Expression Patterns Of F-box Genes TaPP2-A13 And TaSKIP27 In Wheat Response To Stresses And Identification Of Their Interacting Proteins

F-box proteins widely existing in eukaryotic cells constitute one of the largest protein families,are mainly in charge of recognizing specific substrate to be degraded as the component of SCF complex.The functions of some F-box famlily members have been discovered in Arabidopsis thaliana,but less is reported in wheat.Based on screening the wheat stress-related transcripsome database,the present study analyzed the expression patterns of TaPP2-A13 and TaSKIP27 in wheat TcLr15 response to stresses and identified their interacting proteins by some techniques and methods,such as bioinformatic software,qRT-PCR,Yeast 2 Hybird(Y2H),BiFC,Co-IP,fluorescence observation,gene over-expression and subcellular location,etc.Main results obtained are given as follows:1.The full length coding region of the two wheat F-box genes named TaPP2-A13 and TaSKIP27 were obtained,and their predicted proteins were 298 aa and 164 aa in length,respectively.TaPP2-A13 belongs to F-box/PP2 subfamily(FBP),while TaSKIP27 belongs to the subfamily with unknown functional domain(FBU).Both of the two genes are comparatively conservative in the wheat relative species and genus during the evolution.2.After treatment with three hormones,the TaPP2-Al3 gene was significantly up-regulated in wheat TcLr15.Among three treatments,TaPP2-A13 gene showed the earliest expression in TcLr15 treated by ABA,while the strongest expression occurred in TcLr15 treated by SA.TaSKIP27 gene showed stronger up-regulation in TcLr15 after treatment with ABA and SA,but no obvious change was observed in wheat after treatment with MeJA.Under the two abiotic stresses,drought stress(PEG)caused the significant down-regulation of TaPP2-A13 gene,while salt stress induced the TaPP2-A13 gene to up-regulate sharply in a short time.Both drought and salt treatments induced a significant up-regulation of TaSKIP27 expression.In the susceptible combination(TcLr15/05-5-137③)and the resistant(TcLr15/05-19-43②)combinations formed by wheat and leaf rust pathogens,the expression level of TaPP2-A13 in the susceptible combination was much higher than that in the resistant one at all sampled timepoints.The TaSKIP27 gene showed the similar expression patterns in susceptible/resistant combinations,which was down first,then up,down later,but the transcripts in resistant combination at 24 h post inoculation were significantly more than that in susceptible one.3.Two recombinant expression vectors pET-30a-TaPP2-A13 and pET-30a-TaSKIP27 were constructed,and their corresponding proteins were produced successfully in E.coli BL21.The multiclonial antibodys were obtained after immuning the rabbit,which can be used for detecting their corresponding proteins.The recombinant vectors of TaPP2-A13 and TaSKIP27 for determining their subcellular localization were constructed.Both TaPP2-A13 and TaSKIP27 were located in the nucleus and cytoplasm by Fluorescence observation.The vector overexpressing TaSKIP27 was constructed,T2 generation wheat plants with overexpressing TaSKIP27 were obtained when wheat young spikes or callus infected by Agrobacterium tumefaciens.4.11 types of proteins interacting with TaPP2-A13 and 8 types of proteins interacting with TaSKIP27 were obtained by screening yeast AH 109 cDNA library of wheat resistant to leaf rust pathogen(TcLrl5 inoculated with avirolent leaf rust strain 05-19-43②).Y2H further confirmed that protein phosphatase 2C 5(TaPP2C5),S-phase kinase-associated protein 1(TaSKP1),chitinase(TaCHI),SEC1 family protein(TaSLY1)and ribose diphosphate carboxylase small chain(TaRbcS)physically bound with TaPP2-A13,TaSKP1,TaCHI and TaSLY1 physically bound with TaSKIP27.BiFC assay further confirmed that TaSKP1 and TaSLYl interacted with TaPP2-A13 and TaSKIP27,respectively.Co-IP results revealed that TaSKP1 and TaPP2-A13 interacted intracellularly.