Cultural Condition Optimization Of Actinomucor Elegans And Cloning Expression Of Apt1Gene

ATP is very important in all living beings as a carrier molecule for energy. As a medicine, ATP is widely used to cure cancer, progressive muscular atrophy, apoplectic sequelae, myocarditis, coronarythrombosis and hepatitis.This dissertation consist of the identification of Mucor ZGB1using in the experiment, optimization of sacchariferous liquor medium, construction of genetic transformation system for Actinomucor elegans, cloning and experssion of apt1gene in Actinomucor elegans.The Mucor was identified based on morphological observation, physiological and biochemical property analysis,18S and ITS rDNA sequence analysis. It was identified as one mutant strain of Actinomucor elegans and named Actinomucor elegans ZGB1. The position in taxonomy is that Kingdom Fungi、Phylum Zygomycota、Zygomycetes、 Mucorales、Mucoraceae、Actinomucor、Actinomucor elegans。The optimum nitrogen source and inorganic salt((NH4)2SO4and MgSO4·7H2O) were get screening. In the meantime, glucose was replaced by hydrolyzed liquid of corn starch in this fermentation medium, and steepwater was added. Then steepest ascent procedures were applied to define optimal response region of these four factors, finally the optimal factor concentrations were determined by adopting RSM analysis. The results showed that sugar concentration42.13g/L,(NH4)2SO45.60g/L steepwater14.23g/L, MgSO4·7H2O0.92g/L were combined as the most suitable Actinomucor elegans fermentation medium. The thallus yield was increased19.45%. The ATP yield was increased10.83%.The protoplast with higher regeneration activity was obtained using cellulose(2.0%) and snailase(0.5%) to process the spore3h, at30℃.The spore was cultivated4h. PEG and CaCl2mediated protoplast transformation of Actinomucor elegans with plasmid pCB1004-hph was performed to construct the genetic transformation system of Actinomucor elegans.The key enzyme gene apt1for synthesis ATP which from Saccharomyces cerevisiae was cloned by PCR method. The sequence was cloned to corresponding site of pCB1004-Pgpd. Then the expression plasmid pCB1004-Pgpd-apt1which promoted by strong promoter Pgpd was obtained. PEG and CaCl2mediated protoplast transformation of Actinomucor elegans with expression plasmid was performed and ATP overproducing transformat was obtained. The ATP yield and moore conversion were increased44.04%than starting strain.