Cloning of antisense ACC [1-amino-cyclopropane-1-carboxylic acid] oxidase gene from papaya Philippine cultivar 'Davao Solo' for delayed ripening
2004
Perez, M.T.M., Philippines Univ. Los Banos, College, Laguna (Philippines). National Inst. of Molecular Biology and Biotechnology
The partial fragment of ACCX oxidase gene was amplified by primers VF01 and VF02 in the RT-PCR [reverse transcription-polymerase chain reaction] assay using as templates total RNA isolated from papaya Philippine cultivar Davao Solo. The 800 bp fragment generated was cloned in PCR2.1-TOPO vector resulting to putative clones four of which were submitted for DNA sequencing. Two of the clones designated as pDvSI-15 and pDvSI-12 were further analyzed for homology, restriction enzyme sites and insert orientation. The ACC oxidase geneis pDvSI-15 had 98-9 approx/0 homology with Carica papaya ACC oxidase mRNA reported by Neupane, K.R., Mukatira, U.T. and Stiles, J.I. whereas that in pDvSI-12 had 97% homology with Carica papaya ACC oxidase mRNA reported by Chi-Tsai, L. and Ming-Tse, L. Based on information on restriction sites and orientation, the strategies for making the antisense constructs were identified. For pDvSI-15 which was verified to contain the desired fragment in the antisense orientation, Xha I and EcoRV were used to release the fragment which was then ligated into the expression vector pGA643 digested with XhaJ and HpaJ resulting to a construct designated with BamHI and XhaJ, and ligated into pGA03 7. Initial restriction analysis indicate the antisense orientation of the fragment in pGA643 in both clones. The sizes of pGA01 and pGA037 were estimated to be 12.45 and 12.37 kb, respectively. The difference in the sizes of the two constructs was due to the presence of a DNA segment from PCR2.ITOPO vector in pGA01.
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