Yield, Leaf Senescence, and Cry 1 Ac Expression in Response to Removal of Early Fruiting Branches in Transgenic Bt Cotton

Two-year field experiments were conducted at Linqing, Yellow River valley of China, to study the plant response to the removal of early fruiting branches in transgenic Bt (Bacillus thuringiensis) cotton (Gossypium hirsutum L.) from 2003 to 2004. Plants were undamaged and treated by removing two basal fruiting branches (FB) at squaring to form the control and the removal treatment, respectively. The plant height, leaf area (LA), dry weight of fruiting forms (DWFF), the number of fruiting nodes (NFN), photosynthetic (Pn) rate, and levels of leaf chlorophyll (Chl), N, P, K, and Cry 1 Ac protein in mainstem leaves were measured at a 10-or 20-d interval after FB removal, and the sink/source ratio as indicated by NFN/LA and DWFF/LA was determined. FB removal significantly increased the plant height, LA, and plant biomass in both years. Lint yields were increased 7.5 and 5.2% by removal compared with their controls in 2003 and 2004, respectively. Significant increases in boll size (5.7 and 5.1%) were also observed in removal than in control for both years. Either NFN/LA or DWFF/LA was significantly reduced by removal before 40d after removal; however, both NFN/LA and DWFF/LA were significantly enhanced by FB removal at 80d after removal compared to the untreated control. There was no significant difference in fiber quality in the first two harvests between removal and control, but fiber strength and micronarie in the third harvest were significantly improved by FB removal. In terms of leaf Chi, Pn rate, levels of total N, P, and K in late season, leaf senescence was considerably delayed by FB removal. Levels of Cry 1 Ac protein in the fully expanded young leaves were considerably higher in FB-excised plants than in control, indicating FB removal enhanced Cry 1 Ac expression. It is suggested that the yield and quality improvement with FB removal may be attributed to the increased NFN/LA or DWFF/LA in late season and delayed leaf senescence, respectively. FB removal can be a potential practice incorporated into the intensive cultivation system for enhancing transgenic Bt cotton production.     

转双价双Bt基因(Cry1Ac+Cry2Ab)抗虫棉栽培地生存竞争能力研究

为建立国家转双价双Bt抗虫基因棉环境安全评价技术,对转双价双Bt基因(Cry1Ac+Cry2Ab)抗虫棉栽培地生存竞争能力进行研究,结果表明,转双价双Bt基因棉花与对照赣棉11相比,各生育期株高生长具有显著竞争优势,优势值3.48～4.75 cm;吐絮期棉覆盖度竞争优势超对照46.77个百分点,达显著水平;产量竞争力优势籽棉超对照1 013.85 kg/hm2,皮棉超对照433.20 kg/hm2,均未达到显著水平;发芽率竞争优势极显著,超过对照2.75个百分点。与非转基因常规棉相比,转双价双Bt抗虫基因棉生存竞争能力优势显著。     

留叶枝去早果枝对抗虫棉生长发育及产量的影响

为探索留叶枝去早果枝对抗虫棉生长发育和产量的影响,于2004—2005年以两个不同熟性的抗虫棉品系K640和K9918为材料,在济南和临清进行了留叶枝去早果枝试验研究。结果表明,与正常整枝(去叶枝)相比,留叶枝去早果枝显著促进了两个品系的株高增长和叶面积扩展,叶面积系数提高了24%￣94%。早熟品系K640在2004年和2005年分别比正常整枝增产7.4%和15.6%,而中早熟品系K9918的产量在2004年与正常整枝相当、2005年减产12.0%。留叶枝去早果枝显著减少了伏前桃比例,而相应提高了伏桃和秋桃的比例。留叶枝去早果枝对平均衣分影响不大,但显著影响铃数。分析认为,留叶枝去早果枝对棉花生长发育和产量的效应主要是通过提高叶源、降低库源比实现的,只有在叶源相对不足的情况下采取该措施才有增产作用。     

苏云金芽孢杆菌杀虫晶体蛋白对棉铃虫活性分析

利用已克隆的5种Bt Cry基因Cry2Ab4、Cry1Ia8、Cry1Ie1、Cry1Ca7、Cry1Cb2和1种野生菌株HD-73(Cry1Ac)表达的6种Bt Cry杀虫晶体蛋白,对棉铃虫进行生物活性分析,并将Cry1Ac杀虫晶体蛋白分别与其它5种Cry蛋白按1:1的比例组合,对棉铃虫进行生物活性测定。结果表明,单独使用Cry1Ac时对棉铃虫活性最高,LC50为3.16μg·mL-1,其次为Cry2Ab4。Cry1Ac与Cry2Ab4组合对棉铃虫也有较高的活性,LC50为48.70μg·mL-1,该组合对棉铃虫的共毒系数为1.21,有相加作用。Cry1Ac与这5种蛋白的组合对棉铃虫都有较高的毒力。     

两种转Bt基因棉杀虫蛋白Cry1Ac表达量的检测

用EnvirologixCry1Ab/Cry1Ac平板试剂盒检测了两种转Cry1Ac基因的棉花品系 (NUCOTN33B、NUCOTN99B) 以及常规对照品系 (苏棉 12) 不同生育期主茎嫩叶、侧枝嫩叶、蕾及蕾的苞叶中杀虫蛋白Cry1Ac的含量。结果表明, 两种转Bt基因棉主茎嫩叶杀虫蛋白Cry1Ac的含量随生育期的推移呈明显下降趋势, 花铃期 (NUCOTN33B和NUCOTN99B分别为 4 43和 2 93μg·g-1 ) 和吐絮期 (3 87和 2 86μg·g-1 ) 的含量分别降至苗期第 6叶 (7 64和 8 38μg·g-1 )的 58%、35%和 51%、34%; 相同时期的主茎嫩叶和侧枝嫩叶中Cry1Ac的含量均显著高于蕾及蕾的苞叶, 前两者均为后两者的两倍以上。     

利用分子标记辅助选择技术培育抗虫水稻品种的研究

Bt基因是从苏云金芽孢杆菌中克隆出的针对鳞翅目害虫的抗虫基因,经过基因工程方法人工改造已经获得Cry1Ab/Ac、Cry1C等抗虫基因。以云南主栽水稻品种楚粳28为受体,以携带Bt抗虫基因的"华恢1号"、"RJ-5"和"T1C-19"分别作为供体,利用分子标记辅助选择(MAS)技术进行回交育种,选育成携带Bt抗虫基因水稻新品系。结合农艺性状表现,获得了云抗虫稻1号、云抗虫稻2号、云抗虫稻3号Bt蛋白高表达的水稻抗虫品系。这些抗虫品系为云南省抗虫水稻的遗传改良和生产应用提供了基因资源和应用基础。     

用人工合成多肽作为半抗原制备Bt Cry1Ac的单克隆抗体

为了制备Bt Cry1Ac特异性单克隆抗体(MAB),本试验从NCBI获得了Bt Cry1Ac蛋白的氨基酸序列,根据抗原性、亲水性和表位性分析选定Bt Cry1Ac特异性肽段进行人工合成,并将其偶联于匙孔血蓝蛋白（KLH）免疫动物,应用细胞融合技术制备了抗该肽段的杂交瘤细胞34株。通过ELISA和免疫印迹试验从中筛选出与Bt Cry1Ac天然蛋白产生特异性反应的单克隆抗体杂交瘤细胞株一株（6G9）。ELISA和免疫印迹试验结果显示,该克隆株所分泌的MAB能对合成肽和Bt Cry1Ac产生特异性反应,而与同源的Bt Cry1Aa、Bt Cry1Ab天然蛋白均无交叉反应。通过对转基因棉花的ELISA 检测结果表明,本试验所制备的Bt Cry1Ac单克隆抗体能够有效的区分常规棉和抗虫棉,并且能对其中的Bt Cry1Ac蛋白进行特异性的识别。     

二代棉铃虫不防治下SGK321与石远321叶片衰老的比较

以双价转基因抗虫棉SGK321和非抗虫棉石远321为试验材料,在大田条件下研究了二代棉铃虫不防治条件下两品种叶片衰老方面的差异。结果表明,在二代棉铃虫不防治条件下,非抗虫棉石远321棉铃虫危害严重,幼蕾脱落比SGK32I平均每株多6个;受害石远321的单位叶面积载铃量和根冠比显著低于SGK321,叶面积和叶面积系数显著高于SGK321,叶片内细胞分裂素类物质含量在盛花期高于SGK321,而脱落酸含量整个生育期则明显低于SGK．321;与SGK321相比,石远321的主茎功能叶具有较高的SOD活性、较高的叶绿素含量和较低的MDA含量。因二代棉铃虫危害而损失部分早期蕾,改变了非抗虫棉的库源关系,在一定程度上延缓了石远321叶片的衰老。     

Bt基因介导的大豆抗蚜虫表达载体的构建

[目的]构建用于大豆抗蚜虫研究的Bt基因介导的Cry1Ac植物表达载体。[方法]以含有Cry1Ac的质粒pRH201为模板进行PCR扩增,将PCR产物克隆至载体质粒pBS中,将重组质粒转化至大肠杆菌DH5α,并对重组质粒进行酶切和测序鉴定。经验证的Cry1A基因扩增产物双酶切后与pBI121载体连接转化,挑取重组子pBIC121进行BamHⅠ／SnaBⅠ双酶切鉴定。[结果]从pRH201质粒中扩增获得长约3．5kb的Cry1Ac基因序列,对重组质粒pBSCRY的插入片段进行测定,确定了CrylAc片段的全长为3534bp,共编码1176个氨基酸。[结论]构建了Cry1A基因的植物表达载体,为大豆抗蚜虫转基因研究奠定了基础。     

25种南亚热带植物耐阴性的初步研究

该文研究了南亚热带不同生活型植物的耐阴性,为城市绿化植物选择配置提供实验依据.选取25种南亚热带植物的盆栽幼苗作为试验材料,其种类包括目前正在推广的园林植物和采自森林的灌木和地被植物,用LI-6200光合作用测定系统等仪器测定各种植物叶片的形态特征、光合作用和光合-光响应曲线,包括叶片厚度(LT)、叶面积(LA)、比叶面积(SLA)、含水量(LWC)、叶绿素含量(Chl)、叶绿素a/b值(Chl a/b)、光合速率(Pn)、暗呼吸速率(Rd)、光补偿点(LCP)、光饱和点(LSP)和表观量子效率(Φ)等生理指标,并对测定结果进行方差分析、相关分析和聚类分析,比较分析植物的耐阴程度.结果表明,25种植物可分为3类: 第1类为耐阴性较强的硬枝老鸭嘴、百两金、野芋、桢桐、板蓝根、狮子尾、红背桂、虾脊兰、宫粉郁金和黑骨蕨等10种,第2类为耐阴性中等的石柑子、大花鸳鸯茉莉、北江砂仁、崖爬藤、合果芋和华南胡椒等6种,第3类为耐阴性相对较弱的可爱花、鸡爪兰、咖啡、扇蕨、棕叶九尾草、草果、千年健、大叶仙茅和虎舌红等9种.Pn与Chl a/b值和Rd呈显著的正相关,与Chlb呈显著的负相关;LCP与LSP呈显著正相关;Chl b与Chl a和Chl a+b呈极其显著正相关,与Chl a/b呈显著负相关;SLA与LA呈显著正相关,与LWC呈极其显著正相关.      

转Bt(Cry1Ab)基因对水稻光合特性及光合产物积累的影响

 【目的】明确外源Bt基因插入对水稻倒1叶光合速率及光合作用相关生理特性的影响。【方法】以转Bt基因水稻及亲本水稻为试材,研究盆栽及田间条件下转Bt基因及亲本水稻苗期、拔节期、抽穗期和成熟期倒1叶光合特性及其相关光合酶活性、光合产物积累的动态变化。【结果】转Bt基因及亲本水稻生理活动峰值均出现在拔节期。与亲本水稻相比,转Bt基因水稻苗期叶片净光合速率显著低于亲本;而拔节期和抽穗期,转Bt基因水稻倒1叶净光合速率、叶绿素含量和乙醇酸氧化酶活性显著高于亲本水稻（P＜0.05）,且拔节期转Bt水稻倒1叶气孔导度和胞间CO2浓度也显著高于亲本水稻（P＜0.05）;成熟期,转Bt基因与亲本水稻倒1叶光合特性无显著性差异（P＞0.05）。【结论】与亲本水稻相比,外源Bt基因的插入对水稻叶片光合特性产生短暂影响,但这种影响不具有持续性。     

转双价基因棉田昆虫群落的多样性研究

2002-2003年在田间研究了转双价基因(CrylAc CpTI)棉田昆虫群落的多样性,并分析了昆虫群落多样性的时间格局,结果表明,4种类型棉田昆虫群落的多样性指数以双价棉田和Bt棉田最高,其次为常规棉田,常规施药田最低;害虫亚群落多样性指数以双价棉和Bt棉田最高,其次为常规用药田,常规棉田最低;天敌亚群落多样性指数以双价棉田最高,其次为常规棉田和常规用药田,Bt棉田最低,4种类型棉田昆虫群落的多样性时间格局总体变化趋势为5月升高,6月下降,7～9月又有所回升。     

基于三级营养传递Cry1Ac蛋白对普通草蛉酶活力及生长发育的影响

【目的】研究Cry1Ac蛋白通过棉花、棉蚜的传递对普通草蛉酶活性及生长发育的影响,为转Bt基因棉花的安全性评价提供参考和依据。【方法】采用ELISA方法,检测Bt棉、取食Bt棉的棉蚜和捕食棉蚜普通草蛉体内Bt杀虫蛋白含量;通过酶活力测定普通草蛉幼虫取食Bt棉花上棉蚜后其体内的类胰蛋白酶、类胰凝乳蛋白酶和氨肽酶的酶活性;室内生物测定普通草蛉取食Bt棉花上的棉蚜后对其生长发育的影响。【结果】Cry1Ac杀虫蛋白在棉叶中的表达量为788.76 ng/g,远高于棉蚜体内的2.35 ng/g,取食Bt棉花上棉蚜的普通草蛉3龄幼虫体内并未检测到Bt杀虫蛋白;通过三级营养传递的Cry1Ac蛋白对普通草蛉酶活力影响甚微。【结论】棉蚜取食Bt棉花后体内仅能累计微量Cry1Ac蛋白;普通草蛉取食Bt棉花上的棉蚜后,对其体内三种酶活性和生长发育均没有负面影响。     

密度与整枝对抗虫杂交棉产量分布的影响

于2008年在山东临清、嘉祥和金乡三地大田条件下研究了不同密度、不同整枝条件下抗虫杂交棉的库源比例、产量分布和熟相。试验采用裂区设计,主区由去叶枝和留叶枝构成,副区包括3.0、4.5、6.0、7.5株/m^2 4个密度。结果表明：无论是整枝还是密度处理,产量主要分布在棉株下中层（77%～85%）和内围（76%～88%）,上层（15%～23%）和外围（12%～24%）产量分布较少。密度和整枝对棉花产量的空间分布有显著的效应,但两者的互作效应不显著。密度主要影响产量在棉株上的内外分布,随密度升高,产量向内围集中;而整枝则主要影响产量在棉株上的垂直分布,留叶枝使产量上下分布更加分散。土壤肥力和耕作栽培方式的不同可影响叶枝对产量的贡献率,在临清,叶枝对产量的平均贡献率为11.2%;而在嘉祥和金乡,叶枝对产量的平均贡献率分别为19.4%和18.0%。随着密度的升高,叶枝对产量的贡献率显著降低,密度为3.0、7.5株/m^2时3个试验点叶枝对产量平均贡献率分别为24.9%和8.9%。留叶枝未影响棉花熟相,但密度影响熟相。适当提高密度可以降低库源比例,延缓棉花早衰。     

转双Bt基因巨霸杨外源基因表达及抗虫性检测

为提高转基因杨树中Bt基因的表达效率并扩大抗虫谱,以同时转入Cry1Ac和Cry3A基因的转双Bt基因巨霸杨(Populus deltocdes‘55/56’×P.deltocdes‘2KEN8’)株系1年生苗为材料,对外源基因的表达和抗虫性进行检测。经PCR检测,证明目的基因已被整合到巨霸杨基因组中。利用荧光定量PCR和ELISA技术检测了4个转基因株系叶片中Bt基因的转录丰度和毒蛋白表达量。结果表明:不同株系间Bt基因的转录丰度存在显著差异,Cry3A基因的转录丰度显著高于Cry1Ac基因的转录丰度;2种Bt毒蛋白表达量在各株系间存在显著差异,Cry3A毒蛋白质量分数均显著高于Cry1Ac毒蛋白质量分数。各株系对鳞翅目害虫美国白蛾(Hyphantria cunea)幼虫抗虫效果不明显,且无显著差异;对鞘翅目害虫柳蓝叶甲(Plagiodera versicolora)表现出极高抗虫效果,且均显著高于转单基因Cry3A 741杨高抗株系CC84,不同株系间存在显著差异。     

陆地棉品种资源耐复合盐碱性综合评价分析

以119份国内外陆地棉品种资源为试验材料,测定了复合盐碱胁迫下陆地棉的叶绿素(Chl)、株高(PH)、果枝(FB)、果节(FN)、始节高(HFN)、叶面积(LA)、单株结铃数(BN)、铃重(BW)、衣分(LP)共9个农艺性状指标,并采用模糊数学的隶属函数法对陆地棉的耐盐性进行综合评价。结果表明:复合盐碱胁迫下,不同地域陆地棉的Chl、PH、FB、FN、HFN、LA、BN、BW、LP的耐盐系数存在差异,其中LP、BW、Chl差异较小,变异系数为0.078～0.171;而FN、BN、LA差异较大,变异系数为0.34～0.48。综合评价结果显示,119份陆地棉品种资源中,耐盐性为敏感(42.8%)和中耐盐(37.0%)的材料占多数,并筛选得到豫棉15、KK-1047、新陆中69等20份高耐盐及耐盐材料,可以为陆地棉耐盐遗传改良的亲本材料。     

留叶枝对抗虫杂交棉库源关系的调节效应和对叶片衰老与皮棉产量的影响

【目的】去叶枝是我国传统的棉田管理技术，但现有研究认为叶枝具有保留利用的价值。因此进一步探索棉花留叶枝的可行性及对库源关系、叶片衰老和产量、品质的影响与机理十分必要。【方法】于2004～2005年在山东临清比较研究了大田稀植（3.0株/m2）条件下3个转Bt基因抗虫杂交棉去叶枝和留叶枝后的形态和生理变化。【结果】与去叶枝相比，留叶枝棉花的衣分变化不大，全株平均铃重有所降低，铃数显著增多，2004和2005年的皮棉单产分别比去叶枝的对照提高了9.8%和6.2%， 但纤维长度和比强度略低于去叶枝的对照。留叶枝棉株的绿叶比例和冠层叶绿素含量高于去叶枝的棉株，但主茎功能叶的叶绿素含量和光合速率却显著低于对照。留叶枝具有中前期增源、后期扩库的作用，但对中前期的“流”似有不利影响。【结论】抗虫杂交棉在稀植条件下保留叶枝具有一定的增产作用，这与留叶枝增源、扩库的作用有关；留叶枝没有从根本上延缓棉花冠层的衰老，也没有改善纤维品质的作用。     

不同Bt蛋白对捕食性天敌大草蛉的生态安全性评价

【目的】明确转基因棉花中表达Cry1Ac、Cry1F和Cry2Ab蛋白对我国Bt棉田中主要捕食性天敌大草蛉（Chrysopa pallens）的潜在影响。【方法】通过在大草蛉人工饲料中添加高剂量Bt蛋白的方法,评价Cry1Ac、Cry1F和Cry2Ab蛋白对大草蛉重要生命表参数（4龄幼虫体重、幼虫发育历期、化蛹率、蛹重、蛹发育历期、羽化率、成虫体重和产卵量）的影响,以不添加杀虫物质的大草蛉纯人工饲料作为阴性对照,以添加PA（砷酸二氢钾,KH₂AsO₄）的大草蛉人工饲料作为阳性对照;同时,利用双抗体夹心酶联免疫吸附法（DAS-ELISA）测定取食Bt蛋白处理饲料后,大草蛉幼虫、蛹以及成虫体内的Bt蛋白浓度;利用ELISA法和敏感昆虫生物测定法,检测新鲜饲料和在试验环境条件下暴露2 d的处理饲料中Bt蛋白的稳定性和生物活性;此外,利用酶活性测定的方法,比较取食空白对照饲料、Bt蛋白处理饲料或PA处理饲料后大草蛉体内主要消化酶（总蛋白酶、类胰蛋白酶、类凝乳蛋白酶和氨肽酶）、解毒酶（α-乙酸萘酯酯酶、羧酸酯酶、谷胱甘肽-S-转移酶、乙酰胆碱酯酶、酸性磷酸酶和碱性磷酸酶）和保护酶（超氧化物歧化酶、过氧化物酶和过氧化氢酶）的活性。【结果】生命表评价结果表明,在大草蛉人工饲料中添加高浓度单独Bt蛋白或Bt蛋白混合物,对大草蛉4龄幼虫体重、幼虫发育历期、化蛹率、蛹重、蛹发育历期、羽化率、成虫体重和产卵量无显著性不利影响,但饲料中添加PA却能够使大草蛉幼虫和蛹发育历期极显著延长,4龄幼虫体重、化蛹率、蛹重、羽化率、成虫体重和产卵量极显著降低;大草蛉体内Bt蛋白浓度测定结果显示,大草蛉取食Bt蛋白处理饲料后,其幼虫、蛹及成虫体内均含有一定量的Bt蛋白;ELISA和敏感昆虫生物测定结果表明,新鲜准备和在试验环境条件下暴露2 d的Bt蛋白处理饲料,均含有高剂量且具有生物活性的Bt蛋白,因此,大草蛉在整个生物测定过程中接触到的均为高浓度的、具有生物活性的Bt蛋白;此外,取食Bt蛋白处理饲料,对大草蛉幼虫和成虫体内主要消化酶、解毒酶和保护酶活性无显著性影响,但取食PA处理饲料能使大草蛉幼虫和成虫体内氨肽酶活性极显著降低,其他所测消化酶、解毒酶和保护酶活性显著升高。【结论】大草蛉对3种供试Bt蛋白（Cry1Ac、Cry1F和Cry2Ab）不敏感,取食这3种Bt蛋白对大草蛉生长发育及其体内主要消化酶、解毒酶和保护酶活性没有显著不良影响,本研究所建立的生物评价体系还可用于评价其他新型转基因抗虫植物中表达杀虫蛋白对大草蛉的生态安全性。     

Assessment of the potential toxicity of insecticidal compounds to Peristenus spretus,a parasitoid of mirid bugs

With the increased cultivation of Bt crops in China, Apolygus lucorum and other mirid bugs have emerged as important agricultural pests because they are insensitive to the Bt proteins. In addition, the reduction of pesticide applications after planting Bt crops also increases the severity of mirid bug outbreaks. Peristenus spretus is a parasitoid of mirid nymphs, but its sensitivity to Bt proteins is not known. In the current study, we developed a dietary exposure assay to assess the effects of Bt proteins (Cry1Ab, Cry1Ac, Cry1F, Cry2Aa, and Cry2Ab) on P. spretus adults using a diet consisting of a 10% honey solution with or without Bt proteins at 400 µg g^–1 diet. The results showed that the survival and reproduction of P. spretus adults were reduced by the cysteine protease inhibitor E-64 (a positive control) but were not affected by any of the five Bt proteins. The activities of digestive, detoxifying, and antioxidant enzymes in P. spretus were also unaffected by diets containing the Cry proteins, but they were significantly affected by the diet containing E-64. We then developed a tri-trophic bioassay to determine the effects of the five Bt proteins on P. spretus larvae and pupae. In this assay, A. lucorum nymphs fed an artificial diet containing Cry proteins were used as the hosts for P. spretus. The results of the tri-trophic assay indicated that neither the pupation rate nor the eclosion rate of the P. spretus parasitoids were significantly affected by the presence of high concentrations of Bt proteins in the parasitized A. lucorum nymphs. The overall results indicate that these two assays can be used to evaluate the toxicity of insecticidal compounds to P. spretus and that the tested Cry proteins are not toxic to P. spretus.     

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis(Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis(Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins(Cry2 A, Cry1 C, Cry1 Aa, and Cry1 Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride(PMSF), tosyl-L-lysine chloromethyl ketone(TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid(EDTA), PMSF, tosyl phenylalanine chloromethyl ketone(TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane(E-64). EDTA could significantly inhibit the degradation of Cry2 A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1 C and Cry1 Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1 Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.