Functional Analysis Of Arabidopsis PUB19U-box E3Ubiquitin Ligase And Its Interacting Proteins

Environmental stresses, such as extreme temperature, drought, high salt, oxidative stress, usually decrease the quantity and quality of vegetable production heavily. Revealing the mechanism of plant in response to stresses will benefit vegetables genetic improvement of resistance to abiotic stresses by new genes and thinking. Thus these studies have very significant sense to improve the vegetable production and quality as well as develop Chinese characteristic agriculture.Plants will activate diverse cellular processes under stresses. One of those processes is autophagy. Autophagy is the lysosomal degradation pathway extensively existing in eukaryotic cells, which is the major process of intracellular material degradation by the vacuole and lysosome. Autophagy can be induced by a variety of abiotic stress, including lack of nutrition, oxidation, salt stress, drought and other stresses. The damaged proteins will be eliminated by autophagy in plant under stresses such as heat stress and the damaged proteins should be ubiquitinated. But so far, ligases for ubiquitin proteins are not identified in plants.This paper uses Arabidopsis (Arabidopsis thaliana) which is cruciferous vegetables as material to study the structural, functional and related properties of PUB19(Plant U-box Protein) and its interacting proteins by means of bioinformatics and molecular biology. Arabidopsis is cruciferous vegetables like cabbage and kale. They are highly homologous. Hense, research on the model plant-Arabidopsis has imprortant guiding on cruciferous vegetable. The main results were as follows:1. AtPUB19appears to be redundantly involved in abiotic stress responses. AtPUB19is notable for its high up-regulated upon exposure to salt stress, drought, cold and heat stress. More specifically, up-regulation of AtPUB18is also consistently seen with these same treatments (salt stress, drought, cold and heat stress), albeit at much lower levels. We identified Arabidopsis T-DNA inserted mutants, which were called pub19-1and publ9-3. The capability of plant resistance to high temperature significantly decreased because of AtPUB19knockout, which can cause irreversible damage to the plant. However, knockout of AtPUB19showed resistance to drought. Therefore, AtPUB19plays an important role in abiotic stress.2. Using yeast2-hybrid system screen interacting proteins of AtPUB19. Using the full length protein of AtPUB19as bait, we screened for AtPUB19-interacting proteins by yeast-2-hybrid system. After screening the positive yeast clones, we used SD-His medium and X-gal assay to defect and used ONPG qualitative method. After sequencing comparison, two genes were found and they are DUT1(Dutp-Pyrophosphatase-Like1) and LSU3(Response to Low Sulphur3).3. Structural and functional analysis of interacting proteins of AtPUB19. The LSU protein family members share high similarity. They all interact with PUB19specifically, but not its homologous protein PUB18. LSUs and PUB19have been induced to expression during sulfur deficiency stress both in leaf and root. It is the first time to reveal the relationship between AtPUB19and mineral elements. Abiotic stresses such as drought, heat stress and darkness stresses inhibited expression of DUT1. We identified T-DNA insertion-dutl mutants, but failed, showing that T-DNA insertion mutant was unavailable. Overexpression of DUT1to publ9mutants and wild plants showed sensitive to oxidative stress and darkness stresses, indicating the PUB19is related to functionally DUT1.