Characterization of a Novel cry1-Type Gene from Bacillus thuringiensis subsp. alesti Strain LY-99
1
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
B. thuringiensis strain LY-99 belonging to subsp. alesti(H3a3c), was isolated from Chinese tobacco warehouseand showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes fromB. thuringiensis LY-99, an extended multiplex PCRrestrictionfragment length polymorphism (PCRRFLP)method was established by using two pairs ofuniversal primers based on the conserved regions ofthe cry1-type genes to amplify around 2.4 kb cry1-typegene fragments. Then the DNA fragment was clonedinto pGEM-T Easy vector and digested with EcoRIand EcoRV enzymes. Through this method, a knowncry1-type gene was successfully identified from the referencestrain, B. thuringiensis subsp. alesti. In addition,the RFLP patterns revealed that B. thuringiensisLY-99 included a novel cry1A-type gene in addition tocry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novelcry1A-type gene was designated cry1Ah2 (Genbankaccession No DQ269474). An inverse PCR method wasused to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensisLY-99 plasmid DNA including 5’ region andpartial ORF was amplified, and sequence analysisrevealed that cry1Ah2 gene from LY-99 showed89.31% of maximum sequence similarity with cry1Ac1crystal protein gene. In addition, the deduced aminoacid sequence of Cry1Ah2 protein shared 87.80% ofmaximum identity with that of Cry1Ac2. This proteintherefore belongs to a new class of B. thuringiensiscrystal proteins.
Abstract
B. thuringiensis strain LY-99 belonging to subsp. alesti(H3a3c), was isolated from Chinese tobacco warehouseand showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes fromB. thuringiensis LY-99, an extended multiplex PCRrestrictionfragment length polymorphism (PCRRFLP)method was established by using two pairs ofuniversal primers based on the conserved regions ofthe cry1-type genes to amplify around 2.4 kb cry1-typegene fragments. Then the DNA fragment was clonedinto pGEM-T Easy vector and digested with EcoRIand EcoRV enzymes. Through this method, a knowncry1-type gene was successfully identified from the referencestrain, B. thuringiensis subsp. alesti. In addition,the RFLP patterns revealed that B. thuringiensisLY-99 included a novel cry1A-type gene in addition tocry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novelcry1A-type gene was designated cry1Ah2 (Genbankaccession No DQ269474). An inverse PCR method wasused to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensisLY-99 plasmid DNA including 5’ region andpartial ORF was amplified, and sequence analysisrevealed that cry1Ah2 gene from LY-99 showed89.31% of maximum sequence similarity with cry1Ac1crystal protein gene. In addition, the deduced aminoacid sequence of Cry1Ah2 protein shared 87.80% ofmaximum identity with that of Cry1Ac2. This proteintherefore belongs to a new class of B. thuringiensiscrystal proteins.
참고문헌 (34)
- Ben-Dov E., "Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis", Appl Environ Microbiol, 63, pp.4883~4890 (1997)
- Ben-Dov E., "Multiplex PCR screening to detect cry9 in Bacillus thuringiensis strains", Appl Environ Microbiol., 65, pp.3714~3716 (1999)
- Ben-Dov E., "PCR analysis of cry7 genes in Bacillus thuringiensis by the five conserved blocks of toxins", Curr Microbiol., 42, pp.96~99 (2001)
- Birnboim JC., "A rapid alkaline extraction procedure for screening recombinant plasmid DNA", Nucleic Acids Res., 7, pp.1513~1523 (1979)
- Bourque SN., "Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis", Appl Environ Microbiol, 59, pp.523~527 (1993)
- Bravo A., "Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection", Appl Environ Microbiol, 64, pp.4965~4972 (1998)
- Carozzi NB., "Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles", Appl Environ Microbiol., 57, pp.3057~3061 (1991)
- Ceron J., "PCR analysis of the cryI insecticidal family genes from Bacillus thuringiensis", Appl Environ Microbiol., 60, pp.353~356 (1994)
- Ceron J., "Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection", Appl Environ Microbiol., 61, pp.3826~3831 (1995)
- Chang JH., "Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding a-endotoxin Cry1E", Lett Appl Microbiol., 26, pp.387~390 (1998)
- Collins FS., "Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method", Proc Natl Acad Sci USA, 81, pp.6812~6816 (1984)
- Crickmore N., "Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins", Microbiol Mol Biol Rev., 62, pp.807~813 (1998)
- Ferrandis MD., "Distribution of cryI, cryII and cryV genes within Bacillus thuringiensis isolates from Spain", Syst Appl Microbiol, 22, pp.179~185 (1999)
- Ferre J., "Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor", Proc Natl Acad Sci USA, 88, pp.5119~5123 (1991)
- Gleave AP., "Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B. thuringiensis subsp. kurstaki", Appl Environ Microbiol, 59, pp.1683~1687 (1993)
- Grochulski P., "Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation", J Mol Biol., 254, pp.447~464 (1995)
- Herron GA., "Insecticide resistance in Aphis gossypii glover (Hemiptera: Aphididae), a serious threat to Australian cotton", Aust J Entomol., 40, pp.85~91 (2001)
- Hofte H., "Insecticidal crystal proteins of Bacillus thuringiensis", Microbiol Rev., 53, pp.242~255 (1989)
- Huang ZP., "Cloning, characterization and expression of a new cry1Ab gene from Bacillus thuringiensis WB9", Biotechnol Lett., 26, pp.1557~1561 (2004)
- Hwang SH., "A novel class of mosquitocidal a-endotoxin, Cry19B, encoded by a Bacillus thuringiensis serovar higo gene", Syst Appl Microbiol., 21, pp.179~184 (1998)
- Juarez-Perez VM., "Distribution, serological identification, and PCR analysis of Bacillus thuringiensis isolated from soils of Korea", Curr Microbiol., 37, pp.195~200 (1998)
- Kim HS., "Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms", Curr Microbiol., 43, pp.408~413 (2001)
- Yang-Su Kim, "Identification of a Novel Crystal Protein Gene from Bacillus thuringiensis Isolated in Korea", Journal of Asia-Pacific Entomology, 8(3), pp.239~242 (2005), 한국응용곤충학회
- Kuo WS., "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, pp.1369~1377 (1996)
- Lee CS., "Cloning and analysis of deltaendotoxin genes from Bacillus thuringiensis subsp. alesti", J Bacteriol, 173, pp.6635~6638 (1991)
- Maniatis T., "Molecular clonings: a laboratory manual" (1982), Cold Spring Harbor Laboratory
- Porca M., "PCR-based identification of Bacillus thuringiensis pesticidal crystal genes", FEMS Microbiol Rev., 26, pp.419~432 (2003)
- Xu Feng Qi, "Molecular Characterization of a Novel Bacillus thuringiensis Strain from China", International Journal of Industrial Entomology, 11(1), pp.57~61 (2005), 한국잠사학회
- Rosso ML., "Contribution of the 65-kilodalton protein encoded by the cloned gene cry19A to the mosquitocidal activity of Bacillus thuringiensis subsp. jegathesan", Appl Environ Microbiol, 63, pp.4449~4455 (1997)
- Sambrook J., "Molecular cloning: a laboratory manual, 3rd ed" (2001), Cold Spring Harbor Laboratory Press
- Schnepf E., "Bacillus thuringiensis and its pesticidal crystal proteins", Microbiol Mol Biol Rev., 62, pp.775~806 (1998)
- Song FP., "Establishment of PCR-RFLP identification system of cry genes from Bacillus thuringiensis", Sci Agric Sin, 31, pp.19~24 (1998)
- Tabashnik BE., "Cross-resistance of pink bollworm (Pectinophora gossypiella) to Bacillus thuringiensis toxins", Appl Environ Microbiol, 66, pp.4582~4584 (2000)
- Tounsi S., "Cloning and nucleotide sequence of a novel cry1Aa-type gene from Bacillus thuringiensis subsp. kurstaki", Biotechnol Lett., 21, pp.771~775 (1999)
