Expression Of A Leghemoglobin Gene Lba In The Chloroplast Of Chlamydomonas Reinhardtii And Its Effects On Hydrogen Production

The model species Chlamydomonas reinhardtii is the alga of great potential for bio-hydrogen production because of its clearly genetic background, easy culture, rapid growth, easy manipulation, especially its high activity of Fe-H2ase and hydrogen production from water driven by solar energy.To date, the key barrier for the utilization of C. reinhardtii in commercial H2 production is its H2ases easily inhibited by O2, makeing H2 yield very low. Therefore, the study of its metabolic mechanism of hydrogen production at molecular level and the improvment of the efficiency of hydrogen production through genetic engineering methods have been gained lots of attentions. The main stretagies are to improve the O2-tolerance of H2ases or to reduce O2-concentration in cell as well as to improve the electron transfer efficiency.Currently, the low intracellular oxygen-concentration was achieved by the depletion of sulfur in liquid C. reinhardtii cultures due to PSII inactivation resulted from the susceptibility of the D1 reaction center protein by photo-damage and its inability to be repaired under sulfur deprivation. Consequently, under the sulfur-deprived conditions, the PSII-mediated water-splitting reaction and the associated O2 evolution are inhibited. This creates low O2 concentration and in turn releases the oxygen inhibition of H2ase and improves H2 production. However, when PSII is inhibited, electrons from H2O are also inhibited, which is important for hydrogen production, finally H2 production reduced ultimately.To improve H2 production in C. reinhardtii, we developed a new approach by transforming the leghemoglobin gene lba into the chloroplast of C. reinhardtii. Lba protein has high affinity to oxygen and its expression in the chloroplast of C. reinahrdtii may capture more dissolved oxygen in cells than in the wild type and hopefully to improve H2ase activity and H2 yield. It the transgenic algae, both PSII and H2aes probably work at high efficiency at the same time by the partial restoration of PS II activity to increase the supply of electrons, leading to the improvemt of H2 production.The contents and results are as follows:①The RNA of soybean has been extracted from mature root-nodules and was reverse transcripted into cDNA. Then the cloning of leghemoglobin gene lba, lbc1, lbc2 and Flbr was achieved by PCR from cDNA respectively. The expression vector cg401-1-lba was constructed successfully for the transformation into the chloroplast of C. reinhardtii.②The plasmid cg401-1-lba was transformed successfully into the chloroplast of C. reinhardtii by the gene particle method. The transgenic algae were screened under 100mg/L spectiomycin.③The transgenic algae were identified by the PCR and RT-PCR amplification based on total DNA and RNA respectively. The results showed that lba gene was integrated into the DNA of the chloroplast of C. reinhardtii and its transcript was correct.④The expression of lba protein in the transgenic algae were identified by the Western Blotting. The results showed that lba was begun to express on day 3 and reached maximum level on day 5 after anaerobic incubation.⑤By comparison of the growth of wild and transgenic algae, the OD750 of transgenic algae is 2.7, about 20 % (about 106 cell/ml) lower than that of 849. The chlorophyll content of the transgenic algae was also about 3 mg/L lower than that of 849. The results revealed that the transformation of lba gene affected the growth of transgenic algae.⑥In TAP medium, the O2 content of transgenic algal culture was 5 %, 1-4 folds lower than that of 849 and the H2 production in transgenic algal culture was 91μl, 50 % higher than that of 849. In TAP-S medium, the O2 content in transgenic algal culture was 33-56% lower than that of 849 and the H2 production of transgenic algae was 178μl, 29 % higher than that of 849.⑦While no sulfur was in the culture medium, the O2 content of transgenic algae 849-lba was the lowest and the H2 production and the rate of H2 production was highest compared to the other concentration of S. Meanwhile, when the light intension is 30μM ? m-2 ? s-1, transgenic algae 849-lba showed the lowest O2 content and the highest H2 production as well as the rate of H2 production compared to the other light condition.