Isolation of a cDNA encoding a granule-bound 152-kilodalton starch-branching enzyme in wheat

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends res ulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence a lignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide direct ing the polypeptide into plastids. Furthermore, the predicted mature form o f SBEIc was much larger (152 kD) than previously characterized plant SBEI ( 80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like p rotein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a funct ional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of pr oteins produced in developing wheat kernels demonstrated that the 152-kD SB EIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated wi th the starch granules. Proteins similar in size and recognized by wheat SB EI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.