CCCH型锌指蛋白TaZF1调控小麦对赤霉病抗性反应的分子机制研究

Fusarium head blight （FHB）, which is a type epidemic disease in cereal crops, constitutes a serious threat to grain yield and quality causing economic losses as well as food safety. Wheat zinc finger protein plays a important role in resistance to against FHB, but molecular mechanism of zinc finger conferring to FHB resistance is poorly understood. In the wheat-Thinopyrum ponticum shortened segments, we found that the near isogenic lines were resistant and susceptible to FHB, then comparative proteomic analysis by iTRAQ (isobaric tags for relative and absolute quantification) showed that expression of zinc finger and PR5 (pathogenesis-related 5) upaccmulated upon F.graminearum. There are three CCCH type domains on the zinc finger protein TaZF1 sequence, while compared to in the susceptible lines the expression of TaZF1 and PR5 in the resistant lines upaccumulated on the transcription level. Next, TaZF1 was determined to confer to FHB resistance following the silence and over-expression methods. We put forward clearly transcription and functional domain of TaZF1 in vivo and in vitro. Further, we carry out subcellular localization and interaction between TaZF1 and PR5. TaZF1 mediated molecular mechanism of the resistance response may expend knowledge of resistance mechanism, help effectively application of resistance resoure, and promote genetic improvement wheat against FHB.

小麦赤霉病是麦类作物中典型的流行性病害，严重威胁着小麦产量、品质和食品安全。锌指蛋白在抵御小麦赤霉病侵染过程中发挥重要作用，但是锌指蛋白的抗病分子机制还不十分清楚。前期，我们利用小麦和长穗偃麦草杂交并结合ph1b基因诱导创制出赤霉病抗、感近等基因系，通过比较蛋白质组学挖掘出受病原菌诱导表达的锌指蛋白TaZF1和病程相关蛋白PR5。明确TaZF1属于典型的CCCH型锌指蛋白；在转录水平上TaZF1和PR5稳定上调表达。在此基础上，我们拟从瞬时沉默和过表达两个方面鉴定TaZF1的抗病功能，同时分析TaZF1过表达材料受病原侵染后内源性激素的响应；进而从体内、体外两个方面明确TaZF1的转录活性及其与启动子结合的功能区域。并结合锌指蛋白TaZF1与PR5的互作，解析TaZF1参与赤霉病抗性反应的分子机制，拓展对赤霉病抗性机理的认识，有助于赤霉病抗源有效利用，促进小麦抗赤霉病遗传改良。

小麦赤霉病主要是由禾谷镰刀菌引起的毁灭性病害，严重威胁着小麦产量、品质和食品安全。锌指蛋白在调控防卫基因表达和抗性反应中发挥重要作用。本研究首次鉴定了Fhb7近等基因系抗病差异，描述了发病初期小麦麦穗形态变化。从抗病材料中鉴定出11,070个蛋白质和762个差异表达蛋白质，从整体上描绘了小麦赤霉病抗性蛋白质网络图。发现了TaTLP1和CCCH锌指蛋白（TaZF1）在抗病材料中转录水平和蛋白质水平上上调表达。从整体上描绘了赤霉病抗性相关蛋白质基本信息，为进一步研究抗赤霉病基因奠定了坚实基础。TaZF1氨基酸序列具有锌指蛋白的特征结构--三个CCCH结构域，属于典型的CCCH型锌指蛋白。蛋白质TaZF1A与TaZF1B既可以在体内互作，也可以在体外互作。蛋白质TaZF1B与TaTLP1启动子的-654至-6区域存在特异性结合，进而证实了启动子的-654至-6区域是蛋白质TaTLP1的作用元件，并且蛋白质TaZF1B调控TaTLP1的转录表达，从而揭示了锌指蛋白调控抗病基因的分子机制。本研究获得转TaZF1A、TaZF1B基因小麦材料，抗病性鉴定表明，转TaZF1A、TaZF1B基因小麦材料对小麦赤霉病表现出抗性。总之，解析TaZF1参与赤霉病抗性反应的分子机制，丰富对赤霉病抗性机理的知识，为赤霉病抗源有效利用、促进小麦抗赤霉病遗传改良提供了新的思路。