Molecular Cloning And Expression Of Acid Phosphotase Gene In E.coli And Pichis Pastoris

Phytase is a new-type important enzyme product as animal feed additives, which can catalyze phytic acid. Phosphorus presented in crops such as grains, beans, oil-bearing crops is in the form of phytate-phosphorus. The utilization rate of phosphorus in phytate by monogastric animals was very low because of the lack of phytase. Supplementation with phytase is an effective way to increase utilization rate of phosphorus in seed-based animal feed and to reduce phosphate pollution in the environment.Phytase mainly comes from plant and organism, phytase of plant belongs to 6-phytase and it' s optimum pH ranges from 5.0 to 7. 5. Plant phytase can not function effectively in monogastric animals and it is not rich in plant. So research direction about phytase is turned to Aspergillus niger which is rich in acid phytase.This experiment extracted total DNA from Aspergillus niger, using the DNA as template,we get complete phytase gene by PCR amplification. We cloned this gene into pPIC9K vector and transformed this express vector into yeast.Extracting RNA which is not degraded and contain no DNA is basis to do research about molecular cloning and gene expressing analysis. It is very difficult to extract active RNA from Aspergillus niger because of the existance of complex cell wall and endo-Rnase activity. To obtain RNA with high quality, we compared three such different extraction methods as TRIZOL, Isothiocyanate Method and Hot Borate Method. Experiements show that Hot Borate Method is best for the RNA extraction from Aspergillusniger that is rich in Rnase and polysaccharide,and we can get pure and integrate RNA sample which can be used in RT-PCR experiment.The phytase gene was amplified by PCR using Aspergillus niger cDNA as template, and the gene was inserted into the vector pBV220 and pPIC9K. The recombinant vector containing acid phosphorutase gene was transformed into ? coli DH5 a strain. Induced by temperature the acid phosphorutase gene was expressed to be a protein of about 60kD by SDS-PAGE analysis, its optimun reaction pH is 2.5 and its optimum reaction temperature is 55C.Zhang Jianying(Biochemistry and Molecular Biology) Directed by professor Xie Daping Jiao Qinghua...