Construction And Genetic Transformation In Tomato Of SlAMSH3 Fruit-special Overexpression Vector

Ubiquitin-proteasome pathway(UPP) plays an important role in the process of plant growth and development, including hormone synthesis, self-incompatibility, downstream signal perception and transduction, plant morphogenesis, disease resistance, and other epigenetic processes. There is also deubiquitinatin enzyme within the cell dissociating the chain of ubiquitin molecules reversely, such as AMSH3 which is one of deubiquitinating enzymes among MPN(+)/JAMM protease and can combine with ubiquitin molecule of ubiquitinated protein. Through the hydrolysis effect of AMSH3 enzyme on the ester bonds of carboxyl-terminal, peptide bond or isopeptide bond in ubiquitin molecule, Ubiquitin molecule could be specifically spun off from the ubiquitin-proteins substrate or precursor protein, and then enter the cell to involve in plant growth, development, and physiological processes, which recently there has been few studies on ubiquitin and deubiquitin enzymes.In this study, a type of deubiquitination enzymes gene-SIAMSH3 was found in tomato. Compared to the biological information on Sol Genomics Network, there were 81% and 71% similarities between SlAMSH3 in tomato and AtAMSH3 enzyme gene in Arabidopsis thaliana in the levels of nucleic acid and protein respectively. In Arabidopsis, AMSH3 is required for the auxin efflux facilitator PIN2 and can hydrolyze K48- and K63-linked ubiquitin chains and accumulate both ubiquitin chain types in vitro and in vivo. But amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. In molecular cloning, the fruit-specific expression promoter TFM7 is much better than the use of CaMV 35S and other constitutive promoters. Therefore, in the work the SlAMSH3 fruit-specific over-expression vector pBI121-TFM7-SlAMSH3 and the fusion reporter gene expression vector 35s-pART27-S1AMSH3-GFP were constructed and transferred into tomato and tobacco plants, respectly, through a reproducible agrobacterium-mediated transformation. The S1AMSH3 protein subcellular localization was posited by fusion reporter gene localization method. The transgenic plants overexpressing SIAMSH3 in fruit were confirmed by the analysis of semi-quantitative RT-PCR and fluorescence quantitative RT-PCR. To investigate the influence of SIAMSH3 gene on fruit characteristics of tomato, the chlorophyll content of green fruit and the single fruit weight, fruit brix, fruit hardness, lycopene content and β-carotene content of red ripe fruits were determined.The results of this study showed that the SlAMSH3 gene expression level of the transgenic tomato fruits increased four times than the wild type, and S1AMSH3 protein expressed only in the nucleus. The chlorophyll, lycopene, β-carotene and brix content in the transgenic tomato fruit were improved 178.61%,40.53% and 74.86%, respectly, other indicators such as single fruit weight, fruit sugar content, fruit firmness and fruit diameter were no significant difference. Therefore, fruit specific over expression of tomato SIAMSH3 is a useful genetic engineering approach to improve fruit quality, the nutritional value and economic value of tomatoes.