Cloning And Functional Analysis Of Gene FT2 In Rice

Rice(Oryza Sativa)is one of the most important cereal crops in China,stable and sustainable increase of rice yield is benefit for development of China.The number of tillers is one of the key determinants of the yield.However,the molecular mechanisms for tillering in rice are still poorly known.Here we report the isolation and characterization of a rice few tillers mutant few-tillers 2(ft2).To explore the function of FT2,several approaches of genetics,biochemistry and molecular biology were applied.The results of our study are summarizd as following:1.The ft2 mutnant was isolated from a EMS-mutagenized population of indica variety Shuhui 527,which exhibits few tillers and delayed tillering phenotypes.However,no significant growth difference was observed in Arabidopsis Atft2 and wild type.2.To map the FT2 locus,a F2 mapping population derived from a cross between ft2 and japonica cultivar Nipponbare was generated and ft2 locus was finally located in an 4.1 cM DNA region in the top of chromosome 11.Based on the whole genome resequencing,we found a single nucleotide mutation from G to A in the 10 th exon of a candidate gene,leading to the transition of an amino acid changed from Glu to Lys.Compared with WT,the relative expression level of FT2 in the ft2 mutant was significantly up-regulated,this could possibly be caused by feedback regulation mechanisms.Knockdown assay by RNA interference of FT2 gene revealed that the RNAi plants also presented reduced tillerring phenotype.Those data indicated that the candidate FT2 gene may be responsible for the few tillers phenotype in ft2.3.Bioinformatics analysis showed that FT2 belongs to HEAT repeat protein family,which is widely distributed in plant kingdom.In addition,the mutation site detected on FT2 was highly conserved among several plant species.Subcellular locolization analysis revealed that FT2-GFP fusion protein signal was specifically observed in nuclear,which is consistent with the prediction by software,because the typical nucleic-localized signal was found in N-terminal region.We conducted qRT-PCR and western blot to exam the expression of FT2,the results demonstrated that FT2 was broadly expressed in leaf,leaf sheath,stem and root,and dominantly expressed in leaf sheath.4.To explore the function of FT2,yeast-two-hybrid approach was applied to screen the interacting proteins of FT2,and several candidate gene were obtained.One of candidate gene was TOR,which is an atypical Ser/Thr protein kinase and had been known as the target of chemical Rapamycin.Furthermore,luciferase complementation assay verified that FT2 could interact with TOR in its HEAT repeat domian.We also found that ft2 mutant was less sensitive to rapamycin.These results indicated that FT2 might involve in rapamycin signaling transduction via interacting with TOR.However,the mechanism between FT2 and TOR is still unclear,and further studies are needed.