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1.
刘慧  李博  牛林  邱林  王永 《生物安全学报》2018,27(4):255-259
【目的】Bt杀虫蛋白发挥杀虫活性的重要前提是Cry蛋白能够与昆虫中肠上皮细胞刷状缘膜囊(BBMVs)上的受体蛋白结合。在前期获得二化螟氨肽酶N1(Aminopeptidase N,APN1)基因全长序列的基础上,明确二化螟APN1多肽片段与Cry2Aa的结合能力。【方法】将二化螟APN1序列片段在大肠杆菌BL21(DE3)中表达,利用蛋白质单向电泳和ligand blotting技术分析二化螟APN1多肽片段与Cry2Aa的结合能力。【结果】重组载体可在表达菌株BL21(DE3)中表达一个约70 ku的蛋白,纯化后的多肽条带单一,纯度较好。Ligand blot分析结果显示,表达的二化螟APN1多肽片段可以与活化的Cry2Aa杀虫蛋白结合,且结合条带随着重组蛋白上样量的降低而减弱。【结论】APN1多肽片段可以与Cry2Aa结合,为阐明APN1基因的功能奠定基础,也为其他Bt蛋白的受体蛋白相关研究提供新的借鉴。  相似文献   

2.
【目的】Bt杀虫蛋白(Bacillus thuringiensis)具有高度的靶标特异性,已经被广泛用于农业害虫防治。Bt杀虫蛋白要发挥杀虫活性,必须首先与其受体蛋白结合,氨肽酶N(Aminopeptidase N)是一类重要的Bt受体蛋白。因此,分析该受体与Bt杀虫蛋白的结合能力,可为进一步明确不同Bt的分子作用机制、Bt的抗性治理以及新Bt的开发应用等提供借鉴。【方法】本文利用Ligand blot和Elisa方法比较了棉铃虫Helicoverpa armigera中肠APN4(Aminopeptidase N4,APN4)与Cry1Ac、Cry2Aa的结合能力。【结果】原核表达的APN4片段与活化的Cry1Ac、Cry2Aa都可以结合,解离常数(Kd)分别是48.59 nmol/L和21.73 nmol/L。【结论】APN4片段与Cry1Ac、Cry2Aa的结合能力在数量级上不存在显著性差异。  相似文献   

3.
【目的】Cry1A和Cry2A类Bt蛋白通过特异性地与昆虫中肠上的受体蛋白结合而发挥杀虫作用,现已广泛应用于转基因抗虫作物。本研究旨在进一步明确Cry2A类蛋白的作用机制和Cry1A受体蛋白在Cry2A发挥毒力中的作用。【方法】本研究首先提取了棉铃虫Helicoverpa armigera的BBMV,制备了钙粘蛋白(CAD)、氨肽酶N(APN)和碱性磷酸酯酶(ALP)3种受体蛋白的抗体和抗血清;然后,利用Western blot检测BBMV上这3种受体蛋白后,利用抗体封闭技术比较了敏感棉铃虫和Cry1Ac抗性棉铃虫(BtR)中3种受体蛋白的抗血清对Cry1Ac和Cry2Aa毒力的影响。【结果】对敏感品系棉铃虫,这3种已知的Cry1Ac受体蛋白抗血清显著地降低了Cry1Ac和Cry2Aa的毒力。其中APN抗血清对Cry1Ac毒力的影响最大,棉铃虫幼虫的死亡率降低了84.44%;ALP抗血清对Cry2Aa的毒力影响最大,棉铃虫幼虫死亡率比对照降低了71.04%。Cry1Ac对Cry1Ac抗性棉铃虫(BtR)的毒力显著降低,Cry2Aa的毒性也减弱。在Cry1Ac抗性棉铃虫(BtR)中,3种受体抗血清对Cry1Ac的影响比在敏感棉铃虫中的影响小,尤其是CAD和APN抗血清对Cry1Ac毒力的抑制率显著低于在敏感棉铃虫中的抑制作用;CAD和ALP抗血清对Cry2Aa毒力的影响与在敏感棉铃虫中的影响差异不显著,但APN抗血清可以显著降低Cry2Aa对Cry1Ac抗性棉铃虫(BtR)的毒力。【结论】棉铃虫CAD,APN和ALP不仅参与了Cry1Ac的杀虫过程,也对Cry2Aa毒力有一定的影响,而且这3种蛋白可能与棉铃虫对Cry1Ac和Cry2Aa产生抗性及交互抗性相关。  相似文献   

4.
分离和鉴定二化螟Chilo suppresalis幼虫中肠刷状缘膜囊泡(BBMV)中Cry1A毒素的受体蛋白,对于阐明Cry1A毒素作用机理和二化螟抗性机理具有十分重要的意义。为此,本文就Cry1A毒素对二化螟杀虫活性及Cry1Ac与二化螟中肠受体的配基结合进行了研究。结果表明: Cry1Ab对二化螟室内品系(CN)的毒力高于Cry1Ac,而Cry1Ac高于Cry1Aa。配基结合分析表明二化螟CN品系幼虫中肠BBMV中有6个Cry1Ac结合蛋白(分子量分别为50,70,90,120,160和180 kDa), 其中180,160和90 kDa结合蛋白的条带颜色明显深于其他结合蛋白的条带,表明这3个受体蛋白具有较高的结合浓度。同源竞争结合研究表明,180和90 kDa结合蛋白为Cry1Ac的低亲合性结合蛋白,其他4个为高亲合性结合蛋白。为了研究Cry1Ac和Cry1Ab受体结合部位的相互作用,进行了异源竞争结合研究。Cry1Ab可以与Cry1Ac所有的6个结合蛋白进行竞争性结合,与180,120,70和50 kDa结合蛋白具有高亲合性,而与160和90 kDa结合蛋白具有低亲合性。结果显示,Cry1Ac与Cry1Ab在二化螟幼虫中肠BBMV上拥有多个共享的结合位点,但对每个结合位点的亲合性有差异。基于毒素结合部位的相似性,Cry1Ac和Cry1Ab不宜同时用于转基因Bt水稻来控制二化螟。  相似文献   

5.
【目的】长期种植转Bacillus thuringiensis(Bt)基因作物使一些靶标害虫产生了Bt抗性,有研究表明小菜蛾Plutella xylostella的white基因表达下调使其产生了Cry1Ac抗性,由于Pxwhite蛋白与ABC转运蛋白ABCG1同属于ABCG亚家族,我们推测棉铃虫Helicoverpa armigera ABCG1基因(HaABCG1)的表达下调可能与棉铃虫对Cry1Ac毒素的抗性有关。【方法】克隆并分析HaABCG1的开放阅读框(open reading frame,ORF),通过构建HaABCG1基因的表达载体检测HaABCG1蛋白在TnH_5细胞中的亚细胞定位;通过细胞毒力实验验证HaABCG1蛋白与Cry1Ac毒素的关系;利用RNAi技术验证HaABCG1表达下调是否会降低棉铃虫对Cry1Ac毒素的敏感性。【结果】棉铃虫ABCG1基因的开放阅读框长1 896 bp,编码蛋白含631个氨基酸残基,分子质量估计为69.63 k D。离体昆虫细胞表达的HaABCG1蛋白主要定位在细胞的核膜和内质网。通过RNA干扰和生物测定实验发现HaABCG1下调表达不能使棉铃虫在Cry1Ac毒素浓度为0.05μg/m L的人工饲料上正常生长,3 d后处理组和对照组棉铃虫幼虫的体重变化无显著差异。经过细胞毒力实验证明HaABCG1蛋白不介导Cry1Ac毒素对TnH_5细胞的毒力,它既不是Cry1Ac毒素的受体,也不是其他3种Bt毒素Cry1Ca,Cry2Aa和Cry1Fa的受体。【结论】HaABCG1基因表达下调与棉铃虫对Cry1Ac的抗性不相关,HaABCG1不是Cry1Ac毒素的受体。这是首次报道ABCG1基因不参与棉铃虫的Cry1Ac抗性。  相似文献   

6.
通过体外重组的方法,实现了苏云金芽孢杆菌杀虫晶体蛋白Cry1Aa和Cry1Ca的功能性结构域Ⅰ、Ⅱ和Ⅲ的互换,得到了6株苏云金杆菌重组菌株BT-ACC,BT-AAC,BT-ACA,BT-CAA,BT-CCA和BT-CAC。SDS-PAGE和Westernblot分析表明,重组菌株BT-CAA和BT-CCA能表达产生135kDa左右的杂交晶体蛋白Cry1CAA和Cry1CCA,但其蛋白表达量较野生型Cry1Aa和Cry1Ca低。用牛胰蛋白酶对杂交晶体蛋白Cry1CAA、Cry1CCA及野生型Cry1Aa和Cry1Ca进行消化,证明所有晶体蛋白都能产生65kDa的活性毒素。电镜观察发现,野生菌株BT-Cry1Aa和BT-Cry1Ca形成典型的菱形晶体,而重组菌株BT-CCA和BT-CAA则形成球形或颗粒状杂交晶体。纯化晶体的生物测定显示,杂交晶体蛋白Cry1CAA和Cry1CCA对甜菜夜蛾的毒力比野生型晶体蛋白降低3~5倍,对棉铃虫的毒力比野生型晶体蛋白降低了190~260倍。研究结果表明,苏云金杆菌晶体蛋白不同结构域的相互作用会影响杂交晶体蛋白的表达、晶体形态和杀虫活性。  相似文献   

7.
[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.  相似文献   

8.
【目的】Polycalin蛋白是一种新发现的Bt毒素受体,可能与昆虫对Bt的抗性有关。本研究旨在明确小菜蛾Plutella xylostella Polycalin蛋白与Bt Cry1Ac毒素的关系。【方法】本研究通过PCR结合RACE技术从小菜蛾3龄幼虫中肠克隆获得Polycalin蛋白基因的全长cDNA序列,采用实时定量PCR技术研究Polycalin蛋白在小菜蛾不同发育阶段、4龄幼虫不同组织及用不同浓度Cry1Ac毒素饲喂3龄幼虫后的表达量。提取小菜蛾幼虫中肠的刷状缘膜囊泡(BBMV),运用合成多肽的方法制备Polycalin蛋白抗体,利用Western blot和Ligand blot技术对小菜蛾Polycalin蛋白进行鉴定,分析其与Cry1Ac毒素的结合特性。【结果】克隆得到的小菜蛾Polycalin蛋白基因的cDNA序列全长为9 102 bp(GenBank登录号:MF138149),其中,开放阅读框为8 778 bp,编码2 925个氨基酸;预测蛋白质分子量为326.38 kD,等电点为4.39;在推导的氨基酸序列中,前20个氨基酸为N-末端信号肽序列,含有14个N-糖基化位点,67个O-糖基化位点,6个脂质运载蛋白特征序列,6个脂质运载蛋白位点和13个类脂质运载蛋白结构域,并且在第20和21位(TSG-QVV)氨基酸之间存在一个裂解位点,在C-末端存在2个GPI结合位点,具有Bt毒素受体的特征。该蛋白在幼虫期的表达量较高,尤其在3龄幼虫体内表达量最高,在蛹和成虫中的表达量最低;在4龄幼虫的头、胸、腹中均有表达,在幼虫腹部中的表达量最高;3龄幼虫取食不同浓度的Cry1Ac毒素后,Polycalin蛋白的表达量下降,且Cry1Ac毒素浓度越高,下降越明显。通过Western blot检测到小菜蛾中肠刷状缘膜囊泡(BBMV)上存在大约300 kD的Polycalin蛋白条带;Ligand blot实验证明了小菜蛾Polycalin蛋白能与Cry1Ac毒素结合。【结论】本研究首次初步明确了小菜蛾Polycalin蛋白具有与Bt毒素结合的特性,为研究Bt对昆虫的作用机理和利用Bt防治害虫提供一定的理论基础和参考价值。  相似文献   

9.
【目的】在大肠杆菌中表达纯化苏云金芽胞杆菌HD73的转录调控因子Sigma K(σK)。【方法】PCR扩增出苏云金芽胞杆菌HD73中sig K基因的ORF(Open reading frame)装载到带有His标签的表达载体p ET21b上,转入到表达菌株BL21(DE3)中获得重组菌株BL21(p ETsig K),通过SDS-PAGE、镍柱亲和纯化、阴离子交换纯化和凝胶迁移实验(EMSA)等方法对Sigma K蛋白进行提取、纯化和生物活性分析。【结果】正确表达出大小约为27 k D的His-Sigma K蛋白,并获得了纯化的蛋白。EMSA结果表明纯化的His-Sigma K蛋白可以与受其控制的cry1Ac基因启动子结合。【结论】表达和纯化了His-Sigma K蛋白,His-Sigma K具有与受其控制的启动子结合的功能。  相似文献   

10.
[目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.  相似文献   

11.
12.
Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60 kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37 kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.  相似文献   

13.
Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 μg Cry1Ac/g diet but killed by Cry1Ab at 0.5 μg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 μg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.  相似文献   

14.
Toxicity of insecticidal endotoxins produced by Bacillus thuringiensis correlates with the presence of specific proteins in the midgut of susceptible larvae. This study was aimed at identifying and purifying Cry 1A binding proteins from Helicoverpa armigera, an important crop pest of India. B. thuringiensis strain HD 73 which produces Cry 1Ac toxin, specific for H. armigera was used in this study. Toxin-binding proteins from insect larvae were detected by employing a toxin overlay assay using both radiolabelled as well as unlabelled toxin. Detergent-solubilized fractions of larval brush border membranes were subjected to soybean agglutinin (SBA) chromatography, from which N-acetylgalactosamine (NAG)-containing proteins were eluted. Analysis of the SBA-purified proteins indicated that four proteins of approximately 97, 120, 170 and 200 kDa could bind to Cry 1Ac toxin, and three proteins of 97, 170 and 200 kDa proteins could bind to Cry 1Ab. Furthermore, in the presence of excess Cry 1Ab toxin, the labelled Cry 1Ac toxin could bind only to 170 and 200 kDa proteins, implying that Cry 1Ab can also bind the 120 kDa protein. This study therefore demonstrates that in H. armigera, midgut proteins of 97, 120, 170 and 200 kDa have the ability to bind both Cry 1Ab and Cry 1Ac. Furthermore, while the 170 and 200 kDa proteins have higher affinity for Cry 1Ac, the 97 kDa has higher affinity for Cry1 Ab. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

15.
We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site. Received: 12 January 1998 / Accepted: 17 February 1999  相似文献   

16.
First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.  相似文献   

17.
The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R1 binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419–3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R1. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R1 protein is a common high-affinity receptor protein for the Cry1A family of toxins.  相似文献   

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