Production of Hypoallergenic Rice by Gene Engineering

通过基因工程生产低过敏性水稻

• 批准号: 04556012
• 负责人: NAKAMURA Ryo
• 金额: $ 2.82万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04556012/
• 关键词: Food Allergy; Rice Allergenic Protein; Antisense RNA; Transgenic Rice Plants; アレルゲン蛋白質; 低アレルゲン米; 転写調節因子; アンチセンスRNA

Several studies on allergic reaction to cereal grains have suggested that sera from some patients with atopic food allergy contain immunoglobulin E (IgE) reacting with rice proteins, and that soluble proteins, albumins and globulins have a high degree of allergenic activity. We isolated a rice seed protein of about 16kDa with reactivity for IgE in rice-allergic patients, and the 16kDa-protein was later identified as one of the major allergenic in rice seed.A cDNA clone (RA17) encoding the rice major allergenic protei (RAP) was isolated from cDNA library of maturing rice seeds. An antisence RNA strategy was applied to repress RAP gene expression in maturing rice seeds. Some chimeric genes encoding antisence RNAs of RAP under the control of CaMV 35S promoter, rice prolamin promoter and RAP promoter were introduced into rice by electroporation. Protophasts isolated from rice suspension cells were co-transformed with each antisence gene and a hygromycin phosphotransferase (HPT) gene linked to the CaMV 35S promoter, and rice plants were independently regenerated from hygromysin-resistant calli. Total DNA isolated from leaves of the regenerated plants was digested with some restriction enzymes, and Southern blot analyzes were done using an RAP cDNA as a probe. In several clones, an additional band with an expected size of the antisence gene was observed. Selfed seeds from these transgenic plants with the antisence gene were tested for RAP content by SDS-PAGE/immunoblotting and ELISA using a monoclonal antibody specific for RAP.The RAP bands stained with the antibody of seeds from some transgenic plants were much weaker than those of the wild-type control. ELISA analysis for proteins extracted from the trangenic and wild-type rice seeds suggested that RAP content of rice seeds was reduced to about 30% of the original one. Studies on stable transmission and expression of the introduced antisence gene among the transformant progeny are in progress.

对谷物过敏反应的几项研究表明，某些特应性食物过敏患者的血清中含有与大米蛋白反应的免疫球蛋白E（IgE），可溶性蛋白、白蛋白和球蛋白具有高度的致敏活性。从水稻种子中分离到一个分子量约为16 kDa的蛋白，该蛋白是水稻过敏患者的主要过敏原之一，从成熟水稻种子cDNA文库中克隆了编码水稻主要过敏原蛋白（RAP）的cDNA克隆RA 17。利用反义RNA技术抑制水稻成熟种子中RAP基因的表达。利用电转化法将分别由CaMV 35 S启动子、水稻醇溶蛋白启动子和RAP启动子控制的RAP反义RNA嵌合基因导入水稻。从水稻悬浮细胞中分离的原生质体与每个反义基因和连接到CaMV 35 S启动子的潮霉素磷酸转移酶（HPT）基因共转化，并且水稻植株独立地从潮霉素抗性愈伤组织再生。从再生植株的叶片中分离的总DNA用一些限制性内切酶消化，并使用RAP cDNA作为探针进行Southern印迹分析。在几个克隆中，观察到具有预期大小的反义基因的额外条带。用SDS-PAGE/免疫印迹和ELISA方法检测了转反义基因植株自交种子中RAP的含量，结果表明，部分转基因植株种子中RAP的抗体染色条带明显弱于野生型对照。对转基因水稻和野生型水稻种子提取的蛋白质进行ELISA分析表明，水稻种子中RAP含量降低到原始含量的30%左右。导入的反义基因在转基因后代中的稳定传递和表达研究正在进行中。

项目成果

Izumi,H.: "Nucleotide sequence of a cDNA encoding a major aller-genic protein in rice seeds.Homology of the deduced amino acid sequence with members of α-amylase/trypsin inhibitor family." FEBS Lett.302. 213-216 (1992)

Izumi, H.：“编码水稻种子中主要致敏蛋白的 cDNA 的核苷酸序列。推导的氨基酸序列与 α-淀粉酶/胰蛋白酶抑制剂家族成员的同源性。FEBS Lett.302。” 1992）

A.Urisu,et al.: "Rice Protein 16kD-A Major Allergen in Rice Grain Extract." Jpn.J.Allergol.40. 1370-1376 (1991)

A.Urisu 等人：“大米蛋白 16kD-大米提取物中的主要过敏原。”

T. Matsuda, et al.: "Immunochemical Studies on Rice Allergenic Proteins." Agric. Biol. Chem.55. 509-513 (1991)

T. Matsuda 等人：“大米过敏蛋白的免疫化学研究”。

H.Izumi et al.: "Nucleotide Sequence of a cDNA Clone Ecoding A Major Allergenic Protein in Rice Seeds, Homology of the Deduced Amino Acid Sequence with Members of alpha-Amylase/Trypsin Inhibitor Family." FEBS Lett.302. 213-216 (1992)

H.Izumi 等人：“编码水稻种子中主要过敏蛋白的 cDNA 克隆的核苷酸序列，推导的氨基酸序列与 α-淀粉酶/胰蛋白酶抑制剂家族成员的同源性。”

T.Adachi et al.: "Postharvest Biochemistry of Plant Food-Materials in the Tropics." Japan Scientific Societies Press, 15 (1994)

T.Adachi 等人：“热带植物食品材料的采后生物化学”。