A search for new-type photoreceptors in unicellular flagellate algae and true slime mold.

在单细胞鞭毛藻和真正的粘菌中寻找新型光感受器。

• 批准号: 12672087
• 负责人: TAKAHASHI Tetsuo
• 金额: $ 2.11万
• 依托单位: Toho University (2001-2002)Japan Advanced Institute of Science and Technology (2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672087/
• 关键词: unicellular flagellate; Chlamydomonas; Phototaxis; photoreceptor; retinal; eyespot; sensory rhodopsin; phoborhodopsin; ロドプシン; Acop-1; Acop-2

In general, an attempt to clone homologs of plant or bacterial photoreceptors in fungi or in unicellular flagellates is unsuccessful because of their genetical diversity. We therefore combined several new PCR strategies in order to search for true archaeal-type rhodopsin phototaxis receptor(s) that had been physiologically well-established but had not been detected in molecular level.Expressed Sequence Tag (EST) database of Chlamydomonas reinhardtii publicized in March 2000 from Kazusa DNA Research Institute contained a partial cDNA sequence that showed a little similarity in amino acid level to those of F-G helices of bacterial opsins. Using temperature-asymmetric PCR in combination with a highly degenerated primer, we have extended this sequence toward N-terminus using Chlamydomonas genomic DNA as a template. We found a reasonable similarity in the PCR product to DNA sequences corresponding to C-G helices of bacterial opsins. The complete DNA sequence was obtained and deposited in GenBank (accession numbers: AB058890 and AB58891). Using indirect immunofluorescence we also demonstrated the localization of one of these new archaeal-type opsins of Chlamydomonas in a cell-surface region close to the eyespot apparatus by which Chlamydomonas phototaxis is mediated. A homology modeling successfully indicated an archaeal-type folding of the proteins, accounting for previously established stereospecificity of retinal isomers in the phototaxis receptor(s).

一般来说，克隆真菌或单细胞鞭毛中的植物或细菌光感受器的同源物的尝试是不成功的，因为它们的遗传多样性。因此，我们结合了几种新的聚合酶链式反应策略，以寻找真正的古生型视紫红质趋光性受体(S)，该受体在生理上已经建立，但在分子水平上还没有被检测到。2000年3月从Kazusa DNA研究所公布的莱茵衣藻表达序列标签(EST)数据库中包含了一个在氨基酸水平上与细菌Opsins的F-G螺旋略有相似的部分cDNAs。利用温度不对称聚合酶链式反应和高度简并的引物，我们以衣藻基因组DNA为模板，将该序列延伸到N端。我们在PCR产物中发现了与细菌Opsins C-G螺旋相对应的DNA序列的合理相似性。获得完整的DNA序列，并登录GenBank(登录号：AB058890和AB58891)。利用间接免疫荧光法，我们还证明了衣藻的一种新的古生型视蛋白定位于衣藻趋光性所在的眼点附近的细胞表面区。同源模型成功地表明了蛋白质的古生型折叠，解释了先前建立的趋光性受体中视网膜异构体的立体特异性(S)。

项目成果

Nakamura, S.他8名: "Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with multiple eyespots"Rhycological Research. 49. 115-122 (2001)

Nakamura, S. 和其他 8 人：“Chlamydomonas Reinhardtii Dangeard（Chlamydomonadales，Chlorophyceae）突变体具有多个眼点”Rhycological Research 49. 115-122 (2001)。

Kakiuchi, Y., Takahashi, T., Murakami, A., Ueda, T.: "Light irradiation induces fragmentation of the plasmodium, a novel photomorphogenesis in true slime mold Physarum polycepharum : Action spectra and evidence for involvement"Photochem.Photobiol.. 73. 32

Kakiuchi, Y.、Takahashi, T.、Murakami, A.、Ueda, T.：“光照射诱导疟原虫碎裂，这是真正的粘菌多头绒泡菌中的一种新型光形态发生：作用光谱和参与证据”Photochem.Photobiol。

伊関峰生, 高橋哲郎, 鈴木武士, 渡辺正勝: "動く植物-その謎とき-"山村庄亮, 長谷川宏司 編著、大学教育出版. 200 (2002)

Mineo Izeki、Tetsuro Takahashi、Takeshi Suzuki、Masakatsu Watanabe：“移动植物 - 时间之谜”，由 Shosuke Yamamura 和 Koji Hasekawa 编辑，大学教育出版社 200 (2002)。

Nakamura,S. et al.: "A Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with multiple eyespots"Phycol.Res.. 49(June)(in press). (2001)

中村，S.

Nakamura, S., Oihara, H., Tateishi, M., Takahashi, T., Yoshimura, K., Kubota, M., Watanabe, M. and Nakamura, S.: "Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophycease) mutant with multiple eyespots."Rhycological Research. 49. 115-122 (200

Nakamura, S.、Oihara, H.、Tateishi, M.、Takahashi, T.、Yoshimura, K.、Kubota, M.、Watanabe, M. 和 Nakamura, S.：“衣藻莱氏衣藻 (Chlamydomonadales，Chlorophycease) 突变体