Cloning And Expression Of Vitreoscilla Hemoglobin Gene(vgb) In Cephalosporium Acremonium

The Vitreoscilla hemoglobin gene(vgb) with inartificial-low oxygen promoter was cloned and expressed in Cephalosporium acremonium.The Vitreoscilla hemoglobin gene(vgb) can synthesized a kind of protein named VHb when it was in the environment which was absent of oxygen, the vgb gene has now been cloned into many different microbes and also be expressed successfully, its expression has improved the growth of the recombinants and its production when the oxygen is limited in the environment.On the works of the predecessor, we looked up the naturally induced promoter to vgb -low oxygen promoter from Gene Bank, synthesized the low oxygen promoter sequence and added into the vgb structural gene then cloned to pUC18, thus constructed recombined plasmid pUC18-LV.In this paper, in order to clone vgb to Cephalosporium acremonium, The shuttle plasmid pMK₄-LV in E.coli-Bacillus subtilis was constructed on the base of recombined plasmid pUC18-LV, and plasmid pMK₄-LV were transformed into E. coli.successfully, with the inspections of the protein electrophoresis and CO difference spectrum, it is proved that the PMK₄-LV expressed Vitreoscilla hemoglobin(VHb) in E.coli. Because of the expression of the VHb, the bacteria quantity of the E.coli. in low oxygen fermentation increased remarkably.Then, the effects of some factors on the isolation and regeneration of protoplasts of Cephalosporium acremonium were studied, Including the appropriate culture media, growth time of the Cephalosporium acremonium,.the kind and concentrations of enzyme, digesting times and temperatures, and osmotic pressure stabilizers; Then the purified protoplasts were regenerated in A medium.Then the constructed shuttle plasmid pMK₄-LV with chloramphenicol-resistance was transformed into protoplast by the use of electroshock. Some distingctive fungus were filtrated on chloramphenicol culture medium (chloramphenicol concentration 200μg/mL).The vgb gene was successfully recombined into Cephalosporium acremonium genome through the PCR inspection, and the recombinants successfully expressed the Vitreoscilla hemoglobin protein in low-oxygen ferment with the inspections of the protein electrophoresis and CO difference spectrum. Further more, the transformed Cephalosporium acremonium was more ascendant than the original fungus in hight-oxygen fermentation on output of the aimed production, mycelium quantity and fermentation value...