Characterization of the Coat Protein Gene of two Cucumber Mosaic Virus (CMC) Isolates from Plantain and Banana (Musa ssp) | Caracterizacion del gen de la proteina de la capside de dos aislamientos del virus del mosaico del pepino (CMV), obtenidos de platano y banano (Musa spp)

Spanish; Castilian. el presente estudio se adelanto dentro de un proyecto de investigacion para obtencion de plantas resistentes al virus del mosaico del pepino (CMV), mediante la transformacion y expresion del gen de la proteina de la capside de CMV en plantas transgenicas de variedades comerciales de platano y banano (Musa spp). Se trata de una estrategia reciente que ha sido utilizada con exito en varios cultivos comerciales. el CMV fue detectado serologicamente (DaS-eLiSa) en hojas de platano cv. Dominico Harton y de banano cv. Gros Michel que presentaban sintomas de mosaico. el virus se purifico parcialmente a partir de hojas infectadas de Nicotiana tabacum y con la ayuda del microscopio electronico de transmision, se identificaron particulas isometricas de aproximadamente 30 mm de diametro. el peso molecular de la proteina de la capside en geles desnaturalizados de poliacrilamida (SDS-PaGe) fue de 28 kDa. el patron electroforetico del RNa viral de doble cadena (dcRNa) fue similar para los 2 aislamientos de CMV obtenidos de platano y banano. Utilizando la dcRNa como patron, se sintetizo caDN por transcripcion reversa y, a partir de este, se amplificaron por PCR los genes de la proteina de la capside (GPC) de ambos aislamientos. Los productos de la amplificacion de aproximadamente 890 pares de bases (pb), que contenian el GPC, se clonaron en el plasmido vector Bluescript KS (+/-). el analisis de restriccion con endonucleasas y la secuenciacion del GPC de ambos aislamientos, indico que estos pertenecen al subgrupo i (tipificado por el aislamiento DTL). el analisis de la secuencia nucleotidica y de aminoacidos de ambos genes, indico una homologia del 99 por ciento y un alto grado de conservacion con otros miembros del subgrupo i de CMV. el GPC se esta clonando en plasmidos vectores para su transformacion en variedades comerciales de platano y banano en Colombia

English. The final objective of this study is to genetically engineer cucumber mosaic virus (CMV) resistance in edible Musa ssp. in Colombia, by means of expressing the CMV coat protein gene in transgenic plants. This strategy has been successfully employed in other crops. CMV was serologically detected in leaves of commercially grown bananas (cv Gros Michel) and plantains (cv. Dominico-Harton) showing mosaic symptoms. CMV was partially purified from infected tissue of Nicotiana tabacum. Transmission electron microscopy examination showed the presence of viral isometric particles of approximately 30 nm in diameter. The molecular weight of the coat protein subunit was approximately 28 kDa as determined by SDS-PAGE. Analysis of double-stranded RNA (dsRNA) indicated that the isolations present in both varieties had similar dsRNA profiles, characteristic of CMV. cDNA was reverse transcribed using viral dsRNA as the template, and the coat protein genes (CPG) of the two CMV isolates were amplified by the polymerase chain reaction (PCR). The amplified products of approximately 890 bp, containing the CPG, were cloned into the plasmid vector Bluescript KS (+/-). Based on the pattern obtained from the digestion of the amplified CP genes with Msp 1 and DNA sequencing data, the two CMV isolates were classified into the serotype subgroup 1. (typified by isolate DTL). Computer analysis of DNA sequences of the CP genes derived from both CMV isolates showed a 99 porcentage homology between them at the nucleotide and amino acid level as well as a high level of conservation with other members of the CMV subgroup I. The CPG of CMV are now being cloned into plant transformation vectors for their genetic engineering into banana and plantain commercial cultivars