Abstract
Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.
Journal Section:
Research Article
References
1.
Asokan R, Swamy H, Arora D: Screening, diversity and partial sequence comparison of vegetative insecticidal protein (vip3A) genes in the local isolates of Bacillus thuringiensis Berliner. Curr Microbiol 2012;64:365–370.
[PubMed]
2.
Beard C, Court L, Boets A, Mourant R, Van Rie J, Akhurst R: Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection. Curr Microbiol 2008;57:195–199.
[PubMed]
3.
Bhalla R, Dalal M, Panguluri S, Jagadish B, Mandaokar A, Singh A, Kumar P: Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis. FEMS Microbiol Lett 2005;243:467–472.
[PubMed]
4.
Doss V, Kumar K, Jayakumar R, Sekar V: Cloning and expression of the vegetative insecticidal protein (vip3V) gene of Bacillus thuringiensis in Escherichia coli. Protein Expr Purif 2002;26:82–88.
[PubMed]
5.
Estruch J, Koziel M, Yu C, Desai N, Nye G, Warren G: Plant pest control. Patent WO 9844137. 1998.
6.
Estruch J, Warren G, Mullins M, Nye G, Craig J, Koziel M: Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci USA 1996;93:5389–5394.
[PubMed]
7.
Hernández-Rodríguez C, Boets A, Van Rie J, Ferré J: Screening and identification of vip genes in Bacillus thuringiensis strains. J Appl Microbiol 2009;107:219–225.
[PubMed]
8.
Lee M, Miles P, Chen J: Brush border membrane binding properties of Bacillusthuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 2006;339:1043–1047.
[PubMed]
9.
Lee M, Walters F, Hart H, Palekar N, Chen J: The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin. Appl Environ Microbiol 2003;69:4648–4657.
[PubMed]
10.
Liu J, Song F, Zhang J, Liu R, He K, Tan J, Huang D: Identification of vip3A-type genes from Bacillusthuringiensis strains and characterization of a novel vip3A-type gene. Lett Appl Microbiol 2007;45:432–438.
[PubMed]
11.
Pugsley A: The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 1993;57:50–108.
[PubMed]
12.
Rang C, Gil P, Neisner N, Van Rie J, Frutos R: Novel Vip3-related protein from Bacillus thuringiensis. Appl Environ Microbiol 2005;71:6276–6281.
[PubMed]
13.
Rice W: Specific primers for the detection of vip3A insecticidal gene within a Bacillus thuringiensis collection. Lett Appl Microbiol 1999;28:378–382.
14.
Sauka D, Amadio A, Zandomeni R, Benintende G: Strategy for amplification and sequencing of insecticidal cry1A genes from Bacillus thuringiensis. Antonie Van Leeuwenhoek 2007;91:423–430.
[PubMed]
15.
Sauka D, Benintende G: Bacillus thuringiensis: general aspects. An approach to its use in the biological control of lepidopteran insects behaving as agricultural pests. Rev Argent Microbiol 2008;40:124–140.
[PubMed]
16.
Sauka D, Cozzi J, Benintende G: Detection and identification of cry1I genes in Bacillus thuringiensis using polymerase chain reaction and restriction length polymorphism analysis. Curr Microbiol 2006;52:60–63.
[PubMed]
17.
Selvapandiyan A, Arora N, Rajagopal R, Jalali S, Venkatesan T, Singh S, Bhatnagar R: Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis. Appl Environ Microbiol 2001;67:5855–5858.
[PubMed]
18.
Song F, Zhang J, Gu A, Wu Y, Han L, He K, Chen Z, Yao J, Hu Y, Li G, Huang D: Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene. Appl Environ Microbiol 2003;69:5207–5211.
[PubMed]
19.
Thompson J, Higgins D, Gibson T: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–4680.
[PubMed]
20.
Warren G: Vegetative insecticidal proteins: novel proteins for control of corn pests; in Carozzi N, Koziel M (eds): Advances in Insect Control: The Role of Transgenic Plants. London, Taylor and Francis Ltd, 1997, pp 109–121.
21.
Yu X, Zheng A, Zhu J, Wang S, Wang L, Deng Q, Li S, Liu H, Li P: Characterization of vegetative insecticidal protein vip genes of Bacillus thuringiensis from Sichuan Basin in China. Curr Microbiol 2011;62:752–757.
[PubMed]
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