The Cultivation And Physiology-Biochemistry Analysis Of Transgenic Chlorella Sp. MACC/C95 With Phytase Gene

This research was on the basis of successfully transferring the phytase gene into Chlorella sp. MACC/C95 and detecting the phytase expression. Study on the phytase expression stability. Analyse the physiology-biochemistry indexes of transgenic C.sp. The effects of culture condition and culture medium on the growth and phytase activity of transgenic C.sp. were investigated to find out the optimum condition. All of the work in this paper established a good foundation for a large-scale-high-density cultivation. This is a far-reaching meaning for microalgae resource exploitation and phytase application.Transgenic C.sp. was broken up with three methods. Comparing the protein and phytase activity which was detected with these methods from the broken transgenic C.sp., it was found out that transgenic C.sp. was broken up more sufficiently with the ultrasonic disruption method. In the method more protein and higher phytase activity was got. The ultrasonic disruption optimum time was 8min.42 single-clone transgenic algae strains were obtained from antibiotic flat containing G418. Detect the expression of phytase protein when the biomass of these algae strains reach an amount. The expression of phytase protein in transformants was confirmed by phytase activity analysis. The top eight strains in the expression of phytase were selected. After 7 successive subcultures the phytase activity in the eight strains did not decrease gradually. It was proved that phytase gene in C.sp. MACC/C95 expressed stably. The phytase activity of the transgenic C.sp have two peaks at pH 3.8 and pH 6.0 and the optimum temperature of phytase of the transgenic C.sp is 65℃. In the same culture condition, there was no apparent difference in physiology-biochemistry indexes between transgenic and untransgenic chlorella. This indicated that transforming of foreign phytase genes did not change the physiology-biochemistry index of C.sp.The effects of incubation density, temperature, pH and light density on the growth and phytase expression of transgenic C.sp. were investigated. The results of single factor tests showed that the appropriate incubation density for transgenic C.sp. was 0.074 -0.126mg/ml, the appropriate culture temperature was 22.5℃, the appropriate culture pH was 8—10, the appropriate light density was 1000-50001x. The results of 3 factors (temperature, pH and light density)-3 levels orthogonal experiment showed that the appropriate condition for growth of transgenic C.sp. was 22.5℃, pH 9.5, light density 4500±1001x, the appropriate condition for...