Application of RNA interference to Tomato ACC oxidase gene silencing for prelongation of fruit shelf life
2009
Timerbaev, V.R. | Dolgov, S.V., All-Russia Research and Development Inst. of Agricultural Biotechnology, Moscow (Russian Federation)
In order to increase the shelf-life of biotechnological tcmato fruits, binary vectors that trigger the process of RNA interferention have been constructed. A tomato gene for ACC oxidase, a key enzyme of ethylene biosynthesis, was selected as target. The gene construction was based on a full-size sequence of the 4 exon of the 496-bp gene LeACO1. The effect of orientation of the repeats on the efficiency of RNA iterferention was investigated. To this end, two possible expression cassettes with opposite relative positions of the repeats, antisense-sense and sense-antisense were designed. As a result of agrobacterial transformation of the line Yalf tomato, 28 and 19 lines with each of the above orientation, respectively, were obtained. The insertion of a selective gene for kanamycin resistance was confirmed in all lines by PCR, whereas the occurrence of the target insertion was determined in 27 and 18 lines, respectively. A significant delay in fruit ripening in the majority of the analyzed lines with antisense-sense DNA-fragment orientation was observed; the fruits harvested at the blange stage retained good quality during 3-4 months of storage. The fruits with the opposite orientation had the same maturation characteristics as in the control plants. We also observed the dynamics of the development of fruit color during storage under controlled conditions for 140 days.
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