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Sergey Dolgov - One of the best experts on this subject based on the ideXlab platform.
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Tomatoes Expressing Thaumatin Ii Retain Their Sweet Taste After Salting And Pickling Processing
Journal of the science of food and agriculture, 2021Co-Authors: Aleksey Firsov, Lyubov Shaloiko, Oleg Kozlov, Alexander Vainstein, Sergey DolgovAbstract:BACKGROUND Thaumatin II, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), shows promise as a zero-calorie sweetener for use in the food and pharmaceutical industries and for improving the taste of fruit. RESULTS We report on the stability of thaumatin in salted and pickled tomatoes, as well as on the effect of thaumatin on the taste quality of processed tomatoes. Fruit of tomato cv. Yalf, transformed with the thaumatin II gene were salted and pickled and then stored for 6 months. Western blot analysis showed relative thaumatin II stability at salting; its content in processed fruits was 62-83% of the initial level depending in the studied line. In pickled tomatoes, thaumatin II content was decreased by up to 25% of the initial amount. Both salted and pickled tomatoes had a sweet taste with a typical thaumatin aftertaste. Salted tomatoes were characterized as being sweeter than pickled tomatoes. The overall taste of pickled tomatoes was rated by panellists as significantly better compared to that of salted or non-processed ones. CONCLUSION In the present study, we have shown that tomatoes expressing supersweet protein thaumatin II can be used for processing under mild conditions, including salting and pickling. © 2021 Society of Chemical Industry.
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Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Plant Cell Tissue and Organ Culture (PCTOC), 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey DolgovAbstract:Despite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used. The strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.
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Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Plant Cell Tissue and Organ Culture (PCTOC), 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey DolgovAbstract:Key messageThe strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.AbstractDespite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used.
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Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter
Frontiers Media S.A., 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey Dolgov, Tatiana MitiouchkinaAbstract:The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene. The thaumatin II gene from the tropical plant Thaumatococcus daniellii, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein
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Transgenic Plants as Producers of Supersweet Protein Thaumatin II
Sweeteners, 2018Co-Authors: A. P. Firsov, Alexander Pushin, Sergey DolgovAbstract:Thaumatin II is a supersweet protein derived from the West-African plant Thaumatococcus daniellii Benth. It is a perspective low-calorie sugar substitute for food and pharmaceutical industries. Because of the limitations of its natural sources, obtaining recombinant thaumatin using plant-based expression systems is a promising field of research. This review summarizes many years of research focusing on the physicochemical properties of thaumatins I and II, their roles in plants as pathogenesis-related proteins, and the specific characteristics of their taste perception. A special attention is paid to the detailed description of the studies on obtaining transgenic plants that have been transformed with thaumatin II gene in order to improve their agronomic and consumer properties as well as to obtain recombinant thaumatin for industrial use. Further directions of the research focusing on such areas as obtaining transgenic plants to produce recombinant thaumatin and developing the technologies for its isolation and purification are discussed.
Alexander Pushin - One of the best experts on this subject based on the ideXlab platform.
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Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Plant Cell Tissue and Organ Culture (PCTOC), 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey DolgovAbstract:Despite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used. The strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.
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Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Plant Cell Tissue and Organ Culture (PCTOC), 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey DolgovAbstract:Key messageThe strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.AbstractDespite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used.
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Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter
Frontiers Media S.A., 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey Dolgov, Tatiana MitiouchkinaAbstract:The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene. The thaumatin II gene from the tropical plant Thaumatococcus daniellii, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein
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Transgenic Plants as Producers of Supersweet Protein Thaumatin II
Sweeteners, 2018Co-Authors: A. P. Firsov, Alexander Pushin, Sergey DolgovAbstract:Thaumatin II is a supersweet protein derived from the West-African plant Thaumatococcus daniellii Benth. It is a perspective low-calorie sugar substitute for food and pharmaceutical industries. Because of the limitations of its natural sources, obtaining recombinant thaumatin using plant-based expression systems is a promising field of research. This review summarizes many years of research focusing on the physicochemical properties of thaumatins I and II, their roles in plants as pathogenesis-related proteins, and the specific characteristics of their taste perception. A special attention is paid to the detailed description of the studies on obtaining transgenic plants that have been transformed with thaumatin II gene in order to improve their agronomic and consumer properties as well as to obtain recombinant thaumatin for industrial use. Further directions of the research focusing on such areas as obtaining transgenic plants to produce recombinant thaumatin and developing the technologies for its isolation and purification are discussed.
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Transgenic tomato plants as supersweet protein thaumatin II producers
Applied Biochemistry and Microbiology, 2012Co-Authors: A. P. Firsov, Alexander Pushin, I. V. Korneeva, Sergey DolgovAbstract:Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.
Vadim Timerbaev - One of the best experts on this subject based on the ideXlab platform.
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Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Plant Cell Tissue and Organ Culture (PCTOC), 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey DolgovAbstract:Despite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used. The strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.
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Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Plant Cell Tissue and Organ Culture (PCTOC), 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey DolgovAbstract:Key messageThe strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.AbstractDespite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used.
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Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter
Frontiers Media S.A., 2019Co-Authors: Vadim Timerbaev, Alexander Pushin, Sergey Dolgov, Tatiana MitiouchkinaAbstract:The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene. The thaumatin II gene from the tropical plant Thaumatococcus daniellii, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein
Bernard Fritig - One of the best experts on this subject based on the ideXlab platform.
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thaumatin like pathogenesis related proteins are antifungal
Plant Science, 1992Co-Authors: Alison J Vigers, Michel Legrand, Sabine Wiedemann, Walden K Roberts, Claude P Selitrennikoff, Bernard FritigAbstract:Tobacco pathogenesis-related (PR) proteins of group 5, namely PR-S and osmotin, were shown to be serologically related to zeamatin, an antifungal protein of maize seeds. These PR proteins, dubbed thaumatin-like because they show sequence homology with thaumatin, a sweet-tasting protein from fruits of Thaumatococcus daniellii, were demonstrated to have a direct antifungal activity, with specificity for different fungal species. Osmotin was particularly effective in inhibiting the growth of Candida albicans, Neurospora crassa and Trichoderma reesei. PR-S had no detectable activity against these fungi but was found the most potent antifungal protein against the plant pathogen Cercospora beticola. Osmotin caused rapid bursting of the hyphal tips of N. crassa, suggesting that tobacco thaumatin-like PR proteins are antifungal by a membrane permeabilization mechanism similar to that demonstrated previously for zeamatin. These results confirm and extend observations made by Woloshuk et al. [1] who showed that osmotin and a related protein from tomato had antifungal activity against the phytopathogen, Phytophthora infestans.
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Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco as osmotin
Physiological and Molecular Plant Pathology, 1991Co-Authors: Annick Stintzi, Thierry Heitz, Serge Kauffmann, Michel Legrand, Bernard FritigAbstract:A basic PR protein was isolated and characterized from Samsun NN tobacco leaves infected with tobacco mosaic virus. The protein is serologically-related to the pathogenesis-related (PR) proteins R and S, the thaumatin-like (TL) proteins so-called because of their high level of homology with thaumatin, a sweet-tasting protein from the West African shrub Thaumatococcus daniellii Benth. Its amino acid composition and its NH 2 -terminal sequence indicate that this PR protein is in fact osmotin, a protein known to accumulate in tobacco cells in response to osmotic stress. A specific serum was obtained and used in immunoblotting experiments to study the serological relationships of TL-proteins of tobacco and to compare the induction of osmotin and acidic PR proteins R and S during the hypersensitive reaction of tobacco to tobacco mosaic virus.
Augustine C. Okenmuo - One of the best experts on this subject based on the ideXlab platform.
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Fruit Extract of Thaumatococcus daniellii Reduces Oxidative Stress in Rats
The FASEB Journal, 2017Co-Authors: Franklyn Nonso Iheagwam, Shalom Nwodo Chinedu, Opeyemi Christianah Emiloju, Augustine C. OkenmuoAbstract:Thaumatococcus daniellii (Benn.) Benth is a tropical rhizomatous herb recognized as the natural source of the protein sweetner, thaumatin. Thaumatin is extracted from the fruit arils, which constit...