Animal Safety Test of Bacillus thuringiensis BT Protein

[Objectives]To determine the biological safety of BT protein from Bacillus thuringiensis(Bt)fermentation broth to mammals at high doses.[Methods]Healthy mice were randomly divided into 4 groups with 10 mice in each group.The experimental groups were fed with Bt fermentation supernatant at 0.2,0.6 and 1.0 mL/kg,respectively,once a day for 7 consecutive days.The blank control group was fed normally without intragastric administration.[Results]There was no significant difference in blood routine and blood biochemical analysis between the experimental group and the control group.After intragastric administration,the mice were dissected,and no obvious pathological changes were found;the heart,liver,spleen,lung and kidney were taken to make tissue sections,and no pathological changes were found by microscopic observation.[Conclusions]Mice can tolerate high doses of BT protein from B.thuringiensis fermentation broth.

Induced CTL-S15 gene expression by Bacillus thuringiensis declines susceptibility in Spodoptera exigua

It has been reported that C-type lectins(CTLs),which are pattern recognition receptors of the insect innate immunity response,may compete with Cry toxin for the receptor alkaline phosphatase to decrease its toxicity in insects.However,to date,which CTLs affect larval susceptibility to Bt in Spodoptera exigua is not clear.In this study,33 CTL genes were identified from S.exigua.Based on the number of carbohydrate-recognition domains(CRDs)and the domain architectures,they were classified into three groups:(1)nineteen CTL-S(single-CRD),(2)eight immulectin(dual-CRD)and(3)six CTL-X(CRD with other domains).RT-qPCR analysis revealed that expression levels of SeCTL-S15,IML-4 and CTL-X6 were upregulated after challenge with Bt and Cry1Ab.Tissue and developmental stage expression analysis showed that only SeCTL-S15 was mainly expressed in the midgut and larva,respectively.Knockdown of SeCTL-S15 significantly increased Bt susceptibility,as indicated by reduced survival and larval weight.These results suggest that CTL-S15 might play a vital role in the low susceptibility of larvae to Bt in S.exigua.Our results provide new insights into CTL function in insects.

酸橙内生菌Bacillus thuringiensis Bt028产几丁质酶的发酵条件优化

为了提高分离自酸橙果实中的一株内生细菌Bacillus thuringiensis Bt028产几丁质酶的性能。采用单因素实验方法考察了培养基组成和发酵条件对该菌株产几丁质酶的影响,并在此基础上采用响应面方法对该菌株产几丁质酶的摇瓶发酵条件进行了优化。单因素实验结果表明胶体几丁质浓度、培养温度和初始pH对该菌株产几丁质酶影响明显;响应面试验结果表明,当胶体几丁质浓度为0.8%、酵母粉浓度为1%、初始pH为6.9、接种量3%、装液量为100 mL/250 mL、30℃培养48 h时,该菌株产几丁质酶性能最佳,发酵液中几丁质酶酶活可达10.48 U/mL。研究结果为该菌株几丁质酶的进一步开发与应用奠定了基础。

Complete genome sequence of Bacillus thuringiensis Bt185, a potential soil insect biocontrol agent

Bacillus thuringiensis Bt185 and its insecticidal spectrum-expanded engineering strains are considered as potential biocontrol agents to soil insect Holotrichia parallela,Holotrichia oblita or Anomala corpulenta.Here we reported the complete genome of strain Bt185,it harbors eight plasmids,and plasmid p BT1850294 carries three cry8 genes.

Plant biotechnology:a case study of Bacillus thuringiensis(Bt) and its application to the future of genetic engineered trees

Agricultural productivity may be raised in a sustainable way by many different technologies such as biological fertilizers, soil and water conservation, biodiversity conservation, improved pest control, and changes in land ownership and distribution. Of these measures, biotechnology applications probably hold the most promise in augmenting conventional agricultural productivity, because biotechnology applications give not only the need to increase production, but also protect the environment and conserving natural resources for future generations. Biotechnology applications will have the possibilities to increase productivity and food availability through better agronomic performance of new varieties, including resistance to pests; rapid multiplication of disease-free plants; ability to obtain natural plant products using tissue culture; diagnosis of diseases of plants and livestock; manipulation of reproduction methods increasing the efficiency of breeding; and the provision of incentives for greater participation by the private sector through investments. Insect resistance through the transfer of a gene for resistance fromBacillus thuringiensis (Bt) is one of the most advanced biotechnology applications already being commercialized in many parts of the world. This paper reviews the development and the status ofBt technology and application ofBt transgenic plants in current agriculture, and discusses specific issues related to the transfer of the technology to the future of genetic engineered trees with emphasis on conifers. Key words Agricultural productivity - Bacillus thuringiensis - Genetic engineering - Insect resistance - Trees CLC number Q812 - S763.306 Document code A Biography: Tang Wei (1964-), male, Ph. Doctor, Research associate, Department of Biology, Howell Science Complex, East Carelina University, Greenville, NC 27858-4353, USA.Responsible editor: Chal Ruihai

Introduction of Bacillus thuringiensis(Bt)gene does not reduce potassium use efficiency of Bt transgenic cotton(Gossypium hirsutum L.)

Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.

General Evaluation of Biocide （Bt-ASF-1） Produced from Iraqi Isolate of Bacillus thuringiensis

General evaluation of isolate Bacillus thuringiensis （Bt-ASF-1） used as biocide in meddle scale application was conducted. Some morphological and confirmation tests were achieved. The sensitivity tests had been accomplished by diffusion and dilution techniques to determine the response of isolate against the antibiotics. The results of diffusion tests showed to the sensitivity of bacteria to antibiotics of cefixime, erythromycin, gentamicin and tetracycline respectively. It was resistant to trimethoprim sulfonamide （TMP）, bacitracin, penicillin and all its generations, and moderate resistance to nalidixic acid. Minimum Inhibitory Concentration （MIC） for amoxicillin was ranged between 30-40 pg/mL and these results are an approximation of the universal findings. Curing experiments showed the effective role of sodium dodecyl sulfate （SDS） （1.5%） comparing with temperature. The bacterial cells became sensitive to amoxicillin and TMP. The curing by temperature did not differ significantly from control treatment in plasmid pattern or antibiotics response. Plasmid profile referring that curing by SDS has been caused disturbance in beta -lactamase genes through the sensitivity to amoxicillin and remaining resistance to ampicillin. Curing isolate by SDS also became more sensitive to nalidixic acid, erythromycin and tetracycline respectively. It was found from the curing treatments the complexity distribution of r-genes between different plasmid size and chromosome but not effect on their insecticidal ability.

Susceptibility of Ostrinia furnacalis to Bacillus thuringiensis and Bt Corn Under Long-Term Laboratory Selection

The susceptibility of the Asian corn borer, Ostrinia furnacalis to Bacillus thuringiensis (Bt) formulation and Bt corn was evaluated using insect bioassays for 6 years. Four strains of O. furnacalis were developed by laboratory selection from the laboratory strain reared on a non-agar semi-artificial diet. The RR-1 strain was exposed to a commercial formulation of B. thuringiensis subsp. kurstaki (Btk) incorporated into the artificial diet,the RR-2 strain was exposed to Bt corn (MON810) tissue incorporated into the diet, and the SS-1 and SS-2 strains were reared on the standard diet with or without non-Bt corn tissues material. Decreasing susceptibility of O. furnacalis to Bt and to Bt corn were found in each selected strain although the ED50 and larval weight fluctuated from generation to generation. The resistance of Bt-exposed strain (RR-1) to Btk increased 48-fold by generation 39; the Bt corn-exposed strain (RR-2) increased its resistance 37-fold to Btk by generation 24. No larvae of SS-1 survived when they were exposed to the leaves of Bt corn, Bt11 and MON810. However, 2-54% of the RR-1 (generation 46) and RR-2 (generation 20) larvae survived a 3 day-exposure to the leaves of Bt11 and MON810. The survival of both selected strains on Bt corn silk increased by 10-69%, and the larval weights after many generations selection were increased by 15-22% compared with the unselected susceptible strain. The young larvae were much more susceptible to Bt than older larvae. The highest mortality occurred when the larvae were exposed to Bt at the neonate stage. All of the results suggested that ACB could not only develop resistance to Bt preparation but also to Bt corn. Bt had significant effects on the growth and development of Asian corn borer than on the larval mortality. In order to maintain the long-term effectiveness of Bt pesticide and Bt corn, the resistance management should pay much attention to the larvae that may have opportunities to grow and developed on non-Bt corn or alternate hosts before they attack the Bt corn plants and the survival of larvae after Bt application.

Influence of Formate on Bioactivity Material-thuringiensin Synthesized by Bacillus thuringiensis YBT-032

The biological method to synthesize thuringiensin and the influence of formate on thuringiensin biosynthesis were investigated. Addition of 1.00 g/L formate to growth medium of bacillus thuringiensis YBT-032 resulted in significant enhancements in productions of citrate, α -ketoglutarate, intracellular adenine and thuringiensin. These results demonstrate that added formate attends metabolism of cell, facilitates carbon metabolic flux in tricarboxylic acid cycle and hexose monophosphate pathway. As a carbon source, formate facilitates cell growth, increases glucose consumption and enhances the ability of cell to synthesis adenine analogues, and subsequently thuringiensin. Thuringiensin production rate significantly enhanced from 6.44 to 8.46 mg·g^-1 · h and transformation ratio from glucose to thuringiensin increased by 43.30%.

一株苏云金芽孢杆菌(Bacillus thuringiensis)的分离与鉴定

利用醋酸盐对苏云金芽孢杆菌芽孢萌发的选择性抑制特性 ,采用选择性培养基从土壤中分离苏云金芽孢杆菌 .对分离菌株的个体形态、生理生化特点进行了分类研究 ,确定该菌株即为苏云金杆菌 .采用改进的培养基培养伴胞晶体 ,证明该培养基可在

有机溶剂对苏云金芽孢杆菌(Bacillus thuringiensis)几丁质酶的影响

以苏云金芽孢杆菌(Bt)的发酵液为材料,经硫酸铵分级分离、DEAE-32离子交换柱层析分离,获得部分纯化的Bt几丁质酶(EC3. 2. 1. 14)制剂.研究了几种有机溶剂对Bt几丁质酶的影响.结果表明,甲醇、丙三醇、甲醛和戊二醛对几丁质酶有抑制作用;乙醇、丙醇、乙二醇和二甲亚砜在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;二氧六环的浓度低于 5%时,对酶的影响不明显,而高于 5%时,对酶则有激活作用;丙酮对酶有激活作用. 图 1参

苏云金芽胞杆菌(Bacillus thuringiensis)几丁质酶的分离纯化及酶学性质

以苏云金芽胞杆菌为材料 ,通过硫酸铵沉淀分级分离、DEAE - 32离子交换柱层析初步纯化 ,获得比活力为15 0 .5Umg-1的酶制剂 .研究酶催化胶体几丁质水解的反应时间和酶量对酶活力的关系 ,探讨酶催化胶体几丁质的最适条件 .温度和 pH对酶活力的影响和酶的热稳定性及酸碱稳定性的结果表明 :酶催化胶体几丁质水解的的最适pH为 5 .6、最适温度为 5 8℃ .该酶在pH 4 .5～ 6 .5区域稳定 ,而在pH >8能很快失活 ;在 5 5℃以下处理 30min ,酶活力保持不变 ;高于 5 5℃ ,酶快速失活 .图 5参

八字地老虎高毒力苏云金杆菌(Bacillus thuringiensis)菌株的筛选

摘要：苏云金杆菌是应用最广泛的昆虫病原微生物。文章采用叶片浸渍法，室内用50株Bt菌株对4～5日龄八字地老虎幼虫进行生物测定。结果表明，菌株Btl5、Bt43、Bt47、Bt65、Bt66对八字地老虎幼虫有较高毒力，其中以茵株Btl5毒力最高，其对低龄八字地老虎幼虫杀虫效果可达90％~X上，在第5、7、10天毒力回归方程分别为产28．61353x一177．58825（,-=0．944799），y=37．42650x一226．25199（r=0．983670），y=36．72547x一206．81374（r=0．931267）；其中Lcso分别为9~10’、2．4~10’、0．98~10’efu·mL～。在5×10。、1~10。efu·mL。浓度下，其LT50、u、，o和u、，0分别为2．23、2．75、3．73d；4．28、6．19、10．55d。表明该菌株是一株对八字地老虎有很好生物防治应用前景的Bt菌株。

Bacillus thuringiensis营养期杀虫蛋白Vip3与其转基因植物研究进展

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)是目前应用最多的生物杀虫剂。它能够产生多种杀虫因子,其中,最主要的是杀虫晶体蛋白(Insecticidal Crystal Proteins,ICPs)和营养期杀虫蛋白(Vegetative insectici-dal protein,Vip)。当前,大部分商业化利用的转基因作物均为杀虫晶体蛋白类,随着这些转基因作物种植面积的扩大,害虫对这些较为单一的杀虫蛋白产生抗性已成为一个严峻的问题。Vip3是Vip杀虫蛋白中的一类,不形成蛋白晶体,和ICPs在进化上没有同源性;其对鳞翅目、鞘翅目和同翅目等害虫具有毒杀作用,抗虫谱较广。目前,已经把Vip3基因导入了水稻、玉米和棉花等多种作物中,为作物抗虫育种、延缓害虫产生抗性和减少作物产量损失等带来新的前景。

苏云金芽孢杆菌Bacillus thuringiensis LSZ9408菌株发酵特性的研究

本文研究了苏云金芽孢杆菌 LSZ94 0 8菌株的发酵特性 ,2 4 h菌体浓度达最大 ,2 8- 40 h菌体浓度稳定 ,4 0 h后菌体浓度下降 ,发酵液 p H值先降后升 ,最高达 p H7.8,2 0 h达到碳氮源利用高峰 ,2 8h后氮含量回升 ,糖含量基本不变。

苏云金杆菌(Bacillus thuringiensis)与球孢白僵菌(Beauveria bassiana)复配对小菜蛾幼虫(Plutella xylostella)的影响研究

研究不同浓度苏云金杆菌与球孢白僵菌复配混合剂对佳木斯地区小菜蛾幼虫死亡率的影响。用PBS缓冲液将苏云金杆菌与球孢白僵菌稀释成不同浓度梯度的菌悬液,采用正交分析法对苏云金杆菌及球孢白僵菌复配成7种不同浓度梯度的混合剂,并测定不同浓度混合剂对小菜蛾幼虫的死亡率,从而筛选出较适幼虫死亡的LD_(50)的比例及其浓度。经观察分析,在96 h内苏云金杆菌与球孢白僵菌复配为3:1时,小菜蛾幼虫的死亡率达到72%,其中在1×10^(-1)~1×10^(-6) g/mL浓度下幼虫死亡率较高。各混合液对小菜蛾幼虫致死效果不一,其中当苏云金杆菌与球孢白僵菌复配比例为3:1且浓度在1×10^(-1)~1×10^(-6) g/mL时,对昆虫幼虫致死效果明显。

Screening on Synergist of Bacillus thuringiensis Wettable Powder

[Objective] This study aimed to screen the best synergistic material for Bt wettable powder and evaluate their synergistic effect. [Method] The synergism of six different kinds of additives for Bacillus thuringiensis wettable powder （Bt WP） on the 2^nd instar larvae of Plutella xylostella was tested by method of leaf dipping in labora- tory. [Result] The mixtures of Bt with 0.1% ZnCl2, 0.5% ZnCl2, 1.0% ZnCl2, 1.0% MgCI2, 0.5% boric acid, 1.0% boric acid, 0.5% citric acid or 1.0% citric acid all ex- hibited synergistic effect, in which the synergistic effect of mixture containing 0.5% boric acid was the highest, with 17.2 synergistic ratio; followed by the mixture containing 1.0% ZnCl2, with 15.6 synergistic ratio. Moreover, addition of 0.5% boric acid could shorten the median lethal time of Bt wettable powder by about 10 h. After the mixtures of Bt with 0.5% boracic acid or 1.0% ZnCl2 was stored for 15 d at room temperature, toxicities of the two mixtures did not change significantly. [Conclusion] Boracic acid as the synergist of Bt wettable powder could not only increase insecti- cidal effect of Bt, but also accelerate its insecticidal rate. So, boracic acid could improve the disadvantages of Bt wettable powder such as poor insecticidal effect and slow insecticidal speed in a certain degree.

Bacillus thuringiensis P34与海泡石联合阻控蔬菜积累铅和改良土壤的作用

本研究为建立微生物-吸附剂联合钝化修复技术,以期为重金属污染农田的修复和蔬菜安全生产提供试验依据。在南京市栖霞区某矿区周边农田开展田间试验,探究了Bacillus thuringiensis P34及海泡石(S)对辣椒(苏椒五号)和茄子(苏琦一号)的生长、Pb积累和营养品质的影响,评估复配处理(P34+S)改良蔬菜根际土壤理化性质的作用。结果表明,与对照相比,海泡石可显著降低蔬菜果实Pb含量,菌株P34和海泡石复配可进一步显著降低辣椒和茄子果实Pb含量(58%和26%)。与对照相比,复配处理可显著使辣椒果实生物量增加1.5倍,改善蔬菜的营养品质,还能提高根际土壤pH、芽孢细菌数量、蔗糖酶和脲酶活性,显著降低根际土壤DTPA提取态Pb含量,并显著增加>1 mm团聚体的比例、显著减少<0.25 mm团聚体的比例。将B. thuringiensis P34与海泡石联合施用于Pb污染土壤,可显著阻控茄果类蔬菜积累Pb并提高其营养品质,显著改善根际土壤的理化性质,具有修复Pb污染土壤、保障蔬菜安全生产的潜力。

Isolation and Biological Determination of Bacillus thuringiensis with High Toxicity in Soil of Changbai Mountain Area

[Objective] This study was conducted to isolate and screen new Bacillus thuringiensis （Bt） strains against Lepidoptera insecticides. [Method] Bt strains were isolated from soil of Changbai Mountain area by temperature screening method, and highly-toxic Bt stains were then selected by biological determination and toxicity de- termination. [Result] From 150 soil samples, 18 Bt isolates were isolated, with an average isolation rate of 12.0%. Specifically, the isolation rate from mountain field was 8.5 %, and the isolation rate from farmland was 16.2%. The results of activity determination showed that there were17, 5 and 4 strains showing lethality rate over 90% against Plutel/a xylosrel/a, Spodoptera litura and Spodptera exigua, respectively, and among them, strain YNI-1 exhibited high activity against all the 3 kinds of in- sects. The results of toxicity determination showed that strain YNl-lhad the best fast-acting property against S.litura and S. exigua; strains with high toxicity against S. exigua were YN6-2〉YN1-1〉YN4-2〉YN2-6 sequentially; and toxicity of strains against S. /itura was in order of YN4-4〉YN1-1 〉YN6-1 〉YN4-1 〉YN2-1. [Conclusion] According to activity determination and toxicity determination, strain YN1-1 was screened as the target strain with wide spectrum, fast-acting property and high toxi- city.

A Fragment of Cadherin-Like Protein Enhances Bacillus thuringiensis Cry1B and Cry1C Toxicity to Spodoptera exigua(Lepidoptera:Noctuidae)

Cry toxins produced by Bacillus thuringiensis（Bt） are effective biological insecticides against certain insect species.In this study,bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua.We also identified a cadherin-like gene in S.exigua that could enhance the toxicity of Cry1B and Cry1C.The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE）.The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD.BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects.Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages.The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.