苏云金杆菌vip3A基因的克隆、表达及杀虫活性分析

用全长PCR方法从野生型苏云金杆菌（Bacillus thuringiensis ,Bt)菌株S184中克隆了2.3kb大 vip3A基因并进行了序列分析。将vip3A-S184基因插入表达载体pQE30构建了表达质粒pOTP,转化大肠杆菌M15,转化子经1mmol/L IPTG诱导后可表达89kD大小的Vip3A-S184蛋白,并得到Western blot证实。蛋白可溶性试验表明,目的蛋白中约有19％是可溶的,用透射电镜观察到大多数蛋白是以包涵体形式存在的。因此,可以在自然条件下进行目的蛋白的纯化和对家兔进行免疫制备多克隆抗体,用于苏云金杆菌Vip3A蛋白表达的检测。利用IPTG进行诱导培养的菌液对甜菜夜蛾（Spodoptera exigua）,斜纹夜蛾（S.litura)和棉铃虫（Helicoverpa armigera)等3种害虫的初孵幼虫进行生物测定,结果表明,Vip3A-S184蛋白对夜蛾科害虫具有较高的杀虫活性。     

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。     

Expression of vip1/vip2 genes in Escherichia coli and Bacillus thuringiensis and the analysis of their signal peptides

AIMS: To determine the expression time courses and high expression level of Vip2A(c) and Vip1A(c) in Bacillus thuringiensis, and survey their insecticidal toxicity and insecticidal spectrum. METHODS AND RESULTS: A kind of new vegetative insecticidal toxin genes encoded by a single operon from B. thuringiensis had been cloned and sequenced. The individual genes, 5-terminus truncated genes and the operon were respectively expressed in Escherichia coli. Only N-terminus deleted Vip2A(c) and Vip1A(c) proteins could be purified by Ni-NTA agarose, while others were processed and their N-terminal signal peptides were cleaved. The individual genes and the operon were also expressed in B. thuringiensis. Both proteins were mostly secreted into the cell supernatants. The expression level of Vip1A(c) was influenced because of the interruption of vip2A(c) gene on the operon. Bioassays showed that neither separate protein nor both performed any toxicity against tested lepidopteran and coleopteran insects. CONCLUSIONS: Vip2A(c) and Vip1A(c) have similar secretion mechanism in E. coli and B. thuringiensis. Vip1A(c) remained its high expression level only when being expressed with vip2A(c) gene as an operon in B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of vip2A(c) and vip1A(c) genes in E. coli and B. thuringiensis were investigated. This would help to make clear the secretion mechanism of VIP proteins and study the function of ADP-ribosyltransferase Vip2.     

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.     

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.     

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。     

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.     

Vip3A is responsible for the potency of Bacillus thuringiensis 9816C culture supernatant against Helicoverpa armigera and Spodoptera exigua

Culture supernatant of Bacillus thuringiensis 9816C had high toxicity against Helicoverpa armigera and Spodoptera exigua. However, it lost insecticidal activities after being bathed in boiling water for 5 min. Acrystalliferous mutants of Bt9816C (Bt9816C-NP1 and Bt9816C-NP2) cured of its endogenous plasmids no longer possessed vip3A gene and toxicity. The 89 kD protein which existed in Bt9816C supernatant disappeared in the two mutants' supernatant; nevertheless, the two mutants still exhibited hemolytic and phospholipase C activity as Bt9816C did. The vip3A gene of Bt9816C, vip3Aa18, was cloned and expressed in Escherichia coli BL21. Bioassay demonstrated that the recombinant E. coli had high toxicity against S. exigua. Taken together, it suggested that Vip3A protein was responsible for the toxicity of Bt9816C culture supernatants.     

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.     

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。     

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.     

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.     

Unusually High Frequency of Genes Encoding Vegetative Insecticidal Proteins in an Australian <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Collection

Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.     

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.     

Novel Vip3-related protein from Bacillus thuringiensis

A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.     

Characterization,cloning, expression and bioassay of vip3 gene isolated from an Egyptian Bacillus thuringiensis against whiteflies

Throughout the vegetative life of Bacillus thuringiensis, vegetative insecticidal proteins (Vip) are produced and secreted. In the present study, the vip3 gene isolated from Bacillus thuringiensis, an Egyptian isolate, was successfully amplified (2.4 kbp) and expressed using bacterial expression system. The molecular mass of the expressed protein was verified using SDS-PAGE and western blot analysis. Whiteflies were also screened for susceptibility to the expressed Vip3 protein (LC50). In addition, ST₅₀ was determined to assess the kill speed of the expressed Vip3 protein against whiteflies compared to the whole vegetative proteins. The results showed that the potency of whole B. thuringiensis vegetative proteins against whiteflies was slightly higher than the expressed Vip3 protein with 4.7-fold based on LC50 value. However, the ST50 parameter showed no significant difference between both the B. thuringiensis vegetative proteins and the expressed Vip3 alone. The results showed that the vip3 gene was successfully expressed in an active form which showed high susceptibility to whiteflies based on the virulence parameters LC₅₀ and ST₅₀. To our knowledge, this study showed for the first time the high toxicity of the expressed Vip3 proteins of B. thuringiensis toward whiteflies as a hopeful and promising bio-control agent.     

Screening of vip genes from a Spanish Bacillus thuringiensis collection and characterization of two Vip3 proteins highly toxic to five lepidopteran crop pests

A Spanish Bacillus thuringiensis strain collection was screened for the presence of vip genes. One hundred strains from a Canary Island collection were screened for vip1 and vip2 genes and 7% contained potentially novel vip1 and vip2-like genes, as indicated by the low degree of similarity with previously known vip1 and vip2 genes. Four hundred strains from a collection originating from the Spanish mainland were screened for vip3 genes and 14.5% of them contained potentially novel vip3-like genes. Reconstruction of the full-length vip sequences could only be achieved for two vip3 gene variants encoding 789 and 787 amino acid proteins that were designated as Vip3Aa45 and Vip3Ag4, respectively. These proteins showed 82% pairwise identity between them and differed from Vip3Aa1 in the putative signal peptide, two specific proteolytic processing sites and the 66-kDa insecticidal fragment. The purified proteins were tested against nine lepidopteran pest species and displayed toxicity, expressed as mean lethal concentration, for five of them. The two toxins were highly toxic for Lobesia botrana (∼1–2 μg/ml) and Spodoptera littoralis (∼20 ng/cm²), moderately toxic for Spodoptera exigua (∼100–300 ng/cm²), and varied greatly in their toxicity for Mamestra brassicae or Chrysodeixis chalcites, with high toxicity for Vip3Aa45 in M. brassicae (∼40 ng/cm²) and for Vip3Ag4 in C. chalcites (∼45 ng/cm²).     

Characterization of Vegetative Insecticidal Protein vip Genes of Bacillus thuringiensis from Sichuan Basin in China

Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.