Nucleotide Sequence of a Sporamin Gene in Sweet Potato

Sporamin is tissue specific and may account for more than 80% of the soluble protein found in tuberous root of sweet potato (Ipomoea batatas Lam.) (Maeshima et al., 1985). SDS-PAGE analysis indicates that sporamin may be resolved into two bands with molecular masses in the range of 25 kD. These two protein bands were characterized and classified into two subfamilies, based on the homology of their nucleotide sequences (Murakami et al., 1986; Hattori et al., 1989). Within the subfamilies, the homology may be greater than 90%, whereas the cDNAs between the two subfamilies share only 80% homology. Recent studies of two sporamin genes, gSPO-A1 and gSP0-B1, which belong to the two distinct subfamilies A and B, respectively, have shown that they are intronless genes (Hattori and Nakamura, 1988). Comparison of the 5’ flanking region also revea led t h e presence of two conserved sequence blocks, namely Suc box 2 and box 3 (Hattori and Nakamura, 1988; Tsukaya et al., 1991). In this paper, we present a nove1 sporamin gene, gSPOR5-31, which was identified to be an isogene of the gSPO-A1 within subfamily A. A hEMBL3 genomic library comprising 6 X 105 plaqueforming units was constructed with partially Sau3A-digested genomic DNA of tuberous roots. The library was screened by plaque hybridization using the cDNA probe of SP-B (an antisense strand of sporamin cDNA; K.-W. Yeh, unpublished results). One positive signal was obtained during the primary screening. It was purified to a single plaque, from which the DNA was extracted and characterized by restriction endonuclease and Southern blot analysis (Table I). The results showed that the clone (gSPOR5-31) contained an insert DNA of approximately 7 kb. When the insert DNA was digested by BamHI, resolved by agarose gel electrophoresis, and hybridized with the SP-B cDNA probe, only a 2.2-kb fragment clearly showed a strong signal. Therefore, it was subsequently subcloned into pGEM3Z (Promega) at the BamHI site for further studies. The nucleotide sequence of the 2.2-kb BamHI fragment was determined by nested deletion and primer walking. It included an open reading frame of 660 nucleotides corresponding to 220 amino acid residues. The genomic clone of gSPOR5-31 was similar to those of the gSPO-A1 and gSPO-B1 containing no intron. These observations suggest that sporamin genes are intronless. Analysis of the coding