苏云金芽孢杆菌杀虫晶体蛋白超量表达的机制

杀虫晶体蛋白是苏云金芽孢杆菌主要杀虫成分,进一步提高杀虫晶体蛋白的表达量是苏云金芽杆菌高效工程菌构建的主要途径。本文讨论了ｃｒｙ基因启动子活性、ｍＲＮＡ稳定性、不同ｃｒｙ基因间的协同表达发及伴了孢晶体的形成等几个方面在转录水平或转录后水平上对杀虫晶体蛋白表达的影响。     

苏云金杆菌杀虫蛋白的多样性

苏云金杆菌是生物防治中应用最为广泛的一种杀虫剂,其杀虫蛋白具有广泛的多样性。本文就苏云金杆菌杀虫蛋白的基因、基因分布、杀虫蛋白结构以及作用机制的多样性进行了概述。     

昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展

鳞翅目昆虫中肠上皮细胞刷状缘膜(BBM)上的Bt杀虫晶体蛋白毒素受体氨肽酶N(APN)的结构和位点密度的改变是昆虫对Bt毒素的主要抗性机制之一,该文简要综述了APN受体的研究进展。每种昆虫中肠上皮细胞中有数种APNs,彼此间同源性较高,其中部分APNs为crylA家族毒素的功能性受体。不同种类昆虫的APNs受体,甚至同一种昆虫的不同类型APNs,其所结合的毒素种类可能不同。APNs决定该昆虫对crylA类毒素的敏感程度差异。有些抗性昆虫的APNs基因编码区发生了多个点突变。     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue

The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis can be bioassayed in vitro using cultured insect tissue. Larval cells of the spruce budworm, Choristoneura fumiferana, are damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity is accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue is inversely proportional to the amount of toxin added. Tissue cells from the spruce budworm exhibited maximum susceptibility to activated δ-endotoxin after 120 hr incubation. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 μg or less of toxin protein per 2 × 10⁵ insect tissue cells. Activated δ-endotoxin was entomocidal to insects as well, as detemined by mortality studies with second-instar larvae of the European corn borer. Electron microscopic observations of insect tissue treated with activated δ-endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.     

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate‐limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.     

杀蚊苏云金芽孢杆菌及其晶体蛋白研究进展

自从发现苏云金芽孢杆菌Bacillusthuringiensis(Bt)具有杀蚊活性以来,目前已发现多种Bt亚种或血清型对蚊虫具有杀虫活性,同时也发现了一些新的杀蚊晶体蛋白。在对杀蚊晶体蛋白的分子结构进行研究的基础上,对其的作用机理有了一定的了解。近年来利用DNA重组技术显著提高杀蚊晶体蛋白的合成和将不同菌种的杀蚊晶体蛋白进行联合表达,为有效控制蚊虫危害展示广阔前景。     

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

AIM: To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia. METHODS AND RESULTS: A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5.6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10.6% of the isolates contained cry1 genes and 1.1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS-PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively. CONCLUSIONS: Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

苏云金芽胞杆菌转座因子的转座机理及其与杀虫晶体蛋白基因的关系

各种苏云金芽胞杆菌在杀虫毒力和杀虫谱上有很大差异。研究表明,这种特异性的杀虫毒力与存在于苏云金芽胞杆菌内的转座因子有密切关系,不同类型的转座因子其转座方式各异,总的来说可分为３种,即同源重组、转座重组和特异位点重组。这种转座过程的发生往往伴随着苏云金芽胞杆菌杀虫晶体蛋白的变异,这在基因工程菌的构建和杀虫多样性的研究上有着重要意义。     

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.     

Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae.

A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.     

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis

The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.