Cloning and sequence analysis of cDNA encoding thiamin‐binding proteins from sesame seeds

Abstract

The amino acid sequences of the large polypeptides of thiamin-binding proteins (TBPs) from sesame (Sesamum indicum L.) seeds (STBP-I, -II and -III) were analyzed. The large polypeptides of STBP-I, -II and -III had the same amino acid sequences as did their small polypeptides. The peptide sequence information obtained from STBPs was used to synthesize DNA primers for amplification of the gene(s) encoding STBPs. A 200-bp fragment was amplified from cDNA synthesized from RNA from sesame seeds 4 weeks after flowering. The 200-bp fragment was used to clone full-length cDNA(s) encoding STBP(s) with RACE techniques. A 644-bp fragment was amplified, cloned and sequenced. The cDNA was a full-length clone encoding STBP(s). It contained an open reading frame, which defined a 143-residue polypeptide. The identified small and large polypeptide sequences of STBPs exactly matched the sequence encoded within the cDNA clone. These results indicated that the small and large polypeptides of STBPs were encoded on the mRNA as a single large proprotein precursor and that the final mature forms were generated by post-translational processing in the same manner as the other 2S albumins of plant seeds.

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

“…A relatively high proportion of nonphosphorylated thiamine was found in bean germs. This can most probably be explained by the abundance of thiamine storage proteins in seeds and germs [28,29]. Such proteins are thought to retain thiamine in a dormant seed and release it during germination.…”

Biosynthesis of thiamin-binding proteins in developing sesame seeds

“…The amino acid sequences of the large polypeptides were the same, except for the C-terminus of polypeptide chains resulting in differences in molecular mass between STBP-I, -II and -III. On the other hand, cDNA encoding STBPs was isolated and characterized (Watanabe et al 2001). These results proposed the formation of STBP-I, -II and -III from the proprotein precursor by post-translational processing.…”

“…These results proposed the formation of STBP-I, -II and -III from the proprotein precursor by post-translational processing. However, it remains unclear whether the three STBPs are produced from the same large proprotein precursor or encoded by a gene family (Watanabe et al 2001). In this paper, STBPs in developing sesame seeds were analyzed by column chromatography and SDS-PAGE.…”

“…On the other hand, only one cDNA encoding STBPs has been isolated (Watanabe et al 2001); thus, the biosynthesis mechanism of the three STBPs remains unclear. The properties of the three STBPs resemble each other.…”

“…The analysis revealed only one signal band when DNA was digested with EcoR I or Hind III. STBP cDNA have no cleavage site of EcoR I and Hind III (Watanabe et al 2001); thus, the number of hybridizing signal bands indicated the presence of only one copy of STBP in the sesame genome.…”