黄瓜CC型谷氧还蛋白CsGRX1负调控灰霉病抗性反应的分子机制

The Botrytis cinerea is one of the common diseases under the protect cultivation, and the molecular mechanism of defense response in cucumber to Botrytis cinerea was not clear. The CC-type glutaredoxin was the key regulator to the infection of Botrytis cinerea. It was found that the expression of CC type CsGRX1 in cucumber was responsive to the infection of Botrytis cinerea, and the overexpression of CsGRX1 in Arabidopsis was more susceptible to Botrytis cinerea. It was assumed that CsGRX1 was very one of important negative regulators to the infection of Botrytis cinerea. Up to now, we didn’t know the mechanism of how CsGRX1 regulated the response to Botrytis cinerea. The expression pattern of CsGRXs using Real-time PCR, and the suppression function of CsGRXs to the JA signal pathway using transient protoplast expression system and interaction protein analyzing using Yeast-Hybrid system were investigated in 14-1 cucumber seedlings during infection of Botrytis cinerea. The enzyme activity site and ALWL motif of CsGRX1 were mutated respectively and the function of mutated gene was analyzed. The overexpression vector, RNAi vector will be constructed using the gateway technology and then they will be transformated into the cucumber inbred line 14-1 and Arabidopsis respectively. The complementation experiments using grxs13-1 knocked down Arabidopsis was carried out to identified the function of CsGRX1, and the disease resistance, the redox status change and the effects to the JA signal pathway were analyzed to elucidate it's negative regulation mechanism to Botrytis cinerea, which will put the basis of understanding resistance response of cucumber to Botrytis cinerea.

灰霉病是设施黄瓜常见病害，但黄瓜防御灰霉菌侵染的调控机制还不清楚。CC型谷氧还蛋白基因CsGRX是植物应对灰霉菌侵染的关键调控因子，我们发现，黄瓜CC型CsGRX1基因响应灰霉菌侵染、过表达使转基因株感病，推测其在灰霉病抗性反应中起重要的负调控作用，但其机理尚不明确。本项目拟以黄瓜抗灰霉病种质14-1为试材，通过定量PCR分析各CC型CsGRXs对灰霉病的响应、原生质体暂态表达分析各CsGRXs的作用、酵母双杂交分析CsGRX1在灰霉侵染过程中的互作蛋白、CsGRX1基因关键酶活性位点及ALWL结构域突变研究对基因功能的影响、用CsGRX1基因对拟南芥grxs13-1突变体进行互补及CsGRX1基因过表达和抑制表达对黄瓜进行遗传转化，研究CsGRX1对灰霉病抗病性、氧化还原状态、JA含量及JA通路基因表达的影响，阐明CsGRX1负调控灰霉病抗性反应的机制，为改良黄瓜灰霉病抗病性奠定基础。

黄瓜灰霉病是由半知菌亚门葡萄孢属灰葡萄孢菌引起的一种真菌性病害。该病原菌以营腐生生活为主，几乎可以危害黄瓜地上部所有器官，其中以幼果和幼茎受害最重。随着设施栽培面积的扩大和栽培年限的增加，灰霉病有逐年加重的趋势。研究表明，CC型谷氧还蛋白基因GRX在植物灰霉菌侵染防御反应中起重要调控作用。本项目以黄瓜抗病野生型种质和感病突变体为材料进行转录组测序、过表达转基因植株灰霉病抗性分析、酵母双杂交筛选及BIFC验证、原生质体暂态表达分析、GRX关键酶活性位点及ALWL结构域突变等对基因功能的影响，并通过互补实验等研究了CC型谷氧还蛋白基因参与灰霉菌抗性反应的分子机制。结果表明，灰霉菌侵染引起抗病材料中激素信号传导途径和植物与病原体互作的差异基因表现为富集，而在感病材料中调节氧化还原的差异基因表现为富集，其中调节氧化还原的谷氧还蛋白基因CsGRX1在感病突变体中被显著上调。灰霉菌侵染后，16个CC型谷氧还蛋白基因家族成员中与拟南芥谷氧还蛋白基因AtGRX480高度同源的CsGRX1、CsGRX4、CsGRX7等表达明显上调。以CsGRX1为诱饵进行的酵母双杂交筛选到AP2/ERF转录因子CsERF1与其互作，且通过酵母双杂交和BIFC进行了验证。结果表明，CsGRX1可直接结合D亚组bZIP转录因子家族部分成员，或者CsGRX1和AP2/ERF转录因子CsERF1结合，CsERF1再结合D亚组bZIP转录因子家族成员，并引起植物体内活性氧爆发，植物激素JA降低，从而便利于灰霉菌的侵染，导致植物的感病性。利用拟南芥原生质体瞬时表达体系分析了CsGRX1、CsGRX4及CsGRX7重要酶活性位点及C端重要结构域ALWL的作用，结果表明CsGRX1、CsGRX4及CsGRX7可抑制ORA59启动子的表达，即抑制JA信号通路，但酶活性位点和ALWL结构域点突变不影响其与CsERF1和CsbZIPs的互作关系。本项目明确了黄瓜CC型谷氧还蛋白CsGRX1等负调控灰霉病抗性反应的分子机制，为明确黄瓜防御灰霉菌侵染的抗性反应的分子机制奠定基础。....