Abstract
In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction–high resolution melt (PCR–HRM) analysis. A pair of primers was designed according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay for mutant screening using vip3 gene. The PCR–HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes.
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References
Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van RJ, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813
Romeis J, Meissle M, Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat Biotechnol 24:63–71
Beard CE, Court L, Boets A, Mourant R, Van Rie J, Akhurst RJ (2008) Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection. Curr Microbiol 57:195–199
Yu X, Zheng A, Zhu J, Wang S, Wang L, Deng Q, Li S, Liu H, Li P (2011) Characterization of vegetative insecticidal protein vip genes of Bacillus thuringiensis from Sichuan Basin in China. Curr Microbiol 62:752–757
Estruch JJ, Warren GW, Mullins MA (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci USA 93:5389–5394
Lee MK, Walters FS, Hart H, Palekar N, Chen JS (2003) The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab δ-endotoxin. Appl Environ Microbiol 69:4648–4657
Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ (1997) The Bacillus thuringiensis vegetative insecticidal protein Vip3 lyses midgut epithelium cells of susceptible insects. Appl Environ Microbiol 63:532–536
Juarez-Perez VM, Ferrandis MD, Frutos R (1997) PCR-based approach for detection of novel Bacillus thuringiensis cry genes. Appl Environ Microbiol 63:2997–3002
Kuo WS, Chak KF (1996) Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl Environ Microbiol 62:1369–1377
Sauka DH, Cozzi JG, Benintende GB (2006) Detection and identification of cry1I genes in Bacillus thuringiensis using polymerase chain reaction and restriction fragment length polymorphism analysis. Curr Microbiol 52:60–63
Shu C, Yan G, Wang R, Zhang J, Feng S, Huang D, Song F (2009) Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. Appl Microbiol Biotechnol 84:701–705
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LC Green. Clin Chem 49:853–860
Erali M, Voelkerding KV, Wittwer CT (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50–58
Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, Voelkerding KV (2006) Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem 52(3):494–503
Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV (2007) Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 53(8):1544–1548
Montgomery J, Wittwer CT, Palais R, Zhou L (2007) Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc 2(1):59–66
Palais RA, Liew MA, Wittwer CT (2005) Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Anal Biochem 346(1):167–175
Graham R, Liew M, Meadows C, Lyon E, Wittwer CT (2005) Distinguishing different DNA heterozygotes by high-resolution melting. Clin Chem 51(7):1295–1298
Sadowski DC, Broenink L (2008) High-resolution esophageal manometry: a time motion study. Can J Gastroenterol 22:365–368
Vossen RH, Aten E, Roos A, den Dunnen JT (2009) High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat 30:860–866
Acknowledgments
We are grateful to professor Jie Zhang and Fuping Song (Institute of Plant Protection, Chinese Academy of Agricultural Sciences) for the help of this experiment. This study was supported by the national high technology research and development program subject (2011aa10a203), the national new varieties of genetically modified organism special science and technology major (No. 2009ZX08009-030B) and soybean biology ministry of education key laboratory open subject.
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Changlong Shu and Xiaoming He contributed equally to this work.
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Li, H., Shu, C., He, X. et al. Detection and Identification of Vegetative Insecticidal Proteins vip3 Genes of Bacillus thuringiensis Strains Using Polymerase Chain Reaction-High Resolution Melt Analysis. Curr Microbiol 64, 463–468 (2012). https://doi.org/10.1007/s00284-012-0092-9
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DOI: https://doi.org/10.1007/s00284-012-0092-9