Molecular Cloning And Evolution Analysis Of MtPUB Gene In Medicago Truncatula

U-box proteins are widespread in plants shows it play an important role in plant growth and development. Many U-box proteins have the ubiquitin ligase E3 activity and have different physiological and biochemical functions such as identified target proteins in ubiquitin process. AtPUB4 is the protein latest found in Arabidopsis thaliana with E3 ligase activity, studies have shown that AtPUB4 gene can affect growth and development of pollen tapetum cell, using this character may create male sterile materials.Medicago truncatula is the leguminous annual herbaceous plants, diploid, has features like short growth period, small genome, self-pollinate, easy transformation and shorter regeneration time, which was the model plant used for research, the whole genome sequencing has been completed at present. The study of U-box family genes in M.truncatula are not reported until now. In this study, we cloned two U-box genes from M. truncatula and using bioinformatics and biological technology to analysis genetic structure, subcellular localization, prokaryotic expression and gene expression. The plant expression vector constructed was transferred into Arabidopsis thaliana. Finally, we analyzed MtPUB gene family evolution process. The main results of this research are as follows:1. In this study, we obtained MtPUB1 and MtPUB2 cDNA sequences by homologous cloning method form M. truncatula. Each of them contains 2448 bp and 2400 bp and encodes 815 and 799 amino acids. Protein molecular weight was 88.36 KDa and 87.96 KDa. Both of them were the hydrophobic protein, no signal peptide and across the membrane structure.2. Using subcellular localization prediction, MtPUB1 was positioning in the cytoplasm, MtPUB2 was positioning in the mitochondrial matrix. We obtained MtPUB1 protein by construction prokaryotic expression vector.3. Using real-time quantitative PCR analyzed, MtPUB1 relative expression was higher in flower, MtPUB2 relative expression was higher in steam. These two genes were expressed lowest in roots. After NaCl, PEG and ABA induced, the expression level of MtPUB1 was increased, and the change was not obvious under low temperature stress. After NaCl induced expression decreased, the expression level of MtPUB2 was showing a rising trend under the PEG, ABA and cold induced.4. Constructed pBI121-MtPUB1 and pBI121-MtPUB2 vectors were transfered into Atpub4 mutant by agrobacterium mediated. Positive clones were screened by kanamycin and PCR technique,MtPUB1 genetically modified Arabidopsis T1 seeds from transformants were obtained. There was no change in the mutant pollen exine ultrastructure which compared with WT and MtPUB1 transformation Atpub4 mutant by means of scanning electron microscope(SEM).5. It is concluded from phylogenetic analyzed of MtPUB gene family function divergence site,the functional divergence between these two domains focus on 3 motives. According to the analysis of function site and selective site, we obtain 9 functional divergence sites.6. Through analyzed the selection pressure of MtPUB gene sequence,the results showed that MtPUB gene’s function was retained after gene duplication in strong negative selection pressure. The sequence have 114 negative selection site match for consevarive sequence consistent with multiple sequence, presuming this domain binding PEPTIDE that combination of substrate and made MtPUB produced different function.