Expression And Function Analysis Of Diapause Associated Protein3from Antheraea Pernyi

Diapause is a specific physiological phenomenon in living organisms, in which growth andactivity are temporarily suspended in the developmental stage. Many insects make effective useof diapause in their life cycles to overcome unfavorable seasons. It has important significance forindividual survival and populational development, as well as its effects on seasonal demographyof pest insects from the pest management point of view. Chinase oak silkmoth, Antheraea pernyi,is most famous species among wild silkmoths for silk production. And at present, it is used as asource of insect food and for cosmetics. It undergoes a winter diapause as a pupa, which is aclassical organism used for the elucidation of the endocrine mechanism for metamorphosis andpupal diapause.To better understand the molecular mechanism underlying of diapause in A. pernyi, asequence named diapause associated protein3(Ap-dap3) was searched out by NCBI. Sequenceanalysis revealed that this gene full-length cDNA sequence is572bp, and open reading frame is516bp, which encodes171amino acids residues with a predicted molecular mass of18.19kDaand a isoelectric point of6.1. The putative protein has a conserved domain of Copper/Zincsuperoxide dismutase (Cu/Zn-SOD) and high homoloyies with SOD protein from many otherspecies. The novel DAP3gene 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-DAP3fusion protein by Ni-NTA affinitive column,which was highly purified with up to92%purity by software BandScan V5.0. Polyclonalantibody was obtained by immuning BALB/c mice with the purified protein, and the titer reaches1:6400measured by ELISA and the apecificity of the antibody is good.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 analysis the transcription level and expression level of Ap-dap3, respectively.The results showed that both mRNAs and proteins gradually decreased as diapause terminatedand lifed into moth. Analysis of pupae, eggs and1st to5th instar larvae indicated that the mRNAlevel is very high in the larval stage, especially in the1st and5th instar larvae, but the protein level of larval stage is significantly lower than that of pupal stage. In tissues of5th instar larvaeof A. pernyi, DAP3was mainly expressed in the epidermis, followed by the head, hemolymph,and fat body, while the mRNA is abundant in the fat body. At present, the research on DAP doesnot reach a consensus, with some reports considering DAP plays a role in the process of diapauseoccurs, which is highly expressed in hemolymphy or fat body of diapause individual and verylow or no expression in the non-diapause one. The expression profile above does not conform tothis characteristic, which suggests that AP-DAP3might not play the roles of DAPs of traditionaldefinition. We aimed to the opposite direction, speculating DAP3might be associated with thetermination of diapause, but not the induction of diapause.To identify the function of DAP3, the SOD activity of DAP3fusion protein was detectedwith HT Superoxide Dismutase Assay Kit to be approximately667.4U/mg. To further confirmthe SOD activity of DAP3in vivo, we induced the oxidative stress model of pupae by UVirradiation, and detected the mRNA and protein level of DAP3and the change of SOD activity.The results showed that both the mRNA and protein level of DAP3increased in atime-dependent manner by UV irradiation. Furthermore, the SOD activity of the total lysate ofpupae increased significantly at10min post UV irradiation and transiently returned to normallevel afterwards. These results suggested that DAP3might be a novel protein with SOD activitygetting involved in regulation of diapause in A. pernyi. Presently, there is few report about DAPfrom A. pernyi. Our work lays the foundation for the further molecular mechanism study ofdiapause in A. pernyi.