基于PacBio测序数据的蜜蜂球囊菌转录因子、融合基因及RNA编辑事件的鉴定

Identification of transcription factors,fusion genes and RNA editing events in Ascosphaera apis based on PacBio sequencing data

【目的】本研究旨在利用已获得的PacBio单分子实时(single molecule real-time,SMRT)测序数据对蜜蜂球囊菌Ascosphaera apis菌丝(AaM)和孢子(AaS)中的转录因子(TF)、融合基因和RNA编辑事件进行鉴定和分析,以期丰富蜜蜂球囊菌的相关信息,并为进一步探究它们的功能提供理论依据。【方法】利用BLASTx工具将AaM和AaS的全长转录本序列比对到Nr,Swiss-Prot和KEGG数据库以获得一致性最高的蛋白序列,再利用hmmscan软件将上述蛋白序列比对到Plant TFdb数据库以获得TF的分类及注释信息。采用TOFU软件中的fusion_finder.py程序进行融合基因的预测,进而分析融合基因的序列和位置信息。使用SAMtools预测AaM和AaS中的RNA编辑事件,再利用ANNOVAR软件对RNA编辑事件进行注释,进而采用相关生物信息学软件对RNA编辑位点基因进行GO功能和KEGG通路注释。【结果】在AaS中共鉴定到17个TF家族的213个TF,其中C2H2家族包含的TF成员最多。在AaM和AaS中分别鉴定到921和510个融合基因,二者共有的融合基因为510个,特有的融合基因分别为411和0个。在AaM和AaS中分别鉴定到547和191次RNA编辑事件,其中AaM中同义单核苷酸突变的数量最多,AaS中非同义单核苷酸突变的数量最多。此外,在AaM中鉴定到12种碱基替换类型,其中发生C->T的RNA编辑事件数量最多,达到158次;在AaS中鉴定到9种碱基替换类型,其中发生C->T和G->T的RNA编辑事件数量最多,均有42次。AaM和AaS中RNA编辑位点基因分别涉及19和24个GO功能条目;此外还能注释到11和20条KEGG通路。【结论】蜜蜂球囊菌的菌丝和孢子中含有丰富的TF、融合基因和RNA编辑位点;转录因子C2H2家族与蜜蜂球囊菌菌丝和孢子的生长发育和细胞活动具有潜在关联;RNA编辑事件的碱基替换类型在蜜蜂球囊菌和其他物种中具有物种特异性;RNA编辑可能在蜜蜂球囊菌菌丝和孢子的生长和代谢中发挥作用。

【Aim】This study aims to identify and analyze transcription factors(TFs),fusion genes and RNA editing events in Ascosphaera apis mycelium(AaM)and spore(AaS)based on previously obtained PacBio single molecule real-time(SMRT)sequencing data,so as to further enrich the relevant information of A.apis and to offer theoretical basis for further investigation of their function.【Methods】Full-length transcripts in AaM and AaS were respectively aligned against Nr,Swiss-Prot and KEGG databases using BLASTx tool to gain protein sequences with the highest identity,which were then aligned to Plant TFdb database with hmmscan software to obtain the information of classification and annotation of TFs.Additionally,fusion_finder.py program contained in TOFU software was used for prediction of fusion genes,followed by analysis of their sequence and location information.RNA editing events in AaM and AaS were predicted using SAMtools,and then annotation of RNA editing events was performed with ANNOVAR software.Further,GO function and KEGG pathway annotation of genes located at the RNA editing sites was conducted using related bioinformatics software.【Results】A total of 213 TFs from 17 TF families were identified in AaS,and the C2H2 family was the largest.In AaM and AaS,921 and 510 fusion genes were respectively identified.Additionally,in AaM and AaS,there were 510 shared fusion genes,while the numbers of specific ones were 411 and zero,respectively.In total,547 and 191 RNA editing events were respectively identified in AaM and AaS,among them synonymous single nucleotide mutation was the most abundant type in AaM,while nonsynonymous single nucleotide mutation was the most abundant type in AaS.Moreover,12 types of base substitution were identified in AaM,and the number of RNA editing events with C->T was the highest(158),while nine types of base substitution were identified in AaS,and the numbers of RNA editing events with C->T and G->T were the highest(both 42).In AaM and AaS,genes located at the RNA editing sites were involved in 19 and 24 GO functional terms as well as 11 and 20 KEGG pathways,respectively.【Conclusion】There are abundant TFs,fusion genes and RNA editing sites in mycelium and spore of A.apis.C2H2 family of TF has potential relationship with growth,development and cellular activity of mycelium and spore of A.apis.The base substitution types of RNA editing events in A.apis and other species are species-specific.RNA editing may play a role in growth and metabolism of mycelium and spore of A.apis.