| Coccinella septempunctata L.is an excellent predator of many aphid species of fruit trees and crops,with large appetite,wide range of adaptability and high reproductive ability.Diapause is a genetic phenomenon for insects to adapt to the adverse environmental conditions.Using of this physiological characteristic could provide a new technology to control pests by greatly extending the shelf life of natural enemy's products.Rencently,with the development of diapause research,in C.septempunctata,the physiological and biochemical changes and control means of insect diapause have made some progress,while molecular mechanism of diapause is still unclear.In this study,we collected female adults in three different states,i.e.,non-diapause(ND),diapause(D)and diapause termination(DT),for transcriptome sequencing by Illumina Hisq 2500 platform.Through the de novo assembly and gene annotation methods,differentially expressed genes(DEGs)were screened by pairwise comparisons of three sequencing data.Furthermore,candidates of diapause associated genes were filtered to provide support for further study of biological function of genes.To verify the reliability of transcriptome results and understand the change trend of genes during diapause,we carried out the quantative PCR.Moreover,the diapause associated gene juvenile hormone esterase(JHE)was cloned by RT-PCR and RACE(rapid-amplication of cDNA ends),and preliminarily analyzed by bioinformatics.The main results are as follows:1.Transcriptome sequencing for C.septempunctata in three states,ND,D and DT,were carried out using Illumina Hisq 2500 technology.The raw reads were filtered for removing unqualified reads.82820 unigenes were yielded by de novo assembly with an average length of 921 bp.All unigenes assembled were aligned with Nr or uniprot database using BlastX,and 37,872 unigenes were matched.Concurrently,all unigenes were classified for function using COG,GO and KEGG database.By pairwise comparisons of three groups,3,501 and 1,427 DEGs were identified between D and ND and between DT and D,respectively.GO and KEGG enriched analysis for these DEGs revealed that fatty acid synthesis pathway were most abundant.2.Further analyzing for compassion results of two groups,the DEGs which was highly expressed in the diapause stage compared to non-diapause and diapause-terminated stages was defied as diapause associated genes.Total of 443 unigenes were found to be diapause associated genes.The analysis of KEGG enriched pathways were performed on diapause associated genes using KAAS(KEGG Automatic Annotation Server)and found that these genes were mainly involved in carbohydrate metabolism,lipid metabolism and signal transduction.3.According to 8 genes screened from the former study and associated with diapause,the transcriptome results were verified using qPCR.The results showed that qPCR validation and transcriptome sequencing results were basically consistent,indicating that the transcriptome results were credible.While the expression of these genes in 6 time periods during diapause stage were compared,the results showed that most of the genes were highly expressed in diapause 30 or 40d,suggested that ladybird entering diapause maintenance phase were after diapause 30d.The phenomenon also indirectly proved that collection method of diapause sample was reliable.4.JHE is one of the key enzymes in juvenile hormone metabolism.By using RT-PCR and RACE technology,CsJHE genes were cloned.The full-length of this gene was 2317bp,including a 1755bp open reading frame(ORF),encoding 584 amino acids,predicted that the gene encoded a protein with a molecular weight of 66.46ku and theoretical isoelectric point was 8.36.Conserved domain analysis found that the protein encoded by this gene had two complete domains,carboxylesterasse family and esterase_lipases superfamily,and the predicted results of this gene was consistent with other insects.Moreover,construction results of phylogenetic tree showed that C.septempunctata was a closest relative of Harmonia axyridis. |
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