Studies Of Biochemical Characterization And Protein Interaction Factor On Arabidopsis Armadillo/Beta-catenin Repeat Gene

When cells are exposed to environmental stresses such as elevated temperatures, hypoxia and H2O2, the levels of heat shock proteins containing HSP70s which are highly conserved increase and help the cells survive.HSP70s are regulated by various factors, such as cochaperones and NEFs. Fes1p, the S. cerevisiae homologue of people HspBP1, acts as a nucleotide exchange factor (NEF) for Ssa1p and Ssb1p. Deletion of the Fes1p gene in S. cerevisiae moderately impaired normally growth compared to wild-type cells at 37℃; Fes1p was without effect on refolding, whereas HSPBP1 inhibited the refolding of luciferase substantially. Mutants Salk₀21784 of At3g09350 and wild-type Arabidopsis thaliana were pretreated for 2h at 38℃, then heat treated for 4h at 45℃, and then restored at room temperature (25℃) for a week to observe the phenotype. It was found that almost all mutants turn yellow, while wild type lines were green and able to continue to grow. Through the protein sequence and structure analysis, we found expression of At3g09350, which possesses ARM repeat domain, is probably the homologue of Fes1p, so we named it as Fes1p-like protein (AtFes1p).To determine whether AtFes1p plays an important statue and role in the thermotolerance, whether it has a similar role with Fes1p, and whether it interacts with HSP70, GST-AtFes1p, DnaJ, HSP70 and GST-AtFes1p-truncate protein were recombinantly expressed and purified, and the polyclonal antibody against AtFes1p was obtained; using Co-immunoprecipitation and GST-Pull down strategy, it showed that AtFes1p interacts with HSP70 in vivo and in vitro. It is supported that AtFes1p plays an important role in heat tolerance. The main results are shown as follows:1. Identification of T-DNA insertion Arabidopsis thaliana mutantThe mutant Arabidopsis genome DNA was extracted using CTAB minipreparation method, the target fragmen amplified by using polymerase chain reaction (PCR) were subcloned into pGEM-T Easy vector, and then sequenced. Sequencing results showed that Salk₀21784 and Salk₁13165 belong to mutant strain of At3g09350, but Salk₁43747 belongs not to At3g53800.2. Preparation of polyclonal Antibody against AtFes1pThrough immunizing the male New Zealand white rabbits using purified GST-AtFes1p fusion protein, we successfully prepared polyclonal antibody against AtFes1p, which can further determine interaction between AtFes1p and HSP70 in vivo or in vitro, and the AtFes1p expression of different conditions at mutant and wild-type Arabidopsis.3. Protein interaction assays between AtFes1p and HSP70Co-immunoprecipitation (in vivo) was carried out by polyclonal antibody against GST-AtFes1p as a "bait", and then Western-blotting showed that interaction between hsp70 and AtFes1p was determined in wild-type Arabidopsis at 38℃and room temperature (25℃), but in Salk₀21784 not. The GST-Pull down assays ( in vitro) were carried out by maize HSP70 and GST-AtFes1p fusion protein or GST protein purified respectively as a "bait protein" and "target protein" under different conditions. Western-blotting showed that interaction between HSP70 and GST-AtFes1p is not or very little influenced by temperature, KCl and DnaJ, which indicates the interaction is specificity and is reversely tight. So we supposed that AtFes1p plays an important role at heat-sensitive reactions and is essential to the plant growth under high temperature.4. The molecular chaperone activity of AtFes1pFirefly luciferase was thermally inactivated for 10min at 40℃. For the refolding assay, the reaction mixtures containing thermally inactivated firefly luciferase, HSP70 and AtFes1p were incubated in buffer for various periods of time(0min, 30min, 60min) at 30℃.The firefly luciferase activities were measured. Data showed that luciferase activities were promoted by the fusion protein GST-AtFes1p. It is demonstrated that AtFes1p has molecular chaperone activity.