Assessment Of The Alternative Splicing And Regulation Analysis Of Response To Vibrio Harveyi Infection In Japanese Pufferfish

Japanese pufferfish(Takifugu rubripes),which lives in most sea area of Northeast Asia,is a species of economic fish with high commercial value.In recent years,the product source of Japanese pufferfish has gradually changed from wild fishing to artificial cultivation,but various diseases seriously impact the expansion of its breeding scale.Vibrio harveyi is a common conditional pathogen in seawater that would lead to high mortality of Japanese pufferfish.Alternative splicing is one of the most critical ways of post-transcriptional regulation.It is one of the important contributors to the complexity and proteome diversity of eukaryotic transcriptome and plays an important role in the body’s response to environmental changes.In this study,through high-throughput sequencing technology,third-generation sequencing methods were used on spleen tissue of Japanese pufferfish infected with V.harveyi.The spleen m RNA library of Japanese pufferfish before and after V.harveyi infection was constructed and sequenced.A total of 17.38 G sequence data and 9735030 subreads were obtained.By the bioinformatic analysis of the full-length transcript,a total of 54860 full-length non redundant transcripts,3736 novel genes,8571 alternative cleavage and polyadenylation events,4221 lnc RNAs,2759 transcription factors and421 fusion transcripts were identified.Combining NGS transcriptome data of the spleen in T.rubripes under V.harveyi infection,the presence of 153591 alternative splicing events were predicted,which led to the identification of five different types of events including exon skipping,intron retention,variable 3 ’splicing sites,variable 5’ splicing sites,and exon mutual exclusivity.Generated from 950,1181,and 1863 genes,number of differential alternative splicing events in T.rubripes at 12 h,24h and 48 h infected with V.harveyi were filtered by r MATS software to be 974,1196,and 1911.GO functional enrichment analysis results showed that a total of 62 DAS genes were classified to 6 common GO terms at different time points,including innate immune response,ATP bind,tyrosine phosphorylation,etc.KEGG enrichment analysis demonstrated the DAS genes in several signaling pathways like spliceosome,apoptosis,and JAK-STAT pathway.The accuracy and reliability of the experimental results were verified by RT-PCR and q PCR experiments.This study is the first one to analyze the full-length transcriptome of the spleen in T.rubripes.In this study,we identified alternative splicing events of Japanese pufferfish infected with V.harveyi,selected DAS genes,and finished functional enrichment analysis for DAS genes.The study elucidated the regulatory mechanisms after T.rubripes infection with V.harveyi at the level of post-transcriptional regulation,revealed the significant role of alternative splicing during the immune response progress in T.rubripes,and provided support for dissecting the mechanism of immune regulation in T.rubripes.