Nucleotide sequence of cDNA for a putative cysteine protease from rice seeds.

A Cys endopeptidase (SH-EP) is involved in the degradation of storage globulin in cotyledons of germinating seeds of Vigna mungo (Mitsuhashi and Minamikawa, 1989). The structure and expression of the gene for SH-EP have been reported (Akasofu et al., 1990; Yamauchi et al., 1992). Here we isolated a full-length cDNA for a putative Cys protease from a rice seed cDNA expression library using antiserum against SH-EP, and determined its nucleotide sequence (Table I). A Agtll cDNA library was constructed from total poly(A)+ RNA that was prepared from germinating rice seeds grown in the dark for 9 d. The library was screened with a rabbit polyclonal antiserum raised against SH-EP. The initial screening resulted in the isolation of a partial cDNA clone (designated pRP8). Although this pRP8 clone lacked the initiation Met codon, the deduced amino acid sequence showed high homology to the other known Cys proteases. To obtain a full-length clone, the same library was rescreened with the pRP8 cDNA probe, and a cDNA clone (designated pRP80) was isolated. This cDNA is 1440 bp long and contains 1134 bp of an open reading frame, and 93 and 213 bp of 5’ and 3’ untranslated regions, respectively. There is a polyadenylation signal, AATAAA, in the 3‘ untranslated region. Cys proteases are synthesized as prepropeptides and cotranslationally processed to mature enzymes. The pRP80 clone shows a similar feature. The first 24 amino acids deduced from this clone have the feature of typical eukaryotic signal sequence (Von Heijne, 1983). This putative signal peptide is followed by 117 amino acids of prosequence and 237 amino acids of the mature enzyme. The predicted mo1 wt of the mature protein encoded by pRP80 is 25,477. Comparisons of the deduced amino acid sequence of pRP80 revealed high homology to the other reported Cys proteases. The putative mature protein of pRP80 shows similarity to the mature proteins of SH-EP (67%) (Akasofu et al., 1990), EP-B from barley (64%) (Koehler and Ho, 1990), oryzain a from rice (60%) (Watanabe et al., 1991), and aleurain from barley (42%) (Rogers et al., 1985).