Eldridge R, O'Reilly D R, Hammock B D, Miller L K
Department of Entomology, University of Georgia, Athens 30602.
Appl Environ Microbiol. 1992 May;58(5):1583-91. doi: 10.1128/aem.58.5.1583-1591.1992.
Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.
为了探索增强杆状病毒作为害虫生物防治剂特性的可能性，我们测试了表达一种编码保幼激素酯酶的昆虫基因（jhe）的效果。保幼激素酯酶会使保幼激素失活，而保幼激素调控昆虫蜕皮的结果。将编码烟芽夜蛾保幼激素酯酶的cDNA插入苜蓿银纹夜蛾核型多角体病毒的基因组中，使得该基因在一个强的、经过修饰的病毒启动子控制下表达。然而，这种病毒天然编码一种蜕皮甾体UDP - 葡萄糖基转移酶，它会使引发蜕皮的激素蜕皮激素失活。由于蜕皮甾体UDP - 葡萄糖基转移酶可能通过完全阻断蜕皮来掩盖jhe表达的效果，于是构建了缺失或破坏蜕皮甾体UDP - 葡萄糖基转移酶基因的表达jhe的病毒。对感染细胞中的蛋白质进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析，发现了几种细胞内蛋白质以及两种主要的分泌蛋白，它们能与针对天然保幼激素酯酶的抗体发生反应。蛋白质免疫印迹分析结合衣霉素处理表明，糖基化差异导致了多种产物的产生。重组病毒感染的四龄粉纹夜蛾幼虫的血淋巴中保幼激素酯酶活性水平比未感染幼虫中发现的最高水平高40倍。然而，在感染表达jhe的病毒的昆虫与对照病毒感染的昆虫之间，未观察到发育特征、体重增加或死亡时间有显著差异。
