Reserech Of AS Events In Metarhizium Robertsii By RNA-seq

Entomopathogenic fungi are those that can directly invade the body,proliferate and cause death rapidly under host normal physiological condition.They have a wide variety of lifestyles and main life-styles are saprophytes,parasitism,rhizosphere colonization,respectively.Currently,Beauveria spp.and Metarhzium spp.are the most studied among entomopathogenic fungi.Metarhzium spp.has characteristics of easy cultivate and a wide range of insect hosts,therefore,it widely used in biological control of the locusts,citrus gibberella et.al at present.AS enables some genes to produce multiple m RNA isoforms,which is an important mechanism for regulating gene expression and expanding proteome diversity.It is also an important reason for the significant difference of gene and protein in eukaryotic.Although the research of AS is pretty popular in higher organisms,it is lacking to entomopathogenic fungi.Metarhizium robertsii as an important entomogenous fungus,which has rarely research for AS in its transcriptome.In order to further understand AS effect to M.robertsii under different living conditions,we started from a variety of culture conditions to obtain more comprehensive transcriptome sequencing data,and finally obtained the specific data of AS at the transcriptome level.In this study,M.robertsii as the research object,which cultured in various environments,including different developmental stages from solid and liquid situations,ageing,anti-stress,infection and colonization.And tatal RNA from spores or hyphae at different stages was extracted and mixed with equal volume and equal concentration.And then performed to RNA-seq to obtain AS data in M.robertsii.Secondly,the differences of gene expression for different number of m RNA isoforms were analyzed by bioinformatics.Finally,data reliability was verified by RT-PCR.In the end,AS data was uncovered in M.robertsii.The results are as follows:1.Overabundant AS events in M.robertsii:All of common AS events were identified from RNA-seq data.There were four types in these events,including intron retention?IR?,alternative 3' splice site?A3SS?,alternative5' splice site?A5SS?,and exon skipping?ES?.Surprisingly,it was up to 5005 genes involved in 10471 AS events.The proportion of alternatively spliced genes reached up to47.3% of the total number of genes.2.Splicing regulators of M.robertsii and their variable splicing events:In our RNA-seq data,we found 64 genes were correlated with splicing regulators.They were 44 genes encoded SFs,and 7 genes encoded RBPs,3 genes encoded SRs,as well as 10 genes encoded sn RNPs or sn RNPs-like proteins.12 of them shared sequence homology that have been described as splicing regulators in other fungal species by BLASTnr and with an E-value<2E-53.Compare with Aspergillus oryzae,the genes of U2 AF were identified with larger alternatively spliced isoforms.It suggested that gene structure may be closely related with the frequency of AS.3.AS events of protease genes associated with virulence:In protease families,30 genes were expressed as subtilisin and subtilisin-like serine proteases,whereas 55 genes in genome.Thus,more than half of them were expressed.However,it did not reach to one third gene about trypsin and trypsin-like enzymes were presented in our data.By coincidence,half of subtilisin protease genes were underwent AS.Among these alternatively spliced genes,MAA₀4783 not only possessed 2 A3 SS events but also 2 IR events,but others' AS events were belong to IR.By contrast,only one of 10 expressed genes about trypsin family was underwent one AS event,and which belong to IR.Therefore,we deduced that subtilisin family may be more easier undergo AS than trypsin family in M.robertsii.4.Expression and AS events of chitinase genes associated with virulence:Accoroding to 26 chitinase genes in NCBI database,17 of them were expressed in our RNA-seq data.However,about one of third of these expressed genes possessed sequence homology with other Metarhizium spp.by BLASTP homology search against the NCBI database.6 of them were underwent AS events.Unexpected,IR was the common type of AS events for these genes,just MAA₀0983 still had one A3 SS event.We figured up 11 isoforms of the alternatively spliced genes and found that MAA₀2685 encoded the most 3transcript isoforms.In addition,there was a significant difference about style of AS between the chitinase gene chi2 in M.robertsii and a high sequence homology of the chi2 in Metarhizium anisopliae E6.The former retained the first intron,but the latter retained the second intron.5.Functional annotation and classification of alternatively spliced genes:GO functional annotation was performed to alternatively spliced genes and normal spliced genes,respectively.And we found that the most enriched part of alternatively spliced genes were existed in metabolic process of BP?821?,cell?439?and cell part?439?of CC,catalytic activity of MF?870?.But we also found some differences between normalspliced genes and alternatively spliced genes.Enzyme regulator activity which belongs to MF was only identified in alternatively spliced genes?21 genes?.Compared with ⁰.98 %of signaling genes in normal spliced process,³.3% genes were found in alternatively spliced process.KEGG pathway enrichment analysis shown that gene which possessed more isoforms were significant enriched in MAPK signaling pathway and ubiquitin mediated proteolysis pathway.This research found out abundant AS events in M.robertsii,which not only contributes us to change the knowledge that less alternatively spliced genes in lower organisms,but also enables us to further understand the relationship between alternatively spliced genes and biological characters of entomopathogenic fungi.Therefore,this research can provide a reference for AS events about entomopathogenic fungi.