Identification Of Flowering Time Genes And A Protein Interacting With HAF1 In Rice

Rice(Oryza sativa)is an important cereal crop for nearly half of the population of people around the world.Heading date is a vital trait which affects the adaptation to the seasons and regions.Our group has established large rice T-DNA insertional mutant library and lots of heading date mutants were screened such as delayed heading date mutant Heading date Associated Factor 1(HAF1)(Yang et al.,2015).In this study,more flowering genes were mapped with the MutMap method and proteins interacting with HAF1 were identified.The main results are showed as follows:1.Two delayed heading date mutants(N116 and KO)and two never flowering mutants(N108 and MT)were obtained from our T-DNA insertional mutant library.We made sure that these four genes were not allelic with rid1,a never-flowering mutation,by comparing the heading date phenotype of F1 generations crossed by these four mutants with RID1 heterzygous lines(Wu et al.,2008).According to a modified MutMap method,these mutants were crossed with EMSZH11(Zhonghua11 treated with ethylmethanesulfonate but normal phenotype)or Nipponbare.Four segregating F2 population(named N116-E,KO-E,MT-E and N108-NIP)were detected,the phenotypic ratio is in accord with Mendel separation ratio by chi-square test.That meant each mutant line was caused by a recessive mutation in a single locus.From each segregating family,30 mutants and 30 wild-type were chosen out to build DNA pool and subjected to whole-genome sequencing.Analysis of the sequencing data using MutMap method,the results show that N116-E is allelic with Ehd3,caused by a nonsynonymous nucleotide substitution(848:G?T),leading to a Gly to stop coden mutation.KO-E is a MYB transcription factor,which is allelic with AtLUX,caused by a nonsynonymous nucleotide substitution(712:C ? A),leading to a His165 Glu mutation in the myb-like DNA binding-domain.N108-NIP and MT-E are located in the end of chromosome 9 and chromosome 6,respectively.2.HAF1 encodes an E3 ubiquitin ligase containing C3HC4 RING Finger domain(Yang et al.,2015).By yeast two hybrid,BiFC and pull-down essays,we proved HAF1 could interact with HAF1 INTERACTING FACTOR 1(HIF1)in vitro and vivo.We confirmed HAF1 could ubiquit and degrade HIF1 in vitro through ubiqutination and degradation experiments.What's more,we identified HAF1 could degrade HIF1 through 26 S proteasome complex in tobacco.In addition,the inhibited phenotype of hif1-ami,hif1-crispr and overexpression phenotype of HIF1-OX,HIF1-GFP-OX were observed.The results showed that the inhibited plants flowered earlier,the overexpresion plants flowered later.At last,the inhibition plants were crossed with hif1,and finally I confirmed that HIF1 is epistatic to HAF1 by establishing double mutants with haf1.