Cloning and Structural Analysis of Wheat cDNAs Encoding Ndr Protein Kinase Homolog by Data Mining of the EST Database KOMUGI

概要

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In order to gain a better understanding of the nature of nuclear dbf-related (Ndr) protein kinase homologs in monocots, three cDNA clones were isolated from common wheat (Triticum aestivum) by data mining of expressed sequence tag (EST) databases, and their primary structure was determined by reverse transcription-polymerase chain reaction (RT-PCR), 5′-rapid amplification of cDNA ends (RACE), and nucleotide sequencing. Deduced amino acid sequences of the cDNA clones contained all 12 highly conserved subdomains of the eukaryotic Ser/Thr protein kinase, including the ATP-binding site (in subdomain I-II) and the Ser/Thr protein kinase active-site (in subdomain VI). The sequences also contained an insert of 56 amino acids between subdomains VII and VIII, and three conserved Ser/Thr residues, being characteristic of the Ndr family of eukaryotic protein kinases. A sequence comparison among cDNAs from wheat and those from ancestral diploid species (T. boeoticum, Aegilops speltoides and Ae. squarrosa) revealed that at least three homeologous genes were expressed in hexaploid wheat. The results confirmed the usefulness of current EST databases including KOMUGI for gene cloning of wheat, since the use of specific primers designed with information about EST sequences has considerably facilitated the cloning of rare cDNAs such as Ndr.
日本育種学会の論文