| The Oriental Fruit Moth (OFM) Grapholita molesta (Busck) is a serious insect pest of stonefruit and sometimes pip fruit in many parts of the world. However, there are very few research reports about genes involved in development, insecticide resistance, sex pheromone biosynthesis, etc. The genomic resources available for G. molesta are scarce. In this study, firstly, we constructed a de novo transcriptome assembly for the OFM, Grapholita molesta; secondly, we verified the expression of the related genes involed in ecdysone signaling pathway; thirdly, we studied the effect on the related genes involed in ecdysone signaling pathway after juvenile hormone analogue treatment; finally, the molecular characterization and functional analysis of the cDNA encoding ecdysone receptor B1 (EcRB1), ultraspiracle (USP) isform 1 and three isforms of Broad Complex (BrC) were studied. Our main conclusions are as follows:(1) After data cleaning and assembly, we finally obtained 81,898 transcrips and 41,882 unigenes. Transcriptome sequences were subjected to Gene Orthology (GO) and KEGG Orthology (KO) annotations. In the classification of KOG and KEGG pathways, lots of transcript sequences associated with signal transduction were founded, this would provides abundant gene information for our subsequent experiments.(2) A total of 11 non-redundant unigene belong to 6 candidate reference genes were obtained from the transcriptome database. The non-redundant unigene sequences corresponds to each gene were searched through artificial selection. EF-1α was slected as the most stablely reference gene in the condition of different development stages and used for further experiments.(3) A total of 13 non-redundant unigene belong to 8 ecdysone signaling pathway genes were obtained from the transcriptome database. cDNA Library verification indicated that the ecdysone signaling pathway genes expression were consistent with the results in the transcriptome sequencing. The expression of ecdysone signaling pathway genes in different developmental stages indicated that those genes existed a high level expression in the transformation process of larvae-pupae and pupae-adult. Results of juvenile hormone analogue treatment showed that the expression of the ecdysone signaling pathway related genes were inhibited. Juvenile hormone can regulate the expression of those genes, and there exists a mutual regulation between these genes.(4) Bioinformatics analysis of EcRBl and USP1 sequences were conducted. The results of amino acid sequence multiple alignment showed that the amino acid sequence of GmEcRB1 and GmUSP1 genes had a higher identify with other insects. Phylogenetic analysis showed that Lepidoptera species with close relationship clustered together. Tissue expression pattern analysis shows that: GmEcRB1 gene has a tissue-specific, it may be involved in tissue remodeling during metamorphosis. The expression of GmUSPl gene has a stage-specific and tissue-specific. EcRB1 and USP1 gene interference efficiency results showed that the interference effect reached to the best two days after dsRNA injection, Effect on the expression of ecdysone signaling pathway genes after injected GmEcRBl dsRNA and GmUSPl dsRNA shows that GmEcRBl and GmUSP1 can regulate the expression of 20E induced primary response gene. The development statistical shows that EcRB1 and USP1 play important roles in the development of oriental fruit moth metamorphosis, they prolong the pupation period, and influence the development of oriental fruit moth.(5) Bioinformatics analysis of three Broad Complex isform sequences (Z1, Z2 and Z4) were conducted. Amino acid sequence multiple alignment results showed that the amino acid sequence of GmBrCZ1, GmBrCZ2 and GMBrCZ4 genes had a higher identify with other insects. Phylogenetic analysis showed that BrCZl, BrCZ2, BrCZ4 type in different species is divided into those three groups. In each group, G. molesta was clustered with other Lepidoptera insects, such as, Bombyx mori, Spodoptera exigua, etc. The expression of three GmBrC isoforms in different developmental stages indicates that the expression of BrC has a stage-specific. Expression of three GmBrC isoforms in different tissues suggest that they may involved in the formation of epidermis and during metamorphosis. Results of juvenile hormone analogue treatment shows that juvenile hormone can regulate the expression of GmBrC. Interference efficiency shows that the expression of GmBrC in vivo was significantly inhibited after GmBrC dsRNA injected. The development statistical showes that BrC involves in the development of the oriental fruit moth metamorphosis, and influence its development. |
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