The Application Of FMN Binding Proteins In Arabidopsis Mutants Screening

The normal life processes of plants need various environmental factors in a very appropriate state.The presence of suitable environmental factors will help plants grow normally and complete their life circle,while any deficiency of the environmental factors will affect plants growth and development.In fact,this very suitable growth environment condition is rare in nature and all kinds of environmental stresses occur in the growth of plants.Reactive oxygen species(Reactive oxide species,ROS)are crucial signaling molecules in oxidative stress response.In plants,ROS are involved in many processes including seed germination,development,senescence,and high light stress.A large amount of reactive oxygen species will be generated when plant affected by environmental stress.If these excessive reactive oxygen species in plant could not be scavenged in time,the redox equilibrium steady state in the body would be broken.There are some fluorescence probes which can be used to detect biological redox state in the body.Phototropin contains two LOV domains at their N terminal,which are relate to light,oxygen,or voltage and have a binding site for the chromophore flavin mononucleotide(FMN).LOV domains’ C terminal contains a serine/threonine protein kinase,and the light induce the formation of a cysteinyl adduct to the C(4a)carbon of the FMN chromophore,which subsequently breaks down in darkness.This study mainly focuses on the C/A mutant of LOV domains.When C/A in the oxidation state,they become electron donors and emission fluorescence,while in reducing state,they can grasp a large number of electronic and fluorescence will disappear.We constructed FMN binding protein(here we named it FBP1.0)E.Coli expression vector.FBP1.0 proteins were induced in E.Coli and treated with different concentration of H2O2 and DTT.And we found that with the increasing of H2O2,the fluorescence of FBP1.0 increased;With the increasing of DTT,the fluorescence of FBP1.0 is getting weak and disappear.The results of our experiments showed that FBP1.0 protein is sensitive to oxidoreduction.With the extended lighting time,the fluorescence of FBP1.0 became weak gradually,which illustrated FBP1.0 was reduced when treated with light.And spectroscopy analysis of FBP1.0 E.Coli proved the existence of LOV protein and binding with FMN.To further explore the function of FBP1.0 protein,we constructed FBP1.0 plant expression vector and obtained transgenic plants,qPCR found FBP1.0 was highly expressed in the transgenic Arabidopsis thaliana,and the expression is three times of WT.To screen ROS related mutants,we used ethyl methanesulfonate(EMS)mutagenesis to obtain a mutants library and built up a serial preliminary screen methods.The Arabidopsis seedlings were treated with different concentration of H2O2,sorbitol,MV,Salicylic Acid combination,and we found the fluorescence in roots had great difference.Our experimental results indicate that 0.5 mM H2O2 is suitable for mutant screening,and we had got seven potential mutants in preliminary screening in M2.