ISOLATION AND EXPRESSION PATTERN OF A CDNA-ENCODING A CATHEPSIN B-LIKE PROTEASE FROM NICOTIANA-RUSTICA

Sequence analysis of a 1.33 kb clone from a root cDNA library of Nicot iana rustica revealed an open reading frame encoding a protein of 356 amino acids. The deduced protein has high levels of homology to human cathepsin B protease and a cathepsin B-like cysteine protease from whe at but much lower levels of homology with other plant cysteine protein ases. Southern blotting experiments suggest a limited number of cathep sin B-like genes are present in the genome of N. rustica and also that of N. tabacum. RNA analysis involving a range of tissues, harvested f rom both Nicotiana species 4-5 h after the beginning of a 16 h photope riod, revealed the cathepsin B-like gene was being expressed strongly in roots, stem and developing flowers but weakly in mature leaves. Fur ther analysis of RNA extracted from leaf tissue of N, tabacum revealed the gene showed rhythmic expression and also that its expression incr eased in response to wounding. Analysis of leaf tissues harvested duri ng the latter part of a 16 h photoperiod (11 and 16 h after illuminati on commenced) showed that transcript levels were two three times highe r than in leaf tissue harvested either towards the end of the dark per iod or 5 h after illumination commenced. When leaf tissue was wounded at 11:00 (5 h after plants were illuminated), and harvested for RNA ex traction 6 h later, the level of cathepsin B-like transcript in mesoph yll tissue was found to be increased ca. 2-fold relative to the level detected in unwounded controls.