低氮胁迫下黄瓜钙依赖蛋白激酶基因的克隆及表达分析

Cloning and Expression Analysis of Calcium-dependent Protein Kinase (CDPK) Gene in Cucumis sativus L. under Low Nitrogen Stress

在低氮胁迫下,以黄瓜品种津研4号叶片为供试材料,以cDNA为模板,依据黄瓜基因组数据库中Csa002986基因编码区全序列,应用Primer Premier 5.0软件设计引物,克隆得到黄瓜钙依赖蛋白激酶基因(calcium-dependent protein kinase,CDPK)。该基因全长1584bp,编码527个氨基酸。预测该基因编码的蛋白是一个稳定的亲水蛋白,无跨膜结构,无信号肽,存在蛋白激酶结合区、EF手型钙结合区、丝氨酸/苏氨酸蛋白激酶活性位点、N酰基化位点等多个位点。CDPK基因在不同氮浓度处理下黄瓜各部位表达分析结果显示,在无氮条件下,CDPK基因在茎尖表达量最高,其次为叶和茎;在低氮条件下,CDPK基因在茎中表达量最高,其次为茎尖和叶;在正常及高氮条件下,CDPK基因在茎尖表达量最高,其次为茎和叶。CDPK基因在叶中的表达模式分析结果显示,在无氮及低氮胁迫下CDPK基因表达量增加,随着氮浓度的降低,CDPK基因的表达量增加并明显高于对照;在高氮胁迫条件下,CDPK基因的表达量低于对照。

The CDPK was amplified by RT-PCR from a cucumber cultivar‘Jinyan No. 4’leaves cDNA under low nitrogen stress.The sequences of primers were designed based on the Csa002986 gene coding region in the cucumber genome database,which is using Primer Premier 5.0.The gene is 1 584 bp and encoding 527 amino acids.The protein encoded by this gene was calculated to be a stable and hydrophilic protein,no transmembrane structure and no signal peptide,and with the protein kinase domain,and EF-hand calcium-binding domain, and serine/threonine protein kinase active site, and N-myristoylation site,and so on.Expression analysis of CDPK in various parts of cucumber under different nitrogen concentration treatment showed that CDPK gene in shoot apex reached the highest expression level,followed by leaves and stems under the nitrogen-free conditions.CDPK gene expression levels in the stem were the highest,followed by shoot tips and leaves under low nitrogen conditions.CDPK gene in shoot apex were the highest expression level,followed by stems and leaves under normal and high nitrogen conditions.CDPK gene expression patterns analysis in leaves showed that the gene is up-regulated under nitrogen-free and low nitrogen conditions,as the nitrogen concentration decreased,CDPK gene expression is increased and is significantly higher than the normal level.The expression of CDPK gene under high nitrogen condition is lower than the normal levels.