Identification, Cloning And Activities Of Vip3A Genes From Bacillus Thuringiensis

The vip3A-gene types in the Bacillus thuringiensis (Bt.) strains were screened by using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. We identified the cry and vip3A gene types of strains BtAL and Bt41. The vip3A gene in BtAL was cloned, expressed and its toxicity to Spodoptera exigua, Helicoverpa armigera, Plutella xylostella and Ostrinia furnacalis were studied. We obtained vip3A gene fragment of Bt41.PCR-RFLP was used to rapidly identify Bacillus thuringiensis strains harboring known or novel vip3A-type genes. A pair of universal primers was designed based on conserved regions of five vip3A genes. Amplified products (1,456 bps) digested with the Hindâ…¢ and EcoR I enzymes were run on agarose gels so as to confirm different RFLP patterns for used to identify vip3A-type genes of B. thuringiensis strains. The vip3A gene-types of 606 B. thuringiensis strains were screened and three patterns of RFLP were successfully identified. A total of 382 strains harbored vip3A genes, based on a positive PCR signal, and the results indicated that the vip3A gene had a wide range of distribution. Of these strains, agarose gel data confirmed 315 isolates had a vip3Aa type RFLP pattern (860, 260, 190 and 160 bps), conforming to the predicted fragments of vip3Aa1-type genes. 52 strains, including strain BtAL showed a novel vip3A-type RFLP pattern (860, 260, 160, 150 bps and more shorter fragments). A second novel vip3A-type pattern (1,100, 190 and 160 bps) was also identified in 15 isolates including B. thuringiensis Bt41.We found that strain BtAL containing cry1Ab, cry1Ca, cry1Cb, cry1Fb, cry1Bd, cry1Ib, cry2Ab, cry9Eb and novle vip3A genes through identifiing cry and vip3A gene types of plasmid DNA of BtAL. A 2.37kb vip3A gene PCR product was amplified with plasmid DNA of BtAL as template. It was inserted into a vector pET-21b. The nucleotide sequence data of Vip3Aa19 were assigned GenBank accession numbers DQ241674. Sequence analyses showed that vip3Aa19 protein consisted of 789 residues of amino acids with a predicted molecular weight of 88.7 kDa and isoelectric point pH5.17. The Vip3Aal9 protein sequence was compared to all the other Vip3 proteins and shared a 97.6% sequence homology with Vip3Aa12 protein. Soluble protein from Vip3Aa19 was obtained after 18 hours of induction by addition of IPTG (final. Conc. 0.7 mmol/L) and shaking at 130 r/min. The protein showed apparent toxic activity to Spodoptera exigua. LC₅₀ was 1.43 ng per microgram artificial diet. The protein also showed toxic to Helicoverpa armigera andPlutella xylostella with LC50 24.14 ng per microgram artificial diet and 59.82 ug/mL. But the protein showed slight toxicity against the Ostrinia furnacalis and just inhibited the weight growth of Ostrinia furnacalis.We found that strain Bt41 containing crylAa, crylCb, crylle and a novle vip3A type gene through identifiing cry and vip3A gene types of plasmid DNA of Bt41. The l,456bp gene fregment was obtained from Bt41 and shared the highest sequence homology (83%), among known vip3A-type genes, with the vip3Afl (isp3a) (AJ872070) gene. The novel vip3A gene was submitted in GenBank under accession DQ250256.We demonstrated that the novel Vip3Aal9 protein was active against Spodoptera exigua. The lepidopteran insect, Spodoptera exigua, which is a very important pest that attacks many agricultural crops and inflicts heavy damage. The resistance of S. exigua to pesticides has increased in recent years. The Bacillus thuringiensis (Bt) products in the market now had low effectiveness to it. It is necessary to find more toxic Bacillus thuringiensis protein genes against spodoptera exigua. The finding of Vip3Aal9 protein is important for spodoptera exigua control.