Mapping Of Heading Date Genes In Rice And Screening The Proteins Interacting With HAF1

Rice(Oryza sativa) is the main staple food for a large segment of the world population. Owing to determining the areas of rice cultivation and seasonal adaptation, heading date is one of the most important factors determining the yield of rice. A large number of late-flowering mutants were obtained in our lab by using ethyl methanesulfonate(EMS) to mutagenize rice cultivar Kong yu 131(KY131). In this research, we aimed to map some flowering genes with the method of Mut Map to accelerate perfecting the regulation network of rice flowering research by accumulation of genetic material. In addition, HAF1 is an important heading date gene in rice cloned by our lab, which encodes an E3 ubiquitin ligase containing C3HC4 ring finger domain. Using yeast two-hybrid to identify some proteins which interact with HAF1 would contribute to clarify the function of HAF1 in rice flowering regulation network. The main results of this study are present as follows:1. 54 late-flowering mutant lines were identified from the rice variety KY131 EMS mutant library developed in our lab. F2 segregation populations were generated by crossing there mutants with the wild-type plants of cultivar KY131. Among them, 8 populations were already detected phenotypic differences between the mutant and wild type at the F2 generation, and the phenotypic ratio is in accord with Mendel separation ratio by chi-square test. Which means that each mutant line was caused by a recessive mutation in a single locus. The bulked DNAs from recessive mutant F2 progeny in there mutant lines were subjected to whole-genome sequencing respectively. Analysis of there sequencing data using Mut Map method shows that 4 late-flowering mutant lines, B741, B12, A114 and D599, are allelic ehd1 mutants and that the never-flowering phenotype in F345 is caused by a nonsynonymous nucleotide substitution(4989:C→T) in a gene encoding a FAT domain-containing protein, leading to a Leu968 Pro mutation, and that putative positions of causal mutations in the other two late-flowering mutant lines, I459 and E597, are both located in chromosome 10 and several candidate genes were identified respectively.2. Through screening a Gateway-compatible Gal4-AD–TF library of 1589 Arabidopsis transcription factors(TFs) by mating-based yeast-two-hybrid(Y2H) methods, We got 13 Arabidopsis TFs which interact with HAF1(288a.a-667 a.a). After analysis of homologous sequences of there genes, 9 candidate genes were identified in rice which quite possibly interact with HAF1. CIF1 and CIF2 are both proved to interact with HAF1 by yeast-two-hybrid, pull-down assays and bimolecular fluorescence complementation assay or bimolecular luminescence complementation assay. Yeast-two-hybrid assay using a series of truncated CIF2 proteins further demonstrated that the interaction between CIF2 and HAF1 depends on the C terminal of CIF2 in which HLH domain are invoved. Besides, In vitro cell-free degradation assays indicates that HAF1 is specifically responsible for CIF1 and CIF2 protein degradation. In addition, We performed yeast two-hybrid assays between the other CIFs and HAF1, demonstrated that CIF3, CIF4, CIF6, CIF7 are interact with HAF1 in yeast cell.