Study On The Spectral Properties Of Allophycocyanin And Phycobilin And Their Functions On Photosynthesis

Chroococcidiopsis thermalis sp.PCC7203 is a kind of algae capable of adapting to far-red light.The genome sequencing was done in 2012.In cyanobacteria that can adapt to far red light conditions,there are many homologous phycobiliprotein subunits,which are covalently or noncovalently bound to pigment molecules.The phycobiliprotein subunits adapt to the far-red light environment by changing the way of binding to pigments and forming multi-subunit complexes.By homology analyzing,ApcB3?encoding gene Chro₄356?of Chroococcidiopsis thermalis sp.PCC7203 has similarity to ApcB from Synechocystis sp.PCC 6803,ApcD4?encoding gene Chro₄357?homologous to ApcD.ApcB3 and ApcD4 were assembled with phycocyanin PCB with lyase CpcS1 catalytically or auto-catalytically.The cysteine at the possible pigment binding site of ApcD4 was mutated.The cell fluorescence,the absorbance and fluorescence of supernatant obtained after disrupted by ultrasonic and the protein purified by the Ni²⁺-affinity chromatography were measured after in vivo recombination in E.coli.The results showed that ApcD4 binds to phycobilimine PCB in a non-covalently bound form,but the binding force between the two is weak.In the nickel ion affinity chromatography process,the phycobilichimin PCB is detached and fluorescence is only detected in the supernatant..After tyrosine mutation at position 81,phycobilipine PCB could not be bound.When ApcD4 is co-expressed with ApcB3 in E.coli,it can bind to PCB and can be purified by Ni²⁺-affinity chromatography with a red-shifted fluorescence peak.In order to investigate the mechanism of action of ApcD4 and ApcB3 in Cyanobacteria,vector for transformation of ApcD4 and ApcB3 into the location of ApcD and ApcB from Synechocystis sp.PCC 6803 were constructed.Among them,six histidine tags were placed at the C-terminus of ApcD4.Through the extraction of total DNA,PCR confirmed that both ApcD4 and ApcB3 were transformed successfully.No target protein was obtained by Ni²⁺-affinity chromatography.Bdfp1.2,bdfp1.3,bdfp1.2:1.2 fused with mcherry by changing the order of genes and the length of the linker between them.FRET effect was compared by measuring the absorption and fluorescence spectra of purified fusion proteins.The results showed that the FRET is strongest when mCherry is at the N-terminus of BDFP1.2 when 12 amino acids for linker.In order to explore whether phycobiliprotein can improve the photosynthetic efficiency of plants,the fusion fragment with good FRET effect was infected by Agrobacterium and then the physiology data was determined after expression in leaves.The results showed that the photosynthetic efficiency did not improve.In this study,ApcD4 and ApcB3 combined with PCB generated a red-shift spectrum,providing materials for the development of far-red-light probes.The fusion method of mCherry and BDFP1.2 with strong FRET effect was found to provide reference for the fusion of other fluorescent proteins,and provide materials for the development of fluorescent markers and double channel fluorescent markers with large Stokes shift in the far red region.The fusion protein is complementary to the light absorption range of the plant and can be used to transfer plants to improve the photosynthesis efficiency of plants.