| The onion maggot, Delia antiqua (Diptera:Anthomyiidae), is a serious pest of the Liliaceae plants, and is widely distributed over Asia, Europe and North America. It is an ideal model species for the conparative study of winter- and summer-diapause-specific genes. Up to now, there has been little data to file the diapause-specific genes between these two kinds of diapauses. The present study aims to identify and compare these two kinds of diapause-specific genes with Suppression Subtractive Hybridization (SSH) technology, and lay a firmer base to understand the molecular mechanisms of insect diapause, winter-diapause (WD) and summer-diapause (SD). The identification and study of diapause-development-specific genes and stress resistance genes (including hardiness genes to cold, heat, drought and microbial infection, and immuological genes) in the duration of diapause is of significance for the control of agricultural insect pests, and for the gene engineering in agricuture and medicine sciences.In this study, we firstly established a stable laboratory population of the onion maggot, observed the biological characteristics in each stages of normal development, WD and SD, and studied the condition for the induce of WD and SD pupae. We then constructed full-length cDNA libraries of non-diapause (ND), WD and SD pupae. Most importantly, we identified and compared the summer- and winter- diapause- specific genes with SSH, DNA sequencing and bioinformatic analysis. These results are as follows.1. We established a stable laboratory population of the onion maggot, which was introduced from a laboratory colony in Japan, worked out the optimal induce condition for WD and SD, and observed the biological features in pupariation. More important works include:①We designed and manufactured the rearing cage, and adopted the rearing condition of 23±1.0℃,16L:8D photocycle and 50-70% relative humidity (R.H.) for adults. Results showed that the facility and conditions benefit very well for adult growth. We optimized the food recipe for larvae with the products in China in reference to reported recipe in Japan. ②The 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.③15±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.④To 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℃.2. We constructed three full-length cDNA libraries with non-diapause (ND), SD and WD diapausing pupae of the onion maggot, using SMART technique. These libraries laid a base for further cloning and expression analysis of diapause-specific genes. According to the quality evaluation, the titers of primary libraries of ND, SD and WD were 2.3×107 cfu/mL,2.4×107 cfu/mL and 2.4×107 cfu/mL, respectively. The results from random 15 clone sequencing showed that the inserted fragments ranged from 0.4-1.2 kb,0.35 to 2.2 kb and 0.5 to 3 kb, respectively. PCR amplification showed that the average size of inseted fragments were 0.9 kb, 0.9 kb and 1 kb, and the recombination rate was 100%. These results showed a high quality of these three full-length cDNA libraries to meet the requirement of cloning and expression analysis.3. Forward and reverse SSH-cDNA libraries of WD and SD pupae, which contain pre-diapause and diapause in pupae phase, were constructed using SSH. About 150 clones were picked randomly from the forward and reverse libraries, respectively, for DNA sequencing. After discarding the sequences of repeat, redundance and length< 100 bp, the unique ESTs acquired were performed protein homology search, using the BLASX program at NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Only the ESTs with scores> 60, identy>40% and E-value |
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