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ISHS Acta Horticulturae 877: VI International Postharvest Symposium

A UNIVERSAL PRIMER FOR ISOLATION OF FRAGMENTS OF A GENE ENCODING PHYTOENE DESATURASE FOR USE IN VIRUS-INDUCED GENE SILENCING (VIGS) STUDIES

Authors:   L. Yin-Chih, W. Imsabai, M.S. Reid, C. Wagstaff, J. Cai-Zhong
Keywords:   PDS (phytoene desaturase), VIGS (virus-induced gene silencing), TRV (tobacco rattle virus), PCR
Abstract:
We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.
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