苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC50为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

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.     

Cry29A and Cry30A: two novel delta-endotoxins isolated from Bacillus thuringiensis serovar medellin

Two novel crystal protein genes from a highly mosquitocidal Bacillus thuringiensis serovar medellin strain were cloned and sequenced. The corresponding proteins, Cry29A and Cry30A, were nontoxic when tested individually against the mosquito species bioassayed (Aedes aegypti, Culex pipiens and Anopheles stephensi). However, Cry29A synergized the toxicity of Cry11Bb against Aedes aegypti by a four-fold factor.     

Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.     

Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.

Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.     

Cloning and expression of the insecticidal crystal protein gene Cry1Ca9 of Bacillus thuringiensis G10-01A from Taiwan granaries

A new cry gene (cry1Ca9) was cloned and sequenced from a Bacillus thuringiensis isolate native to Taiwan (G10-01A). The cry1C-type gene, designated cry1Ca9, consisted of an open reading frame of 3,567 bp, encoding a protein of 1,189 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 134.7 kDa. The gene sequence was compared against the GenBank nucleotide sequence data base. It was found that the cry1Ca9 gene coded for a 134.7-kDa protoxin which had greater than 99.8% homology with the previously reported cry1Ca1 gene, as only three mismatches were found between the two amino acid sequences. When the Cry1Ca9 toxin was expressed in a crystal-negative strain of B. thuringiensis (cryB-), elliptical crystals were produced. Cell extracts from this recombinant strain appear to have high insecticidal activity against lepidopteran larvae (Plutella xylostella).     

Cryptic endotoxic nature of Bacillus thuringiensis Cry1Ab insecticidal crystal protein

Cry1Ab is one of the most studied insecticidal proteins produced by Bacillus thuringiensis during sporulation. Structurally, this protoxin has been divided in two domains: the N-terminal toxin core and the C-terminal portion. Although many studies have addressed the biochemical characteristics of the active toxin that corresponds to the N-terminal portion, there are just few reports studying the importance of the C-terminal part of the protoxin. Herein, we show that Cry1Ab protoxin has a unique natural cryptic endotoxic property that is evident when their halves are expressed individually. This toxic effect of the separate protoxin domains was found against its original host B. thuringiensis, as well as to two other bacteria, Escherichia coli and Agrobacterium tumefaciens. Interestingly, either the fusion of the C-terminal portion with the insecticidal domain-III or the whole N-terminal region reduced or neutralized such a toxic effect, while a non-Cry1A peptide such as maltose binding protein did not neutralize the toxic effect. Furthermore, the C-terminal domain, in addition to being essential for crystal formation and solubility, plays a crucial role in neutralizing the toxicity caused by a separate expression of the insecticidal domain much like a dot/anti-dot system.     

Selection of reliable reference genes for gene expression studies in Caenorhabditis elegans exposed to crystals (Cry1Ia36) protein of Bacillus thuringiensis

Molecular Biology Reports - Quantitative real time PCR (qRT-PCR) is a nucleic acid quantitative technique and is also considered as a validation tool. The Cry1Ia36 protein isolated from Bacillus...     

Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis(Bt), BBT2-5. The cry8Db gene has 3525 bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3 D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 μg per 1 cm² of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70 kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D’s and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.     

Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.     

A novel recombinant baculovirus expressing insect neurotoxin and producing occlusion bodies that contain Bacillus thuringiensis Cry toxin

A novel recombinant baculovirus, designated AcB5A, was constructed to develop an improved baculovirus insecticide with additional beneficial properties. Bacillus thuringiensis crystal protein gene (cry1–5) and an insect-specific neurotoxin gene (AaIT) were introduced into the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin–cry1–5–polyhedrin under the control of polyhedrin gene promoter, and by insertion of AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus. RT-PCR analysis with total RNA from AcB5A-infected cells indicated that cry1–5 and AaIT genes were normally transcribed. The 150 kDa of polyhedrin–Cry1–5–polyhedrin fusion protein was produced by AcB5A and occluded into polyhedra produced by the recombinant virus. This protein was activated when treated with trypsin to form a crystal protein of approximately 65 kDa. The AcB5A showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the lethal time against Spodoptera exigua larvae compared to those infected with wild-type AcMNPV. The expression level of the fusion protein decreased after in vivo passage as a result of homologous recombination between the two polyhedrin genes.     

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

The insecticidal crystal protein Cry2Ab10 from Bacillus thuringiensis: cloning, expression, and structure simulation

The cry2Ab-type gene was cloned from Bacillus thuringiensis and designated as cry2Ab10. The recombinant Cry2Ab10 protein expressed in E. coli cells shows high toxicity against Plutella xylostella. The protein structure was constructed by homology modeling, and the receptor-binding sites were predicted by a molecular docking method.