The Salt Tolerance Analysis Of Arabidopsis Thaliana E3 Ubiquitin Ligases PUB30 And PUB31 And Screening Their Interacting 14-3-3 Proteins

Land salinization usually decreases the quantity and quality of crop andvegetable production heavily. Revealing the mechanism of plant in response to salt stess make v ery significant sense to crop and vegetable production and quality. Plant will activate diverse cellular processes in stresses. Such as some protein accumulation and degradati on of salt stress pathways. UPP(ubiquitin-26s proteosome pathway) is a highly selectiv e protein degradation pathway in eukaryotic organis MS, and has very important signif icant to regulate the physiological and biochemical environment.E3 ligase is an extremely important enzyme of ubiquitin-26s protease, and U-box ubiquitin ligase was discovered firstly. Plant U-box E3 ligases play important roles in responses to abiotic and biotic stress, responses hormones, growth and development a nd self-incompatibility. So far, various roles of different U-box E3 ligases are still un known.Arabidopsis(Arabidopsis thaliana) is used as the studying materials through molec ular biology and bio-chemistry methods on physiological and protein level by studying E3 ligase PUB30 and PUB31.The main results are as follows:1. Arabidopsis thaliana E3 ubiquitin ligase mutant pub30 and pub31 response to s alt stress. We did bioinformatics analysis for AtPUB30 and its highly homologous AtP UB31 and usedpub30 and pub31 mutant to observe the physiological modification of germination in salt stress in order to research the PUB30 function. The studies show t hat the germination rate of pub30-1 and pub30-3 is higher than col-0, pub31-1 is low er than col-0, pub30-1pub31-1 is between col-0 and pub31-1 on 1/2MS with 150mM NaCl. The result shows that AtPUB30 and AtPUB31 participate in the salt stress toler ance as a negative and positive factors in the germination stage, respectively.2. The physiological response of atpub30-1 complemented line seed in salt stress. On the basis of the original pCAMBIA1305.1-GFP plasmid, we use the In-fusion pla smid construction technology to take pub30, pub31 promoter and pub30, pub31 gene c lone to the upstream and the downstream of GFP, respectively, we gain pub30-1/PUB 30transgenicseeds after the transformation of E.coli, kanamycin resistance screening, pl asmid extraction and identification, Agrobacterium-mediated transformation and activati on, infection, screening, detection of protein expression, but failed to screen pub31-1/P UB31 positive plants, col-0, pub30-1, pub30-1/PUB30 complemented seeds are planted on 1/2MS medium with 150 mM NaCl. We found that the seed germination of pub3 0-1/PUB30 is close to col-0, pub30-1 is higher than col-0 and pub30-1/PUB30. The re sult shows that gene complemented makes the pub30-1 phenotype restored partly whic h further indicate that AtPUB30 participates in the salt stress tolerance as a negative f actor in the germination stage.3. Using the yeast two hybrid system to screening interactions the 14-3-3 proteins of AtPUB30 and AtPUB31. The 14-3-3 is highly conserved molecular chaperone com bined with the phosphorylated amino acid residues. Combined with 14-3-3 proteins wil 1 usually change their stability, enzyme activity, subcellular localization, or easier to c ombine with other proteins. Using the CDS of atpub30 and atpub31, we screen for At PUB30 and AtPUB31-interacting 14-3-3 proteins by yeast two hybrid system MS. And there are three activation markers leucine, tryptophan, histidinol AH 109 and Y187 to indicate that the interactions between target protein. Use defect medium to verify the yeast monoclonal. We screened two positive clones of AtPUB30 and one positive clon es of AtPUB31.The result of experiment shows that the AtPUB30-interacting proteins are OMECRON and OMEGA, and the AtPUB31-interacting protein is IOTA.