Research Of Bovine Prochymosin Gene Expression In Aspergillus Niger

Chymosin is widely used in dairy products especially cheese processing since it can cause protein cohesion and lead milk to condense. The traditional method is to slaughter the newborn calves to extracte chymosin from the fourth stomach. However, this method is expensive because of the worldwide shortage of calves. Meanwhile, it is unsatisfactory to take the place of microbial prolease, plant prolease and other animal prolease. So there is an important research value to produce bovine chymosin using genetic engineering technology.The filamentous fungi aspergillus have the advantages of clear genetic background, rapid growth, expressing many proteins in high levels and secreting the protein products extracellularly. Aspergillus niger is a kind of safe aspergillus which does not produce aflatoxin, so it has received much attention as the"eukaryotic proteins factory"in addition to being widely used in the fermentation industry.In this study, we took the industrial production strain Aspergillus niger used in Glucoamylase Industry as the receptor material to construct the gene replacement vector of bovine chymosin directing towards high expression of glucoamylase gene loci, and transform the gene using different methods. We also designed the direct repeats sequenceat in both ends Hygromycin gene, so made it possible to eliminate the resistance gene in the follow-up study and laid a foundation for constructing the efficient safe engineering strains of Aspergillus niger.The main results are as follows:1.Cloning of gene and homology arm fragmentThe Chymosin gene Cym, Gla5 of the upstream gene of aspergillus niger, the Gla3 and Gla3a of the downstream gene of aspergillus niger were gained by using PCR amplification.2.Construction of VectorWith the overlap extension PCR, get the fragment of Gla5-Cym-Gla3, taking Gla5, Gla3 as the both ends of the homologous arms of Cym. Have constructed the joint vector pSZA, Aspergillus vector pEASY-CYM tansformed by Bovine chymosin gene, Aspergillus vector pSZH-CYM tansformed by Bovine chymosin gene mediated by Agrobacterium, and intermediate vector pEASY-hph,pAN-Gla3a.3.Preparation of Aspergillus niger Protoplast and Optimization of regenerationWe optimized mainly from the choice of medium, cell age, the type of enzyme, hydrolysis time, Osmotic stabilizer, treatment of enzymatic process. The results show that solid PDA medium, 30℃under 0.4% of the snail enzyme, pH 6.0 to 50mM phosphate buffer solution, enzymatic process uses 50rpm protoplast and regeneration rate would get the greatest yield.4. Translation from rennet gene to Aspergillus nigerTranslate plasmid pEASY-CYM directly into Aspergillus niger, using method REMI, and obtained 23 colonies successfully. Efficiency of mycelium as conversion Receptor was about 9cfu/μgDNA, efficiency of protoplast as conversion Receptor was about 2 cfu/μgDNA. Obtained 5 transformants detected by PCR, and the PCR positive rates were 14%, 15%.5. Expression of bovine chymosin gene in Aspergillus nigerMeasured the activity of positive transformants from bovine chymosin gene to Aspergillus niger, and got the the highest expression when the culture time was 6d, enzyme activity was 7.9SU/mL6. Genetic transformation mediated by AgrobacteriumTranslate replacement vector pSZH-CYM into Agrobacterium EHA105 using freeze-thaw method.