Regulation Analysis Of Arabidopsis FB1 And FB2 Genes In The ABA Signal Transduction

ABA plays a key role in plant growth and development,including seed dormancy,germination,seedling growth and flowering,etc.More importantly,ABA make plants tolerate stress related to water deficency,such as drought or salinity.So far,some basic components in ABA signal pathways have been identified,such as PROTEIN PHOSPHATASES TYPE 2Cs(PP2Cs),SUCROSENON FERMENTING1-RELATED PROTEIN KINASEs(Sn RK2s)and ABA-RESPONSIVE ELEMENTSBINDINGFACTORs(ABFs).In 2009,the ABA receptor in plant cells,PYRABACTIN RESISTANCE 1 / PYR-LIKE/REG-ULATORY COMPONENTS OF ABA RECEPTORs(PYR1 / PYLs/RCARs),has been proved.When induced by exogenous ABA,ABA receptor perceive ABA and suppresses the activity of PP2 C to make Sn RK2 dephosphorylation and regulate the expression of downstream genes.The strict control of basic component level in ABA signal transduction pathway is crucial for the steady state of plant cells.Recently,many studies have shown that the central regulators in ABA signaling were degraded by ubiquitin 26 s proteasome system.More than 1400 genes encoding E3 ligase have been found in Arabidopsis genome.These genes can be divided into two groups: single-subunit type and muti-subunit type.As the most important subunits of SCF complex,F-box protein is coded by the rapid expansion eukaryotic gene family.In the N terminal 40-50 amino acid form conservative F-box structure domain.First F-box was discovered from the research for the cell cycle protein F and found to interact with Skp1.In C terminal F-box protein contains one or more highly variable protein-protein interaction domains.Totally 692 and 779 F-box protein sequences were found in Arabidopsis and rice genomes,respectively.However,the functions of most F-box family genes are still unknown.In this study,we cloned two F-box family genes in Arabidopsis and named them FB1 and FB2 individually.The function of these two genes in ABA signaling pathway was analyzed.The main results in this study are as follows:Using the Infusion technology,we successfully cloned the FB1 and FB2 genes,and constructed the T-DNA vector N-p EGAD-LUC expressing FB1 and FB2 respectively.The full length of FB1 and FB2 are respectively 2283 bp and 1337 bp.FB1 contains two exons and one intron,whereas FB2 does not contain any intron.FB1 and FB2 both contain typical F-box structure domain,and have the highest similarity with their counterparts in rape.By dipping inflorescence infection mediated by agro bacterium,the recombinant plasmid were transferred into Arabidopsis thaliana.Through Basta resistance screening,Luminometer instrument instantaneous detection and Western Blot technology,we conformed the transgenic plants successfully over-expressing FB1 and FB2 genes,Then the T3 homozygous were obtained.In addition,from ABRC we bought fb1 and fb2 T-DNA insertion mutants.Using the gene-specific primers fb1/2-F(LP)and fb1/2-R(RP)and the T-DNA-specific primer BP,we identified homozygous mutants.Using Gateway technology,the plasmid expressing YFP-FB1 protein was built and was transiently transformed into Arabidopsis mesophyll protoplasts.The YFP fluorescence signals were examined and the results show that FB1 protein was located in both the nucleus and cytoplasm.The effect of ABA on the seed germination of the transgenic Arabidopsis overexpressing FB1 and FB2 was analyzed.The results showed that the seed germination rate of FB1/2-overexpressed lines were significantly higher than that of the wild type,indicating that overexpression of FB1/2 cause insensitivity to ABA in the transgenic Arabidopsis.The transcript levels of ABA signaling-associated genes were analyzed in FB1/2-overexpressed lines and wild type respectively by q PCR method.The results demonstrated that in FB1/2-overexpressed transgenic lines the mRNA accumulation of ABI1 and ABI2 was increased obviously and the mRNA level of ABI3 and ABI4 was down-regulated.Unexpectedly,the positive regulator of ABA signaling such as ABF transcriptional factors,Sn RK2 and ABI5 were up-regulated at their transcript level,which suggests that degradation of the target protein mediated by FB1 and FB2 may induce the increasing mRNA transcription in a feedback manner.