Abstract
Soybean [Glycine max (L.) Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2), to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME) in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets.
Highlights
Soybeans [Glycine max (L.) Merr.] are the number one protein source for animal feed in the world, accounting for 69% of global protein consumption with poultry and swine—both monogastric animals—being the major consumers (Cromwell, 2012)
We employed two T-DNA construct pMU2T-bar-raffinose synthase 2 (RS2) in which one T-DNA carried plant selectable marker while the other T-DNA contains RS2 silencing cassette (Figure 1). This allowed the independent integration of bar and RS2 transgene cassettes and subsequent segregation of the transgenes (Xing et al, 2000)
The inverted repeat was designed for a complete sequence homology with soybean raffinose synthase 2 (RS2) gene but no significant homology with soybean raffinose synthase 3 (RS3) gene (Figure S1)
Summary
Soybeans [Glycine max (L.) Merr.] are the number one protein source for animal feed in the world, accounting for 69% of global protein consumption with poultry and swine—both monogastric animals—being the major consumers (Cromwell, 2012). One of the major limitations of soy carbohydrates is the presence of the indigestible raffinose family oligosaccharides (RFOs): raffinose, stachyose, and verbascose. These compounds are derived from sucrose, which has a positive effect on metabolizable energy, but due to their α-1,6glycosidic bond, monogastric animals are unable to digest RFOs. Oligosaccharides pass undigested through the upper gut of the animal, and are fermented by anaerobic microbes in the lower gut. Oligosaccharides pass undigested through the upper gut of the animal, and are fermented by anaerobic microbes in the lower gut This fermentation produces carbon dioxide, methane, and hydrogen, causing flatulence and digestive disturbance in the animal. It has been shown that presence of RFOs in animal diets caused the feed to pass quicker through the digestive system, reducing the amount of other nutrients absorbed from the feed (Coon et al, 1990)
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