Study On The Expression And Properties Of Diapause Associated Protein2from Anthereae Pernyi

Diapause associated proteins（DAPs）,which are abundant in diapause insect while little orno in the non-diapause individuals, are considered to be associated with the occurrence andmaintenance of insect diapause. So far, research on DAPs is little and stays in the discoveryphase. The molecular characteristics and the role it plays in the process of diapause is stillunknown.A sequence named diapause associated protein2(Ap-dap2) was searched out by NCBI(Accession number of GenBank is JQ003184.1). Sequence analysis revealed that the openreading frame (ORF) of this gene is834bp, which encodes278amino acids residues with apredicted molecular mass of31.39kD and a isoelectric point of6.01.Alignment of amino acidsequences of DAP2and DAPs or OBPs (ommin-binding proteins) from other species showedhigh homoloyies with OBP of Manduca sexta and Bombyx mori, which suggesting that DAP2may have similar characteristics with OBPs. Signal peptide analysis suggested that DAP2mightbe a secretory protein, which is consistent with the function of OBPs carrying and transferringpigment. Secondary structure prediction indicated that no α helical exist and high content ofβ-fold might form a cylindrical structurel, which may include a ommin-binding-site inside.The Ap-dap2gene from A. pernyi was amplified by RT-PCR and cloned into theprokaryotic expression vector pET-28a (+). The recombinant plasmid was transformed into E.coli BL21(DE3), and induced with IPTG for expression. With the supernatant solution afterultrasonic, we obtained the purified rHIS-DAP2fusion protein by Ni-NTA affinitive column.Polyclonal antibody was obtained by immuning a male New Zealand rabbit with the purifiedprotein, and the titer reaches1:6400measured by ELISA and the apecificity of the antibody isgood.Total RNAs and total proteins were isolated from the A. pernyi individuals of differentdevelopmental stages and different tissues of5th instar larvae. Fluorescence quantitative PCRand Western blot were used for the analysis of the transcription level and expression level ofAp-dap2gene, respectively. The results showed that both mRNAs and proteins graduallydecreased as diapause terminated and developed into moth, which is conform to the characteristic of DAPs in diapause development. In tissues of5th instar larvae of A. pernyi,DAP2was mainly expressed in the hemolymph, while the mRNA is abundant in the fat body,which implied that DAP2might synthesize in the fat body and secreted into hemolymph forfunction.To further clarify the molecular properties of dap2gene, we isolated the natural DAP2protein from pupae using four chomatographic steps including ammonium sulfate precipitation,ion exchange chomatograph, gel-filtration and immune affinity chomatograph. By photometricspectrum scanning, no specific absorption peak was detected in the visible light range. It maybecause the binding of ommin and protein is transient, the protein we isolated was free of ommin,and it also may because the5th instar larvae we chosen are not in the wandering stage, causingno specific ommin binding to the protein. With the PNGase F digestion, we found DAP2to be aglycoprotein. Two-dimensional electrophoresis analysis and mass spectrometry analysissuggested DAP2exists at least thee different kinds of posttranslational modification.The results above implied DAP2might involve into the process of diapause and possess thesimilar properties with OBPs. DAP and OBP were both considered as marker of the diapause,which impling certain internal relation within them. But the the specific roles DAP2played inthe A. pernyi diapause is still remains further study.