Expression Of Diapause Associated Gene And Cloning Of Tn C In Aphidius Gifuensis Ashmead

Aphidius gifuensis Ashmead is an excellent solitary endoparasitoid natural enemy insect,which can control a variety of aphids effectively,especially the aphid Myzus persicae?Sulzer?.But,there are some problems in the production and application,such as long production cycle,short shelf-life and disconnect of supply and demand,which makes it difficult to maximize its biocontrol role at the outbreak of pests.Diapause can evoke unique pattern of gene expression and represent an alternative developmental pathway prompted that result in the sequestration of nutrient reserves,suppression of metabolism,a halt or slowing of development,and the acquisition of increased tolerance to environmental stresses such as low temperature.This attribute plays an important role in the mass production and application of A.gifuensis.It is not only can enhance adaptive capacity and parasitic ability of offspring,but also can increasethe shelf-life of the biological control agents and promote the application of this aphid parasitoid.Recently,deep research has been performed on environmental regulation and physiological biochemistry of diapause in A.gifuensis,while the molecular regulation mechanism of diapause is still unclear.In this study,RNA sequencing werecarried out to compare no-diapause?ND?,diapause?D?and post-diapause?PD?groups of A.gifuensis,which improved the conventional methods of analyzing.Differentially expressed genes?DEGs?were selected by pairwise comparisons of three groups,and the diapause associated genes that specifically expressed in diapause were screened out.To verify the reliability of RNA-seq and the gene expression trends,we carried out the qRT-PCR on 9 candidate genes.Moreover,the diapause associated gene troponin C?TnC?was cloned by RACE?Rapid-amplication of cDNA ends?,and analyzed by bioinformatics.These results laid foundation for the functional verification and further study of diapause associated genes.The main results are as follows:1.We studied the transcriptome characteristics of different developmental states of A.gifuensis.Using RNA sequencing and sequence alignment and annotation,we obtained 47,077 unigenes with an average length of 868 bp,and there are 61.4%of unigenes were matched.By pairwise comparisons of three groups,we obtained 458 DEGs between ND and D,298 DEGs between PD and D,and 806 DEGs between ND and PD,respectively.The structural constituent of cuticle was enriched significantly in GO enrichment of three groups.The cardiac muscle contraction and the antigen processing and presentation were enriched significantlyin KEGG enrichment of three groups.The correlation analysis between proteomics and transcriptome showed that there are 2,245 proteins were correlated,157 proteins were differentially expressed,and 99 differentially expressed proteins have consistent expression trend.2.We analyzed the types and functions of diapause associated genes of A.gifuensis.After further re-screening the DEGs,total of 59 diapause associated genes were obtained,and 46 of them have been annotated.These genes were primarily associated with cold tolerance,autoimmune defense,lipid metabolism,cuticle protein metabolism,transcriptional regulation.Moreover,9 DEGs were selected for Real-time quantitative PCR analysis,and the results were basically consistent with the transcriptome data,which further verified the reliability of RNA-seq results.3.We cloned the significant diapause associated genes of A.gifuensis.TnC exert its biological functions by binding Ca²⁺,which was not only can regulate muscle activity,but also can regulate cytoskeletal system indirectlyby interacting with actin.A total length of 703 bp was obtained by cloning AgTnC,including a 462 bp ORF,encoding 153 amino acids.The highly conserved sequence DXDXSX₆E were found by functional domain prediction and amino acid sequence alignment with other insects,and this conserved sequence isthe characteristic region of EF-hand protein family.AgTnC was up-regulated significantly during diapause.We speculated that it may enhance the function of muscle cytoskeleton,and specific regulatory function remains to be verified by further study.