Expression And Analysis Of Iron-containing Cofactor Protein In Chlamydomonas Reinhardtii

Iron(Fe) and other metals perform a remarkable array of functions that are crucial for life. Proteinscarrying these metals as cofactors mediate diverse biochemical processes. Cup-shaped chloroplast ofChlamydomonas reinhardtii is40%of the total cell volume, which contains fairly amount of iron and isknown to bear folded and assembled complex eukaryotic proteins. Organelles as bioreactor havebecome a hot topic for research application,and have a broad prospect due to the increasing demand forcrop production and chemical compound scavenging. I wish to investigate whether iron-containingcofactor protein FAO1, Cyb5and NifH would be active in Chlamydomonas reinhardtii.Such testingsystem may also be useful for some long-term goals such as introduction of metalloezyme nitrogenasein eukaryotic cell.On the other hand,metabolism and environmental stress significantly influence the final productionin crop plants. Long-chain fatty alcohol oxidase (FAO1) is an important enzyme in ω-oxidationpathway of long-chain fatty alcohol and thought to be existing in many crop plants. It is not only forplant basal metabolism, and may also play an important role in plant signalling and defense. Long-chainfatty alcohol oxidase (FAO1) contains a heme prosthetic group chelating iron. I have successfullyoverexpressed FAO1in E.coli. The enzymatic activity of purified FAO1was tested by a color-linkedassay system. Recently, we cloned the PsaD transit peptide gene, Fao1gene and His-tag geneseparately by polymerase chain reaction (PCR). Then, the3-piece fusion genes were inserted intopDBle, a nuclear expression vector backbone. Such construct was transformed into ChlamydomonasReinhardtii (CW15) by glass beads method, and the recombinant vector have shown to be integratedinto CW15genome. After the correct gene transcription, the translated FAO1would be targeted to thechloroplast through the signal peptide. Then I conducted purification of this protein through an affinitycolumn and subsequently tested its enzymatic activity. In conclusion, a recombinant plasmidpDBle-TFHis was constructed, proven to be integrated into CW15genome and transcribed successfully.Protein purification elution made substrate reaction turn blue. I have completed the modification fromp72B-GX to p72B-MZ and the chloroplast transformation vector p72B-MZ-FAO1was constructedsuccessfully. The studies on FAO1in both prokaryotic and eukaryotic expression system can provideexperimental materials and lay the foundation for studies on FAO1utilization of iron in eukaryoticunicellular Chlamydomonas. If the green algae can be used as biological reactor for FAO, it wouldprovide a good environment for the study of metabolic pathway, related derivatives and possible defensemechanism in higher plants. In addition, we may use the knowledge to transform crops.We also consider Cyb5and nifH as the research objects. The PsaD transit peptide gene, egfp gene,S. pombe cyb5and A. vinelandii Lipmann nifH were cloned by PCR. Nuclear expression vectors wereconstructed based on the vector pDBle and the fusion genes were inserted into pDBle. The vectors weretransferred into C. reinhardtii (CW15) by glass beads and the recombinant plasmids have integrated intoCW15genome. The results showed that the recombinant plasmid pDBle-b5, pDBle-bG, pDBle-TbG and pDBle-THG were constructed correctly, and have been integrated into CW15genome. Now we aredetecting green fluorescence, wishing to obtain the objective protein that localized in the chloroplast.The studies on Cytb5and NifH may provide experimental materials and lay the foundation of furtherstudies on iron trafficking in Chlamydomonas. If cytochrome b5gene can express the red protein inalgae, it could possibly become a visual transformation marker for genetic transformation inchlamydomonas. If the NifH can express active protein in combine with house keeping iron insertiongenes in C. reinhardtii chloroplast, it may pave the way for introducing more iron-containing proteins toplastid, such as nitrogen-fixing genes, and may bring the hope of non-legume crops autonitrogen-fixingone day.