AtMYB32 Regulates The Expression Of AtGA20ox1 And Its Roles In Resistance To Stress In Plant

Gibberellin(GA) is one of the most important hormones in plants, which regulates different stages of plant growth and development, and responses to adversity stresses in plants. GA20ox1 is one of the key enzymes that participating in the synthesis of GA. Now, the transcription factor,AtMYB 32 was screened to interact with the promoter of AtGA20ox1 by yeast one-hybrid. And CPRG assay was applied to measure the interaction intensity between the promoter of AtGA20ox1 and transcription factor AtMYB32 quantitatively. To further study their combination and the biological functions of transcription factor AtMYB32, some related researches have been carried out and the results were as follows:(1) Three over-expressed AtMYB32 transgenetic lines (AtMYB32ox3, AtMYB32ox6 and AtMYB32ox8) were screened out via genomic DNA PCR, real-time fluorescence quantitative PCR and Western blot. The deficiency mutant atmyb32-1 was identified via semi-quantitative PCR and real-time fluorescence quantitative PCR.(2) The recombinant vector pGreen? 0800-LUC-pGA20ox1 was constructed to further verify the combination between transcription factor AtMYB32 and the promoter of AtGA20ox1 through dual luciferase experiment, and transcription factor AtMYB32 was found to inhibit the expression of AtGA20ox1. Their combination target site was found to be the MYB binding site in the AtGA20ox1 promoter through the Chromatin Immunoprecipitation. Thus, transcription factor AtMYB32 inhibits the expression of AtGA20ox1 via binding to the MYB binding site of the AtGA20ox1 promoter.(3) Analysing the basic physical and chemical characters, the orientation in the cells and the expression in different tissues of transcription factor AtMYB32 using bioinformatics.AtMYB32 was confirmed to locate in the nucleus via transient expression of 35S::AtMYB32-GFP agrobacterium solution injecting to tobacco. The expression of AtMYB32 in different tissues and organs in Arabidopsis was analyzed through real-time fluorescence quantitative PCR and consistent with the prediction of gene chip, AtMYB32 was expressed in every tissue and especially higher in flower and pod. The seedling of Col-4 was dealt with 250 mM NaCl and 300 mM mannitol to test the expression of AtMYB32 gene. The results showed that the AtMYB32 expression was induced by NaCl, mannitol and drought. Speculating that AtMYB32 might be involved in the osmotic stress in Arabidopsis.(4) The green rate of cotyledons and root length of the overexpression lines, the deficiency mutant atmyb32-1 and the wild type treated with 150mM NaCl and 300mM mannitol was observed and statistics. And the green rate of cotyledons of overexpression strains was faster than wild type, its root length was longer than wild type.(5) The survival rate under drought treatment, the stomatal aperture in different ABA concentrations and the dehydration rate of the overexpression strains, the deficiency mutant atmyb32-1 and the wild type was statistics. It was showed that the overexpression lines were more drought-resistant, stomatal aperture closure faster and loss of water slower compared with wild-type, while the lost-of-function mutant atmyb32-1 instead. Analyzing the expression of RD29A and RD29B of overexpression lines, the deficiency mutant atmyb32-1 and the wild type treated with 250mM NaCl. It was found that the expression level of RD29A and RD29B was higher in the overexpression strains, while the lost-of-function mutant atmyb32-1 instead..To sum up, the transcription factor AtMYB32 inhibited the expression of AtGA20oxl by binding with the MYB binding site of the promoter of AtGA20oxl and positively regulate the process of osmotic stress response in plant.