Delivery of foreign proteins into insects

• Project/Area Number: 15380043
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied entomology
• Research Institution: Kyoto Institute of Technology
• Principal Investigator: SUGIMURA Yukio Kyoto Institute of Technology, Graduate School of Science and Technology, Professor, 工芸科学研究科, 教授 (20273542)
• Co-Investigator(Kenkyū-buntansha): FURSAWA Toshiharu Kyoto Institute of Technology, Faculty of Textile Science, Professor, 繊維学部, 教授 (70127166) ; NAGAOKA Sumiharu Kyoto Institute of Technology, Faculty of Textile Science, Research Associate, 繊維学部, 助手 (00303933) ; KITAJIMA Sakihito Kyoto Institute of Technology, Graduate School of Science and Technology, Research Associate, 工芸科学研究科, 助手 (70283653) ; HIRAYAMA Chikara National Institute of Agrobiological Sciences, Chief Researcher, 主任研究官 (90370676)
• Project Period (FY): 2003 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥14,700,000 (Direct Cost: ¥14,700,000); Fiscal Year 2005: ¥3,200,000 (Direct Cost: ¥3,200,000); Fiscal Year 2004: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2003: ¥8,000,000 (Direct Cost: ¥8,000,000)
• Keywords: mulberry plant / silkworm / urease gene / uptake of host protein / transgenic mulberry / ウレアーゼの遺伝子発現 / クワ葉タンパクの体内取り組み / トランスポーター / 発現ベクター / ウレアーゼ / タンパクの体内取り込み / 免疫電顕

Research Abstract

1. Selective absorption of mulberry urease to the hemolymph of the silkworm : When urease extracted from mulberry roots was applied to the 6^<th> -the 8^<th> day larvae of the 5^<th> instar stage, more than 8% of total injection amount were uptaked into larvae The uptake efficiency was very low in other ureases derived from different organisms. Immunocytological observation shown that urease entry take place via microvilli surface of midgut cells.
2. Identification of mulberry urease gene : Full-length urease cDNA was isolated and the complete nucleotide sequence was determined, and partially defective urease as well. Deducted amino acid sequence (837 residues) from full-length cDNA had a high similarity with those of other plant ureases. The ure G gene coding urease accessory protein G was also isolated and sequenced partially.
3. Gene expression of urease in mulberry seedlings : High urease activity was detected in roots, whereas cotyledons and hypocotyls had no or little activity of urease. In contract, the expression of urease gene was detected in all of the organs. This suggests that additional gene products including ure G protein are involved in maturation of urease protein.
4. Comprehensive detection of protein species uptaked into silkworms : Biotinylated mulberry leaf proteins were applied to the silkworm larvae. The profile by two dimensional electrophoresis shown that 15 biotinylated proteins were uptaked to the larval hemolymph.
5. Experimental system for generating transgenic mulberry plants : A vector plasmid encoding GUS and HTP genes was constructed to transform mulberry plants by a chimeric gene composed of urease gene and foreign gene.

Research Products

• [Journal Article] Purification and properties of two types of soluble trehalases from embryonic larvae of the silkworm, Bombyx mori. (2006)
  • Author(s): Huang, J.et al.
  • Journal Title: J.Insect Biotech.Sericology 75 Pages: 1-8
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Calcium carbonate deposition in a cell wall formed in mulberry idioblasts (2006)
  • Author(s): Nitta, I.et al.
  • Journal Title: Protoplasma (In press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts. (2006)
  • Author(s): Nitta, I
  • Journal Title: Protoplasma (In press)
• [Journal Article] Purification and properties of two types of soluble trehalases from embryonic larvae of the silkworm, Bombyx mori. (2006)
  • Author(s): Huang, J
  • Journal Title: J.Insect Biotech.Sericology 75 Pages: 1-8
• [Journal Article] Gene expression and localization of trehalase during embryonic development of the silkworm, Bombyx mori (2005)
  • Author(s): Huang, J.et al.
  • Journal Title: Int.J.Wild Silkmoth & Silk 10 Pages: 17-26
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Chemically retted kenaf fibers. (2005)
  • Author(s): Kawahara, Y.et al.
  • Journal Title: 繊維学会誌 61 Pages: 115-117
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori (2005)
  • Author(s): Kurahashi, H.et al.
  • Journal Title: J.Insect Physiol. 51 Pages: 1055-1061
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Sequence variation in the rbcL-accD region in the chloroplast genome of Moraceae (2005)
  • Author(s): Matsuda, Y.
  • Journal Title: Plant Biotech. 22 Pages: 231-233
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Transgenic patchouli plants produced by Agrobacterium-mediated transformation (2005)
  • Author(s): Sugimura, Y.et al.
  • Journal Title: Plant Cell, Tissue and Organ Culture 82 Pages: 251-257
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Gene expression and localization of trehalase during embryonic development of the silkworm, Bombyx mori. (2005)
  • Author(s): Huang, J., Arimatsu, Y., Sadakane, K., Endo, Y., Furusawa, T.
  • Journal Title: Int.J.Wild Silkmoth & Silk 10 Pages: 17-26
  • NAID: 10026887548
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Chemically retted kenaf fibers. (2005)
  • Author(s): Kawahara, Y., Tadokoro, K., Endo, R., Shioya, M., Sugimura, Y., Furusawa, T.
  • Journal Title: Sen'i Gakkaishi 61 Pages: 115-117
  • NAID: 10015507088
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori. (2005)
  • Author(s): Kurahashi, H., Atiwetin, P., Nagaoka, S., Miyata, S., Kitajima, S., Sugimura, Y.
  • Journal Title: J.Insect Physiol. 51 Pages: 1055-1061
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Sequence variation in the rbcL-accD region in the chloroplast genome of Moraceae (2005)
  • Author(s): Matsuda, Y., Yoshimura, H., Kanamoto, H., Ujihara, T., Tomizawa, K-I., Sugimura, Y., Kitajima, S.
  • Journal Title: Plant Biotech. 22 Pages: 231-233
  • NAID: 10028053750
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Transgenic patchouli plants produced by Agrobacterium-mediated transformation. (2005)
  • Author(s): Sugimura, Y., Kadotani, N., Ueda, Y., Shima, K., Kitajima, S., Furusawa, T., Ikegami, M.
  • Journal Title: Plant Cell, Tissue and Organ Culture 82 Pages: 251-257
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Purification and properties of two types of soluble trehalases from embryonic larvae of the silkworm, Bombyx mori. (2005)
  • Author(s): Huang, J., Furusawa, T., Sadakane, K., Sugimura, Y.
  • Journal Title: J.Insect Biotech.Sericology 75 Pages: 1-8
  • NAID: 10017316069
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Sequence variation in the rbcL-accD region in the chloroplast genome of Moraceae. (2005)
  • Author(s): Matsuda, Y.
  • Journal Title: Plant Biotechnology 22 Pages: 231-233
• [Journal Article] Chemically retted kenaf fibers. (2005)
  • Author(s): Kawahara
  • Journal Title: Sen'i Gakkaishi 61 Pages: 115-117
• [Journal Article] Gene expression and localization of trehalase during embryonic development of the silkworm, Bombyx mori. (2005)
  • Author(s): Huang, J.
  • Journal Title: Int.J.Wild Silkmoth & Silk 10 Pages: 17-26
• [Journal Article] Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori. (2005)
  • Author(s): Kurahashi, H.
  • Journal Title: J.Insect Physiology 51 Pages: 1055-1061
• [Journal Article] Transgenic patchouli plants produced by Agrobacterium-mediated transformation. (2005)
  • Author(s): Sugimura, Y.
  • Journal Title: Plant Cell, Tissue and Organ Culture 82 Pages: 251-257
• [Journal Article] Transgenic patchouli plants produced by Agrobacterium-mediated Transformatiion. (2005)
  • Author(s): Sugimura, Y.
  • Journal Title: Plant Cell, Tissue, Organ Culture (印刷中)
• [Journal Article] Chemically retted kenaf fibers (2005)
  • Author(s): Kawahara, Y.
  • Journal Title: 繊維学会誌 (印刷中)
• [Journal Article] Whole-mount silver staining of Arabidopsis and maize : A new method for staining secondary wall thickening in tracheary elements (2004)
  • Author(s): Kitajima, S.et al.
  • Journal Title: Biosci.Biotech.Biochem. 68 Pages: 2627-2629
  • NAID: 130000030897
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Induction of genomic DNA fragmentation by introduction of the BmTRN-1 RNA- binding domain into cultured cells of the silkworm, Bombyx mori. (2004)
  • Author(s): Ohba, T.et al.
  • Journal Title: J.Insect Biotech.Sericology 73 Pages: 15-22
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] New and highly efficient method for silkworm transgenesis using Autographa caifornia nucleopolyhedrovirus and piggyBac transposable elements (2004)
  • Author(s): Yamamoto, M.et al.
  • Journal Title: Biotechnol Bioeng. 30 Pages: 849-853
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] クワ葉細胞への外来遺伝子の導入と発現 (2004)
  • Author(s): 大矢根未来 ら
  • Journal Title: 繊維研究所研究報告 8 Pages: 1-6
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Metabolic shifts in carbohydrate metabolism during embryonic development of non-diapause eggs of the silkworm, Bombyx mori. (2004)
  • Author(s): Sakano, D.et al.
  • Journal Title: J.Insect Biotech.Sericology 73 Pages: 15-22
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Whole-mount silver staining of Arabidopsis and maize : A new wthod for staining secondary wall thickening in tracheary elements. (2004)
  • Author(s): Kitajima, S., Hasegawa, Y., Sugimura, Y.
  • Journal Title: Biosci.Biotech.Biochem. 68 Pages: 2627-2629
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Induction of genomic DNA fragmentation by introduction of the BmTRN-1 RNA- binding domain into cultured cells of the silkworm, Bombyx mori. (2004)
  • Author(s): Ohba, T., Saito, H., Sugimura, Y., Furusawa, T., Hayashi, Y., Kotani, E.
  • Journal Title: J.Insect Biotech.Sericology 73 Pages: 15-22
  • NAID: 10012935477
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements. (2004)
  • Author(s): Yamamoto, M., Yamao, M., Nishiyama, H., Sugihara, S., Nagaoka, S., et al.
  • Journal Title: Biotechnol Bioeng. 30 Pages: 849-853
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression of foreign. genes introduced to mulberry leaf cells. (2004)
  • Author(s): Ohyane, M., Kitajima, S., Katayama, H., Furusawa, T., Sugimura, Y.
  • Journal Title: Bull.Kinugasa Text.Res.Inst. 8 Pages: 1-6
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Metabolic shifts in carbohydrate metabolism during embryonic development of non-diapause eggs of the silkworm, Bombyx mori. (2004)
  • Author(s): Sakano, D., Furusawa, T., Sugimura, Y., Storey, J.M., Storey, K.B.
  • Journal Title: J.Insect Biotech.Sericology 73 Pages: 15-22
  • NAID: 10012935343
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Whole-mount silver staining of Arabidopsis and Maize : A new method for staining secondary wall thickening tracheary elements. (2004)
  • Author(s): Kitajima, S.
  • Journal Title: Biosci.Biotechnol.Biochem. 68 Pages: 2627-2629
  • NAID: 130000030897
• [Journal Article] New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements. (2004)
  • Author(s): Yamamoto, M.
  • Journal Title: Biotechnol, Bioeng. 88 Pages: 849-853
• [Journal Article] De novo gene expression and antisense inhibition in cultured cells of RmTRN-1, cloned from the midgut of the silkworm, Bombyx mori, which is homologous with mammalian TIA-1/R (2003)
  • Author(s): Kotani, E.et al.
  • Journal Title: Gene 320 Pages: 67-79
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] De novo gene expression and antisense inhibition in cultured cells of RmTRN-1, cloned from the midgut of the silkworm, Bombyx mori, which is homologous with mammalian TIA-1/R. (2003)
  • Author(s): Kotani, E., Ohba, T., Niwa, T., Storey, K.B., Storey, J.S., Hara, S., Saito, H., Sugimura, Y., Furusawa, T.
  • Journal Title: Gene 320 Pages: 67-79
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts.
  • Author(s): Nitta, I., Kida, A., Fujibayashi, Y., Katayama, H., Sugimura, Y.
  • Journal Title: Protoplasma (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] ケンブリッジ世界の食物史大百科事典2-主要食物:栽培植物と飼養動物- (2005)
  • Author(s): 杉村順夫
  • Publisher: 朝倉書店
  • Description: 「研究成果報告書概要(和文)」より
• [Book] 世界の食物史大百科事典1 (2004)
  • Author(s): 石毛直道
  • Total Pages: 500
  • Publisher: 朝倉書店
• [Publications] Kotani, E.: "De novo expression and antisense inhibition in cultured cells of BmTRN-1, cloned from the mudgut of the silkworm, Bombyx mori"Gene. 320. 67-79 (2003)
• [Publications] Konno, K.: "Papain protects papaya trees from herbivorous insects : role of cysteine proteases in latex"The Plant J.. 37. 370-378 (2004)