Coat protein-mediated resistance as an approach for controlling an Egyptian isolate of Cucumber mosaic virus (subgroup I)
2008
El-Borollosy, Ali | Mahmoud, Sabry | Khaled, Abdel-Sabour
Cucumber mosaic virus (CMV, cucumovirus) is the most important virus infecting cucurbit crops in Egypt and worldwide causing significant loss in yield quality and quantity. The main target of the present work was to establish a simple controlling system for an Egyptian isolate of such virus (belonging to the subgroup I) via production of tobacco transgenic plants expressing viral coat protein (CP). Coat protein gene (cp) was isolated and amplified using immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) and primers with add-on restriction sites for SmaI and SacI enzymes. The genes were cloned in pBI121 vector plasmid between the CaMV 35S promoter and the nos terminator after removing the Gus gene by restriction enzymes digestion. The new construct was used for Agrobacterium tumefaciens transformation, which was then used for tobacco transformation. Evaluation of transformation success and CP expression degree were confirmed using indirect enzyme-linked immunosorbent assay (I-ELISA) and dot blot immuno-binding assay (DBIA). PCR and RT-PCR were used to study the integration of cp within genetic plant system and to what extent this gene was transcript. It was concluded that in spite of integration success some transformed plants can transcript the gene more than the others do. Plants resistance was tested by challenging with CMV under study and remarkable success was obtained in plants with higher gene transcription and translation degree.
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