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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

Recombination expression vector of pB7FWG2-GmCnx1 and determination of GmCnx1 transgenic soybean plants.(A) Vector contained selective maker gene (bar) coded phosphinothricin acetyltransferase (PAT) and showed resistance to the herbicide phosphinothricin, green fluorescent protein gene (Egfp) and GmCnx1 gene. LB, left border; RB, right border; p35s, promoter; T35S, terminator. (B) T1 transgenic lines were confirmed by coating leaves with Phosphinothricin. (C)T1 transgenic lines were confirmed by bar protein quick dip strip. (D) T1 transgenic lines were confirmed by PCR. (E) Southern blot analysis of transgene copy number in T1 transgenic soybean and WT. Genomic DNA and plasmid DNA was digested with restriction enzyme EcoR Iand HindⅢ. The probe was used for GmCnx1. M, DL2000 marker; +, positive control (plasmid DNA); WT, negative control, non-transgenic plants; 0, blank. L1, L2, L3, L4, L7, L10, L17, L18, L22 and L26 represent the GmCnx1 transgenic line numbers.
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pone.0124273.g002: Recombination expression vector of pB7FWG2-GmCnx1 and determination of GmCnx1 transgenic soybean plants.(A) Vector contained selective maker gene (bar) coded phosphinothricin acetyltransferase (PAT) and showed resistance to the herbicide phosphinothricin, green fluorescent protein gene (Egfp) and GmCnx1 gene. LB, left border; RB, right border; p35s, promoter; T35S, terminator. (B) T1 transgenic lines were confirmed by coating leaves with Phosphinothricin. (C)T1 transgenic lines were confirmed by bar protein quick dip strip. (D) T1 transgenic lines were confirmed by PCR. (E) Southern blot analysis of transgene copy number in T1 transgenic soybean and WT. Genomic DNA and plasmid DNA was digested with restriction enzyme EcoR Iand HindⅢ. The probe was used for GmCnx1. M, DL2000 marker; +, positive control (plasmid DNA); WT, negative control, non-transgenic plants; 0, blank. L1, L2, L3, L4, L7, L10, L17, L18, L22 and L26 represent the GmCnx1 transgenic line numbers.

Mentions: The pB7FWG2-GmCnx1 vector (Fig 2a) was constructed and introduced into soybean via Agrobacterium tumefaciens-mediated transformation (S1 Fig). A total of twenty seven independent putative soybean transgenic lines were generated. Ten positive lines 1, 2, 3, 4, 7, 10, 17, 18, 22 and 26 which had sufficient seeds were selected for subsequent experiments.T1putative transformants were identified by coating leaves with Phosphinothricin (Fig 2b),bar protein quick dip strip (Fig 2c) and PCR (Fig 2d). Southern blot (Fig 2e) of T1 generation revealed successful integration of the target gene into the soybean genome, which showed stable overexpression of GmCnx1. Importantly, the transgene construct was a single copy in line 2, 3, 4 and 7.


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)

Recombination expression vector of pB7FWG2-GmCnx1 and determination of GmCnx1 transgenic soybean plants.(A) Vector contained selective maker gene (bar) coded phosphinothricin acetyltransferase (PAT) and showed resistance to the herbicide phosphinothricin, green fluorescent protein gene (Egfp) and GmCnx1 gene. LB, left border; RB, right border; p35s, promoter; T35S, terminator. (B) T1 transgenic lines were confirmed by coating leaves with Phosphinothricin. (C)T1 transgenic lines were confirmed by bar protein quick dip strip. (D) T1 transgenic lines were confirmed by PCR. (E) Southern blot analysis of transgene copy number in T1 transgenic soybean and WT. Genomic DNA and plasmid DNA was digested with restriction enzyme EcoR Iand HindⅢ. The probe was used for GmCnx1. M, DL2000 marker; +, positive control (plasmid DNA); WT, negative control, non-transgenic plants; 0, blank. L1, L2, L3, L4, L7, L10, L17, L18, L22 and L26 represent the GmCnx1 transgenic line numbers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401665&req=5

pone.0124273.g002: Recombination expression vector of pB7FWG2-GmCnx1 and determination of GmCnx1 transgenic soybean plants.(A) Vector contained selective maker gene (bar) coded phosphinothricin acetyltransferase (PAT) and showed resistance to the herbicide phosphinothricin, green fluorescent protein gene (Egfp) and GmCnx1 gene. LB, left border; RB, right border; p35s, promoter; T35S, terminator. (B) T1 transgenic lines were confirmed by coating leaves with Phosphinothricin. (C)T1 transgenic lines were confirmed by bar protein quick dip strip. (D) T1 transgenic lines were confirmed by PCR. (E) Southern blot analysis of transgene copy number in T1 transgenic soybean and WT. Genomic DNA and plasmid DNA was digested with restriction enzyme EcoR Iand HindⅢ. The probe was used for GmCnx1. M, DL2000 marker; +, positive control (plasmid DNA); WT, negative control, non-transgenic plants; 0, blank. L1, L2, L3, L4, L7, L10, L17, L18, L22 and L26 represent the GmCnx1 transgenic line numbers.
Mentions: The pB7FWG2-GmCnx1 vector (Fig 2a) was constructed and introduced into soybean via Agrobacterium tumefaciens-mediated transformation (S1 Fig). A total of twenty seven independent putative soybean transgenic lines were generated. Ten positive lines 1, 2, 3, 4, 7, 10, 17, 18, 22 and 26 which had sufficient seeds were selected for subsequent experiments.T1putative transformants were identified by coating leaves with Phosphinothricin (Fig 2b),bar protein quick dip strip (Fig 2c) and PCR (Fig 2d). Southern blot (Fig 2e) of T1 generation revealed successful integration of the target gene into the soybean genome, which showed stable overexpression of GmCnx1. Importantly, the transgene construct was a single copy in line 2, 3, 4 and 7.

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