Full-length Cloning And Expression Of Theromacin Gene CDNA In Hyriopsis Cumingii

Hyriopsis cumingii is an important pearl producing animal live in water. In recent years. the disease of Hyriopsis cumingii caused by bacteria and virus lead to big loss in economic producing industry. The theromacin protein is a kind of antimicrobial polypeptides (AMPs). which is a hopeful substitute for antibiotics. In this study, the full length of theromacin gene is cloned by RACE-PCR based on the EST sequence which obtained by constructing a subtractive hybridization cDNA library. Its GeneBank Accession Number is FJ906824.1It has a597bp full length seqence which contains a165bp5’untranslated region (UTR) and a138bp of3’ untranslated region(UTR). In order to constuct a fuse expression plasmid vector and make it successfully expressed. The theromacin gene open reading frame(ORF) was isolated from liver of Hyriopsis cumingii by RT-PCR, it has294bp and coding for91amino acids. The cloned seqence was connected with pUCm-T and transformed into Escherichia coli DH5α, then had the blue-white selection and chose the white colony to submerged cultrue. the bacteria liquid was tested by colony PCR and send to sequencing. Then the plasmid was extraction from the bacteria and treated with BamHⅠ and HindⅢ. subcloned into the expression vector pET32a(+) which was also treated with BamHⅠ and HindⅢ. The recombinant plasmid was transformed into Escherichia coli BL21(DE3), the Escherichia coli BL21(DE3) which contains the recombinant plasmid was cultured and the white colony was choosed to shaking bottle incubating at220r/min,37℃. The bacteria liquid was tested by colony PCR and sequencing. choose the right recombinant expression vector and named it pET32a(+)-Ther. An about19kDa protein was detected which expressed by the pET32a(+) and an about29kDa recombinant fushion protein was detected which expressed by the pET32a(+)-Ther through the SDS-PAGE. induced by1mmol/L of isopropyl beta-D-thiogalactopyranoside at OD6oo0.8.37℃for8h. To detect the mRNA expression of theromacin in different tissues and different conditions, RT-PCR assay was performed using the tissues of mantle, adductor muscle, kidney, gonad. stomach, axe foot, gill, liver, intestine and heart obtained from healthy and artificially infected Hyriopsis cumingii by Aeromonas hydrophila. The result shows that theromacin mRNA were expressed in all tissues with the highest of kidney. Expression in stomach is significance difference expressed with all the tissues except axe foot and intestine(p<0.05).After artificially infected by Aeromonas hydrophila for48hours, the expression of theromacin was increased in mantle, stomach, axe foot, liver, intestine and heart,the expression of theromacin mRNA in liver is about5times of the healthy liver tissue. This experiment was a foundation and research materials of disease-resisted breeding for Hyriopsis cumingii.