Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 initiation: HarDP-C1098 is homologous to Drosophila CG8378, which contains the conserved MYND and SET domains found in human Smyd homologues. Drosophila Smyd represses transcription [45]. Smt3 (SUMO) is a reversible post-translational protein modifier that usually represses the activity of transcriptional activators [46]. Thus, we conclude that the down-regulation of PLAG1 and elongation factor 1 delta and the up-regulation of transcriptional repressors and SUMO lead to the global down-regulation of transcription and translation at dia- pause initiation (Figure 5C). Conclusion In this study, differentially expressed genes at the early pupal stage of diapause- and nondiapause-destined indivi- duals were isolated and identified that may be involved in regulation of diapause initiation. Diapause initiation is an intriguing developmental process with a complex molecu- lar mechanism. Based on the above results, we suggest a possible molecular mechanism for diapause initiation. (1) Transition of metabolism and energy utilization. In addi- tion to a decrease of metabolic activity, metabolic path- ways are also changed in diapause-destined pupae at diapause initiation. Anaerobic metabolism predominates, and sugars and polylols accumulate in the brain. (2) Enhancement of stress resistance. The antifreeze agents glycerol and sorbitol as well as Hsp, GST, and others are heavily synthesized to protect the insect from rigorous environmental conditions. (3) Regulation of cellular devel- opment. The cell cycle is arrested, resulting in repression of pupal development toward adulthood. (4) Repression of transcription and translation. The up-regulation of tran- scriptional repressors, down-regulation of translational activators, and increased protein SUMOylation result in decreases of both gene transcription and protein transla- tion at diapause initiation. This idea awaits detailed experi- mental investigation in the future. Materials and methods Animals H. armigera larvae were reared on an artificial diet at 20° C with a L14:D10 (nondiapause type) and a L10:D14 (dia- pause type) photoperiod. After pupation, the two types of pupae were moved to the same conditions (20°C, L12: D12). Under these conditions, all nondiapause pupae developed toward adults, and more than 95% of diapause type pupae entered diapause. The developmental stages were synchronized at each molt by collecting new larvae or pupae. All tissues were dissected in insect saline con- taining 0.75% NaCl, and stored at -80°C until use. Page 12 of 14 nondiapause-destined individuals at the early pupal stage (diapause initiation) using the PCR-Select™ cDNA Subtraction Kit (Clontech). In the F library, diapause type pupae were used as the “tester”, and nondiapause pupa as the “driver”. In the R library, the “tester” and “driver” were reversed. After pupation, diapause- and nondiapause-destined pupae were incubated with the same condition 20°C and a short daylength (L12:D12) for 2-3 days before dissec- tion. Total RNA from day 1-2 brains of diapause- and nondiapause-destined pupae was isolated using a guani- dinium thiocyanate-chloroform method [47]. The mRNA was obtained according to the manufacturer’s protocols of QuickPrep Micro mRNA Purification Kit (GE health). Double-stranded cDNAs were synthesized from 1.0 μg of polyA+ mRNA and digested with RsaI to obtain shorter blunt-ended cDNA. The “tester” cDNA was sub- divided into two populations, which were ligated to adaptor 1 and adaptor 2R, respectively. Two hybridiza- tions were performed with the tester and driver cDNAs. In the first hybridization, the amount of driver cDNA was 25 times more than the tester cDNA. As a result, cDNAs that were not up-regulated were hybridized by driver cDNAs, only the up-regulated tester cDNAs were left as single strand. In the second hybridization, the amount of driver cDNA was 33 times more than the tester cDNA. As a result, the hybridized cDNAs were eliminated, leaving only the unhybridized cDNAs. The entire population of unhybridized molecules was then subjected to PCR to amplify target cDNA fragments (differentially expressed). Only the molecules of the tes- ter sample, which were ligated to the two different adap- tors, could be amplified exponentially. A second PCR amplification was performed using nested primers to get a low-background, high-level enrichment of the differen- tially expressed sequences. The PCR products were analyzed by 2% agarose gel electrophoresis. Products from the secondary PCRs were inserted into pMD18-T by T/A cloning (TaKaRa). The recombinant plasmid DNAs were transformed into XL-1 blue competent cells. The DNA from recombinant clones was isolated and sequenced (Invitrogen). Bioinformatics analysis All contigs and singlets were annotated according to the GO classification and the hierarchical structure using the Blas- t2GO suite [12]. The Blast2GO program, which assigns the GO terms based on the BLAST definitions, was applied with an E-value < 10-5. If a transcript was annotated with more than one GO category, it was split equally among them. Suppression subtractive hybridization We constructed two subtracted cDNA libraries (F and R) to detect high gene expression in diapause- and RNA extraction and RT-PCR Total RNA was extracted from the brain using the acid- guanidine method [47]. First-strand cDNA was