Effect of a Unique Maize Defense Proteinase on Caterpillar Midgut Structure

当植物受到食草昆虫的攻击时，它们通常会产生蛋白质来保护自己不被吃掉。我们已经证明了一种独特的酶，一种蛋白酶，由gebe mir1编码，当它们受到秋粘虫毛虫的攻击时，在某些抗虫玉米品系中积累起来。我们已经利用基因工程技术将mir1基因转移到其他植物细胞中。当毛毛虫吃了这些转基因植物细胞后，它们的生长速度会减慢60%到80%。似乎幼虫以抗性植物或转基因植物细胞为食，不能利用植物中的营养物质生长。我们的假设是，蛋白酶破坏了昆虫的肠道，阻止了正常的营养利用。本研究项目的目的是确定这种独特的玉米蛋白酶是否会损害昆虫肠道中的特定结构。为了验证这一点，我们将在mir1中进行突变，这将改变酶的特性。一种突变会消除蛋白酶的活性，另一种突变会去除酶的一个独特区域。突变形式的mir1将被转移到植物细胞中。表达突变蛋白酶的转基因植物细胞将被喂给毛毛虫。然后用表达正常和突变蛋白酶的转基因植物细胞喂养幼虫，观察其生长和肠道结构。如果需要蛋白酶活性来延缓毛虫的生长和破坏肠道结构，那么我们预计正常的蛋白酶会伤害毛虫，而突变的形式则不会。此外，我们将在昆虫细胞中表达mir1及其突变形式，制备正常和突变的蛋白酶。纯化的蛋白酶将直接喂给毛毛虫，以确定它们是否攻击昆虫的肠道。如果这种独特的蛋白酶确实能特异性地攻击昆虫的肠道，那么在未来，利用mir1基因改造植物来抵抗昆虫的摄食是可能的。

When plants are attacked by herbivorous insects, they often respond by producing proteins that protect them from being eaten. We have shown that a unique enzyme, a protease, coded for by the gebe mir1, accumulates in certain insect-resistant corn lines when they are attacked by the fall armyworm caterpillar. We have transferred the gene, mir1, into other plant cells using genetic engineering technology. When caterpillars eat these transgenic plant cells, their growth is retarded by 60 to 80%. It appears that caterpillars feeding on resistant plants or transgenic plant cells cannot use the nutrients in the plant for growth. Our hypothesis is that the protease damages the insect gut and prevents normal nutrient utilization. The purpose of this research project is to determine if the unique corn protease damages specific structures in the insect gut. To test this we will make mutations in mir1 that will change the characteristics of the enzyme. One mutation will eliminate protease activity, the other will remove a unique region of the enzyme. The mutated forms of mir1 will be transferred to plant cells. Transgenic plant cells that express mutated proteases will be fed to caterpillars. Then growth and gut structure of caterpillars fed transgenic plant cells expressing normal and mutated proteases will be assessed. If the protease activity is required to retard caterpillar growth and damage gut structure, then we expect that the normal protease will harm the caterpillars and the mutated forms will not. In addition, we will make pure preparations of the normal and mutated proteases by expressing mir1 and its mutated forms in insect cells. The purified proteases will be directly fed to the caterpillars to determine if they attack the insect gut. If the unique protease does specifically attack the insect gut, it may be possible, in the future, to use mir1 to genetically engineer plants to resist insect feeding.