小麦TaCIPK2基因克隆及与TaCBLs蛋白互作分析
发布时间：2019-06-20 07:43
【摘要】：以普通六倍体小麦品种德抗961为材料,根据野大麦HbCIPK2的mRNA序列(GenBank序列号JN831652)设计引物,采用同源克隆方法从小麦中克隆TaCIPK2基因(GenBank序列号KU640381),测序结果显示该基因序列全长1421bp,开放阅读框(ORF)1359bp,编码452个氨基酸残基,预测分子量为50.91kD,pI为9.07。氨基酸序列比对结果表明:该基因与HvCIPK2(KP638475.1)、TaCIPK2(KJ561791.1)、HbCIPK2(JN831652.1)的相似度分别为94.69%、96.93%、95.80%,具有高度同源性;系统进化分析表明它们位于相同进化支上,亲缘关系最近。采用Real-timePCR方法分析不同逆境胁迫处理下TaCIPK2基因表达的特异性,200mmol/LNaCl高盐胁迫处理小麦幼苗后根和叶部TaCIPK2基因均上调表达;4℃低温胁迫处理后根部TaCIPK2基因上调表达,而叶部下调表达;100μmol/LABA胁迫处理后根和叶中TaCIPK2均上调表达。为检测TaCIPK2与TaCBL1、TaCBL2、TaCBL6、TaCBL7的相互作用,构建酵母表达载体pGADT7-TaCIPK2,转化酵母Y187感受态细胞;同理pGBKT7-TaCBL1、pGBKT7-TaCBL2、pGBKT7-TaCBL6、pGBKT7-TaCBL7转化到酵母细胞Y2Hgold中,都没有出现自激活活性和毒性现象。共转化的二倍体酵母,只有pGADT7-TaCIPK2×pGBKT7-TaCBL2和pGBKT7-53×pGADT7-T在SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA培养基板上长出的菌落呈现蓝色,说明TaCIPK2能够与TaCBL2相互作用,激活下游报告基因HIS3、AUR1-C、MEL1和ADE2的表达,该研究结果对进一步研究TaCIPK2的功能有一定的指导意义。
[Abstract]:Using common hexaploid wheat variety Dekang 961 as material, primers were designed according to the mRNA sequence of wild barley HbCIPK2 (GenBank serial number JN831652). The TaCIPK2 gene (GenBank serial number KU640381) was cloned from wheat by homologous cloning method. The sequencing results showed that the full length of the gene sequence was 1421bp, the open reading frame (ORF) 1359bp, encoding 452amino acid residues, the predicted molecular weight was 50.91kD, Pi was 9.07. The results of amino acid sequence alignment showed that the similarity between the gene and HvCIPK2 (KP638475.1), TaCIPK2 (KJ561791.1) and HbCIPK2 (JN831652.1) was 94.69%, 96.93% and 95.80%, respectively, which had high homology, and the phylogenetic analysis showed that they were located on the same evolutionary branch and were closely related to each other. The specificity of TaCIPK2 gene expression under different stress treatments was analyzed by Real-timePCR. The expression of TaCIPK2 gene in roots and leaves was up-regulated after 200mmol/LNaCl high salt stress, the expression of TaCIPK2 gene in roots was up-regulated while the expression in leaves was down-regulated after 4 鈩，
本文编号：2503052
[Abstract]:Using common hexaploid wheat variety Dekang 961 as material, primers were designed according to the mRNA sequence of wild barley HbCIPK2 (GenBank serial number JN831652). The TaCIPK2 gene (GenBank serial number KU640381) was cloned from wheat by homologous cloning method. The sequencing results showed that the full length of the gene sequence was 1421bp, the open reading frame (ORF) 1359bp, encoding 452amino acid residues, the predicted molecular weight was 50.91kD, Pi was 9.07. The results of amino acid sequence alignment showed that the similarity between the gene and HvCIPK2 (KP638475.1), TaCIPK2 (KJ561791.1) and HbCIPK2 (JN831652.1) was 94.69%, 96.93% and 95.80%, respectively, which had high homology, and the phylogenetic analysis showed that they were located on the same evolutionary branch and were closely related to each other. The specificity of TaCIPK2 gene expression under different stress treatments was analyzed by Real-timePCR. The expression of TaCIPK2 gene in roots and leaves was up-regulated after 200mmol/LNaCl high salt stress, the expression of TaCIPK2 gene in roots was up-regulated while the expression in leaves was down-regulated after 4 鈩，
本文编号：2503052
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