High-level expression of a foreign gene by a recombinant baculovirus with an expanded host range

The usefulness of host range expanded viruses as an expressionvector system was investigated by following the expression ofthe E. coli lacZ gene. The host range expanded recombinantviruses were obtained from Sf-21 or BmN-4 cells coinfected withAutographa californica and Bombyx mori nuclearpolyhedrosis viruses. Among the host range expanded viruses,RecB-8 and RecS-B6 have similar enzyme digestion profiles butdifferent infection characteristics in cells. Therefore, tostudy the foreign gene expression efficiency of these twoviruses, we constructed recombinant viruses RecB8-LacZ andRecSB6-LacZ containing the lacZ gene instead of the polyhedringene. Also, the host range expanded recombinant AcNPV, Bac-BH,containing lacZ gene in the polyhedrin gene locus was constructedby substitution of the 0.6 kb region within the helicase gene ofBacPAK6 with that of BmNPV. -Galactosidase expressionefficiency by these viruses were determined and compared in Sf-21and BmN-4 cells. The result showed that Bac-BH has highexpression efficiency only in Sf-21 cells, whereas RecB8-LacZhas high expression efficiency both in Sf-21 and BmN-4 cells.Also, in BmN-4 cells, -galactosidase expressionefficiency of RecB8-LacZ was higher than that of recombinantBmNPV (BmK1-LacZ containing lacZ gene in polyhedrin gene locus).In addition, the expression efficiency was not correlated withvirus titer.     

Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects

A newly engineered strain, denominated BIOT185, was constructed through integrating the cry8Ca2 gene into the endogenous plasmid of BT185 (contains cry8Ea1) by homologous recombination. The thermosensitive plasmid vector was eliminated by the rising temperature of recombinant cultures. No antibiotic gene or other unnecessary genes were introduced to the new strain. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry8Ca2 gene was expressed normally and produced a 130-kDa protein in the BIOT185 strain. Bioassay results showed that the new strain had high toxicity to the pests Anomala corpulenta and Holotrichia parallela, which often damage the same fields.     

Molecular characterisation of Bacillus thuringiensis isolates from the Egyptian soils

Molecular characterisation of nine different Bacillus thuringiensis isolates from the soil of different Egyptian governorates and with varying activities against some lepidopterous insects was carried out using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD)-PCR analysis. Molecular weights of the major components of the crystal proteins of the tested strains revealed that those strains with bands 39 and 141 KDa would be possibly potent against the cotton leafworm Spodoptera littoralis (Biosduval) (Lepidoptera: Noctuidae), those with bands 39–73 and 104–178 KDa showed toxicity against the American bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and those with bands 25–3 and 135 KDa may be toxic to the pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae). PCR analysis indicates that the frequency of the cry 1 genes predominated 72.41% of isolates amplifying cry 1 gene. DNA fingerprinting-based randomly amplified polymorphic DNA (RAPD) techniques proved to be a reliable method for identification of different B. thuringiensis strains at the DNA level.     

Surface display of recombinant proteins on Bacillus thuringiensis spores

The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically.     

Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.     

解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究

植物真菌病害给农业生产带来了巨大损失,因此对生物农药的开发迫在眉睫。从堆肥中分离得到一株解淀粉芽孢杆菌,它具有强烈的抗真菌活性。其发酵液经硫酸胺沉淀得到粗提液,粗提液经Hiprep 26/10 Desalting,HiLoad 26/10 Q Sepharose和HPLC多步柱层析,分离纯化得到一种抗真菌活性物质。ESI-MS质谱法测得其分子量为1498 Da。经活性检测发现,该纯物质对尖孢镰刀菌、草莓蛇病菌等植物病原真菌具有很强的抑制作用,对毛霉、黑曲霉等食品腐败菌也有抑制作用。经过显微镜观测,该物质可造成草莓蛇病菌菌丝生长异常,表现在菌丝弯曲,顶端膨大,分生孢子数量减少。     

Estimation of the spectrum of the aerial dispersion of Bacillus thuringiensis

The present paper estimates the number of viable spores of Bacillus thuringiensis per droplet and reveals the importance of this data to improve B. thuringiensis treatments. For a given diameter, droplets from Futura formula contained two times more spores than droplets from the formulas used to date. It is preferable to have B. thuringiensis droplets larger than those of chemical insecticides. A Grumman AgCat aircraft calibrated to give the desired larger droplets resulted in successful B. thuringiensis treatments.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

苏云金芽胞杆菌高效表达载体的构建

[目的]通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性,筛选出一个强启动子,利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)高效表达载体.[方法]利用启动子融合lacZ技术检测了4种启动子的转录活性.通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证.[结果]构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E4个启动子融合报告基因lacZ的表达载体,经β-半乳糖苷酶活性分析得知,启动子活性从高到低依次为Pcry8E＞Pcry1A＞Pcry4A＞Pcry3A.选取cry8E启动子,以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b,将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上,分别转入无晶体突变株HD-73-,获得菌株HD-8E1Ac和HD-422-1Ac.扫描电子显微镜观察显示,HD-8E1Ac菌株可以形成菱形晶体,说明正确表达了cry1Ac基因.SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b.对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性,且菌株活性高于HD-422-1Ac.[结论]利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b,该载体可正确表达cry1Ac基因,其表达效率高于被广泛应用的pSXY422b.     

A Bacillus thuringiensis host strain with high melanin production for preparation of light-stable biopesticides

The bacterium Bacillus thuringiensis produces a crystal protein with insecticidal properties; however, crystal proteins can be damaged by ultraviolet (UV) radiation. The aim of this study was to improve the stability of the insecticidal crystal protein (ICP) by constructing a mutant line that expresses high levels of the UV light-protecting pigment, melanin. BMB181, a B. thuringiensis mutant with high melanin production, was obtained after sub-culturing BMB171 for several generations at 42 °C. The melanin yield by BMB181 (without tyrosine supplementation) reached 8.55 mg/ml. The electroporation efficiency of BMB181 reached 10⁶ CFU/μg when a 6.7-kb foreign plasmid was used. Microscopic and SDS-PAGE analyses revealed that ICP (CryIAc10; GenBank: AAA73077.1), which is highly toxic to Lepidoptera, was synthesized efficiently by strain BMB181. The insecticidal properties of a recombinant line derived from strain BMB181, designated BMB32 (cry1Ac10/BMB181), was tested against the cotton bollworm, Helicoverpa armigera. After UV irradiation for 4 h, BMB32 had a half maximal inhibitory concentration value of 1.37 μg/ml, whereas the control line BMB31 (cry1Ac10/BMB171) had a median lethal dose value of 25.85 μg/ml. These results indicate that the B. thuringiensis mutant is a candidate for industrial scale production of light-stable insecticides.     

The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host

Host-pathogen interactions are central components of ecological networks where the MAPK signaling pathways act as central hubs of these complex interactions. We have previously shown that an insect hormone modulated MAPK signaling cascade participates as a general switch to trans-regulate differential expression of diverse midgut genes in the diamondback moth, Plutella xylostella (L.) to cope with the insecticidal action of Cry1Ac toxin, produced by the entomopathogenic bacterium Bacillus thuringiensis (Bt). The relationship between topology and functions of this four-tiered phosphorylation signaling cascade, however, is an uncharted territory. Here, we carried out a genome-wide characterization of all the MAPK orthologs in P. xylostella to define their phylogenetic relationships and to confirm their evolutionary conserved modules. Results from quantitative phosphoproteomic analyses, combined with functional validations studies using specific inhibitors and dsRNAs lead us to establish a MAPK “road map”, where p38 and ERK MAPK signaling pathways, in large part, mount a resistance response against Bt toxins through regulating the differential expression of multiple Cry toxin receptors and their non-receptor paralogs in P. xylostella midgut. These data not only advance our understanding of host-pathogen interactions in agricultural pests, but also inform the future development of biopesticides that could suppress Cry resistance phenotypes.     

Molecular and phenotypic characterisation of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L

Twelve Bacillus thuringiensis strains were isolated from intestinal tracts of Cydia pomonella larvae during epizootics in different laboratory insect culture lines. Phenotypic and genetic similarity of these isolates, together with two cultured from Leucoma salicis larvae and 14 reference B. thuringiensis strains were determined. The epizootic bacteria did not form a single group based on numerical analysis of biochemical properties. Simple RAPD method with only one primer does not allow estimating the genetic similarity of B. thuringiensis strains. We propose a novel strategy based on combining several DNA patterns obtained by RAPD technique with different primers for B. thuringiensis typing. Majority of infections in the C. pomonella culture lines were caused by bacteria with different genotypes. However, two isolates cultured from infected insects at different time (one strain was isolated in 1990 and the other in 1992) had identical DNA fingerprint that suggested a possibility of these bacteria to survive in the laboratory and to cause infections in different years. The results of SDS-PAGE of whole-cell proteins revealed a possibility to apply protein profile analysis in epidemiological investigations of infections caused by B. thuringiensis. Strains with identical DNA patterns had very similar whole-cell protein profiles.     

Isolation and characterisation of a novel bacteriocin produced by Bacillus thuringiensis strain B439

Bacillus thuringiensis strain B439 produces a bacteriocin-like inhibitory substance in its growth medium. This antimicrobial peptide, referred to as thuricin 439, acts as a bacteriocidal peptide and exhibits an apparent narrow range of inhibitory activity, essentially only affecting growth of Bacillus cereus and B. thuringiensis strains. It remains active over a relatively wide pH and temperature range, showing no loss of activity following heat treatments up to 80 degrees C. Purification of thuricin 439 was achieved using several chromatographic steps, which resulted in the identification of two peptides with inhibitory activity. These two peptides were shown to possess identical N-terminal sequences, but different molecular masses.     

The cost of resistance to Bacillus thuringiensis varies with the host plant of Trichoplusia ni

Selection for resistance to insecticides, diseases and parasitoids is assumed to be costly and often requires tradeoffs with reproductive fitness. The costs of resistance, however, are often difficult to measure. Cabbage looper, Trichoplusia ni, a generalist Lepidopteran herbivore, has become highly resistant following the extensive use of the microbial insecticide, Bacillus thuringiensis kurstaki (Bt) in vegetable greenhouses. We compared the growth rate, pupal size and survival of resistant, susceptible and hybrid T. ni larvae fed on tomato, bell pepper and cucumber. Performance was best on cucumber and worst on pepper, and the magnitude of fitness costs associated with Bt resistance increased with declining host plant suitability. This supports the hypothesis that in this system, resistance costs are condition dependent and are greatest in the most stressful environment. Management strategies that rely on the presence of fitness costs to reduce the frequency of resistance genes must consider this variation and should be more successful on crops that are less suitable food plants. In general, condition dependence should be considered in studies designed to measure the costs of resistance.     

苏云金芽胞杆菌标记重组菌株的构建与杀虫基因水平转移

利用SOE法将构建的绿色荧光蛋白基因gfp和苏云金芽胞杆菌的杀虫晶体蛋白基因cry1Ac10的嵌合基因克隆到穿梭载体pAD4412上获得重组质粒pBMBZGC10,再通过电转化法导入苏云金芽胞杆菌无质粒突变株CryB中获得重组菌株CryB(pBMBZGC10).将重组菌株CryB(pBMBZGC10)的发酵液按30、60和90 ml共3个浓度梯度,菌数约为10.7～10.8·ml^-1,分次喷洒供试植株小白菜、蕹菜和番茄,结果表明,cry1Ac10基因没有向供试土壤细菌、真菌和放线菌转移,也未在供试植物根、茎和叶中检测到该基因.     

Characterization of a Bacillus thuringiensis strain with a broad spectrum of activity against lepidopteran insects

The insect pathogen Bacillus thuringiensis is suitable for use in biological control, and certain strains have been developed as commercial bioinsecticides. The molecular and biological characterization of a Bacillus thuringiensis subsp. aizawai strain, named HU4‐2, revealed its potential as a bioinsecticide. The strain was found to contain eight different cry genes: cry1Ab, cry1Ad, cry1C, cry1D, cry1F, cry2, cry9Ea1, and a novel cry1I‐type gene. Purified parasporal crystals from strain HU4‐2 comprised three major proteins of 130–145 kDa, which were tested for their insecticidal potency to four species of Lepidoptera (Helicoverpa armigera, Spodoptera exigua, S. littoralis, and S. frugiperda) and three species of mosquito (Culex pipiens pipiens, Aedes aegypti, and Anopheles stephensi). The crystal proteins were highly toxic against all the species of Lepidoptera tested, moderately toxic against two of the mosquito species (C. pipiens and Ae. aegypti), but no toxicity was observed against a third species of mosquito (An. stephensi) at the concentrations used in our study. The LC₅₀ values of the HU4‐2 Bt strain against H. armigera larvae (5.11 µg/ml) was similar to that of HD‐1 Bt strain (2.35 µg/ml), the active ingredient of the commercial product Dipel®. Additionally, the LC₅₀ values of the HU4‐2 Bt strain against S. littoralis, S. frugiperda, and S. exigua (2.64, 2.22, and 3.38 µg/ml, respectively) were also similar to that of the Bt strain isolated from the commercial product Xentari® for the same three species of Spodoptera (1.94, 1.34, and 2.19 µg/ml, respectively). Since Xentari® is significantly more toxic to Spodoptera spp. than Dipel® and, reciprocally, Dipel® is significantly more toxic against H. armigera than Xentari®, we discuss the potential of the HU4‐2 strain to control all these important lepidopteran pests.     

Detection and characterisation of a Bacillus thuringiensis crystal protein with nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus

Nematicidal Bacillus thuringiensis (Bt) strains were isolated from forests in Zhejiang, China for further characterisation. PCR analysis was performed with nine pairs of primers specific for cry1, cry2, cry3, cry4, cry5, cry6, cry9, cry11 and cry13 to characterise and classify cry gene groups from Bt isolates. The isolates from individual cry groups were tested for nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus, which is implicated in pine wilt disease. PCR identified 14 different categories of cry gene combinations, indicating a large diversity of cry genes. The cry1 gene was by far the most abundant in Bt isolates and was found in 68% of samples. The Bt isolates zjfc85 and zjfc392 were from two distinct classes, but shared the same cry5 amplification profile and the same ~130 kDa protein; they had the highest nematicidal activity against pinewood nematode during the 48 h exposure tests, resulting in 90 and 59% mortality (9% of mortality under control conditions), respectively. The ~130 kDa Cry protein from isolate zjfc85 was purified and named as Cry5Ba3. Bioassay results indicated pinewood nematode was highly susceptible to Cry5Ba3 and exhibited profound growth abnormalities after exposure to Cry5Ba3. Our results are a novel finding and provide a potential strategy to manage pine wilt disease caused by B. xylophilus based on a nematicidal Bt.     

D-阿洛酮糖3-差向异构酶重组枯草芽孢杆菌构建及固定化应用

D-阿洛酮糖3-差向异构酶(D-allulose-3-epimerase)是异构化D-果糖生成D-阿洛酮糖(D-allulose)的关键酶.为提高D-阿洛酮糖3-差向异构酶的热稳定性并获得可重复使用的D-阿洛酮糖3-差向异构酶重组枯草芽孢杆菌固定化细胞,N端融合双亲短肽,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,...