Research On The Mechanism Of Diapause Bio-clock Protein Based On Structure And Expression Of Gene Ea4 In Silkworm, Bombyx Mori

Bombyx mori is a kind of model insect of Lepioloptera with diapausing during its egg stage. Diapause inducement is due to temperature, illumination rhythm and other environment elements in the incubation stage of the parent eggs, but not its temporal environment conditions. However, the level of diapause hormone (DH) secreted by female parent pupae's suboesophageal ganglion (SG) and the temperature after ovipositing are the direct factors in baby eggs diapausing determination. There is a special esterase A4 related to energy metabolism in silkworm diapause eggs, its transient ATPase activity depend on low temperature, hydrochloric acid stimulating and other environment activation elements, and has a characteristic change in its protein spatial structure, so called (Time Interval Measuring Enzyme,TIME).It is found that protein time measuring in silkworm is a new measuring manner.In order to investigate the regulation mechanism of diapause bio-clock EA4 in silkworm, we used Bombyx mori C108 strain as our material, have cloned the ea4 whole gene sequence, analyzed EA4 structure characteristic and its molecular evolution; analyzed ea4 expression from larva to moth stage in different diapause eggs and different issues. EA4 has stable SOD activity in silkworm eggs, and gene cat in Rat is a clock-controlled gene of circadian rhythm gene clock. So we investigated the expression of gene sod, cat and silkworm diapause eggs characteristic energy metabolism related gene sdh.Based on ea4 gene ORF(NCBI:DQ086424) and its whole cDNA sequence(NCBI:EF121773) released in our laboratory, we cloned and aligned the 3 introns sequence and gained the whole ea4 sequence which is 10 265 bp (NCBI:EF121772), including 4 exons (23 bp,69 bp,204 bp and 309 bp) and 3 introns (4 889 bp,3 521 bp and 1 250 bp). The joint between intron and exton is according to the GT-AG rules. Protein homology analysis showed that EA4 has no serine activity core domain of carboxylesterase (Carboxylesterase type-B serine active site : FGGdpelItLfGeSAG),but it has Cu/Zn-SOD core domain,and has high homology(48%-50%) with Cu/Zn-SOD of M.musculus, B.taurus and D.rerio. However, molecular evolution analysis indicated that the evolution distances between EA4 and Cu/Zn-SOD of M.musculus, D.rerio, C.elegans and D.melanogaster, especially Cu/Zn-SOD of silkworm itself are far. So we speculated that EA4 is a new esterase derived from Cu/Zn-SOD family and is evolving toward esterase family. Also it is may be the reasons that EA4 have both SOD activity and transient esterase activity.RT-PCR and embryo in situ hybridization method research showed that as to parent eggs under different temperature, illumination rhythm and other diapause inducement conditions, the expression of ea4 has no development stage difference from larva to moth after incubation. During eggs shaping stage in the fifth larva, gonad and fat body, also midgut, hemocyte and silk gland have gene ea4 transcription product and have no issue difference. Zygote hasn't completed diapause eggs such as virgin eggs and eggs after oviposited 0.5 hour and so on, there are many ea4 mRNA coming from female parent.Diapause eggs after oviposited 24 hours have much ea4 mRNA, then treated with hydrochloric acid immediately, during the embryo activation and development stage, ea4 keeps a high transcription activity. EA4 is synthesized specially from larva to moth and then stored in eggs. The regulation of TIME-EA4 is not depended on circadian rhythm.Relationship between sod, cat and ea4 gene expression and different diapausing and different activate environment showed that, though incubation temperature and illumination rhythm of parent eggs are the diapause inducement conditions of baby eggs, sod, cat and ea4 gene expression from larva to moth after incubation have no time-spatial difference and rhythm difference among different inducement conditions. The effects of incubation temperature and illumination rhythm diapausing inducements on silkworm eggs may be not through the regulation of sod, cat and ea4, or may be not in the stage from larva to moth, or even baby eggs. Whether gene cat is a circadian rhythm related gene of silkworm depends on further expression research in parent eggs. Although the eggs treated by hydrochloric acid is the same as non-diapause eggs, hardly have the reaction of hepatin change into sorbitolum and glycerine, eggs treated by hydrochloric acid and diapause eggs both have high sdh transcription activity while non-diapause eggs haven't. The expression of sdh may depend on the inducement elements in parent eggs diapause determination stage. In non-diapause eggs after oviposited 24 to 88 hours and eggs after treated by hydrochloric acid 24-168 hours, gene sod (sod and sod+ea4) has high transcription activity, but it is contrast to the diapause eggs after oviposited 0 to 72 hours and non-diapause eggs after oviposited 0 to 24 hours, which is similar to the H2O2 metabolism in different diapausing eggs.