Functional Study Of Kelch Motif-containing F-box Proteins In Tartary Buckwheat Secondary Metabolic Regulation

The kelch motif-containing F-box(KFB)proteins is one of the components of E3 ubiquitin ligase family member SCF(Skp1-Cullin-F box)complex,which specifically recognizes the target protein and promotes its degradation by ubiquitination pathway to regulate various cellular activities.It has been reported that the KFB protein affects the production of metabolites by controlling the stability of secondary metabolism-related enzymes to achieve its regulation on secondary metabolic pathways.In this study,Real-time quantitative PCR(qRT-PCR)analysis was performed on 15 KFB genes of tartary buckwheat(named FtKFB1,FtKFB2,FtKFB3,FtKFB4,FtKFB5,FtKFB6,FtKFB7,FtKFB8,FtKFB9,FtKFB10,FtKFB11,FtKFB12,FtKFB13,FtKFB14,FtKFB15)in the local transcriptome database to study the tissue-specific expression of KFB gene in tartary buckwheat.The amino acid sequence of FtKFB1-FtKFB15 and two Arabidopsis thaliana KFB proteins(At1g80440,At2g44130)with function of secondary metabolic regulation in the Uniprot protein database were compared.FtKFB7 and FtKFB14 had 44% and 41%identity to At1g80440 and At2g44130,respectively.We speculate that FtKFB7 and FtKFB14 may be involved in secondary metabolic regulation.The interaction between FtKFB7 ?FtKFB14 with phenylalanine ammonia lyase(FtPAL)and rutin degrading enzyme(FtRDE)of tartary buckwheat was verified by yeast two-hybrid system and GST pull-down assay.By the construction of plant expression vector,the overexpression of FtKFB7 and FtKFB14 genes was realized in tobacco.The total flavonoids content of transgenic positive plants was detected to further explore the regulation mechanism of FtKFB protein on secondary metabolism.The results suggested:(1)qRT-PCR analysis in different tissues of tartary buckwheat(roots,stems,leaves,flowers,seeds)showed that FtKFB4,5,6,7,8,9,10,11 had the highest expression level in leaves which were 10.0,7.1,21.3,7.7,13.4,18.4,1.5,and 2.4times higher than in roots,respectively;FtKFB2,3,13,and 14 expressed highest in seeds which were 1.3,8.5,7.0 and 10.1 times the expression level in roots,respectively.FtKFB12 had the highest expression level in stems,which were 2.1times that in roots;FtKFB1 expressed highest in roots,and the expression level of FtKFB15 was similar in stems or roots.It indicated that there are differences in theexpression level of different FtKFB genes in different tissues of tartary buckwheat.(2)The experiment constructed bait recombinant vectors pGBKT7-FtKFB7,pGBKT7-FtKFB14 and prey recombinant vectors pGADT7-FtPALB,pGADT7-FtPALN,pGADT7-FtRDE,then transformed yeast Y187 competent cells,and pGBKT7-Lam+pGADT7-T was used as negative control,pGBKT7-53+pGADT7-T as positive control.The yeast two-hybrid system was verified to have no self-activating effect.The tested results of X-?-Gal indicated that there was interaction between FtKFB7 and FtRDE,FtPALN,FtPALB,while FtKFB14 only had interaction with FtPALB.The interaction between FtKFB and FtPAL or FtRDE was confirmed by yeast two-hybrid assay,which laid a foundation for subsequent research.(3)The recombinant expression vectors pGEX-6p-1-FtKFB7 and pGEX-6p-1-FtKFB14 were constructed and purified by GST affinity chromatography to obtained FtKFB7 and FtKFB14 proteins.The PALB protein was successfully purified by cobalt ion chelate chromatography.The crude extraction of FtRDE enzyme was purified from the tartary buckwheat seeds.GST pull-down and western blot assays showed that FtKFB7 and FtPALB or FtRDE proteins were successfully detected in the pull-down elution buffer,further confirming the interaction of FtKFB with FtPAL and FtRDE in vitro.(4)In order to further study the function of FtKFB protein in plant secondary metabolism,the plant expression vectors pCAMBIA3301-eGFP-FtKFB7 and pCAMBIA3301-eGFP-FtKFB14 were constructed.Then we transformed tobacco cultivar SR-1(Nicotiana tabacum cv.Petit Havana SR-1)by agrobacterium to achieve overexpression of FtKFB7,FtKFB14.In this experiment,the T0 transgenic positive plants of FtKFB7,FtKFB14 and pCAMBIA3301-eGFP were obtained by Basta resistance selection and PCR detection,and the total flavonoids in T0 transgenic plants of FtKFB14 and pCAMBIA3301-eGFP were preliminarily determined.The results indicated that the overexpression of FtKFB14 may regulate flavonoid content negatively but further verification is needed.This experiment created conditions for an in-depth understanding of the effects of FtKFB proteins on plant secondary metabolites.