Cloning of cDNA for pyruvate, Pi dikinase from maize leaves

Abstract

To obtain molecular probes for studies of gene regulation in photosynthetic tissues of maize, we have cloned DNA complementary to poly(A)+RNA extracted from green leaves by insertion into plasmid pBR322 and transformation of E. coli, strain RR1. Colonies were screened by sequential hybridisation with 32p-labeled single stranded cDNA synthesized from pooled aliquots of poly(A)⁺RNA fractionated by sucrose density centrifugation. Among the clones bearing cDNA homologous to high molecular weight poly(A)⁺RNA, we identified one with an insert of 440 base pairs homologous to mRNA for pyruvate, Pi dikinase, a C-4 carbon cycle protein localized in mesophyll cells of the leaf. Our work indicates that the dikinase subunits are synthesized in the cytoplasm as precursors approximately 13,000 daltons larger than the mature peptide subunits. Leaves of seedlings illuminated during growth have higher levels of pyruvate, Pi dikinase mRNA than leaves of dark-grown plants.