Interactions of Insecticidal Toxin Gene Products from Xenorhabdus nematophilus PMFI296

Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined. The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E. coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens. The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P. rapae and P. brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H. virescens. When each of these three genes was expressed individually in E. coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes xptB1 and xptC1 were expressed in the same E. coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P. rapae and P. brassicae. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H. virescens. Individual gene disruptions in X. nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E. coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes. The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.     

Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC(50)] of 2 to 6 microg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

昆虫病原线虫共生菌BP品系杀虫毒素基因簇中各基因与杀虫活性的关系

昆虫病原线虫共生菌Xenorhabdus nematophilus BP的多个杀虫毒素基因集中在一起形成一个约40kb的基因簇。为研究这个基因簇中各基因与杀虫活性的关系,对该共生菌粘粒文库中5个粘粒克隆XnBP76、XnBP83、XnBP203、XnBPp378 和XnBP414及XnBP83的3个亚克隆插入DNA片段的基因结构和它们对棉铃虫的杀虫活性进行了比较,结果显示,xptB1, xptC1和xptA2 3个基因或后两者的联合表达产物具有最强的杀虫效果,缺失其中的任何1个或2个会使杀虫活力大幅度地下降或完全消失;而xptD1和xptA1的缺失对毒素基因簇的表达产物的杀虫活力影响很小;杀虫毒素的物理混合没有明显的增效作用。     

Sequence Analysis of Insecticidal Genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC₅₀] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P_L promoter were shown to have insecticidal activity (LC₅₀ of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.     

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。     

A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein

Only one of the four lepidoptera-specific crystal protein subclasses (CryIC) Bacillus thuringiensis was previously shown to be highly toxic against several Spodoptera species. By using a cryIC-derived nucleotide probe, DNA from 25 different strains of B. thuringiensis was screened for the presence of homologous sequences. A putative crystal protein gene, considerably different from the cryIC gene subclass, was identified in the DNA of strain 4F1 (serotype kenyae) and cloned in Escherichia coli. Its nucleotide sequence was determined and appeared to contain several features typical for a crystal protein gene. Furthermore, the region coding for the N-terminal part of the putative toxic fragment showed extensive homology to subclass cryIA sequences derived from gene BtII, whereas the region coding for the C-terminal part appeared to be highly homologous to the cryIC gene BtVI. With an anti-crystal protein antiserum, a polypeptide of the expected size could be demonstrated in Western immunoblots, onto which a lysate of E. coli cells harboring the putative gene, now designated as BtXI, had been transferred. Cells expressing the gene appeared to be equally toxic against larvae of Spodoptera exigua as recombinant cells expressing the BtVI (cryIC)-encoded crystal protein. However, no toxicity against larvae of Heliothis virescens, Mamestra brassicae, or Pieris brassicae could be demonstrated. The nucleotide sequence analysis and the toxicity studies showed that this novel crystal protein gene falls into a new cryl gene subclass. We propose that this subclass be referred to as cryIE.     

Photorhabdus toxins: novel biological insecticides.

Following concerns over the potential for insect resistance to insecticidal Bacillus thuringiensis toxins expressed in transgenic plants, there has been recent interest in novel biological insecticides. Over the past year there has been considerable progress in the cloning of several alternative toxin genes from the bacteria Photorhabdus luminescens and Xenorhabdus nematophilus. These genes encode large insecticidal toxin complexes with little homology to other known toxins.     

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.     

Cloning of Baeyer-Villiger monooxygenases from Comamonas,Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.     

Relationships between parasitoid host range and host defence: a comparative study of egg encapsulation in two related parasitoid species

Abstract. Parasitoid host range may proceed from traits affecting host suitability, traits affecting parasitoid foraging behaviour, or both. We tested the hypothesis that encapsulation can be used as a reliable indicator of parasitoid host range in two closely related larval endoparasitoids of Lepidoptera. Cotesia glomerata (L.) (Hymenoptera: Braconidae) is gregarious and a generalist on several species of Pieridae, whereas C. rubecula (Marshall) is solitary and specific to Pieris rapae (L.). We determined the effects of host species ( Pieris brassicae (L.), P. napi (L.) and P. rapae ) (Lepidoptera: Pieridae) and host developmental stage (early first, second and third instar) on encapsulation of parasitoid eggs. Host species and parasitoid species, as well as the resulting interaction between these two factors had significant effects on encapsulation of Cotesia eggs. Encapsulation in Pieris hosts was much lower for C. glomerata (<34%, except for second and third instar of P. rapae ) than for C. rubecula (>32%), even when the latter was parasitizing P. rapae. Encapsulation increased with the age of the larvae, although the only significant difference was for C. glomerata. Overall, P. rapae showed a stronger encapsulation reaction than P. brassicae and P. napi. Encapsulation levels of C. glomerata corresponded well to patterns of female host species and host age preference for oviposition and parasitoid larval performance. In contrast, percentages of encapsulation of C. rubecula were not consistent with host preference and host suitability. We argue that encapsulation alone is unlikely to provide a sufficient explanation for C. glomerata and C. rubecula host range.     

Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human

E,E,E-Geranylgeranyl diphosphate (GGPP) is an important precursor of carotenoids and geranylgeranylated proteins such as small G proteins. In this study, we have identified mouse and human GGPP synthase genes. Sequence analysis showed that mouse and human GGPP synthases share a high level of amino acid identity (94%) with each other, and share a high level of similarity (45-50%) with GGPP synthases of lower eukaryotes, but only weak similarity (22-31%) to plant and prokaryotic GGPP synthases. Both of the newly identified GGPP synthase genes from mouse and human were expressed in Escherichia coli, and their gene products displayed GGPP synthase activity when isopentenyl diphosphate and farnesyl diphosphate were used as substrates. The GGPP synthase activity of these genes was also confirmed by demonstrating carotenoid synthesis after co-transformation of E. coli with a plasmid expressing the crt genes derived from Erwinia uredovora, and a plasmid expressing either the mouse or human GGPS1 gene. Southern blot analysis suggests that the human GGPS1 gene is a single copy gene.     

His/GST-HDAC1在大肠杆菌中表达的研究

目的:对His/GST-HDAC1在大肠杆菌BL-21中的表达进行研究。方法:HDAC1的完整基因片断被克隆到pColdⅠ载体和pGEX载体上,并在其N末端分别联有His和GST;采用大肠杆菌BL21对HDAC1进行表达;采用亲和色谱对HDAC1进行纯化;用SDS-PAGE和蛋白质印迹来验证表达和纯化效果;对HDAC1活性进行测定。结果:多数HDAC1存在于大肠杆菌BL-21细胞裂解液的沉淀组分和纯化过程中的未结合组分中,小部分HDAC1可从细胞裂解液的上清液中得以纯化,但未显现出酶活性。用FPLC对HDAC1进行进一步分离,结果表明,HDAC1发生了分子聚集,使得它们的分子量大于正常分子量。结论:活性His/GST-HDAC1不能用大肠杆菌BL21成功表达。     

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.     

小桐子果壳提取物杀虫活性的生物测定

以水、乙醇、正丁醇、乙酸乙酯、氯仿和石油醚为溶剂,采用冷浸法对小桐子果壳进行粗提,并分别测定各粗提物对豌豆长管蚜和菜青虫的毒力,从中筛选出毒力最强的粗提物进一步对菜青虫进行作用方式的测定.6种溶剂粗提物对豌豆长管蚜和菜青虫的毒力测试结果表明:三氯甲烷提取物和乙醇提取物的活性显著高于其它溶剂提取物,且两种提取物对豌豆长管...     

Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.     

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。     

几种十字花科蔬菜害虫生态位的研究

以Levins生态位宽度指数和Pianka生态位重叠指数估计发生在同一营养阶层的 4种主要十字花科植物害虫营养生态位、时间生态位和空间生态位的生态位宽度和重叠度。结果表明小菜蛾、黄曲条跳甲、菜粉蝶和小猿叶甲 4种昆虫的生态位并不完全重叠。     

A toxin protein from Xenorhabdus nematophila var. pekingensis and insecticidal activity against larvae of Helicoverpa armigera

Xenorhabdus nematophila var. pekingensis, which is highly virulent for many insects, is a symbiotic bacterium of Steinernema carpocapsae isolated from Beijing soil in China. Previous studies demonstrated that the bacterium had high antifeedant activity against larvae of Helicoverpa armigera, Plutella xylostella and Spodoptera exigua. Herein, we report the purification, molecular cloning and antifeedant activity of an intracellular toxic protein from the bacterium. The purified protein displayed a single band and a relative molecular weight of over 212 kDa determined by SDS-PAGE. We designated the protein as XnAFP2. Peptide segments were obtained by MALDI-TOF and covered 40% of the amino acid sequence of a toxin protein from X. nematophilus PMFI1296. The full cDNA sequence encoding for XnAFP2 (Genbank accession number FJ222606) was amplified from X. nematophlia var. pekingensis and consists of 7575 bp. The gene showed homology with up to 99% identity to the A2 gene from X. nematophila strain BP (GenBank accession number AY282763) and 92% identity to the insecticidal toxin xptA2 gene from X. nematophila PMFI 1296 (GenBank accession number AJ308438). The protein caused a rapid cessation in feeding and reduction in larval weight of H. armigera. When fed to third instar larvae of H. armigera in an artificial diet at 6.0 µg/g (w/w) toxin protein, growth reduction reached 97.9%. The insecticidal protein greatly decreased fourth instar larval weight, lengthened larval stage, and reduced pupation and emergence rates. The antifeedant rate in choice and no-choice leaf disk tests against fifth instar larvae was 78.4 and 87.6% in 24 h, respectively.