‘南通小方柿’GA2ox基因的克隆、亚细胞定位及表达分析

【目的】从地方矮化品种‘南通小方柿’(Diospyros kaki Linn.cv.Nantongxiaofangshi)中分离赤霉素合成关键酶基因GA2ox,并进行亚细胞定位及表达分析,为进一步探索其矮生性状形成机理及选育新型矮生品种奠定基础。【方法】以‘南通小方柿’幼嫩叶片为材料,通过改良的CTAB法提取总RNA,利用简并引物克隆GA2ox家族中2个基因的片段,采用3′和5′RACE方法得到基因的全长c DNA序列,分别命名为Dk GA2ox1和Dk GA2ox2。通过生物信息学方法分析结构特征,利用GFP进行亚细胞定位,实时荧光定量RT-PCR技术研究其在发芽期、展叶期、枯顶期、开花期、生理落果期、果实着色期、果实成熟期中的表达特性。【结果】生物信息学分析Dk GA2ox1和Dk GA2ox2全长c DNA序列分别为1 318 bp和1 267 bp,分别含有198 bp和61 bp的5′非翻译区及97 bp和172 bp的3′非翻译区。编码332个和334个氨基酸,与毛白杨(JX102472.1)、夹竹桃(AY594292.1)、烟草(AB125232.1)、矮牵牛(GU059939.1)、苹果(FJ571521.1)、梨(JF441168.1)、葡萄(JQ608472.1)的相似度达73%—77%。含有保守20G-Fe(Ⅱ)-Oxy蛋白结构域,高度保守的2-酮戊二酸结合位点(Dk GA2ox1:Arg-272、Ser-274;Dk GA2ox2:Arg-271、Ser-273)和Fe2+结合位点(Dk GA2ox1:His-205、Asp-207、His-262;Dk GA2ox2:His-204、Asp-206、His-261),有赤霉素2-氧化酶蛋白家族共同的结构特点。Dk GA2ox1和Dk GA2ox2编码蛋白分子量分别为36 596.1 Da和37 544.2 Da,均为稳定蛋白,无信号肽,无跨膜结构域,无明显疏水区,属于C19-GAoxs。瞬时表达载体的构建及洋葱表皮细胞转化后,洋葱表皮细胞的瞬时表达结果显示,Dk GA2ox1编码蛋白定位于细胞核和细胞质中。Dk GA2ox1和Dk GA2ox2在开花期表达最丰富,并在7个典型物候期中均高于乔化品种‘大方柿’。【结论】南通小方柿中赤霉素2-氧化酶基因的表达与其矮生性状有关。

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

【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.