Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 II–dependent genes. The 80-kDa mcm3-associated pro- tein (Har-NP387) interacts with MCM3, which is a fac- tor that allows the DNA to undergo a single round of replication per cell cycle and is required for DNA repli- cation and cell proliferation [17]. GTP-binding nuclear protein ran (Har-NP284) is involved in chromatin con- densation and cell cycle control. MCM9 (Har-NP831), as a DNA replication licensing factor, participates in cell cycle regulation. Septin 2 (Har-NP1044) is required for the progression through mitosis [18]. Transcription fac- tor dp-2 (TFDP2, HarNP-798) can stimulate E2F-depen- dent transcription and promote the transcription of a number of genes whose products are involved in cell cycle regulation or in DNA replication [19]. Transcription and translation Six genes related to transcription and translation were also found in the two SSH libraries (Table 1E). Two genes, CG8378 (HarDP-C1098), which is predicted to have transcriptional repressor activity, and SUMO (HarDP-A589), which always represses the activity of transcription factors, were up-regulated at diapause initiation. In contrast, four genes were down-regulated in diapause-type pupae: Pleomorphic adenoma gene 1 (HarNP-730) is a transcription factor whose activation results in up-regulation of target genes, such as Insulin- like growth factor (IGF) [20]. Elongation factor 1 delta (HarNP-642) facilitates the events of translational elon- gation, resulting in promotion of protein biosynthesis. Oocyte zinc finger protein xlcof22 (HarNP-418) func- tions in transcriptional regulation. Reptin (HarNP-905) acts as a transcriptional activator, and also as an essen- tial cofactor for the normal function of Myc, so it is required for cellular proliferation and growth [21]. Discussion The insect brain is the center of developmental control and serves as the repository of the diapause program [22]. In pupal diapause species, photoperiodic signal is perceived by larval brain during diapause induction. Then gene expression changes affected by photoperiod are first present in diapause preparation phase which follows diapause induction to regulate specific metabo- lism for diapause [3,6,22]. It is well known that after pupation, a shut-down of prothoracicotropic hormone (PTTH) in the brain and ecdysteroids in the prothoracic gland cause diapause initiation [6]. Meola and Adkisson demonstrated that the shut-down of PTTH is found in day 0 of pupal brain of Helicoverpa zea, a closely related species to H. armigera [23]. Thus, these differentially expressed genes isolated from the two libraries in day 1-2 pupal brain of H. armigera for diapause initiation are in response to hormones, but not photoperiodic signal. Page 8 of 14 In H. armigera, the photosensitive stage for diapsuse induction is from 5th instar to early stage of 6th instar. This is little different compared to H. armigera popula- tion from Okayama (Japan), whose photosensitive stage for diapause induction is the early fifth instar [24]. After pupation, H. armigera diapause-type pupae are trans- ferred into L14:10D photoperiod, all pupae will enter diapause, and all pupae will develop without diapause even if nondiapause-type pupae are transferred into L10:14D photoperiod (data not shown). Apparently, photoperiod regime does not affect pupal diapause or development. The most remarkable characteristic of insect diapause is strong metabolic suppression. For example, in dia- pausing pupae of the flesh fly, Sarcophaga argyrostoma, the metabolic rate is approximately 90% lower than in nondiapause counterparts [25]. Therefore, diapause was thought to represent a shutdown in gene expression. However, Joplin et al. [4] and Flannagan et al. [5] demonstrated that diapause should be a unique develop- mental pathway rather than a simple shutdown of gene expression. Recently, the proteomic analysis of the brain at diapause initiation has been reported, suggesting that the expression of many diapause-specific genes in the brain accompanies certain down-regulated genes [10,11]. Thus, identification of diapause-associated genes at dia- pause initiation is the first step to understand the com- plex process of diapause. In the present paper, we isolated 304 diapause-specific mRNAs from H. armigera brain using SSH, and the subset of these genes with sequences similar to known genes in GenBank were classified according to their functions. Furthermore, we evaluated their mRNA expression at diapause initiation by RT-PCR and Northern blot analysis, and investigated the expression patterns of four important genes by RT- PCR and Western blot analysis, showing that these genes may be associated with diapause initiation. From the SSH F library, we found a high percentage of undescribed sequences (61.3%). Some sequences may correspond to 3’ or 5’ untranslated regions (UTRs), so it is impossible to find their homologues in protein data- bases. However, most of these undescribed sequences can be classified as novel genes related to H. armigera pupal diapause initiation, because only a few genes related to developmental arrest have been identified. The large percentage of unknown (novel) genes in the F library shows that diapause is a complex physiological process involving a number of unknown genes in the regulation of developmental arrest. We also constructed an R library to identify specific genes expressed in nondiapause individuals. The up- regulated gene expression in nondiapause pupae identi- fied from the R library usually corresponded to down- regulated expression in diapause-type pupae (Figure