野生大豆CDPK基因保守区的克隆及序列分析

CDPK(Ca2+-dependent,calmodulinindependentproteinkinase)是植物钙信号传导过程中关键的蛋白激酶,在逆境胁迫信号传导过程中起到重要作用。研究基于同源序列候选基因的策略,根据已经公布的多个CDPK序列的比对结果,在保守性较高的区域,设计两对巢式简并引物,结合降落PCR和巢式PCR在野生大豆中克隆。对获得的序列片段进行序列分析,发现该序列具有激酶活性的功能区域和CDPK特有的EF手性结构区,并且与VrCDPK(U08140)序列达到94%的相似性,可以确定该片段是CDPK基因的保守序列。结果证明,野生大豆中也存在CDPK基因,并为通过RACE等方法获得野生大豆全长CDPK基因奠定了基础。

Clone the conserved sequence of CDPK from Glycine soja and analysis the sequence

CDPK(Ca2+-dependent,calmodulin independent protein kinase) is the crucial protein kinase in plant calcium signal transduction,and play important role in osmotic stresses.Here based on the strategy with homologous sequences clone new gene.According to the published DNA sequences,alignment them,designed two pairs nest-primers in the higher homologous regions of CDPKs,combinated touch-down PCR and nest PCR to clone the conserved sequence of CDPK from Glycine soja.Eventually have gotten wild soybean CDPK conserva-tive sequence,through analysing,it included the conserved catalytic domain that is typical of serine/threonine protein kinases and a Ca2+-binding domain that contains three typical EF hand structures those different from other protein kinases.What′s more,the DNA sequence exhibited significant similarity to VrCDPK(U08140),reaches 94% similarity.Result has proved,also have CDPK gene in wild bean,and have established foundation to get the all length genes through the methods such as RACE in wild soybean.