Cloning And Functional Analysis Of Tomato E3Ubiquitin Ligase Gene, SLBAH1

In natural environments, plant faces challenges associated with various microorganism. In this long-term evolutionary process of interaction and mutual influence, the plant gradually gained a complex series of defense mechanisms to protect themselves. This innate immunity exist not only in plants, but also in insects and vertebrates as their common pathogen infection defense mechanism. By specific recognition of cell wall, metabolites and other components of pathogens, plants can either directly or indirectly identify the corresponding pathogen, activates defense response signal to produce a series of defenses and hypersensitivity reactions substances, and eventually produce resistance. In the process of the plant defense response, the plant itself will occure important metabolic changes, in order to maintain and regulate normal physiological processes of cells. Among them, the regulation of the ubiquitin-mediated protein is a widespread regulatory mechanism, and it plays an important role in the regulation of normal cell function and response of plants to environmental signals. In this study, a ubiquitin ligase gene from tomato was cloned and the functions of the gene was studied. The main results are as follows:1. According to BAH1gene sequence of Arabidopsis, a EST sequence was got from tomato by searching the tomato genome. The primers were designed on the basis of EST sequence. And the full length cDNA sequence of SLBAH1gene was obtained by3’-RACE amplification. The predicted SLBAH1gene contained1002nucleotide and encoded333amino acids. Function prediction found that the protein contained a SPX domain and a RING domain.2. Based on the SLBAH1gene sequence, the primers were designed to construct three prokaryotic expression vectors. They are separately contain the whole sequence, the lack of RING domain and only RING domain of the gene sequence. Enzyme activity showed that the fusion protein SLBAH1and SLBAH1R had E3ubiquitin ligase activity in vitro. However, the SLBAH1R protein did not show out any E3ubiquitin ligase activity.3.35S-S1BAH1::GFP fusion protein was constructed, and plasmid vector was shoot into the onion epidermis paved in MSo by particle gun then the fluorescence microscope was used to observe. Subcellular localization analysis showed that, SLBAH1protein localized in the cytoplasm and nucleus.4. By RT-PCR, organ-specific gene expression characteristics of tomato SLBAH1biotic and abiotic stress were studied and analyzed. The results showed that, SLBAH1expression levels in roots was much higher than in leaves and stems; and was regulated by Botrytis cinerea. And it was induced in the early stages of pathogen infection then was reduced after expression defense signaling molecules. ABA, SA and MeJA also inducee different degree of gene expression of SLBAH1. The above results showed that, SLBAH1gene may play role in self-defense of disease.5. Leaves of tomato seedling were inoculated with Botrytis cinerea in vitro. When there were chlorosis in PDS silenced leaves, extracted total RNA from pTRV S1BAH1processed tomato seedlings, using Real Time PCR method to detect SIBAH1gene expression levels. The results showed that the gene expression of SIBAH1reduced from30%to70%. Biomorphic observation showed that gene silencing occurs on SLBAH tomato plants leaves lesion diameter was significantly less than the control treatment of tomato leaves.To sum up, the down-regulates expression of SIBAH1gene can significantly improve plant resistance against Botrytis cinerea, which play negative regulatory role in pathogenic microorganisms and abiotic threats. These results, for the people to further understand the function of RING (really interesting new gene, RING) family proteins in plant disease resistance but also provide a theoretical basis for further research and genetic resources in tomato resistance gene engineering.