Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.)

Abstract

Two types of genes (Adk‐a, and Adk‐b) encoding for adenylate kinase (AK, EC 2.7.4.3.) were isolated from the cDNA library constructed from poly(A)⁺ RNA of rice (Oryza sativa L.). Two cDNAs were heterogeneous at 5′ and 3′ ends of non‐coding sequences and had possible polyadenylation signals. One of the genes, Adk‐a, had 1154 bp sequences encoding 241 amino acid residues, while the other type, Adk‐b, contained 1085 bp sequences encoding for 243 amino acid residues. Homology between Adk‐a and Adk‐b was 73.7% in nucleotide sequences, and 90.8% in amino acid level. Two genes showed about 53% homology to bovine mitochondrial adenylate kinase (AK2) at nucleotide and amino acid levels. Concerning the codon usage of rice AK genes, T was abundant at the third position of a codon in the reading frames. In order to examine the enzyme activity of the protein encoded by the rice cDNA, Adk‐a was cloned into an expression vector, pUC119, which was introduced into Escherichia coli strain CV2, a temperaturesensitive mutant of adenylate kinase. We found that the transformant carrying the rice Adk‐a gene in the sense orientation recovered cell growth at nonpermissive high temperature (42°C) and expressed enzyme activities higher than the untransformed CV2 and the transformant possessing Adk‐a cDNA in the antisense orientation. These observations suggest that rice Adk‐a codes a biologically active enzyme. Furthermore, sucrose was found to regulate the transcription of AK genes in rice cell cultures. Organ related accumulation of mRNA in whole plants was also found.