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 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.
Peterbauer
T
T
Institute of Plant Physiology, University of Vienna, A-1091 Vienna, Austria.
Mucha
J
J
Mayer
U
U
Popp
M
M
Glössl
J
J
Richter
A
A
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D009844:Oligosaccharides; D011994:Recombinant Proteins; C005695:stachyose; D005700:Galactosyltransferases; C113913:galactinol -raffinose galactosyltransferase