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，一种被称为Mir1-CP的半胱氨酸蛋白酶，它在抗虫玉米品系中迅速积累，以应对毛毛虫的攻击。抗虫玉米系是利用原产于安提瓜的植物材料进行常规育种培育而成的。Mir1-CP是独一无二的，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。Mir1-CP似乎在毛毛虫进食后一小时内被动员起来，并在伤口部位积聚。当毛毛虫以含有Mir1-CP的植物组织为食时，它们的生长速度会降低约50%至80%。这种生长减少是由于营养利用受损。为了研究Mir1-CP对毛虫的影响，研究人员检查了昆虫抵抗食物中潜在有毒成分的第一道防线，即营养基质（PM）。PM是一种膜状结构，由蛋白质和几丁质组成。它包裹着食物丸，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Mir1-CP的植物材料为食的昆虫的pm严重受损，出现许多穿孔、裂缝和孔洞。对PM的损害可能会扰乱消化系统，阻碍毛虫的生长。因此，较小的毛虫有较长的生命周期，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Mir1-CP如何损害毛虫的中肠结构。它能降解PM蛋白吗？它会攻击特定的PM蛋白吗？它是否通过与几丁质或其他PM成分结合来阻止PM的形成？研究人员将使用两种不同的策略来测试Mir1-CP对PM的影响。首先，毛毛虫将被喂食转基因植物材料，这些材料要么表达正常的Mir1-CP，要么表达已突变且没有酶活性的Mir1-CP，要么表达缺少最后25个氨基酸的Mir1-CP。将对食用这些转基因品系的毛虫的外观、中肠通透性和PM结构进行研究。研究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破坏毛虫的中肠和PM。其次，研究人员将在异种系统中表达Mir1-CP和突变形式，这将允许大量纯化每种Mir1-CP形式。纯化后的蛋白质将被喂给毛毛虫，看看它们是否能产生与植物中表达的蛋白质相同的效果。此外，从毛虫上解剖的PM将与三种Mir1-CP形式一起孵卵，以确定它们是否对PM结构有直接影响，或降解PM蛋白质。最终，这项研究将允许确定Mir1-CP是否可以作为一种环境友好的昆虫防治剂。每年全球15%的农作物损失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芽孢杆菌（Bt）毒素的转基因作物来控制害虫。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抗药性。通过了解Mir1-CP如何阻碍毛虫的生长，科学家们将来可能会使用Mir1-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.