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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。
According to the sequence of HbCIPK2 mRNA of GenBank (GenBank accession number JN831652), the gene of TaCIPK2 was cloned from wheat by homologous cloning (GenBank accession number KU640381). The sequencing results The full length of this gene was 1421bp and contained 1359bp of open reading frame (ORF) encoding 452 amino acid residues with a predicted molecular weight of 50.91kD and a pI of 9.07. The results of amino acid sequence alignment showed that the similarity of this gene with that of HvCIPK2 (KP638475.1), TaCIPK2 (KJ561791.1) and HbCIPK2 (JN831652.1) was 94.69%, 96.93% and 95.80%, respectively. Phylogenetic analysis shows that they are located on the same clade and have the closest genetic relationship. Real-time PCR was used to analyze the specificity of TaCIPK2 gene expression under different stress conditions. TaCIPK2 gene was up-regulated in roots and leaves after treated with 200 mmol / L NaCl at high salt stress. TaCIPK2 gene was up- While the leaves were down-regulated. After treatment with 100μmol / L LABA, TaCIPK2 was up-regulated in roots and leaves. To detect the interaction of TaCIPK2 with TaCBL1, TaCBL2, TaCBL6 and TaCBL7, the yeast expression vector pGADT7-TaCIPK2 was constructed and transformed into yeast Y187 competent cells. Similarly, pGBKT7-TaCBL1, pGBKT7-TaCBL2, pGBKT7-TaCBL6 and pGBKT7-TaCBL7 were transformed into yeast Cell Y2Hgold, no self-activation activity and toxicity. Co-transformed diploid yeast with only pGADT7-TaCIPK2 × pGBKT7-TaCBL2 and pGBKT7-53 × pGADT7-T grown on SD / -Ade / -His / -Leu / -Trp / X-α-Gal / AbA medium The blue colonies showed that TaCIPK2 interacted with TaCBL2 and activated the expression of downstream reporter genes HIS3, AUR1-C, MEL1 and ADE2. The results of this study may be helpful to further study the function of TaCIPK2.