Structure, Temporal And Spatial Expression Of Gene Of Diapause Bioclock Protein Esterase A4 Of The Silkworm, Bombyx Mori

Domestic silkworm (Bombyx mori) is an egg diapause insect. In the production ofsilkworm, low-temperature chilling and hydrochloric acid treatment methods are used toterminate the diapause development. After above treatments, the spatial structure ofEsterase A4 (EA4) will be changed, and EA4 will get a transient burst AYPase activity.Thus it is named Time Interval Measuring Enzyme-Esterase A4 (TIME-EA4), acting as adispause duration clock in diapause eggs of the silkworm, Bombyx mori.Based on the ORF sequence of ea4 released by my laboratory (NCBI accessionnumber: DQ086424), we use rapid amplification cDNA end (RACE) to get the full lengthcDNA with the material of silkworm variety C108. The results show that the full lengthcDNA consists of 606 base pairs, and ORF is 519 base pairs coding 172 amino acids. Analigment of cDNA sequence with the whole genomic sequences of Bombyx mori onNational Center for Biotechnology Information (NCBI) revealed that there were 4 exonsand 3 introns. According to assembling and filling in GAPs of full genome data, thewhole sequence of ea4 gene was known except the first intron was uncertain.The expression of ea4 in different tissues of the fifth instar larva and pupa stage isresearched by nucleic acid hybridization and semi-quantitative RT-PCR method. WithDIG-High Prime labeled ea4 cDNA, the result of dot blotting shows that there was notissue and sex specificity of expression. With Actin3 gene as the positive control, theexpression profile of ea4 gene was detected by RT-PCR. Results showed that there wasno stage specificity in different tissues. Expression of ea4 is at higher level in silkgland,hemopoietic organs, midgut and fat body while the expression in ovary was lower. Wecome to conclusion that EA4 may be synthesized nonspeeificly by parents and thenstored in the eggs.Based on the transient burst of ATPase activity and SOD activity of EA4 during thetimer period, we researched the structure of ea4 gene, EA4 protein and SOD protein from Bombyx mori and Bombyx mandarina so that the bioclock mechanism of EA4 will befurther understood at gene expression level. The homology of ea4 ORF from Bombyxmori and Bombyx mandarina is 97%. And they share 99% at amino acid level. Thededuced amino acid sequence Cu/Zn-sod from Bombyx mandarina showes 100%identity with that of Bombyx mori. The homologies of EA4 with Bombyx mori andCu/Zn-SOD of Bombyx mori and Bombyx mandarina are 46%.The SOD active site including metal-binding amino acid ligands and a disulfidebond are completely identical in EA4 and SOD protein. The EA4 N-terminal region islow identities with SODs. The region is flexible because ofβ-sheet structure. The uniqueregion for EA4 is deduced to be the core domain to measure the transient burst of ATPaseactivity probably. The rigid core of EA4 has probably the SOD activity. According toanalyzing the structure of EA4 and SOD, EA4 of Bombyx mori is a specific easterase thatmay stem from SOD family. In a word, structure of EA4 will be changed and get atransient burst of ATPase activity treated by low-temperature chilling and hydrochloricacid. Then a time-dependent ATPase measures tne diapause duration of Bombyx daipauseeggs.