Cloning of Glutathione Peroxidase PmGPX6 Gene from Pinus massoniana and the Study on Drought Tolerance of Transgenic Arabidopsis thaliana

CAI Qiong; DING Gui-jie; WEN Xiao-peng

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Cloning of Glutathione Peroxidase PmGPX6 Gene from Pinus massoniana and the Study on Drought Tolerance of Transgenic Arabidopsis thaliana

1.
Guizhou Institute for Forest Resources & Environment, Guizhou University, Guiyang 550025, Guizhou, China
2.
College of Forestry, Guizhou University, Guiyang 550025, Guizhou, China
3.
Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, Guizhou, China

Received Date: 2015-09-29

Abstract

[Objective] To clone the glutathione peroxidase gene from Pinus massoniana and evaluate its gene functions. [Methods] The gene was cloned using RACE methods. Quantitative real-time PCR was used to analyze the gene expression in P. massoniana under drought stress. Transgenic Arabidopsis thaliana lines were obtained by dipping flowering plants. The phenotypes and root developments of wild-type and transgenic A. thaliana were evaluated. The activity of green fluorescence protein on root in transgenic A. thaliana plants was studied using fluorescence microscopy. [Results] The designated PmGPX6 was 871 bp in length with an open reading frame (513 bp), capable of encoding a predicted protein of 170 amino acids. The sequencing analysis indicated that the deduced amino acids shared 95% of identity with PtCBL from Pinus tabulaeformis. The expression levels of PmGPX6 were higher in roots than in stems and leaves, and the PmGPX6 expression increased in the initial 15 days, and then decreased under drought stress. No differences in the phenotypes and root lengths were observed between the wild-type and transgenic A. thaliana under normal conditions containing 0% PEG 6000. Nevertheless, the root lengths of transgenic A. thaliana were longer than those of the wild-type lines under drought stress. Bright green fluorescence was observed in transgenic A. thaliana under fluorescence microscopy, indicating that PmGPX6 is expressed efficiently. [Conclusion] These findings indicate that the PmGPX6 gene may play an important role under drought stress.
- Pinus massoniana,
- glutathione peroxidase,
- transgenic Arabidopsis thaliana,
- drought tolerance

References

[1]	Bela K, Horváth E, Gallé á, et al. Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses [J]. J Plant Physiol, 2015, 176 (1):192-201.
[2]	Ahmad P, Jaleel C A, Salem M A, et al. Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress [J]. Crit Rev Biotechnol, 2010, 30 (3): 161-175.
[3]	Bérczi A, Moller I M. Redox enzymes in the plant plasma membrane and their possible roles [J]. Plant Cell Environ, 2000, 23 (12): 1287-1302.
[4]	Rodriguez Milla M A, Maurer A, Rodriguez Huete A, et al. Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways [J]. Plant J, 2003, 36 (5): 602-615.
[5]	Chang C C, Slesak I, Jordá L, et al. Arabidopsis chloroplastic glutathione peroxidases play a role in cross talk between photooxidative stress and immune responses [J]. Plant Physiol, 2009, 150 (2): 670-683.
[6]	Miao Y C, Lv D, Wang P C, et al. An Arabidopsis glutathione peroxidase functions as both a redox-transducer and a scavenger in abscisic acid and drought stress responses [J]. The Plant Cell, 2006, 18 (10): 2749-2766.
[7]	丁贵杰, 周志春, 王章荣, 等. 马尾松纸浆用材林培育与利用[M]. 北京:中国林业出版社. 2006.
[8]	韩文萍,丁贵杰,鲍斌.不同种源马尾松对干旱胁迫的生理生态响应[J].中南林业科技大学学报, 2012, 32(5):25-29.
[9]	胡晓健,欧阳献,喻方圆.干旱胁迫对不同种源马尾松苗木生长及生物量的影响[J].江西农业大学学报, 2010, 32(3):510-516.
[10]	施积炎, 丁贵杰, 袁小凤. 不同家系马尾松苗木水分参数的研究[J]. 林业科学, 2004, 40 (3): 51-55.
[11]	施积炎, 丁贵杰, 袁小凤. 不同家系马尾松维持水分平衡能力及综合评价[J]. 上海交通大学学报:农业科学版, 2004, 22 (2): 143-148.
[12]	袁小凤, 丁贵杰, 施积炎. 土壤干旱胁迫对不同品系马尾松水分利用效率的影响[J]. 浙江师范大学学报:自然科学版, 2008, 31 (3): 328-331.
[13]	Fan F H, Cui B W, Zhang T, et al. The temporal transcriptomic response of Pinus massoniana seedlings to phosphorus deficiency [J]. PLoS ONE, 2014, 9: e105068. doi:10.1371/journal.pone.0105068.
[14]	Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR [J]. Nucleic Acids Res, 2001, 29 (9): 2002-2007.
[15]	崔武, 刘炜, 郑海燕. 将外源DNA直接导入根瘤土壤农杆菌的新方法[J]. 植物生理学通讯, 1995, 31(1):48-49.
[16]	Clough S J, Bent A F. Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal, 1998, 16 (6): 735-743.
[17]	Noctor G, Gomez L, Vanacker H, et al. Interactions between biosynthesis, compartmentation, and transport in the control of glutathione homeostasis and signaling [J]. Journal of Experimental Botany, 2002, 53 (372):1283-1304.
[18]	Li T, Yang X D, Liu J Y. Tissue and induction expression profiles of rice phospholipid hydroperoxide glutathione peroxidase at protein level [J]. Prog Biochem Biophys, 2009, 36 (1): 77-82.
[19]	张丽丽, 徐碧玉, 刘菊华, 等. 香蕉谷胱甘肽过氧化物酶基因MaGPX的克隆和表达分析[J]. 园艺学报,2012,39 (8):1471-1481.
[20]	Nahar K, Hasanuzzaman M, Alam M M, et al. Glutathione-induced drought stress tolerance in mung bean: coordinated roles of the antioxidant defence and methylgolyoxal detoxification systems [J]. AoB Plants, 2015, 7 (1). pii: plv069. doi: 10.1093/aobpla/plv069.
[21]	Passaia G, Margis-Pinheiro M. Glutathione peroxidases as redox sensor proteins in plant cells [J]. Plant Sci, 2015, 234: 22-26. doi: 10.1016/j.plantsci.2015.01.017. Epub 2015 Feb 7.
[22]	周立敬,周宜君,高飞,等. 拟南芥谷胱甘肽过氧化物酶的生物信息学分析[J].中央民族大学学报:自然科学版, 2010, 19 (2): 11-17.
[23]	马亭亭, 周宜君, 高飞, 等. 盐芥谷胱甘肽过氧化物酶基因(THGPX6)的克隆及表达分析[J]. 植物遗传资源学报, 2012, 13 (2): 252-258.
[24]	肖蓉,罗慧珍,张小娟,等. 干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因(ZjGPX)的差异表达及功能分析[J]. 中国农业科学, 2015, 48 (14): 2806-2817.
[25]	Wang R G, Chen S L, Zhou X Y, et al. Ionic homeostasis and reactive oxygen species control in leaves and xylem sap of two poplars subjected to NaCl stress [J]. Tree Physiol, 2008, 28 (6): 947-957.
[26]	王菲菲, 丁明全, 邓澍荣, 等. 胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析[J]. 基因组学与应用生物学, 2012, 31 (3): 231-239.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Cloning of Glutathione Peroxidase PmGPX6 Gene from Pinus massoniana and the Study on Drought Tolerance of Transgenic Arabidopsis thaliana

1. Guizhou Institute for Forest Resources & Environment, Guizhou University, Guiyang 550025, Guizhou, China

2. College of Forestry, Guizhou University, Guiyang 550025, Guizhou, China

3. Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, Guizhou, China

Received Date: 2015-09-29

Abstract: [Objective] To clone the glutathione peroxidase gene from Pinus massoniana and evaluate its gene functions. [Methods] The gene was cloned using RACE methods. Quantitative real-time PCR was used to analyze the gene expression in P. massoniana under drought stress. Transgenic Arabidopsis thaliana lines were obtained by dipping flowering plants. The phenotypes and root developments of wild-type and transgenic A. thaliana were evaluated. The activity of green fluorescence protein on root in transgenic A. thaliana plants was studied using fluorescence microscopy. [Results] The designated PmGPX6 was 871 bp in length with an open reading frame (513 bp), capable of encoding a predicted protein of 170 amino acids. The sequencing analysis indicated that the deduced amino acids shared 95% of identity with PtCBL from Pinus tabulaeformis. The expression levels of PmGPX6 were higher in roots than in stems and leaves, and the PmGPX6 expression increased in the initial 15 days, and then decreased under drought stress. No differences in the phenotypes and root lengths were observed between the wild-type and transgenic A. thaliana under normal conditions containing 0% PEG 6000. Nevertheless, the root lengths of transgenic A. thaliana were longer than those of the wild-type lines under drought stress. Bright green fluorescence was observed in transgenic A. thaliana under fluorescence microscopy, indicating that PmGPX6 is expressed efficiently. [Conclusion] These findings indicate that the PmGPX6 gene may play an important role under drought stress.

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[1]	Bela K, Horváth E, Gallé á, et al. Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses [J]. J Plant Physiol, 2015, 176 (1):192-201.
[2]	Ahmad P, Jaleel C A, Salem M A, et al. Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress [J]. Crit Rev Biotechnol, 2010, 30 (3): 161-175.
[3]	Bérczi A, Moller I M. Redox enzymes in the plant plasma membrane and their possible roles [J]. Plant Cell Environ, 2000, 23 (12): 1287-1302.
[4]	Rodriguez Milla M A, Maurer A, Rodriguez Huete A, et al. Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways [J]. Plant J, 2003, 36 (5): 602-615.
[5]	Chang C C, Slesak I, Jordá L, et al. Arabidopsis chloroplastic glutathione peroxidases play a role in cross talk between photooxidative stress and immune responses [J]. Plant Physiol, 2009, 150 (2): 670-683.
[6]	Miao Y C, Lv D, Wang P C, et al. An Arabidopsis glutathione peroxidase functions as both a redox-transducer and a scavenger in abscisic acid and drought stress responses [J]. The Plant Cell, 2006, 18 (10): 2749-2766.
[7]	丁贵杰, 周志春, 王章荣, 等. 马尾松纸浆用材林培育与利用[M]. 北京:中国林业出版社. 2006.
[8]	韩文萍,丁贵杰,鲍斌.不同种源马尾松对干旱胁迫的生理生态响应[J].中南林业科技大学学报, 2012, 32(5):25-29.
[9]	胡晓健,欧阳献,喻方圆.干旱胁迫对不同种源马尾松苗木生长及生物量的影响[J].江西农业大学学报, 2010, 32(3):510-516.
[10]	施积炎, 丁贵杰, 袁小凤. 不同家系马尾松苗木水分参数的研究[J]. 林业科学, 2004, 40 (3): 51-55.
[11]	施积炎, 丁贵杰, 袁小凤. 不同家系马尾松维持水分平衡能力及综合评价[J]. 上海交通大学学报:农业科学版, 2004, 22 (2): 143-148.
[12]	袁小凤, 丁贵杰, 施积炎. 土壤干旱胁迫对不同品系马尾松水分利用效率的影响[J]. 浙江师范大学学报:自然科学版, 2008, 31 (3): 328-331.
[13]	Fan F H, Cui B W, Zhang T, et al. The temporal transcriptomic response of Pinus massoniana seedlings to phosphorus deficiency [J]. PLoS ONE, 2014, 9: e105068. doi:10.1371/journal.pone.0105068.
[14]	Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR [J]. Nucleic Acids Res, 2001, 29 (9): 2002-2007.
[15]	崔武, 刘炜, 郑海燕. 将外源DNA直接导入根瘤土壤农杆菌的新方法[J]. 植物生理学通讯, 1995, 31(1):48-49.
[16]	Clough S J, Bent A F. Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal, 1998, 16 (6): 735-743.
[17]	Noctor G, Gomez L, Vanacker H, et al. Interactions between biosynthesis, compartmentation, and transport in the control of glutathione homeostasis and signaling [J]. Journal of Experimental Botany, 2002, 53 (372):1283-1304.
[18]	Li T, Yang X D, Liu J Y. Tissue and induction expression profiles of rice phospholipid hydroperoxide glutathione peroxidase at protein level [J]. Prog Biochem Biophys, 2009, 36 (1): 77-82.
[19]	张丽丽, 徐碧玉, 刘菊华, 等. 香蕉谷胱甘肽过氧化物酶基因MaGPX的克隆和表达分析[J]. 园艺学报,2012,39 (8):1471-1481.
[20]	Nahar K, Hasanuzzaman M, Alam M M, et al. Glutathione-induced drought stress tolerance in mung bean: coordinated roles of the antioxidant defence and methylgolyoxal detoxification systems [J]. AoB Plants, 2015, 7 (1). pii: plv069. doi: 10.1093/aobpla/plv069.
[21]	Passaia G, Margis-Pinheiro M. Glutathione peroxidases as redox sensor proteins in plant cells [J]. Plant Sci, 2015, 234: 22-26. doi: 10.1016/j.plantsci.2015.01.017. Epub 2015 Feb 7.
[22]	周立敬,周宜君,高飞,等. 拟南芥谷胱甘肽过氧化物酶的生物信息学分析[J].中央民族大学学报:自然科学版, 2010, 19 (2): 11-17.
[23]	马亭亭, 周宜君, 高飞, 等. 盐芥谷胱甘肽过氧化物酶基因(THGPX6)的克隆及表达分析[J]. 植物遗传资源学报, 2012, 13 (2): 252-258.
[24]	肖蓉,罗慧珍,张小娟,等. 干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因(ZjGPX)的差异表达及功能分析[J]. 中国农业科学, 2015, 48 (14): 2806-2817.
[25]	Wang R G, Chen S L, Zhou X Y, et al. Ionic homeostasis and reactive oxygen species control in leaves and xylem sap of two poplars subjected to NaCl stress [J]. Tree Physiol, 2008, 28 (6): 947-957.
[26]	王菲菲, 丁明全, 邓澍荣, 等. 胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析[J]. 基因组学与应用生物学, 2012, 31 (3): 231-239.

Cloning of Glutathione Peroxidase PmGPX6 Gene from Pinus massoniana and the Study on Drought Tolerance of Transgenic Arabidopsis thaliana

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通讯作者: 陈斌, bchen63@163.com

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Cloning of Glutathione Peroxidase PmGPX6 Gene from Pinus massoniana and the Study on Drought Tolerance of Transgenic Arabidopsis thaliana

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