Postharvest Fruit Ripening Isolation And Identification Of Differentially Expressed Genes In

The aim of the research was to isolate and identify the genes specifically expressed, and to know the function of the genes in the fruit of ripening bananas at last. Firstly, the Scion Image digital analysis was applicable for rapid quantification of total RNA and cDNA for SMART-PCR , which came from the fruit of preclimacteric bananas (M.AAA Group Cavendish) stored in air alone for Oh, 24h, 48h, and 72h (No.0, No.l, No.2, No.3). The result showed the quantity of mRNA from banana fruit stored for 48h increased obviously. The expression of genes has changed at transcription level during the post-harvest 48 hour. Accordingly the study decided two-directional (forward and backward) Suppression Subtractive Hybridization (SSH) was performed on cDNA from banana fruit stored in air alone for Oh and 48h. In forward SSH I regarded the fruit of bananas stored for Oh as Driver, and the fruit of bananas stored for 48h as Tester. In backward SSH the reverse material was used. Two subtracted cDNA libraries have been established with the forward and the backward -ubtracted cDNAs. The forward subtracted cDNA library contained 312 individual clones, and the backward contained 339 individual clones. 200 positive clones have been screened from the forward subtracted cDNA library by PCR. After sequenced, these cDNA clones were searched for homology in the GenBank database. The result of BLASTn and BLASTx showed that 19 cDNA clones scored to known banana genes, and 105 cDNA clones scored between 60% and 90% homology to known genes including 73 genes encoded known protein. 80 cDNA fragments showed very low or no homology to any known genes. There were two possibilities: 1. Some represented novel genes; 2. Others were located near 3' terminal of full-length cDNAs so that the homology with known genes (of other organisms) could not be detected.A series of cDNA clones including 200 SSH inserts amplified by PCR was arrayed .The cDNA microarrays were hybridized by Digoxigenin-labeled cDNA probes prepared from ds-cDNA for SMART- PCR from banana fruit stored for Oh and 48h, and images were captured using a camera using uviphoto V.10. Every cDNA microarray could be use to compare gene expression patterns independently. The microarray image showed there were 19 cDNA fragments expressed at high levels and 17 cDNA fragments expressed at low levels. Two microarray images were analyzed by ScanAlyze 2.0 software. The 2chip/0chip ratio showed the difference of gene expression between banana fruit stored for Oh and 48h. After stored for 48h , the expression of 55 cDNA fragments increased obviously, and the expression of 7cDNA fragments decreased. The copy of BR-1 clone was analysed, the BR-1 in banana genome was a singe copy gene by Southern Blotting.The research set up a way to indentify the transcription level of genes expressed in banana fruit in post-harvest early phase. Combining SSH and cDNA microarrays isolated the genes in the post-harvest banana fruit and studied the expression change. The research contributed to set up a way to isolate and indentify genes specifically expressed in the course of banana ripening, at the same time, to get novel genes. Initially studied genetics base of banana attribute forming and ripening.