以荷花品种‘台城拂翠’(Nelumbo nucifera ‘Taicheng Focui’)嫩叶为材料,采用简并引物PCR 和RACE 技术得到荷花γGCS 基因的全长cDNA 序列,命名为NnγGCS。序列分析结果表:NnγGCS 基因cDNA 序列的全长为1 801 bp,其开放阅读框(ORF)长度为1 569 bp,编码522 个氨基酸残基。NnγGCS 蛋白质的理论相对分子质量为59 159. 0,理论等电点为pI 6. 27,稳定指数为39. 60;该蛋白质无跨膜结构Ⅱ,但具有1 个保守的GCS2 结构Ⅱ,并被定位于细胞质和叶绿体中,说明NnγGCS 蛋白质较稳定,并属于谷氨酰半胱氨酸连接酶家族。系统进化分析结果表明:荷花NnγGCS 氨基酸序列与龙眼(Dimocarpus longan Lour.)和黄瓜(Cucumis sativus Linn.)等双子叶植物γGCS氨基酸序列的亲缘关系较近,与水稻(Oryza sativa Linn.)等单子叶植物γGCS 氨基酸序列的亲缘关系较远。实时荧光定量PCR 扩增结果显示:NnγGCS 基因在荷花各器官中均能表达,且在嫩叶中的相对表达量最高、在茎中的相对表达量最低。不同浓度镉胁迫条件下,NnγGCS 基因在荷花嫩叶和须根中的相对表达量及表达趋势差异较大;NnγGCS 基因的相对表达量在嫩叶中总体表现为随胁迫时间延长先下降后上升,在须根中表现为200 μmol·L^-1 镉胁迫1 h 和400 μmol·L^-1 镉胁迫12 h 时显著高于初始水平、而在其他胁迫时间与初始水平差异不明显。亚细胞定位结果显示:NnγGCS 基因能够在洋葱(Allium cepa Linn.)表皮细胞的细胞质中表达,说明该基因编码的蛋白质在细胞质中发挥作用。研究结果显示一定浓度的镉胁迫能够诱导NnγGCS 基因的表达。

  Taking tender leaf of Nelumbo nucifera ‘Taicheng Focui’as materials, full-length cDNA sequence of γGCS gene from N. nucifera was obtained by degenerate primer-PCR and RACE technologies, the cDNA sequence is named as NnγGCS. The results of sequence analysis show that full-length of cDNA sequence of NnγGCS gene is 1 801 bp, its open reading frame. (ORF) length is 1 569bp, which encodes 522 amino acid residues. Theoretical relative molecular mass of NnγGCS protein is 59 159. 0, theoretical isoelectric point is pI 6. 27, and stability index is 39. 60. The protein has no transmembrane structure domain but with one conserved GCS2 domain and the protein is localized in cytoplasm and chloroplast, indicating that NnγGCS protein is stable and belonging to glutamate cysteine ligase modulatory family. The phylogenetic analysis result shows that NnγGCS amino acid sequence of N. nucifera has a close relationship with γGCS amino acid sequence of dicotyledon including Dimocarpus longan Lour. and Cucumis sativus Linn., etc, and has a distant relationship with γGCS amino acid sequence of monocotyledon including Oryza sativa Linn., etc. The amplification results of real-time fluorescence quantitative PCR show that NnγGCS gene can be expressed in various organs of N. nucifera,  and the relative expression in tender leaf is the highest, that in stem is the lowest. Under cadmium stress with different concentrations, differences in relative expression and expression trend of NnγGCS gene in tender leaf and fibrous root of N. nucifera are great. Relative expression of NnγGCS gene in tender leaf generally appears firstly decreasing and then increasing with prolonging of stress time, that in fibrous root appears significantly higher than original level when 200 μmol·L^-1 Cd stress for 1 h and 400 μmol·L^-1 Cd stress for 12 h, while without obvious difference with the original level at other stress times. Subcellular localization result shows that NnγGCS gene can express in cytoplasm of epidermal cells of Allium cepa Linn., meaning that the protein encoded by this gene plays a role in cytoplasm. It is suggested that Cd stress with a certain concentration can induce NnγGCS gene expression.