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

寡孢根霉几丁质酶I是一种聚酶，属于接合菌丝状真菌。为了研究真菌几丁质酶i的表达对转基因植物抗真菌活性的影响，我们从chil基因中删除内含子和3'区编码亲频率，并将其引入一些重要的植物中，并分析了这些植物的表型。首先，将上述修饰的chil基因置于花菜花叶病毒35S启动子和野碱合成酶（Nos）基因终止子之间，通过农杆菌叶盘体系导入烟草。从两个转化子中分离出总dna。通过PCR和Southern blot分析证实了这些基因组dna中child基因的整合。用抗几丁质酶I抗体检测其幼叶裂解物中几丁质酶I的表达。这些裂解物的几丁质酶活性比对照植物高3 ~ 4倍。然后，在其叶片上感染菌核菌（Sclerotinia sclerotiorum）和灰葡萄孢菌（Botrytis cirerea）。与对照植株相比，这两个转化植株的症状得到了显著抑制。然后，将修饰后的chil基因置于玉米泛素基因启动子和Nos基因终止子之间，通过粒子轰击法将含潮霉素磷酸转移酶基因的质粒作为选择标记共转化到水稻中。获得转化体123个，并再生到植株上。制备总dna作为PCR和Southern blot分析的底物。在24个转化子中证实了child基因的整合。Western blot检测到4个转化子叶片裂解物中存在43 kDa的弱信号。这些转化体再生到植株上，并感染稻瘟病病菌。与对照植物相比，这些植物的抗真菌活性似乎有所提高。我们还将转基因的chil基因导入番茄和玻璃中，目前正在进行转基因番茄和玻璃愈伤组织的再生试验。少

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