Identification Of Alternative Splicng In Plutella Xylostella And Macrocentrus Cingulum

Alternative splicing(AS)is the main mechanism that increases the biodiversity of proteins that can be encoded by the genome and regulates gene expression.Large-scale analysis of alternative splicing becomes possible along with accomplish of many insects'genome,rapid advances in high-throughput sequencing technology and continuous improvement in transcriptome assembly algorithm.In our research,we build and optimize the prediction workflow of alternative splicing in insects,and prediction and analysis was also been made for alternative splicing in Plutella xylostella and Macrocentrus cingulum.The results are as follows:1.Construction and optimization of alternative splicing prediction in insectsDue to the low quality of insects genome assembly and less RNA-Seq data,traditional methods cannot be applied to agricultural insects,so we construct and optimize the alternative splicing prediction in insects.The whole process includes read quality filtering,Transcriptome assembly,ORF prediction,evaluation of assembly quality,gene annotation,and isoforms differential expression analysis.In the process of pipline construction,we compared different parameters combination and pick the optimal one.The most reliable gene prediction result was screened out by ORF prediction based on conservation of protein domains.2.Alternative splicing analysis of Plutella xylostella genesThe pest P.xylostella(Lepidoptera:Yponomeutidae)has become the most destructive pest of economically important food crops,including rapeseed,cauliflower,cabbage and some other crucifer plant family.Recently,the total cost of damage and management wordwide was estimated at $4-5 billion every year.This insect has developed resistance to all classes of insecticide,making it increasingly difficult to control(You et al.,2013).Here,we use the public avaiable RAN-Seq data(include egg,larva,pupa,adult,the larva of chlorpyrifos resistance,the larva of fipronil resistance,midgut and the larva of Bt sensitive RNA-Seq data)to predicted 10,701 alternative splicing genes,67.7%of all multiple exon genes.We find the AS gene have longer isoforms,CDS and 3'-UTR than without AS gene.More than 65%protein genes have more than 80%integrity,and it's more easy to find AS events as a longer gene assembly.There 323 resistance genes among 13 category,and 73.0%of them are AS genes.The traditional expression differential analysis general focus on genes,the gene's expression is total expression of it's isoforms.We compare expression in isoform level,find 17 Isoforms related to resistance differential expressed between sensitive and chlorpyrifos resistance strain,and 24 isoforms related to resistance differential expressed between sensitive and fipronil resistance strain.We also find 79,98,26,85 isoforms specific express in egg,larva,pupa and adult of P.xylostella.This chapter describe the general picture of alternative splicing in P.xylostella,and provide data for alternative splicing analysis of P.xylostella.The result indicated that the insecticide resistance have related to alternative splicing.The expssion differential analysis in isoform level is more accurate than in gene level.3.Alternative splicing analysis of Macrocentrus cingulum genesThe Macrocentrus cingulum(Hymenoptera:Braconidae)is a polyembryonic endoparasitoid parasitoid of Ostrinia furnacalis,it is used to control O.furnacalis.Here,we use the RNA-Seq data of egg,larva,male and female to predict 4580 AS genes,58.3%of multiple exon genes,and about half of them only have 2 isoforms.The gene annotation result indicate that M.cingulum have most similar genes with Megachile rotundata and Nasonia vitripennis.We find the intron of M.cingulum is short than it's exon,The AS genes's loci,isoforms,CDS and 3'-UTR are longer than without AS genes.The AS and without AS genes have similar 5'-UTR and exon length,and the AS genes have more exon number than without AS genes.The transcriptome assembly quality assessment indicate more than 71.3%protein genes have more than 80%integrity.