Yi Pang

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.

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli.

Aims: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli.

Highly Toxic and Broad-Spectrum Insecticidal Bacillus thuringiensis Engineered by Using the Transposon Tn917 and Protoplast Fusion

The chromosome of the Bacillus thuringiensis strain S184 that was toxic against the third instar larvae of Spodoptera litura with the LC50 of 9.74 μg/ml was successfully integrated into two genes of cyt1Aa and cry11Aa using the transposon Tn917, yielding the primary engineered strain TnX. The strain TnX was highly toxic to the third instar larvae of Culex pipiens fatigans with the LC50 of 5.12 ng/ml which was 1.82-fold higher than that of B. thuringiensis subsp. israelensis, but lowly toxic to lepidopterous larvae. By the protoplast fusion of the strain TnX and the strain S184-Tetr (resistance to tetracycline), the target engineered strain TnY was obtained. Against the third instar larvae of S. litura, the strain TnY LC50 was of 4.68 μ g/ml and increased by 2.08-fold in comparison with the parent strain S184. Against the third instar larvae of C. pipiens fatigans, the strain TnY LC50 was of 103.20 ng/ml. The two target genes of cyt1Aa and cry11Aa integrated into the chromosome were extremely stable and had little possibility of a second transposition. It was unclear whether some factors existing in the parent strain, S184, contributed to the high toxicity of the strains TnX and TnY.

Cloning and expression of the binary toxin gene from Bacillus sphaericus IAB872 in a crystal-minus Bacillus thuringiensis subsp. israelensis.

Bacillus sphaericus IAB872 has high toxicity against susceptible Culex spp. and medium larvicidal activity against binary toxin-resistant Culex spp. Sequence analysis revealed that the sequence of the binary toxin gene from IAB872 was totally identical to that of the reference strain 2362. The recombinant plasmids were introduced into an acrystalliferous B. thuringiensis strain 4Q7, and the resulting transformants produced parasporal inclusion composed of 51 kDa and 42 kDa proteins during sporulation. SDS-PAGE and Western blot further confirmed that B. thuringiensis transformants were able to express the binary toxin in a high level. Toxicity bioassays showed that they performed high toxicity to susceptible Culex spp. larvae, but no toxicity to resistant Culex larvae. It was, therefore, suggested that other unknown toxins perhaps existed in the wild strain IAB872 except the binary toxin. Their modes of action might be different from that of binary toxin, and they were possibly responsible for the activity of the wild strain IAB872 against resistant mosquito larvae.

Coexpression of cyt1Aa of Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus binary toxin gene in acrystalliferous strain of B. thuringiensis.

Abstract. The cyt1Aa gene of Bacillus thuringiensis subsp. israelensis and binary toxin gene of Bacillus sphaericus C3-41 were introduced into an acrystalliferous strain of B. thuringiensis independently and in combination by using shuttle vector pBU4. SDS-PAGE and Western blot analysis proved that cyt1Aa and binary toxin genes coexpressed during the sporulation of the recombinant. Transformant strain expressing the Cyt1Aa and binary toxin proteins in combination was more toxic to susceptible and resistant Culex pipiens quinquefasciatus than the transformants expressing Cyt1Aa protein or binary toxin proteins independently. It was suggested that large amount of production of Cyt1Aa protein and binary toxin proteins possibly interacted synergistically, thereby increasing its mosquitocidal toxicity significantly.

Efficient expression of vip184ΔP gene under the control of promoters plus Shine-Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis

Aims:  To compare vip184ΔP gene expression time course and Vip184 protein yield under the control of promoters and Shine‐Dalgarno (SD) sequences of vip184, cry3A and cry1A gene from Bacillus thuringiensis respectively.

Phylogenetic analysis of Bacillus thuringiensis based on PCR amplified fragment polymorphisms of flagellin genes

Phylogenetic analysis of 35 strains of Bacillus thuringiensis was performed based on PCR amplified fragment polymorphisms of flagellin genes (PCR-AFPF). From coefficient matrix, the genetic distance coefficient D among them ranged from 0.14286 to 0.81818. Ten clusters were divided by the taxonomic threshold of 0.42 from the UPGMA dendrogram, demonstrating that the genetic dissimilarities existed among 35 strains of B. thuringiensis corresponding to 35 different serovars. This method could provide a fast, convenient and accurate way to classify all subspecies of B. thuringiensis including strains that can not be classified using the H-antigen method.

Reduction of resistance of Culex pipiens larvae to the binary toxin from Bacillus sphaericus by coexpression of cry4Ba from Bacillus thuringiensis subsp. israelensis with the binary toxin gene

The cry4Ba gene from Bacillus thuringiensis subsp. israelensis and the binary toxin gene from B. sphaericus C3-41 were cloned together into a shuttle vector and expressed in an acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. Transformed strain Bt-BW611, expressing both Cry4Ba protein and binary toxin protein, was more than 40-fold more toxic to Culex pipiens larvae resistant to B. sphaericus than the transformed strains expressing Cry4Ba protein or binary toxin protein independently. This result showed that the coexpression of cry4Ba of B. thuringiensis subsp. israelensis with B. sphaericus binary toxin gene partly suppressed more than 10,000-fold resistance of C. pipiens larvae to the binary toxin. It was suggested that production of Cry4Ba protein and binary toxin protein interacted synergistically, thereby increasing their mosquito-larvicidal toxicity.

Expression of the full-length and 3'-spliced cry1Ab gene in the 135-kDa crystal protein minus derivative of Bacillus thuringiensis subsp. kyushuensis.

Bacillus thuringiensis produces a 130–135-kDa insecticidal protein in the form of bipyramidal crystal which is toxic to lepidopteran larvae. Part of the C-terminal region of the native Cry1Ab was replaced by a heterologous sequence of Cry11Aa C-terminus to get a 3′-spliced cry1Ab gene. The full-length cry1Ab and 3′-spliced cry1Ab, which were both cloned into the E. coli–B. thuringiensis shuttle expression vector pHZB1, were expressed in a 135-kDa crystal protein minus derivative of B. thuringiensis subsp. kyushuensis (4U1-Cry−135). The crystal shape of Cry1Ab proteins from both recombinants was regularly bipyramidal, while the crystal size of the intact Cry1Ab was approximately fivefold larger than the 3′-spliced Cry1Ab. In addition, these two kinds of Cry1Ab proteins had similar toxicity against Argyrogramma agnata larvae.

A replication origin of Bacillus thuringiensis

Abstract. A replication origin of Bacillus thuringiensis (Bt) was found in a Bacillus thuringiensis–Escherichia coli shuttle vector of pHT3101. Deletion analysis showed that the replication origin was segregationally stable at suitable temperature for Bt growth. The fragment containing the replication origin was cloned in pUC18 and sequenced. It was 261 base pairs in length, located in the open reading frame 2 (ORF2) of BTSPB sequence. The 261-bp fragment was cloned in pBR322, creating an improved Bt-E. coli shuttle vector pBR261, which contained two resistance genes responsible for ampicillin and tetracycline. Our study showed that the replication origin structure could be recognized by replication protein of host cells.