利蜜蜂MAPK信号通路相关基因和全长转录本鉴定及分析

Identification and analysis of MAPK signaling pathway-related genes and full-length transcripts in Apis mellifera ligustica

【目的】利用纳米孔(Nanopore)测序数据鉴定意大利蜜蜂Apis mellifera ligustica丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)信号通路相关基因和全长转录本,并发掘MAPK信号通路相关基因的可变多聚腺苷酸化(Alternativepolyadenylation,APA)位点及可变剪接(Alternative splicing,AS)事件,以期加深对意大利蜜蜂MAPK信号通路的认识,为持续深入开展相关基因和剪接体(Isoform)的功能研究提供数据资源和基础。【方法】采用Blast工具将所有全长转录本比对到Nr数据库和KEGG数据库,再根据注释信息筛选出MAPK信号通路相关基因和全长转录本。利用gffcompare软件将MAPK信号通路相关全长转录本与西方蜜蜂参考基因组(Amel_HAv3.1)上注释的转录本进行比较以优化已注释基因结构。采用TAPIS pipeline预测APA位点,并通过MEME软件鉴定所有APA位点上游50 bp的基序(motif)。使用Astalavista软件鉴定AS事件并统计不同类型的AS事件数目。通过PCR验证MAPK信号通路相关基因的AS事件。【结果】共鉴定到意大利蜜蜂MAPK信号通路相关的83个基因与443条全长转录本。同时延长了1个基因的5′UTR和3′UTR,延长了12个基因的5′UTR,延长了10个基因的3′UTR。共鉴定到52个MAPK信号通路相关基因含有1个及以上的APA位点,并在APA位点上游鉴定到2个motif。共鉴定到MAPK信号通路相关的13个基因的21次AS事件,其中最丰富的类型为外显子跳跃(Exonskipping,ES)。PCR结果证实了骨形态发生蛋白受体1b基因的4次ES事件和EtsDNA结合蛋白pokkuri基因的1次ES事件的真实性。【结论】首次鉴定到意大利蜜蜂MAPK信号通路相关的83个基因和446条全长转录本,优化了该信号通路中西方蜜蜂参考基因组已注释的21个基因的结构,发掘出MAPK信号通路相关基因的406个APA位点和52次AS事件,证实了MAPK信号通路相关基因AS事件的真实性。

[Aim]To identify genes and full-length transcripts associated with the mitogen activated protein kinase(MAPK)signaling pathway in Apis mellifera ligustica,and explore alternative polyadenylation(APA)sites and alternative splicing(AS)events in MAPK signaling pathway-related genes.[Methods]All full-length transcripts were aligned to the Nr and KEGG databases,after which MAPK signaling pathway-associated genes and full-length transcripts were screened out.MAPK signaling pathway-associated full-length transcripts were compared with those annotated in the reference genome of A.mellifera(Amel_HAv3.1)to optimize the structures of annotated genes.APA sites were predicted using the TAPIS pipeline,followed by identification of motifs 50 bp upstream of all APA sites.AS events were identified using Astalavista software followed by statistics on the number of various kinds of AS events.PCR was used to validate AS events in genes related to the MAPK signaling pathway.[Results]In total,83 genes and 443 full-length transcripts relevant to the MAPK signaling pathway were identified.5′UTR and 3′UTR of one gene were prolonged,and 5′UTR of 12 genes and 3′UTR of 10 genes were prolonged,respectively.52 genes associated with the MAPK signaling pathway contained one or more APA sites and there were two motifs upstream of the APA sites.Twenty-one AS events in 13 genes related to the MAPK signaling pathway were identified,of which the most abundant was exon skipping(ES).PCR confirmed the authenticity of four ES events in the Bone morphogenetic protein receptor type-1B gene and one ES event in the Ets DNA-binding protein pokkuri gene.[Conclusion]Eighty-three genes and 446 full-length transcripts associated with the MAPK signaling pathway in A.m.ligustica were identified for the first time,structures of 21 annotated genes in the A.mellifera reference genome were optimized,406 APA sites and 52 AS events of genes related to MAPK signaling pathway were discovered,and the authenticity of AS events in MAPK signaling pathway-associated genes was verified.