The Characterization Of Zinc Finger Protein AtZFP6 From Arabidopsis Thaliana Regulate Stress And Ineract Protein

AtZFP6 (AT5G18650.1), is a C3HC4-type zinc finger protein which contains multiple finger-like protrusions, and is also a E3-type ubiquitin ligase. The length of the gene is 804 bp, encoding 268 amino acids. The study had mainly carried on the proteins interacted with AtZFP6 protein, the tolerant ability of AtZFP6 to stress, and the regulation of AtZFP6 to floral development. First of all, we built AtZFP6 gene to plant expression vector pROK Ⅱ; After we got the plant over expression vector 35S:AtZFP6, we transformed flowers of wild-type Arabidopsis thaliana with Agrobacterium, which is though Agrobacterium-mediated floral dip method plasmid into wild-type Arabidopsis plants. With the selection method, we got the T3 generation homozygous over expression lines, OE1 and OE2. At the same time, we used the method of three primers to select of homozygous mutant lines SALK1 (CS24967) and SALK2 (CS82893).In order to study whether AtZFP6 associated with stress, we used OE1, OE2, SALK1, SALK2,35 s (arabidopsis transformed with empty pROK Ⅱ, control) and WT (wild type) seeds were respectively done with different concentration gradient JA, SA, ABA and NaCl solutions, after that we calculated the germination rates, root length and fresh weight, then analyed tolerant ability of AtZFP6 responded to the different stress; The experimental results showed that the germination rates, root length and fresh weight of these lines had no significant differences under JA, SA, ABA and NaCl stressed. While indicated that AtZFP6 gene in arabidopsis stress process didn’t work at all. Then we observed the whole life cycle of OE1, OE2, SALK1, SALK2,35s and WT lines, and analyzed the influence of AtZFP6 on plant growth and development; Phenotypic observation results indicated that the AtZFP6 could observablely delay the florescence in Arabidopsis thaliana. Yeast double hybridization technique was used to study the proteins interacted with AtZFP6 protein; we transformed AtZFP6 into the pGBKT7 vector, as Bait carrier. At the same time we set transcription factors from different family into pGADT7-Rec, formed the Prey library. Then we transformed Bait carrier and Prey library into Y2H and Y187 yeast competent cells respectively, and we selected the positive transformed yeasts to Yeast Two Hybird. There were a total of 13, these proteins interacted with AtZFP6 including AT3G17609.2, AT3G56850.1 and AT4G34590.1 from bZIP family, AT4G11140.1, AT3G60490.1 and AT4G17490.1 from ERF family, AT5G62470.2 and AT4G05100.1 from MYB family, AT1G69120.1 and AT1G22130.1 from MADS-box family, AT5G07100.1 from WRKY family, AT5G43650.1 from bHLH family and AT4G21080.1 from Dof family. To study AtZFP6 localization in cells,we built fluorescence localization expression vector 35S:AtZFP6-GFP, and instantaneously transformed into onion epidermal cells with particle gun technology, observe AtZFP6 gene expression, the results showed that the AtZFP6 gene expressed in the nucleus and the cytoplasm.