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首页> 外文期刊>Breeding science >Cloning and structural analysis of wheat cDNAs encoding Ndr protein kinase homolog by data mining of the EST database KOMUGI.
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Cloning and structural analysis of wheat cDNAs encoding Ndr protein kinase homolog by data mining of the EST database KOMUGI.

机译：通过EST数据库KOMUGI的数据挖掘，对编码Ndr蛋白激酶同源物的小麦cDNA进行克隆和结构分析。

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摘要

To gain a better understanding of the nature of nuclear dbf-related (Ndr) protein kinase homologues in monocots, three cDNA clones were isolated from common wheat (Triticum aestivum) by data mining of expressed sequence tag (EST) databases, and their primary structures were 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 A. squarrosa [A. tauschii]) revealed that at least three homologous 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..
机译：为了更好地了解单子叶植物中核dbf相关（Ndr）蛋白激酶同源物的性质，通过对表达序列标签（EST）数据库及其主要结构进行数据挖掘，从普通小麦（Triticum aestivum）中分离了三个cDNA克隆。通过逆转录-聚合酶链反应（RT-PCR），cDNA末端的5'-快速扩增（RACE）和核苷酸测序来确定。 cDNA克隆的推导氨基酸序列包含真核Ser / Thr蛋白激酶的所有12个高度保守的亚域，包括ATP结合位点（在亚域I-II中）和Ser / Thr蛋白激酶的活性位点（在亚域VI中） ）。该序列还包含在亚结构域VII和VIII之间的56个氨基酸插入片段，以及三个保守的Ser / Thr残基，这是真核生物蛋白激酶Ndr家族的特征。小麦和祖先二倍体物种（T. boeoticum，Aegilops speltoides和A. squarrosa [A. tauschii]）cDNA的序列比较表明，在六倍体小麦中表达了至少三个同源基因。该结果证实了包括KOMUGI在内的当前EST数据库对于小麦基因克隆的有用性，因为使用设计有EST序列信息的特异性引物大大促进了稀有cDNA（如Ndr。）的克隆。

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