家蚕氨肽酶和类钙粘蛋白与苏云金芽孢杆菌Cry1Ac毒素相互作用（英文）

苏云金芽孢杆菌Bacillusthuringiensis生产的晶体毒素被广泛用作农林害虫的杀虫剂。鳞翅目昆虫受体蛋白是阐明其与晶体毒素相互作用的重要模式。文中纯化了苏云金芽孢杆菌的晶体毒素蛋白,质谱鉴定为Cry1Ac毒素,然后重组表达家蚕氨肽酶N (BmAPN6)和类钙粘蛋白(CaLP)结合结构域。利用免疫共沉淀、Far-Western印迹和酶联免疫吸附试验,证明Cry1Ac毒素蛋白和BmAPN6之间的相互作用。在Sf9细胞中,对Cry1Ac毒素的细胞毒活性分析,表明BmAPN6参与Cry1Ac毒素诱导的细胞形态异常和裂解死亡。文中也利用相同的方法,对钙粘蛋白的3个结合位点CR7、CR11和CR12进行相互作用分析,结果表明3个重复结构域是CaLP的Cry1Ac结合位点。上述结果表明,BmAPN6和CaLP可作为Cry1Ac毒素致病的功能性受体,为进一步揭示晶体毒素的致病机制和基因编辑增强家蚕抗病性提供了研究靶标。     

RNAi介导的棉铃虫氨肽酶N基因Haapnl和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N(aminopeptidase N,APN)和钙粘蛋白(cadherin)是存在于鳞翅目昆虫中肠刷状缘膜囊(brush border membrane vesicles,BBMV)上Bt毒素Cry1A的受体.本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapnl和钙粘蛋白基因Ha_BtR双链RNA(dsRNA)注入棉铃虫4龄幼虫体内,以研究这两种受体基因沉默后对Cry1Ac毒力的影响.结果表明:注射dsRNA(1 μg/头)进行基因沉默后,Haapnl mRNA表达量比注射缓冲液(elution solution,ES)的对照下降了30%～49%,Ha_BtR mRNA表达量下降了30%～37%.注射Haapnl dsRNA的幼虫在40和70 μg/cm2 Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫,而在100和170 μg/cm2 Cry1Ac原毒素处理下两者死亡率无显著差异;Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异.当同时注射Haapnl及Ha_BtR dsRNA后,干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降.本研究进一步证明了棉铃虫Haapnl和Ha_BtR均是Bt毒素Cry1Ac的功能受体,这两种受体蛋白共同参与Cry1Ae的毒杀作用过程.该结果也提示.Haapnl或Ha_BtR基因产生突变都可能导致棉铃虫对CrylAc产生抗性.     

RNAi介导的棉铃虫氨肽酶N基因Haapn1和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N（aminopeptidase N, APN）和钙粘蛋白（cadherin）是存在于鳞翅目昆虫中肠刷状缘膜囊（brush border membrane vesicles, BBMV）上Bt毒素Cry1A的受体。本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapn1和钙粘蛋白基因Ha_BtR双链RNA（dsRNA）注入棉铃虫4龄幼虫体内, 以研究这两种受体基因沉默后对Cry1Ac毒力的影响。结果表明: 注射dsRNA（1 μg/头）进行基因沉默后, Haapn1 mRNA表达量比注射缓冲液（elution solution, ES）的对照下降了30%~49%, Ha_BtR mRNA表达量下降了30%~37%。注射Haapn1 dsRNA的幼虫在40和70 μg/cm² Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫, 而在 100 和 170 μg/cm² Cry1Ac原毒素处理下两者死亡率无显著差异; Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异。当同时注射Haapn1及Ha_BtR dsRNA后, 干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降。本研究进一步证明了棉铃虫Haapn1和Ha_BtR均是Bt毒素Cry1Ac的功能受体, 这两种受体蛋白共同参与Cry1Ac的毒杀作用过程。该结果也提示, Haapn1或Ha_BtR基因产生突变都可能导致棉铃虫对Cry1Ac产生抗性。     

家蚕氨肽酶 (BmAPN5) 与黑胸败血芽孢杆菌伴孢晶体 (PC) 毒素相互作用

氨肽酶N(APN)属于锌金属肽酶M1(Peptidase_M1)家族的成员,不仅参与蛋白水解过程,而且也作为毒素受体参与病原微生物的致病过程。家蚕氨肽酶家族含有16个成员,其中BmAPN4结合黑胸败血芽孢杆菌产生的伴孢晶体(PC)毒素,为研究该基因家族其他成员是否与PC毒素结合,参与其致病过程。本文克隆家蚕中肠特异表达的氨肽酶家族成员BmAPN5基因,全长3 313 bp,编码953个氨基酸,含有1个锌金属肽酶M1和ERAP1_C结构域。构建原核表达载体,表达和纯化获得可溶性GST-BmAPN5重组蛋白。Far-Western blotting、免疫共沉淀和ELISA等实验结果表明BmAPN5和活化的PC毒素相互结合。通过构建BmAPN5细胞转染载体,转染Sf9细胞系,与PC毒素共孵育,导致细胞形态改变和裂解死亡;同时,乳酸脱氢酶含量测定结果 (LDH)表明BmAPN5参与PC毒素致病过程,导致细胞裂解死亡,使细胞培养基中的乳酸脱氢酶升高。上述结果表明BmAPN5作为一种功能性受体,PC毒素与其相互作用,参与了病原物的致病过程,为进一步揭示病原微生物黑胸败血芽孢杆菌与宿主相互作用的致病机制研究奠定了基础。     

一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响

为探讨苏云金芽孢杆菌Bacillus thuringiensis（Bt）杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明：N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.     

钙粘蛋白与苏云金芽胞杆菌晶体毒素 互作机制研究进展

昆虫钙粘蛋白是苏云金芽胞杆菌晶体毒素的重要受体之一,本文简述了昆虫钙粘蛋白种类、结构特征和在昆虫体内的分布,对昆虫钙粘蛋白与苏云金芽胞杆菌毒素相互结合位点进行了较深入讨论;详细论述了苏云金芽胞杆菌晶体毒素作用孔洞模式和信号转导模式,并简要介绍了钙粘蛋白与昆虫抗性的关系.     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1AC毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1C产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1Ac 毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1c 产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

粉纹夜蛾类Cry1Ac受体氨肽酶NcDNA片段的克隆与分析

设计简并引物,采用RT-PCR方法对粉纹夜蛾Trichoplusia ni(Hubner)细胞系BTI-TN-5B1-4的氨肽酶N(aminopeptidase N,APN)基因cDNA片段进行了克隆和序列分析,通过两对引物扩增出了两种氨肽酶N基因的cDNA片段,大小分别为188 bp和564 bp,分别命名为AS188(GenBank登录号:CD809324)和AS564(GenBank登录号:CD809326).对这两个片段推导的氨基酸序列进行同源性分析,结果表明两者与已报道的鳞翅目昆虫中肠的Cry1Ac毒素受体氨肽酶N有较高的同源性.     

昆虫类钙粘蛋白与Bt Cry1A蛋白之间的相互作用

类钙粘蛋白(cadherin-likeprotein)位于昆虫中肠刷状缘膜囊泡(brushbordermembranevesicles,BBMV)上,是苏云金芽孢杆菌(Bacillusthuringiensis,Bt)产生的杀虫晶体蛋白(BtCry蛋白)的主要受体之一。它能够与BtCry蛋白结合,引起细胞膜的渗透性发生改变,促进BtCry蛋白对敏感昆虫的毒杀作用。类钙粘蛋白基因的突变还能导致敏感昆虫对BtCry蛋白产生抗性。因此,研究昆虫类钙粘蛋白与BtCry蛋白之间的相互作用,将有助于揭示BtCry蛋白杀虫作用机理。文章对昆虫类钙粘蛋白种类、结构特征、在昆虫体内的分布、及其与BtCry蛋白之间的相互作用等方面的研究现状进行详细论述。     

GalNAc pretreatment inhibits trapping of Bacillus thuringiensis Cry1Ac on the peritrophic membrane of Bombyx mori

Bombyx mori (ShunreixShogetsu) is sensitive to Cry1Aa and resistant to Cry1Ac, both insecticidal proteins of Bacillus thuringiensis. Cry1Aa passed through the peritrophic membrane (PM) much faster (0.37 microg/mm2 PM/h) than Cry1Ac (0.05 microg/mm2 PM/h) during the initial observation period. Both Cry1Aa and Cry1Ac bound to the PM but only the binding of Cry1Ac was specifically inhibited by N-acetylgalactosamine (GalNAc). When Cry1Ac was pretreated with GalNAc, Cry1Ac permeated the PM much faster. These results suggested that Cry1Ac bound a PM protein via GalNAc on a sugar side chain. The role of the PM on Cry1Ac resistance of B. mori was briefly discussed.     

Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N

The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Location of the Bombyx mori 175kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin

To identify and gain a better understanding of the cadherin-like receptor-binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin-like protein-binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175kDa cadherin-like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, amino peptidaseN1, from Bo.mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N-(9-acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175-binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175-binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo.mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.     

昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展

鳞翅目昆虫中肠上皮细胞刷状缘膜(BBM)上的Bt杀虫晶体蛋白毒素受体氨肽酶N(APN)的结构和位点密度的改变是昆虫对Bt毒素的主要抗性机制之一,该文简要综述了APN受体的研究进展。每种昆虫中肠上皮细胞中有数种APNs,彼此间同源性较高,其中部分APNs为crylA家族毒素的功能性受体。不同种类昆虫的APNs受体,甚至同一种昆虫的不同类型APNs,其所结合的毒素种类可能不同。APNs决定该昆虫对crylA类毒素的敏感程度差异。有些抗性昆虫的APNs基因编码区发生了多个点突变。     

Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dissociated epithelial cells from Bombyx mori

Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with labelled 125I-Cry1Ab revealed a marked reduction in reversible and irreversible binding when anti-BtR175 (a cadherin-like protein) was used for BBMV pre-treatment. By contrast, the anti-APN1 antibody specifically affected the irreversible binding, while the reversible binding component was not affected. This is the first time that binding of Cry1Ab to APN1 and to a cadherin-like protein from BBMVs in solution has been shown. Dissociated epithelial cells from the Kin strain were used to test the inhibitory effect of the antibodies on the cytotoxicity of Cry1Ab. Pre-incubation of the cells with the anti-BtR175 antibody conferred protection against Cry1Ab, but not the anti-APN1 antibody. Therefore our results seem to support the two models of the mode of action of Cry1Ab in Lepidoptera, depending on whether BBMVs or intact dissociated cells are used, suggesting that both pathways may co-operate for the toxicity of Cry1A toxins in vivo.     

Purification of the Cry1Ac protein of Bacillus thuringiensis and assessment against the Plutella xylostella and soil microbial community

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is the most commonly used to develop insect‐resistant living modified organisms (LMOs). Insecticidal proteins produced in transgenic plants are released into the soil from the roots. In this study, possible effects of crystal 1Ac (Cry1Ac) protein on the soil microbial community in Korea were studied. To purify the insoluble Cry1Ac protein expressing Escherichia coli cells, we performed repeated sonication and PBS washing of the insoluble part and Cry1Ac protein was isolated in soluble form from the insoluble form using 100 mM Na₂CO₃ buffer (pH 9.6) without affinity bead. Also, size‐exclusion chromatography (SEC) was performed to increase the purity of the isolated Cry1Ac protein. The final protein product was identified as Cry1Ac protein through MALDI‐TOF. Insecticidal activity of Cry1Ac protein was demonstrated through the death of Plutella xylostella treated with Cry1Ac protein. Purely isolated Cry1Ac protein showed the same insecticidal activity as Cry1Ac expressed in LM crops. To investigate the change of soil microbial distribution using maize field soils treated with Cry1Ac protein, we isolated high quality metagenomic DNAs from buffer‐ and Cry1Ac protein‐treated soil groups, and analyzed the distribution of soil microorganisms through next‐generation sequencing (NGS) analysis. NGS results showed a similar microbial distribution in both buffer‐ and Cry1Ac protein‐treated samples. These results suggest a useful risk assessment method for domestic targeted insect and soil microorganisms using the Cry1Ac protein.