Functional Study Of Different Haplotype OsIDEF1 Genes In Iron Uptake Of Rice

Iron,as one of the most important nutrients of rice,participates in various physiological and biochemical processes of plant cells.Rice,as one of the main food crops in China,is widely cultivated.However,due to the different soil environment for rice cultivation,a large part of rice is facing direct or indirect iron deficiency stress.There is very little iron which most exists as Fe³⁺in rice.That seriously affects the normal growth and metabolism of rice.The expression of Os IDEF1 is increased in rice under iron-deficiency conditions.This transcription factor can recognize the downstream cis-acting element IDE1 and induce the expression of downstream genes.By studying Os IDEF1,the molecular mechanism of rice under iron-deficiency stress can be deeply explored,and the theoretical basis for solving the iron-deficiency problem caused by it under saline-alkali conditions is provided.Previous studies in our laboratory found that Os IDEF1 has three different haplotypes,including the shortest type Jigeng 88（J88）,the next type Changbai 9（C9）and the longest type IR64.The gene lengths of these three haplotypes differ by 12 bp in sequence,and the protein sequences differ by 4 amino acids.It was found that the mutation site mainly exists in the HN region combined with divalent metal ions through the analysis of the protein structure.Therefore,does the mutation sites affect the function of the gene?In this study,phenotypic analysis was carried out to identify the gene function of different haplotypes of Os IDEF1 by transgenic materials and substitution lines.The Os IDEF1 function of three different gene lengths was deeply mined through the related transcriptional activation and metal ion binding ability experiments.The results were as follows:1.Os IDEF1 gene transgenic material acquisition and phenotype analysisThe CRISPR/cas9 vector and overexpression vector of Os IDEF1 gene were constructed,and two stable gene knockout and overexpression lines were obtained.The gene knockout and overexpression lines were treated with iron deficiency stress,and it was found that the gene knockout mutant grew significantly weaker than the wild type and had yellow leaves,while the overexpression mutant had the opposite phenotype.2.Construction and phenotypic analysis of three haplotype substitution lines of Os IDEF1Two different background substitution lines were constructed by hybridization:Koshihikari（KS）background and Jigeng 88（J88）background.After obtaining stable lines,the genotypes of individual plants were identified by PCR.Under the same background,individual plants of different haplotypes were selected for iron deficiency stress treatment.After observing the phenotypes,it was found that the phenotypes of the two different backgrounds showed that the shorter the gene length of Os IDEF1,the stronger the gene function.3.Transcriptional activation ability of different haplotype transcription factor Os IDEF1In order to verify the transcriptional activation ability of different haplotypes of Os IDEF1,the cis-acting element IDE1,which can act on the downstream Os IRO2promoter,was selected as the transcriptional activation site.After connecting with LUC-related vectors,different haplotypes of Os IDEF1 were used for transcriptional activation.In the two systems of protoplasts,it was found that the shorter the gene length,the stronger the transcriptional activation ability.4.Analysis of the binding ability of different haplotypes of Os IDEF1 to divalent metal ionsThree different haplotypes of Os IDEF1 were obtained by purifying the protein after prokaryotic expression.After quantification,the Os IDEF1 protein bound on the resin was eluted by chelating Fe²⁺,and the protein concentration was verified by Western Blot.It was found that the shorter the length of the Os IDEF1 gene,The stronger the binding ability to divalent iron ions.In summary,we identified three haplotypes of Os IDEF1 genes in rice varieties;each genotype differed by 12 bp and differed by 4 amino acids;the in vivo and in vitro activities were preliminarily confirmed:the shorter the haplotype,the greater its effect on iron.The stronger the absorption function;this will provide a theoretical basis for molecular assisted breeding in the future.