Studies On Autoassemblying Of Phycocyanin Of Spirμlina Platensis And Functional Comparison Of Some Phycocyanobilin Lyases

In this thesis, genes for the apoprotein (phycocyaninαsubunit; cpcA) and the heterodimeric lyase (cpcE and cpcF) that catalyzes chromophore attachment were cloned from blue-green alga (or cyanobacteria) Spirμlina platensis FACHB314. Gene for enzyme(heme oxygenase 1;hoxl) required for the conversion of heme to biliverdin was cloned from Synechocystis sp.PCC6803; and gene for enzyme (3Z-phycocyanobilin:ferredoxin oxidoreductase; pcyA) necessary for the conversion biliverdin to PCB was from Spirμlina platensis FACHB314. The expression of these five genes in Escherichia coli. BL21 to produce fluorescent holo-a-phycocyanin. We also found that the apo-CpcA had the function of autoassemblying with PCB in E. colis train that lacks cpcE and cpcF, but the products had very low fluorescence yields and the fluorescence intensity was only 34% of the E. colis train with cpcE and cpcF.Gene for the apoprotein(phycocyaninβsubunit; cpcB) was cloned from Spirμlina platensis FACHB314; there lyase genes cpcT,cpcU and cpcS(7002) were cloned from Synechococcus sp. PCC 7002; the lyase gene cpcS(6803) were cloned from Synechocystis sp. PCC6803.The recombinant experimental results show that CpcU/CpcU(7002) as a heterodimer catalyzes the connection of phycocyaninpsubunit and phycocyanobilin in E.coli BL21, which finally produced fluorescentβ-subunit PC,and its fluorescence intensity was 110.1; the fluorescence intensity of the E.coli BL21 with CpcT lyase was only 52.71; and the fluorescence intensity of the E.coli BL21 with CpcS(6803) lyase was 81.69. Also found that the apo-CpcB had the function of autoassemblying with PCB in E. colis train that lacks lyase, and the products had a high fluorescence yields and the fluorescence intensity was close to the BUS(7002). The expression of AUS (7002) shows that Synechococcus lyase CpcU/ CpcS (7002) also in charge of correct addition of PCB to CpcA. Two genes cpcBA,ussBcpcBA were cloned from Spirμlina platensis FACHB314. Various expression E.coli strains were constructed, which were BA,BA-EF,BA-US(7002),uBA,uBA-EF,uBA-US(7002), and analyzed their spectroscopy characteristic. The experimental results show that the expression strain uBA-EF was best, and its the maximum emission peak at 640nm, and fluorescence intensity was the highest (369.2). It suggests that the Construction of expression vector of a fluorescent phycocyanin in rice can reference uBA-EF.This paper studies the autoassemblying of phycocyanin and the difference of the catalytic activity of various lyase, explores the mechanism of fluorescent and biological activities in PC and provides a theoretical and experimental basis for the expression of a fluorescent phycocyanin in rice.