Spore stage expression of a vegetative insecticidal gene increase toxicity of Bacillus thuringiensis subsp. aizawai SP41 against Spodoptera exigua.
To enhance the toxicity of the Bacillus thuringiensis subsp. aizawai strain SP41 (SP41), the vegetative insecticidal protein (Vip) gene vip3A from SP41 was redirected to the sporulation stage by replacing its native promoter with the strong promoter P19 of the cry11Aa operon. Compared to the wild type, SP41 with PVIP (vip3A with its native promoter and ter) had the relative expression ratios of 457, 548, and 290 at 8, 14, and 20 h of cultivation, respectively, as measured by quantitative reverse transcription polymerase chain reaction (PCR). SP41 transformed by P19VIP (vip3A controlled by P19 promoter with vip3A ter) showed higher expressions (23, 2055, 1831) at the same time points. SP41 with P19VIP20 (vip3A controlled by P19 promoter and containing P20 and operon ter) had the lowest expression levels (3, 11, 9) at any time point. SDS-PAGE analysis of proteins in the culture supernatant of the P19VIP at 8, 14, and 20 h demonstrated a significant increase in Vip3A at the sporulation stage. Using the surface contamination bioassay, the 50% lethal concentration (LC(50)) of whole culture of PVIP, P19VIP, and P19VIP20 at 20 and 48 h of cultivation against Spodoptera exigua larvae were (68.3, 21.2, and 60.2 microg cm(-2)) and (69.8, 41.8, and 74.6 microg cm(-2)), respectively, compared with 86.6 and 104.4 microg cm(-2) for SP41. The results showed that Vip from P19VIP, expressed at spore stage at 20 and 48 h, can increase the toxicity of SP41 for 4.1- and 2.5-fold, respectively.
Thamthiankul Chankhamhaengdecha
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Department of Biology, Faculty of Science, Mahidol University, RamaVI, Bangkok 10400, Thailand.
Tantichodok
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Panbangred
Watanalai
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D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001681:Biological Assay; D015964:Gene Expression Regulation, Bacterial; D020128:Inhibitory Concentration 50; D007306:Insecticides / Q000378:metabolism*; D008969:Molecular Sequence Data; D010572:Pest Control, Biological / Q000379:methods*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera / Q000254:growth & development / Q000382:microbiology*; D013171:Spores, Bacterial / Q000378:metabolism*; D018675:Toxicity Tests
D001426:Bacterial Proteins; D007306:Insecticides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C447139:Vip3A protein, Bacillus thuringiensis