Transcriptome Sequencing Of Megalurothrips Usitatus And Identification Of Olfactory Related Protein Gene

Olfactory proteins of insects have great importance in the process of communication with the outer world.The odorant-related proteins mainly consist of six proteins,namely odorant binding protein,chemosensory proteins,odorant receptors,sensory neuron membrane proteins,ionotropic receptor and odorant degradation enzyme respectively.The bean flower thrips,Megalurothrips usitatus(Thysanoptera,Thripidae)is a major pest of leguminous plants in South China.In this study,different developmental stages of M.usitatus were sampled for transcriptome analysis through RNA-Seq sequencing.Four olfactory-related proteins of M.usitatus were cloned and their expression in different developmental stages was analyzed.The aim of this study was to find out olfactory-related proteins in this insect pest and interpretation of olfactory behavior molecular mechanisms of M.usitatus.1.The larvae,pupa,female and male adults M.usitatus were collected for transcriptome sequening.Total RNA was extracted from each sample.The cDNA library was sequenced on the Illumina sequencing platform.Finally,we produced 45,683,836 clean reads with the average length of 150 bp,and obtained 6,852,575,400 bases.These reads were further assembled into 78,474 unigenes with the average length of 1,904 bp,which range from 200~26,358 bp.N50 length of unigene was 3,742 bp.Based on similarity analysis,we obtained 54,271 high quality unigenes after BLAST against databases.Species distribution analysis showed that the matching degree with Zootermopsis nevadensis was the highest,which was 19.95%.2.We identified 9,000~19,000 significantly differential expressed genes from pairwise developmental stages(FDR?0.001,fold-change>2)using expression profiled analysis methods.These unigenes were assigned to GO functional classification consisting of biological process,cellular component and molecular function.KEGG pathway analysis of DEGs showed that 7,000~14,000 unigenes were involved in 296~299 metabolic or signaling pathways.Genes related to reproductive metabolic pathways,regulation and control of circadian rhyrhm-fly,nucleotide replication and transcription,protein translation and modification,phosphatidylinositol signaling system etc.,were up-regulated or down-regulated to some extent.Thus,these genes may play an important role in the growth and development,physiological rhythms of M.usitatus.3.We cloned and identified three olfactory-related protein genes with full-length cDNA and one cDNA fragment from M.usitatus,named MusiCSP1,MusiCSP2,MusiOBP1 and MusiOBP2.The full-length cDNA of MusiCSP1,MusiCSP2,MusiOBP1 genes are 639 bp,708 bp and 645 bp,and contained 405 bp,405 bp and 414 bp open-reading frame encoding putative proteins of 134,134,137 amino acids with a molecular weight of 15.185,15.065,14.754 k Da and an isoelectric point of 7.57,8.21,4.15,respectively.The deduced amino acid sequence of MusiCSP1,MusiCSP2,MusiOBP1 contained putative signal peptide of 19 amino acid residues at the N-terminus.The cDNA fragment of MusiOBP2 gene was of 1,014 bp,which did not contain the open-reading frame encoding.4.Research on expression profiling of olfactory-related protein genes of M.usitatus showed that MusiOBP1 in one-day-old adult females had the highest expression,followed by first and second-instar nymphs and one-day-old adult males,with the lowest expression in prepupa.MusiOBP2 in one-day-old adult females had the highest expression,followed by first and second-instar nymphs,with the lowest expression in pupa.MusiCSP1 in prepupa had the higher expression,followed by first-instar nymphs and adults,with the lowest expression in prepupa.MusiCSP2 in prepupa and one-day-old adult males had the highest expression,followed by second-instar nymphs,with the lowest expression in one-day-old adult females.