Breeding of plants resistant to fungal infections by introduction of a chitinase gene from filamentous fungi.

通过引入丝状真菌的几丁质酶基因来培育抗真菌感染的植物。

• 批准号: 07556021
• 负责人: TAKAGI Masamichi
• 金额: $ 5.63万
• 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 (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556021/
• 关键词: Rhizopus; chitinase; glass; tomato; rice

Chitinase I of Rhizopus, oligosporus, belonging to zygomycete filamentous fungi, is a pae-pro enzyme. We have cloned chil gene encoding chitinase I.To investigate the effects of expression of fungal chitinase on anti-fungal activity of the transgenic plant, we deleted introns and 3'-region encoding pro-wequence from chil gene and introduced it into some important plants and analyzed phenotypes of the plants. First, the modified chil gene described above was placed between the 35S promoter of Cauliflower mosaic virus and nopaline synthase (Nos) gene terminator and was introduced it into tobacco by the Agrobacterium tumefaciens leaf disc system. Total DNAs were isolated from two of the transformants. Integration of chil gene in these genome DNAs was confirmed by PCR and Southern blot analysis. The expressions of chitinase I in lysates from the young leaves of them were detected by Western blot analysis with anti-chitinase I antibody. Chitinase activities in these lysates were three- to f … More our-fold higher than that from the control plant. Then, the discomycete pathogens Sclerotinia sclerotiorum and Botrytis cirerea were infected on the leaves of them. The symptoms observed with these two transformants were remarkably suppressed as compared with leaves of the control plants. Next, the modified chil gene was placed between the maize ubiquitin gene promoter and Nos gene terminator and was cotransformed into rice with a plasmid containing hygromycin phosphotransferase gene as a selectable marker by the method of particle bombardment. 123 transformants were obtained and regenerated to plants. Total DNAs of them were prepared and used as substrates of PCR analysis and Southern blot analysis. Integration of chil gene was confirmed in 24 transformants. A weak signal of 43 kDa was detected in lysates of the leaves of 4 transformants by Western blot analysis. These transformants were regenerated to plants and infected by Magnaporthe grisea which could cause rice blast disease. The anti-fungal activity of these plants seemed to raise as compared with that of control plants. We also introduced the modified chil gene into tomato and glass and are now trying the regeneration of these transgenic tomato and glass calli. Less

少孢根霉几丁质酶Ⅰ是一种产于放线菌纲丝状真菌的几丁质酶。我们克隆了几丁质酶I的编码基因chil，为了研究真菌几丁质酶的表达对转基因植物抗真菌活性的影响，我们将chil基因的内含子和3 '端编码区缺失，并将其导入一些重要植物中，分析了植物的表型。首先，将上述修饰的chil基因置于花椰菜花叶病毒的35 S启动子和胭脂碱合酶（Nos）基因终止子之间，并通过根癌土壤杆菌叶盘系统将其导入烟草。从两个转化体中分离总DNA。PCR和Southern杂交分析证实chil基因已整合到这些基因组DNA中。用抗几丁质酶I抗体进行Western blot分析，检测几丁质酶I在它们幼叶裂解液中的表达。几丁质酶活性分别为3 ~ 100 mg/L， ...更多信息 比对照植物高出10倍。然后，将盘状真菌病原菌核盘菌（Sclerotinia sclerotiorum）和番茄灰霉病菌（Botrytis cirerea）感染到它们的叶片上。与对照植物的叶子相比，用这两个转化体观察到的症状被显著抑制。然后，将修饰的chil基因置于玉米泛素基因启动子和Nos基因终止子之间，并通过粒子轰击法与含有潮霉素磷酸转移酶基因作为选择标记的质粒共转化到水稻中。共获得123个转化子，并再生植株。制备它们的总DNA并用作PCR分析和Southern印迹分析的底物。在24个转化子中证实了chil基因的整合。通过Western印迹分析，在4个转化体的叶的裂解物中检测到43 kDa的弱信号。这些转化子再生为植株，并被稻瘟病菌感染，可以引起稻瘟病。与对照植物相比，这些植物的抗真菌活性似乎有所提高。我们还将修饰过的chil基因导入番茄和玻璃中，目前正在尝试这些转基因番茄和玻璃愈伤组织的再生。少

项目成果

Terakawa T,Takaya N,Noriuchi H,Koike M,Takagi M: "gal chitinase gene from Rhizopus oligosporus confers the antifungal ty to the transgenic tobacco." Plant Cell Reports.(in press). (1997)

Terakawa T、Takaya N、Noriuchi H、Koike M、Takagi M：“来自少孢根霉的半乳糖几丁质酶基因赋予转基因烟草抗真菌能力。”

寺川輝彦 他: "A fungal chitinase gene from Rhizopus oligosporus confers the antifungal activity to the transgenic tobacco." Plant Cell Rep.(印刷中).

Teruhiko Terakawa 等人：“来自少孢根霉的真菌几丁质酶基因赋予转基因烟草抗真菌活性。”（正在出版）。