Functional Analysis Of Arabidopsis Receptor-Like Kinase IOS1 And Study Of Its Interaction Protein

Iron is an essential nutrient for animals and plants,and the lack of iron will directly affect the growth and development of plants and animals.Although the content of Fe in the soil is very abundant,most of the Fe existing in the soil cannot directly be absorbed and utilized.Therefore,studying the mechanism of plant responsing to iron deficiency is necessary to improve the healthy diet of plants and humans.Previous studies of this subject have found that the Arabidopsis receptor-like kinase IOS1 can bind to Fe3+,regulate the accumulation of callose,affect the permeability of plasmodesmata,limit the movement of iron-responsive transcription factors(such as PYE)and affect its regulation of downstream genes(such as FRO,IRT1,etc.).Therefore,it is speculated that IOS1 may act as a regulator upstream of the iron signal transduction process,or as a subunit of the iron receptor.However,IOS1 has no kinase region in intracellular.So how to transmit the signal arise our interest.In this thsis,Arabidopsis thaliana plants such as WT,iosl,pdlp8 and ios1 pdlp8 were used as materials to study IOS1 function through immunoprecipitation,transient transformation of protoplasts,transgenic and yeast two-hybrid methods,and search for and identify partial interaction proteins.The main results are as follows1.Phenotypic analysis of IOS1 related plant materialsIn order to further study the function of IOS1,we used IOS1 related plant materials iosl,pdlp8,ios1 pdlp8,ios1-D and WT to observe and determine some quantitative and qualitative phenotypes under different conditionsIron content,the acidification ability,ferric chelate reductase activity assay of iosl pdlp8 was higer than WT,so ios1 pdlp8 showed tolerance to iron deficiency.The root length of ios1-D and WT have no difference with the treatment of MS+10 ?M estradiol,MS-Mn+10 ?M estradiol and MS-Zn+10 ?M estradiol respectively.2.We found 258 proteins that may interact with IOS1 by immunoprecipitation and mass spectrometry.Through informatics analysis,we searched for 32 genes that might be related to iron and ordered mutants of these genes.We identified the seeds of homozygous mutants of 27 genes,and a large number of homozygous mutants seeds of 18 genes have been obtained.Phenotypic experiments of two of these mutants under iron deficiency were studied:Under iron-deficiency conditions,there was no difference in root elongation of lem3.3 compared to wild type,and there was a significant difference in root elongation of iim compared to wild type.It indicates iim is insensitive to iron deficiency.We found lem3.3 and iim leaves yellowing more slightly than WT.3.Interaction between IOS1 and LEM3.1Amino acid sequence analysis:AtIOS1 consists of a longer extracellular domain,a single transmembrane domain and a shorter intracellular domain.IOS1 and PDLP8 form one subclade and share 52%amino acid similarity.However,IOS1 and PDLP1 share 30%amino acid similarity.AtLEM.3.1 includes two extracellular domain,two transmembrane regions and a longer intracellular domain.AtLEM3.1 has two homologous genes in Arabidopsis.Phylogenetic analysis showed that AtLEM3.1 is similar to AtLEM3.2The interaction research results between IOS1 and LEM3.1 showed that IOS1,PDLP1 and PDLP8 can interact with LEM3.1 and did not interact with LEM3.2 by bimolecular fluorescence complementation,yeast two-hybrid also prove the interaction of IOS1,PDLP1,PDLP8,and LEM3.1.The localization result showed IOS1,PDLP1,PDLP8 and LEM3.1 were localized on the cell membrane.4.Further study of the relationship between IOS1 and LEM3.1 through genetic methods,using CRISPR/Cas9 technology to construct lem 3.1 lem 3.2 lem 3.3 triple mutant and Gateway method to build IOS1,PDLP1,PDLP8,LEM3.1 overexpression vector,screening of genetical materials is under way.Study of the IOS1 location:It was observed that IOS1 was indeed localized in the plasmodesmata when roots were stained with PI and treated with 5%NaCl.Observing the fluorescence of IOS1 point mutation material(C98S/C101S/C200S/C203S),we found that the four cysteine mutations of IOS1 did not affect the normal location of IOS1.