Cloning And Expression Of The DaHSP23 Gene During Diapause In The Onion Maggot, Delia Antiqua

Diapause is a developmental strategy widespread among insects and their arthropod relatives. It allows insects to survive in harsh summers, winters, dry seasons and other unfavorable conditions, to exploit seasonal resources, and to synchronize the growth pace of their populations. The diapause program is expected to associate with diapauses-specific genes expression. Therefore, wide screening of diapauses-specific genes is of great importance in understanding of the molecular mechanisms of diapause, such as sHSP, HSP60, HSP70, HSP90. Studies in different experimental systems have revealed a variety of functions for the sHSP under stress conditions, including basic chaperoning activity, cytoskeleton protection and modulation of the apoptotic process. Contrasting with the classical definition of heat shock proteins as polypeptides induced by stress, cell-specific expression of sHSP in the absence of stress has been reported during the development of a wide range of organisms such as Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus, human and so on. In the pupal diapause of the flesh fly Sarcophaga crassipalpis, 23 kDa heat shock protein transcripts are expressed at very high levels as part of the normal diapause program, indicating that HSP23 plays an essential role during this overwintering developmental arrest. However in the blow fly Lucilia sericata, HSP23 transcripts was consistently low, and there were no differences in the expression levels in normal condition or during diapause, which indicates that expression of HSP23 is not regulated in response to diapause. Thus, up-regulation of HSP23 would not be a common feature of diapause insects. However, it is still necessary to investigate the expression of other copies of small HSPs before validating this conclusion.Delia antiqua is a good model species for diapauses research because summer diapause (SD) and winter diapause (WD) can be easily induced in the same stages in laboratory. HSP90 and HSP70 were identified in the summer and winter diapause pupae of D. antiqua responding to temperature and stresses.In present study, we firstly cloned and characterized the full length cDNA of HSP23 gene from Delia antiqua, DaHSP23, by RACE PCR, quantitative real-time PCR and sequence analyses. It has been deposited in GenBank under accession HQ392521.1andADX36150. These results are as follows.①The full-length cDNA, 904 bp long, and an open reading frame (ORF) of 561bp (133~693), encoding a protein of 186 amino acids with a calculated molecular weight of 20.9 kDa and theoretical isolectric point of 6.42. For DaHSP23 intron analysis, and by comparing the sequence isolated from genomic DNA with cDNA sequence of DaHSP23, it is identical to the sequence of DaHSP23 cDNA, indicating that there is no intron in the coding region of DaHSP23 gene. Homology analysis revealed that DaHSP23 shares more than 64% identity with known HSPs of other Diptera insects. The conservedα-crystallin domain was identified to range a.a. 65 to 142, which is characteristic feature of sHSPs. The InpA region for posttranslational modification, protein turnover and chaperones was identifid to range a.a. 12 to 158 of DaHSP 23.②We adopted the rearing condition of 23±1.0℃, 16L:8D photocycle and 50-70% relative humidity (R.H.) for adults. The conditions of 20±0.5℃, 16L:8D photocycle and 50-70% R.H. were used and worked well for the rearing of non-diapausing larval and pupal development. 15±0.5℃with a 16L:8D photocycle and 50-70% R.H. to rear larve and pupae were for the induction and termination of the WD. The results showed that 100% pupae could enter WD with the rearing condition. To induce SD, the larve and pupae were reared under 25±0.5℃with a 16L:8D photocycle and 50-70% R.H., with which over 95% pupae entered SD. With the intensity enhance of illumination, 100% SD pupae could acquired. The optimized temperatrue for the termination of SD is 16℃. We selected 7 diapause sampling points during the diapause, namely: non-diapause sampling points in the non-diapause after pupation 1,3,5,7,9,11,13 days; Winter-diapause sampling point is in the winter diapause after pupation 3,4,29,54,79,103,114 days; Summer diapause sampling points in the summer diapause after pupation 0.5 1,5,9,10,11,17 days.③HSP23 mRNA levels were normalized with those of 18S rRNA in the same samples quantified in the same manner, and the final relative mRNA levels of HSP23 were averages of three replicates by Bio-rad iCycler iQ5. DaHSP23 transcription is up-regulated in both summer diapauses (SD) and winter diapauses (WD) pupae with higher mRNA levels during WD than SD, while decreased when diapause was terminated. These results suggested that DaHSP23 is up-regulated in both SD and WD, and it may play an essential role in over-wintering and over-summering developmental arrest.