Functional Analysis And Overexpression Of LeMBF1 In Tomato

Transcriptional coactivators play a crucial role in eukaryotic gene expression by communicating between transcription factors and/or other regulatory components and the basal transcription machinery. They are divided into two classes: transcriptional coactivator that recruit or possess enzymatic activities that modify chromatin structure and transcriptional coactivator that recruit the general transcriptional machinery to a promoter where a transcription factor(s) is bound. Multiprotein bridging factor 1 (MBF1) belong to the Second class. MBF1 is a highly conserved transcriptional coactivator involved in the regulation of diverse processes such as endothelial cell differentiation, hormone-regulated lipid metabolism, central nervous system development, and His metabolism. MBF1 proteins from different organisms interact with transcription factors such as c-Jun, GCN4, and ATF1, or with different nuclear receptors, and link them with the TATA-binding protein. The plant MBF1s participate regulating involved in the development of organisms and the response to environment conditions. The expression of MBF1 in transgenic plants regulates a variety of signal transduction pathways and augments the accumulation of a number of defense transcripts. MBF1 protein could be used to enhance the tolerance of plants to different multiple stresses, such as high temperature, drought, pathogen invasion dyeing. In order to cultivate a high resistance to transgenic crops has provided a new path. However there is not relative report on MBF1 gene in tomatoes. 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. Getting regenerated tomatoMBF1 gene was ligated to pDH51 cut with PstI. 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, named after pB1N19-MBF1. AC++ 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.2. Southern blotting analysisThe Southern blotting analysis indicated that all transformants had been transformed. Eight lines displayed multiple copies and three lines displayed single copy.3. Northern analysisThe line 04, 05, 08 and 09 were chosen for Northern analysis with LeMBF1 as probe suggested that the LeMBF1 is over expressed in the 4 lines. 4. Down-stream gene analysisWe cloned tomato Actin, PR1, PR2, PR4, PR6, Pti4, HSP90, Apx2, CHS and Zat gene from tomato. The expression level of PR1, PR2, PR4, PR6, Pti4, HSP90, Apx2, CHS and Zat were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that the expression levels of HSP90, Apx2, Zat, PR1, PR4 and PR6 obviously increased and the expression levels of CHS, PR2 and Pti4 showed no change in MBF1-overexpressing plants. Therefore, we suggested that expression of LeMBF1 upregulates the expression levels of HSP90, Apx2, Zat, PR1, PR4 and PR6.5. Biotic stress experimentsBrevibacliius brevis was infiltrated into AC++ and transgenic plant leaves. Bacteria was extracted from leaves, plated on agar plates, and scored for cfu cm-2. The result showed that MBF1-overexpressing plants were more resistant than wild type plants to bacterial growth. AC++ and transgenic plant leaves were inoculated with freshly harvested Fusarium graminearum conidial spore suspension. All samples were harvested. The above cloned PR genes were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that LeMBF1, PR1, PR2, PR4, PR6 and Pti4 mRNA level enhanced in AC++ plants. The expression levels of LeMBF1, PR2, PR6 and Pti4 obviously increased in transgenic plants, however, PR1 mRNA level weakly decreased. Compared with AC++ plants, expression of LeMBF1, PR2, PR4 and Pti4 mRNAs were significantly higher in transgenic plants, however, PR1 and PR6 mRNAs levels were lower. LeMBF1 expression enhances the tolerance of transgenic plants to pathogen by activating the expression of several PR genes and Pti4.6. High temperature experimentsAC++ and transgenic plants were incubated at 39°C. Survival rate of AC++ and transgenic plants in response to heat stress, the result showed enhanced basal thermotolerance of MBF1-expressing plants. Leaves were harvested. LeMBF1, PR1, HSP90, Apx2, CHS and Zat were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that the expression levels of LeMBF1, PR1, HSP90, Apx2, CHS and Zat obviously increased in AC++ and transgenic plants. Compared with AC++ plants, expression of LeMBF1, PR1, HSP90, Apx2 and Zat mRNAs were significantly higher in transgenic plants. CHS mRNAs levels was higher within 30 min, thereafter, became lower. LeMBF1 expression enhances the thermotolerance of transgenic plants by activating the expression of PR1, HSP90, Apx2 and Zat.