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Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Zhou Z, He H, Ma L, Yu X, Mi Q, Pang J, Tang G, Liu B - PLoS ONE (2015)

Bottom Line: Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression.The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV).Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

View Article: PubMed Central - PubMed

Affiliation: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.

ABSTRACT
Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic tree of GmCnx1 gene and alignment of amino acid residues from different species.(A) Phylogenetic tree constructed by multiple sequence alignments of the Glycine max Cnx1 and those of other Cnx1 proteins. The sequences used are: Medicago truncatula Cnx1, Theobroma cacao Cnx1, Morus notabilis Cnx1, Malus domestica Cnx1, Prunus mume Cnx1, Arabidopsis thaliana Cnx1,Cucumis melo Cnx1,Hordeum vulgare Cnx1, Zea mays Cnx1, Triticum urartu Cnx1, Aegilops tauschii Cnx1, Micromonas sp Cnx1 and Micromonas pusilla Cnx1(Accession nos AET00019, EOY33170, EXB53803, XP_008385638, XP_008231326, EEH56197, XP_008451034, AAF73075, ABB30174, EMS55337, EMT03164, ACO61591 and AED92917.1, respectively). (B) Through the analysis of two Moco domains and alignment of amino acid residues, encoding protein sequences of GmCnx1, Medicago truncatula Cnx1, Cucumis melo Cnx1, Theobroma cacao Cnx1 and Arabidopsis thaliana Cnx1 had high sequence similarity.
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pone.0124273.g001: Phylogenetic tree of GmCnx1 gene and alignment of amino acid residues from different species.(A) Phylogenetic tree constructed by multiple sequence alignments of the Glycine max Cnx1 and those of other Cnx1 proteins. The sequences used are: Medicago truncatula Cnx1, Theobroma cacao Cnx1, Morus notabilis Cnx1, Malus domestica Cnx1, Prunus mume Cnx1, Arabidopsis thaliana Cnx1,Cucumis melo Cnx1,Hordeum vulgare Cnx1, Zea mays Cnx1, Triticum urartu Cnx1, Aegilops tauschii Cnx1, Micromonas sp Cnx1 and Micromonas pusilla Cnx1(Accession nos AET00019, EOY33170, EXB53803, XP_008385638, XP_008231326, EEH56197, XP_008451034, AAF73075, ABB30174, EMS55337, EMT03164, ACO61591 and AED92917.1, respectively). (B) Through the analysis of two Moco domains and alignment of amino acid residues, encoding protein sequences of GmCnx1, Medicago truncatula Cnx1, Cucumis melo Cnx1, Theobroma cacao Cnx1 and Arabidopsis thaliana Cnx1 had high sequence similarity.

Mentions: The phylogenetic tree (Fig 1a) was constructed by Mega software version 5.0 using the neighbor-joining method [27].As expected, Glycine maxCnx1 and Medicago truncatulaCnx1both belonging to Legume, Papillionoideae, had the closest genetic relationship. Two Moco domains in protein coded by Glycine max Cnx1 gene were found by online SMART software of EMBL (http://smart.embl-heidelberg.de/). The locations of the two domains were confirmed by DNAMAN software. A comparison of amino acid sequences (Fig 1b) revealed that Glycine max Cnx1 shared high sequence similarity with the Cnx1 gene of Medicago truncatula, Cucumis melo, Theobroma cacao and Arabidopsis thaliana. The sequence similarity of the proteins coded by the Cnx1 genes was higher than 60% and amino acid sequences at theCnx1 conserved domains in particular were highly conserved.


Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Zhou Z, He H, Ma L, Yu X, Mi Q, Pang J, Tang G, Liu B - PLoS ONE (2015)

Phylogenetic tree of GmCnx1 gene and alignment of amino acid residues from different species.(A) Phylogenetic tree constructed by multiple sequence alignments of the Glycine max Cnx1 and those of other Cnx1 proteins. The sequences used are: Medicago truncatula Cnx1, Theobroma cacao Cnx1, Morus notabilis Cnx1, Malus domestica Cnx1, Prunus mume Cnx1, Arabidopsis thaliana Cnx1,Cucumis melo Cnx1,Hordeum vulgare Cnx1, Zea mays Cnx1, Triticum urartu Cnx1, Aegilops tauschii Cnx1, Micromonas sp Cnx1 and Micromonas pusilla Cnx1(Accession nos AET00019, EOY33170, EXB53803, XP_008385638, XP_008231326, EEH56197, XP_008451034, AAF73075, ABB30174, EMS55337, EMT03164, ACO61591 and AED92917.1, respectively). (B) Through the analysis of two Moco domains and alignment of amino acid residues, encoding protein sequences of GmCnx1, Medicago truncatula Cnx1, Cucumis melo Cnx1, Theobroma cacao Cnx1 and Arabidopsis thaliana Cnx1 had high sequence similarity.
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Related In: Results  -  Collection

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pone.0124273.g001: Phylogenetic tree of GmCnx1 gene and alignment of amino acid residues from different species.(A) Phylogenetic tree constructed by multiple sequence alignments of the Glycine max Cnx1 and those of other Cnx1 proteins. The sequences used are: Medicago truncatula Cnx1, Theobroma cacao Cnx1, Morus notabilis Cnx1, Malus domestica Cnx1, Prunus mume Cnx1, Arabidopsis thaliana Cnx1,Cucumis melo Cnx1,Hordeum vulgare Cnx1, Zea mays Cnx1, Triticum urartu Cnx1, Aegilops tauschii Cnx1, Micromonas sp Cnx1 and Micromonas pusilla Cnx1(Accession nos AET00019, EOY33170, EXB53803, XP_008385638, XP_008231326, EEH56197, XP_008451034, AAF73075, ABB30174, EMS55337, EMT03164, ACO61591 and AED92917.1, respectively). (B) Through the analysis of two Moco domains and alignment of amino acid residues, encoding protein sequences of GmCnx1, Medicago truncatula Cnx1, Cucumis melo Cnx1, Theobroma cacao Cnx1 and Arabidopsis thaliana Cnx1 had high sequence similarity.
Mentions: The phylogenetic tree (Fig 1a) was constructed by Mega software version 5.0 using the neighbor-joining method [27].As expected, Glycine maxCnx1 and Medicago truncatulaCnx1both belonging to Legume, Papillionoideae, had the closest genetic relationship. Two Moco domains in protein coded by Glycine max Cnx1 gene were found by online SMART software of EMBL (http://smart.embl-heidelberg.de/). The locations of the two domains were confirmed by DNAMAN software. A comparison of amino acid sequences (Fig 1b) revealed that Glycine max Cnx1 shared high sequence similarity with the Cnx1 gene of Medicago truncatula, Cucumis melo, Theobroma cacao and Arabidopsis thaliana. The sequence similarity of the proteins coded by the Cnx1 genes was higher than 60% and amino acid sequences at theCnx1 conserved domains in particular were highly conserved.

Bottom Line: Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression.The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV).Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

View Article: PubMed Central - PubMed

Affiliation: Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.

ABSTRACT
Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

No MeSH data available.


Related in: MedlinePlus