| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 97 === F-box protein is a subunit of E3 ubiquitin ligase SCF complex (S-phase kinase-associated protein; SKP, Cullin 1; CUL1, F-box protein complex). The F-box domain of F-box protein interaction with the SCF component Skp1. And F-box protein often include additional interaction domain capable of protein-protein interaction with target protein in carboxy-terminal. Target proteins were recognized and ubiquitinated by F-box protein of SCF complex. The ubiquitinated proteins alter protein function or degraded by the 26S proteasome. The Arabidopsis genome encodes about 700 putative F-box proteins which may involve in plant growth, development, hormone signaling and stress response.
Our lab made a F-box gene T-DNA insertion mutant which insert to 95 bp upstream of At5g50220’s start codon. Use database search indicate At5g50220 translates a F-box protein. So in this thesis named this gene and mutant FOX1 (F-box protein 1) and fox1-1.To discover the FOX1 mRNA that retains predicted intron by RT-PCR and restriction enzyme digestion. And the FOX1 translates a protein which only contains a F-box domain, suggesting that FOX1 protein may competes SKP binding site with other F-box protein and inhibits SCF complex ubiquitinates target protein. FOX1 expression in the fox1-1 mutant is higher than wild-type, but FOX1 transcription start site is changed since T-DNA insertion, causing FOX1 protein doesn’t have function in fox1-1. So fox1-1 is a loss-of-function mutant. Similar to wild-type, salt stress induced FOX1 gene expression in fox1-1, guessing FOX1 gene of fox1-1 resulting a new promoter which contains stress-responsive elements since T-DNA insertion.
In order to understand the function of FOX1 in osmotic stress, I analyses the physiological characterization of fox1-1 different with wild-type under ABA, salt and drought stress. First, I compared the fox1-1 mutants and wild-types under salt stress, and found fox1-1 mutants were weaker than wild-types. Then, I detected the variation of chlorophyll fluoresce Fv/Fm’ at the same condition, and Fv/Fm’ of fox1-1 decrease faster than wild-type. To measure ABA and salt stress inhibited efficiency of germination and early seedling growth in fox1-1 mutants were slightly lower than wild-type, suggesting that knockout mutant of FOX1 showed ABA-insensitive phenotype and reduced salt tolerance. Both salt and drought stress induce osmotic stress, and fox1-1 mutant is weaker than wild-type under drought stress, indicate that FOX1 involve in drought stress, suggesting that FOX1 involve in osmotic-induced ABA signaling pathway.
The above results indicate FOX1 inhibit SCF complex ubiquitination. FOX1 gene expression induced under salt stress, suggesting that FOX1 inhibit SCF complex degrades the positive regulator of ABA signaling by ubiquitination cycle. Causing accumulation the positive regulators of ABA stimulate the ABA signaling and increased tolerance to osmotic stress. And I construct overexpression of FOX1 in transgenic Arabidopsis plants in order to understand functions of FOX1 in the future.
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