Cloning And Prokaryotic Expression Of Diapause Bioclock Protein Esterase A4 Of Silkworm, Bombyx Mori.

The diapause bioclock protein of Bombyx silkworm is count down clock which depends on esterase A4 (EA4), EA4 is the first confirmed time interval measuring enzyme.A 308bp of ea4 sequence was got by degenerate primer RT-PCR, the primers were designed according to the determined amino acids and the total RNA of fat body and ovary from 3 days old female pupa was used for the template. The consistence of 1-144nt and 145-308nt of the sequence with 5595-5738nt of WGS (AADK01019126.1) and 8636-8473nt of WGS (AADK01014467.1) are 95% and 98% respectively. A pair of special primers was designed according to the cDNA sequence of ea4, and used the total RNA of fat body and ovary of female pupa as the template for RT-PCR, then got a 519bp sequence which is consist with the predicted sequence completely and the consistence of the predicted amino acids with the determined is 98%, which proved to get the overall cDNA of ea4. Designed another pair of special primers according to the DNA sequence of ea4, and used the DNA of Bombyx silkworm as the template for PCR, the link sequence between WGS (AADK01019126.1) and WGS (AADK01014467.1) were gained, naturally, the genome of ea4 was clear, the full length DNA of ea4 is 5281bp including 3 extrons and 2 introns, they are at the same chromosome.Clone the ea4 gene to the express vector pET28a(+) which hold the sequence express tag of His₆ for construct the fusion express vector pET28a(+)-ea4. Transform the fusion vector to E.coli BL₂₁ and induced by IPTG, the fusion protein was expressed, which set the foundation of the protein function for In vitro research.