Cellular Localization of Wheat High Molecular Weight Glutenin Subunits in Transgenic Rice Grain.

Yeong-Min Jo,Jong-Yeol Lee,Kyoungwon Cho,Sun-Hyung Lim,Young-Mi Kim,Hye-Jung Lee,Jin Kim

doi:10.3390/ijms18112458

Abstract

Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T3 generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice.

Highlights

Seed storage proteins (SSPs) in cereal grains serve as a source of nitrogen during germination and play important roles in food processing [1]
Wheat glutenins are synthesized in the endoplasmic reticulum (ER), which are assembled via ER chaperones, and transported to protein storage vacuoles (PSVs) either directly or via the Golgi apparatus [6,7,8]
The extracted total SSPs (10 μg) and gliadin fraction were all separated on 12.5% SDS-PAGE gels, stained using Coomassie brilliant blue (CBB) staining solution (0.1% (w/v) CBB R-250, 45% (v/v) methanol, and 45% (v/v) glacial acetic acid) for 3 h, and destained in 10% methanol, 10% glacial acetic acid, and 80% distilled H2O

Summary

Introduction

Seed storage proteins (SSPs) in cereal grains serve as a source of nitrogen during germination and play important roles in food processing [1]. Glutelins account for 60–80% of total SSPs, whereas the major SSPs in other cereals are prolamins, including hordeins in barley, secalins in rye, avenins in oats, and gluten proteins that are composed of gliadin and glutenin subunits in wheat [2] These prolamins are responsible for the elastic properties of dough made from these cereals. Recent studies have shown that allelic forms of HMW-GSs that are present in bread wheat cultivars contribute towards bread-making quality, and they are assigned a quality score from one to five that facilitates the prediction of a cultivar’s bread-making suitability Based on these quality scores, the 1Dx5 glutenin subunit has a large positive effect on wheat dough elasticity [2,5]. Wheat glutenins are synthesized in the endoplasmic reticulum (ER), which are assembled via ER chaperones, and transported to protein storage vacuoles (PSVs) either directly or via the Golgi apparatus [6,7,8]

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