• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

NF-YC15转录因子调控木薯抗细菌性枯萎病的分子机制研究

Molecular Mechanism of NF-YC15 Transcription Factor in Regulating Resistance to Bacterial Blight of Cassava

  • 摘要:
    目的 探明NF-YC15转录因子调控木薯(Manihot esculenta)抗细菌性枯萎病的分子机制。
    方法 利用酵母双杂交筛选MeNF-YC15的互作蛋白,通过双分子荧光互补、Pull down等试验进一步进行互作验证。在木薯中构建超表达植株,通过接种细菌性枯萎病致病菌后的表型鉴定,分析蛋白互作如何调控木薯的抗病能力。采用转录组测序,鉴定MeNF-YC15调控的下游抗病基因,通过定量PCR试验分析各超表达植株中下游基因的表达水平,解析MeNF-YC15通过蛋白互作调控木薯抗病的分子机制。
    结果 筛选到1个MeNF-YC15的候选互作蛋白,茉莉酸信号转导途径中的关键抑制因子-茉莉酸ZIM结构域蛋白(Jasmonate ZIM-domain2.2, MeJAZ2.2),并验证了这两个蛋白在植物体内外是相互作用的。进一步通过抗病功能分析发现MeNF-YC15超表达植株的细菌数和病斑面积显著低于对照,MeJAZ2.2超表达植株的细菌数和病斑面积显著高于对照。MeNF-YC15-MeJAZ2.2共超表达株系的细菌数量和病斑面积显著高于MeNF-YC15超表达植株,且显著低于MeJAZ2.2超表达植株。进一步的研究结果表明MeNF-YC15正调控3个茉莉酸下游抗病基因WRKY68Matrix metalloproteinase-3MMP-3)、Leucine-rich repeat receptor-like kinasesLRR-RLK)的表达。同时,MeJAZ2.2能够抑制WRKY68MMP-3LRR-RLK的表达。
    结论 MeNF-YC15与MeJAZ2.2的蛋白互作共同调控茉莉酸下游的抗病基因的表达,通过茉莉酸途径调控木薯对细菌性枯萎病的抗性,为解析MeNF-YC15通过植物激素调控木薯抗病的分子机制提供新的方向与思路。

     

    Abstract:
    Objective Molecular mechanism of NF-YC15 transcription factor associated with the regulation of bacterial blight resistance of Manihot esculenta was investigated.
    Methods Yeast two-hybrid was employed to determine the interacting proteins with the MeNF-YC15 transcription factor. Bimolecular fluorescence complementation and pull-down experiments were conducted to verify the interactions. On overexpressed cassava, bacterial blight pathogens were inoculated to study disease resistance of the plant. Through transcriptome sequencing, downstream disease-resistant genes regulated by MeNF-YC15 were identified, and the molecular mechanism analyzed by quantitative PCR.
    Results The key inhibitor of jasmonic acid ZIM domain protein MeJAZ2.2 in the signal transduction pathway was identified to be the sole protein interacting with MeNF-YC15, which was further verified in vivo and in vitro in plants. The bacteria and disease lesion areas on the significantly taller MeNF-YC15-overexpressed plants were significantly smaller than those on the MeNF-YC15-MeJAZ2.2-co-overexpressed counterparts and control. MeNF-YC15 activated the downstream disease resistant WRKY68, matrix metalloproteinase-3 (MMP-3), and leucine-rich repeat receptor-like kinases (LRR-RLK) of jasmonic acid, and in contrast, MeJAZ2.2 inhibited the expressions.
    Conclusion MeNF-YC15 and MeJAZ2.2 antagonistically regulated the expression of downstream disease-resistant genes of jasmonic acid and the resistance of cassava plant to the bacterial blight through the jasmonic acid pathway.

     

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