一种新的Bt杀虫基因HJC原核表达蛋白活性的鉴定

HJC基因是由2个Bt基因(cry1Ab和vip3)经过人工融合而成,具有更广谱的杀虫活性,可延缓害虫产生交互抗性的时间。将已构建好的携带HJC基因的重组质粒pET28a-HJC转化到大肠杆菌BL21中诱导表达。该HJC融合蛋白主要以包涵体形式存在,变性条件下使用镍亲和层析柱对其进行纯化,并经尿素梯度透析复性后,进行免疫反应活性及美国白蛾杀虫活性测定。Western blot结果显示,该原核表达蛋白与转HJC基因水稻中的HJC蛋白有相同的免疫反应性,对美国白蛾也有一定的杀虫活性,可以替代植物外源蛋白进行转HJC基因产品的食用安全性评价。     

苏云金芽胞杆菌营养期杀虫蛋白基因特性分析

营养期杀虫蛋白是在苏云金芽胞杆菌营养期中发现的一种非晶体状胞外杀虫蛋白 .通过Southern杂交的基因定位实验 ,证实了营养期杀虫蛋白基因并非位于染色体和环型质粒上 ;在 2 0个不同血清型的 2 3种菌株中 ,营养期杀虫蛋白基因的存在率为 5 6 5 % (13 2 3) .同时对随机选择的 3个不同亚种菌株进行基因克隆和序列分析 ,表明其基因具有相当高的同源性 .运用生物信息技术 ,对营养期杀虫蛋白多肽进行了功能结构域比对分析 ,发现这 789多肽蛋白的N端 31至 15 0残基之间存在一种趋化信号传导因子相似序列 ,C端 5 36至 6 6 6残基之间则存在着纤维素结合结构域相似肽 .上述结果提示 ,营养期杀虫蛋白基因在苏云金芽胞杆菌天然菌株中高度保守并较为广泛分布 ,其编码蛋白的特殊结构提示了营养期杀虫蛋白潜在的功能特性 ,为进一步研究营养期杀虫蛋白奠定了基础 .     

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.     

Introduction of a Lepidopteran-Specific Insecticidal Crystal Protein Gene of Bacillus thuringiensis subsp. kurstaki by Conjugal Transfer into a Bacillus megaterium Strain That Persists in the Cotton Phyllosphere

A lepidopteran toxin gene of the entomopathogen Bacillus thuringiensis subsp. kurstaki HD-1 was introduced into a cotton leaf-colonizing Bacillus megaterium strain, RS1, by conjugal transfer. Rifampin- and nalidixic acid-resistant colonies obtained after cell mating were screened for crystal production by microscopy. A transcipient, B. megaterium RS1-43, was selected by this procedure. Southern blot hybridization with both total DNA and HindIII-digested DNA of the transcipient showed positive signals with a cryIA-specific probe, suggesting the transfer of the lepidopteran-specific cryIA(a) gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis confirmed the presence of the 134-kDa toxic crystal protein specific to lepidopteran larvae in the transcipient. Survival studies with cultures of the transcipient at both vegetative and postvegetative growth stages on cotton, under field conditions, suggested that the bacterium persisted on the leaf surfaces for more than 28 days, with a gradual decline in the population level with time, while the donor, B. thuringiensis subsp. kurstaki, disappeared completely after 7 days following inoculation. An in situ differential crystal-staining technique indicated the production of crystals by the transcipient on cotton leaf surfaces for about 30 days. Leaf bioassays of cotton plants inoculated with a single spray of the transcipient showed 75- to 96% mortality to the first-instar larvae of Heliothis armigera up to 21 days, and this single spray conferred total protection to the plants for about 30 days by causing an antifeeding effect on the remaining larvae.     

Host Range of an Insecticidal Crystal Protein of Bacillus thuringiensis subsp. kurstaki Produced in Escherichia coli

A crylA(c)-like gene of Bacillus thuringiensis subsp. kurstaki strain HD1 was over-expressed in Escherichia coli from a multicopy plasmid. Biological toxicity tests conducted on the larvae of three lepidopteran insects showed that the host range of transgenic E. coli HB 101 (pRT 200) was a subset of the host range of B. thuringiensis kurstaki HD1. Both were toxic to the larvae of Helicoverpa armigera (Gram pod borer) and Bombyx mori (Silkworm). However. though the sporecrystal formulation of HDl was toxic to the larvae of Phthorimaea operculella (Potato tuber moth). the transgenic E. coli was not. Product of St toxin gene other than crylA(c) present In HD1 may be responsible for Its toxicity to the larvae of P. opercuiella.     

农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究

以杂交育种中广泛使用的优良玉米自交系340、4112为材料,用带有质粒pGBIL04（Pactin-Bt-Tnos）的根癌农杆菌LBA4404转化其幼胚及其初始愈伤组织,共培养3天后,在含PPT的培养基上连续筛选培养3代,然后分化获得再生植株。PCR检测证明目的基因已整合到再生植株的基因组中。实验结果表明幼胚预培养后形成的新鲜的初始愈伤组织是比较适宜的转化受体,结果还发现将共培养温度降到22℃可以提高农杆菌介导的玉米遗传转化的筛选频率。 Abstract:Excellent inbred-lines of maize,340 and 4112,which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens.The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04.After 3 days of co-cultivation,the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations,then plants were regenerated.It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants.The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22℃.     

Expression of 135-kDa Insecticidal Protein Gene from Bacillus thuringiensis in the Yeast Saccharomyces cerevisiae

Bacillus thuringiensis subsp. aizawai produces 130-kDa and 135-kDa (CrylA(a)) insecticidal proteins. When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed. When the 5′-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast. The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.     

苏云金杆菌cry 1C基因检测及其与杀虫活性的关系

对实验室分离保存的 5 4株苏云金芽孢杆菌的H 血清型、杀虫晶体蛋白质 ,杀虫基因cry 1C和对甜菜夜蛾的活性进行了检测 ,分析了它们之间的关系。结果表明 ,有 2 8株菌株含有cry 1C基因 ,携带有cry 1C基因的菌株的晶体蛋白质主要为 135ku左右 ,它们对甜菜夜蛾均有较高的毒性 ,这些菌株的鞭毛抗原血清型主要分布在H 5和H 7。     

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.     

苏云金芽胞杆菌cry2Ad基因的克隆及其表达产物的活性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)SBT2是我国新分离出的一株野生菌株.扫描电镜显示该菌株产生双锥体形晶体.琼脂糖凝胶电泳发现其质粒图谱含有5个条带.聚丙烯酰胺凝胶电泳显示此菌株产生130 kD晶体蛋白.利用PCR-RFLP法进行杀虫基因类型鉴定,发现其含有cry1Aa、cry1Da、cry1Hb、cry1Jb、cry1Ka 、cry1Ib、基因.Cry2Ad蛋白的活性至今未见研究报道,本研究克隆和测序了该基因.并对其进行了表达.生物活性测定结果表明其表达产物对舞毒蛾(Lymantria dispar)、棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)、小菜蛾(Plutella xylostella)有低活性;对大猿叶甲(Colaphellus bowringi)无活性.     

苏云金芽孢杆菌营养期杀虫蛋白VIP3研究进展

的报道.本文从VIP3的类型和杀虫活性、作用机理、vip3基因的定位和分离、vip3基因重组和转vip3基因植物等方面详细介绍了VIP3近十年来的研究进展.     

Interaction of Bacillus thuringiensis Vegetative Insecticidal Protein with Ribosomal S2 Protein Triggers Larvicidal Activity in Spodoptera frugiperda

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.Insecticidal proteins produced by strains of Bacillus thuringiensis can broadly be classified into two major categories based on their site of accumulation. Category I consist of proteins that are deposited as crystals in sporangia and are referred to as insecticidal crystalline proteins (ICPs). The second category consists of recently described group of insecticidal proteins, called vegetative insecticidal proteins (8). These proteins are synthesized during the vegetative growth of Bacillus cells and are secreted into the culture medium. Irrespective of the site of accumulation of insecticidal proteins, their ingestion by susceptible insect larvae leads to disruption and lysis of epithelial tissue from the midgut, resulting in larval death (12). The mechanism of lysis of gut epithelial tissue by ICPs has been investigated in detail in several insects (16). Ingestion of ICPs triggers a sequence of biochemical cascade that involves its solubilization and subsequent activation by gut proteases. The activated toxin interacts with specific receptors located at the midgut epithelial tissue. In this sequence of events, the interaction with the receptor is the most significant event since subsequent to interaction, pore formation is initialized, and that leads to lysis of epithelial cells. The identification and characterization of receptors from various insect larvae has led to the identification of following molecules as receptor to ICPs, such as cadherinlike protein (21), glycosyl phosphatidylinositol (GPI)-anchored aminopeptidase N (APN) (1, 9, 11, 17, 19, 20), a GPI-anchored alkaline phosphatase (10, 14), and a 270-kDa glycoconjugate (see references 2, 7, 9, and 16 and references therein for an extensive list of receptors). In addition, certain glycopeptides have been identified as lysis-initiating receptor molecules. Although there is extensive information about the receptor-toxin interaction for ICPs, negligible work has been done toward the identification of receptors to vegetative insecticidal proteins. The ultrastructural changes induced at the midgut epithelial tissue, upon ingestion of ICPs or Vip3As, are common (12). Both ICPs and Vip3As interact at the epithelial layer of midgut, enlarging the affected cells due to osmotic imbalance and eventually causing lysis. In spite of inflicting nearly identical structural damage, the interacting receptor for the Vip3A is not identical (12). In fact, the receptor to Vip3As has not yet been characterized.Our group has been working on the identification, cloning, and evaluation of vegetative insecticidal proteins from strains of B. thuringiensis held in our collection. We have characterized the Vip3A (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"Y17158","term_id":"3135740","term_text":"Y17158"}}Y17158) class of protein and evaluated its toxicity profile (2, 8, 18). Vip3A is active against larvae of Spodoptera litura, among several other lepidopteran pests. In a parallel series of experiments, we identified APN as a receptor to the B. thuringiensis protein Cry1C in S. litura. The heterologously expressed APN did not interact with Vip3A, suggesting that Vip3A toxicity in this insect is not through interaction with APN (1). Our preliminary results on the toxicity of Vip3A revealed that purified insecticidal protein could lyse Sf21 cells, suggesting the presence of receptors in the insect cell line. In the present study, we identified the Vip3A interacting protein in Sf21 cells and the larvae of S. litura. The specificity of the interaction has been examined by a combination of ex vivo and in vitro assays. These assays identified ribosomal S2 protein as the interacting partner of Vip3A. The functional significance of S2-Vip3A protein interaction was examined by monitoring the reduction in Vip3A toxicity in Sf21 cells and larvae of S. litura by the RNA interference-induced knockdown of S2 protein. The results of these experiments are discussed in the context of colocalization of the S2-Vip3A protein interacting complex by confocal microscopy.     

苏云金芽孢杆菌菌株YBT1535转cry1C基因菌的构建

利用电脉冲将ｃｒｙ１Ｃ基因转子苏云金孢杆菌野生菌株ＹＢＴ１５３５,筛选得到３个转化子。质粒电泳、ＰＣＲ扩增及Ｓｏｕｔｈｅｒｎ杂交结果均证明,基因ｃｒｙ１Ｃ已转入菌株ＹＢＴ１５３５。生物测定结果表明,３个转化子对甜菜夜蛾的毒力比出发菌ＹＢＴ１５３５均有显著的提高,转化子ＹＢＴ１５３５－１和ＹＢＴ１５３５－３对小菜蛾和棉铃虫的生物活性与出发菌ＹＢＴ１５３５相近,而转子化子ＹＢＴ１５３５－２则有一定幅度     

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.     

苏云金杆菌抗虫基因cryI Ac 转化辣椒的研究

通过农杆菌介导法将杀虫结晶蛋白基因cryIAc导入到辣椒子叶外植体中,经卡那霉素筛选、组织化学法检测GUS活性以及PCR、southern杂交分析证实cryIAc基因已整合进辣椒核基因组中。     

Only Part of the Protoxin Gene of Bacillus thuringiensis subsp. berliner 1715 Is Necessary for Insecticidal Activity

[This corrects the article on p. 708 in vol. 49.].     

Degradation of the parasporal crystal produced by Bacillus thuringiensis var. kurstaki

Degradation products of the parasporal crystals of Bacillus thuringiensis var. kurstaki obtained by treatment with alkali, gut juice from larvae of Bombyx mori, and various plant and mammalian enzymes were compared for elution pattern, approximate molecular weight (MW), and toxicity. The results indicated that with alkaline treatment the most toxic extract was obtained with 0.05–0.1 M NaOH. Toxicity was found associated mainly with a protein peak of 230,000 MW although other toxic peaks were found in the tailing. Heat-treated midgut juice from larval B. mori gave similar results. After digestion of parasporal crystals with clarified midgut juice, five peaks causing toxicity and having MW of approximately 235,000, 67,000, 30,200, 5000, and 1000, respectively, were identified. Treatment of B. thuringiensis δ-endotoxin with α-chymotrypsin gave peaks causing mortality of approximate MW 235,000, 34,000, 5000, and 1000. Trypsin, pronase, carboxypeptidase, and enterokinase digests of the B. thuringiensis δ-endotoxin gave toxic components ranging from 235,000 to 30,000 MW. The protein protoxin molecules are digested to give small toxic subunits that may be of practical value for structural determinations and for molecular mode of action studies.     

苏云金芽胞杆菌cry2Ab、cry9Ea基因的表达及活性分析

旨在为农业害虫防治提供更多的苏云金芽胞杆菌基因资源,从中国吉林市龙潭山土壤样品中分离得到野生菌株命名为Bt LTS-7,扫描电镜显示该菌株产生晶体形状为双锥体和正方体,聚丙烯酰胺凝胶电泳显示该菌株产生130 kD和71 kD的晶体蛋白,通过PCR鉴定出该菌株中含有cry2Ab和cry9Ea基因,并成功克隆到了这两个新基因,并被Bt国际命名委员会正式命名为cry2Ab28和cry9Ea9,将两个基因分别在大肠杆菌Rosetta( DE3)中表达,并进一步对其表达蛋白进行杀虫活性测定.结果显示,Cry2Ab28蛋白对棉铃虫(Helicoverpa armigera)初孵幼虫具有杀虫活性,LC50为32.45 μg/mL.Cry9Ea9蛋白对小菜蛾初孵幼虫(Plutella xylostella)具有较高杀虫活性,LC50为0.77μg/mL.