Jach G, Görnhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C
Max-Planck Institut Für Züchtungsforschung, Abeteilung Genetische Grundlagen der Pflanzenzüchtung, Cologne, Germany.
Plant J. 1995 Jul;8(1):97-109. doi: 10.1046/j.1365-313x.1995.08010097.x.
cDNAs encoding three proteins from barley (Hordeum vulgare), a class-II chitinase (CHI), a class-II beta-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed antifungal proteins in vivo.
编码来自大麦（Hordeum vulgare）的三种蛋白质的cDNA，即一种II类几丁质酶（CHI）、一种II类β-1,3-葡聚糖酶（GLU）和一种I型核糖体失活蛋白（RIP），在花椰菜花叶病毒35S启动子的控制下在烟草植株中表达。通过Northern和Western印迹分析在转基因植株中检测到了转移基因的高水平表达。CHI和GLU中的前导肽导致这些蛋白质在烟草叶片的细胞间隙中积累。天然存在于大麦胚乳细胞胞质溶胶中的RIP，以其原始的胞质形式表达或与植物分泌肽融合（spRIP）表达。真菌感染试验表明，在每种情况下单个基因的表达都增强了对土传真菌病原体立枯丝核菌的抗性，立枯丝核菌可感染包括烟草在内的多种植物物种。为了创造一种类似于“多基因”耐受性的情况，传统育种经验表明这种耐受性能为作物提供更持久的保护，将其中几个抗真菌基因进行组合，并评估它们在植物中共表达对真菌攻击的抗性。构建了串联排列编码RIP和CHI以及GLU和CHI的基因的转基因烟草品系。在立枯丝核菌感染试验中，共表达大麦转基因GLU/CHI或CHI/RIP的烟草植株的表现表明，与表达单个大麦转基因至相似水平的相应同基因系相比，对真菌攻击的抗性显著增强。数据表明共表达的抗真菌蛋白在体内具有协同保护作用。
