Cloning Characterization And Genetic Transformation Of MBF1 Transcriptional Coactivator In Tomato

Adversity-stresses, like drought, salinity, cold, flood, and so on, it can influence plant growth, distribution, production capacity and the quality of crops. Acclimation of plants to adversity conditions is mediated by a complex network of transcription factors and other regulatory factors (such as controlling multiple defense enzymes and proteins). It has been demonstrated that transcriptional coactivator has close relationship with transcriptional factors and can enhance the combination of transcriptional factors and cis-acting element. Multiprotein bridging factor 1(MBF1) is a transcriptional co-activator mediating transcriptional activation by bridging between an activator and a TATA-box binding protein (TBP). It was found in silkworm, drosophila melanogasteral at first, then in plant species. It was reported that the expression of MBF1 in Arabidopsis (Arabidopsis thaliana) can augment its defense ability to adversity-stresses, but the function of MBF1 in tomato has not been clear yet. We cloned the tomato MBF1 gene and plan to overexpresse this gene in tomato to analysize its function. The main results are as follows:1. Being cloned the gene coding MBF1 in tomato.We cloned LeMBF1 gene from tomato by RT-PCR. LeMBF1 shares approximately 82% homology at the putative amino acid level with AtMBF1. The mRNA sequence of LeMBF1 was deposited in GenBank and GenBank Accession Number is EF051474.2. Being detected the expression level of the LeMBF1 by Northern blotting.The total RNA samples that extracted from the materials which were treated with ethylene and different environmental stresses (such as drought, water recovery, wounding and high temperature) were probed by the LeMBF1. The results of northern blotting showed that the expression of LeMBF1 was effected by high temperature, drought and wounding stress. The expression level of LeMBF1 was also detected in Nr (Never-ripening, Nr) tomato and rin(ripening inhibitor, rin) tomato. There was no evidence to show that ethylene could effect the expression of LeMBF1 in tomato seedlings and mature green fruits.3. Being constructed the plant binary over-expression vector pBINl9-MBF1.The LeMBF1 gene was ligated to pDH51 vector that digested by Pst I to construct the plasmid pDH51-MBF1. pDH51-MBF1 was then analyzed by PCR and restricted enzyme digest. A fragment including 35S promoter, LeMBF1 and 35S terminator was digested from pDH51-MBF1 with EcoR I, and then ligated to pBIN19 that digested by EcoR I to construct the plasmid pB1N19-MBF1. The pB1N19-MBF1 was then analyzed by PCR and restricted enzyme digested.4. Gained the transgenic tomato.The LeMBF1 was transformed into AC + + tomato cotyledon explants by Agrobacterium LBA4404. A few putative transgenic plantlets have regenerated from the tomato calli after being screened with 50 mg/L Kan and 500 mg/L Sm.