- Email: [email protected]
Genetic basis of tomato marker assisted selection in Belarus
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
cytotoxic effect of the compounds, which followed an exponential curve with increasing concentrations. Acknowledgements: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCSUEFISCDI project number PN-II-ID-PCE-2011-3-0226. http://dx.doi.org/10.1016/j.jbiotec.2015.06.076 Determination of cytotoxic and proliferative effects of 2-amino-5-nitrobenzonitrile on AGS cancer cell line Mustafa Anar 1,∗ , Hamit Emre Kizil 3 , Guleray Agar 2 1
Department of Molecular Biology and Genetics, Ataturk University, Erzurum, Turkey 2 Department of Biology, Ataturk University, Erzurum, Turkey 3 Vocational School of Health Services, Bayburt University, Bayburt, Turkey E-mail address: [email protected] (M. Anar). In this study, we aimed to determine the cytotoxic and proliferative effects of 2-amino-5-nitrobenzonitrile on AGS cancer cell line and healthy HUVEC cells. RTCA (Real-Time Cell Analyser) – SP (Single Plate) system was used to evaluate the cell viability of HUVEC cell line to be used as control. We investigated cytotoxic effects by LDH (Lactate Dehydrogenase Assay) test on HUVEC cell line. Proliferative effects of 2-amino-5-nitrobenzonitrile were investigated by WST-1 (water soluble tetrazolium salts) test. After the incubation of HUVEC cells, 12.5 g/mL, 25 g/mL, 50 g/mL, 100 g/mL, 200 g/mL doses of 2-amino-5-nitrobenzonitrile were added to wells and whole doses of 2-amino-5-nitrobenzonitrile did not show toxic effects on cell proliferation of HUVEC. LDH and WST-1 assays were performed on AGS cells and different doses was used. High doses decreased the cell proliferation and showed cytotoxic effects on AGS cells. The results showed that 50 g/mL, 100 g/mL and 200 g/mL doses have cytotoxic and antiproliferative properties. Therefore, 2-amino-5-nitrobenzonitrile can has anticancer potential and it will be used for cancer treatment after the advanced research. http://dx.doi.org/10.1016/j.jbiotec.2015.06.077 Genetic basis of tomato marker assisted selection in Belarus Alexander Kilchevsky ∗ , Olga Babak, Victoria Adzhieva, Natalya Nekrashevich, Sergei Malyshev Institute of Genetics and Cytology at the National Academy of Sciences of Belarus, Minsk, Belarus E-mail address: [email protected] (A. Kilchevsky). Development of hybrids with a high fruit quality and disease resistance is one of the urgent trends in tomato breeding in Belarus. We have used our own methods for DNA identification of fruit quality genes nor, rin, norA (long shelf life), og, Del, r, hp-1, hp-2dg, B from S. pennellii, ogc, t, gf-3 (increased carotenoid content) and the techniques known from scientific literature for resistance genes to Cladosporium fulvum (cf-2, cf-4, cf-5, cf-9), to Fusarium oxysporum (I-2), to Meloidogyne incognita (Mi-1.2) in order to identify the breeding material according to the allele composition. Hybrids, combining alleles of the genes under study, were developed in the result of a number of topcrosses. The forms with interested allele combinations were selected in the F2 generation, using МAS
S29
METHODS: homo- and heterozygotes by the carotenoid content genes and the genes extending fruit shelf life, hybrid combinations with two carotenoid content genes and one long shelf life gene and hybrids with a complex of the fruit quality and disease resistance genes. Now work on the development of the lines combining the given groups of genes in a homozygous stay is conducted. The unique model tomato forms with different combinations of fruit quality genes are used to study on peculiarities of carotenoid accumulation in the fruit and to develop of valuable hybrids for agriculture. http://dx.doi.org/10.1016/j.jbiotec.2015.06.078 Tomato bushy stunt virus-based vector for transient expression of human G-CSF in Nicotiana benthamiana plants Laura S. Abeuova 1 , Herman B. Scholthof 2 , Erlan M. Ramankulov 1 , Shuga A. Manabayeva 1,∗ 1 National Center for Biotechnology of Republic of Kazakhstan, Astana, Kazakhstan 2 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, USA
E-mail address: [email protected] (S. A. Manabayeva). The use of plant viral vectors for transient expression of foreign proteins in plants offers a useful tool for the production of proteins such as biopharmaceuticals. Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates the proliferation and differentiation and function of neutrophil granulocyte progenitor cells. The objective of this work was the development of an advanced Tomato bushy stunt virus (TBSV)-based protein production system to produce human G-CSF in non-transgenic plants. We evaluated the potential of a TBSV vector system for efficient expression of the recombinant human G-CSF protein in Nicotiana benthamiana plants. For this purpose, we developed a TBSV derived viral vector driven by the CaMV 35S promoter that can be delivered as DNA. In this vector, the CP gene was replaced by the codon optimized rhG-CSF gene that was synthesized by a PCR-based gene synthesis method. To facilitate purification of the rhG-CSF from plant materials, the protein sequence was modified by the addition of the short amino acid tag (6 x His). Four days after infiltration expression of rhC-CSF was verified by protein extraction followed by western blots procedures. The rhG-CSFx6His was isolated using metal affinity chromatography on Ni2+-NTA agarose. The concentration of purified rhG-CSF was determined by ELISA. The results indicate that the TBSV-based protein production system is suitable for transient production of biopharmaceuticals in plants. http://dx.doi.org/10.1016/j.jbiotec.2015.06.079 Transient expression of Acidothermus cellulolyticus endoglucanase E1 by a Tomato bushy stunt virus-based plant expression vector Aiganym T. Makenova 1 , Herman B. Scholthof 2 , Erlan M. Ramankulov 1 , Shuga A. Manabayeva 1,∗ 1 National Center for Biotechnology, Astana, Kazakhstan 2 Department of Plant Pathology and Microbiology, Texas A&M University, College station, USA
E-mail address: [email protected] (S. A. Manabayeva). Acidothermus cellulolyticus E1 endoglucanase is one of the most active cellulases known. The use of plant viral vectors for tran-
cytotoxic effect of the compounds, which followed an exponential curve with increasing concentrations. Acknowledgements: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCSUEFISCDI project number PN-II-ID-PCE-2011-3-0226. http://dx.doi.org/10.1016/j.jbiotec.2015.06.076 Determination of cytotoxic and proliferative effects of 2-amino-5-nitrobenzonitrile on AGS cancer cell line Mustafa Anar 1,∗ , Hamit Emre Kizil 3 , Guleray Agar 2 1
Department of Molecular Biology and Genetics, Ataturk University, Erzurum, Turkey 2 Department of Biology, Ataturk University, Erzurum, Turkey 3 Vocational School of Health Services, Bayburt University, Bayburt, Turkey E-mail address: [email protected] (M. Anar). In this study, we aimed to determine the cytotoxic and proliferative effects of 2-amino-5-nitrobenzonitrile on AGS cancer cell line and healthy HUVEC cells. RTCA (Real-Time Cell Analyser) – SP (Single Plate) system was used to evaluate the cell viability of HUVEC cell line to be used as control. We investigated cytotoxic effects by LDH (Lactate Dehydrogenase Assay) test on HUVEC cell line. Proliferative effects of 2-amino-5-nitrobenzonitrile were investigated by WST-1 (water soluble tetrazolium salts) test. After the incubation of HUVEC cells, 12.5 g/mL, 25 g/mL, 50 g/mL, 100 g/mL, 200 g/mL doses of 2-amino-5-nitrobenzonitrile were added to wells and whole doses of 2-amino-5-nitrobenzonitrile did not show toxic effects on cell proliferation of HUVEC. LDH and WST-1 assays were performed on AGS cells and different doses was used. High doses decreased the cell proliferation and showed cytotoxic effects on AGS cells. The results showed that 50 g/mL, 100 g/mL and 200 g/mL doses have cytotoxic and antiproliferative properties. Therefore, 2-amino-5-nitrobenzonitrile can has anticancer potential and it will be used for cancer treatment after the advanced research. http://dx.doi.org/10.1016/j.jbiotec.2015.06.077 Genetic basis of tomato marker assisted selection in Belarus Alexander Kilchevsky ∗ , Olga Babak, Victoria Adzhieva, Natalya Nekrashevich, Sergei Malyshev Institute of Genetics and Cytology at the National Academy of Sciences of Belarus, Minsk, Belarus E-mail address: [email protected] (A. Kilchevsky). Development of hybrids with a high fruit quality and disease resistance is one of the urgent trends in tomato breeding in Belarus. We have used our own methods for DNA identification of fruit quality genes nor, rin, norA (long shelf life), og, Del, r, hp-1, hp-2dg, B from S. pennellii, ogc, t, gf-3 (increased carotenoid content) and the techniques known from scientific literature for resistance genes to Cladosporium fulvum (cf-2, cf-4, cf-5, cf-9), to Fusarium oxysporum (I-2), to Meloidogyne incognita (Mi-1.2) in order to identify the breeding material according to the allele composition. Hybrids, combining alleles of the genes under study, were developed in the result of a number of topcrosses. The forms with interested allele combinations were selected in the F2 generation, using МAS
S29
METHODS: homo- and heterozygotes by the carotenoid content genes and the genes extending fruit shelf life, hybrid combinations with two carotenoid content genes and one long shelf life gene and hybrids with a complex of the fruit quality and disease resistance genes. Now work on the development of the lines combining the given groups of genes in a homozygous stay is conducted. The unique model tomato forms with different combinations of fruit quality genes are used to study on peculiarities of carotenoid accumulation in the fruit and to develop of valuable hybrids for agriculture. http://dx.doi.org/10.1016/j.jbiotec.2015.06.078 Tomato bushy stunt virus-based vector for transient expression of human G-CSF in Nicotiana benthamiana plants Laura S. Abeuova 1 , Herman B. Scholthof 2 , Erlan M. Ramankulov 1 , Shuga A. Manabayeva 1,∗ 1 National Center for Biotechnology of Republic of Kazakhstan, Astana, Kazakhstan 2 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, USA
E-mail address: [email protected] (S. A. Manabayeva). The use of plant viral vectors for transient expression of foreign proteins in plants offers a useful tool for the production of proteins such as biopharmaceuticals. Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates the proliferation and differentiation and function of neutrophil granulocyte progenitor cells. The objective of this work was the development of an advanced Tomato bushy stunt virus (TBSV)-based protein production system to produce human G-CSF in non-transgenic plants. We evaluated the potential of a TBSV vector system for efficient expression of the recombinant human G-CSF protein in Nicotiana benthamiana plants. For this purpose, we developed a TBSV derived viral vector driven by the CaMV 35S promoter that can be delivered as DNA. In this vector, the CP gene was replaced by the codon optimized rhG-CSF gene that was synthesized by a PCR-based gene synthesis method. To facilitate purification of the rhG-CSF from plant materials, the protein sequence was modified by the addition of the short amino acid tag (6 x His). Four days after infiltration expression of rhC-CSF was verified by protein extraction followed by western blots procedures. The rhG-CSFx6His was isolated using metal affinity chromatography on Ni2+-NTA agarose. The concentration of purified rhG-CSF was determined by ELISA. The results indicate that the TBSV-based protein production system is suitable for transient production of biopharmaceuticals in plants. http://dx.doi.org/10.1016/j.jbiotec.2015.06.079 Transient expression of Acidothermus cellulolyticus endoglucanase E1 by a Tomato bushy stunt virus-based plant expression vector Aiganym T. Makenova 1 , Herman B. Scholthof 2 , Erlan M. Ramankulov 1 , Shuga A. Manabayeva 1,∗ 1 National Center for Biotechnology, Astana, Kazakhstan 2 Department of Plant Pathology and Microbiology, Texas A&M University, College station, USA
E-mail address: [email protected] (S. A. Manabayeva). Acidothermus cellulolyticus E1 endoglucanase is one of the most active cellulases known. The use of plant viral vectors for tran-