Nucleotide sequence analysis of a cDNA encoding chloroplastic fructose-1,6-bisphosphatase from pea (Pisum sativum l.).

Fructose-1,6-bisphosphatase (EC 3.1.3.11) catalyzes the dephosphoric reaction of Fru-1,6-bis P to Fru-6-P and Pi. Two isozymes exist in higher plants, one in the cytosol and the other in the chloroplast. The latter is a pivotal enzyme for photosynthetic carbon dioxide assimilation. It is active in the light but almost inactive in darkness. The lightmediated activation of the enzyme is attributed to the increase of pH, Mg2+ level, and reducing power in the stroma of the chloroplast upon light illumination (Buchanan, 1980). Recently we purified and characterized chloroplastic and cytosolic fructose-1,6-bisphosphatase f rom pea (Pisum sativum L.) leaves (Cho and Hahn, 1991; Lee et al., 1994). Polyclonal antibodies were raised from the purified enzymes and used to screen a cDNA library. Here we report a cDNA sequence of pea chloroplastic fructose1,6-bisphosphatase. A cDNA library was constructed in Agtll using cDNA synthesized with polyadenylated mRNA isolated from 2-week-old green pea leaves. The library was then screened with a polyclonal antibody raised in rabbit against the purified pea chloroplast fructose-l,6-bisphosphatase. Among 15 positive clones, the clone with the largest cDNA insert (FD5) was subcloned and sequenced. The nucleotide sequence of the cDNA showed it to be a full-length cDNA of 1371 bp that contained 5’ (39 bp) and 3’ (110 bp) untranslated regions. The 3’ noncoding region was followed by the poly(A) tail. The open reading frame of the cDNA contained 1221 bp corresponding to a polypeptide of 407 amino acids. Amino acid sequence analysis suggested that the 50 amino acid residues from the N terminus were a transit peptide that is necessary for transport into the chloroplast (Raines et al., 1988). The polypeptide had significantly higher homology with the deduced amino acid sequence of the chloroplast fructose-l,6bisphosphatase than with that of the cytosolic enzyme (Table I) (Raines et al., 1988; Ladror et al., 1990; Marcus and