Cloning And Expression Analysis Of Genes Related To Banana Fruit Ripening

The banana (Musa acuminate L. AAA group) fruit undergoes a postharvest ripening process, which plays an important role in improving the quality and extending the shelf life of bananas. To manipulate postharvest banana ripening, a better understanding of the mechanism of postharvest ripening is necessary.To isolate differentially expressed genes at the early stage of postharvest banana ripening, a forward suppression subtractive hybridization (SSH) cDNA library was constructed in our laboratory. A total of 289 clones in the SSH library were sequenced. BLASTX results revealed that 191 cDNAs had signifficant sequence homologies with known sequences in the NCBI database. The identified cDNAs encoded proteins involved in cellular processes such as: metabolism; protein destination and storage; protein synthesis; signal transduction; transport and intracellular traffic; cell structure, growth, and division; transcription and post-transcription; and disease and defense.According to these identified cDNAs, we designed 3' and 5' RACK (Rapid Amplification of cDNA Ends) primers. Four novel genes had been cloned from banana by RACE techniques, which included: the banana aquaporin gene (named with MaAQPl), the banana ubiquitin conjucting ezyme gene (MaUCE), tow glyoxalase I genes (MaGL014, MaGLOl8). The banana Aseorbate Peroxidase gene(MaAPX) was also obtained by the same method, whose GSPs (Gene Specific Primers) was designed according to the gene in NCBI(accession number: AF146521.1).Semi-quantitative RT-PCR results revealed that the genes were expressed in roots, stems, leaves, floral buds and fruits. The expressing level of MaAQPl gene was the highest in leaves than other organs. The expression patterns of MaAQPl and MaAPX were similar. They decreased gradually during the first stages of normal ripening fruits or induced by ethene and reached their lowest points at the time when ethene had its respiratory peak. MaAQPl and MaAPX expressed intensively in the fruits which were treated by KMnO4. It showed that MaAQPl and MaAPX were probably involved in the regulation of fruit development. The expression of MaGLOl8 and MaGLO14 failed slightly in the first four days, and then had upregulation in the rest days of fruit ripening.Green fluorescent protein (GFP) is a new reporter gene and is widely used in plant research. The banana aquaporin gene (MaAQPl) was fused with GFP gene, and constructed intopCAMBlAI304 under the control of the 35S promoter. The plasm id (pCambMaAQP-GFP) was transformed into onion epidermal cells by particle bombardment. Bright green fluorescence was visualized in the membrane with bombardment of pCambMaAQP-GFP by fluorescence microscopy, whereas the 35S controled GFP plasmid showed the whole cell distribution of GFP. The result suggested that the MaAQP1 gene might function as a water channel in the membrane.The information generated in this study provides new clues to aid in the understanding of banana ripening.