Comparative Proteomic Analysis of Two Varieties of Genetically Modified (GM) Embrapa 5.1 Common Bean (Phaseolus vulgaris L.) and Their Non-GM Counterparts.

The genetically modified (GM) common bean event Embrapa 5.1 was commercially approved in Brazil in 2011; it is resistant to golden mosaic virus infection. In the present work grain proteome profiles of two Embrapa 5.1 common bean varieties, Pérola and Pontal, and their non-GM counterparts were compared by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). Analyses detected 23 spots differentially accumulated between GM Pérola and non-GM Pérola and 21 spots between GM Pontal and non-GM Pontal, although they were not the same proteins in Pérola and Pontal varieties, indicating that the variability observed may not be due to the genetic transformation. Among them, eight proteins were identified in Pérola varieties, and four proteins were identified in Pontal. Moreover, we applied principal component analysis (PCA) on 2-DE data, and variation between varieties was explained in the first two principal components. This work provides a first 2-DE-MS/MS-based analysis of Embrapa 5.1 common bean grains.

Development of an event-specific hydrolysis probe quantitative real-time polymerase chain reaction assay for Embrapa 5.1 genetically modified common bean (Phaseolus vulgaris).

A genetically modified (GM) common bean event, namely Embrapa 5.1, resistant to the bean golden mosaic virus (BGMV), was approved for commercialization in Brazil. Brazilian regulation for genetically modified organism (GMO) labeling requires that any food containing more than 1% GMO be labeled. The event-specific polymerase chain reaction (PCR) method has been the primary trend for GMO identification and quantitation because of its high specificity based on the flanking sequence. This work reports the development of an event-specific assay, named FGM, for Embrapa 5.1 detection and quantitation by use of SYBR Green or hydrolysis probe. The FGM assay specificity was tested for Embrapa 2.3 event (a noncommercial GM common bean also resistant to BGMV), 46 non-GM common bean varieties, and other crop species including maize, GM maize, soybean, and GM soybean. The FGM assay showed high specificity to detect the Embrapa 5.1 event. Standard curves for the FGM assay presented a mean efficiency of 95% and a limit of detection (LOD) of 100 genome copies in the presence of background DNA. The primers and probe developed are suitable for the detection and quantitation of Embrapa 5.1.

A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.

The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean.

Development of plasmid DNA reference material for the quantification of genetically modified common bean embrapa 5.1.

The genetically modified (GM) common bean Embrapa 5.1 was recently approved for commercialization. The reliable detection and quantification of GM organisms is strongly dependent on validated methods as well as calibration systems. This work presents the development of a calibrant plasmid for Embrapa 5.1 common bean detection. The reaction parameters were determined and compared for both the plasmid DNA (pDNA) and the genomic DNA (gDNA). PCR efficiencies for pDNA were 81% for the construction-specific assays and 76% for the taxon-specific assay, whereas for gDNA efficiencies were 94 and 93%, respectively. The limits of detection (LOD) in both qPCR assays were 10(2) and 10(3) copies of gDNA and pDNA per PCR reaction, respectively. This is sufficient to detect 0.067 and 0.67% of GM common bean in 100 ng of DNA, respectively, which is in agreement with detecting the 1% GM content required by the Brazilian legislation.

Primers and probes development for specific PCR detection of genetically modified common bean (Phaseolus vulgaris) Embrapa 5.1.

The genetically modified common bean Embrapa 5.1, developed by Brazilian Agricultural Research Corporation (Embrapa), is the first commercial GM plant produced in Latin America. It presents high resistance to the Bean golden mosaic virus. In this work, primers and probes targeting a taxon-specific reference DNA sequence for the common bean (Phaseolus vulgaris L.) and a construct-specific DNA sequence of Embrapa 5.1 GM common bean were successfully developed. The primers and probes showed high specificity for the target detection. Both methods showed suitable efficiency and performance to be used as an endogenous target for detection of common bean DNA and for construct-specific detection of GM common bean Embrapa 5.1, respectively. Both real-time PCR assays proved to be valuable for future assessment of interlaboratory studies.

Proteomic analysis of four Brazilian MON810 maize varieties and their four non-genetically-modified isogenic varieties.

Profiling techniques have been suggested as a nontargeted approach to detect unintended effects in genetically modified (GM) plants. Seedlings from eight Brazilian maize varieties, four MON810 GM varieties and four non-GM isogenic varieties, were grown under controlled environmental conditions. Physiological parameters (aerial part weight, main leaf length, and chlorophyll and total protein contents) were compared, and some differences were observed. Nevertheless, these differences were not constant among all GM and non-GM counterparts. Leaf proteomic profiles were analyzed using two-dimensional gel electrophoresis (2DE) coupled to mass spectrometry, using six 2DE gels per variety. The comparison between MON810 and its counterpart was limited to qualitative differences of fully reproducible protein spot patterns. Twelve exclusive proteins were observed in two of four maize variety pairs; all of these leaf proteins were variety specific. In this study, MON810 leaf proteomes of four varieties were similar to non-GM counterpart leaf proteomes.