结合三代测序与二代测序技术揭示蜜蜂球囊菌孢子转录组的复杂性

Unraveling the complexity of transcriptome in Ascosphaera apis spore: based on third-generation and next-generation sequencing

摘要:

【目的】蜜蜂球囊菌(Ascosphaera apis)是一种专性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在利用PacBio单分子实时(single molecule real-time，SMRT)测序技术对蜜蜂球囊菌孢子(AaS)中基因的可变剪切(alternative splicing，AS)和可变多聚腺苷酸化(alternative polyadenylation，APA)以及长链非编码RNA (long non-coding RNA，lncRNA)进行鉴定和分析，进而揭示蜜蜂球囊菌孢子中转录组的复杂性。【方法】采用Suppa软件对蜜蜂球囊菌孢子中基因的AS事件进行鉴定。通过RT-PCR对不同类型的AS事件进行验证。采用TAPIS pipeline对蜜蜂球囊菌孢子基因的APA位点进行鉴定。利用MEME软件分析孢子全长转录本的poly (A)剪接位点上游50 bp的序列特征并鉴定motif。联用CPC和CNCI软件和比对Swiss-prot数据库的方法预测lncRNA，取三者的交集作为高可信度的lncRNA集合。进一步比较lncRNA和mRNA的转录本长度，外显子数量与长度，内含子长度，GC含量，AS事件数量。【结果】在蜜蜂球囊菌孢子中共鉴定到2 609次AS事件，包括1 227次(47.03%)内含子保留(retained intron，RI)，842次(32.27%)可变3ʹ剪切(alternative 3ʹsplice sites，A3)，415次(15.91%)可变5ʹ剪切(alternative 5ʹsplice sites，A5)，85次(3.26%)可变起始外显子(alternative first exons，AF)，35次(1.34%)外显子跳跃(skipping exon，SE)，4次(0.15%)可变末端外显子(alternative last exons，AL)和1次(0.04%)互斥外显子(mutually exclusive exons，MX)。通过RT-PCR证实了不同类型AS事件(RI、A5、SE和A3)的可靠性。共鉴定出5 552个基因含APA位点，其中含有>5个APA位点的基因数量最多，达到2 197个，其次为含有1个APA位点的基因，数量为1 149个；此外，含有2、3、4和5个APA位点的基因数量分别为782、596、477和351个。蜜蜂球囊菌孢子中全长转录本的上下游序列具有明显的碱基倾向性，U和A分别在转录本的下游和上游富集。共鉴定到953条lncRNA。与mRNA相比，上述lncRNA的外显子数量更少且长度更短，内含子长度更短，转录本长度更短，GC含量更低，AS事件数量更少。【结论】本研究全面解析了AaS中的AS、APA和lncRNA，研究结果丰富了蜜蜂球囊菌的基础生物学信息，深入揭示了蜜蜂球囊菌孢子中转录组的复杂性，并为进一步探究不同剪接异构体在病原孢子和病原侵染中的分子功能打下了基础。

Abstract:

[Objective] Ascosphaera apis is a lethal fungal pathogen of honey bee larvae. This study aims to identify and analyze alternative splicing (AS) and alternative polyadenylation (APA) of genes and long non-coding RNAs (lncRNAs) in A. apis spore (AaS) by PacBio single molecular real-time (SMRT) sequencing, further unveiling the complexity of AaS's transcriptome. [Methods] AS events of genes in AaS were identified with Suppa, and AS events of various types were confirmed based on RT-PCR. TAPIS pipeline was utilized to explore APA sites of AaS genes. MEME was employed to investigate statistics of sequences at 50 bp upstream of poly(A) splicing sites and identify the motifs. The lncRNAs were predicted based on CPC, CNCI, and Swiss-prot database, and the intersection was regarded as lncRNA dataset. Then the transcript length, exon number and length, intron length, GC content, and AS event number were compared between lncRNAs and mRNAs. [Results] A total of 2 609 AS events were identified in AaS, including 1 227 (47.03%) retained introns (RI), 842 (32.27%) alternative 3ʹ splice sites (A3), 415(15.91%) alternative 5ʹ splice sites (A5), 85 (3.26%) alternative first exons (AF), 35 (1.34%) skipping exons (SE), 4 (0.15%) alternative last exons (AL), and 1 (0.04%) mutually exclusive exon (MX). The reliability of various AS events such as RI, A5, SE, and A3 was validated via RT-PCR. Additionally, 5 552 genes containing APA sites were identified, among which genes with more than 5 APA sites were the most abundant (2 197), followed by genes with 1 APA site (1 149). The numbers of genes with 2, 3, 4, and 5 APA sites were 782, 596, 477, and 351, respectively. Moreover, upstream and downstream sequences of full-length transcripts in AaS had apparent base bias, with U and A respectively enriching at upstream and downstream. In total, 953 lncRNAs were identified, including 247 bidirectional lncRNAs, 171 long intergenic RNAs, 154 anti-sense lncRNAs, 141 sense lncRNAs and 9 intronic lncRNAs. Compared with mRNAs, these lncRNAs had fewer and shorter exons, shorter introns, shorter transcripts, lower GC content and fewer AS events. [Conclusion] In this study, we analyzed the AS, APA, and lncRNA in AaS. The findings enrich the basic biological information of A. apis, unravel the complexity of transcriptome in AaS, and lay a foundation for further exploration of the molecular function of various splicing isoforms in pathogenic spores and during pathogen infection.