RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds

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• 作者：Shiho Kurokawa (1) (2)
  Masaharu Kuroda (3)
  Mio Mejima (1)
  Rika Nakamura (4)
  Yuko Takahashi (1)
  Hiroshi Sagara (5)
  Natsumi Takeyama (1)
  Shigeru Satoh (2) (6)
  Hiroshi Kiyono (1) (7)
  Reiko Teshima (4)
  Takehiro Masumura (2) (6)
  Yoshikazu Yuki (1) (7)
  
• 关键词：Rice ; Allergen ; Localization ; MucoRice ; Mucosal vaccine
• 刊名：Plant Cell Reports
• 出版年：2014
• 出版时间：January 2014
• 年：2014
• 卷：33
• 期：1
• 页码：75-87
• 全文大小：947 KB
• 作者单位：Shiho Kurokawa (1) (2)
  Masaharu Kuroda (3)
  Mio Mejima (1)
  Rika Nakamura (4)
  Yuko Takahashi (1)
  Hiroshi Sagara (5)
  Natsumi Takeyama (1)
  Shigeru Satoh (2) (6)
  Hiroshi Kiyono (1) (7)
  Reiko Teshima (4)
  Takehiro Masumura (2) (6)
  Yoshikazu Yuki (1) (7)
  
  1. 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
  2. Laboratory of Genetic Engineering, Graduate School of Life and Environmental Science, Kyoto Prefectural University, 1-5, Nakaragi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan
  3. Crop Development Division, NARO Agricultural Research Center, 1-2-1, Inada, Joetsu-shi, Niigata, 943-0193, Japan
  4. National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan
  5. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
  6. Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Research Center, 74, Kitainayazuma, Ohji, Seika-cho, Soraku-gun, Kyoto, 619-0244, Japan
  7. International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
  
• ISSN：1432-203X

文摘

Key message 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. Abstract 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.