Studies On Bacillus Thuringiensis WB9

Bacillus thuringiensis (Bt) is an entomopathogen and has been used as an insecticidal agent for decades in commercial agriculture, forest management, and mosquito control. Its insecticidal activity is primarily due to the insecticidal crystal proteins (ICPs) formed during the stationary and sporulation phase of the growth cycle. More than 3000 insect species within 16 orders were demonstrated to be susceptible to different Bt ICPs. The search for novel Bt isolates and new cry genes is an ongoing effort worldwide. Herein, we reported a novel strain isolated from soil and its biochemical characteristic, toxicity to Plutella xylostella and Spodoptera exigua, genes to encode active factors, as well as cloning and expression of cry1Ab17, optimization of fermentation media and control of Ectropis obligua.A method was developed to isolate Bacillus thuringiensis from soil samples collected from Wuyi Mountain in southern China. Twelve Bt strains were obtained from 200 soil samples and the proportion was 6.0%. Bioassay showed that WB9 was highly and moderately toxic to Plutella xylostella and Spodoptera exigua, respectively.The biochemical characteristics of WB5, WB7 and WB9 strains were tested by using Bt kurstaki 8010 and 8311, and aizawai No.1 as controls. The results indicated that the presentations of biochemical reactions of WB9 and 8010, and WB5 and No.1, were the same. Thus, it can be deduced that the potential status of serovar of WB5 and WB9 were aizawai and kurstaki, respectively.PCR-RFLP and PCR technology were used to analyse the encoding genes for active factors of WB9. The results showed that WB9 contained fivedifferent oy1 genes, cryiAa, aylAb, CAyiCb, ay1Fa and cryiGa, and also the v/p3A, /?b/A, bceT and entS genes. However, no chi gene was found. The PCR products of wp3A, hbIA, bceT and entS were purified and directly sequenced. Homology comparison revealed that nucleotide sequences of wp3A and hbIA of WB9 had 99% and 96-98% identity with those of the known wp3A and hbIA of Bt, respectively, and bceT of WB9 had 97% identity with the known bceT of Bacillus cereus (Be), whereas entS of WB9 had 94-99% identity with the known entS of Bt and Be.The entire coding region of the cry1Ab-WB9 gene was produced by PCR with primers F1 and R1. PCR product was purified, cloned into pMD18-T and sequenced. Nucleotide sequence of cry1Ab-WB9 had been registered in GenBank (accession no. AY646166). It consists of 3471 bases which encoded a protein of 1156 residues with a calculated molecular weight of 130.5 kDa and an pi value of 5.04. Amino acids of Cry1Ab-WB9 comprised 30.8% of hydrophilic amino acids, 45.2% of hydrophobic amino acids, 12.9% of acidic amino acids and 11.1% of basic amino acids.The deduced amino acid sequence of Cry1 Ab-WB9 showed 95.4% to 99.7% identity with those of the known CrylAb proteins. Four residues (Proi7o, Gly449, Gly796 and Glya63) of Cry1Ab-WB9 were found to be different from those of the respective corresponding position in all known CrylAb proteins. In addition, Cry1Ab-WB9 was one residue longer than each known CrylAb (except for Cry1Ab2). The Cry1Ab-WB9 had been named as Cry1Ab17 by the Bt delta-endotoxin nomenclature committee and become a new member of Bt Cry toxins.The nucleotide and amino acid representative sequences of Cry1Aa^ b CryiAdx Cry1Ae> CrylAg and the known CrylAb proteins weredownloaded from GenBank and aligned with those of Cry1Ab17 by using CLUSTAL X. A phylogenetic tree was constructed using PAUP 4.0 (2000 replicates, unrooted tree) to evaluate the biological evolution of these proteins.With PredictProtein for the prediction of structure, it indicated that 31.96% of helix, 27.69% of sheet and 40.35% of loop in secondary structure of Cry1Ab17. Moreover, SignalP 3.0 server (http://www.cbs.dtu.dk/ services/ SiqnalP) and 2 Zip server (http://www.2zip. molqen. de) were used to analyze the presence and location of signal peptide cleavage sites and Leucine Zipper in the amino acid sequence,respectively. The results showed that no signal peptide or Leucine Zipper was found in Cry1Ab17.Based on the structure assignment of CryiAa, the three domains of Cry1Ab17 were proposed here. Domain I consists of 221 residues (Tyr33 to Arg253) with a calculated molecular weight of 25.6 kDa and an pi value of 4.76. Domain II consists of 198 residues (Arg265 to Phe462) with a calculated molecular weight of 21.9 kDa and an pi value of 7.21. Domain III consists of 147 residues (Asn464 to Thr6io) with a calculated molecular weight of 15.8 kDa and an pi value of 10.83. Domain I, II and III of Cry1Ab17 were 96.8%, 68.2% and 100% identical to the corresponding domains of CryiAa.The recombinant expression plasmid pET-29a-cry1Ab17 was constructed by insertion of c/y1Ab17 gene into the expression vector pET-29a, and then was transformed into E. coli BL21 to express the Cry1Ab17 under the induction of 1 mmol/L IPTG. SDS-PAGE showed that the molecular weight of the expressed product was about 130 kDa, which corresponds with the calculation by sequence. Bioassay revealed that the expressed Cry1Ab17 extracted from the culture medium was highly toxic to...