| Bananas are the best-characterized climacteric fruits. A large body of evidence has demonstrated that ethylene is the hormone of catalyzing climacteric fruit ripening. After the starting of ethylene biosynthesis in fruits, ethylene production increased fast. Ethylene was perceived by its receptor and Ethylene signal was transferred to target genes that related to fruit ripening, resulting in fruit ripening. According to these, three major strategies are explored to delay climacteric fruit ripening: 1) limiting / reducing ethylene biosynthesis in fruits, 2) decreasing the sensitivity of fruit to ethylene, and 3) blocking ethylene signal transduction pathways between ethylene and its downstream target genesCloning of DNA related to ACS / ACO were obtained firstly in tomato fruits and then demonstrated that they encoded ACS / ACO protein, which catalyzed formation of ACC / ethylene, respectively. So far, modulation of ACS activity has proved to be a potential approach.Genes of ethylene receptor were cloned and characterized firstly in Arabidopsis Thaliana. Then five cDNAs of putative ethylene receptor gene were obtained using the homology with the ethylene receptor genes of Arabidopsis Thaliana from tomato. NR was the first gene that confirmed to be a ethylene receptor gene from fruit. Mutant harboring mutation in NR gene displayed ethylene insensitivity due to loss of ethylene binding activity by the nutant receptor, resulting in "never ripening" fruits in natural conditions.A cDNA of putative ACS gene was obtained from banana fruits in our laboratory and nowstudy on its functional identification is undergoing by using the anti-sense RNA. Cloning of putative ethylene receptor gene was reported, but identification and characterization of its expression and function has not been documented so far.RNAi, which was discovered in 1998 by Andrew Fire et al, turned out to be a mechanism of regulation of gene expression on post-transcription level in many organisms ranged from prokaryotic organisms to plants and mammals. RNAi has been used as powerful tool for functional identification of genes because of its nature that shuts down sequence-specific gene expression and that exists in organism widely.Functional identification of obtained cDNA is fundaments of applying them to genetic engineering. Forasmuch the genetic regeneration accepter system of banana for gene transform is not yet established to date , it is difficult that identifies the function of the published cDNA of putative ACS gene and ethylene receptor gene by using anti-sense RNA technology.Here , we has cloned the cDNA of putative ethylene receptor gene ORF sequence, and then analyzed its differential expression. In addition, used dsRNA-mediated RNAi methodology to further explore functional analysis of the cloned banana cDNA. At meantime, siRNA assays for published banana ACS cDNA were also conducted.Results and conclusions:1. Two cDNA fragments corresponding to ethylene receptor gene ORF were obtained by RT-PCR using total RNA isolated from banana fruits as a template. One cDNA clone showed 99% of homology to the ORF sequence of a published putative ethylene receptor gene, and it had the same size as the reported gene. The other one displayed 97% of identity with the published sequences but with a missing region corresponding to nucleotide 193 to 1036. Such a truncated form ethylene receptor cDNA was likely generated through alternative splicing, therefore may represent a novel form of ethylene receptor gene in banana.2. Southern blot analyses using probe derived from the larger clone revealed an identical single band after hybridization with genomic DNAs isolated from two wild species and two varieties of bananas, indicating that the cloned cDNA exists widely in banana genomes; RT-PCR analyses were further carried out to delineate the expression profile of the cloned ethylene receptor cDNA. The results have demonstrated that its expression was prominent atexpression pattern of the cloned ethylene receptor c... |
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