Neuhaus J M, Flores S, Keefe D, Ahl-Goy P, Meins F
Friedrich Miescher-Institut, Switzerland.
Plant Mol Biol. 1992 Aug;19(5):803-13. doi: 10.1007/BF00027076.
Vacuolar class I beta-1,3-glucanases (EC 3.2.1.39) are believed to be important in the induced defense reaction of plants to fungal infection. We used antisense transformation to test this hypothesis and to identify other possible physiological functions of this enzyme. Nicotiana sylvestris plants were transformed with antisense constructions containing the region from position 27 to 608 of the coding sequence of the basic, vacuolar beta-1,3-glucanase gene GLA of tobacco regulated by cauliflower mosaic virus 35S RNA expression signals. Plants homozygous for this transgene showed a marked, ca. 20-fold reduction in the constitutive expression of class I beta-1,3-glucanase antigen in their leaves. RNA blot analysis indicated that the antisense plants expressed low levels of the sense transcript of the host beta-1,3-glucanase gene and the antisense transcript of the transgene. Immune blot analysis of plant extracts indicated that only expression of the N. sylvestris homologue of class I tobacco beta-1,3-glucanase and not the acidic, class II isoforms of the enzyme was blocked in the antisense plants. Class I isoforms of beta-1,3-glucanase and chitinase were coordinately induced in leaves of untransformed and empty-vector-transformed N. sylvestris plants treated with ethylene or infected with the fungal leaf pathogen Cercospora nicotianae. In antisense plants, chitinase but not beta-1,3-glucanase was induced under these conditions indicating that antisense transformation effectively blocks constitutive as well as induced expression of class I beta-1,3-glucanase. Under greenhouse conditions, antisense plants developed normally and were fertile. The plants did not exhibit increased susceptibility to C. nicotianae infection. These results suggest that expression of the beta-1,3-glucanase isoform blocked by antisense transformation is not necessary for 'housekeeping' functions of N. sylvestris nor defense against the fungal pathogen tested.
液泡I类β-1,3-葡聚糖酶（EC 3.2.1.39）被认为在植物对真菌感染的诱导防御反应中起重要作用。我们利用反义转化来检验这一假设，并确定该酶的其他可能生理功能。用含有受花椰菜花叶病毒35S RNA表达信号调控的烟草基本液泡β-1,3-葡聚糖酶基因GLA编码序列第27至608位区域的反义构建体转化了林烟草植株。该转基因的纯合植株在其叶片中I类β-1,3-葡聚糖酶抗原的组成型表达显著降低，约降低了20倍。RNA印迹分析表明，反义植株中宿主β-1,3-葡聚糖酶基因的正义转录本和转基因的反义转录本表达水平较低。对植物提取物的免疫印迹分析表明，在反义植株中，仅I类烟草β-1,3-葡聚糖酶的林烟草同源物的表达被阻断，而该酶的酸性II类同工型的表达未被阻断。在用乙烯处理或感染真菌叶病原体烟草尾孢菌的未转化和空载体转化的林烟草植株的叶片中，I类β-1,3-葡聚糖酶和几丁质酶同工型被协同诱导。在反义植株中，在这些条件下几丁质酶被诱导，但β-1,3-葡聚糖酶未被诱导，这表明反义转化有效地阻断了I类β-1,3-葡聚糖酶的组成型表达以及诱导表达。在温室条件下，反义植株正常生长且可育。这些植株对烟草尾孢菌感染的易感性并未增加。这些结果表明，被反义转化阻断的β-1,3-葡聚糖酶同工型的表达对于林烟草的“管家”功能以及对所测试的真菌病原体的防御并非必需。
