菊花CmMLO17调控链格孢菌侵染的分子机制

Chrysanthemum（Chrysanthemum morifolium）is one of the ten traditional famous flower and one of the four important cut flowers all over the word, which is of high ornamental and economic values. The black leaf spot disease caused by the fungi Alternaria tenuissima, is one of the most severe diseases during chrysanthemum production. We have previously screened candidate CmMLO17 from full transcripts generated from chrysanthemum infected by Alternaria tenuissima, and get the stably over-expressed and RNAi of CmMLO17 in chrysanthemum. When CmMLO17 was stably over-expressed in chrysanthemum, the plants showed an increased sensitivity to A. tenuissima, and the RNAi of CmMLO17 in chrysanthemum lead to resistant to A. tenuissima. Based on the above, we will carry out the research how chrysanthemum CmMLO17 responds to A. tenuissima. The main contents are as follows: First, we aim to identify its function by analyzing the expression pattern of the relevant CmMLO17 and its subcellular localization; Second, we will screen and verify genes regulated downstream by CmMLO17 by bio informatics analysis; Third, we will try to obtain a clear post-translational regulatory mechanism of CmMLO17 by using the yeast-two-hybrid to screen the proteins interacting with CmMLO17, and by BiFC assays, together with Co-IP assays to validate the interation both in vitro and in vivo. Taking all these experimental evidence, we will identify the function and clarify the mechanism of CmMLO17 involved in the response of chrysanthemum to the infection of A. tenuissima. Our proposal can enrich molecular mechanisms of chrysanthemum in response to pathogen infection, which meanwhile will provide a theoretical basis for the control of leaf spot disease caused by A. tenuissima and the disease resistant breeding of Chrysanthemum.

菊花作为我国十大传统名花和世界四大切花之一，在花卉生产中占有重要地位；菊花黑斑病是其生产上的重要病害之一，严重影响菊花的观赏价值和经济效益。本申请项目是在筛选出受链格孢菌强烈诱导表达的CmMLO17基因，获得CmMLO17转基因菊花，初步明确CmMLO17基因参与黑斑病菌侵染菊花的功能基础上，开展菊花CmMLO17响应黑斑病菌侵染的调控机制研究，具体包括三个方面：一是通过组织表达特征、亚细胞定位分析明确CmMLO17基本功能；二是通过生物信息学筛选CmMLO17下游基因，并描述病原菌防御调控网络；三是通过酵母双杂交技术筛选CmMLO17互作蛋白，并进行验证；同时利用菊花转基因技术，组织表达特征分析等技术手段明确CmMLO17互作蛋白对链格孢菌的响应情况。本项目的实施可阐明CmMLO17参与菊花对链格孢菌侵染的响应机制，丰富菊花抗病分子机理，并为科学防治菊花黑斑病及菊花抗病育种提供理论指导。

菊花（Chrysanthemum morifolium）是我国十大传统名花和世界四大切花之一，具有很高的观赏与经济价值。菊花生产过程中常会有很多病害发生，其中由链格孢属真菌引起的黑斑病是最严重病害之一。本研究挖掘黑斑病抗性相关基因并揭示其抗病分子机理，对菊花黑斑病抗性分子改良具有重要的理论和实践价值。 研究发现，CmMLO17在链格孢菌侵染时响应强烈，构建了基因沉默表达载体pMDC32-amiCmMLO17并进行菊花遗传转化，成功获得了9个沉默株系。实时荧光定量结果显示，沉默株系中CmMLO17基因的表达量均降低。转基因植株接种链格孢菌发现，CmMLO17基因沉默增强了菊花对链格孢菌的抗性。随后，以CmMLO17的C端为“诱饵”，利用酵母双杂交系统筛选菊花-链格孢菌互作的文库，获得候选基因CmKIC。KIC是与类驱动蛋白钙调素结合蛋白KCBP互作的蛋白。利用转录组数据并结合RACE-PCR的方法克隆得到CmKIC，通过酵母双杂交、双分子荧光互补（BiFC）技术证实了CmMLO17和CmKIC互作的真实性，实验结果显示互作发生在细胞膜上。亚细胞定位分析发现，CmKIC是细胞膜和细胞核双定位。组织定量结果表明，CmKIC在花中的表达量最高，其次是茎和根。链格孢菌接种处理后发现，CmKIC受到强烈诱导表达。另外，构建了CmKIC的超表达载体pMDC43-CmKIC和基因沉默载体pMDC32-amiCmKIC，并进行菊花遗传转化和转基因植株抗性接种鉴定，发现CmKIC基因超表达提高了菊花对链格孢菌的敏感性，而基因沉默则显著增强了对链格孢菌的抗性。以上结果表明，CmMLO17和CmKIC可能通过互作来共同调控Ca2+信号路径基因的表达，从而负向调控植物的抗病性。综上，CmMLO17通过与钙调素结合蛋白CmKIC互作介导了菊花对链格孢菌的感病性。