Abstract
The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.
Highlights
RNA interference (RNAi) is a recent technique that is used to knock-out or knock-down targeted gene expression
The target site for the RNAi constructs development was aimed at the 3′untranslated regions (UTR) regions of ACC oxidase 1 (ACO1) and ACC oxidase 2 (ACO2), respectively
Many studies reported the use of the 3′UTR region of the gene of interest to generate dsRNA [8,9,10] because it is more specific and effective, and is believed to affect mRNA stability and translational regulation [11]
Summary
RNA interference (RNAi) is a recent technique that is used to knock-out or knock-down targeted gene expression. Discovered the introduction of a pigment-producing gene under the control of the cauliflower mosaic virus 35S promoter suppressed expression of both the introduced gene and its homologous endogenous gene Among the major papaya varieties produced in Malaysia for export is Eksotika papaya, which resulted from a cross between Subang 6 and the Hawaian Sunrise Solo It was released by the Malaysian Agricultural and Research Development Institute (MARDI) in 1987 [6]. Manipulation of the gene(s) involved in papaya fruit ripening was targeted in order to improve the shelf-life of future harvests of Eksotika papaya to more than 4 keeping days as exhibited by a normal Eksotika papaya
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