Cloning Diapause Hormone Gene Of Helicoverpa Helicoverpa Assulta (Guenée) And Its Expression In Schizosaccharomyces Pombe

With primers designed according to sequence(AY052417) of diapause pupae of oriental tobacco budworm(Helicoverpa assulta Guenee) registered on GenBank, a double-strand cDNA fragment of DH(diapause hormone) gene was obtained from RT-PCR amplification and was further cloned into pGEM-T Easy vector.The fragment was digested by two restriction enzyme BamH I / Nhe I and then was linked with pESP-2 plasmid which was subjected to same enzymes as insert fragment. Then pESP-DH was transformed into Schizosaccharomyces pombe SP-Q01. By checking the transformed bacterial colonies, we had seclected the positive clone successfully. The sequence showed that the reading frame was correct.After being induced in EMM medium and cultured for some periods, the GST-DH fusion protein was checked by SDS-PAGE(SDS polyacrylamide gel electrophoresis) and Western blotting . The molecular weight of fusion protein was about 49KDa with a GST tag which provided a convenient way for further separation and purification of this protein.By bioinformatics analysis of diapause hormone gene, molecular weight is 22343.2Da, theoretical pI is 9.42,formula is C986H1546N282O298S7,estimated half-life is beyond 20 hours in yeast vivo, total negatively charged residues (Asp + Glu) is 13.9%,total positively charged residues (Arg + Lys) is 17.7%, the instability index (II) is computed to be 46.03 .According to the statistic,the protein was recognized as unstable. By scanning Prosite data base, we found 4 Pyrokinins signatures.The phylogenetic tree shows they have high homology among Helicoverpa assulta Helicoverpa armigera and Helicoverpa zea.