苏云金芽孢杆菌及其杀虫晶体蛋白 作用机制的研究进展

综合叙述了苏云金芽胞杆菌Bacillus thuringiensis和杀虫晶体蛋白的作用机制及在不同水平上解释这些机制的一些流行模型和有关亚分子结构的作用。     

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。     

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.     

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。     

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.     

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P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

一株苏云金杆菌噬菌体的形态结构及其蛋白质性质

从武汉微生物药厂苏云金杆菌发酵裂解液中分离到1株具有独特形态结构的苏云金杆菌噬菌体GP-1。电镜观察发现,这株噬菌体的头部呈长六棱柱状,具一短直尾和一“衣领”状结构,并首次发现了“衣领”状结构是由8～10个颗粒亚单位组成。该株噬菌体所具有的这8～10个颗粒亚单位对于噬菌体牢固地吸附于宿主表面应具有很强的促进作用,对于进一步研究噬菌体与宿主之间的关系提供一个结构上的证据。该株噬菌体的蛋白经SDS-聚丙烯酰胺凝胶电泳法测定,呈现一条主带,分子量为58892 D,一条次主带和七条次带,表明该株噬菌体的蛋白是由9种蛋白质构成。     

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.     

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。     

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。