High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing

Abstract

We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein ⌬9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine 6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications.Although cotton (Gossypium hirsutum) is primarily grown for fiber production, it is also the world's sixth largest source of vegetable oil. Cottonseed oil is typically composed of about 26% palmitic acid (C16:0), 15% oleic acid (C18:1), and 58% linoleic acid (C18:2). The relatively high level of palmitic acid provides a degree of stability to the oil that makes it suitable for high-temperature frying applications, but is nutritionally undesirable because of the low-density lipoprotein cholesterol-raising properties of this saturated fatty acid (Cox et al., 1995). Although cottonseed oil has recently been shown to lower total serum cholesterol compared with corn (Zea mays) oil (Radcliffe et al., 2001), it did so by lowering the level of the desirable high-density lipoprotein cholesterol without reducing the level of the undesirable lowdensity lipoprotein cholesterol, presumably because of its significant content of palmitic acid. Furthermore, cottonseed oil is sometimes hydrogenated to achieve the very high stability required in deepfrying food service applications or to provide the solidity required for margarine hard stock. Unfortunately, the hydrogenation process results in the production of trans-fatty acids,...

Physicochemical properties of seeds and oil from an F2 population of Jatropha curcas × Jatropha integerrima

“…For oil composition, a highly negative correlation was observed between the two major unsaturated fatty acids; oleic and linoleic acids (r = −0.97). Our finding agrees with the report of Liu et al 21 implying that there could be common genetic factors affecting these two compositions. Although a high level of oleic acid suppresses the level of linoleic acid, developing high oleic genotypes is desirable for physic nut oil as a raw material for biodiesel.…”

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hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

“…First, such high-level silencing enables down-regulation of endogenous genes to be attempted in difficult species that have relatively low-transformation rates. For example, we have already used the efficiency of hpRNA constructs to separately down-regulate both ⌬9-desaturase and ⌬12-desaturase in cotton, a poorly transformable species, to produce novel high-stearic and high-oleic cottonseed oils (Liu et al, 2002). Second, by reducing the population size needed to recover a good silencing transformation, the use of ihpRNA constructs will facilitate the removal by segregation of selectable marker genes that have been inserted at separate loci to the silencing transgene, such as by using twin T-DNA vectors (Matthews et al, 2001).…”

Biofuels Get in the Fast Lane: Developments in Plant Feedstock Production and Processing

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