Bouchard Edith, Cloutier Conrad, Michaud Dominique
Département de Biologie, Centre de recherche en horticulture, Université Laval, Cité Universitaire (Québec), Canada G1K 7P4.
Mol Ecol. 2003 Sep;12(9):2439-46. doi: 10.1046/j.1365-294x.2003.01919.x.
We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.
我们最近观察到，在转基因马铃薯中表达的水稻半胱氨酸蛋白酶抑制剂——水稻胱抑素I（OCI），不会通过以该植物为食的食草猎物影响双斑臭虫捕食者（双斑青步甲）的生长和发育。在此，我们沿着马铃薯→食草动物→捕食者这条食物链监测了重组OCI的抑制活性，以确定没有产生影响是否与重组胱抑素抑制捕食者蛋白酶后其消化的补偿反应有关。在确认OCI存在于植物中，并被马铃薯甲虫幼虫以活性形式摄入后，定量和电泳分析使我们能够确定，重组胱抑素（约占叶片总可溶性蛋白的0.8%）完全与猎物中肠内一种约30 kDa的目标蛋白酶结合，形成一种在免疫印迹上能用抗OCI多克隆抗体检测到的十二烷基硫酸钠（SDS）稳定复合物。尽管在甲虫中肠中明显没有游离的、残留的OCI，但当猎物取食转基因植物时，捕食者中已知包括对OCI敏感活性的消化蛋白酶活性却受到了负面影响改变。在第三营养级的这种抑制过程伴随着捕食者的补偿反应，即从头合成丝氨酸型蛋白酶。总体而言，我们的数据表明OCI与捕食者对OCI敏感的蛋白酶之间的亲和力：（i）与OCI和马铃薯甲虫30 kDa敏感蛋白酶之间的亲和力一样强（或更强）；（ii）比这些酶与植物内源性OCI同源物——马铃薯甲虫取食诱导植物产生马铃薯多胱抑素之间的亲和力更强。我们的结果还表明，捕食性生物能够使其消化代谢适应其食草猎物摄入的植物抗消化蛋白的存在。在更广泛的背景下，这项研究强调不仅要监测表达蛋白酶抑制剂（PI）的植物对目标食草昆虫的抑制作用，还要监测对环境中可能捕食这些害虫的生物的抑制作用。
