Study On The Control Of Hydrogen Production By Chlamydomonas Reinhardtii By Artificial MicroRNA Targeting D1 Gene

Hydrogen(H2) is considered as the most attractive and clean alternative fuels. Photobio- H2 production driven by solar energy has been observed in green algae Chlamydomonas reinhardtii since 1939. However, the development of algal H2 production has lagged mainly due to the oxygen(O2) hypersensitivity of hydrogenases that catalyze the reaction of H2 formation. In 2000, by affecting the D1 protein of photosystem II in C. reinhardtii, the sulfur-deprived algal culture discovered by Melis et al. prolonged significantly the H2 evolving duration. Nevertheless, it is still very difficult to employ this technique when we scale up the culture in application. Moreover, without changing the culture medium will be more applicable and more economic way for the future practice.Here we presented a study with a successful H2 releasing regulation in C. reinhardtii by expressing an artificial mi RNA(ami RNA) targeting D1 gene. The results obtained were as followings: 1. ami RNA targeting D1 gene was successfully designed based on an onlinetool WMD3( http://wmd3.weigelworld.org/cgi-bin/webapp.cgi); 2. An ami RNA-D1 expression vector was constructed under the control of a heat-inducible promoter, which utilized endogenous Cre-MIR1162 as expression backbone. This recombinant plasmid was next transformed into C. reinhardtii cells by glass-bead method; 3. The expression of ami RNA-D1 in transformants increased 15 folds compared to that of untreated algae after heat shock; 4. The expression of target gene D1 was decreased 73% after heat shock intransformants; 5. Yield value that reflects the PS II activity detected by Phyto-PAM techniquedecreased 53% after heat shock in transformants; 6. After two continous heat shock treatments, H2 production and O2 consumption intransformants were both higher than the wild type group. The H2 production rateof transgenic algae after heat shock reached the maximum level, which was 2.3folds more than the wild type group. 7. The total H2 production yield of transgenic algae in a 7-day cultivation was 57%more than the wild type group.In conclusion, we have constructed a transgenic green algae C. reinhardtii, which could produce H2 continuously without imposing a nutrient stress in culture medium. This transgenic alga provides a new strategy in regulating the H2 production under normal culture conditions.