Characterization Of A Banana MaBTB Gene In Transgenic Tomato

BTB protein was found in many eukaryotes, including various species from yeast to human. BTB protein was characterized in that the N-terminal domain containing BTB domain.BTB domain was a protein-protein interaction motif for wide distribution, and protein containing BTB domain regulated a variety of physiological and biochemical processes, such as transcriptional repressor, cytoskeletal regulation, gating of ion channel, protein ubiquitination.A full length cDNA named MaBTB has been isolated from banana by SSH combined with RACE technology. It was sequenced that contains BTB (Bric-a-brac, Tramtrack and Broad complex) domain and MATH (meprin and TRAF homology) domain. RT-PCR analyzed all of organs like root, stem, leaf, flowers and fruits. The result showed that the gene is expressed in all of organs. For expression analysis of the gene of postharvest banana when naturally ripened, induced ripened by ethylene and inhibited ripened by1-MCP, results showed that expression of MaBTB was associated with postharvest banana ripening and was induced by ethylene.In the study a plant expression vector pCAMBIA1302-MaBTB with green fluorescent proteins was transformed into onion epidermal cells by gene gun method. And it was transformed into tomato by Agrobacterium infection method. Through the analysis of the gene expression in tomato and relevant impact on the quality, further identified its possible function. The results of the study are as follows.1. The result of transient expression of MaBTB-GFP protein in onion epidermal cells showed that MaBTB was localized in cell nucleus. It was characteristic of transcription factors.2. In order to analysis transactivation of the MaBTB protein, MaBTB coding sequences were cloned into the DNA-BD vector pGBKT7to construct fusion plasmids pGBKT7-MaBTB, pGBKT7-MaBTB-MATH and pGBKT7-MaBTB-BTB. These plasmids were transformed into the yest strain AH109harboring the LacZ and HIS3reporter genes. The transformed yeast culture was spotted onto SD/-Trp or SD/-Trp/-His or SD/-Trp/X-a-gal. All the yeast colonies on SD/-Trp were positive, meaning that all the plasmids were transformed into AH109cells. But the fusion plasmids could not survive on SD/-Trp/-His and there were not blue colonies on SD/-Trp/X-a-gal in addition to the positive control. The results indicated that expression of the reporter gene was not activated by MaBTB. We supposed that the role of the gene in plants may be through protein-protein interactions. 3. As a result,35transgenic plants have been obtained. The plants were detected by molecular techniques including PCR Southern blot and RT-PCR. It confirmed that the banana MaBTB gene has been successfully inserted into tomato genome, and expressed in tomato.4. The related physiological parameters of selecting B21, B3, B25, B12transgenic tomato lines were measured. The sugar and vitamin C content of tomato fruit during red ripe period were measured, and the results show that compared with the wild-type turn sugar and vitamin C content of transgenic tomato fruit significantly increased.5. The ethylene emission measurement of in turn color of (BR) and red ripe stage (RM) of transgenic tomato fruit showed that ethylene production of the transgenic tomato fruit were more than that of the wild type, and ethylene production of turn color was higher than that of red ripe stage.6. Extracting the RNA from the transgenic tomato fruit during the stage of BR and RM, the ripening-related gene expression of transgenic tomato fruit was analyzed by the quantitative PCR methods. We mainly chose six key genes ACO1, ACS2, E8, CNR, GMP, GLDH associated with the maturation and quality. The results showed that the gene expression of ACO1, ACS2, E8and CNR significantly became larger compared with the wild-type. ACO1and ACS2gene expression of transgenic lines B21, B3and B12tomato fruit in BR stage was higher than that of red ripe stage, while it was opposite for B25strain E8and CNR gene expression of transgenic tomato lines in BR stage were higher than that of the RM stage. GMP and GLDH gene expression of the transgenic tomato fruit did not change significantly compared with that of the wild-type tomato fruit.In conclusion, in this study the results of subcellular localization and transcriptional activation analysis of MaBTB gene initially revealed its possible mechanisms of action. And through the study of transgenic tomato it showed the important role of MaBTB gene on the regulation of fruit ripening.