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Chilean journal of agricultural research

On-line version ISSN 0718-5839

Chilean J. Agric. Res. vol.70 no.2 Chillán June 2010

http://dx.doi.org/10.4067/S0718-58392010000200004 

Chilean Journal of Agricultural Research 70(2):213-220 (April-June 2010)

RESEARCH

Application of Oligonucleotide Microarray for the Detection and Genotyping of cry Genes in Bacillu thuringiensis

Aplicación de Microarreglo de Oligonucleótidos en la Detección y Tipificación Génica de Genes cry en Bacillu thuringiensis

Liu Xu-Guang1, 2, Song Fu-Ping1, Wen Si-Yuan3, Wang Sheng-Qi3, Huang Da-Fang4, and Zhang Jie1*

1 Institute of Plant Protection (CAAS), State Key Laboratory for Biology of Plant Diseases and Insect Pests, Beijing 100094, China. *Corresponding author (jzhang@ippcaas.cn).
2Lianyungang Teachers College, Lianyungang 222206, China.
3Beijing Institute of Radiation Medicine Science, Beijing 100850, China.
4Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Beijing 100081, China.


ABSTRACT

We have developed a parallel, rapid, high-throughput oligonucleotide microarray-based assay for the reliable detection and genotyping of three cry genes (cry1, cry2 and cry9) in Bacillus thuringiensis (Bt). After the non-polymerase chain reaction (PCR), amplified Bt genomic DNA were fluorescent-labeled using a random primer.   The corresponding oligonucleotide probes were designed for the different cry genes that can hybridize Bt genomic DNA after cluster analysis and were printed on glass slides. This microarray has unambiguously detected and identified the cry genes in 10 isolates and reference Bt. Our data demonstrates that the microarray assay is simple and rapid for the detection and genotyping of genes. This type of assay is also a potentially valuable tool for identification and characterization of bacterial functional genes in general.

Key words: oligonucleotide microarray, cry genes, genotyping, random primer labeling, bioinformatics.


RESUMEN

Se desarrolló un ensayo paralelo, rápido, de alto rendimiento, basado en microarreglo de oligonucleótidos para la detección y tipificación génica confiable de tres genes cry (cry1, cry2 y cry9) en Bacillus thuringiensis (Bt). Después de la no reacción de cadena polimerasa (PCR), el ADN genómico de Bt amplificado se marcó con fluorescencia usando primer al azar. Las correspondientes sondas de oligonucleótido fueron diseñadas por los diferentes genes cry en ADN genómico de Bt que pueden ser hibridados después de análisis cluster y se imprimieron en portaobjetos de vidrio. Este microarreglo ha detectado e identificado sin ambigüedad los genes cry en 10 aislamientos y Bt de referencia. Nuestros datos demuestran que el microarreglo es simple y rápido para la detección y tipificación génica de genes. Este tipo de ensayo es además una herramienta potencialmente valiosa para identificación y caracterización de genes funcionales bacterianos en general.

Palabras clave: microarreglo de oligonucleótidos, genes cry, tipificación génica, marcación con primer al azar, bioinformática.


INTRODUCTION

Bacillus thuringiensis (Bt) is capable of producing highly poisonous crystal proteins (also called β-endotoxin) that kill Lepidoptera insects and other agricultural pests. More than 375 crystal protein genes have already been cloned and sequenced. Based on amino acid sequence similarity, Crickmore (2003) categorized these endotoxins into 55 groups (1st level), 106 subgroups (2nd level), 173 types (3rd level), and 375 subtypes (4th level). Bt has become the most effective biopesticide in the biological control of agricultural pests. With the development of biotechnology, new β-endotoxin genes are continually being isolated and cloned. Along with the development in the study of toxin structure and functions, genetic modification of the toxin genes has increased the effectiveness and specific targeting ability of these endotoxins and widened the application perspective. Identification of Bt resources is essential to predict the pesticide effect, rapid screening for desirable isolates, and cloning of novel toxin genes.

Current detection and identification methods of Bt cry genes are based on polymerase chain reaction (PCR), including PCR-restriction fragment length polymorphism (RFLP) (Song et al., 2003), PCR-single-strand conformation polymorphism (SSCP), PCR-sequencing, and Multiplex PCR. These methods are rapid and sensitive. However, they become complicated, tedious, and time-consuming for analyzing multiple genes.

The development of DNA microarray has enabled the rapid, parallel, and high throughput detection and identification of microbes (Gabig and Wegrzyn, 2001; Bodrossy et al., 2003). DNA microarray is not only manufactured by light-directed in situ synthesis, but is also produced by DNA probe fixation (PCR products, synthetic oligonucleotides) on solid surfaces. This microarray is then hybridized with nucleic acid samples. Information about the nucleotide sequences can be rapidly and accurately obtained on a large scale (Small et al., 2001; Hwang et al., 2003). In this study, we report the development of an oligonucleotide microarray that can be used to detect and identify cry1, cry2, and cry9 genes in Bt.

MATERIALS AND METHODS

Strains and culture media
The 10 Bt strains used (G03, HD-73, and Ly30, etc.) are collections from the authors' laboratory. These strains are cultured in Luria-Bertani (LB) medium (containing tryptone, yeast extract, sodium chloride, and microbiologically tested agar).

Total DNA isolation
Bacillus thuringiensis strains were activated in LB at 30 ºC overnight. Activated bacteria were diluted in LB (1:100), incubated at 37 ºC for 4 h, and shaken (230 rpm) until OD600 = 2.0. Cells were collected by centrifugation and then resuspended in solution I (0.3 mol L-1 sucrose, 25 mmol L-1 Tris-HCl [pH8.0], 25 mmol L-1 EDTA, 50 mg mL-1 lysozyme). For each 100 mL culture, 3 mL of solution I was used. Suspension was incubated at 37 ºC for 1 to 2 h and then solution II was added (0.1 mol L-1 NaCl, 0.1% sodium dodecyl sulphate [SDS], 0.1 mol L-1 Tris-HCl [pH 8.0]). Bacterial cells were disrupted by repeated freeze-thaw until the solution became clear. Lysate was extracted twice with phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1). DNA was precipitated by adding 1/10 volume of 3 mol L-1 sodium acetate (pH adjusted to 5.2 with glacial acetic acid) and 2 × volume of absolute ethanol. Precipitate was separated by spinning, washed twice with 70% ethanol, once with absolute ethanol, and dried in a vacuum. DNA was resuspended in ultrapure water, analyzed with 0.7% agarose gel electrophoresis (5 V cm-1), and quantified with UV spectrophotometry (DU® 640, Beckman Coulter, Fullerton, California, USA).

Oligo probe design and synthesis
Nucleotide sequences for cry1, cry2, and cry9 were retrieved from the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) and saved in the local BioEdit database. Probes were designed by Vector NTI, Array Designer 2.0 software after eliminating non-coding sequences in these genes.

Oligonucleotide probes were synthesized with the conventional phosphoramidite method with ABI 8089 (USA). Oligos were released by incubation with concentrated ammonium at 55 ºC for 15 h followed by column purification. Probes were linked by a spacer (PEG-phosphoramidite) to modified 3'-amine.

Chip manufacture
In the microarray, each cry gene was distinguished by three corresponding oligonucleotide probes (Figure 1, Table 1). Probes were mixed with a printing solution (3 × SSC [sodium chloride/sodium citrate], 0.01% SDS) concentrated to 40 μmol L-1 and printed on glass slides with Cartisan (Cartesian Technologies, Delaware, USA). Each print has a volume of about 0.5 nL. The distance between two spots was 500 μm and spot diameter was 200 µm. Each spot was printed twice. Printing temperature was controlled at 23 ºC and relative humidity was 90%. Slides were kept at room temperature for at least 24 h before hybridization. Sixty-three probes and empty controls were arranged in a 10 × 13 array. The location of the probes is listed in Table 1. A1A2 was the empty control and M9M10 was the negative control.

Figure 1. Strategy for designing a probe to detect and determine the genotype of Bt cry1, cry2, and cry9 genes.

Table 1. Probes for detection and typing of Bt cry1, cry2,and cry9 genes.



Random labeling of genomic DNA (Brown, 2000)
Genomic DNA was quantified by spectrophotometry, labeled with high-concentration Klenow enzyme and random primer. Labeling volume was 50 µL containing 5 µL of 10× buffer, 30 µg of genomic DNA, 5 µL of 10 × dNTP mix (dATP, dGTP, dCTP each 1.2 mmol L-1, 0.6 mmol L-1 dTTP), 3 μL of Cy3 or Cy5 dUTP (Amersham), 5 μL of hexamer primer, 50 U μL-1 of Klenow DNA polymerase (Invitrogen or New England Biolabs [NEB]). Reaction mix was incubated at 37 ºC for 2 h, inactivated in boiling water for 10 min, and chilled on ice. Labeled DNA was analyzed with 0.7% agarose gel electrophoresis and Typhoon 9410 scanner (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The probe was precipitated and suspended in 2 µL ultrapure water and 8 µL hybridization buffer (6 × SSC, 0.5% SDS, 5 × Denhart's reagents, 0.1 μg μL-1 salmon sperm DNA).

Hybridization and washing
The chip was pretreated with hybridization buffer at room temperature for 1 h, then hybridized with 10 µL probe. It was placed in a box and incubated at 56 ºC for 2 h, and washed with buffers A (1 × SSC, 0.2% SDS), B (0.2 × SSC), C (0.1 × SSC) for 1 min each. It was finally spun dried  (Wen et al., 2003).

Microarray scanning and data analysis
The microarray was scanned with GenePix 4000 (Axon Instrument, Foster City, California, USA) at 530 nm excitation, 585 nm (Cy3) and 650 nm (Cy5) emission. PMT was 700 to 800. Power level was 33% to100%. Image resolution was 10 µm in 16 bit tiff format. Signal intensity was the mean value of the two spots after background subtraction.

RESULTS

Oligonucleotide probes for gene detection and typing
Nucleotide sequences (131 entries) of cry1, cry2, and cry9 were retrieved from the NCBI database. After removing non-coding sequences, they were deposited in a local Bioedit database for local BLAST. Sequences were aligned with the strategy described in Figure 1 using ClustalW from the Vector NTI package (Invitrogen, Carlsbad, California, USA). Thirteen sequences, cry1Aa1~cry1Aa13, were first aligned and a consensus sequence cry1Aa was obtained. Probes were designed by Array Designer 2.0 based on the consensus sequence. Melting temperature of the probe was 70 ± 5 ºC, sizes were 40to 50 bp, hairpin maximum DG was -3.0 kcal mol-1, self-dimer maximum DG was -6.0 kcal mol-1, and the run/repeat (dinucleotide) maximum length was 4 bp. A specific oligonucleotide probe, 01Aa, for cry1Aa gene detection and typing was obtained after the probe sequences were searched with local BLAST to ensure specificity. Those with E value smaller than 1e-10 and specificity score greater than 70 were selected (Liu et al., 2004). Similarly, consensus sequences of cry1Ab…cry1Ag were obtained and corresponding oligonucleotide probes 01Ab…01Ag obtained. After consensus, cry1A was obtained by aligning the sequences of cry1Aa…cry1Ag, and the 01A probe was obtained. In the same way, we got the consensus sequences cry1B…cry1K and the 01B…01K probes. Furthermore, we got a consensus sequence cry1 by aligning the sequences of cry1A…cry1K, and 01 probes were obtained.

In this method, the cry1Aa1 gene could be distinguished by three probes: 01, 01A, and 01Aa, and we called this method TPPS (Three-Ply Position System).

We designed a total of 63 probes. Information regarding the probes (sequences, Tm, size, GC%, and location on chip) is listed in Table 1.

Random labeling of genomic DNA
Genomic DNA was fragmented during fluorescent labeling. By controlling the amount of random primer, the labeled probe was concentrated in a smear band of 1000 bp ~ 150 bp. Since the non-fluorescent bands cannot be registered in the Typhoon scanner, the smear band (II) image in Figure 2 is different from the other two lanes. This figure shows that genomic DNA was randomly fragmented and successfully labeled with fluorescent dye.

Figure 2. Random labeling of prepared products and total DNA.

 

Hybridized fluorescent images
Figure 3
shows the hybridized fluorescent images with partial Bt of Ly30, G03, and HD-73. The cry genes in these strains were examined with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).

Figure 3. Fluorescent images of microarray hybridized with Ly30 (A), G03 (B), and HD73 (C). Ly30 contains cry1Aa, cry1Ac, cry1Ia, cry2Aa, and cry2Ab. G03 contains cry1Aa, cry1Ac, cry1Ca, and cry2Ab. HD-73 contains cry1Ac.


The fluorescence signal was weaker than that obtained from the PCR-based probe because of the small size of the oligonucleotide probe. After optimizing hybridization parameters, cry genes in the different strains can be appropriately distinguished with the corresponding probes. The result of chip hybridization is consistent with PCR-RFLP (Yao et al., 2003).

DISCUSSION

Nowadays, for an important agricultural pest such as Lepidoptera insects, Bt is widely and effectively used as a critical biopesticide in biocontrol. It is significantly necessary to screen a Bt isolate with high toxicity and clone its novel insecticidal toxin genes to construct genetically engineered bacteria and transgenic plants. Among the insecticidal toxin genes against Lepidoptera, the most important are the cry1, cry2, and cry9 genes. In this study, we used an oligonucleotide microarray to detect and identify cry1, cry2, and cry9 genes in Bt.

At this time, methods for the detection and identification of Bt cry genes are based on PCR. One method is multiplex PCR (Bourque et al., 1993; Jaoua et al., 1996) and another is the two-step multiplex PCR (Ceron et al., 1995; Juarez-Perez et al., 1997). These methods, which include more than one primer, are applied to detect known cry sequences. However, it becomes less reliable than separate PCR (Lin et al., 2000). Another method is PCR-RFLP can identify known and unknown cry genes with only several pairs of primers. However, it becomes complicated, tedious, and time-consuming for analyzing multiple genes. It also ignores some variances in the conserved region (Yu et al., 1998). More importantly, PCR does not allow the rapid, parallel, and high throughput detection and identification of microbes. F. Song identified 14 known cry genotype with three universal primers and the corresponding enzymes, in the 3rd –level (Song et al., 2003); scholars from Israel obtained  cry genes by PCR using five universal primers, and then identified 20 cry genotypes (Ben-Dov et al., 1997) by using specific primers. In this study, 45 known cry genotypes can be identified by probes. It fully reflects that the DNA microarray is an advantage.

The design and selection of probes are essential for the quality and application of a microarray. It is particularly important to use bioinformatic tools to explore the details of related genes in designing and optimizing probes.

The probes should be able to not only detect, but also correctly classify cry genes. There are currently 375 known cry sequences. The similarity among the 4th level sequences is greater than 95%. It is not possible to design an oligonucleotide probe that is able to correctly detect and type all cry genes at this level. As a result, we designed probes to distinguish sequences at the 1st, 2nd, and 3rd levels with a reasonable specificity level. At the 3rd level, some sequences have a 75% similarity. It is difficult to design proper specificity probes Consequently, we relaxed the criteria, such as Tm, length, E value, and specificity score, and even designed two separate probes for some sequences (for example, cry1Ab). It is not possible to find one or two probes that could detect all cry1 genes so we designed three probes which contain almost all the cry1 genes (Liu et al., 2004).

In this study, the detection and typing of cry genes were based on TPPS. Any cry gene in the NCBI can hybridize with three corresponding probes. If a new cry gene exists in a strain, it will be unable to hybridize with all corresponding three-tier probes. Analysis of the hybridization profile can reveal the new cry gene. If a strain has more than one type of new cry genes (i. e. cry1Aa and cry1A), this method will be unable to reveal them (Jaroslaw et al., 2005).

Fluorescence labeling of genomic DNA is another key step of chip-based detection. It is not possible to design one or a few sets of primers for labeling all cry genes simultaneously by PCR amplification because there are a large number of diverse cry genes. Thus, conventional PCR-based chip detection is not applicable here.

Labeling of genomic DNA usually employs random primed nucleotide synthesis. This method can generate short fluorescent-labeled DNA. Due to its complexity, genomic DNA was first sheared with an ultrasonic processor (Vibra-Cell) before labeling. Therefore, it is not necessary to sonicate DNA prior to labeling. Since a large amount of genomic DNA is used for labeling, a high concentration of Klenow DNA polymerase should be used. If not, low efficiency of labeling results. In addition, the amount of random primer should be optimized so that the product is 1000 bp or shorter. A large DNA fragment could cause the hybridization signal to decrease due to the spatial effect.

Since the random labeling method does not have a selective amplification step of target genes, the content of target genes is very low, leading to a low hybridization signal on the oligo chip. To counteract these drawbacks, it is necessary to use several dozen or even one hundred micrograms of genomic DNA to label them. In addition, the labeled target has to be concentrated and purified before hybridization. High PMT and power settings are necessary during array scanning. These operations lead to high background and increased difficulty level (Volokhov et al., 2002; Wang et al., 2003). Sometimes inconsistent results may be obtained using a chip to detect genes in the same strain. However, it may be reduced by a higher hybridization temperature. This inconsistency may be caused by random primed DNA labeling which requires multiple trials to determine the cry genes in Bt.

Although sensitivity and consistency need to be improved, our study demonstrates that hierarchical oligonucleotide microarray is a high throughput assay capable of realistically reflecting all functional genes without PCR amplification of the target fragment (Wu et al., 2001; Zhou, 2003). Compared to the PCR method, the chip-based assay has more application prospects.

CONCLUSIONS

Our study demonstrates that hierarchical oligonucleotide microarray is a high throughput assay capable of realistically reflecting all functional genes without PCR amplification of the target fragment, although the sensitivity and consistency need to be improved.

ACKNOWLEDGEMENTS

This study was supported by 973 Project (Nº 2009CB118902, 2007CB109203), and 863 Plan (Nº 2006AA10A212), China.

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Received: 28 October 2008.                        Accepted: 22 April  2009.

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