Cloning of CrFtsZ2 Gene from Chlamydomonas Reinhardtii and Expression in E.coli

Abstract: FtsZ, a functional protein existed in prokaryotes and plants controlled the division of prokaryotic cells and plastids in plants. In £. coli, formation of FtsZ into a cytokinetic ring – Z ring at the midcell division site is the first step in the assembly of the bacterial cell division machinery. The discovery of FtsZ protein in plants provided us a chance to study the molecular mechanism controlling the division of chloroplasts in plant cells. The chloroplast plays importantance roles in plant. The ancestors of chloroplast and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. At present,it has been identified that The chloroplast divorces by FtsZ protein yet, but the gene encoding FtsZ protein had transfered to nucleus genome from chloroplast genome. In contrast to most of bacteria which encode a single FtsZ protein, plants encode multiple proteins that group into two families: FtsZl and FtsZ2. It was considered that the divergences of FtsZl and FtsZ2 fiamilies happened in forepart evolution of land plants.The CrFtsZ2 cDNA encoding the prokaryotic cell division protein FtsZ homologoues has been isolated from Chlamydomonas reinhardtii by reverse transcription polymerase chain reaction(RT-PCR) . The full-length CrFtsZ2 cDNA is 1815bp, encoding proteins of 435 amino acids. CrFtsZ2 has 10 introns and 11 extrons. Protein sequence alignments indicate that the CrFtsZ2 is similar to other FtsZ proteins and contain the conserved GTP binding motif. CrFtsZ2 also show a typical N-terminal extension with the typical character of the plastid trisit peptide (PTP). Sequence data alignment with the higher plants suggest that the CrFtsZ2 is the members of FtsZ2 family. Genomic structure comparisons of FtsZl and FtsZ2 show that extron length and intron position are conserved, especially within each clade. Our data indicate that the divergence of the FtsZl and FtsZ2 families occurred long before the evolution of land plants, after the divergence of red and green lineages diverged. The results are consistent with proposals that the two FtsZ families evolved distinct functions during the evolution of the chloroplast division apparatus, and indicate that genetic and functional differentiation occurred much earlier than previously hypothesized. Overexpression of the CrFtsZ2-GFP fusion proteins in E. coli also resulted in filamentous phenotype that is similar to the phenotype of EcFtsZ2 mutants. These results suggest that CrFtsZ2 can recognize the signals for division site positioning in bacteria and can interact with the bacterial division machinery…
Key words: Chlamydomonas reinhardtii; CrFtsZ2; expression in prokaryote phylogenetic analysis

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