Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase

Stachyose is the major soluble carbohydrate in seeds of a number of importa nt crop species. It is synthesized from raffinose and galactinol by the act ion of stachyose synthase (EC 2.4.1.67). We report here on the identificati on of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna a ngularis Ohwi et Ohashi). Based on internal amino acid sequences of the enz yme purified from adzuki bean, oligonucleotides were designed and used to a mplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence co mprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was ampli fied 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 polyc lonal antibodies raised against stachyose synthase purified from adzuki bea n and was shown to be a functional stachyose synthase with the same catalyt ic 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.