Construction Of A System For The Transformation Of Foreign Genes In Chlorella Sp. And Expression Of Phytase

It is the first time to report that the phytase gene was transferred and expressed stably in Chlorella sp. MACC/C95, a kind of unicellular eukaryotic green marine algae. Acco- rding to its characteristic, Chlorella sp. MACC/C95 can be a good bioreactor for large- scale producing foreign proteins such as phytase. The transformation of this phytase gene into this kind of alga could be an efficient way to supply the phytase. After researching the sensitivities of Chlorella sp. to 10 antibiotics, Determined the suitable selectable markers in genetic engineering of Chlorella sp.. Then by using proper antibiotics mixtures eliminate associated bacteria obtained axenic Chlorella sp. and using gus gene optimized the electroporation condition of the whole Chlorella sp. cell. On the basis of the optimal electroporation condition, the phytase gene was transferred into Chlorella sp. and obtained transgenic algae for the first time. All of the work in this paper established a good foundation to widely produce phytase in low cost. The research contents and results in detail were stated as follows:Study on sensitivities of Chlorella sp. to 10 antibiotics. The result showed that Chlorella sp. was not sensitive to streptomycin, kanamycin, cefalothin. neomycin, gentamycin and penicillin, they cannot inhibit the growth of Chlorella sp. in the range of experimental concentration. It relatively sensitive to hygromycin and geneticin, and very sensitive to zeocin and chloramphenicol. LD₅₀ and 95% confidence ranges of these four reagents in both seawater culture and decreasing NaCl concentration medium were calculated respectively. The conclusion is that G418 is the most suitable regent if using nptâ…¡ gene as selection marker for Chlorella sp. genetic engineering. Moreover, the selection concertration in solid culture is 140.5μg/mL.Determining effects of those antibiotics which cannot inhibit the growth of Chlorella sp. on the bacteria obtained from the culture of Chlorella sp.. On this basis, the effects of different antibiotics combination & concentration on the bacteria and their host-Chlorella sp.were also researched. The conclusion is that the bacteria is very sensitive to 5 antibiotics (gentamycin, penicillin, kanamycin, cefalothin and streptomycin ). By first selecting mould-free algae colony on the plate, then treated the liquid culture of those colony two times with the finial concentration of 100 μg/mL of this 5 antibiotics combination, axenic Chlorella sp. were obtained. Then the effect of incubation density, temperature, pH and light density on growth of axenicChlorella sp. were investigated. The results showed that the appropriate incubation density for Chlorella sp. was about 4.27x106cells/mL, the appropriate culture temperature for Chlorella sp. was 25±5°C, the appropriate culture light density for Chlorella sp. was 3.0xl03¹.0xl04lx, and the appropriate culture pH for Chlorella sp. was about pH 10. The optimum concentrations of 5 nutrient factors (nitrogen, phosphorus, carbon, microelements, vitamins Bi and B12) were obtained by orthogonal experiment, that is : KNO30.5 g/TU NaHCO30.2 g/I^ VB121.0 ug/L> KH2PO4 0.02 g/I^ VB! 0.3 mg/L. Addition of lmL/L ill microelements with this optimal medium, the cell growth speed and biomass productivity increased significantly.Using gus gene to optimize the electroporation condition of Chlorella sp. The whole cells of Chlorella sp. were treated with plasmid pBI121 which bears fi-glucuronidase under the control of the CaMV35S promoter. The effects of several factors: pulse voltage, pulse times, pulse duration time, pulse cycle, concentration of carrier DNA, osmosis treatment, concentration of plasmid DNA and cell growth phase were studied. The result showed that maximal GUS activity was obtained under conditions that: pulse voltage 5kV, total number of pulse 211 , pulse duration time 0.05s, and pulse cycle 90. Osmosis treatment can improve the GUS activity notably. When Chlorella sp. cells growed in exponential phase, plasmid DNA added in concentration of 6p.g/mL and carrier DNA added in concentration of 150p.g/mL, the maximum GUS activity was obtained.Transferred the phytase genes into the whole cell of Chlorella sp. with the optimal electroporation condition. PCR and Southern blotting analysis indicated that the phytase gene was integrated into the Chlorella sp. genome. The expression of phytase protein in transformants was confirmed by RT-PCR and phytase activity analysis. The activities of phytase expressed in the transgenic Chlorella sp. have two peaks at pH 3.8 and pH 6.0; the optimum temperature of it had been improved from 55°C to 65°C.