王杨铭, 王晓琼, 郑贝贝, 韩玲玲, 刘松涛, 张亚菲, 许海霞, 程西永. 2023: 小麦TaCIPK15基因克隆及与TaCBLs蛋白的互作分析. 南方农业学报, 54(4): 982-992. DOI: 10.3969/j.issn.2095-1191.2023.04.002
引用本文: 王杨铭, 王晓琼, 郑贝贝, 韩玲玲, 刘松涛, 张亚菲, 许海霞, 程西永. 2023: 小麦TaCIPK15基因克隆及与TaCBLs蛋白的互作分析. 南方农业学报, 54(4): 982-992. DOI: 10.3969/j.issn.2095-1191.2023.04.002
WANG Yang-ming, WANG Xiao-qiong, ZHENG Bei-bei, HAN Ling-ling, LIU Song-tao, ZHANG Ya-fei, XU Hai-xia, CHENG Xi-yong. 2023: Cloning of TaCIPK15 gene and interaction analysis with TaCBLs protein in wheat. Journal of Southern Agriculture, 54(4): 982-992. DOI: 10.3969/j.issn.2095-1191.2023.04.002
Citation: WANG Yang-ming, WANG Xiao-qiong, ZHENG Bei-bei, HAN Ling-ling, LIU Song-tao, ZHANG Ya-fei, XU Hai-xia, CHENG Xi-yong. 2023: Cloning of TaCIPK15 gene and interaction analysis with TaCBLs protein in wheat. Journal of Southern Agriculture, 54(4): 982-992. DOI: 10.3969/j.issn.2095-1191.2023.04.002

小麦TaCIPK15基因克隆及与TaCBLs蛋白的互作分析

Cloning of TaCIPK15 gene and interaction analysis with TaCBLs protein in wheat

  • 摘要: 【目的】克隆小麦丝氨酸/苏氨酸蛋白激酶(CIPK) TaCIPK15基因,并分析该基因编码的蛋白与小麦钙调素B类似蛋白(TaCBLs)的互作关系,为深入研究TaCIPK15基因在小麦逆境胁迫应答机制中的作用提供参考。【方法】通过PCR扩增小麦TaCIPK15基因编码区(CDS)全长序列,对其进行生物信息学分析,采用实时荧光定量PCR(qRTPCR)研究其对逆境的响应,通过酵母双杂交技术和双分子荧光互补技术研究TaCIPK15与TaCBLs的互作关系。【结果】克隆获得的小麦TaCIPK15基因CDS序列全长1302 bp,编码433个氨基酸残基,与NCBI数据库中参考序列XR_006454427.1相似性为100%,蛋白分子量为48.48 kD,原子总数为6896,分子式为C2166H3487N599O628S16,理论等电点(pI)为9.38,不具备跨膜结构和信号肽。TaCIPK15蛋白含有CIPK家族保守NAF结构域和激活环,与大麦HvCIPK15(XP_044946251)氨基酸序列最为相似且亲缘关系最近。盐胁迫(NaC1)下TaCIPK15基因呈下调表达趋势;在冷胁迫(4℃)下,根系中TaCIPK15基因上调表达;脱落酸(ABA)处理后,根系和叶片中12 h的表达量均上调。TaCIPK15与TaCBL1和TaCBL2在酵母双杂交试验中能在四缺培养基SD/-Trp/-Leu/-Ade/-His/X-α-gal/ABA上长出蓝色菌斑;在双分子荧光互补试验中,TaCIPK15与TaCBL1在细胞核上检测到黄色荧光,TaCIPK15与TaCBL2在细胞质和细胞膜上检测到黄色荧光。【结论】TaCIPK15基因属于CIPK家族,TaCIPK15与TaCBL1和TaCBL2存在互作关系且在细胞内的互作位置不同,并在小麦响应高盐胁迫、冷胁迫和ABA胁迫的Ca2+信号转导过程中发挥作用。

     

    Abstract: 【Objective】The purpose of the study was to clone the wheat serine / threonine protein kinases(CIPK) TaCIPK15 gene and analyze the interaction between the protein encoded by TaCIPK15 gene and wheat calcineurin B-like protein(TaCBLs),so as to provide reference for the in-depth study of the function of TaCIPK15 gene in the mechanism of response to adversity stress in wheat.【Method】The full length of coding region sequence(CDS)of wheat TaCIPK15 gene was amplified by PCR and analyzed by bioinformatics methods. The real-time fluorescence quantitative PCR( qRTPCR)method was used to study its response to adversity,and the interaction between TaCIPK15 and TaCBLs was studied by yeast two-hybrid and bimolecular fluorescence complementation techniques.【Result】The CDS sequence of wheat TaCIPK15 gene obtained by cloning was 1302 bp in length,encoding 433 amino acid residues,which was 100% similar with the reference sequence XR_006454427.1 in the NCBI database. TaCIPK15 protein had a molecular weight of 48.48 kD,with a total number of atoms of 6896,a molecular formula of C2166H3487N599O628S16,and theoretical isoelectric point(pI) of 9.38. TaCIPK15 protein had no transmembrane structure and signal peptide. TaCIPK15 protein contained conserved NAF structural domain and activation loop of CIPK family,and the amino acid sequence was most similar and the closest relative to that of barley HvCIPK15(XP_044946251). The expression of TaCIPK15 gene was down-regulated under salt stress(NaCl). The expression of TaCIPK15 gene was up-regulated in root under cold stress. The expression was up-regulated in root and leaf at 12 h after abscisic acid(ABA)treatment. TaCIPK15,TaCBL1 and TaCBL2 showed the ability to grow blue plaques on four-deficient media SD/-Trp/-Leu/-Ade/-His/X-α-gal/ABAin yeast two-hybrid test. In the bimolecular fluorescence complementation test,TaCIPK15 and TaCBL1 detected yellow fluorescence in the nucleus,and TaCIPK15 and TaCBL2 detected yellow fluorescence in the cytoplasm and cell membrane.【Conclusion】The TaCIPK15 gene belongs to CIPK gene family. TaCIPK15 interacts with TaCBL1 and TaCBL2 and has different interaction positions in cells. TaCIPK15 gene plays a role in Ca2+ signaling transduction in wheat in response to high salt stress,cold stress and ABA stress.

     

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