Brants A, Earle E D
Cornell University, Department of Plant Breeding, Ithaca, NY 14853-1901, USA e-mail:
Plant Cell Rep. 2001 Jan;20(1):73-78. doi: 10.1007/s002990000286.
Calli and cell suspensions were obtained from tobacco plants transformed with an endochitinase-encoding cDNA from the biocontrol fungus Trichoderma harzianum. Calli from four primary transformants had high levels of endochitinase activity, like the plants from which they were derived. Endochitinase activity was also detected in the medium surrounding the calli and in the medium from transgenic cell suspensions. Western blots demonstrated the presence of the expected 40-kDa T. harzianum protein in transgenic samples but not in controls. These results indicate that the fungal enzyme is secreted and that the fungal signal peptide in the cDNA construct functions in plant cells. A cell suspension medium in which the protein concentration was increased up to 34-fold by ammonium sulfate precipitation inhibited germination of Penicillium digitatum spores. Some inhibition of spore germination was also observed in concentrated medium from control suspensions, probably due to the secretion and concentration of endogenous enzymes.
愈伤组织和细胞悬浮液取自用来自生防真菌哈茨木霉的编码内切几丁质酶的cDNA转化的烟草植株。来自四个初级转化体的愈伤组织具有高水平的内切几丁质酶活性，与它们所源自的植株相似。在愈伤组织周围的培养基以及转基因细胞悬浮液的培养基中也检测到了内切几丁质酶活性。蛋白质免疫印迹表明，在转基因样品中存在预期的40 kDa哈茨木霉蛋白，而在对照中则不存在。这些结果表明，真菌酶被分泌出来，并且cDNA构建体中的真菌信号肽在植物细胞中发挥作用。通过硫酸铵沉淀使蛋白质浓度提高到34倍的细胞悬浮液培养基抑制了指状青霉孢子的萌发。在对照悬浮液的浓缩培养基中也观察到了对孢子萌发的一些抑制作用，这可能是由于内源酶的分泌和浓缩所致。
