Detection of potyviruses by non-radioactive nucreic acid probe.

用非放射性核酸探针检测马铃薯Y病毒组。

• 批准号: 02660043
• 负责人: NATSUAKI Tomohide
• 金额: $ 1.47万
• 依托单位: Utsunomiya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660043/
• 关键词: non-radioactive probe; Potyviruses; clover yellow vein virus; turnip mosaic virus; distinction of strains; ウエスタンブロット法; ウイルスRNAーcDNAクロ-ニング; ウイルス系統分類; フォトビオチン標識プロ-ブ

Potyvirus group is one of the biggest group in plant viruses and very important in agriculture. The purpose of this research is to make a specific nonradioactive nucleic acid probe for detecting each polyvirus and its strains. A virus, designated NFU, isolated from bean plants(Phaseolus vulgaris)with severe vine necrosis(Tsurugare-byo)in Fukushima Prefecture was compared with a seed-borne isolate of bean yellow mosaic virus(BYMV-SB)from broad bean. Based on the results of inoculation test, serological test and western blotting, NFU was identified as a strain of clover yellow vein virus(CYVV-NFU). CYVV-RNA was extracted from purified virus preparations and complementary DNA was prepared by the procedure of Gubler and Hoffman. The CDNA was ligated into Smal site of PUC19 plasmid. The E. coli transformed by plasmids containing CDNA inserts were immunoscreened with CYVV antiserum. Three immunopositive clones were obtained. In hybridization test using CDNA probe of CYVV-RNA labeled with photobiotin, a non-radioactive chemical, CYVV was detected very easily and sensitively. Two strains of turnip mosaic virus(TUMV)were also studied. Radish and cabbage strains were distinguished by host range but not by serological method. The CDNA clones were obtained and used as non-radioactive probes.

马铃薯Y病毒组是植物病毒中最大的类群之一，在农业生产中具有重要的意义。本研究的目的是制备一种特异性的非放射性核酸探针，用于检测每种多病毒及其毒株。从福岛县发生严重蔓坏死病的菜豆（Phaseolus vulgaris）上分离到一种病毒，命名为NFU，并与蚕豆种子传的菜豆黄花叶病毒（BYMV-SB）进行了比较。通过接种试验、血清学试验和免疫印迹试验，初步鉴定NFU为一株三叶草黄脉病毒（CYVV NFU）。从纯化的病毒制备物中提取CYVV-RNA，并通过Gubler和霍夫曼的方法制备互补DNA。将cDNA连接到PUC 19质粒的Smal位点。急诊用CYVV抗血清对转化的大肠杆菌进行免疫筛选。获得三个免疫阳性克隆。在使用非放射性化学物质光生物素标记的CYVV-RNA的CDNA探针进行杂交试验中，CYVV非常容易且灵敏地被检测到。对芜菁花叶病毒（TUMV）的两个株系进行了研究。萝卜和甘蓝菌株可通过寄主范围区分，但不能通过血清学方法区分。获得cDNA克隆并用作非放射性探针。

项目成果

Sano, H.: "Molecular cloning of turnip mosaic virus RNA with using photobiotin-labeled probe." Ann. Phytopath. Soc. Japan. 56. 100 (1990)

Sano, H.：“使用光生物素标记探针对萝卜花叶病毒 RNA 进行分子克隆。”

佐野 博,夏秋 知英,奥田 誠一,寺中 理明: "フォトビオチンプロ-ブを用いてのカブモザイクウイルスRNAのクロ-ニング" 日本植物病理学会報. 56. 100 (1990)

Hiroshi Sano、Tomohide Natsuaki、Seiichi Okuda、Yoshiaki Teranaka：“使用光生物素探针克隆萝卜花叶病毒 RNA”日本植物病理学会杂志 56. 100 (1990)。

長谷川 睦己,梶 和彦,夏秋 啓子,夏秋 知英,奥田 誠一,寺中 理明: "福島県で発生したインゲンマメつる枯病" 日本植物病理学会報. 57. 72 (1991)

Mutsumi Hasekawa、Kazuhiko Kaji、Keiko Natsuaki、Tomohide Natsuaki、Seiichi Okuda、Yoshiaki Teranaka：“福岛县发生的普通豆藤枯病”日本植物病理学会通报 57. 72 (1991)。