| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 95 === Starch is the major carbohydrate reserve in higher plants. In the starch biosynthetic pathway, starch phosphorylase (EC 2.4.1.1, SP) catalyzes a reversible reaction between synthesis and degradation of starch with bidirectional activities. Mungbean (Vigna radiata L.) starch is the most special that amylose content was 15-30% higher than normal level, which implied that SP plays an important role in amylose synthesis. Previous studies have identified the tryptic fragments of mungbean 105-kDa SP by MALDI-TOF and conducted immunological analysis. The objective of this study was to further clone the full-length cDNA of SP in order to investigate the characteristics and function of recombination SP in the future. Based on the internal amino acid sequence of N- and C-terminal of mungbean SP, degenerate primers were designed as gene-specific primers (GSP). The developing mungbean seed (Vigna radiata cv. Tainan no.5) was used as the material to extract its total RNA, and mRNA was purified and used as the template in RT-PCR. First, a 1819 bp sequence of a middle fragment was obtained, and then primers were designed from its internal sequence. The 5’-nucleotide conserved region sequences from fave bean, sweet potato and potato were used to design primers which were coupled with internal GSPs for RT-PCR amplification. Sequences containing the start codon ATG of 5’ 640 bp and 3’ sequences containing the stop codon TAG of 997 bp were obtained successfully. The full-length cDNA of the mungbean SP which possessed nucleotides of 2961 bp in length, containing complete open reading frame (ORF) that covers from start codon to stop codon was designated as Vrsp. Vrsp encodes a polypeptide of 987 amino acids with predicted molecular mass of 111 kDa and pI of 5.38. Putative protein sequences possess the motifs of starch binding site, L-78, catalytic site and pyridoxal phosphate (PLP) binding site. In addition, Vrsp includes the L-78 insertion sequence, it belongs to the known L-type isoform.
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