Isolation And Characterization Of Fruit-specific Expressed CDNA And Promoter In Banana (Musa Spp.)

Bananas and plantains (Musa spp) are globally important fruit crop, especially in developing tropic and sub-tropic countries, because they feed tens of millions in Central America and Asia. Besides that, banana trade is an essential source of income for many national economies. Therefore, the United Nations Food and Agriculture Organization ranks bananas as the world's fourth most important food crop. Banana postharvest ripening plays an important role in influencing fruit quality and shelf life. As a result, many researches on molecular biological and physical mechanism of banana postharvest ripening have been carried out. Banana fruits are typical climacteric fruits, with many features in ripening like tomatoes, avocado, mango and others. Many experiments demonstrate that banana fruit ripening is regulated by ethylene, companied by aspiratory climacteric. In the view of molecular biology, protein de novo synthesis is necessary for many of physiological changes during postharvest. For the reason of that, by cloning the transcripts of the proteins related to fruit ripening, it would be possible to understand the genes related to fruit ripening and their expression patterns.Differential screening of cDNA libraries representing ripening banana pulp led to the isolation of groups of differential expressed mRNAs. Identification of these transcripts partial sequence analysis indicates that proteins encoded by these genes involved in ethylene biosynthesis, aspiration, starch mobilization, cell wall breakdown and other mobilizations. In this study, cDNA microarrays combined with suppression subtractive hybridizaiton (SSH) were carried out successfully for isolating fruit-specific expressed cDNA. 287 cDNA fragments from SSH were printed onto a glass plate so that a cDNA chip was made. By using labeled RNA from root, leaf and stem, and fruit as probes to screen the chip, as a form of reverse Northern blot, 32 cDNA clones were identified as fruit-specific expressed in banana. Sequencing of these 32 cDNAs showed that 4 cDNAswere homology to those genes related to signal transduction, 4 clones were homology with those genes encoding ripening associated protein, 3 clones were homology with those genes encoding protein kinase, 9 clones were homology with those genes encoding some enzymes which took part in some metabolisms in banana fruit ripening, 8 clones were homology with those genes encoding different proteins with different functions and also 4 clones had no homologues in NCBI. The fruit-specific expressed cDNA in the chip were confirmed to be fruit-specific by using Northern blot, in which 3 clones selected randomly were used as probes to hybridize to RNA from root, stem and leaf, and fruit. The results of Northern blot suggested that those cDNAs expressed not only specially in fruit, but also the quantity of expression were just similar to those in the chip. It is the first report that so many clones have been isolated from banana and so important cDNA have been obtained, because just total 36 clones had been obtained previously.The gene encoding a -1,4-glucan phosphorylase has been firstly cloned in this study. As for the importance in banana fruit ripening, the full length of the gene was obtained by using rapid amplification of cDNA 5' and 3' end and RT-PCR was used to examine the expression patterns in different organs and different stages of fruit ripening. In order to determine the ripening stages, ethylene biosynthesis and CO₂ production in postharvest banana were measured. Combining the result of RT-PCR with different ripening stages represented by ethylene biosynthesis and CO₂ production, it could be conclude that a -1,4-glucan phosphorylase gene expression happened much earlier than the bust of ethylene biosynthesis and CO₂ production. However, the function of this gene in fuit ripening was still needed to further reseach.A highly fruit-specific expressed promoter takes an important role in expressing foreign protein in fruit, which make banana be possible to be used as bioreactor to produce oral pharmaceutical proteins and chemicals in this way. For the reason of that a highly fruit-specific expressed gene promoter called BanLec gene promoter was cloned in this study. It was demonstrated tha BanLec gene expressed with a very high level in banana fruit in our previous reseach. BanLec gene promoter was isolated by using chromosome walking and a 670 nt DNA with basic elements of promoter, such as TATA-box, CAAT-box,...