Cloning and Expression Analysis of GA2ox1 Gene from Camellia lipoensis

XIAO Zheng; LI Ji-yuan; FAN Zheng-qi; LI Xin-lei; YIN Heng-fu

Volume 29 Issue 1

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Cloning and Expression Analysis of GA2ox1 Gene from Camellia lipoensis

1.
Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, Jiangsu, China
2.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China

Received Date: 2015-03-31

Abstract

Gibberellin 2 oxidase is one of the key enzymes in GA biosynthesis and metabolism, and GA2ox genes are often used to dwarf plant engineering breeding. In this study, the gibberellin 2 oxidase gene was cloned from the stems of Camellia lipoensis using RT-PCR and RACE methods. The full length of the GA2ox gene, named ClGA2ox1, is 1 371 bp (GenBank accession No. KJ502290), which contains a 1 002 bp open reading frame encoding 333 amino acid residues, contains a 5'-UTR with 59 bp and 3'-UTR 310 bp. The putative protein molecular weight is 37.31 kD and its theoretical isoelectric point is 5.92. The sequence includes GA2ox superfamily domain and the family signature amino acid residues. Homologous alignment shows that it shares 80% of amino acid identities with GA2ox from other plants in GenBank. The phylogenetic tree constructed on the basis of amino acid sequences of GA2ox suggests that the relationship between C. lipoensis and Nicotiana tabacum is the most intimate. The results showed that the ClGA2ox1 gene expression level in root, young stem, two-year-old stem, young leaf, mature leaf and seed of C. lipoensis were different. The ClGA2ox1 transcripts were the most abundant in two-year-old stem, followed by new stem and root, and the lowest transcripts were found in the young leaf. This study may provide references for further research on the functional characteristics of ClGA2ox1 and its molecular mechanism involved in the regulation of plant growth.
- Camellia lipoensis,
- GA2-oxidase,
- real-time PCR,
- cloning

References

[1]	Radi A, Lange T, Niki T, et al. Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants[J]. Plant Physiol, 2006, 140(2): 528-536.
[2]	Pimenta Lange M J, Liebrandt A, Arnold L, et al. Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L.[J]. Phytochemistry, 2013(90): 62-69.
[3]	Lee D, Lee I, Kim K, et al. Expression of gibberellin 2-oxidase 4 from Arabidopsis under the control of a senescence-associated promoter results in a dominant semi-dwarf plant with normal flowering[J]. Journal of Plant Biology, 2014, 57(2): 106-116.
[4]	Hedden P, Phillips A L. Gibberellin metabolism: new insights revealed by the genes[J]. Trends Plant Sci, 2000, 5(12): 523-530.
[5]	Sakamoto T, Miura K, Itoh H, et al. An overview of gibberellin metabolism enzyme genes and their related mutants in rice[J]. Plant Physiol, 2004, 134(4): 1642-1653.
[6]	Hedden P, Thomas S G. Gibberellin biosynthesis and its regulation[J]. Biochem J, 2012, 444(1): 11-25.
[7]	Dijkstra C, Adams E, Bhattacharya A, et al. Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species[J]. Plant Cell Reports, 2008, 27(3): 463-470.
[8]	Biemelt S, Tschiersch H, Sonnewald U. Impact of altered gibberellin metabolism on biomass accumulation, lignin biosynthesis, and photosynthesis in transgenic tobacco plants[J]. Plant Physiol, 2004, 135(1): 254-265.
[9]	Zhou B, Lin J, Peng W, et al. Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana[J]. Molecular Breeding, 2012, 29(1): 115-127.
[10]	Studzinska A K, Gardner D S, Metzger J D, et al. The Effect of PcGA2ox overexpression on creeping bentgrass (Agrostis stolonifera L.): performance under various light environments[J]. HortScience, 2012, 47(2): 280-284.
[11]	余勇, 葛文东, 邹世慧, 等. 异源表达拟南芥赤霉素 2-氧化酶基因对矮牵牛形态发育的影响[J]. 园艺学报, 2014, 41(10): 2065-2074.
[12]	Huang J, Tang D, Shen Y, et al. Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.)[J]. J Genet Genomics, 2010, 37(1): 23-36.
[13]	Thomas S G, Phillips A L, Hedden P. Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation[J]. Proceedings of the National Academy of Sciences, 1999, 96(8): 4698-4703.
[14]	Schomburg F M, Bizzell C M, Lee D J, et al. Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants[J]. The Plant Cell Online, 2003, 15(1): 151-163.
[15]	常丽, 赵小英, 郭明, 等. GA2ox1氧化酶基因的克隆及原核表达[J]. 西北植物学报, 2010, 30(06): 1099-1104.
[16]	Chen Q, Ya H, Li S, et al. Isolation and analysis of homoeologous genes encoding gibberellin 2-oxidase 3 isozymes in common wheat[J]. Journal of Genetics, 2012, 91(3):1-9.
[17]	Otani M, Meguro S, Gondaira H, et al. Overexpression of the gibberellin 2-oxidase gene from Torenia fournieri induces dwarf phenotypes in the liliaceous monocotyledon Tricyrtis sp[J]. Journal of Plant Physiology,2013,170(16): 1416-1423.
[18]	Lange T, Kappler J, Fischer A, et al. Gibberellin biosynthesis in developing pumpkin seedlings[J]. Plant Physiol, 2005, 139(1): 213-223.
[19]	Zhao H, Dong J, Wang T. Function and expression analysis of gibberellin oxidases in apple[J]. Plant Molecular Biology Reporter, 2010, 28(2): 231-238.
[20]	张宏达,任善湘. 中国植物志:第49卷,第3分册[M]. 北京: 科学出版社, 1998: 1-152.
[21]	Gasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPASy server, in The proteomics protocols handbook[M].Totowa, NJ: Humana Press, 2005:571-607.
[22]	Dyrløv Bendtsen J, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0[J]. Journal of Molecular Biology, 2004, 340(4): 783-795.
[23]	Guermeur Y, Pollastri G, Elisseeff A, et al. Combining protein secondary structure prediction models with ensemble methods of optimal complexity[J]. Neuro Computing, 2004, 56: 305-327.
[24]	Tamura K, Dudley J, Nei M, et al. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599.
[25]	肖政, 李纪元, 范正琪, 等. 荔波连蕊茶肌动蛋白基因全长 cDNA 的克隆及其表达分析[J]. 林业科学研究, 2014, 27(3): 374-380.
[26]	王西成, 王晨, 房经贵, 等. 葡萄VvGA2ox1基因克隆、亚细胞定位及时空表达分析[J]. 南京农业大学学报, 2013,36(1): 29-34.
[27]	Yamaguchi S. Gibberellin metabolism and its regulation[J]. Annu Rev Plant Biol, 2008, 59(4): 225-251.
[28]	Gargul J M, Mibus H, Serek M. Constitutive overexpression of Nicotiana GA2ox leads to compact phenotypes and delayed flowering in Kalanchoë blossfeldiana and Petunia hybrida[J]. Plant Cell, Tissue and Organ Culture (PCTOC), 2013, 115(3): 407-418.
[29]	Lo S F, Yang S Y, Chen K T, et al. A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice[J]. Plant Cell, 2008, 20(10): 2603-2618.
[30]	Rieu I, Eriksson S, Powers S J, et al. Genetic analysis reveals that C₁₉-GA2-oxidation is a major gibberellin inactivation pathway in Arabidopsis[J]. Plant Cell, 2008, 20(9): 2420-2436.
[31]	屠煦童, 张仕杰, 陈小云, 等. ‘南通小方柿’GA2ox基因的克隆、亚细胞定位及表达分析[J]. 中国农业科学, 2015 ,48(1): 197-206.
[32]	王成祥, 李长生, 侯蕾, 等. 花生赤霉素2-氧化酶基因的克隆和表达研究[J]. 山东农业科学, 2013, 45(1): 14-18.

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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 and Expression Analysis of GA2ox1 Gene from Camellia lipoensis

1. Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, Jiangsu, China

2. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China

Received Date: 2015-03-31

Abstract: Gibberellin 2 oxidase is one of the key enzymes in GA biosynthesis and metabolism, and GA2ox genes are often used to dwarf plant engineering breeding. In this study, the gibberellin 2 oxidase gene was cloned from the stems of Camellia lipoensis using RT-PCR and RACE methods. The full length of the GA2ox gene, named ClGA2ox1, is 1 371 bp (GenBank accession No. KJ502290), which contains a 1 002 bp open reading frame encoding 333 amino acid residues, contains a 5'-UTR with 59 bp and 3'-UTR 310 bp. The putative protein molecular weight is 37.31 kD and its theoretical isoelectric point is 5.92. The sequence includes GA2ox superfamily domain and the family signature amino acid residues. Homologous alignment shows that it shares 80% of amino acid identities with GA2ox from other plants in GenBank. The phylogenetic tree constructed on the basis of amino acid sequences of GA2ox suggests that the relationship between C. lipoensis and Nicotiana tabacum is the most intimate. The results showed that the ClGA2ox1 gene expression level in root, young stem, two-year-old stem, young leaf, mature leaf and seed of C. lipoensis were different. The ClGA2ox1 transcripts were the most abundant in two-year-old stem, followed by new stem and root, and the lowest transcripts were found in the young leaf. This study may provide references for further research on the functional characteristics of ClGA2ox1 and its molecular mechanism involved in the regulation of plant growth.

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Reference (32)

[1]	Radi A, Lange T, Niki T, et al. Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants[J]. Plant Physiol, 2006, 140(2): 528-536.
[2]	Pimenta Lange M J, Liebrandt A, Arnold L, et al. Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L.[J]. Phytochemistry, 2013(90): 62-69.
[3]	Lee D, Lee I, Kim K, et al. Expression of gibberellin 2-oxidase 4 from Arabidopsis under the control of a senescence-associated promoter results in a dominant semi-dwarf plant with normal flowering[J]. Journal of Plant Biology, 2014, 57(2): 106-116.
[4]	Hedden P, Phillips A L. Gibberellin metabolism: new insights revealed by the genes[J]. Trends Plant Sci, 2000, 5(12): 523-530.
[5]	Sakamoto T, Miura K, Itoh H, et al. An overview of gibberellin metabolism enzyme genes and their related mutants in rice[J]. Plant Physiol, 2004, 134(4): 1642-1653.
[6]	Hedden P, Thomas S G. Gibberellin biosynthesis and its regulation[J]. Biochem J, 2012, 444(1): 11-25.
[7]	Dijkstra C, Adams E, Bhattacharya A, et al. Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species[J]. Plant Cell Reports, 2008, 27(3): 463-470.
[8]	Biemelt S, Tschiersch H, Sonnewald U. Impact of altered gibberellin metabolism on biomass accumulation, lignin biosynthesis, and photosynthesis in transgenic tobacco plants[J]. Plant Physiol, 2004, 135(1): 254-265.
[9]	Zhou B, Lin J, Peng W, et al. Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana[J]. Molecular Breeding, 2012, 29(1): 115-127.
[10]	Studzinska A K, Gardner D S, Metzger J D, et al. The Effect of PcGA2ox overexpression on creeping bentgrass (Agrostis stolonifera L.): performance under various light environments[J]. HortScience, 2012, 47(2): 280-284.
[11]	余勇, 葛文东, 邹世慧, 等. 异源表达拟南芥赤霉素 2-氧化酶基因对矮牵牛形态发育的影响[J]. 园艺学报, 2014, 41(10): 2065-2074.
[12]	Huang J, Tang D, Shen Y, et al. Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.)[J]. J Genet Genomics, 2010, 37(1): 23-36.
[13]	Thomas S G, Phillips A L, Hedden P. Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation[J]. Proceedings of the National Academy of Sciences, 1999, 96(8): 4698-4703.
[14]	Schomburg F M, Bizzell C M, Lee D J, et al. Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants[J]. The Plant Cell Online, 2003, 15(1): 151-163.
[15]	常丽, 赵小英, 郭明, 等. GA2ox1氧化酶基因的克隆及原核表达[J]. 西北植物学报, 2010, 30(06): 1099-1104.
[16]	Chen Q, Ya H, Li S, et al. Isolation and analysis of homoeologous genes encoding gibberellin 2-oxidase 3 isozymes in common wheat[J]. Journal of Genetics, 2012, 91(3):1-9.
[17]	Otani M, Meguro S, Gondaira H, et al. Overexpression of the gibberellin 2-oxidase gene from Torenia fournieri induces dwarf phenotypes in the liliaceous monocotyledon Tricyrtis sp[J]. Journal of Plant Physiology,2013,170(16): 1416-1423.
[18]	Lange T, Kappler J, Fischer A, et al. Gibberellin biosynthesis in developing pumpkin seedlings[J]. Plant Physiol, 2005, 139(1): 213-223.
[19]	Zhao H, Dong J, Wang T. Function and expression analysis of gibberellin oxidases in apple[J]. Plant Molecular Biology Reporter, 2010, 28(2): 231-238.
[20]	张宏达,任善湘. 中国植物志:第49卷,第3分册[M]. 北京: 科学出版社, 1998: 1-152.
[21]	Gasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPASy server, in The proteomics protocols handbook[M].Totowa, NJ: Humana Press, 2005:571-607.
[22]	Dyrløv Bendtsen J, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0[J]. Journal of Molecular Biology, 2004, 340(4): 783-795.
[23]	Guermeur Y, Pollastri G, Elisseeff A, et al. Combining protein secondary structure prediction models with ensemble methods of optimal complexity[J]. Neuro Computing, 2004, 56: 305-327.
[24]	Tamura K, Dudley J, Nei M, et al. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599.
[25]	肖政, 李纪元, 范正琪, 等. 荔波连蕊茶肌动蛋白基因全长 cDNA 的克隆及其表达分析[J]. 林业科学研究, 2014, 27(3): 374-380.
[26]	王西成, 王晨, 房经贵, 等. 葡萄VvGA2ox1基因克隆、亚细胞定位及时空表达分析[J]. 南京农业大学学报, 2013,36(1): 29-34.
[27]	Yamaguchi S. Gibberellin metabolism and its regulation[J]. Annu Rev Plant Biol, 2008, 59(4): 225-251.
[28]	Gargul J M, Mibus H, Serek M. Constitutive overexpression of Nicotiana GA2ox leads to compact phenotypes and delayed flowering in Kalanchoë blossfeldiana and Petunia hybrida[J]. Plant Cell, Tissue and Organ Culture (PCTOC), 2013, 115(3): 407-418.
[29]	Lo S F, Yang S Y, Chen K T, et al. A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice[J]. Plant Cell, 2008, 20(10): 2603-2618.
[30]	Rieu I, Eriksson S, Powers S J, et al. Genetic analysis reveals that C₁₉-GA2-oxidation is a major gibberellin inactivation pathway in Arabidopsis[J]. Plant Cell, 2008, 20(9): 2420-2436.
[31]	屠煦童, 张仕杰, 陈小云, 等. ‘南通小方柿’GA2ox基因的克隆、亚细胞定位及表达分析[J]. 中国农业科学, 2015 ,48(1): 197-206.
[32]	王成祥, 李长生, 侯蕾, 等. 花生赤霉素2-氧化酶基因的克隆和表达研究[J]. 山东农业科学, 2013, 45(1): 14-18.

Cloning and Expression Analysis of GA2ox1 Gene from Camellia lipoensis

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通讯作者: 陈斌, bchen63@163.com

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Cloning and Expression Analysis of GA2ox1 Gene from Camellia lipoensis

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