Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (1): 197-206.doi: 10.3864/j.issn.0578-1752.2015.01.19

• RESEARCH NOTES • Previous Articles    

Cloning, Subcellular Localization and Expression Analysis of Gibberellin 2-Oxidase Gene in Diospyros kaki Linn. cv. Nantongxiaofangshi

TU Xu-tong1, ZHANG Shi-jie1, CHEN Xiao-yun1, LI Ning-ning1, XIN Lu1, XUE Xiao-hui2, ZHANG Zhen1, QU Shen-chun1   

  1. 1Department of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2Haian Agricultural Commission of Jiangsu Province, Haian 226600, Jiangsu
  • Received:2014-03-27 Online:2015-01-01 Published:2015-01-01

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Abstract: 【Objective】 This paper aims to isolate the cDNA of GA2ox from Diospyros kaki Linn. cv. Nantongxiaofangshi, and do some preliminary study on their functions and expression level, in order to lay a foundation for further exploration of the dwarf mechanism and breeding of new dwarf cultivars. 【Method】 Total RNA was extracted from leaves of ‘Nantongxiaofangshi’ persimmon by improved CTAB method. Twofragments were identified from cDNA of ‘Nantongxiaofangshi’ by degenerate primers, and their full length cDNA were acquired by RACE amplification and named as DkGA2ox1 and DkGA2ox2, respectively. Gene structure characteristics were analyzed using the bioinformatics software. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern during pre-budding period, budding period, leaf expanding period, tip buds dying period, flowering period, physiological fruit-falling period, fruit coloring period, fructescence and abscission period. 【Result】 The full length cDNA of DkGA2ox1 and DkGA2ox2 were 1 318 bp and 1 267 bp, respectively, containing 5′ untranslated region (UTR) with lengths of 198 bp and 61 bp, 3′ UTR with lengths of 97 bp and 172 bp, and coding region with lengths of 999 bp and 1 005 bp which encoded 332 and 334 amino acids. The two amino acid sequences shared 73%-77% in homology compared with Populus tomentosa (JX102472.1), oleander (AY594292.1), tobacco (AB125232.1), Petunia (GU059939.1), apple (FJ571521.1), pears (JF441168.1) and grapes (JQ608472.1). The conserved structural domain analysis revealed that DkGA2ox1 and DkGA2ox2 had the typical functional domains of GA2ox protein, containing Fe2+ binding sites (DkGA2ox1: His-205, Asp-207, His-262; DkGA2ox2: His-204, Asp-206, His-261) and 2-oxoglutarate binding sites (DkGA2ox1: Arg-272, Ser-274; DkGA2ox2: Arg-271, Ser-273), as well as the 2OG-Fe(II)-Oxy protein domains. The protein molecular weights were 36 596.1 Da and 37 544.2 Da, respectively. Both of them are stable proteins, have no signal peptide, transmembrane domains, and significant hydrophobic region, as well as belong to C19-GAoxs. After construction of transient expression vector and onion epidermal cell transformation, subcellular localization assays showed that the GA2ox1 protein was located in the nucleus and cytoplasm. The quantitative RT-PCR results showed that the highest expression levels of DkGA2ox1 and DkGA2ox2 were detected in florescence, and all higher than those in vigorous cultivar ‘Dafangshi’ during all the 7 phenological periods. 【Conclusion】 The expression of gibberellin 2-oxidase genes in ‘Nantongxiaofangshi’ is related with the dwarf trait.

Key words: persimmon, gibberellins 2-oxidase, gene clone, expression analysis, subcellular localization

[1]    Sasaki A, Ashikari M, Ueguchi-Tanaka M, Itoh H, Nishimura A, Swapan D, Ishiyama K. Saito T, Kobayashi M, Khush G. Green revolution: a mutant gibberellin-synthesis gene in rice, Nature, 2002, 416(6882): 701-702.
[2]    Sui J M, Guo B T, Wang J S, Qiao L X, Zhou Y, Zhang H G, Gu M H, Liang G H. A new GA-insensitive semidwarf mutant of rice (Oryza sativa L.) with a missense mutation in the SDG gene. Plant Molecular Biology Reporter, 2012, 30(1): 187-194.
[3]    Spielmeyer W, Ellis M H, Chandler P M. Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene. Proceedings of the National Academy of Sciences of the USA, 2002, 99(13): 9043-9048.
[4]    Aya K, Ueguchi-Tanaka M, Kondo M, Hamada K, Yano K. Nishimura M, Matsuoka M. Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. The Plant Cell Online, 2009, 21(5): 1453-1472.
[5]    Olszewski N, Sun T P, Gubler F. Gibberellin signaling biosynthesis, catabolism, and response pathways. The Plant Cell Online, 2002, 14(suppl 1): S61-S80.
[6]    Harberd N P, King K E, Carol P, Cowling R J, Peng J, Richards D E. Gibberellin: inhibitor of an inhibitor of...? BioEssays, 1998, 20(12): 1001-1008.
[7]    Richards D E, King K E, Ait-ali T, Harberd N P. How gibberellin regulates plant growth and development: a molecular genetic analysis of gibberellin signaling. Annual Review of Plant Biology, 2001, 52(1): 67-88.
[8]    De Jong M, Mariani C, Vriezen W H. The role of auxin and gibberellin in tomato fruit set. Journal of Experimental Botany, 2009, 60(5): 1523-1532.
[9]    Schwechheimer C. Understanding gibberellic acid signaling-are we there yet? Current Opinion in Plant Biology, 2008, 11(1): 9-15.
[10]   MacMillan J. Occurrence of gibberellins in vascular plants, fungi, and bacteria. Journal of Plant Growth Regulation, 2001, 20(4): 387-442.
[11]   Hedden P, Phillips A L. Manipulation of hormone biosynthetic genes in transgenic plants. Current Opinion in Biotechnology, 2000, 11(2): 130-137.
[12]   Sakamoto T, Morinaka Y, Ishiyama K, Kobayashi M, Itoh H, Kayano T, Iwahori S, Matsuoka M, Tanaka H. Genetic manipulation of gibberellin metabolism in transgenic rice. Nature Biotechnology, 2003, 21(8): 909-913.
[13]   Ross J J, Reid J B, Swain S M, Hasan O, Poole A T, Hedden P, Willis C L. Genetic regulation of gibberellin deactivation in Pisum. The Plant Journal, 1995, 7(3): 513-523.
[14]   Schomburg F M , Bizzell C M , Lee D J, Zeevaart J A, Amasino R M. Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. The Plant Cell Online, 2003, 15(1): 151-163.
[15]   Biemelt S, Tschiersch H, Sonnewald U. Impact of altered gibberellin metabolism on biomass accumulation, lignin biosynthesis, and photosynthesis in transgenic tobacco plants. Plant Physiology, 2004, 135(1): 254-265.
[16]   Dayan J, Schwarzkopf M, Avni A, Aloni R. Enhancing plant growth and fiber production by silencing GA 2‐oxidase. Plant Biotechnology Journal, 2010, 8(4): 425-435.
[17]   Appleford N E, Wilkinson M D, Ma Q, Evans D J, Stone M C, Pearce S P, Powers S J, Thomas S G , Jones H D, Phillips A L. Decreased shoot stature and grain α-amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat. Journal of Experimental Botany, 2007, 58(12): 3213-3226.
[18]   Dijkstra C, Adams E, Bhattacharya A, Page A, Anthony P, Kourmpetli S, Power J, Lowe K, Thomas S, Hedden P. Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species. Plant Cell Reports, 2008, 27(3): 463-470.
[19]   Agharkar M, Lomba P, Altpeter F, Zhang H N, Kenworthy K, Lange T. Stable expression of AtGA2ox1 a low‐input turfgrass (Paspalum notatum Flugge) reduces bioactive gibberellin levels and improves turf quality under field conditions. Plant Biotechnology Journal, 2007, 5(6): 791-801. in
[20]   Busov V B, Meilan R, Pearce D W, Ma C, Rood S B, Strauss S H. Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature. Plant Physiology, 2003, 132(3): 1283-1291.
[21]   El-Sharkawy I, Kayal W E, Prasath D, Fernandez H, Bouzayen M, Svircev A M, Jayasankar S. Identification and genetic characterization of a gibberellin 2-oxidase gene that controls tree stature and reproductive growth in plum. Journal of Experimental Botany, 2012, 63(3): 1225-1239.
[22]   Gargul J, Mibus H, Serek M. Constitutive overexpression of Nicotiana GA 2 ox leads to compact phenotypes and delayed flowering in Kalanchoë blossfeldiana and Petunia hybrida. Plant Cell, Tissue and Organ Culture (PCTOC), 2013, 115(3): 407-418.
[23]   蒋德新, 薛晓晖, 盛炳成, 章镇, 戴文浩, 徐长宝. 珍稀矮生型柿品种—南通小方柿. 江苏农业科学, 1992, 8(1): 49-50.
Jiang D X, Xue X H, Sheng B C, Zhang Z, Dai W H, Xu C B. The rare dwarf persimmon cultivar-‘Nantongxiaofangshi’. Jiangsu Agricultural Science, 1992, 8(1): 49-50.
[24]   蔡斌华, 张计育, 高志红, 渠慎春, 佟兆国, 靡林, 乔玉山, 章镇. 一种改良的提取草莓属叶片总RNA的方法. 江苏农业学报, 2009, 24(6): 875-877.
Cai B H, Zhang J Y, Gao Z H, Qu S C, Tong Z G, Mi L, Qiao Y S, Zhang Z. An improved method for isolation of total RNA from the leaves of Fragaria spp. Jiangsu Journal of Agricultural Sciences，2009, 24(6): 875-877. (in Chinese)
[25]   刘金戈. 八棱海棠和水稻抗逆相关转录因子的克隆及功能鉴定[D]. 南京: 南京农业大学, 2007.
Liu J G. Cloning and functional analysis of transcription factor involved in abiotic stress in Malus robusta rehd and Oryza sativa L. [D]. Nanjing: Nanjing Agricultural University, 2007. (in Chinese)
[26]   Fleet C M, Sun T P. A DELLAcate balance: the role of gibberellin in plant morphogenesis. Current Opinion in Plant Biology, 2005, 8(1): 77-85.
[27]   罗霞, 康宗利, 樊金娟. 赤霉素的信号转导途径. 植物生理学通讯, 2007, 43(1): 191-194.
Luo X, Kang Z L, Fan J J. GA Signal transduction pathway. Plant Physiology Communications, 2007, 43(1): 191- 194. (in Chinese)
[28]   Thomas S G, Phillips A L, Hedden P. Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proceedings of the National Academy of Sciences of the USA, 1999, 96(8): 4698-4703.
[29]   Martin D N, Proebsting W M, Hedden P. The SLENDER gene of pea encodes a gibberellin 2-oxidase. Plant Physiology, 1999, 121(3): 775-781.
[30]   Sakamoto T, Kobayashi M, Itoh H, Tagiri A, Kayano T, Tanaka H, Iwahori S, Matsuoka M. Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice. Plant Physiology, 2001, 125(3): 1508-1516.
[31]   戴文浩. ‘南通小方柿’矮生机理的研究[D]. 南京: 南京农业大学, 1998.
Dai W H. Study on dwarfness and dwarfing mechanisn of ‘Xiao Fang Shi’ persimmon[D]. Nanjing: Nanjing Agricultural University, 1998. (in Chinese)
[32]   Emanuelsson O, Heijne G. Prediction of organellar targeting signals. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2001, 1541(1): 114-119.
[33]   Sun T P. Gibberellin metabolism, perception and signaling pathways in Arabidopsis. The Arabidopsis Book/American Society of Plant Biologists, 2008, 6(1): 1-28.
[34]   Huang J, Tang D, Shen Y, Qin B, Hong L, You A, Li M, Wang X, Yu H, Gu M. Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.). Journal of Genetics and Genomics, 2010, 37(1): 23-36.
[35]   Ueguchi-Tanaka M, Ashikari M, Nakajima M, Itoh H, Katoh E, Kobayashi M, Chow T Y, Yue-ie C H, Kitano H, Yamaguchi I. Gibberellin insensitive dwarf1 encodes a soluble receptor for gibberellin. Nature, 2005, 437(7059): 693-698.
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