Detection of stress resistance genes in transgenic maize by multiplex and touchdown polymerase chain reaction

M. A. Bannikova © 2015 M. A. Bannikova; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited Molecular and Cell Biotechnologies ISSN 0233-7657 Biopolymers and Cell. 2015. Vol. 31. N 5. P. 362–370 doi: http://dx.doi.org/10.7124/bc.0008F8


Introduction
Maize is one of the most common cereal, forage and silage crops in the world. The transgenic plants of maize have been cultivated in the open system for more than 15 years, taking over 50 % of arable land, suitable for this crop.
Not one, but several genes with their regulatory sequences are usually inserted into the maize genome. Such complex transformants, remarkable for their unique combination of genetic construction and genomic DNA of a transgenic plant, were called transformation events ( Table 1).
According to different literature sources 5-10 % of the maize plants, cultivated in Ukraine, contain additional transgenes/transformation events. The majority of Ukrainian laboratories, analyzing the samples for transgenes (http://www.quality.ua), detect the presence of 35S promoter or NOS terminator whereas the Joint Research Centre as European Union Reference Laboratory for GM Food and Feed (JRC) elaborated the protocols for quantitative detection of transgenes using Real-Time PCR [1]. Thus, our task was to develop a methodology of the fast, reliable, accurate and relatively cheap detection of the maize transformation events and included transgenes.

Plant material
The reference samples of maize, containing the corresponding transformation events, and the experimental selection samples of maize of the Researchand-Production Farm Mais Company (Ukraine) were studied in the work. All the samples were germinated for further DNA isolation.

Isolation and purification of DNA
Maize germ plants were ground in ceramic mortars with CTAB (20 g/l CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na 2 EDTA, pH 8.0) and 1.4-mercaptoethanol. RNA treatment and triple purification from proteins and enzymes were performed using chloroform. The total DNA was salted out with isopropyl alcohol and washed with ethyl alcohol. The precipitate was dissolved in TE buffer, pH 8.0. The method of electrophoresis of nucleic acids in agarose gel was used to check the presence and quality of total plant DNA after the isolation procedure. The concentration of nucleic acids was measured spectrophotometrically. The purity of the total DNA was determined by the absorption ratio at the wavelength of 230, 260, 280 nm. The DNA concentration of the samples was normalized to 30 ng/µl [2].

Electrophoresis
The electrophoresis of amplification products was conducted according to [4]. 400 ng of molecular mass marker O'GeneRuler TM DNA Ladder Mix (Thermo Scientific) were used as a marker. The gel plate was kept in ethidium bromide for the visualization of amplicons, shot by GelDoc TM (Bio-Rad) and processed using the GIMP graphic editor.

Results and Discussion
The genetically modified maize, containing individual transgenes, is an uncommon case. Practically all the transgenes are included in transformation events. The maize transformation events contain the of the plant DNA sample and primers to the reference gene (adh1; zein -for MON863 and T25) and to the transformation event under study. The concentration of primers was selected for each specific case depending on the specificity of binding to the DNA matrix ( Table 2). The reaction mixture with 1 µl of TE buffer (pH 8.0) was used instead of the DNA sample as a negative control for all the cases.

Polymerase chain reaction (PCR)
PCR was conducted according to [3]. The conditions for the annealing stage were estimated in accordance with the properties of the oligonucleotide primers and their optimization was performed empirically.  genes of resistance to insect pests and the genes of tolerance to herbicides (Table 1). Gene pat (bar) encodes the enzyme phosphinothricin N-acetyltransferase, determining the tolerance of plants to herbicides on the basis of phosphinothricin (PPT) -BASTA TM , glufosinate, phosphinothricin.
Our task was to determine the presence of sequences of transformation events or the genes thereof. Multiplex polymerase chain reaction (mPCR) and touchdown PCR, which allow the amplification of several DNA sites of specific length in one reaction, were developed to shorten the time, improve the reliability and quality of the PCR analysis [5][6][7][8][9][10]. This method presupposes the use of two (or more) pairs of oligonucleotide primers, specific to transformation events, and one pair of primers, specific to the maize reference gene adh1 (in some cases zein). Therefore, the selection of specific primers was conducted for each individual gene/transformation event ( Table 2). mPCR was used to detect the transformation events BT176, MON810, MON88017, DAS1507, MIR604, DAS59122, GA21, NK603 whereas no reliable results were obtained for detecting transformation events Bt11, MON863, MON89034 and T25. Therefore, the method of touchdown PCR was adapted to improve the quality and specificity of the reaction, to increase the amount of the amplified product and to neutralize the non-specific signals for the transformation events Bt11, MON863, MON89034 and T25.
The adh1 gene was used as a reference gene to detect all transformation events with the exception of MON863 and T25 [6,7]. The amplicons of the events MON863 and T25 have sizes of 234 bp and 209 bp respectively, similar to the amplicon of adh1 (231 bp), that makes it complicated to distinguish and identify them using the gel-electrophoresis method. Thus, zein (277 bp) was used as a reference gene ( Table 2).
The mPCR procedure of detection of the transformation events BT176, MON810, MON88017, DAS1507, MIR604, DAS59122, GA21, NK603 was as follows. The time of synthesis of the fragments of target genes and the temperature of DNA renaturation with oligonucleotide primers were selected individually for each transformation event ( Table 4). The final synthesis of the fragments of target genes in all the reactions was conducted at 72 °С for 10 min. The method of touchdown PCR, used to detect the transformation events BT11, MON863, MON89034, T25, was similar to mPCR. The difference was that the initial annealing temperature for primers was 15 °C higher than the expected melting temperature, during the first 15 cycles the annealing temperature was gradually (by 1 °C) decreased until reaching the temperature, optimal for the tested pair of primers, and the next 21 cycles were performed at the optimal temperature, ensuring the exponential increase in the amount of target amplicon only.
PCR products were fragments of maize reference genes adh1 or zein and the target fragments of genes/ /transformation events/genetic constructions of specific length ( Table 2, Figures 1 and 2).
It is evident in Figures 1 and 2 that the obtained amplicons correspond to the expected ones, referred in Table 2.
Maize adh1 or zein referenсe genes only were detected in the test samples; this testifies to the presence and quality of plant DNA in the samples and the absence of detected genes/transformation events in the genetic material of plants. Both marker genes of maize adh1 or zein and the amplicons of the expected size were detected in the reference samples, which testified to the adequacy of the selected conditions of mPCR and the presence of transformation events. The negative controls did not contain any fragments. This was the evidence of the purity of DNA reagents as well as the proper quality of reaction performance.
The method may be applied in the mass analysis of maize samples. Neither transgenes, nor transformation events were detected among 200 samples of the experimental forms of maize of RPF Mais Company, which were analyzed.