Screening And Identification Of Interacting Proteins Of FLO2 And Other Regulatory Proteins

In this laboratory,four chemiluminescence mutants heat],enpigl,enpig2,and bsl were screened by the LUC luminescence imaging system.In addition,the mutant flo2 was obtained.In the previous research work,the heat1 mutant showed heat-sensitive Phenotypes enpigl and enpig2 mutants showed a phenotype with enhanced expression of foreign genes,bs1 mutants showed a disease-sensitive phenotype,and flo2 mutants showed an early flowering phenotype.HEAT1 and ENPIG2 are confirmed to be splice factor-related proteins,ENPIG1 is a spliceosome-related protein,and the function of BS1 protein is unknown.I screened the library through yeast two-hybrid experiments,and obtained proteins that may interact with the above proteins.I then used yeast point-to-point and two-molecule fluorescence complementary experiments to further verify these results.For the FLO2 protein,it is not a nuclear protein.The library screened out 12 proteins in the reading frame.There were two proteins that regulate flowering(LD,BRR2).These two interacting proteins are involved in the flowering inhibitor FLC.In the peer-to-peer verification of regulation and control,it was confirmed that FL02 interacts with another protein FLO1 in the family,and V1 and V2 proteins respectively.Later,FLO2 and V1 were investigated for functional domains.The Vastl domain of FLO2 protein interacts with the V1 protein,and the C domain of the V1 protein interacts with the FLO2 protein.HEAT1 was used as the bait protein.After screening the library,25 correctly expressed' proteins were obtained,5 of which were localized in the nucleus.One of these proteins was related to the splicing of mRNA.HEAT1 may regulate heat resistance by regulating the splicing of mRNA.mechanism.Further verification using BiLC method revealed that there were 4 proteins interacting with HEAT1,including the protein JAZ1involved in disease regulation and the protein NF-YC4 involved in flower regulation.Since ENPIG1 has been verified to be a nuclear protein,after experimental sequencing and comparison,29 correctly expressed proteins were obtained,of which 22 were localized in the nucleus.These proteins have various functions and some are constitutive.Proteins,there are also splicing factors that regulate splicing,as well as proteins that regulate flowering through apparent modification,which is consistent with previously published results.After performing point-to-point verification through different methods,it was found that ENPIG1 interacts with six proteins,two of which are epigenetic proteins(SUVH2,DMS3).This provides directions for ENPIG1 to regulate the expression of foreign genes.One is involved in mRNA splicing(SR45a,SCL33,ATSF1).This is consistent with previous reports.In addition,it is involved in regulating salt stress proteins(ATP1).ENPIG2 protein is also a protein located in the nucleus.In the library screening work,34 proteins in the reading frame were obtained,of which 14 were nucleoproteins,and one protein(RPT2A)was involved in epigenetic regulation,The phenotype of the rpt2a mutant in regulating foreign gene expression is completely opposite to that of enpigl,which may prove that the former is negatively regulating the latter.In addition,previous people have verified that ENPIG2 interacts with nine epigenetic regulated proteins.I verified that the main functional domain of ENPIG2 is M1 through yeast two-hybrid.BS1 has gone through the yeast library screening process in the early stage.I used BiLC to verify the interaction between BS1 and different epigenetic related proteins.I verified that BS1 interacts withSUVH9 andDRM2,both of which are involved in DNA methylation.Important protein,which may confirm that BS1 may regulate the expression of related downstream genes through epigenetic methods to regulate disease resistance mechanisms.