The Construction of Halo-Tolerant Higher Plants which were Transformed by Marine Alga Na^+-ATPase gene.

海藻Na + -ATP酶基因转化耐光高等植物的构建。

• 批准号: 01400001
• 负责人: FUJII Tadashi
• 金额: $ 10.62万
• 依托单位: Tsukuba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01400001/
• 关键词: Raphidophyceae; Na^+-ATPase; halo-tolerant; ラフィド藻; イオン輸送; Heterosigma akashiwo; Na^<++>-activated ATPase; PCR(ポリメラ-ゼ・チェィン・リアクション); cDNA

The ability which can export intracellular sodium ions can be transformed usual higher plants to halotolerant. The Na^+ K^+-ATPase of animal cells or Na^+-ATPase of raphidophycean H. akashiwo was able to transport Na ions. The Na^+-ATPase of H. akasiwo was consequently proper enzyme for the transformation. A DNA between conserved parts of eucaryotic ATPase was amplified from H. akasiwo cDNA library with PCR method. The Na^+-ATPase of H. akashiwo was cloned with the amplified DNA as a probe. Two kinds of ATPase molecules was cloned consequently. The amino acid sequences and the secondary structures analysis showed the homology to other species' ATPases(70% : protozoan L. donovani, 40-50% : higer plants and fungi, 20-30% : higher animals). This suggests that the ATPase amino acid sequences tend to conserve among systematical close to species rather than the functional characters (ion selectivity, etc). Rescreeing was needed to the reason why the two cDNA clones were short of complete coding regions. The most expressing cell period of the genes was determined with northern hybridization and 5' end probe was successed to get the enough length cDNA (3.4kb).

植物细胞内钠离子的输出能力可以使普通高等植物转化为耐盐植物。动物细胞的Na^+-K^+-ATP酶或针孢藻H. Akashiwo能够运输Na离子。H.因此，Akasiwo是用于转化的合适酶。从H.用PCR方法扩增akasiwo cDNA文库。H.以扩增的DNA为探针克隆了赤实。同时克隆了两种ATP酶分子。氨基酸序列和二级结构分析表明，该蛋白与其它物种的ATP酶同源性为70%（原生动物L. donovani，40-50%：高等植物和真菌，20-30%：高等动物）。这表明ATP酶的氨基酸序列在系统接近的物种中趋于保守，而不是功能特征（离子选择性等）。这两个cDNA克隆缺乏完整的编码区，需要重新筛选。采用北方杂交法确定基因的最高表达细胞期，并成功获得5'端探针，获得足够长度的cDNA（3.4kb）。

项目成果

和田 雅人・佐藤 忍・浦山 修・原 諭吉・井川 洋二・藤伊 正: "Immunological homology between Na^+ーactivated ATPase from marine alga Heterosigma akashiwo and animal Na^+,K^+ーATPase." Biochemical and Biophysical Research Communications.

Masato Wada、Shinobu Sato、Osamu Urayama、Yukichi Hara、Yoji Ikawa、Tadashi Fujii：“来自海洋藻类 Heterosigma akashiwo 的 Na^+ー激活 ATP 酶与动物 Na^+、K^+ーATP 酶之间的免疫同源性。”通讯。

和田雅人,佐藤忍,浦山修,原諭吉,藤伊正: "Immunological homology between Na^+-activated ATPase from Marine alga Heterosigma akashiwo and animal Na^+,K^+-ATPase" Biolchemical and Biophysical Research Communications.

Masato Wada、Shinobu Sato、Osamu Urayyama、Yukichi Hara、Masa Fujii：“来自海洋藻类 Heterosigma akashiwo 的 Na^+ 激活的 ATP 酶与动物 Na^+、K^+-ATP 酶之间的免疫同源性”生物化学和生物物理研究通讯。

和田 雅人・佐藤 忍・笠毛 邦宏・藤伊 正: "Presence of a Na^+ーactivated ATPase in the Plasma Membrane of the Maline Raphidophycean Heterosigma akashiwo" Plant Cell Physiolgy. 30(6). 923-928 (1989)

Masato Wada、Shinobu Sato、Kunihiro Kasage 和 Tadashi Fujii：“Maline Raphidophycean Heterosigma akashiwo 质膜中 Na^+ 激活的 ATP 酶的存在”植物细胞生理学 30(6)。

和田 雅人・佐藤 忍・笠毛 邦宏・藤伊 正: "Presence of a Na^+ーactivated ATPase in the Plasma Membrane of the Marine Raphidophycean Heterosigma akashiwo" Plant Cell Physiolgy. 30(6). 923-928 (1989)

Masato Wada、Shinobu Sato、Kunihiro Kasage 和 Tadashi Fujii：“海洋针藻异形藻质膜中 Na^+ 激活的 ATP 酶的存在”植物细胞生理学 30(6)。

和田 雅人・佐藤 忍・浦山 修・原 諭吉・井川 洋二・藤伊 正: "Immunological homology between Na^+ーactivated ATPase from marine alga Heterosigma akashiwo and animal Na^+,K^+ーATPase" Biochemical and Biophysical Research Communications.

Masato Wada、Shinobu Sato、Osamu Urayyama、Yukichi Hara、Yoji Ikawa、Tadashi Fujii：“来自海洋藻类赤潮异形藻的 Na^+ー激活的 ATP 酶与动物 Na^+,K^+ーATP 酶之间的免疫同源性”生物化学和生物物理研究通讯。