Cloning And Expression Of Vegetative Insecticidal Proteins (VIPs) From Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is a spore-forming gram-positive bacterium. During sporulation, the intracellular insecticidal crystal proteins(Cry proteins) are produced as phase-bright inclusion. These proteins are toxic to insect larvae in the orders Lepidopetera, Duptera and Coleopteoptera. The Cry proteins from B. thuringiensis have been developed as one of the most successful biological agents in agriculture to control insect pests. The vegetative insecticidal proteins(VIPs) which are secreted outside the cells during mid-logarithmic growth were found recently. Two novel VIPs which are toxic to Helicoverpa armigera were identified from Bt-LS1 and Bt-LS8 isolates respectively. The characteristics of Bt-LSl and Bt-LS8, the cloning and expression of VIP3A were reported in this study.PCR Detection and bioassay from Vip3A proteins were carried out among 99 wild Bacillus thuringiensis,which were isolated by our lab. A pair of specific primers was designed according to the sequences of known vip3A genes. The vip3A-type genes in 99 wild strains were assayed by PCR amplification. The results appeared that 55 strains contain vip3A gene. Bioassay showed that the strains of Bt LS1 and LS8 have high toxicity toward Helicoverpa armigera and Spodoptera exigua. The morality rates against Helicoverpa armigera (1st instar) were 29.6% and 55.1% respectively. The weight inhibitory rates against Helicoverpa armigera (second star larvae) were (78.7 ±6.6)% and (50.4 + 2.4)% respectively, and the weight inhibitory rates against Spodoptera exigua (1st instar) were 45.1% and 43.2% respectively.The two isolates, which were highly toxic to Lepidoptera pests, Bt-LSl and Bt-LS8 were chosen and studied on molecular characterization systematically. The vip3A genes amplified from isolates were cloned into pBluescript SK(+) and two vip3A genes obtained. Vip3A-LSl protein, 88.6kD protein, was composed of 790 amino acids deduced from 2370 bps nucleotide sequences. Vip3A-LS8 protein, 88.5kD protein, was composed of 2370 bps nucleotide sequences. Alignment of these 2 putative Vip3A proteins with 10 others showed that vip3A are highly conservative, the homologous rate is 98.6% to 99.8%. The GenBank accession number were DQ016968 and DQ016969 respectively.The two vip3A genes were inserted into expression plasmid pET-21b and transformed into Escherichia coli strain BL21 respectively. After being induced by IPTG, expression products about 89kD protein were performed by SDS-PAGE analysis. The results indicated that vip3A genes could be expressed normally in cloned and expressed in E coli.Bioassay of expression products of these genes showed that Vip3A-LS1 andVip3A-LS8 proteins were toxic to the larvae of Helicoverpa armigera and Spodoptera exigua. The intracellular protein crude extracts from these genes have high toxicity to Helicoverpa armigera. The inhibition is lower than other reported genes. The results indicated that some amino acids change had great effect on the protein toxicity.