Evaluating the Effect of a Unique Maize Cysteine Proteinase on Caterpillar Midgut Structure

这项研究的重点是一种独特的蛋白质降解酶，一种名为Mir 1-CP的半胱氨酸蛋白酶，它在抗虫玉米品系中迅速积累，以应对毛虫的攻击。 抗虫玉米品系通过常规植物育种从源自安提瓜的植物材料开发。Mir 1-CP是唯一的，因为酶的最后25个氨基酸的序列在目前可用的数据库中没有匹配。Mir 1-CP似乎在毛虫进食后1小时内被动员，并在伤口部位积聚。当毛虫以含有Mir 1-CP的植物组织为食时，它们的生长减少约50%至80%。这种生长减少是由于营养利用受损。为了研究Mir 1-CP对毛虫的影响，研究人员检查了昆虫对食物中潜在有毒成分的第一道防线，即围食基质（PM）。PM是由蛋白质和几丁质组成的膜状结构。它包围食物团，保护昆虫中肠免受化学和物理损伤，并在消化和营养吸收中发挥积极作用。扫描电子显微镜（SEM）显示，以含有Mir 1-CP的植物材料为食的昆虫的PM严重受损，具有许多穿孔、裂缝和孔洞。 对PM的破坏可能会扰乱消化系统并阻碍毛虫的生长。因此，较小的毛虫具有较长的生命周期，并且更容易受到环境中的寄生虫和捕食者的影响。本研究项目的主要目标是确定Mir 1-CP如何破坏毛虫中肠结构。它能降解PM蛋白吗？ 它是否攻击特定的PM蛋白？它是否通过与甲壳素或其他PM成分结合来防止PM形成？研究人员将使用两种不同的策略来测试Mir 1-CP对PM的影响。 首先，给毛虫喂食表达正常Mir 1-CP、已突变且无酶活性的Mir 1-CP或缺失最后25个氨基酸的Mir 1-CP的转基因植物材料。将检查食用这些转基因品系的毛虫的中肠和PM结构的外观和渗透性。 结果将揭示蛋白酶活性或最后25个氨基酸的存在是否需要损害毛虫中肠和PM。 其次，研究者将在异源系统中表达Mir 1-CP和突变形式，这将允许纯化大量的每种Mir 1-CP形式。 纯化的蛋白质将被喂给毛虫，看看它们是否会产生与植物中表达的蛋白质相同的效果。此外，将从毛毛虫上切下的PM与三种Mir 1-CP形式一起孵育，以确定它们是否对PM结构有直接影响或降解PM蛋白。 最终，这项研究将允许确定Mir 1-CP是否可以用作环境友好的昆虫控制剂。每年世界上15%的农作物损失都是由食草昆虫造成的。这些损失是主要的经济和生态问题，减少了对不断增长的世界人口的粮食供应。美国农民一直在通过种植表达苏云金杆菌（Bt）毒素的转基因作物来控制毛虫害虫。目前，Bt毒素非常有效，但科学家预测昆虫最终会对这种毒素产生抵抗力。通过了解Mir 1-CP如何阻碍毛毛虫的生长，科学家们可能能够在未来使用Mir 1-CP作为Bt毒素的替代品。

The focus of this research is a unique protein-degrading enzyme, a cysteine protease called Mir1-CP that rapidly accumulates in insect resistant corn lines in response to caterpillar attack. The insect resistant corn lines were developed by conventional plant breeding from plant material that originated in Antigua. Mir1-CP is unique because the sequence of the enzyme's last 25 amino acids has no matches in the currently available databases. Mir1-CP appears to be mobilized within one hour of caterpillar feeding and accumulates at the wound site. When caterpillars feed on plant tissues containing Mir1-CP, their growth is reduced approximately 50% to 80%. This growth reduction is due to impaired nutrient utilization. To investigate the effect of Mir1-CP on caterpillars, the investigators examined the insect's first line of defense against potentially toxic components in food, the peritrophic matrix (PM). The PM is a membrane-like structure that consists of proteins and chitin. It surrounds the food bolus, protects the insect midgut from chemical and physical damage and plays an active role in digestion and nutrient absorption. Scanning electron microscopy (SEM) revealed that PMs of insects that feed on plant material containing Mir1-CP were severely damaged with numerous perforations, cracks, and holes. Damage to the PM is likely to disrupt the digestive system and stunt caterpillar growth. Consequently, the smaller caterpillars have longer life-cycles and are more susceptible to parasites and predators in the environment.The major goal of this research project is to determine how Mir1-CP damages the caterpillar midgut structure. Does it degrade PM proteins? Does it attack specific PM proteins? Does it prevent PM formation by binding to chitin or other PM components? The investigators will use two different strategies for testing Mir1-CP's effect on the PM. First, caterpillars will be fed transgenic plant material that expresses either normal Mir1-CP, Mir1-CP that has been mutated and has no enzyme activity, or Mir1-CP that is missing the last 25 amino acids. The appearance and permeability of the midgut and PM structure of caterpillars that eat these transgenic lines will be examined. The results will reveal whether protease activity or the presence of the last 25 amino acids is needed to damage the caterpillar midgut and PM. Second, the investigators will express Mir1-CP and the mutated forms in a heterologous system that will permit purification of large amounts of each Mir1-CP form. The purified proteins will be fed to caterpillars to see if they result in the same effects as those expressed in plants. In addition, PMs that have been dissected from caterpillars will be incubated with the three Mir1-CP forms to determine if they have a direct effect on PM structure, or degrade PM proteins. Ultimately, this research will permit determination of whether Mir1-CP can be used as an environmentally friendly insect control agent. Insect herbivory is responsible for 15% of the world's crop losses each year. These losses are major economic and ecological problems that decrease the supply of food to a growing world population. US farmers have been controlling caterpillar pests by planting transgenic crops that express Bacillus thuringiensis (Bt) toxin. Currently, Bt-toxin is highly effective, but scientists predict that insects will eventually become resistant to the toxin. By understanding how Mir1-CP stunts caterpillar growth, scientists may be able to use Mir1-CP as an alternative to Bt-toxin in the future.