Development of transgenic rice (Oryza sativa L.) expressing wheat high-and low-molecular-weight glutenin subunit proteins

Abstract

We isolated transformation-competent artificial chromosome (TAC) clones harboring a high-molecularweight glutenin subunit (HMW-GS) gene and a low-molecular-weight GS (LMW-GS) gene from wheat genomic DNA, and developed transgenic rice lines with these genes using a transformation system mediated by Agrobacterium tumefaciens. We developed two lines of transgenic rice, one expressing the gene coding for HMW-GS and the other expressing the gene coding for LMW-GS. By crossing these transgenic lines, a novel line harboring both genes was developed and the expression of GS proteins was analyzed. This is the first study indicating that the two kinds of wheat GSs, HMW-GS and LMW-GS, accumulated in rice endosperm. In all the transgenic lines, the introduced GS genes were expressed in the rice endosperm, and the expressed proteins were processed at the same site as the mature GS protein in wheat seeds, forming insoluble polymeric proteins similar to those found in wheat. It was suggested that the protein-processing system was conserved between rice and wheat, and that it was possible to produce wheat gluten using the proteinprocessing system of rice.

Development of Marker-free Transgenic Rice Expressing the Wheat Storage Protein, Glu-1Dy10, for Increasing Quality Processing of Bread and Noodles

“…Protein extraction and Western blot T1 generation seeds were frozen in liquid nitrogen and then ground to powder using a mortar and pestle. Total storage proteins in the rice endosperm were extracted with 50mM Tris-HCl (pH 8.0) containing 2% SDS, 50% of 1-propanol and 1% of dithiothreitol, as described [4]. Amount of extracted total proteins was measured by Nanodrop Spectrophotometer (ND-1000, Thermo Fischer Scientific, Wilmington DE, USA).…”

Genetic Transformation of a High Molecular Weight Glutenin (Glu-1Dx5) to Rice cv. Fatmawati

“…Total storage proteins in the rice endosperm were extracted with 50mM Tris-HCl (pH 8.0) containing 2% of SDS, 50% of 1-propanol and 1% of dithiothreitol, as described by Araki et al 2008. Proteins were precipitated in cold 80% acetone.…”

“…The protein detected in our transgenic T 2 endosperm was similar in size to the wheat Glu-1Dx5 product detected in transformed maize plants (Sangtong, 2002). Araki et al (2008) suggested that the protein-processing system was conserved in both rice and wheat, and that it was possible to produce wheat glutenin using the proteinprocessing system of rice. The gene we transferred to rice was slightly different from that reported by Araki et al (2008), since we used 8.2 kb of the genomic DNA of Glu-1Dx5 that includes the native promoter of the Glu1Dx5 gene.…”

“…Araki et al (2008) suggested that the protein-processing system was conserved in both rice and wheat, and that it was possible to produce wheat glutenin using the proteinprocessing system of rice. The gene we transferred to rice was slightly different from that reported by Araki et al (2008), since we used 8.2 kb of the genomic DNA of Glu-1Dx5 that includes the native promoter of the Glu1Dx5 gene. The present results showed the expected activity of the wheat promoter for Glu-1Dx5 gene in rice endosperm, although endosperm specificity was not clear.…”

“…Therefore, transfer of this HMW glutenin especially Glu-1Dx5 gene into rice plants is expected to improve rice flour quality traits. Recently, transgenic rice lines with a HMW glutenin gene from wheat have been produced by the Agrobacterium method (Araki et al, 2008). Particle bonbardment is another 【Short Report】 genetic transformation method.…”