Analysis Of Structure, Expression And Function Of Bombyx Mori Diapause Receptor Gene

Silkworm, Bombyx mori, is an important economic insect and model organism as well. It is of great theoretical and practical significance to study the molecular mechanism of diapause of the silkworm. Experiencing a long process of evolution, silkworm has formed the character of embryonic diapause which is determined by both genetic characters and environmental conditions. Silkworm varieties can be classified into univoltine, bivoltine and polyvoltine ones according to their diapause characters. Diapause character of bivoltine silkworm is influenced by the environment where their parental generation lives: when bivoltine silkworm eggs (parental generation) are incubated at 25℃in lightness, the moths lay diapause eggs; by contrast, when the eggs are incubated at 15℃in darkness, the moths lay non-diapause eggs. The character of embryonic diapause of the silkworm is closely related to diapause hormone (DH) synthesized and secreted by subpharyngeal ganglion (SG) in pupal stage. DH can function after the diapause hormone receptors (BmDHR) binds to it. In order to explore the molecular mechanism of how incubation temperature regulates diapause of bivoltine silkworm, we preliminarily studied the structure, expression profile and functional characteristic of Bmdhr gene.1. Analysis of Bmdhr gene structureBmdhr mRNA-1 ~ Bmdhr mRNA-5 sequence (accession No.: AB164386 ~ AB164390) were downloaded from the NCBI, and blasted in the silkworm genome database online. Their open reading frames (ORFs) and the amino acid sequences were analyzed with the DNASTAR software package, and then the transmembrane domain of BmDHR was analyzed via TMHMM service v. 2.0. The results showed that Bmdhr was a single copy gene with 33.79 kb nucleotides in length consisting 7 exons and 6 introns. The 5 mRNA transcripts were resulted from the same pre-mRNA through different splicing patterns; Bmdhr mRNA-1 and Bmdhr mRNA-2 are the same receptor since they encode the identical amino acid sequence. BmDHR-1 contains 7 transmembrane regions, and BmDHR-3, BmDHR-4 and BmDHR-5 contains 5 transmembrane regions, respectively.2. Spatiotemporal expression patterns of Bmdhr gene and influence of incubation temperature on gene expressionEggs of bivoltine silkworm strain Qiufeng were divided into two groups with the half egg batch method. One group was incubated at 15℃in darkness and the other at 25℃in lightness, respectively. By using real-time fluorescent quantitative PCR, we analyzed the influences of incubation temperature on the transcription of Bmdhr mRNAs in different developmental stages and various tissues. The results showed that Bmdhr mRNA-1 was mainly expressed in ovaries of pupae; its mRNA level quickly reached the peak at day 4 of pupation when the pupae were the most sensitive to diapause hormone, and the peak mRNA level of the group incubated at 25℃was significantly higher than that of the group incubated at 15℃. Bmdhr mRNA-4 was mainly expressed in hemolymph of silkworm at every developmental stage. Especially, its expression level from incubation at 25℃in lightness was 7.7 times to that at 15℃in darkness, implying that BmDHR-4 might be a key factor in determining whether the next generation eggs fall into diapause or not. Expression level of Bmdhr mRNA-5 was high in ovaries of pupae at 2~3 d after pupation, and its mRNA level in the group incubated at 15℃was higher than that of the group incubated at 25℃. However, the expression of Bmdhr mRNA-5 at 3 d after pupation was reduced and up to 4~5 d there was no significant difference between the two groups.3. Characterization of Bmdhr gene promoterBased on bioinformatics analysis, Bmdhr promoters which contain different gene regulatory sequences were cloned from silkworm genome DNA, and were inserted into pGL3.0 basic vector to construct reporter plasmids. Activities of the promoter sequences with the different lengths were analyzed through the comparison of influences of different concentrations of juvenile hormone analogue (JHA), molting hormone (MH) and DH on them. The results showed that a negative regulatory element might exist between -941～-1364 nt in Bmdhr promoter range. In contrast to the control, the promoter activity was significantly reduced at MH with concentration of 4μg/mL, and it was significantly increased at the rest concentrations (1, 2, 6, 8 and 10μg/mL). 2, 4, 6 and 10μg/mL of JHA could significantly enhance the promoter activity; nevertheless, 1μg/mL of JHA significantly downregulated the promoter activity and 8μg/mL of JHA extremely significantly downregulated it. When the mixture of MH and JHA concentration were 1, 2, 4 and 6μg/mL, promoter activity was increased significantly and the activity was the highest at 1μg/mL; nonetheless, under the condition of 8 and 10μg/mL mixtrue, promoter activity did not change significantly. At DH concentration between 10 ~ 60 nM, promoter activity was significantly increased: it peaked at proximate 20 nM; the promoter activity did not change significantly when the concentration is greater than 60 nM.4. Eukaryotic expression and function analysis of BmDHR Using the pcDNA3.1 vector as the backbone, we constructed IE-1 promoter-BmDHR expression plasmid, and respectively detected impacts of BmDHR on the Bmtrehalase promoter activity. The results show that BmDHR-1, BmDHR-3 BmDHR-4 and BmDHR-5 independent and large expression can increase the activity of Bmtrehalase promoter. Different ratios (1:3, 1:1 and 3:1) of BmDHR-1 / BmDHR-5 can increase Bmtrehalase promoter activity, with the activity inhibited under the increase of BmDHR-5 ratio. The Bmtrehalase promoter activity under BmDHR-1/BmDHR-5 ratio of 1: 3 is closed to the activity of BmDHR-1 alone.These results provide experimental data for elucidating the molecular mechanism of silkworm diapause.