昆虫中肠Bt毒素受体氨肽酶N的研究进展

氨肽酶N(APN)是昆虫中肠中主要的Bt毒素受体,它与Cry毒素特异结合后,毒素插入细胞膜,在膜上形成孔洞,细胞裂解,最终导致昆虫死亡.APN的变异导致昆虫对Bt敏感性下降甚至产生抗性.就APN的结构特征、分类、APN与Cry毒素的相互作用机制以及APN与昆虫Bt抗性的关系作一综述.     

Bt毒素受体钙黏蛋白的酶联免疫检测方法

报道了3种鳞翅目昆虫(棉铃虫、斜纹夜蛾、二化螟)中肠细胞膜上Bt毒素受体--钙黏蛋白的酶联免疫(ELISA)检测方法.在这3种鳞翅目幼虫中肠BBMV(brush border membrane vesicles)粗提液中,棉铃虫和斜纹夜蛾的总蛋白含量相近,而二化螟中肠BBMV的总蛋白含量较低,大约为棉铃虫的一半.采用棉铃虫钙黏蛋白合成多肽的多克隆抗体对3种幼虫的BBMV进行了ELISA分析,建立了ELISA检测的方法与条件,当用该抗体检测二化螟幼虫的BBMV时灵敏度很高,其次是斜纹夜蛾与棉铃虫幼虫.     

棉铃虫氨肽酶N基因片段克隆、表达和内源蛋白检测

氨肽酶N（APN）是苏云金芽孢杆菌杀虫毒素Cry在昆虫中肠中的一个重要受体。研究氨肽酶N在昆虫中肠中的分布特征对于阐明Cry毒素的杀虫机理和昆虫对Cry毒素的抗性机理具有重要的意义。通过RT-PCR的方法从棉铃虫中肠上皮细胞中克隆得到氨肽酶N的基因片段APN1551,并诱导表达纯化得到其重组蛋白APN517。以此蛋白为抗原,制备其抗血清。用该抗血清能检测到棉铃虫中肠上皮细胞中的APN蛋白。为研究Cry毒素的作用机理奠定基础。     

Cry2Aa毒素对小菜蛾的杀虫机理

为研究苏云金杆菌Cry2Aa毒素的生物活性及杀虫机理,先利用浸叶法测定了商品化的Cry2Aa毒素对小菜蛾的杀虫活性,并用石蜡包埋切片后进行免疫组化分析,再用ELISA法测定了Cry2Aa毒素与小菜蛾刷状缘膜囊泡(BBMV)的结合曲线,最后通过Ligand blot法和肽指纹质谱对小菜蛾BBMV与Cry2Aa毒素的结合蛋白进行了分离与鉴定。结果表明,Cry2Aa毒素对小菜蛾的半数致死浓度(LC_(50))为27.90μg/ml。Cry2Aa毒素与小菜蛾中肠上皮细胞存在结合,与小菜蛾BBMV的表观结合亲和力为266.6 nmol/L。小菜蛾BBMV与Cry2Aa毒素存在5条主要结合条带,其中分子量2.45×10~5上方的主结合条带酶解后得到ATYSEGPNGSVR片段,通过数据库比对分析,匹配到一个分子量为3.32×10~5的小菜蛾未鉴定蛋白,其功能注释为脂质运载蛋白。     

小菜蛾碱性磷酸酯酶受体表达与分子模拟

【目的】利用原核表达小菜蛾(Plutella xyllostella)中肠膜结合碱性磷酸酯酶(membrane-bound alkaline phosphatase,mALP)并经Ligand blot验证其具有与Cry1Ac毒素结合的能力;通过同源建模和分子对接研究Cry1Ac-mALP的结合模式,预测毒素和受体结合区域及关键氨基酸位点(热点残基),为了解毒素-受体互作机制及分子改造增强Cry毒素活性的研究打下基础。【方法】针对小菜蛾mALP全长设计引物,并以小菜蛾c DNA为模板扩增mALP基因,双酶切后用T4连接酶连接至pET-26b原核表达载体,将构建的pET-26b-mALP载体转化Trans1-T1克隆感受态,挑取克隆并提取质粒后进行PCR、双酶切和测序验证,将验证无误的重组质粒转化E.coliBL21(DE3)表达感受态细胞,进行诱导表达。将诱导表达后的mALP转至PVDF膜上,通过Western blot和Ligand blot分别验证mALP是否成功表达以及是否具有与Cry1Ac毒素结合的能力。对mALP进行同源建模、分子动力学模拟以及模型评价,获得的mALP最佳三维结构与Cry1Ac毒素利用Patch DOCK和Fire Dock程序进行分子对接试验,对确定的最佳毒素-受体复合物进行结合区域和结合氨基酸位点分析,并通过计算机辅助的丙氨酸突变扫描试验确定毒素和受体参与的关键氨基酸残基。【结果】扩增出小菜蛾mALP基因并克隆至pET-26b原核表达载体,转化E.coli BL21(DE3)表达感受态后挑取阳性克隆提取质粒后进行PCR、双酶切和测序均显示构建正确。通过原核表达和Western blot验证成功表达了mALP蛋白,并经Ligand blot试验证实了原核表达的mALP具有和Cry1Ac毒素结合的能力。利用同源建模成功获得了mALP的三维结构,通过Patch DOCK和Fire Dock分子对接程序,获得毒素和受体的对接复合物,通过溶剂可及表面积变化计算和Ligplot分析,确定毒素结构域Ⅱ和结构域Ⅲ均参与了受体结合,并且毒素和受体均以疏水结合和氢键结合模式参与结合,最后通过热点残基预测发现Cry1Ac毒素和mALP中分别有3个氨基酸残基(376ASN、443SER和486SER)和4个氨基酸残基(452ARG、499THR、502TYR和513TYR)是参与互作的关键氨基酸位点。【结论】经原核表达的小菜蛾mALP同样具有与Cry1Ac毒素结合的能力,并利用分子模拟技术预测了小菜蛾mALP三维结构及与Cry1Ac毒素结合模式。     

亚洲玉米螟miR-1a-5p表达与Cry1Ab蛋白敏感性的关系

【目的】探索亚洲玉米螟miR-1a-5p表达与Cry1Ab蛋白敏感性的关系,为揭示miRNA在昆虫Bt抗性产生过程中的作用提供依据。【方法】通过荧光定量PCR,研究亚洲玉米螟Cry1Ab抗性品系(ACB-AbR)和Bt敏感品系(ACB-BtS)表皮及中肠组织中miR-1a-5p的表达差异。以Sf9细胞为研究对象,通过转染miR-1a-5p促进剂(Mimics)或抑制剂(Inhibitor)改变细胞中miR-1a-5p的表达量,测定不同处理细胞对Cry1Ab蛋白的敏感性,探讨miR-1a-5p表达量改变后Sf9细胞对Cry1Ab蛋白的敏感性变化。通过荧光素酶报告试验,研究miR-1a-5p对潜在靶标基因Unigene12370_All的调控作用。【结果】ACB-AbR表皮组织中miR-1a-5p的表达量显著高于ACB-BtS表皮组织,ACB-AbR中肠组织中miR-1a-5p的表达量显著低于ACB-BtS中肠组织;ACB-AbR和ACB-BtS幼虫表皮组织中miR-1a-5p的表达量均显著高于其中肠组织。Sf9细胞miR-1a-5p表达量升高导致Sf9细胞对Cry1Ab蛋白的敏感性增强,反之亦然。荧光素酶报告试验结果提示,miR-1a-5p与靶标基因Unigene12370_All可以结合。【结论】亚洲玉米螟miR-1a-5p表达量的变化与其对Cry1Ab蛋白敏感性的改变有关。     

Bt Cry1类毒素共性结构域的分析、表达及鉴定

【目的】分析定位Bt Cry1类毒素Cry1Ab、Cry1Ac、Cry1B、Cry1C、Cry1F的共性结构域,克隆并表达共性结构域蛋白,为筛选Bt毒素广谱抗体及建立广谱检测方法打下基础。【方法】利用生物信息学和分子模拟技术,通过SWISS-MODEL同源建模分别对5种Cry1类毒素进行三维建模,并结合Ramachandran plot、ERRAT和Verify3D方法评价模型构象的合理性。通过分析比对5种Cry1类毒素的三维结构,确定DomainⅠ区域作为5种Cry1毒素的共性结构域。以含Cry1Ac基因的苏云金芽孢杆菌库斯塔克亚种为模板设计引物,PCR扩增获得共性结构域DomainⅠ基因,将其经NcoⅠ和NotⅠ双酶切连接至原核表达载体pET-26b(+),构建原核表达载体pET-26b-DomainⅠ。重组质粒经菌液PCR、双酶切以及测序鉴定验证正确后,转化至E.coli BL21(DE3),经终浓度为1 mmol·L~(-1)的IPTG在20℃下诱导表达16h后检测共性结构域蛋白的表达情况。离心收集诱导表达的大肠杆菌菌液,进行超声波破碎处理,收集上清及沉淀,采用SDS-PAGE分析融合蛋白的表达。利用His-Trap HP镍亲和柱纯化上清中的可溶性融合蛋白,经SDS-PAGE电泳、Western blot和ELISA试验验证纯化的共性结构域蛋白的生物活性。【结果】基于氨基酸序列及三维空间比对分析,发现5种Cry1类毒素的DomainⅠ的序列一致性最高,而且它们的DomainⅠ三维结构几乎完全重合,确定DomainⅠ区域作为5种Cry1毒素的共性结构域,通过PCR、双酶切及测序鉴定成功构建原核表达载体pET-26b-DomainⅠ,经IPTG诱导表达、His-Trap HP镍亲和柱纯化获得了可溶性的DomainⅠ共性结构域蛋白,SDS-PAGE和Western blot证实表达的共性结构域蛋白的分子量约为33.4 kD,且能与抗His标签鼠单克隆抗体发生特异性反应,ELISA试验证实共性结构域蛋白与5种Cry1类毒素特异性抗体均具有很强的结合能力,抗原表位分析结果显示共性结构域蛋白具有和完整的Cry蛋白存在多个潜在抗原表位位点的特征,抗原表位区域所占的比例分别为48.4%和63.6%,表明共性结构域蛋白具有良好的免疫原性和免疫反应性。【结论】基于分子模拟与分子克隆技术,成功定位及表达纯化获得共性结构域蛋白,为下一步利用共性结构域为靶标分子制备广谱特异性识别Cry1类毒素抗体打下基础。     

Bt(Cry1F)毒素多克隆抗体制备及其检测应用

通过免疫新西兰大白兔,制备并纯化Bt(Cry1F)毒素多克隆抗体,建立针对Cry1F毒素检测的免疫学分析方法,用于室内分析毒素在农产品和土壤中的残留情况。采用皮下注射法,经4次级联免疫,获得免疫血清效价为1:2 500 000,经柱纯化后,测得抗体浓度为4.70 mg/m L;并以其建立的针对Cry1F毒素的间接非竞争时间分辨荧光免疫分析方法(TRFIA),测得线性检测范围在0.04~2 000 ng/m L,最低检测灵敏度为0.03 ng/m L,对5种供试毒素类似物(Cry1Ab、Cry1Ac、Cry1B、Cry1C、Cry2A)均具一定交叉识别能力。分别以稻米和黄土为基质,进行添加回收试验,测得批内、批间的稳定性和重复性均达到室内仪器检测要求。     

棉铃虫中肠特异性结合蛋白ABCC1对Cry1Ac毒力的影响

【目的】 研究棉铃虫（Helicoverpa armigera）中肠蛋白ABCC1（HaABCC1）与Cry1Ac的结合特性及对Cry1Ac毒力的影响,明确HaABCC1在Cry1Ac杀虫机制中的作用。【方法】 分析HaABCC1基因序列,设计引物,通过原核表达得到HaABCC1两个跨膜区片段的蛋白,与Cry1Ac进行Ligand blot试验,验证其与Cry1Ac的体外结合特性;利用RNAi技术干扰棉铃虫幼虫的HaABCC1,在3龄幼虫腹部注射siABCC1,比较HaABCC1的表达量及Cry1Ac处理后棉铃虫死亡率的变化;通过细胞转染将ABCC1导入Sf9细胞系中,确定pAc-ABCC1重组质粒转入Sf9细胞后,用细胞生物测定的方法比较Cry1Ac处理后细胞死亡率的变化;比较敏感品系（96S）和Cry1Ac抗性品系（BtR）棉铃虫的HaABCC1基因全长序列,并通过荧光定量RT-PCR检测HaABCC1在抗、感棉铃虫中的表达量。【结果】 HaABCC1跨膜区TMD1和TMD2在Escherichia coli BL21（DE3）感受态细胞中成功表达,两个HaABCC1跨膜区片段蛋白均能与活化的Cry1Ac在体外结合;棉铃虫注射siABCC1后,HaABCC1的表达量显著下降,与未注射的棉铃虫、注射DEPC水和siEGFP的棉铃虫相比,用活化的Cry1Ac蛋白处理HaABCC1被干扰的棉铃虫,其幼虫死亡率显著降低,表明棉铃虫幼虫的HaABCC1被干扰后,能显著降低Cry1Ac对棉铃虫的毒力;用活化的Cry1Ac蛋白处理成功转入HaABCC1的Sf9细胞,与对照Sf9细胞相比,细胞的死亡率明显上升,表明将HaABCC1导入Sf9后能显著提高Cry1Ac处理后的细胞死亡率;抗性品系（BtR）与敏感品系（96S）棉铃虫的HaABCC1氨基酸序列没有差别,但抗性品系BtR棉铃虫HaABCC1的表达量显著降低。【结论】 HaABCC1是Cry1Ac的特异性结合蛋白,可能是Cry1Ac的功能受体蛋白,并可能参与对Cry1Ac的抗性机制。     

利用基因工程抗体建立的新型双抗夹心ELISA法检测Cry1Ac毒素

为明确新型双抗夹心ELISA方法检测Cry1Ac毒素的效果,分别用纯化后的抗Cry1Ac单链抗体和anti-Cry1Ac兔多克隆血清(Anti-Cry1Ac-PAbs)作为捕获抗体和检测抗体,通过方阵滴定确定其最佳工作浓度,建立双抗夹心ELISA法。再用优化后的双抗夹心ELISA方法对检测Cry1Ac毒素的敏感性、特异性和回收率进行鉴定。结果表明,新型双抗夹心ELISA法最低检测限为0.91 ng/ml,线性检测范围为1.04 ng/ml~3.49μg/ml。5种稻米中Cry1Ac毒素的平均回收率为69.42%~87.95%,变异系数为1.19%~6.50%。     

苏云金芽孢杆菌cry1Ea基因的克隆及 生物信息学分析

不同来源的苏云金芽孢杆菌能产生多种多样的晶体(Cry)蛋白.基于这个特性,人们可以通过基因工程的手段向工程菌中转入编码多种Cry毒素的基因来控制虫害.通过DNA重组技术,从BtHZM2菌株中克隆出了cry1Ea基因,对其进行了生物信息学分析,同源比对结果表明.cry1Ea8基因的核苷酸序列与已知cry1Ea的同源性为99.77%～99.91%.对应的氨基酸序列同源性为99.49%～99.74%.对cry1Ea8基因的分析还揭示出了cry1Ea8及其编码蛋白的一些生物和理化性质.结构域预测表明,Cry1Ea8由3个结构域组成,其中N-末端螺旋状结构域与膜插入与孔隙形成有关,而第二和第三个结构域与受体的结合有关.该研究为转基因抗虫植物和微生物杀虫工程菌的构建提供了新的基因来源.     

Engineering modified Bt toxins to counter insect resistance

The evolution of insect resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins that are widely used in sprays and transgenic crops. Resistance to Bt toxins in some insects is linked with mutations that disrupt a toxin-binding cadherin protein. We show that susceptibility to the Bt toxin Cry1Ab was reduced by cadherin gene silencing with RNA interference in Manduca sexta, confirming cadherin's role in Bt toxicity. Native Cry1A toxins required cadherin to form oligomers, but modified Cry1A toxins lacking one alpha-helix did not. The modified toxins killed cadherin-silenced M. sexta and Bt-resistant Pectinophora gossypiella that had cadherin deletion mutations. Our findings suggest that cadherin promotes Bt toxicity by facilitating toxin oligomerization and demonstrate that the modified Bt toxins may be useful against pests resistant to standard Bt toxins.     

Genome editing of the SfABCC2 gene confers resistance to Cry1F toxin from Bacillus thuringiensis in Spodoptera frugiperda

ATP-binding cassette transporter C2(ABCC2) is known to be a receptor for Bacillus thuringiensis(Bt) toxins in several lepidopteran insects. Mutations in the ABCC2 gene have been genetically linked to field-evolved resistance to the Cry1 F toxin from Bt in Spodoptera frugiperda. Here we generated a SfABCC2 knockout strain of S. frugiperda using the CRISPR/Cas9 system to provide further functional evidence of the role of this gene in susceptibility and resistance to Cry1 F. Results from bioassays showed that the SfABCC2 knockout S. frugiperda strain displayed 118-fold resistance to Cry1 F compared with the parental DH19 strain, but no resistance to Vip3 A toxin from Bt. These results provide the first reverse genetic evidence for SfABCC2 as a functional receptor for Cry1 F.     

Identification of a gene associated with Bt resistance in Heliothis virescens

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used for pest control. Bt-resistant insect strains have been studied, but the molecular basis of resistance has remained elusive. Here, we show that disruption of a cadherin-superfamily gene by retrotransposon-mediated insertion was linked to high levels of resistance to the Bt toxin Cry1Ac in the cotton pest Heliothis virescens. Monitoring the early phases of Bt resistance evolution in the field has been viewed as crucial but extremely difficult, especially when resistance is recessive. Our findings enable efficient DNA-based screening for resistant heterozygotes by directly detecting the recessive allele.     

Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme

The development of resistance is the main threat to the long-term use of toxins from Bacillus thuringiensis (Bt) in transgenic plants. Here we report the cloning of a Bt toxin resistance gene, Caenorhabditis elegans bre-5, which encodes a putative beta-1,3-galactosyltransferase. Lack of bre-5 in the intestine led to resistance to the Bt toxin Cry5B. Wild-type but not bre-5 mutant animals were found to uptake toxin into their gut cells, consistent with bre-5 mutants lacking toxin-binding sites on their apical gut. bre-5 mutants displayed resistance to Cry14A, a Bt toxin lethal to both nematodes and insects; this indicates that resistance by loss of carbohydrate modification is relevant to multiple Bt toxins.     

The Minimal Active Fragment of the Cry1Ai Toxin is Located Between 36I and 605I

The novel cry1Ai gene that cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21 (DE3), and the insecticidal activity against 2nd- instar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36I and 605I, which is smaller than the regions previously reported for Cry1A. The first two amino acids (34T and 35P) on helix α-1 and whole helix α-2 of domain I and sheet β-32 of domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.     

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.     

Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain

Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene.Because Cry1 Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops.The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments.The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin.In this paper, three media for crystal production and the method for Cry1 Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1 Ac protoxin.The results showed that the 1/2 LB(Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1 Ac protoxin in 300 m L 1/2 LB medium was(112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method.It will be a reference for other Cry protoxin preparation, especially for larger number.