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

Abstract

SummaryTwo 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 safiwa 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 colistrain 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.

Primary structure of maize chloroplast adenylate kinase

“…The plant enzyme is longer at both termini, but shorter by four residues in the insertion (possibly the loop before strand PS is shorter) and by seven residues in the two long helices thereafter. As published earlier, the other two plant AK sequences from a rice cDNA library [3] are more related to the mitochondria1 AK2-type from yeast (52%) and mammals (e.g. 51% for bovine AK2) than to maize chloroplast AK (34% and 35%, respectively).…”

“…Although the sequences of some 30 adenylate kinases are known, as well as five well-resolved X-ray structures with different substrates in different conformations [2], so far we have little structural information on the enzymes from plants. Only recently two very similar cDNA sequences from rice were cloned, both showing the highest similarity to mammalian mitochondria1 AK2 [3].C, plants, like maize, have a particularly efficient CO, fixation by the C, cycle at the expense of ATP to AMP conversion in the pyruvate phosphate dikinase reaction. Because of the reversibility of the latter reaction, these plants need an efficient AMP recycling ; not surprisingly, an approximately 50-fold higher adenylate kinase (AK) content than in C, plants was found [4, 51.…”

“…Although the sequences of some 30 adenylate kinases are known, as well as five well-resolved X-ray structures with different substrates in different conformations [2], so far we have little structural information on the enzymes from plants. Only recently two very similar cDNA sequences from rice were cloned, both showing the highest similarity to mammalian mitochondria1 AK2 [3].…”

Functional Analysis for Rolling Leaf of Somaclonal Mutants in Rice (Oryza sativa L.)

“…RT-PCR (reverse transcriptase-PCR) was used to analyze the mutants containing the retrotransposon elements as determined by the PCR and Southern analysis [2]. The total RNAs that were from the putative rice plants (leaves) using guanidine thiocyanate were isolated [11].…”

“…Meanwhile, there are about 1,000 retrotransposons with 32 classes in the rice genome that existed [6] and, LTR (long terminal repeats) retrotransposon with a minimum of 59 classed which composed 17% of the rice entire genome, but research about the evolution or introduced time is insufficient [3]. The reverse transcription modulator (RTs; Reverse Transcriptase) of Tos family with 20 classes caused somaclonal variation because it was activated at the tissue culture which were only found in the japonica variety 'Nipponbare' [9][10][11][12][13][14][15][16][17][18]. However, very little is known about the ecotype and copy number of the rice species or kind of Tos.…”