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马铃薯羧肽酶抑制剂与Cry1Ac毒素混用对棉铃虫中肠蛋白酶活性及杀虫活性的影响
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引用本文：陈文波,臧中旭,孙龙龙,郜晓妍,谢桂英,赵新成.马铃薯羧肽酶抑制剂与Cry1Ac毒素混用对棉铃虫中肠蛋白酶活性及杀虫活性的影响.植物保护学报,2021,48(5):1147-1155
DOI：10.13802/j.cnki.zwbhxb.2021.2021867
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作者单位E-mail
陈文波 河南农业大学植物保护学院昆虫系, 郑州 450002  
臧中旭 河南农业大学植物保护学院昆虫系, 郑州 450002  
孙龙龙 河南农业大学植物保护学院昆虫系, 郑州 450002  
郜晓妍 河南农业大学植物保护学院昆虫系, 郑州 450002  
谢桂英 河南农业大学植物保护学院昆虫系, 郑州 450002  
赵新成 河南农业大学植物保护学院昆虫系, 郑州 450002 xincheng@henau.edu.cn 
中文摘要:为研发转多价抗虫棉，利用蛋白酶专性底物测定马铃薯羧肽酶抑制剂（potato carboxypeptidases inhibitor，PCI）和Cry1Ac毒素单用及混用后棉铃虫Helicoverpa armigera幼虫中肠内总蛋白酶、类胰蛋白酶、类胰凝乳蛋白酶和羧肽酶的活性、羧肽酶A（carboxypeptidase A，CPA）和羧肽酶B（carboxypeptidase B，CPB）基因的相对表达量及对棉铃虫的杀虫活性。结果表明，饲喂20 μg/cm2 PCI+1 μg/cm2 Cry1Ac、40 μg/cm2 PCI+5 μg/cm2 Cry1Ac含药饲料后，棉铃虫3龄幼虫中肠内CPA活性分别为0.20 U/mg和0.16 U/mg，显著低于对照；总蛋白酶活性均为0.15 OD·min-1·mg-1，与对照差异不显著；类胰蛋白酶活性均为0.45 μmol·min-1·mg-1，均显著低于其他处理，CPB基因表达量较对照显著下调。饲喂20 μg/cm2 PCI、40 μg/cm2 PCI、1 μg/cm2 Cry1Ac、5 μg/cm2 Cry1Ac、20 μg/cm2 PCI+1 μg/cm2 Cry1Ac、40 μg/cm2 PCI+5 μg/cm2 Cry1Ac含药饲料后，CPB活性分别为0.26、0.26、0.24、0.26、0.27和0.26 U/mg，与对照差异不显著；棉铃虫3龄幼虫中肠内类胰凝乳蛋白酶活性分别为0.48、0.39、0.42、0.41、0.40和0.45 μmol·min-1·mg-1，6者之间差异不显著，但均显著低于对照；棉铃虫3龄幼虫中肠内CPA基因表达量均较对照显著上调。饲喂20 μg/cm2 PCI+0.02 μg/cm2 Cry1Ac、40 μg/cm2 PCI+0.02 μg/cm2 Cry1Ac、20 μg/cm2 PCI+0.06 μg/cm2 Cry1Ac、40 μg/cm2 PCI+0.06 μg/cm2 Cry1Ac含药饲料后，棉铃虫初孵幼虫实际校正死亡率分别为66.54%、68.28%、77.74%和85.76%，均高于其预期校正死亡率，表明PCI和Cry1Ac两者对杀虫活性表现为独立作用和增效作用，两者可共同用于棉铃虫幼虫的防治。
中文关键词:棉铃虫  羧肽酶抑制剂  Cry1Ac毒素  蛋白酶活性  杀虫活性
 
Effects of potato carboxypeptidases inhibitor combined with Cry1Ac toxin on protease activities and insecticidal activity against cotton bollworm Helicoverpa armigera
Author NameAffiliationE-mail
Chen Wenbo Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China  
Zang Zhongxu Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China  
Sun Longlong Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China  
Gao Xiaoyan Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China  
Xie Guiying Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China  
Zhao Xincheng Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China xincheng@henau.edu.cn 
Abstract:In order to develop insect-resistance pyramided cotton varieties, the activities of total proteases, trypsin-like proteases, chymotrypsin-like proteases and carboxypeptidases were determined using the specific substrates, and the expression levels of carboxypeptidase A (CPA) and B (CPB) genes in the third instar larvae of cotton bollworm Helicoverpa armigera fed on different concentrations of potato carboxypeptidases inhibitor (PCI), Cry1Ac or PCI+Cry1Ac were also investigated, and the effects of PCI toward insecticidal activity of Cry1Ac against the newly hatched larvae of H. armigera were further tested in bioassay. The results showed that when the third instar larvae of H. armigera were fed on an artificial diets containing 20 μg/cm2 PCI+1 μg/cm2 Cry1Ac and 40 μg/cm2 PCI+5 μg/cm2 Cry1Ac, respectively, the activities of CPA were 0.20 U/mg and 0.16 U/mg,which were significantly lower than those in the control; the activities of total protease were 0.15 OD·min-1·mg-1, which had no significant difference from those in the control; the trypsin-like enzyme activities were 0.45 μmol·min-1·mg-1, which were significantly lower than those in other groups; the expression levels of CPB significantly lowered than those in the control. When the third instar larvae of H. armigera were fed on an artificial diets containing 20 μg/cm2 PCI, 40 μg/cm2 PCI, 1 μg/cm2 Cry1Ac, 5 μg/cm2 Cry1Ac, 20 μg/cm2 PCI+1 μg/cm2 Cry1Ac and 40 μg/cm2 PCI+5 μg/cm2 Cry1Ac, the activities of CPB were 0.26, 0.26, 0.24, 0.26, 0.27 and 0.26 U/mg, which had no significant difference from those in the control; chymotrypsin-like protease activities were 0.48, 0.39, 0.42, 0.41, 0.40 and 0.45 μmol·min-1·mg-1, respectively, among which were no significant difference, but they were all significantly lower than the control; the expression levels of CPA significantly increased compared with those in the control. When the newly hatched larvae of H. armigera were fed on an artificial diets containing 20 μg/cm2 PCI+0.02 μg/cm2 Cry1Ac, 40 μg/cm2 PCI+0.02 μg/cm2 Cry1Ac, 20 μg/cm2 PCI +0.06 μg/cm2 Cry1Ac and 40 μg/cm2 PCI+0.06 μg/cm2 Cry1Ac, the observed corrected mortalities were 66.54%, 68.28%, 77.74% and 85.76%, which were higher than its expected corrected mortality, the results suggested that there were independent and synergistic interactions between PCI and Cry1Ac toxin, their combination can be used to in resistance pyramid to the pest H. armigera larvae.
keywords:Helicoverpa armigera  carboxypeptidases inhibitor  Cry1Ac toxin  protease activity  insecticidal activity
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