Screenning Of The Regulation Genes Of The Key Photorespiration Enzyme AtCAT2 And Studies On Its Protein Interaction

Photorespiration metabolism is the second major metabolic flux besides the Calvin cycle in C3 plants.As a key enzyme in photorespiration,Catalase?C AT?mediatesthe removal of the most H₂O₂in plants,thus plays as an important regulating factor in plant signal transduction and physiological processes.There are threeCAT genes in Arabidopsis,CAT1,CAT2 and CAT3,CAT2 and CAT3are related to photorespiration metabolism.In this study,the regulation genes of CAT2 were screened and studied through enhancer and suppressor mutants from EMS mutagenized cat2-1 M₂ pools;on the other hand,the interaction between At GOX and AtCAT was studied bybimolecular fluorescence complementation?BiFC?,yeast two-hybrid system and pull down technologies.Results were as followings:1.Screenning ofthe regulation genes of CAT2To obtain the mutants that enhance or revert the phenotype of cat2-1,we screened the M2 generation seeds of EMS mutagenizedcat2-1under the 24 h light and high light conditions(1500?mol.m^-2.s^-1).We discovered a mutant named enhancer2 that enhances the phenotype of cat2-1?stunted rosettes,serious leaf yellowing and deleyed development?.Genetic analysis showed that this mutant is a recessive mutant controlled by one gene.Through map-based cloning and genome resequencing technology,thechlorophyll synthesis gene?At GUN5?and the unknown function gene?At5G16950?were found the candidate gene to cause the phenotype of enhancer2.The phenotype of enhancer2 is verified caused by At GUN5 mutation through complementation,and has nothing to do with At CAT2 by genetic analysis.A partially reverttedmutant named suppressor4?stunted rosettes,dark green leaves and longer root than cat2-1?was also identified from the screening.The chlorophyll content in suppressor4is higher than Col-0,and its maximum photosynthesis efficiency?Fv/Fm?is greater than that ofcat2-1,close to the level of Col-0.Through genetic analysis,the phenotype of suppressor4 is regulated by CAT2 and its suppressor gene.2.Interaction between GOX and C ATStudies had shown that GLO physically interacted with CAT in rice leaves,and that the interaction could be disassociated by salicylic acid?SA?,besides the association and disassociation between the two proteins could regulate the H₂O₂ content of peroxidase in rice.At GOX1 and AtGOX2 were verified interacted with AtCAT2 and AtCAT3,respectively,by using BiFC technology,this is consistent with the conclusion in rice.However,AtC AT2 was only detected in one sample in the pull down of prokaryotic expressed between At GOX and AtCAT,thus the prokaryotic expression system is not suitable to study theinteraction between At GOX and AtCAT.The results of pull down in vivo showed that another protein can be detected in the over-expression plants of At GOX or AtC AT after the protein was been purified.By using yeast two-hybrid system,a weak interaction between At GOX and AtCAT was identified.In summary,there are interactions between GOX and CAT.