The Functional Analysis Of Carbonic Anhydrase Gene In Rice (Oryza Sativa L.) Under Stress

Carbonic anhydrase (CA, EC 4.2.1.1), a zinc-containing metalloenzyme, the enzyme has been found in most organisms including animals, plants, archaebacteria, eubacteria. CA plays an important role in photosynthesis. But some researchers found that CA responded to environmental stresses. For catalyzing the reversible inter-conversion of CO₂ and HCO₃^- (CO₂ +H₂O ←→ HCO₃^- + H⁺), we focused in Carbonate stress(NaHCO₃, Na₂CO₃) for plant in the alkali soil (NaHCO₃, Na₂CO₃) in the northeast of China. Using molecular biological methods, the relationship between CA and stress was studied in rice plant. Those results were as followed:1. The results of comparative experimentation showed that the germination rates and the growth of rice were affect by the NaHCO₃ and NaCl stresses.Rice roots were more sensitive to NaHCO₃ than the shoot. Compare with NaCl stress, Na+, pH value, and HCO₃^- were supplied by NaHCO₃ stress.2. To understand the responsive relationshio between rice CA and environmental stress, experiment studied the profile of rice CA expression by Northren blot analysis using rice seedlings under several stresses. The results showed that the levels of CA transcripts were increased under stresses from NaCl, NaHCO₃, and PEG and the intensity of salt and drought stresses influenced the expression of rice CA. Moreover, CA activity also increased under NaHCO₃, Na₂CO₃, NaCl and PEG₆₀₀₀ stresses. These results suggested CA responded to environmental stresses.3. In order to research the relation between CA gene and environmental stresses better, the ORF of rice CA was constructed into plant expressional vector pBI121, and introduced into Arabidopsis thaliana using an Agrobacterium-mediated system. The result of northern blot proved that the CA gene was transferred into the Arabidopsis genome. Transgenic Arabidopsis overexpressing the rice CA gene showed greater salt tolerance at seedling stage than wild-type plants in the 1/2 MS medium with 5 mM NaHCO₃, 50 mM NaCl, and 100 raM NaCl.4. The CA gene and GFP were together cloned into plant expressional vector pBI121, then the plasimed pBI121-CA was transformed into Arabisopsis. Using the fluorescence dye-Mito Tracker Red CmxRos to dye the Arabidopsis protoplast, and observing the cell by Confocal Laser Scanning Bioligical Microscopr (OLYMPUS). The result indicated that the CA gene encoding protein lied in Arabidopsis mitochondria, this result is identical with predictive result initially.5. The CA gene was cloned into the prokaryotic expression vector pGEX-6p-3, the plamid pGEX-6p-3-CA was transformed into E. coli BL21. After the inducement with IPTG and theGST-CA fusion protein was finally purdied by Glutathione-Sepharose 4B affinity column and incubateing ATP solution. After cleavage of the GST tag, native CA protein was obtained, and analyzed by SDS-PAGE. The yields of the purified recombinant rice CA were about 19 mg g-1 dry cells. The recombinant rice CA protein after the cleavage of the GST tag had CO2 hydration activity and no detectable esterase activity in vitro. Zinc is helpful for C dehydration activity of produced rice CA from E. coli. AZ and N3" were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ > N3" > NO3" > I> Br> Cl". The amount of CA proteins was enough for antibody production, which will be invaluable in future studies of the function of rice CA.