Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins

[This corrects the article on p. 1821 in vol. 59.].     

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.     

Bacillus thuringiensis Vip3 mutant proteins: Insecticidal activity and trypsin sensitivity

Vip3 is a novel insect toxin isolated from Bacillus thuringiensis (Bt), and could be used as an alternative toxin for Bt δ-endotoxins for transgenic insect control. Vip3 mutants with deletion, addition or mutations at the very end of the C-terminus were generated. The deletion and addition of a few amino acid residues at the C-terminus totally abolished the insecticidal activity. The mutation of the last two residues from IK to LG also resulted in the total loss of its insecticidal activity; however, the mutation from IK to LR increased its activity substantially against beet armyworm. Interestingly, all the inactive mutants were found to be highly sensitive to trypsin digestion, while the active mutant generated a trypsin-resistant polypeptide of 62-kDa upon trypsin digestion. However, this 62-kDa polypeptide expressed in E. coli from the 5' end truncated vip3 gene was biologically inactive and sensitive to trypsin digestion. Thus, the N-terminal part of the protein is required to form the 62-kDa trypsin resistant core. This study suggested that the 62-kDa peptide core could be a hallmark of active insecticidal Vip3 proteins.     

苏云金杆菌营养期杀虫蛋白的研究

营养期杀虫蛋白 (vegetativeinsecticidalproteins ,VIPs)是苏云金杆菌 (Bacillusthuringiensis,Bt)在对数生长中期分泌的一类新型杀虫毒蛋白。VIPs主要分为VIP1、VIP2和VIP3三种。VIP1和VIP2构成二元毒素 ,对鞘翅目叶甲科的昆虫具有杀虫特异性 ;而VIP3对鳞翅目昆虫具有较广谱的杀虫活性。VIP1和VIP2的杀虫作用机理还不清楚 ;VIP3通过诱发细胞凋亡 ,最终导致昆虫死亡 ,这种作用机理与Bt杀虫晶体蛋白的作用机理完全不同 ,这为筛选新的杀虫活性物质提供了新的思路。vip基因现已被应用于转基因杀虫植物的构建 ,得到高效抗虫的多价转基因玉米。此外 ,VIPs嵌合蛋白的构建、vip及其融合基因导入其它许多宿主微生物等方面的研究也具有诱人的潜在应用前景。     

苏云金杆菌Cyt类杀虫晶体蛋白及其特征

本文综述了国内外有关苏云金杆菌Cyt类杀虫晶体蛋白的分类、杀虫特性、作用机理 ;具有分子伴侣功能的 2 0kDa蛋白对cyt基因在大肠杆菌和苏云金杆菌中的表达的影响 ;以及利用Cyt类蛋白控制害虫对苏云金杆菌抗性的意义。     

Insecticidal activity of Bacillus laterosporus

Strains of Bacillus laterosporus demonstrated pathogenic activity for second-instar larvae of the mosquito, Culex quinquefasciatus, but failed to demonstrate detectable pathogenicity against larvae of the cabbage looper, Trichoplusia ni. Of 29 strains of the bacterium screened, 16 displayed pathogenicity for mosquito larvae. One of the most pathogenic strains, NRS 590, also demonstrated pathogenic activity for larvae of the mosquito, Aedes aegypti, and for larvae of the black fly, Simulium vittatum. The pathogenicity for Culex larvae was associated with the cell mass rather than with the culture supernatant. A suspension of ultraviolet irradiation-killed cells demonstrated no loss in pathogenic activity, an indication that the pathogenicity is toxin mediated. The toxic substance produced by NRS 590 was found to be resistant to heating at 96°C for 10 min. The toxin was not associated with the heat-resistant, bacterial endospore or with the associated paraspore since a suspension consisting primarily of spores was not toxic to mosquito larvae. Toxic activity in stationary phase cells of NRS 590 was associated with the cell's particulate fraction rather than with the soluble fraction.     

苏云金芽孢杆菌营养期杀虫蛋白VIP3研究进展

的报道.本文从VIP3的类型和杀虫活性、作用机理、vip3基因的定位和分离、vip3基因重组和转vip3基因植物等方面详细介绍了VIP3近十年来的研究进展.     

苏云金芽胞杆菌6618杀虫菌株的分离和鉴定

从我国不同地区采集118份土样,利用温度筛选法分离获得一株苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)6618,镜检观察发现该菌株能产生典型的菱形晶体,PCR分析表明其含有cry1类杀虫基因。采用SDS-PAGE和质谱分析发现该菌株主要产生130 k D原毒素,其组分由Cry1Ae和Cry1Ac原毒素组成。基因序列分析表明该菌株的cry1Ac基因为已知的cry1Ac1,而cry1Ae为新型杀虫基因。毒力生测表明该菌株对棉铃虫(Helicoverpa armigera)幼虫具有显著的杀虫效果。筛选获得的高毒力Bt菌株6618为丰富我国苏云金芽胞杆菌储备和研发新型高效生物杀虫剂提供了菌株资源。     

苏云金芽胞杆菌营养期杀虫蛋白基因特性分析

营养期杀虫蛋白是在苏云金芽胞杆菌营养期中发现的一种非晶体状胞外杀虫蛋白 .通过Southern杂交的基因定位实验 ,证实了营养期杀虫蛋白基因并非位于染色体和环型质粒上 ;在 2 0个不同血清型的 2 3种菌株中 ,营养期杀虫蛋白基因的存在率为 5 6 5 % (13 2 3) .同时对随机选择的 3个不同亚种菌株进行基因克隆和序列分析 ,表明其基因具有相当高的同源性 .运用生物信息技术 ,对营养期杀虫蛋白多肽进行了功能结构域比对分析 ,发现这 789多肽蛋白的N端 31至 15 0残基之间存在一种趋化信号传导因子相似序列 ,C端 5 36至 6 6 6残基之间则存在着纤维素结合结构域相似肽 .上述结果提示 ,营养期杀虫蛋白基因在苏云金芽胞杆菌天然菌株中高度保守并较为广泛分布 ,其编码蛋白的特殊结构提示了营养期杀虫蛋白潜在的功能特性 ,为进一步研究营养期杀虫蛋白奠定了基础 .     

利用整合载体构建苏云金芽胞杆菌杀虫工程菌

利用由苏云金芽胞杆菌整合子Tn4 4 30衍生出的整合载体pBMB F7E ,克隆了对鳞翅目夜蛾科昆虫有毒力的杀虫晶体蛋白基因cry1C ,获得了整合重组质粒pBMB FLC。用电脉冲法将该重组质粒转入对鳞翅目昆虫高毒力的野生菌株YBT80 3 1,在 4 6℃下 ,经过约 12 0代转接培养后 ,筛选出在染色体基因组上整合了cry1C基因的重组子 ,整合频率约为 3 4× 10 -5。Southernblotting验证了cry1C在菌株YBT80 3 1中于染色体的不同的位点整合。生物测定结果显示 ,重组子BMB80 3 Z对小菜蛾的毒力与出发菌株无明显差异 ,而对甜菜夜蛾则较出发菌株高。     

Interaction between Functional Domains of Bacillus thuringiensis Insecticidal Crystal Proteins

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.     

Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.     

苏云金杆菌抗虫基因cryI Ac 转化辣椒的研究

通过农杆菌介导法将杀虫结晶蛋白基因cryIAc导入到辣椒子叶外植体中,经卡那霉素筛选、组织化学法检测GUS活性以及PCR、southern杂交分析证实cryIAc基因已整合进辣椒核基因组中。     

苏云金芽孢杆菌4.0718菌株的杀虫晶体蛋白基因分析

根据苏云金杆菌(Bacillus thuringiensis)cry1、cry2和cry3型基因的保守区分别设计了3对通用引物Un1(d)/Un1?、Un2(d)/Un2?和Un3(d)/Un3?,以Bt4.0718菌株质粒DNA为模板进行PCR扩增,通过扩增产物片段的分子量大小来确定该菌株所含有的杀虫晶体蛋白基因类型。随后根据上述3类cry基因的高变区设计特异引物再次进行PCR鉴定。结果表明:Bt4.0718菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Cb、cry2Ac和新基因cry4.5等6种基因类型。这一结果为利用该菌株构建高效广谱杀虫工程菌提供了客观依据。     

Insecticidal activity of mixtures of Bacillus thuringiensis crystals with plant oils of Sinapis alba and Azadirachta indica

The main purpose of this study was to determine the insecticidal activity of mixtures of Bacillus thuringiensis crystalline toxins and plant substances that could contribute to create a new bioinsecticide of high efficiency. We decided to determine the toxicity of crystals of B. thuringiensis MPU B9 isolate, azadirachtin (NeemAzal), mustard oil and their mixtures against lepidopteran pests: Cydia pomonella, Spodoptera exigua and Dendrolimus pini. The interaction of the components in mixtures against insects was evaluated and their utility in plant protection was assessed. The crystals and plant substances showed synergistic effect against all insect species. The occurrence of synergism between two components depended on the ratio of their concentration in mixtures. Slight amount of one of the components added to the mixture led to enhanced toxicity of the preparation. The supplementation of mustard oil with B. thuringiensis toxins from up to 0.3% of the mixture resulted in more than twofold higher observed mortality of L1 larvae of D. pini in comparison with expected mortality. The beneficial insecticidal effect of the mustard oil and crystals mixture was also noted when crystals comprised up to 1.3% and from 7 to 20% against L1 caterpillars of C. pomonella and L3 larvae of S. exigua, respectively. The insecticidal efficient effect was achieved when mixture of B. thuringiensis crystals and azadirachtin was used against the three species of lepidopteran pests. The combinations in which components interacted synergistically against caterpillars are potentially useful for making a new bioinsecticide for controlling lepidopteran pests in plant protection.     

苏云金芽孢杆菌杀虫晶体蛋白与DNA分子的相互作用

苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,简称Ｂｔ)在形成芽孢的同时能够产生伴孢晶体 ,其中含有一种或几种杀虫晶体蛋白 (ＩＣＰｓ,ＩｎｓｅｃｔｉｃｉｄａｌＣｒｙｓｔａｌＰｒｏｔｅｉｎｓ) ,即δ 内毒素[1] 。伴孢晶体进入敏感昆虫的消化道后发生溶解并释放出 2 7～ 1 40ｋＤ的原毒素。在中肠蛋白酶的作用下 ,原毒素被激活为 2 3～ 70ｋＤ的毒性多肽[2～ 4 ] 。随后毒素与中肠刷状缘膜泡 (ＢＢＭＶ ,ＢｒｕｓｈＢｏｒｄｅｒＭｅｍｂｒａｎｅＶｅｓｉｃｌｅ)上的特异受体发生结合并且在细胞膜上形成孔道 ,破坏细胞…     

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.