一种新的对鳞翅目昆虫具有广谱活性的苏云金杆菌杀虫蛋白──Vip3A

1996年Estruch等报道了一种由苏云金杆菌BacillusthuringiensisBerliner（下略为Bt）AB88株和AB424株产生的新杀虫蛋白,分别称为Vip3A（a）和Vip3A（b）[详见Proc．Natl。AcadSci.USA93（11）：5389～5394]。Vip3A杀虫蛋白有以下几个特点：（1）它是在Bt的营养体生长阶段产生并分泌到培养液中的。（2）它是不耐热的,95℃处理20分钟便失活。（3）它由791个氨基酸组成,分子量为88．5kD。Vip3A（b）是Vip3A（a）的同系物,两者98％的氨基酸相同,只有5个氨基酸不同。（4）Vip3A杀虫蛋白基因可以在大肠杆菌＆cherichiacoliCastel…     

新vip3Aa基因的克隆、表达及其序列分析

克隆了Bt9816C的vip3A基因,并将测序结果提交到GenBank(序列号:AY945939)。该基因是一个新的vip3Aa基因,Bt杀虫晶体蛋白命名委员会将其命名为vip3Aa18。在大肠杆菌BL21中表达了该基因,生物测定结果表明纯化的Vip3Aa18蛋白对棉铃虫和甜菜夜蛾具有很高的杀虫活性。序列分析结果显示Vip3Aa18C端536至667位氨基酸残基间是一个糖类结合域,推测可能参与Vip3Aa18与敏感昆虫中肠受体结合;N端272至292位氨基酸残基间存在一个跨膜螺旋,可能与Vip3Aa18形成穿孔有关。此外,Vip3Aa18还可能具有一个二硫键。这些特殊区域和位点可能与其功能密切相关。     

苏云金芽孢杆菌vip3A基因的鉴定、克隆及活性分析

【目的】鉴定我国自行分离的苏云金芽孢杆菌（Bacillus thuringiensis, Bt）菌株中vip3A基因的分布和基因型,从其中对鳞翅目幼虫表现高毒力的Bt菌株中克隆vip3Aa基因。【方法】利用PCR-RFLP方法确定vip3A基因的分布和鉴定基因型,利用PCR方法克隆vip3A全长基因。【结果】171株野生型Bt菌株中共鉴定出63株含有vip3A基因,均与vip3Aa1类基因相似。从TF9和Bt16菌株中克隆得到2个vip3Aa基因,分别构建了携带vip3Aa基因的表达载体p30vip-26和p30vip-27,SDS-PAGE和Western Blot分析表明,IPTG诱导后均可表达88 kDa左右的Vip3A蛋白,蛋白可溶性分析表明约10%可溶。这两种基因序列已被国际Bt基因命名委员会分别正式命名为vip3Aa26和vip3Aa27。生物测定结果显示,Vip3Aa27蛋白对粉纹夜蛾（Trichoplusia ni）、甜菜夜蛾（Spodoptera exigua）和棉铃虫（Helicoverpa armigera）3种重要鳞翅目害虫初孵幼虫的毒力较高,LC50值分别为0.125 μg/mL,0.238 μg/mL和9.238 μg/mL。而Vip3Aa26蛋白仅对粉纹夜蛾有活性,LC50值为4.423 μg/mL。【结论】本研究中的Vip3Aa27蛋白对粉纹夜蛾、甜菜夜蛾和棉铃虫幼虫均能表现高杀虫活性,而Vip3Aa26蛋白仅对粉纹夜蛾幼虫有活性,实验结果表明Vip3A蛋白个别氨基酸的变化对其杀虫活性影响很大。     

Bt9816C培养上清中杀虫活性成分的研究*

Bt98 1 6C的培养上清对棉铃虫和甜菜夜蛾具有很高的杀虫活性。热敏实验表明 β -外毒素和ZwittermicinA不是其主要杀虫活性成分。Bt981 6C的无晶体株保留有卵磷酯酶C和溶血素活性 ,但是不再具有vip3A基因 ,其培养上清也丧失了杀虫活性。SDS -PAGE结果显示Bt981 6C培养上清中有Vip3A蛋白特征大小的 89kD的蛋白 ,而其无晶体株正好缺失了该蛋白。以上结果表明Bt981 6C培养上清中主要的杀虫活性成分是Vip3A蛋白。     

新vip3Aa基因的克隆、表达及其序列分析

克隆了Bt9816C的vip3A基因,并将测序结果提交到GenBank (序列号：AY945939)。该基因是一个新的vip3Aa基因, Bt杀虫晶体蛋白命名委员会将其命名为vip3Aa18。在大肠杆菌BL21中表达了该基因,生物测定结果表明纯化的Vip3Aa18蛋白对棉铃虫和甜菜夜蛾具有很高的杀虫活性。序列分析结果显示Vip3Aa18 C端536至667位氨基酸残基间是一个糖类结合域,推测可能参与Vip3Aa18与敏感昆虫中肠受体结合;N端272至292位氨基酸残基间存在一个跨膜螺旋,可能与Vip3Aa18形成穿孔有关。此外,Vip3Aa18还可能具有一个二硫键。这些特殊区域和位点可能与其功能密切相关。     

新vip3Aa基因的克隆、表达及其序列分析

克隆了Bt9816C的vip3A基因，并将测序结果提交到GenBank (序列号：AY945939)。该基因是一个新的vip3Aa基因, Bt杀虫晶体蛋白命名委员会将其命名为vip3Aa18。在大肠杆菌BL21中表达了该基因，生物测定结果表明纯化的Vip3Aa18蛋白对棉铃虫和甜菜夜蛾具有很高的杀虫活性。序列分析结果显示Vip3Aa18 C端536至667位氨基酸残基间是一个糖类结合域，推测可能参与Vip3Aa18与敏感昆虫中肠受体结合；N端272至292位氨基酸残基间存在一个跨膜螺旋，可能与Vip3Aa18形成穿孔有关。此外，Vip3Aa18还可能具有一个二硫键。这些特殊区域和位点可能与其功能密切相关。     

苏云金杆菌辅助蛋白P20对营养期杀虫蛋白Vip3A表达的影响

为检测苏云金杆菌辅助蛋白P20对Vip3A表达和杀虫活性的影响,将p20基因与vip3A基因相连构建了重组质粒pHVP20,然后电激转化至Bt中进行了共表达,以仅携带vip3A基因的质粒pHPT3作为对照质粒。Westernblot结果显示,当vip3A基因和p20基因在Bt无晶体缺陷株CryB中共表达时,Vip3A蛋白的最大表达量约是其在CryB(pHPT3)菌株中单独表达的1.5倍。生物测定结果表明,CryB(pHVP20)和CryB(pHPT3)菌株对初孵斜纹夜蛾幼虫的LC50值分别为48.79μg/mL和78.00μg/mL,这说明P20蛋白可以促进vip3A基因在Bt中的表达,但对提高Vip3A蛋白的杀虫毒力没有显著性帮助。     

苏云金芽孢杆菌vip3A基因的检测及保守性分析

Vip3A蛋白是苏云金芽孢杆菌(Bacillus thuringiensis,Bt)在营养期分泌的一类新型杀虫蛋白。用PCR方法从114个Bl菌株和41个Bl标准菌株中筛选到39株即约25％的菌株含有vip3A基因。利用所制备的Vip3A蛋白的多克隆抗体对以上含有vip3A基因的Bt菌株进行Western印迹分析,发现多数PCR反应为阳性的菌株都产生89kD大小的蛋白,其中有4株没有Vip3A蛋白的表达。从以上菌株中挑选2个对夜蛾科害虫具有较高和较低毒力的菌株,即S101和6ll,并分别进行vip3A基因的克隆和测序,再与GenBank上所登录的其它6个全长vip3A基因和2个已报道的但未登录GenBank的vip3A基因进行核苷酸和氨基酸序列比较,结果表明,vip3A是一个极其保守的基因。将以上所克隆的2个却3A基因即vip3A—S101和vip3A-611分别插入表达载体pQE30构建了表达质粒pOTP-S101和pOTP-6ll,转化到大肠杆菌M15,经lmmol／L IPTG诱导后均表达89kD大小的Vip3A蛋白。蛋白可溶性试验表明,Vip3A-S101和Vip3A-611分别有48％和35％的蛋白是可溶的。将Vip3A-S101和Vip3A-6ll蛋白和已报道的Vip3A—S184蛋白对初孵斜纹夜蛾(Spodoptera litura)幼虫进行生物测定,结果表明,3个Vip3A蛋白对斜纹夜蛾幼虫毒力没有显著性差异,这说明了Vip3A个别氨基酸的变化对蛋白的杀虫活性没有影响。     

苏云金芽胞杆菌Vip3AaAdAa嵌合蛋白的构建及其杀虫活性分析

Vip3A类杀虫蛋白是一类新型杀虫蛋白,在氨基酸序列上与研究较为深入的Cry蛋白没有同源性,且昆虫受体结合位点与Cry蛋白也没有竞争关系。利用同源重组技术针对对甜菜夜蛾具有高活性的Vip3Aa39与无活性的Vip3Ad开展研究,成功构建了三个vip3AaAdAa型嵌合基因,将其转入大肠杆菌中表达嵌合蛋白Vip3AaAdAa1、Vip3AaAdAa2、Vip3AaAdAa3,并将这三种嵌合蛋白对甜菜夜蛾进行了杀虫活性测定。研究表明,三种嵌合蛋白对甜菜夜蛾具有杀虫活性,Vip3AaAdAa3蛋白的LC_(50)值比Vip3Aa39增加了1.4倍,Vip3AaAdAa1蛋白的LC_(50)值比Vip3Aa39增加了2.7倍,嵌合蛋白Vip3AaAdAa1和Vip3AaAdAa3的杀虫活性均低于Vip3Aa39;Vip3AaAdAa2的杀虫活性与Vip3Aa39差异不显著。     

苏云金芽孢杆菌vip3A基因的检测及保守性分析

Vip3A蛋白是苏云金芽孢杆菌（Bacillus thuringiensis,Bt）在营养期分泌的一类新型杀虫蛋白。用PCR方法从114个Bt菌株和41个Bt标准菌株中筛选到39株即约25%的菌株含有vip3A基因。利用所制备的Vip3A蛋白的多克隆抗体对以上含有vip3A基因的Bt菌株进行Western印迹分析,发现多数PCR反应为阳性的菌株都产生89 kD大小的蛋白,其中有4株没有Vip3A蛋白的表达。从以上菌株中挑选2个对夜蛾科害虫具有较高和较低毒力的菌株,即S101和611,并分别进行vip3A基因的克隆和测序,再与GenBank上所登录的其它6个全长vip3A基因和2个已报道的但未登录GenBank的vip3A基因进行核苷酸和氨基酸序列比较,结果表明,vip3A是一个极其保守的基因。将以上所克隆的2个vip3A基因即vip3AS101和vip3A611分别插入表达载体pQE30构建了表达质粒pOTP-S101和pOTP-611,转化到大肠杆菌M15,经1 mmol/L IPTG诱导后均表达89 kD大小的Vip3A蛋白。蛋白可溶性试验表明,Vip3A-S101和Vip3A-611分别有48%和35%的蛋白是可溶的。将Vip3A-S101和Vip3A-611蛋白和已报道的Vip3A-S184蛋白对初孵斜纹夜蛾 (Spodoptera litura) 幼虫进行生物测定,结果表明,3个Vip3A蛋白对斜纹夜蛾幼虫毒力没有显著性差异,这说明了Vip3A个别氨基酸的变化对蛋白的杀虫活性没有影响。     

Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene

AIMS: To search for novel Vip3A proteins for controlling insect pests. METHODS AND RESULTS: A pair of universal primers was designed based on the conserved regions of five vip3A genes. Amplified products were digested with the HindIII and EcoR enzymes so as to confirm different restriction fragment length polymorphism (RFLP) patterns used to identify vip3A-type genes. The vip3A gene types of 606 Bacillus thuringiensis strains were screened and three patterns of RFLP were successfully identified. Two novel vip3A genes were found and one of these, vip3Aa19, was further characterized and its product was confirmed toxic to Spodoptera exigua, Helicoverpa armigera and Plutella xylostella larvae. Partial sequences of another novel vip3A-type gene were obtained that shared 83% homology with that of the vip3Af1 gene. CONCLUSIONS: A polymerase chain reaction (PCR)-RFLP system we developed could be used for identifying novel vip3A-genes from B. thuringiensis strains. A novel Vip3A protein was found to have a broader insecticidal spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported method is a powerful tool to find novel Vip3A proteins from large-scale B. thuringiensis strains. The novel Vip3A protein may be used to control insect pests or resistant insect pests by constructing genetically engineered strains or transgenic plants.     

Efficient expression of vip184DeltaP gene under the control of promoters plus Shine-Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis

AIMS: To compare vip184DeltaP gene expression time course and Vip184 protein yield under the control of promoters and Shine-Dalgarno (SD) sequences of vip184, cry3A and cry1A gene from Bacillus thuringiensis respectively. METHODS AND RESULTS: Derived from the shuttle vector pHT3101, recombinant plasmids pHPT3, pHTP3A(Delta)P and pHTP1A(Delta)P were constructed with the native vip184 gene and the vip184(Delta)P gene, either under the control of promoters and SD sequences of cry3A or cry1A genes. When the above plasmids were transformed into an acrystalliferous B. thuringiensis strain Cry(-)B, their expression time course were consistent with those of vip184, cry3A and cry1A gene respectively. The maximum yields of Vip184 protein were increased when under the control of promoters plus SD sequences of cry3A and cry1A gene. CONCLUSIONS: The results showed that both cry3A and cry1A promoter/SD sequence combinations were able to enhance synthesis of Vip184 and change its expression time course. SIGNIFICANCE AND IMPACT OF THE STUDY: Both cry3A and cry1A promoter/SD systems offer a method for improving the expression efficacy of the vip184 gene in B. thuringiensis and it is possible to co-express the vip184 gene and cry genes and accumulate Vip184 in the form of inclusion bodies by these systems in order to construct novel useful B. thuringiensis engineered strains.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

Effects of Mycobacterium bovis on monocyte-derived macrophages from bovine tuberculosis infection and healthy cattle

A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance.     

Increase of the Bacillus thuringiensis Secreted Toxicity Against Lepidopteron Larvae by Homologous Expression of the vip3LB Gene During Sporulation Stage

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein.     

Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid

A novel vip3-type gene named vip3LB has been isolated from Bacillus thuringiensis strain BUPM95. The corresponding secreted vegetative insecticidal protein was active against the lepidopteran insect Ephestia kuehniella. The vip3LB gene was shown, for the first time, to be carried by the large plasmid containing the cry1Ia genes of B. thuringiensis. The nucleotide sequence predicted a protein of 789 amino acids residues with a calculated molecular mass of 88.5kDa. Both nucleotide and amino acid sequences similarity analysis revealed that vip3LB is a new vip3-type gene, presenting several differences with the other vip3-type genes. Heterologous expression of the vip3LB under the control of the strong promoter P(BAD) was performed in Escherichia coli and the produced protein conferred insecticidal activity against Ephestia kuehniella. This novel vegetative insecticidal protein Vip3LB could be a very useful biological control agent.     

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.