两种鳞翅目幼虫对Bt敏感性的差异及其机理的探讨

本文用生物鉴定和ＳＤＳ－ＰＡＧＥ等方法研究了两种鳞翅目昆虫家蚕与菜粉蝶幼虫对４株苏云金杆菌的敏感性差异及其机理。生物鉴定结果表明菜粉蝶幼虫对Ｂｔ的敏感性比家蚕低。用两种昆虫的胃液体外活化伴孢晶体，ＳＤＳ－ＰＡＧＥ结果表明，菜粉蝶幼虫胃液对伴孢晶体的活化能力低，不能将无杀虫活性的原毒素在短时间内转化为毒性肽。将伴孢晶体称经家蚕胃液化再饲喂菜粉蝶幼虫，同样条件下死亡率提高３－５倍。当菜粉蝶幼虫胃液的ｐ     

球形芽直菌杀蚊毒素的作用方式和蚊虫的抗性特征

球形芽孢杆菌是一种对蚊幼虫有毒杀作用的好气芽孢杆菌。高毒力菌株在孢形成过程中能产生有５１．４和４１．９ｋＤａ蛋白组成的伴孢晶体。蚊幼虫取食芽孢／晶体混合物后,晶体蛋白被降解形成活性蛋白而结合到中肠上皮细胞的特异性结合位点上,最后导致蚊幼死亡;低毒力菌和部分高毒力菌株在其营养体生长阶段可产生可溶性的１００、３０．５和３８．５ｋＤａ的Ｍｔｘ毒素。该毒晶体毒素其它杀虫纱无同源性,其杀蚊人艇机理不祥。在实     

粘虫颗粒体病毒对苏云金杆菌δ-内毒素的酶解活化作用

粘虫颗粒体病毒(Psedualetia unipuncta granulovirus,PuGV-Ps)对苏云金杆菌(Bacillus thuring-iensis,Bt)具有增效作用.为明确其增效机制,采用SDS-PAGE方法研究PuGV-Ps对Bt δ-内毒素的降解活化作用.结果表明,碱性条件下晶体蛋白和PuGV-Ps共同孵育,130kD的δ-内毒素被进一步酶解为分子量为110、87、61、47 kD等多种不同的肽链,其酶解活化程度随缓冲液pH的升高不断加深,在pH值10.7的0.1 mol/L Na2CO3缓冲液中,δ-内毒素完全降解,并产生具有一定抗蛋白酶继续降解的分子量为47、60和61 kD的活性片段.用氨苯磺胺偶氮酪蛋白为底物测定PuGV-Ps中总蛋白酶活性的结果表明,PuGV-Ps在pH值为7.38～10.38时均具有蛋白酶活性,且蛋白酶活性随pH升高而显著提高.4种蛋白酶抑制剂均可抑制PuGV-Ps的蛋白酶活性,且以STI的抑制作用最强.SDS-PAGE试验同样显示了STI可以抑制PuGV-Ps对δ-内毒素的酶解活化.     

苏云金素对昆虫细胞株的毒力试验

苏云金杆菌伴孢晶体毒素对昆虫细胞株的毒性以及用昆虫细胞株测定伴孢晶体毒素的含量,国内外都发表了一些研究报告,并有所进展。然而,苏云金素对昆虫细胞株的毒性尚未见这方面的报告,本文将简报这一研究结果。     

对夜蛾科幼虫高毒力的苏云金芽孢杆菌菌株及其晶体蛋白特性

本文对自德国土壤中分离的８株苏云金芽孢杆菌进行毒力测定和晶体蛋白特性的研究。生物测定结果表明，８株Ｂｔ中除Ｓ２１－１、Ｓ２１－１３对甜菜夜蛾无毒性外，对夜蛾科的４种供试昆虫均有很高的毒效，４８小时校正死亡率高达８０％～１００％；扫描电镜观察和ＳＤＳ－ＰＡＧＥ晶体蛋白电泳结果可知，Ｓ１１－６和Ｓ２１－１３２个菌株只有一种菱形晶体和一条相应的１３５ＫＤａ主带，其余６个菌株均有菱形和方形两种晶体，以及两条相应的１３５ＫＤａ和６５ＫＤａ主带。另对晶体的特性与其毒力的关系进行了讨论。     

苏云金芽孢杆菌的分类及生物活性蛋白基因

本文综述叙述了苏云金苏孢杆菌鞭毛抗原血清亚种的分类、生理生化鉴定，伴孢晶体对无脊椎动物４个门中的生物以及节肢动物门中９个目昆虫的生物活性，生物活性蛋白及其其编码这些蛋白基因原定位和分类等方面的新动态。     

苏云金芽胞杆菌晶体毒素的多样性

苏云金芽胞杆菌是生物防治中应用最为广泛的一种杀虫剂,它对多种昆虫和其他一些无脊椎动物具有特异的毒杀活性。苏云金芽胞杆菌的杀虫活性主要来自于菌体在形成芽胞期间生成的伴胞晶体毒素,这些晶体毒素在结构和生物学功能方面表现高度的多样性。本文介绍了苏云金芽胞杆菌晶体毒素的分类、命名方法,深入讨论了晶体毒素的氨基酸序列、三维结构、靶标生物和杀虫机理的多样性。评价了各种分类方法的特点,并展望了晶体毒素作用机制研究和未来晶体毒素新基因的发掘。     

Bt制剂防治棉铃虫“五忌”

Ｂｔ制剂防治棉铃虫“五忌”一忌选用伴孢晶体含量低或过期的产品。因为Ｂｔ制剂杀虫的主要有效成份是伴孢晶体，而不是芽孢的数量，而过期的产品中伴孢晶体毒性蛋白大部分已经钝化，丧失了杀棉铃虫活性。二忌在温度较低的条件下使用。Ｂｔ制剂防治棉铃虫的最佳温区是２５...     

紫外线对Bt伴孢晶体的损伤和腐植酸的保护作用

用SDS-PAGE电泳分析和生物测定等方法研究了紫外线对苏云金芽孢杆菌（Bacillus thuringiensis,简称Bt）伴孢晶体的损伤及腐植酸对Bt伴孢晶体的保护作用。结果表明，紫外线对Bt伴孢晶体有明显的损伤，伴孢晶体紫外线辐射3h后溶解性能及生物活性基本丧失。腐植酸对紫外线有较强的吸收作用，能有效地减轻紫外线对Bt伴孢晶体的损伤。     

提高Bt农药的防治效果

提高Ｂｔ农药的防治效果（湖南省农科院植保所长沙４１０１２５）谭荫初Ｂｔ是苏芸金芽孢杆菌（Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ）的简称。这是一种能在细胞内形成菱形伴孢晶体的芽孢杆菌。伴孢晶体是能毒害昆虫的肠道而使之瘫痪致死的强毒性蛋白质结晶。芽孢...     

昆虫对生物农药的抗性机制及对策

本文综述了昆虫对B.t等生物杀虫剂的抗性机制及延缓昆虫抗性发展所应采取的措施。昆虫通过下列不同机制产生抗生：1）昆虫的血淋巴对B.t等生物杀虫剂的营养细胞的抑制作用。2）各种来源的蛋白酶对毒素蛋白的过度降解作用。3）昆虫中肠沉淀蛋白对毒素蛋白的沉淀作用。4）中肠上皮修复能力增强。5）中肠的吸附位点对毒素蛋白的亲和力下降。通过加强对B.t菌株的选育，合理科学的用药方式及采用不同的模式进行植物基因操作以提高杀虫蛋白的表达和活性等综合措施，减缓和降低昆虫抗性的发展。     

Digestive proteases of Papilio demoleus : Compartmentalization and characterization

Digestive proteolytic activities were studied in the larval midgut of Papilio demoleus by using azocasein, specific substrates and inhibitors regarding enzymatic allocation in the alimentary canal. The highest proteolytic activity was found in the midgut rather than foregut and hindgut that highest proteolytic activity was observed in soluble fraction. All specific proteases were almost equally distributed in both the anterior and posterior midgut except for trypsin- and chymotrypsin-like proteases, which showed the highest activity in the anterior midgut. Two peaks were observed in alkaline and acidic pHs when general proteolytic activities were assayed using azocasein in soluble and membrane-bound fractions. Specific substrates revealed the presence of trypsin-like, chymotrypsin-like, elastase, cathepsins B and L as well as two exopeptidases. These findings were confirmed using specific inhibitors including PMSF, TLCK, TPCK, SBTI, cystatin, DTT, E-64, EDTA and phenanthroline. Determination and characterization of the insect’s digestive proteases will enable development of pest control by designing protease inhibitors. This procedure will help us to designate an efficient pest control method with the lowest impact on the environment.     

昆虫对Bt杀虫蛋白的抗性机制及治理策略

转Bt基因抗虫作物在害虫综合防治中发挥重要作用，是新一代植物保护产品，其抗虫性能和经济效益已得到普遍肯定。然而由于转基因抗虫植物在整个生育阶段高水平的表达Bt杀虫蛋白，有可能会导致害虫对Bt杀虫蛋白产生抗性，抗性的产生将严重影响Bt植物的应用。田间已经发现小菜蛾Plutella xylostella（Linnaeus）对Bt杀虫剂产生了抗性，实验室人工汰选条件下已经有多种害虫对Bt杀虫蛋白产生了抗性，并对抗性产生的机制进行了研究，目前主要认为害虫产生抗性与杀虫蛋白与中肠细胞上受体蛋白结合能力的改变以及Bt杀虫蛋白在中肠的水解作用的变化有关，但抗性产生的机制还与其它因素有关，估计是多方面因素造成的，因此，抗性机制的产生还不十分清楚。目前已经制订了“高剂量＋庇护所”和多基因策略等一系列有效抗性治理策略，这些策略还需要在深入研究并阐明抗性机制的基础上，进一步完善，以实现Bt抗虫植物在害虫防治中的可持续利用。     

羧肽酶在Bt抗性棉铃虫中肠的表达分析

本研究利用实时定量PCR技术检测了对Bt(Bacillus thuringiensis)Cry1Ac敏感和抗性品系棉铃虫Helicov-erpa armigera中肠羧肽酶(carboxypeptidase)的mRNA表达,发现在抗性品系中羧肽酶表达量明显下降(约2.6倍);中肠汁液中羧肽酶酶活测定结果表明,抗性品系羧肽酶酶活性同样显著下降约2.2倍。羧肽酶作为重要的消化性外切酶(exopeptidase),能够消化摄取食物中的大量可吸收的蛋白质,抗性品系羧肽酶表达量显著下降表明该种群消化食物的能力降低,这可能会导致与抗性适合度相关的多种生理指标的下降。另外,体外成功表达棉铃虫羧肽酶,为从蛋白和抗体水品进一步研究羧肽酶在棉铃虫抗性适合度中的作用奠定基础。     

棉铃虫APN基因家族系统进化与功能分析

氨肽酶N（APN）是锌金属蛋白酶M1家族的成员,鳞翅目昆虫中肠刷状缘膜囊泡（brush border membrane vesicles,BBMV）细胞膜上的APN不仅在蛋白消化吸收过程中起着重要的作用,而且是Bt的重要受体蛋白。本研究通过PCR技术克隆得到7条棉铃虫APN基因全长序列,HaAPN1～7（Genbank登录号为MT002819～MT002825）。经生物信息学分析,HaAPN1～7全长为2592～3099 bp,编码863～1032个氨基酸,分子量为110～115 kDa,等电点为4.7～6.4,N端信号肽为15～20 aa。系统进化分析结果表明,HaAPN1～7分属于Class 1～7类别。RT-qPCR结果表明,在敏感棉铃虫5龄幼虫中肠中APN2的表达量最高,APN7的表达量最低;Cry1Ac抗性品系棉铃虫中各类APN的表达趋势与敏感品系一致。取食Cry1Ac、Cry2Ab和Vip3Aa蛋白后棉铃虫中肠APN活性显著降低,取食Cry1Ac和Vip3Aa蛋白后中肠液和BBMV中APN活性都显著降低,而取食Cry2Ab蛋白后只有BBMV上APN活性显著降低;棉铃虫对Cry1Ac、Cry2Ab和Vip3Aa产生耐受性后,中肠液和BBMV中APN活性也都发生显著变化。因此,不同类别的棉铃虫APN在Bt的杀虫机制中可能起到不同的作用,APN活性变化可能与Bt蛋白的降解及抗性演化相关。     

Prohibitin,an essential protein for Colorado potato beetle larval viability,is relevant to Bacillus thuringiensis Cry3Aa toxicity

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30 kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828 pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5 days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.     

Midgut juice of Plutellaxylostella highly resistant to Bacillusthuringiensis Cry1Ac contains a three times larger amount of glucosinolate sulfatase which binds to Cry1Ac compared to that of susceptible strain

Midgut juice of Plutellaxylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SDS–PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SDS-PAGE bound to Cry1Ac and soybean agglutinin, and K_D for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, K_m and V_max were estimated and involvement of the enzyme in the PXR resistance was discussed.     

Activity of gut proteinases from Cry1Ab-selected colonies of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae)

Susceptibility to the Cry1Ab protoxin and toxin from Bacillus thuringiensis (Berliner) and activity of gut proteinases were assessed in both susceptible and Cry1Ab-selected colonies of European corn borer, Ostrinia nubilalis (Hubner). Resistance in two different selected colonies was at least 6- and 15-fold for the Cry1Ab protoxin and 108- and 484-fold for the Cry1Ab toxin. Activities of trypsin-like, chymotrypsin-like and elastase-like proteinases were variable among the colonies tested and not indicative of a major contribution to Cry1Ab resistance. Activation of the 130-kDa Cry1Ab protoxin occurred rapidly in all colonies, with no apparent differences among colonies. In addition, there were no apparent changes in activated Cry1Ab processing, indicating that proteolytic degradation was not associated with resistance. These results suggest that mechanisms other than proteolytic activation of protoxin and toxin degradation, such as target site modification may be involved in the resistance to B thuringiensis Cry1Ab in these O nubilalis colonies.     

类钙粘蛋白对Cry3A毒素杀虫活性的影响

将克隆的黄粉虫类钙粘蛋白基因片段在大肠杆菌中表达纯化,并分析其对Cry3A毒素杀虫活性的影响,从而探讨黄粉虫类钙粘蛋白对Cry3A毒素杀虫活性的增效作用。黄粉虫类钙粘蛋白基因片段在大肠杆菌中表达产物为包涵体,经尿素变性后,用亲和纯化法可得到较纯的重组蛋白;随着类钙粘蛋白片段与Cry3A质量比值的升高,Cry3A毒素对黄粉虫的杀虫活性也逐步提高,当其比值为1时,活性达到最大,提高了2.5倍,而黄粉虫类钙粘蛋白片段对Cry3Am的杀虫活性却没有显著性影响。研究表明,黄粉虫类钙粘蛋白对Cry3A毒素的杀虫活性具有增效作用。