| Metamorphosis is an important feature in holometabolous insects.They undergo four different stages along their life time:egg,larva,pupa and adult.They possess unique adaptability to environment in each of the developmental stages,which contributes most to their widely spreading all over the world.Under the synergy of molting hormone(20E)and juvenile hormone(JH),a large number of response genes are up-regulated or down-regulated in accordance with propriate time and tissues.The insects molting and metamorphosis are initiated and executived then.Striped stem borer,Chilosuppressalis belongs to Lepidoptera and goes through complete metamorphosis.C.suppressalis is an important agricultural pest,causing huge losses on rice production.The continuously use of chemical pesticides not only causes resistance year after year but also increases the pollution to the environment.Study on metamorphosis of C.suppressalis will fill in blank in the agricultural pests in metamorphosis.It can provide potential targets or a theoretical basis for gene-silencing based pest control.1.Expression profile of protein coding genes at seven developmental stagesA total of 22,197 transcripts were obtained from stage-mixed sample via large-scale sequencing technology.Seven developmental stages were seclected and they are:ageing larvae,prepupae,early pupae,compound eye formation stage,pretarsus formation stage,pupae elongation stage and adult.The expression profile of 22,197 transcripts across multiple stages was detected by microarray.In time-development analysis,487 genes were up-regulated with time sequence.There were 795 genes were down-regulated,however.In time-specific analysis,622 genes were highly differentially expressed and they were clustered into 6 expression patterns.There were 79,70,154,and 79 genes highly expressed at ageing larvae,early pupae,compound eye formation and pretarsus formation stages,respectively.In the mean while,there were 182 genes highly expressed at ageing larvae-prepupae-early pupae stages and 58 genes highly expressed at pupae elongation-adult stage.Functional enrichment analysis were carried out based on 622 genes by KEGG and Blast2Go.Results showed that genes related with substances metabolism and energy use mainly expressed at ageing larvae-prepupae-early pupae stages.However,genes related with signal transduction,cell contents and development were highly expressed at ompound eye formation stage and pupae elongation-adult stage.Hormone related gene were found mostly at pupation and early pupae stage.Nine hormones related genes were verified using quantitative PCR and expression trend of eight genes showed perfect match with microarray.2.Functional study of SLC22A1 gene at pupationThe most differentially expressed genes in 6 expression patterns were:1)ageing larvae stage,tyrosinase;2)ageing larvae-prepupae-early pupae stages,pancreatic triglyceride lipase;3)early pupae stage,solute carrier family member 1,SLC22A1;4)compound eye formation stage,Domon domain containing protein;5)pretarsus formation stage,cuticle protein 19;6)pupae elongation stage-adult stage,solute carrier family 2.SiRNA was designed based on the six genes and injected to specimen at appropriate time to study potential function.The mRNA expression of SLC22A1 was dropped to 43.8%compared with control group(p=0.02486)24 hours after injection with siRNA.Besides,88.9%of treated specimen showed defects of milky and soft epidermis at abdomen,back or head.The pupae became shriveled and died in one week.This phenotype was similar to a mutant of "white pupa" in Mediterranean fruit fly.We proposed that SLC22A1 be involved in the transportation of tyrosine hemolymph to pupa epidermis.Further analysis revealed that a total of 27 solute carrier family gene were highly differentially expressed in different stages.They belong to 10 subfamilies:SLC2A,SLC16A,SLC22A,SLC26A,SLC27A,SLC30A,SLC46A,SLC36A,Orct2 and Orct-like family.The result indicated that the SLC family genes participate in process of pupation,pupal development and emergence of C.suppressalis.However,there were no phenotype observed in RNA interference experiment of other five genes.3.The discovery and expression profile analysis of microRNA genesA small RNA library was constructed from stage-mixed sample of C.suppressalis.And 103 conserved and 197 non conserved csu-miRNAs were discovered using a bioinformatics pipeline.54 csu-miRNAs were differentially expressed across seven developmental stages using microarray.The csu-miRNA of which signal value was greater than 500 in at least one developmental stage was considered reliable.Twenty-one csu-miRNAs met the criterion.There were 5,4,1,4,1 and 6 csu-miRNAs highly expressed in ageing larvae,prepupae,early pupae,compound eye formation stage,pupae elongation and adult stages,respectively.2116 genes were predicted to be targeted.KEGG enrichment analysis of these target genes found more genes in three pathways(amino acid metabolism,glycan biosynthesis and metabolism,carbohydrate metabolism)in early pupa stage than that in other stages.The result indicated that more genes related with substances metabolism and energy use maybe negative-regulated by csu-miRNA in early pupa stage.Quantitative PCR was performed to validate the results of microarray.10 of 21 were confirmed and they were:1)decreased gradually,csu-miR-275,csu-miR-6094;2)decreased first and increased then,csu-miR-34,csu-miR-281-5p;3)increased first and then decreased,csu-miR-263a,and csu-novel-193 csu-miR-2779;4)increased gradually:csu-miR-14,csu-miR-277 and csu-miR-989a.4.Functional study of csu-miR-277.csu-miR-6094 and csu-miR-275Csu-miR-277 started to highly express in the middle-pupa stage and reach to top at adult stage.Mimics of csu-miR-277 was injected at ageing larvae stage.This resulted in a"lost" of prepupae stage in-30%treated specimen.These specimen underwent shorter duration of prepupae stage than control group and showed precocious metamorphosis to some extent.Imaginal disk growth factor(IDGF)was one of target genes of csu-miR-277.Molecular cloning resulted in 2,126 bp sequence of csuIDGF including 1,326 bp CDS and 781 bp 3’UTR with poly(A).The expression of csuIDGF showed decrease trend from ageing larvae to adult.The expression of csuIDGF and csu-miR-277 showed negative correlation(P=0.0164).Dual luciferase report gene vector system validated that csu-miR-277 targeted 3’UTR of csuIDGF.Inhibitor of csu-miR-277 was injected at compound eye formation stage.The treated specimen molted to moth as normal as control group.The pupae were dissected at 48 hours and 72 hours after injection.The head,fat body and wing of inhibitor injected specimen were also the same as control group.Besides,both csu-miR-6094 and csu-miR-275 highly expressed at ageing larvae stage.Mimics of the two csu-miRNAs were injected at 2nd day of 4th instar,respectively.However,the treated group developed normally.They did not pupae nor show precocious metamorphosis.In view of the complexity of development of miRNA,further research is needed.5.Network regulation analysis of mRNA-miRNAWe tried to carry out correlation analysis based on the expression profile of both mRNA and miRNA microarray.The genome of C.suppressalis was used to get reliable 3’UTR of mRNA in transcriptome data set.There were 46 csu-miRNAs and 205 mRNAs in the result of positive correlation.Eight csu-miRNAs and ten mRNA were predictied to be in a network.There were 39 csu-miRNAs and 110 mRNAs in the result of negative correlation.Moreover,the expression of SLC25A27 was negatively correlated with the expression of csu-miR-11 and 7 SLC family genes and 6 csu-miRNAs were in positive correlation.In summary,we studied the expression profiles of large-scale protein-coding genes and miRNAs across seven developmental stages from ageing larvae to adult in C.suppressalis.Many genes were differentially high-expressed at pupation,pupal development and adult processes,showing they played important roles in metamorphosis.Besides,correlation analysis of mRNA and miRNA microarray resulted in positive,negative and regulatory networks.This will provide basis for further research.Insect metamorphosis can provide ideas for pest control,20E and JH agonists and inhibitors have been used as insecticide.But gene silencing based methods appiled in pest control is rear.The research work in this paper will provides a theoretical basis and guidance. |
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