Screening And Validation Of IQM5-interacting Proteins In Arabidopsis

Previous studies of our research group showed that AtIQM5 encoded by At5g57010 is a calmodulin-binding protein(CaMBP)with an IQ motif;IQM5 plays an important role in the regulation of plant flowering by inhibiting FLC expression,and the transformation of plants from infancy to adulthood.However,the molecular-regulation mechanism of IQM5 involved in the above process needs to be further studied.In this study,yeast two hybrid assay and protein affinity capture technology are used to find the interaction protein of IQM5,so as to further understand the molecular mechanism of IQM5 inhibiting FLC expression and regulating plant flowering,and to provide a reference for a comprehensive understanding of the biological function of IQM5.The main results are as follows:1)Yeast two hybrid technique was used to verify that IQM5 is a calmodulin binding protein with an IQ motif.The results showed that IQM5 could interact with CaM1,CaM2,CaM3,CaM4,CaM6 and CaM7 in yeast cells;The above studies were further verified by Luciferase complementation assay(LCA).2)The complementary line of iqm5-1 with a point mutation in IQ motif which is the key domain to binding with CaM was constructed and the flowering phenotype was observed in long day.The results showed that the above line and iqm5-1 were basically the same in flowering time and the number of rosette leaves,showed a late-flowering phenotype,which may be due to the inability of IQ motif mutation to receive calmodulin signals,suggesting that IQM5 mediates CaM signal to regulate plant flowering.3)The constructed bait vector p GBKT77-IQM5 and prey vectors habouring Arabidopsis c DNA library were transformed into yeast competent cells to screen candidate interaction proteins.The results showed that IQM5 was able to interact with CAT2 and CIB22 in yeast cells;Luciferase complementation assay(LCA)was further used to verify that CIB22 also interacts with IQM5 in plant cells.4)To know the genetic relationship between CIB22 and IQM5,CRISPR/cas9genome editing technology was used to construct CIB22 single mutant and iqm5-1cib22 double mutant.The transformed plants have been infected by Agrobacterium tumefaciens,which lays a material foundation for subsequent phenotypic observation experiments and related gene expression analysis.5)The protein affinity capture technology was used to screen IQM5 interacting proteins in Arabidopsis.The plant binary vectors with GFPpCAMBIA1305.1-P_IQM5:GFP-IQM5,pCAMBIA1305.1-P_35S:GFP-IQM5, pCAMBIA1305.1-P_IQM5:GFP and pCAMBIA1305.1-P_35S:GFP were transformed into iqm5-1 mutant,and homozygous strains were obtained by hygromycin resistance screening..The results of molecular identification of transgenic lines showed that the homozygous transgenic lines carrying GFP-IQM5 fusion protein had been successfully constructed.The results of flowering phenotype analysis showed that transformation of pCAMBIA1305.1-P_IQM5:GFP-IQM5 was ale to rescue the late-flowering phenotype of iqm5-1.It is suggested that GFP-IQM5 fusion protein with normal function can be produced in transgenic plants.While the protein extracted from transgenic plant seedlings was detected by Western Blot with GFP antibody,no positive results were obtained.This study confirmed that IQM5 is a calmodulin binding protein in yeast and plants,which mediates calmodulin signal and regulates flower formation.Atthe same time,CIB22 and CAT2 can bind to IQM5 in yeast cells and plant cells.