垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析

Cloning and functional analysis of trehalose-6-phosphate synthase gene from Selaginella pulvinata

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3223bp,包括一个2790bp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(tps1△)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。

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 3223 bp long,containing an open reading frame（ORF） of 2790 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-phosphate synthase mutant（tps1△）,transformed by the ORF of SpTPS1 gene,can restore growth on the mediam supplemented with glucose as a sole carbon source.This result indicated that SpTPS1 of S.pulvinata encodes for an activative protein and is hopeful to be applied in transgeneic improvement of abiotic stress tolerance in plant.