CLONING AND CHARACTERIZATION OF THE CALRETICULIN GENE FROM RICINUS-COMMUNIS L

A full-length cDNA encoding a calreticulin-like protein was isolated b y immune-screening a germinating castor bean endosperm cDNA library wi th antisera raised to the total lumenal fraction of purified plant end oplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to th ose reported for the mammalian calreticulin. Calcium overlays and immu ne blot analysis confirmed the endoplasmic lumenal identity of this re ticuloplasmin. Probing protein blots of endoplasmic reticulum subfract ions with radio-iodinated calreticulin showed specific associations wi th various polypeptides including one identified as the abundant retic uloplasmin protein disulfide isomerase. Characterization of the corres ponding genomic clones revealed that calreticulin is encoded by a sing le gene of 3 kb in castor. The full genomic sequence reveals the prese nce of 12 introns, 12 translated exons, and one exon containing the la st three amino acids of the translated sequence and the 3'-untranslate d region of the gene. Northern blot analysis of RNA isolated from vari ous organ tissues showed a basal constitutive level of expression thro ughout the plant, but more abundant mRNA being detected in tissues act ive in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5'-untranslated genomic sequence fused to the beta-glucuronidase reporter gene (GUS) showed a more localized pa ttern of expression. Activity being localized to the vasculature (phlo em, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinatin g seed.