Cloning, Expression and Functional Analysis of CaAPX Gene from Camellia azalea

WANG Jiang-ying; FAN Zheng-qi; YIN Heng-fu; LI Xin-lei; WU Bin; LI Ji-yuan

Volume 29 Issue 4

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Cloning, Expression and Functional Analysis of CaAPX Gene from Camellia azalea

1.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Key Laboratory of Tree Breeding of Zhejiang Province, Hangzhou 311400, Zhejiang, China
2.
Flower Research Center of Lianyungang Academy of Agricultural Sciences, Lianyungang 222006, Jiangsu, China

Received Date: 2015-05-12

Abstract

[Objective] Using molecular biology techniques to investigate the expression patterns of CaAPX gene in Camellia azalea, and to carry out the functional analysis of CaAPX in tobacco under temperature stress. [Method] On the basis of homologous sequences of C. japonica, an ascorbate peroxidase (APX) gene was isolated from the tender leaf in C. azalea by the 3', 5'-RACE technology named CaAPX. The full-length cDNA of CaAPX is 1 097 bp, containing a 753 bp ORF which encodes 250 amino acids. [Result] Quantitative real-time PCR analysis indicated that the CaAPX was expressed differently in all examined camellia tissues, and the expression levels in order were immature green fruit, tender leaf, flower, leaf bud, seed embryo, petal and flower bud. The highest expression level of CaAPX was in immature green fruit, approximately ranging 2.68 to 11.44 times as high as that of the other six groups. It was also found that the expression level was notably up-regulated in leaves of camellia plants subjected to abnormal temperatures. Furthermore, the APX activity in transgenic plants increased by 158%-309%, and the AsA content increased by 167%-256% as higher as wild type plants. In addition, the over expression of CaAPX enhanced cold and heat stress tolerance in transgenic plants under temperature stresses. [Conclusion] The over expression of CaAPX from C. azalea could enhance the cold and heat stress tolerance in transgenic tobacco, and which also provide a basis in molecular breeding of resistance adversity of Camellias in the future.
- Camellia azalea,
- gene clone,
- temperature stress,
- expression analysis,
- transgenic tobacco

References

[1]	闵天禄. 世界山茶属的研究[M]. 昆明: 云南科学技术出版社, 2000: 35-44.
[2]	庄瑞林. 中国山茶[M]. 海口: 海南人民出版社, 1989.
[3]	Sofo A, Tuzio A C, Dichio B, et al. Influence of water deficit and rewatering on the components of the asccorbate-glutathione cycle in four interspecific Prunus hybrids [J]. Plant Science, 2005, 169(2): 403-412.
[4]	Suzuki N, Mittler R. Reactive oxygen species and temperature stress: A delicate balance between signaling and destruction[J]. Physiologia Plantarum, 2006, 126(1): 45-51.
[5]	Lu Z, Liu D, Liu S. Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic Arabidopsis[J]. Plant Cell Reports, 2007, 26(10): 1909-1917.
[6]	Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants [J]. Plant Physiol Biochemistry, 2010, 48(12): 909-930.
[7]	Foyer C H, Halliwell B. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism [J]. Planta, 1976, 133(1): 21-25.
[8]	Jablonski P P, Anderson J W. Light dependent reduction of hydrogen peroxide by ruptured pea chloroplasts [J]. Plant Physiology, 1981, 67(6): 1239-1244.
[9]	Hossain M A, Asada K. Purification of dehydroascorbate reductase from spinach and its characterization as a thiol enzyme [J]. Plant and Cell Physiology, 1984, 25(1): 85-92.
[10]	Yutaka S, Toyotaka M, Hideyuki F, et al. Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings [J]. Journal of Experimental Botany, 2001, 52(354): 145-151.
[11]	Tang L, Kwon S Y, Kim S H, et al. Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature [J]. Plant Cell Reports, 2006, 25(12): 1380-1386.
[12]	Kim M D, Kim Y H, Kwon S Y, et al. Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes [J]. Physiologia Plantarum, 2010, 140(2): 153-162.
[13]	朱高浦, 李纪元. 珍稀濒危植物张氏红山茶研究进展(综述)[J]. 亚热带植物科学, 2009,38(3):83-89.
[14]	Horsch R B, Fry J E, Hoffmann N L, et al. A simple and general method for transferring genes into plants [J]. Science, 1985, 227(4691): 1229-1231.
[15]	Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts [J]. Plant and Cell Physiology, 1981, 22(5): 867-880.
[16]	Kampfenkel K, van Montagu M, Inze D. Extraction and determination of ascorbate and dehydroascorbate from plant tissue [J]. Analytical Biochemistry, 1995, 225(1): 165-167.
[17]	Sairam R K, Srivastava G C. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress [J]. Plant Science, 2002, 162(6): 897-904.
[18]	韩立敏. 丹参APX和GPX基因克隆及其表达分析[D]. 西安: 陕西师范大学, 2007: 22-44.
[19]	孙云, 林玉玲, 赖钟雄, 等. 茶树品种及萎凋过程中叶片APX基因表达的qPCR分析[J]. 福建农林大学学报: 自然科学版, 2012,41(4): 476-481.
[20]	邵巍. 龙眼胚性培养物胞质型apx基因克隆及其表达研究[D]. 福州: 福建农林大学, 2008: 84-95.
[21]	许传俊, 孙叙卓, 李玲, 等. 蝴蝶兰抗坏血酸过氧化物酶基因克隆及其表达研究[J]. 园艺学报, 2012, 39(4): 769-776.
[22]	刘慧春. 红掌‘阿技巴马’低温相关基因AOX、CAT和APX的表达分析、功能验证及遗传转化体系的构建[D]. 杭州: 浙江大学, 2012: 100-129.
[23]	陈莉, 辛海波, 孙向荣, 等. 百合APX基因的克隆及转LlAPX提高拟南芥耐盐性[J]. 园艺学报, 2010, 37(12): 1983-1990.
[24]	Kornyeyev D, Dmytro K, Barry A, et al. Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpresion genes encoding chloroplast-targeted antioxidant enzymes [J]. Physiologia Plantarum, 2001, 133(1):323-331.
[25]	Battraw M J, Hall T C. Histochemical analysis of CaMV35S Promoter β-glucuronidase gene expression in transgenic rice plants [J]. Plant Molecular Biology, 1990, 15(4): 527-538.
[26]	Lim S, Kim Y H, Kim S H, et al. Enhanced tolerance of transgenic sweetpotato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress [J]. Molecular Breeding, 2007, 19(3): 227-239.
[27]	王雨水. 低温锻炼对冷胁迫下油茶幼苗光合速率与抗氧化酶活性的影响[J]. 福建林业科技, 2011, 38(1): 41-46.
[28]	安辽原, 牛向丽, 黄龙翔, 等. 过量表达OsAPX1基因增强水稻的抗热和抗氧化能力[J]. 中国农业科技导报, 2012,14(5): 28-32.
[29]	马箐. 过量表达毛白杨APX基因增强植物对非生物胁迫的抗性[D]. 济南: 山东师范大学, 2012: 37-59.

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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, Expression and Functional Analysis of CaAPX Gene from Camellia azalea

1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Key Laboratory of Tree Breeding of Zhejiang Province, Hangzhou 311400, Zhejiang, China

2. Flower Research Center of Lianyungang Academy of Agricultural Sciences, Lianyungang 222006, Jiangsu, China

Received Date: 2015-05-12

Abstract: [Objective] Using molecular biology techniques to investigate the expression patterns of CaAPX gene in Camellia azalea, and to carry out the functional analysis of CaAPX in tobacco under temperature stress. [Method] On the basis of homologous sequences of C. japonica, an ascorbate peroxidase (APX) gene was isolated from the tender leaf in C. azalea by the 3', 5'-RACE technology named CaAPX. The full-length cDNA of CaAPX is 1 097 bp, containing a 753 bp ORF which encodes 250 amino acids. [Result] Quantitative real-time PCR analysis indicated that the CaAPX was expressed differently in all examined camellia tissues, and the expression levels in order were immature green fruit, tender leaf, flower, leaf bud, seed embryo, petal and flower bud. The highest expression level of CaAPX was in immature green fruit, approximately ranging 2.68 to 11.44 times as high as that of the other six groups. It was also found that the expression level was notably up-regulated in leaves of camellia plants subjected to abnormal temperatures. Furthermore, the APX activity in transgenic plants increased by 158%-309%, and the AsA content increased by 167%-256% as higher as wild type plants. In addition, the over expression of CaAPX enhanced cold and heat stress tolerance in transgenic plants under temperature stresses. [Conclusion] The over expression of CaAPX from C. azalea could enhance the cold and heat stress tolerance in transgenic tobacco, and which also provide a basis in molecular breeding of resistance adversity of Camellias in the future.

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Reference (29)

[1]	闵天禄. 世界山茶属的研究[M]. 昆明: 云南科学技术出版社, 2000: 35-44.
[2]	庄瑞林. 中国山茶[M]. 海口: 海南人民出版社, 1989.
[3]	Sofo A, Tuzio A C, Dichio B, et al. Influence of water deficit and rewatering on the components of the asccorbate-glutathione cycle in four interspecific Prunus hybrids [J]. Plant Science, 2005, 169(2): 403-412.
[4]	Suzuki N, Mittler R. Reactive oxygen species and temperature stress: A delicate balance between signaling and destruction[J]. Physiologia Plantarum, 2006, 126(1): 45-51.
[5]	Lu Z, Liu D, Liu S. Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic Arabidopsis[J]. Plant Cell Reports, 2007, 26(10): 1909-1917.
[6]	Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants [J]. Plant Physiol Biochemistry, 2010, 48(12): 909-930.
[7]	Foyer C H, Halliwell B. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism [J]. Planta, 1976, 133(1): 21-25.
[8]	Jablonski P P, Anderson J W. Light dependent reduction of hydrogen peroxide by ruptured pea chloroplasts [J]. Plant Physiology, 1981, 67(6): 1239-1244.
[9]	Hossain M A, Asada K. Purification of dehydroascorbate reductase from spinach and its characterization as a thiol enzyme [J]. Plant and Cell Physiology, 1984, 25(1): 85-92.
[10]	Yutaka S, Toyotaka M, Hideyuki F, et al. Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings [J]. Journal of Experimental Botany, 2001, 52(354): 145-151.
[11]	Tang L, Kwon S Y, Kim S H, et al. Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature [J]. Plant Cell Reports, 2006, 25(12): 1380-1386.
[12]	Kim M D, Kim Y H, Kwon S Y, et al. Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes [J]. Physiologia Plantarum, 2010, 140(2): 153-162.
[13]	朱高浦, 李纪元. 珍稀濒危植物张氏红山茶研究进展(综述)[J]. 亚热带植物科学, 2009,38(3):83-89.
[14]	Horsch R B, Fry J E, Hoffmann N L, et al. A simple and general method for transferring genes into plants [J]. Science, 1985, 227(4691): 1229-1231.
[15]	Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts [J]. Plant and Cell Physiology, 1981, 22(5): 867-880.
[16]	Kampfenkel K, van Montagu M, Inze D. Extraction and determination of ascorbate and dehydroascorbate from plant tissue [J]. Analytical Biochemistry, 1995, 225(1): 165-167.
[17]	Sairam R K, Srivastava G C. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress [J]. Plant Science, 2002, 162(6): 897-904.
[18]	韩立敏. 丹参APX和GPX基因克隆及其表达分析[D]. 西安: 陕西师范大学, 2007: 22-44.
[19]	孙云, 林玉玲, 赖钟雄, 等. 茶树品种及萎凋过程中叶片APX基因表达的qPCR分析[J]. 福建农林大学学报: 自然科学版, 2012,41(4): 476-481.
[20]	邵巍. 龙眼胚性培养物胞质型apx基因克隆及其表达研究[D]. 福州: 福建农林大学, 2008: 84-95.
[21]	许传俊, 孙叙卓, 李玲, 等. 蝴蝶兰抗坏血酸过氧化物酶基因克隆及其表达研究[J]. 园艺学报, 2012, 39(4): 769-776.
[22]	刘慧春. 红掌‘阿技巴马’低温相关基因AOX、CAT和APX的表达分析、功能验证及遗传转化体系的构建[D]. 杭州: 浙江大学, 2012: 100-129.
[23]	陈莉, 辛海波, 孙向荣, 等. 百合APX基因的克隆及转LlAPX提高拟南芥耐盐性[J]. 园艺学报, 2010, 37(12): 1983-1990.
[24]	Kornyeyev D, Dmytro K, Barry A, et al. Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpresion genes encoding chloroplast-targeted antioxidant enzymes [J]. Physiologia Plantarum, 2001, 133(1):323-331.
[25]	Battraw M J, Hall T C. Histochemical analysis of CaMV35S Promoter β-glucuronidase gene expression in transgenic rice plants [J]. Plant Molecular Biology, 1990, 15(4): 527-538.
[26]	Lim S, Kim Y H, Kim S H, et al. Enhanced tolerance of transgenic sweetpotato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress [J]. Molecular Breeding, 2007, 19(3): 227-239.
[27]	王雨水. 低温锻炼对冷胁迫下油茶幼苗光合速率与抗氧化酶活性的影响[J]. 福建林业科技, 2011, 38(1): 41-46.
[28]	安辽原, 牛向丽, 黄龙翔, 等. 过量表达OsAPX1基因增强水稻的抗热和抗氧化能力[J]. 中国农业科技导报, 2012,14(5): 28-32.
[29]	马箐. 过量表达毛白杨APX基因增强植物对非生物胁迫的抗性[D]. 济南: 山东师范大学, 2012: 37-59.

Cloning, Expression and Functional Analysis of CaAPX Gene from Camellia azalea

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通讯作者: 陈斌, bchen63@163.com

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Cloning, Expression and Functional Analysis of CaAPX Gene from Camellia azalea

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