Preliminary Study On Diapause Regulation By HSP70 And Nitric Oxide Synthase In Trichogramma Dendrolimi

Diapause is a phenomenon of developmental stagnation exhibited by insects in response to adverse external conditions.Insects can undergo diapause in various periods,but for a population of insects,the period of diapause is usually fixed.Insects under diapause show a significant reduction in metabolic rate and a significant increase in resistance to the external environment.These have led to an extension of insect lifespan.Insect diapause is a dynamic process,artificially divided into six stages.Trichogramma dendrolimi is an important egg parasitic wasp and has good control effect on the eggs of many important pests of Lepidoptera.In recent years,diapause technology has been successfully applied to the factory production of T.dendrolimi.Five different developmental stages(Diapause prepupae;Diapause terminating;Diapaused pupa,Normally developed prepupa stage,Non-diapaused pupa,NDp)were selected.The sequence characteristics of heat shock protein 70(HSP70)gene and nitric oxide synthase(NOS)gene were analyzed and gene expression levels at different developmental stages were analyzed using quantitative real-time PCR and western blot.The results are as follows:1.The full-length sequence of HSP70(Genbank No.MN709377)was cloned by RACE technology,with a length of 2,666 bp.The length of the open reading frame(ORF)was 1,992 bp,and the length of 5 'UTR and 3' UTR were 49 bp and 625 bp,respectively.Encoding 663 amino acid residues,containing 3 conserved regions of the HSP70 family,with molecular weight of 73 kDa and theoretical isoelectric point(pI)of the sequence of 5.5,it is a hydrophilic protein without signal peptide and transmembrane structure.Through phylogenetic analysis,the HSP70 sequence of T.dendrolimi was the most similar to those of T.chilonis and T.pretiosum.The expression level of HSP70 in the diapause prepupae was significantly higher than other developmental stages.2.The partial sequence of NOS(Genebank No.MN650600)with a length of 1,014 bp was cloned.The sequence encodes a total of 337 amino acids residues,which are hydrophilic proteins,with no signal free peptides and transmembrane structures.Through quantitative analysis by qPCR,it was found that the expression level of NOS was the highest in the individuals of diapaused pupae.Specific antibody of NOS was produced,and the result of Western blot showed that the expression trend of NOS at the protein level was the same as that at the transcription level.To sum up,the results of this study suggested that HSP70 and NOS played a certain role in diapause maintenance and diapause termination,respectively,providing basic data for the study of the molecular mechanism of diapause of T.dendrolimi.