Low Density DNA Microarrays in GMO analyses - testing of system
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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S489 Upon the crosses, the putative phenotype lines will be analyzed by several techniques such as SEM (scanning electron microscope), histochemistry, in-situ hybridization, confocal microscopy, and GUS staining. Our perspectives are that if we increase cell proliferation in the vascular meristem, we expect to obtain trees producing more wood without changing the characteristics of the xylem and/or phloem. doi:10.1016/j.jbiotec.2010.09.748 [P-P&F.45] Low Density DNA Microarrays in GMO analyses - testing of sys- tem Jan Hodek 1,∗ , Jaroslava Ovesna 1 , Katerina Demnerova 2 1 Crop Research Institute, Czech Republic 2 Institute of Chemical Technology, Czech Republic Keywords: GMO; DNA Microarrays; PCR; food and feed analyses Genetically modified (GM) food and feed are monitored within European Community. GMO (Genetically Modified Organism) han- dling arises under the control of the competent authorities in each country. Control laboratories involved in GMO analyses use internationally validated methods recognised by ISO and validated through Community Reference Laboratory (CRL). Mostly are used methods based on DNA analyses such as PCR and real-time PCR, especially in cases of GMO analyses in processed food or feed. How- ever, published validated methods do not cover all spectrum of worldwide used GMO. Capturing of unknown GMOs could be done by PCR amplification of general used regulation elements, if they are presented in transgenic cassete. These step-by-step PCR analyses use to be time consuming. DNA Microarrays are high-throughput technique based on hybridization of unknown sample DNA and DNAs of known sequence (probe), which are immobilized on a glass support. On the glass support could be immobilized as many as hundreds of such probes. In our study we have tested the throughput of our in house developed DNA Microarrays based on control amplicons for several plant genes, transgenes and regulation elements used in GMOs. We focused on the fluorescent labeled total genomic DNA as a template DNA. Acknowledgements The work was supported by the project of the Ministry of Agriculture of the Czech Republic (PUV) NAZV 1B 44068 and CZ002700604 doi:10.1016/j.jbiotec.2010.09.749 [P-P&F.46] Obtaining of protoplasts from leaves and suspension cells of three tomato’s genotypes A.I. Urrea T, C. Restrepo O, C. Botero G ∗ Universidad de Antioquia, Colombia Keywords: Solanum lycopersicum; protoplast; suspension cells; Cal- lus The aim of this research was to determinate the protoplast isolation efficiency from leaf mesophyll and suspension cells of three tomato’s genotypes with different resistance grade to Phy- tophthora infestans. We developed a protocol with high percentage of viable protoplasts and appropriate number of them to evaluate the defense response against pathogens at cellular level and thus the possibility to achieve an early selection of new cultivars either conventional improvement or by biotechnological methods. doi:10.1016/j.jbiotec.2010.09.750 [P-P&F.47] Carboxypeptidase activity and bioinformatics analyses of CP-III gene of Theobroma cacao L C. Bautista 1,∗ , M.L. Sánchez 2 , X.M. Boldo 3 , M.E. Jaramillo 2 1 Colegio de Postgraduados Campus Tabasco, Mexico 2 Escuela Nacional de Ciencias Biológicas-IPN, Mexico 3 Universidad Juárez Autónoma de Tabasco, Mexico Keywords: Carboxypeptidase; Theobroma cacao; Fermentation; Germination Serine carboxypeptidases (SCPs) are members of the / hydro- lase family of proteins, which makes use of a Ser-Asp-His catalytic triad to cleave the carboxyterminal peptide bonds of their proteins or peptides substrates. The carboxypeptidase (tcCP) of Theobroma cacao L. has been involved in the formation of flavor precursors during the fermentation process. In this work, the tcCP activity was detected in both germination and fermentation processes of cacao seeds using N-Benzoil-L-Tirosina--NA as proteolytic substrate. The levels of tcCP activity increases during the phase of growth corresponding to the begining of visible morphological changes, as well as the lack of a enzyme-inhibitor system described in other organisms. The tcCP enzyme detected in fermented seeds, indicates that it is a heat-stable enzyme. Bioinformatics analyses indicated that CP-III is a high expression gene witch code for a carboxipeptidase-type III protein predicted of 508 amino acids approximately, molecular weight of 56.439 Da and a theoretical pI of 4.915. This protein is rich in Leu, Ala, Gly, Ser and Val amino acids. The codon usage frequency and the CAI value correspond to high expression genes with respect to Car- boxypeptidase C gene of Oryza sativa and SCPL49 of Arabidopsis thaliana. Catalytic triad indicated that this enzyme is a counter- part SCPs to the described ones in other organisms. The prediction of hydrophobicity, helical membranous regions and subcellular location, showed that this protein can be located in the vacuo- lar membrane. The constructed similarity tree revealed that the predicted amino acid sequence of gene CP-III of T. cacao L is more related to the Carboxypeptidase C (GenBanK access number gene: NM187300) and Serine Carboxypeptidase III (GenBanK access num- ber protein: P37891) of O. sativa, as well as with the SCPL49 of A. thaliana (GenBanK access number gene: NM111876) respectively. doi:10.1016/j.jbiotec.2010.09.751
Transcript of Low Density DNA Microarrays in GMO analyses - testing of system
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S489
Upon the crosses, the putative phenotype lines will be analyzedby several techniques such as SEM (scanning electron microscope),histochemistry, in-situ hybridization, confocal microscopy, and GUSstaining.
Our perspectives are that if we increase cell proliferation in thevascular meristem, we expect to obtain trees producing more woodwithout changing the characteristics of the xylem and/or phloem.
doi:10.1016/j.jbiotec.2010.09.748
[P-P&F.45]
Low Density DNA Microarrays in GMO analyses - testing of sys-tem
Jan Hodek 1,∗, Jaroslava Ovesna 1, Katerina Demnerova 2
1 Crop Research Institute, Czech Republic2 Institute of Chemical Technology, Czech RepublicKeywords: GMO; DNA Microarrays; PCR; food and feed analyses
Genetically modified (GM) food and feed are monitored withinEuropean Community. GMO (Genetically Modified Organism) han-dling arises under the control of the competent authorities ineach country. Control laboratories involved in GMO analyses useinternationally validated methods recognised by ISO and validatedthrough Community Reference Laboratory (CRL). Mostly are usedmethods based on DNA analyses such as PCR and real-time PCR,especially in cases of GMO analyses in processed food or feed. How-ever, published validated methods do not cover all spectrum ofworldwide used GMO. Capturing of unknown GMOs could be doneby PCR amplification of general used regulation elements, if they arepresented in transgenic cassete. These step-by-step PCR analysesuse to be time consuming.
DNA Microarrays are high-throughput technique based onhybridization of unknown sample DNA and DNAs of knownsequence (probe), which are immobilized on a glass support. Onthe glass support could be immobilized as many as hundreds ofsuch probes. In our study we have tested the throughput of our inhouse developed DNA Microarrays based on control amplicons forseveral plant genes, transgenes and regulation elements used inGMOs. We focused on the fluorescent labeled total genomic DNAas a template DNA.
Acknowledgements
The work was supported by the project of the Ministry ofAgriculture of the Czech Republic (PUV) NAZV 1B 44068 andCZ002700604
doi:10.1016/j.jbiotec.2010.09.749
[P-P&F.46]
Obtaining of protoplasts from leaves and suspension cells ofthree tomato’s genotypes
A.I. Urrea T, C. Restrepo O, C. Botero G ∗
Universidad de Antioquia, ColombiaKeywords: Solanum lycopersicum; protoplast; suspension cells; Cal-lus
The aim of this research was to determinate the protoplastisolation efficiency from leaf mesophyll and suspension cells ofthree tomato’s genotypes with different resistance grade to Phy-tophthora infestans. We developed a protocol with high percentage
of viable protoplasts and appropriate number of them to evaluatethe defense response against pathogens at cellular level and thusthe possibility to achieve an early selection of new cultivars eitherconventional improvement or by biotechnological methods.
doi:10.1016/j.jbiotec.2010.09.750
[P-P&F.47]
Carboxypeptidase activity and bioinformatics analyses of CP-IIIgene of Theobroma cacao L
C. Bautista 1,∗, M.L. Sánchez 2, X.M. Boldo 3, M.E. Jaramillo 2
1 Colegio de Postgraduados Campus Tabasco, Mexico2 Escuela Nacional de Ciencias Biológicas-IPN, Mexico3 Universidad Juárez Autónoma de Tabasco, MexicoKeywords: Carboxypeptidase; Theobroma cacao; Fermentation;Germination
Serine carboxypeptidases (SCPs) are members of the �/� hydro-lase family of proteins, which makes use of a Ser-Asp-His catalytictriad to cleave the carboxyterminal peptide bonds of their proteinsor peptides substrates. The carboxypeptidase (tcCP) of Theobromacacao L. has been involved in the formation of flavor precursorsduring the fermentation process. In this work, the tcCP activity wasdetected in both germination and fermentation processes of cacaoseeds using N-Benzoil-L-Tirosina-�-NA as proteolytic substrate.The levels of tcCP activity increases during the phase of growthcorresponding to the begining of visible morphological changes,as well as the lack of a enzyme-inhibitor system described in otherorganisms. The tcCP enzyme detected in fermented seeds, indicatesthat it is a heat-stable enzyme.
Bioinformatics analyses indicated that CP-III is a high expressiongene witch code for a carboxipeptidase-type III protein predictedof 508 amino acids approximately, molecular weight of 56.439 Daand a theoretical pI of 4.915. This protein is rich in Leu, Ala, Gly,Ser and Val amino acids. The codon usage frequency and the CAIvalue correspond to high expression genes with respect to Car-boxypeptidase C gene of Oryza sativa and SCPL49 of Arabidopsisthaliana. Catalytic triad indicated that this enzyme is a counter-part SCPs to the described ones in other organisms. The predictionof hydrophobicity, helical membranous regions and subcellularlocation, showed that this protein can be located in the vacuo-lar membrane. The constructed similarity tree revealed that thepredicted amino acid sequence of gene CP-III of T. cacao L is morerelated to the Carboxypeptidase C (GenBanK access number gene:NM187300) and Serine Carboxypeptidase III (GenBanK access num-ber protein: P37891) of O. sativa, as well as with the SCPL49 of A.thaliana (GenBanK access number gene: NM111876) respectively.
doi:10.1016/j.jbiotec.2010.09.751
