Detection and Identification of cry1I Genes in Bacillus thuringiensis Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

Sauka, Diego Herman; Cozzi, Jorge; Benintende, Graciela Beatriz

doi:10.1007/s00284-005-0171-2

Cited by 12 publications

(16 citation statements)

References 14 publications

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“…There were no products identified using primer set two or three. Thus, the HD1 strain was determined to contain four cry1-type genes, cry1Aa, cry1Ab, cry1Ac, and cry1Ia, which corresponded with known results (10,20). Strain HD29 produced a cry1Ab gene RFLP pattern (Fig.…”

Section: Resultssupporting

confidence: 71%

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1 -Type Genes

Shu

Liu

Zhou

et al. 2013

Appl Environ Microbiol

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The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera. Bacillus thuringiensis, which is a Gram-positive bacterium, is known for its specific toxicity toward insect pests (1). This toxicity is largely attributed to the insecticidal crystal proteins encoded by the cry genes (2-4). The cry1-type genes of B. thuringiensis are highly toxic to lepidopteran pests, and some genes have been used to develop plants with resistance to insect pests (5-7). Because of their potential application and commercial value, much research has focused on the discovery of novel cry1 genes; to date, approximately 258 cry1 genes have been cloned and named, and 50 distinct holotypes have been classified (http://www.lifesci .sussex.ac.uk/home/Neil_Crickmore/Bt/). Previous research has revealed that cry1 genes are typically found in clusters; for example, B. thuringiensis strains HD12 and HD525 contain at least four different cry1-type genes (8).PCR is a simple and convenient investigative method and has been widely used to identify the vast variety of cry genes by the use of different primers (9-12). PCR-restriction fragment length polymorphism (PCR-RFLP) is a modified PCR technique that is generally used for the identification of known and unknown cry1-type genes (except for cry1I-type genes), and of parts of the cry7-type and cry9-type genes, according to the fragment lengths of digested PCR-amplified products described by Kuo and Chak (8). Some primers have been designed for the identification of cry1-type genes (13) and cry8-type genes (14) based on the PCR-RFLP method. However, it is becoming difficult to identify novel cry1-type genes using the PCR-RFLP method (8) because of the increase in the numbers of cry1-type genes.To resolve this problem, an improved PCR-RFLP method was designed to directly identify the fourth class of cry1-type genes by dividing...

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Section: Resultssupporting

confidence: 71%

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1 -Type Genes

Shu

Liu

Zhou

et al. 2013

Appl Environ Microbiol

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“…Novel primers for the specific detection of vip3A genes were designed following a previously described methodology [Sauka et al, 2006[Sauka et al, , 2007. Primers used for amplification of a vip3Ab,vip3Ad,vip3Ae,vip3Af,vip1Ag and vip1Ah genes were as follows: DS3AF (forward; 5 -GTG AAA ACA AGT GGC AGT G-3 ) and DS3AR (reverse; 5 -TCC GCT TCA CTT GAT TCT ACT-3 ).…”

Section: Detection Of Vip3a Genesmentioning

confidence: 99%

“…The DNA templates for PCR were obtained as previously described [Sauka et al, 2006]. Five microliters of supernatant was used in each reaction.…”

Section: Detection Of Vip3a Genesmentioning

confidence: 99%

New Variants of Lepidoptericidal Toxin Genes Encoding <b><i>Bacillus thuringiensis</i></b> Vip3Aa Proteins

Sauka

Rodríguez²,

Benintende³

2012

Microb Physiol

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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.

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“…Twin strains were previously discarded to avoid overestimated distribution of frequencies, using sodium dodecyl sulphate-polyacrylamide gels and PCR (Sauka et al, 2006 (Benintende & Cozzi, 1996), until complete autolysis was observed at 340 rpm and 30°C. Spore-crystal complexes were obtained by centrifugation for 15 min at 12,000 g and 4°C, and pellets were freeze-dried.…”

Section: Bioensaios De Alimentação Induzida Para Determinar a Atividamentioning

confidence: 99%

Induced-feeding bioassays for detection of Bacillus thuringiensis insecticidal activity against Epilachna paenulata (Coleoptera)

Sauka

Monella

Benintende

2010

Pesq. agropec. bras.

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-The objective of this work was to establish and test the induced-feeding bioassay in order to detect Bacillus thuringiensis insecticidal activity against Epilachna paenulata (Coleoptera: Coccinellidae). Larvae were induced to swallow high concentrations of spore-crystal suspensions of seven exotic and 30 Argentine B. thuringiensis strains. The great majority of strains showed no toxicity to E. paenulata larvae, and observed mortality was lower than 30%. Induced-feeding bioassay is feasible, and should be used for prospecting strains that produce right combinations of Cry proteins needed to an efficient pest control.

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Detection and Identification of cry1I Genes in Bacillus thuringiensis Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

Cited by 12 publications

References 14 publications

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1 -Type Genes

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1 -Type Genes

New Variants of Lepidoptericidal Toxin Genes Encoding <b><i>Bacillus thuringiensis</i></b> Vip3Aa Proteins

Induced-feeding bioassays for detection of Bacillus thuringiensis insecticidal activity against Epilachna paenulata (Coleoptera)

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