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

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

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