Peterbauer T, Mucha J, Mayer U, Popp M, Glössl J, Richter A
Institute of Plant Physiology, University of Vienna, A-1091 Vienna, Austria.
Plant J. 1999 Dec;20(5):509-18. doi: 10.1046/j.1365-313x.1999.00618.x.
Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.
水苏糖是许多重要农作物种子中的主要可溶性碳水化合物。它是由棉子糖和肌醇半乳糖苷在水苏糖合酶（EC 2.4.1.67）的作用下合成的。我们在此报告从赤小豆（Vigna angularis Ohwi et Ohashi）种子中鉴定出一个编码水苏糖合酶的cDNA。根据从赤小豆中纯化得到的该酶的内部氨基酸序列设计寡核苷酸，并通过RT-PCR从赤小豆cDNA中扩增相应序列，随后进行cDNA末端快速扩增（RACE-PCR）。完整的cDNA序列包含3046个核苷酸，其中包括一个编码857个氨基酸残基多肽的开放阅读框。通过PCR扩增整个编码区，将其构建到杆状病毒表达载体pVL1393中，并导入草地贪夜蛾（Sf21）昆虫细胞进行异源表达。重组蛋白与针对从赤小豆中纯化得到的水苏糖合酶产生的多克隆抗体具有免疫反应性，并且显示出与天然对应物具有相同催化特性的功能性水苏糖合酶。在种子发育中期，水苏糖合酶mRNA高水平瞬时积累，并且该酶也存在于成熟种子和萌发过程中。
