Florack D, Allefs S, Bollen R, Bosch D, Visser B, Stiekema W
Department of Molecular Biology, DLO Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, Netherlands.
Transgenic Res. 1995 Mar;4(2):132-41. doi: 10.1007/BF01969415.
Cecropin B is a small antibacterial peptide from the giant silkmoth Hyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco via Agrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance to Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P. syringae pv. tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin B in vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.
天蚕素B是一种来自大蚕蛾（Hyalophora cecropia）的小抗菌肽。为了揭示该肽在将细菌抗病性导入作物方面的潜力，制备了几种天蚕素B基因构建体，用于在细胞质中表达或分泌。所有构建体都克隆到植物表达载体中，并通过根癌农杆菌导入烟草。一个缺少氨基末端信号肽的cDNA来源的天蚕素B基因构建体在转基因植物中的mRNA水平表达较差，而含有包含昆虫信号肽的全长cDNA来源构建体的植物，其天蚕素B - mRNA水平有所增加。在含有植物基因来源信号肽构建体的植物中发现了最高表达。在任何转基因植物中都未检测到天蚕素B肽。这很可能是由于植物内源性蛋白酶对该肽的降解所致，因为化学合成的天蚕素B肽在各种植物细胞提取物中几秒钟内就会降解。通过添加特定的蛋白酶抑制剂以及在添加肽之前将提取物煮沸，可以防止这种降解。此外，与阳离子、两性离子或非离子洗涤剂相比，阴离子洗涤剂可以防止这种降解。然而，对转基因烟草植株进行了对青枯假单胞菌（许多作物细菌性萎蔫病的病原体）和烟草野火病菌（细菌性野火病的病原体）的抗性评估，这两种病菌在体外对天蚕素B高度敏感。未发现抗性。这些实验表明，在烟草中导入和表达天蚕素B基因不会导致可检测到的天蚕素B蛋白水平以及对细菌感染的抗性，很可能是由于内源性蛋白酶对该蛋白的降解。
