| In anaerobic conditions, the hydrogenase (H2ase) in the Chlamydomonas reinhardtii produces hydrogen using electrons and protons which come from photosynthesis and organic decomposition and releases into the extracellular. The H2ase activity of C. reinhardtii is high and C. reinhardtii grows fast, is easy to culture, has clearly genetic background and easy for experiment. Therefore, C. reinhardti is the model algal species for the study of biohydrogen production by using solar energy and water and has great potential to be utilized in the industry in the future.But the H2 ase of C. reinhardtii is extremely oxygen (O2)-sensitive enzyme, while the oxygen is the main product of photosynthesis, which leading to the low hydrogen yield and limiting the development of its industrial hydrogen production. Currently the method to lower the oxygen content in C. reinhardtii cells is to remove sulfate in the culture medium to suppress the photosynthetic system II (PSII) activity, thereby reducing the oxygen produced by photolysis of water. But this method also inhibites electronic production by the photolysis of water, which limiting the electron source for the hydrogen production and finally leading to the low hydrogen production of C. reinhardtii. Therefore, to improve the efficiency of hydrogen production of C. reinhardtii requires not only reducing the oxygen concentration inside the cells but also maintaining the supply of electrons.Leghemoglobin ( Lb) in soybean root nodules has a high affinity with oxygen and capabilities. It can reduce intracellular oxygen concentration and regulate respiration, thus enable the oxygen-sensitive nitrogenase enzyme in the soybean root nodules has a highly efficient nitrogen-fixing efficiency. In several biofermentation engineering researches, the hemoglobin has been applied to express in vitro and increased production of the cultures. Lb consists of two parts. One part is globulin (globin), encoded by soybean (Glycine max). Another part is encoded by the symbiotic Bradyrhizobium japonicum. In the B.japonicum, hemH gene encodes the ferrochelatase which catalyzes the final step in heme synthesis and is necessary in the heme (heme) synthesis. The precursors of heme are similar to those of chlorophylls in C. reinhardtii and higher plants via the 5-aminolevulinic acid (ALA) pathway. Therefore, in C. reinhardtii cells there are adequate precursors for heme synthesis.This study attempts to transfer the soybean rhizobia hemH gene and soybean lba gene into the chloroplasts of C. reinhardtii strain cc 849 to synthesize the active Lb, which would help to reduce the oxygen content inside the chloroplast and increase activity of H2ase. With the helps of partial restoration of PSⅡactivity, the improvement of the electron supply from PSII maight increase the hydrogen yield of the transgenic algae. The work is a new attempt to improvement of hydrogen yield by using the characteristics of leghemoglobin.The main results of this thesis are as follows:1. Total DNA of B. japonicum has been extracted. The coding region of both the ferrochelatase gene, hemH, from B. japonicum, and the leghemoglobin gene, lba, from Glycine max, were transferred into chloroplast of C.reinhardtii. Chloroplast transformation vector cg401-1-hemH-lba was constructed.2. Transgenic C. reinhardtii was gotted by the gene particle method and screened on the solid spectinomycin mediu. Subcultured under liquid-solid medium with spectinomycin repeatedly to maintain the transgenic algae.3. Transgenic C. reinhardtii were identified by the PCR and RT-PCR amplification based on total DNA and RNA respectively. PCR results confirmed hemH and lba coding region DNAs have been integrated into part of algal chloroplast genomes. RT-PCR results showed that hemH and lba genes are transcribed at the RNA level.4. Western blotting results confirmed that ferrochelatase and lba protein expressed successfully in C. reinhardtii chloroplasts both in the normal culture condition and in the anaerobic condition. The maximum expression level appeared on the sixth day of normal culture condition and on the fifth day of the anaerobic condition, respectively.5. By comparison of the growth of wild and transgenic, the OD750 of transgenic algae is 3, about 14% slightly lower than that of strain cc849. But, the chlorophyll content of the transgenic algae was about 40 mg/L, higher than that of strain cc849. The results revealed that the transformation of hemH-lba gene affected the growth and chlorophyll content of transgenic algae.6. Using UV spectrophotometer to scan the heme extraction of strain cc849 and transgenic alga hemH-lba between 500nm and 600nm, there is a small absorption peak in 550nm. The result showed that transgenic alga hemH-lba synthesized small quantity of heme.7. To compare the amount of hydrogen yield, oxygen content and hydrogen production rate of strain cc849 and transgenic alga hemH-lba at different sulfur concentrations of 0, 12.5, 25, 50, the results show that in various sulfur concentration, the oxygen consumption rate of transgenic alga hemH-lba are larger than that of strain cc849 and hydrogen production increased compared to wild algae 849.8. Through the orthogonal experiment of light intensity, chlorophyll content, sulfur content (three factors and three levels), the best conditions for hydrogen production of transgenic alga hemH-lba and wild algae 849 were identified respectively. In culture conditions at 25℃, 60μmol m-2 s-1 light intensity, 0.5mg bottle-1 of the chlorophyll content and , 12.5μmol L-1 of sulfur content favored transgenic alga hemH-lba's hydrogen production. While it was 60μmol m-2 s-1 light intensity, 0.5mg bottle-1 of the chlorophyll content, 0mol L-1 of the sulfur content best for strain cc849's hydrogen production.9. According to codon bias of the chloroplast DNA of C. reinhardtii, the nuclear acids of hemH gene and lba gene were modified to make the AT content reaching 63%. The modified hemH gene and lba gene were also successfully transformed into the chloroplast of C. reinhardtii.
|
PDF Full Text Request