Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.
Arch Insect Biochem Physiol. 2012 Mar;79(3):153-81. doi: 10.1002/arch.21017.
The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.
科罗拉多马铃薯甲虫（Leptinotarsa decemlineata）是世界上许多地区马铃薯最重要的害虫。它成功的主要原因之一在于其幼虫能够对抗植物防御化合物。幼虫通过用对抑制剂不敏感的半胱氨酸蛋白酶替代对抑制剂敏感的消化半胱氨酸蛋白酶来适应马铃薯叶片中产生的蛋白酶抑制剂（PIs）。为了更深入地了解幼虫适应植物防御的基础，我们使用喂食甲基茉莉酸诱导或未诱导的马铃薯叶片的 L. decemlineata 幼虫的肠道 cDNA 创建了一个“抑制性消减杂交”文库。从文库中随机选择 400 个克隆，通过 DNA 微阵列杂交筛选与适应相关的克隆。使用定量 PCR 和酶活性测量进一步检查了选定的昆虫消化酶系统的基因表达变化。我们鉴定了两组新的消化半胱氨酸蛋白酶，肠内酶 D 和肠内酶 E。肠内酶 D 代表一组结构上不同的消化半胱氨酸蛋白酶，其中测试成员对诱导的植物防御有强烈的上调反应。此外，我们发现其他消化酶也参与了适应，即纤维素酶、丝氨酸蛋白酶和内切多聚半乳糖醛酸酶。此外，保幼激素结合蛋白样（JHBP-like）基因被上调。所有研究的基因都特异性地在幼虫肠道中表达。与早期报道基于 PI 富集人工饲料的实验的研究相比，我们的结果增加了对昆虫在自然条件下适应的理解。
