Influence of Incubation Temperature on Expression of Diapause Hormone Receptor Genes in Bivoltine Bombyx mori Variety and Structural Features of the Gene

Diapause of bivoltine silkworm(Bombyx mori) eggs is influenced by the living environment of their parental generation.When bivoltine silkworm eggs(parental generation) are incubated at 25 ℃ in lightness,the moths will lay diapause eggs.In contrast,when the eggs are incubated at 15 ℃ in darkness,the moths will lay non-diapause eggs.For probing into the molecular mechanism of how incubation temperature regulates diapause in bivoltine silkworm,bioinformatic analysis was carried out on the 5 cDNAs of diapause hormone receptor gene,namely Bmdhr cDNA-1 to Bmdhr cDNA-5,which were cloned from the ovary of B.mori.The results showed that these 5 cDNAs of Bmdhr gene were from the same mRNA transcript through different splicing patterns.Among them,Bmdhr cDNA-1 and Bmdhr cDNA-2 encoded the same amino acid sequence.Amino acid sequence encoded by Bmdhr cDNA-4 had 99.2% identity with that of BmDHR-1.Eggs 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,the influences of incubation temperature on the transcription of Bmdhr mRNA were analyzed in different developmental stages and in various tissues.The results showed that:Bmdhr mRNA-1 was expressed mainly in ovaries of pupae and its mRNA level quickly reached the peak level at day 4 after pupation during which the pupae were most sensitive to diapause hormone,and the peak mRNA level of the group incubated at 25 ℃ was significantly higher than that incubated at 15 ℃.Bmdhr mRNA-4 was expressed mainly in hemolymph of silkworm at various developmental stages.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 determining whether the next generation eggs fall into diapause.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 incubated at 25 ℃.Yet from 3 d the expression of Bmdhr mRNA-5 was reduced and up to 4~5 d there was no significant difference between the two groups.These results provide experimental data for elucidating the molecular mechanism of silkworm diapause.