Dafang Huang

Identification of cry1I-Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry1I-Type Gene

ABSTRACT A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis δ-endotoxin nomenclature committee.

Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn borer and other lepidopteran insects.

A novel cry1A was cloned from Bacillus thuringiensis strain BT8 and expressed in the B. thuringiensis acrystalliferous mutant HD73(-). The gene, designated cry1Ah1, encoded a protein with a molecular weight of 134 kDa. Reverse transcriptase-PCR and Western blotting showed that Cry1Ah was expressed in the host strain BT8. The toxin expressed in HD73(-) exhibited high toxicity against lepidopteran larvae of Ostrinia furnacalis, Helicoverpa armigera, Chilo suppressalis, and Plutella xylostella. The 50% lethal concentrations (LC(50)s) were 0.05, 1.48, 0.98 microg g(-1) and 1.52 microg mL(-1), respectively. The LC(50)s of Cry1Ah were significantly lower than that of Cry1Ac for H. armigera, C. suppressalis, and O. furnacalis, and lower than that of Cry1Ab and Cry1Ie for Ostrinia furnacalis. The high toxicity against a range of pest species makes this novel toxin a potential candidate for insect biocontrol.

Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer : Holotrichia parallela

A new Bacillus thuringiensis strain, Bt185, was isolated from HeBei soil samples in China. Observations after transmission electron microscopy found that the strain produced spherical parasporal inclusions similar to that of the B. thuringiensis subsp. japonensis Buibui strain, which showed toxicity to both Anomala corpulenta and Popillia japonica. The plasmid profile seen on an agarose gel revealed that Bt185 contained six large bands of 191 kb, 161 kb, 104 kb, 84 kb, 56 kb, and 37 kb. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis revealed one major band with an estimated molecular mass of 130 kDa. Polymerase chain reaction–restriction fragment length polymorphism results showed that a novel cry8-type gene sequence was found in the Bt185 strain. When we screened for this novel gene sequence, an additional novel cry8-type gene was isolated, having a partial sequence of 2340 bp and encoding a protein of 780 amino acids. Bioassay results showed that Bt185 had no toxicity against several Coleopteran and Lepidopteran pests. However, Bt185 exhibited toxicity against larvae of the Asian cockchafer, Holotrichia parallela. This is the first report of the occurrence of a Bacillus strain that has insecticidal activity against Holotrichia parallela larvae.

Improving Toxicity of Bacillus thuringiensis Strain Contains the cry8Ca Gene Specific to Anomala corpulenta Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73−), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC50) of the two mutants were 0.2334 × 108 and 0.2591 × 108 colony-forming units g−1, considerably lower than the LC50 of the wild-type strain HBF-1 (0.9583 × 108 CFU g−1) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 108 CFU g−1).

Complete Genome Sequence of Bacillus thuringiensis subsp. kurstaki Strain HD73

ABSTRACT Bacillus thuringiensis is a Gram-positive bacterium that produces intracellular protein crystals toxic to a wide variety of insect larvae. We report the complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 from the Centre OILB (Institut Pasteur, France), which belongs to serotype 3ab and is toxic to lepidopteran larvae.

Structure and Regulation of the gab Gene Cluster, Involved in the γ-Aminobutyric Acid Shunt, Are Controlled by a σ54 Factor in Bacillus thuringiensis

The structure and regulation of the gab gene cluster, involved in gamma-aminobutyric acid (GABA) shunt, were studied by characterizing gabT and gabD genes cloned from Bacillus thuringiensis. Deletions of the gabT and gabD genes in B. thuringiensis strain HD-73 did not affect the growth of mutant strains in rich culture media, but the growth of a gabT deletion mutant strain was reduced in basic media (containing 0.2% GABA). Genome analysis indicates that the structure of the gab gene cluster in B. thuringiensis HD-73 is different from that in Escherichia coli and Bacillus subtilis but is common in strains of the Bacillus cereus group. This suggests that the gene cluster involved in GABA shunt is specific to the B. cereus group. Based on reverse transcription-PCR and transcriptional fusion analysis, we confirmed that the gabT and gabD genes belong to different transcriptional units, while the gabD and gabR genes form an operon. We also demonstrated that the gabR gene plays a positive regulatory role in gabD and gabT expression. The GabR protein may be a sigma(54)-dependent transcriptional activator, according to a conserved domain search in the NCBI database, and it is highly conserved in the B. cereus group. The -24/-12 consensus sequence of a promoter upstream from gabT suggests that the promoter can be recognized by a sigma(54) factor. Further analysis of the genetic complementation studies also suggests that the expression of the gabT gene is controlled by a sigma(54) factor. Thus, the expression of the gab cluster is regulated by a sigma(54) factor by way of the transcription activator GabR.

Identification of the Promoter in the Intergenic Region between orf1 and cry8Ea1 Controlled by Sigma H Factor

ABSTRACT Bacillus thuringiensis Cry8Ea toxin is specifically toxic to larvae of the Asian cockchafer, Holotrichia parallela. Here we investigated the mechanism of transcriptional regulation of the cry8Ea1 gene. Reverse transcription-PCR (RT-PCR) results indicated that cry8Ea1 and an upstream gene (orf1) were cotranscribed. Transcriptional fusions with the lacZ gene demonstrated that transcription of the cry8Ea1 gene started from two promoters: P orf1 , which is located upstream of the orf1 gene, and P cry8E , located in the intergenic region mapping between orf1 and cry8Ea1. Of the known, similar orf1-cry operons, this is the first report of the existence of a promoter in the intergenic region between the orf1 and cry genes. The transcriptional activity of P orf1 was found during sporulation in B. thuringiensis subsp. kurstaki HD-73 and was almost abolished in the sigE mutant, while the transcriptional activity of P cry8E was detected after the end of the exponential phase in HD-73 and was considerably lower in the sigH mutant. The transcription start sites generated by the two cry8Ea1 promoters were determined by the 5′ -SMARTer rapid amplification of cDNA ends (RACE) method. The −35 and −10 regions of P orf1 and P cry8E showed high sequence similarity with the σE and σH promoters, respectively. These results indicated that P orf1 is controlled by the σE factor and P cry8E by the σH factor.

Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects

A newly engineered strain, denominated BIOT185, was constructed through integrating the cry8Ca2 gene into the endogenous plasmid of BT185 (contains cry8Ea1) by homologous recombination. The thermosensitive plasmid vector was eliminated by the rising temperature of recombinant cultures. No antibiotic gene or other unnecessary genes were introduced to the new strain. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry8Ca2 gene was expressed normally and produced a 130-kDa protein in the BIOT185 strain. Bioassay results showed that the new strain had high toxicity to the pests Anomala corpulenta and Holotrichia parallela, which often damage the same fields.

Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation.

AbstractThe advantages of gene ‘stacking’ or ‘pyramiding’ are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG2-epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG2, were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.

Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain T03B001

Three cry9 genes, cry9Da4, cry9Eb2, and cry9Ee1, were cloned from Bacillus thuringiensis strain T03B001 using a high-resolution melting analysis method. All three cry9 genes were overexpressed in Escherichia coli Rosetta (DE3), and the expressed products Cry9Eb2 and Cry9Ee1 were shown to be toxic to Plutella xylostella and Ostrinia furnacalis, but not to Helicoverpa armigera or Colaphellus bowringi. The bioassay of Cry9Eb2 and Cry9Ee1 against Cry1Ac-resistant P. xylostella strains indicated that both novel Cry9 toxins exhibited no cross-resistance with Cry1Ac. Cry9Eb2 and Cry9Ee1 can be applied not only for P. xylostella and O. furnacalis control, but also for the Cry1Ac-resistance management of pests.