Zhu Y C, Oppert B, Kramer K J, McGaughey W H, Dowdy A K
Grain Marketing and Production Research Center, U.S. Department of Agriculture, Manhattan, KS 66502-2736, USA.
Insect Biochem Mol Biol. 1997 Dec;27(12):1027-37. doi: 10.1016/s0965-1748(97)00089-1.
Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.
肠道蛋白酶参与苏云金芽孢杆菌产生的杀虫毒素的溶解和激活，也可能与抗性发展有关。在印度谷螟（Plodia interpunctella）的一种对Bt（昆虫杀灭型）具有抗性的品系中，观察到类胰凝乳蛋白酶活性比Bt敏感品系低约三倍。由于类胰凝乳蛋白酶参与印度谷螟中Bt原毒素的激活，我们比较了印度谷螟Bt敏感品系和Bt抗性品系中类胰凝乳蛋白酶的cDNA序列、mRNA表达水平和基因组DNA。为了分离编码类胰蛋白酶原的cDNA，使用载体引物和对应于丝氨酸蛋白酶活性位点保守序列的简并引物，通过聚合酶链反应（PCR）扩增Bt敏感品系的cDNA文库来制备探针。该探针用于筛选抗性和敏感品系的cDNA文库。每个品系的cDNA克隆预测的氨基酸序列与类胰凝乳蛋白酶序列具有相似性，并且与烟草天蛾（Manduca sexta）的类胰凝乳蛋白酶最为相似。印度谷螟Bt敏感品系和Bt抗性品系中假定的类胰蛋白酶原蛋白的cDNA具有相同的846个核苷酸的开放阅读框。编码的蛋白质包含丝氨酸蛋白酶活性位点的氨基酸序列基序、二硫键桥半胱氨酸残基以及酶原激活和信号肽。仅在非翻译区观察到这些cDNA之间的差异，在Bt抗性品系中发生了鸟嘌呤取代腺嘌呤的情况。Southern和Northern印迹分析表明，两个品系中类胰蛋白酶原的基因组组织和mRNA表达没有重大差异。这些数据表明，印度谷螟Bt敏感品系和Bt抗性品系中类胰蛋白酶原基因及其转录相似。
