Cloning Of MADS-box Gene, Analysing Its Expression At Different Ripening Stages Of Banana Fruit

Banana was a kind of typical respiration climacteric fruit, which had important theoretical and practical meanings to regulate its ripening and to study the mechanism of ripening. MADS-box gene was a gene family and the encoded protein was transcription factor, more and more researches showed that it played important functions to the development of flowers, the development of fruits and the fruits ripening. Our laboratory had already obtained the SSH fragment of MADS-box gene by constructing the SSH library of banana fruit and found it might be associated with fruit ripening by the result of chip analysis, so it was very need to research it further.Designing the PCR primers on the base of the SSH fragment and screened a 888bp fragment named MuMADSl and another 553bp fragment named MuMADS2 by the PCR method from banana fruit cDNA library. The results of Blastx showed that both had highly homologous with MADS-box genes, the analysis of conserved regions of MuMADSl indicated that it had a very conserved MADS -box region and a littler conserved K-box region, which was identical with the typical structure of the MADS-box gene, the result of Genscan indicated that MuMADSl included an integrated ORF and there were 235 amino acids. The result of Southern blot showed that the copy number of MuMADSl was not high in the banana genome. In order to research the expression of MuMADS1, firstly, we measured the respiration and ethylene, the results showed that the rise of the climacteric of both intiated on the 15rd day after harvest and both reached to the peaks on the 25rd day after harvest, based on the results, we selected the fruit of 0rd , 4rd, 8rd, 12rd, 18rd, 20rd and 25rd as materials for expression, at the same time ,we selected the flower, root, stem and leaf as materials for expression. And then extracted the RNA of all and reverse transcripted them to cDNA as the templates for expression. The result of RT-PCR showed that MuMADS1 just expressed in the flower and fruit but not in the root, stem and leaf. In order to further analyse the expression in flower and the different stages during the fruit ripening, we used the method of real time PCR to research the expression based on the different cDNA as templates and SYBR Green as dye,the result showed that, the...