Isolation And Characterization Of Relate Genes Regulated By Soybean GmbHLH30Transcription Factor Under Al Stress

Al toxicity is the most important factor which limits crop growth on acidic soils. It inhibits the elongation of plant roots and affects the nutrients and water uptake, thus inhibiting the aboveground growth. In recent decades, numerous of studies for Al toxicity and Al-tolerance mechanisms of plants on the world suggests that plants alleviate the effects of Al toxicity by external exclusion mechanism and internal tolerance mechanisms, and a large number of genes in response to Al stress were identified, of which a lot of Al stress response genes were transcription factor, but only function of Stop1transcription factor was identified. Therefore, there is little information for the functional mechanism of transcription factors involved in the Al Stress. Our previous studies identified the GmbHLH30transcription factor induced by Al stress from forward SSH cDNA library of Al-resistance black beans (RB). The resistance of the tobacco over-expressed GmbHLH30to Al toxicity was enhanced. In this project, we will apply the fusion gene of reporter gene GFP and GmbHLH30gene for analyzing GmbHLH30protein subcellular localization and expression patterns. The proteins binding with GmbHLH30in roots of RB under Al stress was separated by immunoprecipitation. The target genes regulated by GmbHLH30was identified by DNA microarray technology. The expression profiling of each target gene in RB roots were analyzed under Al stress, and the function of GmbHLH30was further validated in the model plant transgenic system. The functional mechanism of GmbHLH30in RB roots in response to Al stress was clarified. The following results were obtained in this study:The proteins binding with GmbHLH30were isolated and identified by co-immunoprecipitation and proteins spectrum technology. The three known proteins binding with GmbHLH30were obtained, i e. WD40proteins, ATP-citrate synthase beta chain protein1-like and pyruvate decarboxylase isozyme1-like.The peptides were synthesized with the specific amino acid sequence (GGLNMYPGAEVSPIMN) of GmbHLH30, and get the GmbHLH30specific antibody was obtained by immunizing New Zealand rabbits. The expression level analysis of GmbHLH30in RB roots under Oh and8h Al stress, respectively, using Far-Western technology showed that the expression levels of GmbHLH30was induced by Al stress. The strong green fluorescence in root cell nucleus of RB under8h Al stress was observed with FITC method, indicating that GmbHLH30transcription factors located in the nucleus. Meanwhile, the GmbHLH30pKGWFS7-GmbHLH30plant expression vector was constructed, and transgenic tobacco plants were obtained by transferring to wild-type tobacco. We observed the green fluorescent in the cell nuclei of the transgenic tobacco root under8h50μM Al stress under a fluorescence microscope. This also demonstrated that GmbHLH30transcription factors targeted to the cell nucleus.The homozygous of T-DNA insertion mutants was screened out by designing primers and detecting GmbHLH30mutation t on the levels of RNA and the DNA. Meanwhile, transgenic Arabidopsis over-expressed pK2G7-GmbHLH30transfer vector was screened. There were1923genes in which gene expression changeable times≥3in Arabidopsis mutant under8h50μM Al stress by microarray hybridization analysis, of which688were up-regulated and1235were down-regulated. While there were2697genes in which gene expression exchangeable times≥3in the wild-type Arabidopsis under8h Al stress, of which948were up-regulated and1749were down-regulated. There were719same genes in which gene expression exchangeable times≥3in wild-type and mutant Arabidopsis thaliana under8h50μM Al stress. The genes selected from these genes which up-regulated more than3times in wild-type Arabidopsis and down-regulated more3times in Arabidopsis mutant were clustered, of which signal transduction genes accounted for3%, the energy metabolism-related genes with44%, cell growth, structure and plant growth-related genes (8%), stress defense-related genes (21%), protein synthesis, modification and degradation-related genes(5%), the rest is unknown (19%). Genes that up-regulated more than three times in wild-type Arabidopsis were clustered, of which the signal transduction genes accounted for7%, energy metabolism related genes(16%), cell growth, structure and plant growth related genes (10%), stress defense related gene(13%), channel protein and transport related genes (6%), protein modification, synthesis and degradation related genes (5%), photosynthesis related genes (1%) and unknown genes (42%).3interesting genes were selected from these genes and the expression levels of the genes were analyzed by RT-PCR. The expression profilings are consistent with the microarray results, indicating the reliability of the chip.