| The F-box/Kelch(FBK)subfamily ranks fourth in wheat F-box family,and is one of the larger subfamilies in plant F-box family.The wheat F-box family were subjected to the systematic classification and identification in our previous study,and 58 TaFBK members were obtained.With the release of the wheat genome database(IWGSC v1.0)in 2017.are there more FBK proteins being annotated?According to previous reports,members of the FBK subfamily enhanced the susceptibility in Arabidopsis against nematodes,and participated in phenylalanine ammonia-lyase(PAL)degradation and regulating level of polyphenol,as well as controlling light morphogenesis and flowering time.Do FBK proteins play roles in wheat response to abiotic stresses and resistance to infection of leaf rust pathogens?What is the detailed molecular mechanism?To answer these questions,the present study selected more seed sequences to thoroughly search against the IWGSC v1.0 database.According to the search results,different types of FBK genes were selected to further obtain their complete coding regions,analyze their sequence characteristics and expression patterns under biotic/abiotic stresses,verify functions of transgenic wheat plants in response to stresses,screen and validate partner proteins,etc.The main results are as follows:1.A total of 10 seed sequences(2 F-box,2 F-box-like and 6 Kelch)were used to search and identify wheat FBK(TaFBK)subfamily members against the IWGSC v1.0 database.68 TaFBK genes encoding 74 TaFBK proteins were obtained in the present study:TaFBK proteins are predicted to mainly locate in the nucleus and cytoplasm:Both F-box and Kelch domains share weak sequence similarity,moreover,the amino acids in the Kelch domains are less conservative than that in F-box domains;TaFBK proteins were divided into 5 categories and 7 evolutionary clades according to their functional domains.The number of Keleh domains is the main criterion to classify the FBK subfamily:The 68 TaFBK genes are unevenly distributed on 20 wheat chromosomes(except chromosome 2B),and fragment duplication is the main way to expand the subfamily members.TaFBK genes showed tissue-specific expression,and participated in wheat response to drought,heat and drought+heat stresses according to the expression pattern based on FPKM(Fragments per kilobase per million mapped reads)value,and heat stress caused TaFBK genes stronger transcript change.2.The complete coding regions of the four TaFBK genes with different type of Kelch domain were isolated from wheat TcLr15.Phylogenetic analysis showed that these four proteins shared higher sequence similarity with homologous proteins from wheat related species,such as Aegilops tauschii and Brachypodium distachyon,and were grouped in a different clade with dicotyledonous and woody plants.The results of qRT-PCR indicated that more transcript of TaFBK50 and TaFBK34 were accumulated in wheat flag leaves,the expression of TaFBK71 was relatively higher in pistils and young leaves,and TaFBK19 transcript accumulated more in young wheat leaves,which demonstrated that the four TaFBK genes showed tissue specific expression;After infected by leaf rust pathogens with different virulence,the four TaFBK genes showed different expression levels and patterns in wheat TcLr15.Among them,the greater changes happened in transcripts of TaFBK19 and TaFBK34.After inoculation with the susceptible leaf rust strain 05-5-137?(compatible combination)for 96 h,the expression level of TaFBK19 in TcLr15 quickly increased,which was significantly higher than that of the resistant combination and the control.The expression level of TaFBK34 in wheat inoculation with the resistant leaf rust strain 05-19-43?(incompatible combination)for 24 h increased strongly to around 3.6 times higher than that of the 0 h sample and the susceptible combination;The four genes presented different expression patterns in response to three plant hormone treatments.TaFBK19,TaFBK50 and TaFBK34 were more affected,while TaFBK71 less changed by treatment of these hormones;The effect of NaCl treatment on the expression of the four TaFBK genes in wheat was greater than that of PEG 6000 treatment,especially on expression of TaFBK19 and TaFBK34.Fluorescence observation revealed that all of the four TaFBK proteins were located in the nucleus and/or cytoplasm.3.Three lines with overexpressing TaFBK34 for three times higher when comparing to the wild type Fielder were obtained in wheat.Phenotype observation showed that the overexpression wheat lines enhanced the salt tolerance at the three-leaf stage compared to the wild type,and the expression levels of TaPOD and TaAPX in the two overexpression lines were significantly higher than those in the wild type when treating with 300 mM NaCl for 48 h.However,the seed germination rate of overexpression wheat lines did not improve significantly after salt stress.There was no significant difference between the overexpression lines and wild type under the treatment of leaf rust pathogens and drought.4.Three assays of Yeast two-hybrid(Y2H).Bimolecular Fluorescence Complementation(BiFC)and Co-immunoprecipitation(Co-IP)confirmed that TaFBK 19 interacted with Skp1/ASK1-like protein(TaSkp1),TaPAL and ADP-ribosylation factor 2-like isoform X1(TaARL2),TaFBK34 interacted with TaPAL and TaARL2.Moreover,TaFBK19 bound to TaSkp1 via the F-box domain,TaFBK19 and TaFBK34 recognized TaARL2 and TaPAL by the Kelch domain(s).respectively.TaFBK50 physically interacted with SEC1 family transport protein SLY1(TaSLY1),TaSkpl and Chitinase 2(TaChi)proteins when testing by Y2H,no any fluorescence signal was observed in further BiFC assay;TaFBK71 was tested to interact with Skpl and SLY1 in both Y2H and BiFC.The results obtaining from the present study will give some evidence for dissecting the functions and intercating network of Kelch type F-box proteins in wheat suffered from abiotic stresses,such as salt and drought,and infected by leaf rust pathogen,moreover,supply for some reference for broadening the knowledge of roles of FBK subfamily in wheat. |
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