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The assessment of biochemical parameters with grapevine in correlation with organic fertilization of the soil
New Biotechnology · Volume 25S · September 2009
3.2.32 The assessment of biochemical parameters with grapevine in correlation with organic fertilization of the soil C. Bejan National Institute of Research and Development for Biotechnology in Horticulture Stefanesti, Arges County, Romania
Maintaining and soil fertility improvement are important aims for grapevine growing, taking into account that, in most situations, this species are grown on poor nutritive soils. In these circumstances, efficient systems of fertilization, based on maximum utilization of organically fertilizers, are of present interest. The goal of this study is to explore the correlations between the type of organic fertilizer and grapevine performances expressed in accumulation of biochemical compounds which are involved in plant growth and development. The study was conducted with two grapevine cultivars — Cabernet Sauvignon and Zweigelt — used for high quality red wines production, from vineyards belonging to the National Research and Development Institute for Biotechnology in Horticulture Stefanesti-Arges. Our results revealed the positive effect of soil manuring with organic fertilizer upon the biosyntheses processes in leaves over the vegetative period of main primary metabolites (soluble glucides, chlorophyll pigments and carotenes) and secondary metabolites (proteins, polyphenols, auxins). As a result, woody materials collected from the organic fertilized areas proved to have a better maturation at the beginning of the winter repose period. This was correlated with a better buds viability from yield canes. doi:10.1016/j.nbt.2009.06.665
3.2.33 Remediation of zinc and copper by transgenic tobacco plants with metal transporter genes from Neurospora crassa S. Eapen ∗ , P. Dixit, S. Singh, P. Maruthi Mohan, V. Ramachandran Bhabha Atomic Research Centre, Mumbai, India
Zinc and copper are essential elements for growth and development of plants and uptake of these mineral elements by plants is a part of mineral nutrition. Plants have homeostatic mechanisms to maintain the correct concentrations of these metals in their cellular compartments. It is possible to enhance the uptake of these metals by introducing efficient genes for metal uptake from other organisms into plants. Plants with enhanced ability for metal uptake can be developed by introduction of metal transporter genes from other sources. Development of transgenic plants with high efficiency of metal uptake will help in removing toxic levels of these metals from contaminated soils and solutions. With this objective, two metal transporter genes, namely a zinc transporter gene and a copper transporter gene from Neurospora crassa were cloned and individually introduced into plant expression vec-
ABSTRACTS
tor pCAMBIA 1301 having hph as a selectable marker and uidA as a reporter gene and transgenic tobacco plants developed by Agrobacterium mediated gene transfer. The transgenic nature of plants was confirmed by PCR and Southern blot hybridization, while expression of transgene was confirmed by reverse transcription PCR. Transgenic plants with zinc transporter gene, when grown in media supplemented with radiolabelled ZnCl2 , showed enhanced accumulation of zinc in roots (11 times). Uptake of copper was studied in transgenic plants with copper transporter gene and the transgenic plants could take up more copper (2 times) compared to control plants. The present studies have shown that it is possible to develop transgenic plants with improved efficiency for remediation of metals like zinc and copper. doi:10.1016/j.nbt.2009.06.666
3.2.34 High-level of expression, purification and characterization of active Artin M Lectin in different E. Coli hosts M.C.D.S. Pranchevicius 1,∗ , L. Oliveira 2 , N.C. Avanci 3 , J.C. Rosa 4 , A.C. Quiapim 3 , V. Cossalter 3 , M.S. Brito 3 , E.A. Aragão 5 , T.L. Ferreira 5 , M.C. Roque Barreira 2 , M.H.S. Goldman 3 1
Department of Biology, FFCLRP/USP, School of Medicine, UFT/TO, 1, Brazil Department of Molecular and Cell Biology, FMRP/USP, Brazil 3 Department of Biology, FFCLRP/USP, Brazil 4 Department of Molecular and Cell Biology, FMRP/USP, Protein Chemistry Center, FMRP/USP, Brazil 5 Department of Chemistry, FFCLRP/USP, Brazil 2
Artin M (KM+) is a D-mannose-binding lectin from Artocarpus integrifolia seeds that induces interleukin (IL)-12 production by macrophages and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis, and Paracoccidioides brasiliensis infections. Considering its potential use as an immunotherapeutic molecule, this study was designed to produce high-level of active Artin M, in E. coli system, for both research and pharmaceutical purposes. The Artin M cDNA was inserted in pET29a+ vector and expressed in E. coli strains BL21(DE3), BL21(DE3) Codon Plus-RP and BL21(DE3) Rosetta. The cells were induced under the given optimized conditions, 0.5 mM IPTG at 20o C for 19 h, and resulted in the accumulation of large amounts of soluble Artin M in the three E. coli strains. The recombinant Artin M proteins (rArtin Ms) were functionally active, as shown by one-step purification through immobilized D-mannose affinity chromatography, which resulted in 22.4 mg/l, 17.9 mg/l and 14.1 mg/l of purified Artin M for BL21(DE3) Codon Plus-RP, BL21(DE3), BL21(DE3) Rosetta, respectively. The apparent molecular mass of the native Artin M and rArtin Ms were estimated by gel filtration and showed that the native protein is a tetramer and the rArtim Ms are monomers. The exact molecular weight and the identity were verified with mass spectrometry analysis and N-terminal sequencing by automated Edman degradation. To verify whether the soluble rArtin Ms have folded in a similar way to the native Artin M, we characterized the rArtin Ms by circular dichroism (CD) and fluorescence spectroscopy. The CD spectrum of rArtin Ms indicated that the global fold comprised -sheet strucwww.elsevier.com/locate/nbt S293
3.2.32 The assessment of biochemical parameters with grapevine in correlation with organic fertilization of the soil C. Bejan National Institute of Research and Development for Biotechnology in Horticulture Stefanesti, Arges County, Romania
Maintaining and soil fertility improvement are important aims for grapevine growing, taking into account that, in most situations, this species are grown on poor nutritive soils. In these circumstances, efficient systems of fertilization, based on maximum utilization of organically fertilizers, are of present interest. The goal of this study is to explore the correlations between the type of organic fertilizer and grapevine performances expressed in accumulation of biochemical compounds which are involved in plant growth and development. The study was conducted with two grapevine cultivars — Cabernet Sauvignon and Zweigelt — used for high quality red wines production, from vineyards belonging to the National Research and Development Institute for Biotechnology in Horticulture Stefanesti-Arges. Our results revealed the positive effect of soil manuring with organic fertilizer upon the biosyntheses processes in leaves over the vegetative period of main primary metabolites (soluble glucides, chlorophyll pigments and carotenes) and secondary metabolites (proteins, polyphenols, auxins). As a result, woody materials collected from the organic fertilized areas proved to have a better maturation at the beginning of the winter repose period. This was correlated with a better buds viability from yield canes. doi:10.1016/j.nbt.2009.06.665
3.2.33 Remediation of zinc and copper by transgenic tobacco plants with metal transporter genes from Neurospora crassa S. Eapen ∗ , P. Dixit, S. Singh, P. Maruthi Mohan, V. Ramachandran Bhabha Atomic Research Centre, Mumbai, India
Zinc and copper are essential elements for growth and development of plants and uptake of these mineral elements by plants is a part of mineral nutrition. Plants have homeostatic mechanisms to maintain the correct concentrations of these metals in their cellular compartments. It is possible to enhance the uptake of these metals by introducing efficient genes for metal uptake from other organisms into plants. Plants with enhanced ability for metal uptake can be developed by introduction of metal transporter genes from other sources. Development of transgenic plants with high efficiency of metal uptake will help in removing toxic levels of these metals from contaminated soils and solutions. With this objective, two metal transporter genes, namely a zinc transporter gene and a copper transporter gene from Neurospora crassa were cloned and individually introduced into plant expression vec-
ABSTRACTS
tor pCAMBIA 1301 having hph as a selectable marker and uidA as a reporter gene and transgenic tobacco plants developed by Agrobacterium mediated gene transfer. The transgenic nature of plants was confirmed by PCR and Southern blot hybridization, while expression of transgene was confirmed by reverse transcription PCR. Transgenic plants with zinc transporter gene, when grown in media supplemented with radiolabelled ZnCl2 , showed enhanced accumulation of zinc in roots (11 times). Uptake of copper was studied in transgenic plants with copper transporter gene and the transgenic plants could take up more copper (2 times) compared to control plants. The present studies have shown that it is possible to develop transgenic plants with improved efficiency for remediation of metals like zinc and copper. doi:10.1016/j.nbt.2009.06.666
3.2.34 High-level of expression, purification and characterization of active Artin M Lectin in different E. Coli hosts M.C.D.S. Pranchevicius 1,∗ , L. Oliveira 2 , N.C. Avanci 3 , J.C. Rosa 4 , A.C. Quiapim 3 , V. Cossalter 3 , M.S. Brito 3 , E.A. Aragão 5 , T.L. Ferreira 5 , M.C. Roque Barreira 2 , M.H.S. Goldman 3 1
Department of Biology, FFCLRP/USP, School of Medicine, UFT/TO, 1, Brazil Department of Molecular and Cell Biology, FMRP/USP, Brazil 3 Department of Biology, FFCLRP/USP, Brazil 4 Department of Molecular and Cell Biology, FMRP/USP, Protein Chemistry Center, FMRP/USP, Brazil 5 Department of Chemistry, FFCLRP/USP, Brazil 2
Artin M (KM+) is a D-mannose-binding lectin from Artocarpus integrifolia seeds that induces interleukin (IL)-12 production by macrophages and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis, and Paracoccidioides brasiliensis infections. Considering its potential use as an immunotherapeutic molecule, this study was designed to produce high-level of active Artin M, in E. coli system, for both research and pharmaceutical purposes. The Artin M cDNA was inserted in pET29a+ vector and expressed in E. coli strains BL21(DE3), BL21(DE3) Codon Plus-RP and BL21(DE3) Rosetta. The cells were induced under the given optimized conditions, 0.5 mM IPTG at 20o C for 19 h, and resulted in the accumulation of large amounts of soluble Artin M in the three E. coli strains. The recombinant Artin M proteins (rArtin Ms) were functionally active, as shown by one-step purification through immobilized D-mannose affinity chromatography, which resulted in 22.4 mg/l, 17.9 mg/l and 14.1 mg/l of purified Artin M for BL21(DE3) Codon Plus-RP, BL21(DE3), BL21(DE3) Rosetta, respectively. The apparent molecular mass of the native Artin M and rArtin Ms were estimated by gel filtration and showed that the native protein is a tetramer and the rArtim Ms are monomers. The exact molecular weight and the identity were verified with mass spectrometry analysis and N-terminal sequencing by automated Edman degradation. To verify whether the soluble rArtin Ms have folded in a similar way to the native Artin M, we characterized the rArtin Ms by circular dichroism (CD) and fluorescence spectroscopy. The CD spectrum of rArtin Ms indicated that the global fold comprised -sheet strucwww.elsevier.com/locate/nbt S293