Functional Analysis Of FOF2 Gene Working In Abiotic Stress Pathway In Arabidopsis Thaliana

F-box protein is one of curcial subunits of SCF complex,acting as identification of the substrate in ubiquitin degradation pathway.The protein family plays an important role in regulating the process of plant growth and development.In previous studies,our laboratory had found that F-box protein FOF2 worked in regulation of plant flowering.By using bioinformatics methods on the structure of FOF2 gene promoter,found many abiotic-relative cis element,so speculating FOF2 may take part in abiotic stress.The research by analyzing difference of FOF2 gene expression under abiotic stress,the influence of FOF2 overexpress or mutant on plant abiotic stress resistance,as well as FOF2 target protein screening and interacting,system-studied the function and mechanism of response to abiotic stress in plant resistance.1.Firstly,using bioinformatics combined with real-time gene expression detection method is analyzed.The results showed that the promoter of FOF2 exist relative response elements of abiotic stress,and there are many Ubiquitin the sites.Real-time PCR analysis showed expression of FOF2 in wild type,found the levels induced by ABA.?drought?NaCl and cold stress.2.Under drought stress,the tolerance of overexpression lines(MycFOF2oxl?MycFOF2ox2?MycFOF2ox6)>wild type(Col-4)?mutant lines(fof2)<wild type(Col-0);Under exogenous ABA stress,compared overexpression lines/wild type(Col-4)or mutant lines/wild type(Col-0),which included the length of root,seed germination rate,stomatal aperture?water loss rate,proline,SOD and electrolyte loss rate,had significant difference.3.Under drought stress,Q-PCR analysis RAB18?RD22 and CBF4 in overexpression lines and wild type or mutant lines and wild type.The three genes expressed higher in overexpress lines compared wild type,while was lower in mutant fof2.In salt stress processing,determination of salt stress related genes SOS?,SOS2 and SOS3,the three genes expressed lower in overexpress lines compared wild type,while was higher in mutant fof2;The downstream genes CBF1 CBF3 and COR15a were measured under cold stress:the three genes expressed higher in overexpress lines compared wild type,lower in mutant fof2.Data suggests that FOF2 may are regulating role played in abiotic stress.4.To verify FOF2 drought-resistant ability interact with F-box function structure domain,builting delected F-box strain of expression lines(FOF2?F),the determination of the same RAB18,RD22 and CBF4 genes expression level,and exogenous ABA treatment,to F-box expression compared with wild type.In the main root,germination act as negative dominant mutation phenotype.Further illustrates,FOF2 function depend on the F-box function structure domain under drought stress.5.The method of Yeast two-hybrid found FOF2 interactions with VOZ1 and VOZ2 proteins.BiFC and Co-IP method were confirmed that the interaction relationship between them.In addition,BiFC experiments show that the position of their interactions within the cell nucleus,suggested FOF2 function may be work in the nucleus.In order to further study the relationship between FOF2 and VOZs,through the proteasome ubiquitin analysis:mixing Myc-FOF2 and VOZs-Flag express in tobacco plants then deal with MG132,ubiquitin hybridization results showed that the antibody VOZ2-Flag in the protein ubiquitin is most obvious.Half of in vitro degradation analysis:GST-VOZ2 fusion protein mixed the total protein of overexpression lines,wild type and the mutant lines,then by using Western blotting and ImageJ software analysis the degradation rate,and we get the degradation in overexpression lines is higher than the wild type.To sum up,FOF2 involved in ubiquitin protein degradation of VOZ2.6.To discuss the upstream and downstream relationships between FOF2 and VOZs,cross-fertilized mutant fof2 and voz1/2,and got triple mutants vozl/2/fof2.We measured the RAB18,RD22 and CBF4 gene expression,and under exogenous ABA treatment,the phenotypic vozl/2/fof2 in root elongation and seed germination were similar to double mutant vozl/2,which illustrates the FOF2 upstream VOZ1/2.To sum up,FOF2 through regulated the level of VOZs protein to effect on abiotic stress.