Ilam Hardy) genomic DNA by the PCR, using primers com- plementary to a pin2 genomic sequence (Keil et al., 1986). The amino acid sequence of pin2-CM7 ...
Plant Physiol. (1994) 106: 1681
Plant Gene Register
Genomic Nucleotide Sequence of a Proteinase lnhibitor II Gene Colleen Murray* and John T. Christeller The Horticultural and Food Research lnstitute of New Zealand Limited, Batchelar Research Centre, Tennent Drive, Private Bag 11 030, Palmerston North, New Zealand
PI-I1 is a potent inhibitor of endopeptidases, exhibiting a dual specificity toward trypsin and chymotrypsin (Gustafson and Ryan, 1976). This protein is encoded by a small multigene family and expression is regulated by both developmental and environmental signals. PI-I1 is constitutively expressed in potato tubers and in flowers of potato and tomato plants. In leaves, synthesis of the inhibitor is induced in response to severe mechanical damage (Ryan and An, 1988) such as insect chewing. Plant protease inhibitors can serve as protective agents against herbivorous insects (Broadway and Duffey, 1986). A nove1 PI-I1 gene (pin2-CM7) has been isolated in our laboratory (Table I). The gene was isolated from potato (var Ilam Hardy) genomic DNA by the PCR, using primers complementary to a pin2 genomic sequence (Keil et al., 1986). The amino acid sequence of pin2-CM7 exhibits 86% homology with two PI-I1 genes isolated from potato (Keil et al., 1986; Sanchez-Serrano et al., 1986) and 80% homology with a PI-I1 gene from tomato (Graham et al., 1985). Based on comparison with these published data, the N-terminal portion of the pin2-CM7 gene appears to encode a 31-residue signal peptide, necessary for co-translational import into the ER, from which the inhibitor is directed into the vacuole (Graham et al., 1985; Sanchez-Serrano et ai., 1986).
Table 1. Characteristics of the Dotato PI-/I gene
Organism: Solanum tuberosum var Ilam Hardy. Gene; Gene Product: pin2-CM7; Pl-11. Techniques: lsolation of gene using PCR and primers complementary to a genomic sequence (Keil et al., 1986). Complete dideoxy sequencing of both strands. Method of Identification: Comparison of sequence with published sequences (Graham et al., 1985; Keil et al., 1986; Sanchez-Serranoet al., 1986). Features of Gene Structure: An open reading frame of 462 bp interrupted by an intron of 119 bp.
Features of Protein Sequence: An open reading frame of 462 bp encoding a polypeptide of 154 amino acid residues. The P1 sites for trypsin and chymotrypsin, which interact with the primary binding site ( F l ) of the endopeptidases, occur at bases 225 to 227 and 396 to 398, respectively (based on genomic sequence homology). The sequence includes an N-terminal 31-residue signal peptide (based on genomic sequence homology). Antibodies: Polyclonal rabbit. Subcellular Location: Vacuolar (Walker-Simmonsand Ryan, 1977).
Received May 11, 1994; accepted May 26, 1994. Copyright Clearance Center: 0032-0889/94/106/1681/01. The EMBL accession number for the sequence reported in the article is X78275.
Gustafson G, Ryan CA (1976) Specificity of protein tumover in tomato leaves: accumulation of proteinase inhibitors, induced with the wound honnone PIIF. J Biol Chem 251: 7004-7010 Keil M, Sanchez-SerranoJ, Schell J, Willmitzer L (1986) Primary structure of a proteinase inhibitor I1 gene from potato (Solanum tuberosum). Nucleic Adds Res 1 4 5641-5650 Ryan C, An G (1988) Molecular biology of wound-induable proteinase inhibitors in plants. Plant Cell Environ 11: 345-349 Sanchez-Serrano J, Schmidt R, Scell J, Willmitzer L (1986) Nucleotide sequence of proteinase inhibitor I1 encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mo1 Gen Genet 203 15-20 Walker-Simmons M, Ryan CA (1977) Immunological identification of proteinase inhibitors I and I1 in isolated tomato leaf vacuoles. Plant Physiol60 61-63
LITERATURE CITED
Broadway R, Duffey S (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larva1 Heliothis zea and Spodoptera exigua. J Insect Physiol 3 2 827-833 Graham JS, Pearce G, Merryweather J, Titani K, Ericsson LH, Ryan CA (1985) Wound-induced protease inhibitors from tomato leaves. 11. The cDNA-deduced primary structure of pre-inhibitor 11. J Biol Chem 260 6561-6564
Corresponding author; fax 64-6-354-6731. Abbreviation: PI-11, proteinase inhibitor 11.
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