Mazumdar-Leighton S, Broadway R M
Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
Insect Biochem Mol Biol. 2001 Apr 27;31(6-7):633-44. doi: 10.1016/s0965-1748(00)00168-5.
Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.
像棉铃虫和小地老虎这样的鳞翅目昆虫在摄入像大豆胰蛋白酶抑制剂（STI）这样的膳食植物蛋白酶抑制剂后，会在中肠中产生对STI不敏感的胰蛋白酶[百老汇，R.M.，《昆虫生理学杂志》43(9) (1997) 855 - 874]。在本文中，研究了膳食STI对中肠丝氨酸蛋白酶相关家族——胰凝乳蛋白酶的影响。在以含1% STI的饲料喂养的棉铃虫和小地老虎幼虫中检测到了对STI不敏感的中肠胰凝乳蛋白酶，而在以含0% STI的饲料喂养的幼虫中存在对STI敏感的胰凝乳蛋白酶。这些胰凝乳蛋白酶不受甲苯磺酰苯丙氨酰氯甲基酮（TPCK，一种哺乳动物胰凝乳蛋白酶的诊断抑制剂）的影响，但被抑肽酶完全抑制。从分别以含0% STI或1% STI饲料喂养的每个物种的幼虫构建了四个中肠cDNA文库。分离出了六个编码不同前体胰凝乳蛋白酶的全长cDNA（三个来自棉铃虫，三个来自小地老虎），它们具有某些使其与哺乳动物对应物区分开来的序列基序。Northern印迹显示，与在含0% STI和1% STI饲料上饲养的昆虫相比，一些胰凝乳蛋白酶mRNA的检测水平较高，而另一些则下调。Southern杂交表明（与哺乳动物一样）这两个物种都含有几个胰凝乳蛋白酶基因。首次对棉铃虫的一个全长胰凝乳蛋白酶基因进行了测序，并推断出有四个内含子。首次对小地老虎的三个胰凝乳蛋白酶基因和两个胰蛋白酶基因的5'上游区域进行比较，表明存在假定的TATA框和调控元件。然而，这些上游区域缺乏一致的基序，这表明可能存在多种转录因子来调控编码消化蛋白酶的基因，以及与摄入蛋白酶抑制剂相关的复杂反应机制。
