Site-directed Mutagenesis Transformation Of Cyanobacteria Phytochrome Slr1393GAF3and Heme Oxygenase HO1

There are lots of light-harvesting pigment-proteins in Synechoystis PCC6803,it has a high homology with phytochrome,the study found that gene slr1393is similar to the phytochrome.The study found that the gene slr1393in Synechocystis PCC6803is similar to the phytochrome,and it has a similar effect of reversible conversion,so we called it cyanobacteria phytochrome.Three consecutive GAF domain of the gene in gene slr1393,the separate GAF3owned completly reversible effect of reversible conversion.528cysteine of GAF3combined with algae blue pigment.the complex forms a reddish green reversible pigment-protein. The protein has some special optical properties as a fluorescent probe.The major purpose of this subject is filtering out the protein as a fluorescent probe by site-directed mutagenesie method.We elected a number of important mutations site by homology comparison method, and constructed mutations physical particles by site-directed mutagenesie method, the sites are V23Q, Y24H, R25E, N71K, A76K, A76C, D87I, E81Y, H89Y, S73H. The results:the fluorescence quantum yield and molar extinction coefficient of the mutant (A76K) increased1.22, on the contrary, the mutants (H89Y, S73H, N71K) of cells are no activity, and others have no significant difference.In this subjrct we studied the reaction mechanism of heme oxygenase HO-1in cyanobacteria.We identify the mutation sites,the sites are H17F,E134C,E134H,G134D, constructed mutations physicalparticles by site-directed mutagenesie method.Measuring with wild-type ang mutants fluorescence and UV absorption spectra and comparison of their characterisrics found that the mutant (H17F) is no activity,the mutant(E134C) of cells became red and its UV absorption spectra has significant difference,others have no significant difference.The experimental confirmed that histidine of17is the center of the activity of HO-1protein.