Transcriptome-Based Global Gene Expression Profile And Temporal Changes Of Specifically Highly Expressed Genes Of Bombyx Mori

Bombyx mori is an important lepidopteran model insect with complete metamorphosis.The whole life span of B.mori contains four major stages: egg,larva,pupa and moth.Silkworm farming in China has a history of more than five thousand years,and people already have a clear and systematic understanding of the physiological changes of silkworms.At the early twenty century,with the deciphering of the silkworm genome,the association of various silkworm physiological characteristics with the information of the genome,transcriptome,and proteome has been gradually carried out.However,the global gene expression profile during silkworm development and the associations among these genes and traits remain poorly understood.Therefore,this research collected ten temporal samples at four developmental stages: egg,larva,pupa,and moth to do RNA sequencing and bioinformatics analyses,to clarify the genes temporal changes in different stages,and to infer the correlation between some genes and traits.Also on the basis of transcriptome data and previous researches,we selected some core genes for temporal analysis,and validated these genes by real-time PCR.The main results obtained were as follows:1.From the high-throughput RNA sequencing results of ten stages: eggs of 24 h after oviposition(Ed),eggs of 24 h after artificial egg-hatching(E),larvae from first to fifth instar(L1～L5),the fourth and the eighth day of pupa(P4 and P8),and moth(M),a total of 15745 genes were detected.The number of specifically expressed genes at Ed,E,L1～L5,P4,P8 and M stages are respectively 758,570,151,158,102,217,89,485,745 and 1001.2.Results showed that 758 genes specifically expressed in Ed stage were not expressed after acid immersion at E stage,and 570 new specific genes started to express.According to GO and KEGG enrichment analysis results of these specifically expressed genes,structural constituent of cuticle is most related to Ed stage.In order to get more accurate and credible results,the genes with higher expression levels were selected.The top 100 up-regulated and down-regulated genes were selected to do PPI.As a result,5 down-regulated proteins were found to have interaction relationships,suggesting the 5 proteins have a certain relationship with the diapause egg stage.According to enrichment analysis results of specifically expressed genes from L1 to L5,speculated that some specific genes of larvae stage are related to hormones,protein synthesis,reproductive development and so on.The L2 stage-specific genes are most related to hormone action,the L3 stagespecific genes are mainly used for self-repair and respond to external stimuli,and the L4 stage-specific genes are mainly used for amino acid modification,protein synthesis and other functions.PPI of L1～L3 specifically-expressed genes found 6core proteins,while PPI of L4～L5 found another 9 core proteins.The results of enrichment analysis showed that the 485 specifically expressed genes in P4 stage mainly performed enzymatic activities and cholesterol metabolism,and the 745 specifically expressed genes in P8 stage were mainly involved in neuroactive ligand-receptor interactions and protein digestion and absorption.Also,ProteinProtein Interaction Networks were done to the specifically-expressed genes of P4,P8 and M stages respectively,finding 7,6 and 6 core proteins separately.3.Based on the temporal analysis of the transcriptome data,12 genes were screened and performed PCR verification.The results showed that the transcriptome data results were most consistent with the PCR verification results.Egg stage highly expressed genes BGIBMGA002381(BMHSC70-4)and BGIBMGA003608(EF-1A),larva stage highly expressed genes BGIBMGA013945,BGIBMGA002259(MLC-2),BGIBMGA004074(BMORCPG19),BGIBMGA007469(EIF5A),BGIBMGA010571,BGIBMGA014298 and BGIBMGA003326,pupa stage highly expressed genes BGIBMGA012305(IGFLP),BGIBMGA004045(CSP4)and BGIBMGA009714(BMORCPH1),moth stage highly expressed genes BGIBMGA009647(BMO.7284)and BGIBMGA014360(IRP1),further discussion and analyzation of these genes’ functions suggested that these genes function in specific periods through whole life.