Cloning Of MBF1 Transcriptional Coactivator Gene From Tomato, Constraction Of Plant Expression Vector And Transforming Tomato

Adversity-stresses, like drought, salinity, cold, flood, and so on, are the important environmental factors which influence plant growth, distribution, productivity and quality of crops. Acclimation of plants to adversity conditions is mediated by a complex network of transcription factors and other regulatory genes such as by 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 basic transcriptional elements. 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. Some reports said the expression of MBF1 in Arabidopsis (Arabidopsis thaliana) can augment its defense ability to adversity-stresses, but not to have relative report on MBF1 gene in tomatos. We overexpressed this gene in tomato to check its defense ability and know clearly about the principal in molecular level. The main contents and results of the experiment are as follows:1. Cloning of MBF1 gene in tomatoAC++ total RNA from fruits as template, get cDNA by reverse transcription, then acquire 632bp fragment by PCR and named LeMBF1. The mRNA sequence of LeMBF1 was deposited in GenBank and the GenBank Accession Number is EF051474. Comparing it with that in Arabidopsis the similarity is 56% in DNA level and 82% in amino acid level.2. Northern blotting checking the expression level of the LeMBF1The specific MBF1 gene fragment of tomato was cloned and as a probe to hybridize with total RNA separated from the materials treated with ethylene and different environmental stresses, such as drought, water recovery, wounding and temperature, respectively. The results show that the expression of LeMBF1 was induced by high temperature and drought, while restrained by water recovery; After treated by wounding stress, the expression level of LeMBF1 gene was induced in wild type tomato leaves, while its expression level had no distinctively changes in Nr(Never-ripening, Nr) tomato leaves and was restrained in rin(ripening inhibitor, rin) tomato leaves; The expression level of LeMBF1 in wild type tomato fruit at mature green stage did not distinctively change when treated with exogenous ethylene. This result demonstrated that ethylene could not induce LeMBF1 mRNA expression at this stage.3. Construction of plant binary over-expression vector pBINl9-MBF1MBF1 gene was ligated to pDH51 vector and cut with PstI by T4 DNA ligase, ligate product was verified as positive by PCR and enzyme cutting, named after pDH51-MBF1. A fragment including 35S promoter, MBF1 gene and 35S terminator was excised from pDH51-MBF1 with EcoRI, the fragment was ligated to pBIN19 cut with EcoRI, ligate product was verified was verified by PCR and enzyme cutting, named after pB1N19-MBF1.4. Getting regenerated tomatoAC++ tomato cotyledon as explants, the MBF1 transformation of tomato was mediated by Agrobacterium LBA4404. Some regenerated tomatos have been obtained after screened by 50 mg/L Kan and 500 mg/L Sm.