Elucidation of molecular mechanism of habituation of cultured plant cells

阐明培养植物细胞适应的分子机制

• 批准号: 13440237
• 负责人: NAGATA Toshiyuki
• 金额: $ 9.28万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13440237/
• 关键词: Habituation; Auxin; Tobacco; Chromatography; Cell division; Auxin starvation; Cell culture; Glycoprotein; タバコ培養細胞株BY-2; オーキシン信号伝達; クロマトグラフィー; 細胞増殖; 細胞分泌; 信号伝達; タバコ培養細胞

In this study we have conduced the elucidation of the molecular mechanism of auxin-habituation using tobacco 2B-13 cells. In fact, tobacco BY-2 cells require auxin for their proliferation, while 2B-13 cells do not require it for growth. After various kinds of trials, we found that the addition of culture filtrates from the 2B-13 cells could induce cell division in the auxin-starved tobacco BY-2 cells. However, an active factor for inducing cell division was not ascribed to lower molecular mass molecules such as plant hormones, but the active fractions was found to be higher molecular mass glycoproteins. Thus, purification of this active fraction from the culture filtrates was conducted by hydroxyapatite chromatography, affinity chromatography and gel filtration, which revealed that the glycoprotein was ascribed possibly to a single protein of the molecular mass of 30 kDa in SDS-PAGE. On the other hand, seeking for similar factors in the culture filtrates of tobacco BY-2 cells revealed that there were two glycoproteins after the gel filtration. Intriguingly, these glycoproteins had molecular masses of 25 kDa and 40 kDa, respectively, which were apparently different from the 30 kDa glycoprotein mentioned above.Since such high molecular mass proteins that could be replaced with auxin have not been identified thus far, these proteins should be quite novel. It is intriguing to determine at which step of the auxin signaling pathway these proteins would be located. On the other hand, relationship among these three proteins is also interesting, which could be answered by the determination of amino acid sequences of these proteins, which is under study.

本研究以烟草2B-13细胞为材料，对生长素驯化的分子机制进行了初步的研究。事实上，烟草BY-2细胞的增殖需要生长素，而2B-13细胞的生长不需要生长素。经过各种试验，我们发现添加来自2B-13细胞的培养物可以诱导生长素饥饿的烟草BY-2细胞的细胞分裂。然而，诱导细胞分裂的活性因子并不归因于低分子量的分子，如植物激素，但活性组分被发现是高分子量的糖蛋白。因此，通过羟基磷灰石层析、亲和层析和凝胶过滤从培养物中纯化该活性级分，这表明糖蛋白可能归因于SDS-PAGE中分子量为30 kDa的单一蛋白质。另一方面，在烟草BY-2细胞的培养物中寻找相似的因子，发现凝胶过滤后有两种糖蛋白。有趣的是，这些糖蛋白的分子量分别为25 kDa和40 kDa，这与上述30 kDa的糖蛋白明显不同，因为迄今为止还没有鉴定出可以被生长素取代的高分子量蛋白，所以这些蛋白应该是相当新颖的。确定这些蛋白质位于生长素信号通路的哪个步骤是有趣的。另一方面，这三种蛋白质之间的关系也很有趣，这可以通过确定这些蛋白质的氨基酸序列来回答，这是正在研究中的。

项目成果

Kumagai, F., Nagata, T., Yahara, N., Moriyama, Y., Horio, T., Yamato, M., Naoi, K., Hashimoto, T., Hasezawa, S.: "γ-Tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells."Eur.J.Cell Biol.. 82. 43-51 (2

Kumagai, F., Nagata, T., Yahara, N., Moriyama, Y., Horio, T., Yamato, M., Naoi, K., Hashimoto, T., Hasezawa, S.：“γ-微管蛋白分布在烟草 BY-2 细胞 M/G1 界面的皮质微管重组过程中。“Eur.J.Cell Biol.. 82. 43-51 (2

Nagata, T., Bajaj, Y.P.S: "Somatic Hybridezation and in Crop Improvements II. In : Biotechnology in Agriculture and Forestry (Nagata, T., Bajaj, Y.P.S. eds.)"Springer-Verlag.

Nagata，T.，Bajaj，Y.P.S：“体细胞杂交和作物改良 II。见：农业和林业生物技术（Nagata，T.，Bajaj，Y.P.S. 编辑）”Springer-Verlag。

Kuwabara, A. et al.: "Identification of factors that cause heterophylly in Ludwigia arcuata"Plant Biology. 3. 98-105 (2001)

Kuwabara, A. 等人：“导致 Ludwigia arcuata 异源性的因素的鉴定”植物生物学。

Kuwabara, A., Tsukaya, H., Nagata, T.: "Identification of factors that cause heterophylly in Ludwigia arcuata"Plant Biology. 3. 98-105 (2001)

Kuwabara, A.、Tsukaya, H.、Nagata, T.：“导致 Ludwigia arcuata 异源性的因素的鉴定”植物生物学。

Kumagai, F. et al.: "γ-Tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells"Eur. J. Cell Biol.. 81. 1-9 (2002)

Kumagai，F.等人：“烟草BY-2细胞中M/G1界面处皮质微管重组过程中的γ-微管蛋白分布”Eur. J. Cell Biol.. 81. 1-9 (2002)