Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
Plant Cell Rep. 2014 Jan;33(1):75-87. doi: 10.1007/s00299-013-1513-3. Epub 2013 Oct 2.
RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.
RNAi 介导的 MucoRice-CTB-RNAi 种子中内源储存蛋白的抑制不仅影响了过表达 CTB 和 RAG2 过敏原的水平，还影响了 CTB 和 RAG2 的定位。我们实验室先前使用霍乱毒素 B 亚单位（CTB）过表达系统开发了一种基于纯化水稻的口服霍乱疫苗（MucoRice-CTB）。最近，通过使用 RNAi 抑制内源储存蛋白 13-kDa 亲油蛋白和谷蛋白的产生，开发了 MucoRice-CTB-RNAi 的高级版本，以增加 CTB 的表达。与野生型（WT）水稻相比，MucoRice-CTB-RNAi 种子中 RAG2（一种主要的水稻过敏原）的α-淀粉酶/胰蛋白酶抑制剂样蛋白水平降低。为了研究储存蛋白的 RNAi 介导抑制是否影响过表达 CTB 和主要水稻过敏原的定位，我们生成了一种不含 CTB 的 RNAi 系（MucoRice-RNAi），并研究了 MucoRice-CTB、MucoRice-CTB-RNAi、MucoRice-RNAi 和 WT 水稻中两种储存蛋白、CTB 和五种主要过敏原的基因表达、蛋白产生和定位。在所有系中，均检测到了乙醛酸酶 I 在细胞质中的存在，并且在糊粉粒中发现了 52-和 63-kDa 球蛋白样蛋白。在 WT 中，RAG2 和 19-kDa 球蛋白主要定位于胚乳细胞的 II 型蛋白体（PB-II）中。谷蛋白 A 的敲低导致 PB-II 的部分破坏，并伴随着 RAG2 向质膜/细胞壁和细胞质的重新定位。在 MucoRice-CTB 中，CTB 定位于细胞质和 PB-II 中。在 MucoRice-CTB-RNAi 中，CTB 的产量是 MucoRice-CTB 的六倍，并且与 RAG2 相似，定位于质膜/细胞壁和细胞质中。我们的研究结果表明，MucoRice-CTB-RNAi 中 CTB 的重定位可能导致 RAG2 的下调。
