垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及其功能分析.pdfVIP

HEREDITAS (Beijing) 2010 5 , 32(5): 498―504 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2010.00498 垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析 林荆, 付凤玲, 蒋伟, 牟禹, 雍太明, 李晚忱 , 625014 海藻糖-6-磷酸合成酶(Trehalose-6-phosphate synthse, TPS)是植物海藻糖合成途径的关键酶, 在旱生卷柏 等复苏植物对逆境胁迫应答中起重要作用。文章以我国特有旱生植物垫状卷柏(Selaginella pulvinata)为材料, 采 用同源扩增与RACE 技术相结合的方法克隆了海藻糖-6-磷酸合成酶基因SpTPS1, cDNA 全长3 223 bp, 包括一 个2 790 bp 的开放阅读框, 推导的氨基酸序列与模式物种的海藻糖-6-磷酸合成酶具有较高的序列相似性, 催化 活性中心保守位点基本一致。酵母功能互补实验证明, 用 SpTPS1 基因开放阅读框转化的海藻糖合成酶基因突 变(tps 1△) 酵母菌株, 可恢复在以葡萄糖作为唯一碳源培养基上的生长, 说明垫状卷柏海藻糖-6-磷酸合成酶基 因SpTPS1 的编码蛋白具有生物活性, 可应用于植物抗逆性的转基因改良。 垫状卷柏; 海藻糖-6-磷酸合成酶; 基因克隆; 功能 Cloning and functional analysis of trehalose -6-phosphate synthase gene from Selaginella pulvinata LIN Jing, FU Feng-Ling, JIANG Wei, MU Yu, YONG Tai-Ming, LI Wan-Chen Maize Research Institute , Sichuan Agricultural University, Ya’an 625014, China Abstract: Trehalose-6-phosphate synthase, a key enzyme in trehalose synthesis pathway of plant, plays an important role in response to abiotec stress in xerophilous rock lily and other resurrection plants. In this study, homologous amplification and RACE technique were used to clone gene SpTPS1 for trehalose-6-phosphate synthase from Selaginella pulvinata, which is an endemic xerophilous plant in China. The full-length cDNA is 3 223 bp long, containing an open reading frame (ORF) of 2 790 bp. Protein sequence comparison showed that the pupative amino acid sequence of this ORF shares high similarity to trehalose-6-phosphate synthases of mode speciese, especially at the conserved sites of catalytic activity centers. Yeast functinal complementation test showed that trehalose-6-phosph