Screening Target Genes Regulated By WRKY6 In Arabidopsis Thaliana Under Low Phosphorus Condition By DIGE Technique

WRKY proteins are plant-specific transcription factors and they can bind with the W-box.They play an important roles in response to biotic and abiotic stress.Researches showed that WRKY 6 can bind to the PHO1 promoter and inhibit the expression of PHO1 under low phosphorus stress.We guess that the WRKY6 may regulate other genes except PHO1.This experiment want to find the target genes regulated by WRKY6 through the DIGE technology.In this study three species material the wild type(WT),the WRKY6 over expression plant and WRKY6 knockout mutant were chosen for low Pi(3 uM)treatment 3 d.We find the possible target genes regulated by WRKY6 through protein extraction,the two-way electrophoresis,the gel analysis,mass spectrometry identification and the biological informatics.The main results were presented as follows:1.High quality proteins of Arabidopsis thaliana leaves were extracted using BPP method,and these proteins were up to the standard of proteomic studies in the next step.High repeatability gel maps of 2-DE were obtained using GAP staining method.The mass spectrum success rate was very high.Comparing the 2-DE maps of the three materials,we obtained 131 differentially expressed protein spots.The 112 spots were successfully identified using MALDI TOF and MALDI TOF/TOF mass spectrometry.These proteins were divided into 14 categories by EggNOG function classification.And they were main involved in carbohydrates,protein translation modifications.The subcellular localization predictor(CELLO analysis)indicated that,58 proteins were located to in the cytoplasm,8 proteins outside the cell,3 protein on the inner membrane protein,11 proteins in the outer membrane,32 protein in the Periplasmic space.2.The metabolic pathway analysis show that the identified proteins were main involved in 8 different metabolic pathways,which are carbon fixation in photosynthesis,carbon metabolism,glyoxylate dicarboxylate metabolism,glycolysis gluconeogenesis,fructose and mannose metabolism,nitrogen metabolism,biosynthesis of amino acids,glutathione metabolism.3.The gene promoter sequences analysis in the WRKY6 over expression plant,show that there are 4 genes whose promoter has 4 W-box sequences,3 genes have 3 W box sequences,6 genes have 2 W-box sequences.26 genes have 1 W-box sequences,29 genes have no W-box sequences.4.Q-PCR shows that translationally-controlled tumor protein-like protein,glycine-rich RNA-binding protein 8,ATP synthase gamma chain,chloroplast precursor and nitrilase I were both increased in the protein expression and gene expression levels in the WRKY6 overexpression plant leaves.Dehydroascorbate reductase,tryptophan synthase alpha chain and HSP60 were both increased in protein expression and gene expression levels in the WRKY6 overexpression plant roots.Only jacalin-like lectin domain-containing protein was decreased in both levels in the WRKY6 overexpression plant roots...