Abstract
Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.
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References
Beron CM, Curatti L, Salerno GL (2005) New strategy for identification of novel cry-type genes from Bacillus thuringiensis strains. Appl Environ Microbiol 71:761–765
Choi SK, Shin BS, Kong EM, Rho HM, Park SH (2000) Cloning of a new Bacillus thuringiensis cry1I-type crystal protein gene. Curr Microbiol 41:65–69
Kuo W, 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
Orita M, Suzuki Y, Sekiya T, Hayashi K (1989) Rapid and sensitive detection of point mutation and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
Porcar M, Juárez-Pérez VM (2003) PCR-based identification of Bacillus thuringiensis pesticidal crystal genes. FEMS Microbiol Rev 26:419–432
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806
Tounsi S, J’Mal A, Zouari N, Jaoua S (1999) Cloning and nucleotide sequence of a novel cry1Aa-type gene from Bacillus thuringiensis subsp kurstaki. Biotechnol Lett 21:771–775
Acknowledgements
This work was supported by NNSFC (National Natural Science Foundation of China), Grant No. 40601046, NCETFJ (Program for New Century Excellent Talents in Fujian Province University), and NSFFC (Natural Science Foundation of Fujian, China), Grant No. B0510011.
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Lin, Y., Fang, G. & Peng, K. Characterization of the highly variable cry gene regions of Bacillus thuringiensis strain ly4a3 by PCR-SSCP profiling and sequencing. Biotechnol Lett 29, 247–251 (2007). https://doi.org/10.1007/s10529-006-9224-2
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DOI: https://doi.org/10.1007/s10529-006-9224-2