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棉子糖(raffinose)作为一种重要的可溶性碳水化合物广泛存在于高等植物中,并且参与植物抵抗各种非生物逆境胁迫。棉子糖合成酶(raffinose synthase,RS,EC2.4.1.82)是催化肌醇半乳糖苷和蔗糖产生棉子糖的关键酶。本研究通过同源比对克隆到了一个小麦(Triticum aestivum)的棉子糖合成酶基因(triticum aestivum raffinose synthse,TaRS),该基因定位于小麦3B染色体上。序列分析显示,TaRS基因具有一个完整的开放阅读框(2 349 bp),包含两个保守模体,即Kx D和Rxxx D,属于糖苷水解酶超家族GHD(glycoside hydrolases,clan D)。在进化关系上,小麦TaRS蛋白与山羊草(Aegilops tauschii)的棉子糖合成酶XM020298559.1亲缘关系最近(相似度为98.47%)。Southern杂交结果表明,TaRS基因在中国春小麦基因组中存在至少4个拷贝。亚细胞定位结果显示,TaRS蛋白定位在小麦叶肉细胞的细胞膜上。对该基因进行组织表达特异性分析发现,TaRS在小麦的根、茎、叶和种子中都有表达,但在叶片中的表达量最高。将TaRS在大肠杆菌(Escherichia coli)中进行异源表达,通过高效液相色谱(high performance liquid chromatography,HPLC)法检测发现,TaRS能够以蔗糖和肌醇半乳糖苷为底物在体外合成棉子糖,并且反应的最适pH在8.0左右。此外,TaRS的表达受到脱水、42℃高温、高盐和4℃低温4种胁迫的诱导,分别于胁迫12、1、1和48 h时表达量达到最大。本研究为进一步利用TaRS基因进行小麦抗逆育种提供了理论依据。
Raffinose, an important soluble carbohydrate, is widely found in higher plants and participates in plant resistance to various abiotic stresses. Raffinose synthase (RS, EC 2.4.1.82) is a key enzyme that catalyzes the production of raffinose from galactinol and sucrose. In this study, we cloned a triticum aestivum raffinose synthse (TaRS) gene from wheat (Triticum aestivum) by homology alignment, which locates on chromosome 3B of wheat. Sequence analysis showed that the TaRS gene has a complete open reading frame (2 349 bp) and contains two conserved motifs, Kx D and Rxxx D, belonging to glycoside hydrolases (clan D). In evolutionary relation, the wheat TaRS protein has the closest genetic relationship with the raffinose synthase XM020298559.1 of Aegilops tauschii (the similarity is 98.47%). Southern hybridization results showed that there are at least 4 copies of TaRS gene in Chinese spring wheat genome. Subcellular localization results show that TaRS protein is located on the cell membrane of wheat mesophyll cells. Tissue expression analysis of this gene showed that TaRS was expressed in wheat roots, stems, leaves and seeds but highest in leaves. TaRS was heterologously expressed in Escherichia coli. High performance liquid chromatography (HPLC) assay showed that TaRS could synthesize cotton in vitro with sucrose and galactinol as substrates Sugar, and the optimum pH of the reaction is around 8.0. In addition, the expression of TaRS was induced by four kinds of stress: dehydration, high temperature of 42 ℃, high salt and low temperature of 4 ℃. The expression of TaRS reached the maximum at 12,1,1 and 48 h stress respectively. This study provided the theoretical basis for the further utilization of TaRS gene in wheat breeding.