| F-box protein,whose N-terminal usually contains a conserved F-box domain,can bind to Skp1 protein to form SCF(SKP1-Cullin-F-box)complex,which participates in the ubiquitin-26 S protease system(UPS)as a class of E3 ubiquitin ligases.Then,target proteins can be specifically recognized and degraded by protein-protein interaction regions with structural diversity at their C-terminal.According to the different c-terminal domains,they can be divided into different subfamilies,such as the common FBK subfamily with Kelch domain at its C-terminal.FBA subfamily with F-box associated domain at c-terminal.F-box proteins regulate cellular activities by ubiquitination and degradation of target proteins,respond to plant biological or abiotic stress,and participate in plant growth and development,etc.Therefore,functional studies of F-box proteins mainly use protein interaction research techniques to screen target proteins for degradation and explore their functions.Mulberry tree(Morus alba L.) is a Morus L.plant in Moraceae.Its fruits mature through three stages: green fruit stage,color turning stage and mature stage.Fruit color is one of the main traits to measure the degree of fruit maturity,and it is also closely related to the quality of mulberry.In the early stage,we identified two differentially expressed F-box genes in mulberry with different fruit colors,and explored whether they played a role in fruit coloring.We preliminarily identified and analyzed the F-box gene family of Mulberry,laying a foundation for further functional exploration of candidate genes MaFBA380 and MaFBK70.The expression levels of target genes in mature fruits of 7 mulberry varieties with different fruit colors and fruits at different development stages were analyzed,so as to analyze the correlation between target genes and fruit coloring.The target proteins of ubiquitination degradation of MaFBA380 and MaFBK70 were screened by protein interaction study technique to preliminarily explore their functions.Transgenic tobacco lines with overexpression were obtained by leaf disk transformation method,and pure and mutant arabidopsis homolog lines with MaFBA380 and MaFBK70 genes were obtained.Then,the function of target genes was further studied by transgenic materials,so as to explore the relationship between mulberry F-box proteins MaFBA380 and MaFBK70 and fruit coloring.The main results obtained are as follows:1.Identification and analysis of f-Box protein family in Mulberry164 members were retrieved from the Proteome database of Chuanmulberry by using f-box hidden Markov model.15 C-terminal domains were identified and divided into 16 subfamilies according to the c-terminal domains.Phylogenetic analysis of mulberry F-box proteins showed that proteins with the same or similar C-terminal domains were clustered together.2.Expression analysis and cloning of MaFBA380 and MaFBK70 in MulberryThe expression patterns of the two candidate genes identified by us,MaFBA380 and MaFBK70,were analyzed.The results showed that in the mature fruits of different varieties,the expression level of MaFBA380 in mulberry varieties with purple and red mulberry color was significantly higher than that of white varieties.At different fruit development stages,the expression level of MaFBA380 in green fruit stage was low.The expression level in mature fruit was high and the difference was significant.MaFBK70 was highly expressed in the "pearl white" variety with white mulberry fruit color,and the gene expression level increased significantly in mulberry fruit color transition stage.The coding sequences of MaFBA380 and MaFBK70 were cloned from different varieties and compared with the electronic sequences of Mulberry.The results showed that there was no large fragment loss or premature termination of transcription in the two gene sequences in different varieties of Mulberry.The similarity of gene sequences was very high,and the possibility of protein function difference caused by natural mutation was excluded.The protein domains of the two genes were analyzed,and it was found that both genes had conserved F-box domains at the N-terminus.Among them,MaFBA380 had an FBA domain at the C-terminus,while MaFBK70 had three repeated Kelch domains at the C-terminus.3.Mulberry MaFBA380,MaFBK70 function verificationThree tobacco-positive plants with overexpression of MaFBA380 and MaFBK70 were obtained,and the phenotypes of the tobacco plants were observed.The results showed that the flowers of the transgenic tobacco plants with two genes were still white,there was no anthocyanin accumulation in the leaves and stems,and the seed coat color was not significantly deepened,but the leaf color was somewhat lighter than that of the wild type.They showed a lighter light green color,but there was no obvious change in other traits.The expression levels of anthocyanin synthesis pathway genes were detected,and it was found that the expression levels of some genes were significantly up-regulated in MaFBA380 transgenic tobacco,while there was no significant difference in MaFBK70 overexpressed plants.The chlorophyll content of transgenic tobacco plants was lower than that of wild type,but the difference was not significant.T-dna insertion mutants of arabidopsis homologous genes of MaFBA380 and MaFBK70 were obtained,and three homozygous mutants of fbk70 and fba380 were identified by triple primer method.Phenotypes of the mutants were observed,and there was no obvious accumulation or degradation of anthocyanins and chlorophyll,nor other dwarfing phenotypes.Quantitative analysis of anthocyanin synthesis related genes in fbk70 mutant showed no significant difference.The expression levels of anthocyanin synthesis pathway genes in rosette leaves of fba380 were detected,and CHS,CHI,PAL,MYB114 and other genes were significantly down-regulated.4.Protein interaction study techniques were used to screen the target proteins of MaFBA380 and MaFBK70First,in order to explore whether MaFBA380 and MaFBK70 are involved in UPS pathway degradation of target proteins,yeast two-hybrid technology was used to verify the interaction between two F-box proteins and Ma Skp1 A,indicating that both proteins can bind to Skp protein to form E3 ubiquitin ligase.Then their target proteins were identified,and four proteins(Ma Mal D,Ma Lhc1,Ma CTP and Ma RPN11)that may interact with MaFBA380 protein were selected as candidate proteins.Meanwhile,three candidate target proteins of MaFBK70(Ma GS,Ma RRP,Ma PPO)were screened,and their interactions were verified by yeast two-hybrid technology.The results show that MaFBA380 may interact with Ma Lhc1,Ma CTP,Ma RPN11,and MaFBK70 may interact with Ma GS,Ma RRP,Ma PPO,but the interaction between MaFBA380 and Ma Lhc1,Ma RPN11 may be weak.These results indicate that MaFBA380 may regulate the synthesis and accumulation of anthocyanin on the one hand,and affect the synthesis and accumulation of chlorophyll by interacting with chlorophyll A-B binding protein Lhc1 to degrade it on the other hand,thus coordinating the contents of chlorophyll and anthocyanin to regulate fruit color transformation.However,MaFBK70 may affect the synthesis of chlorophyll through the interaction with Ma GS and thus regulate the fruit color transformation,but has little correlation with anthocyanins.. |
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