桃褐腐病菌Mona元件的生物学功能验证及其调控MfCYP51基因超量表达的分子机理研究

Brown rot is one of the most important diseases of peach, it can cause damage on both pre- and post-harvest peach.Becasue DMI fungicides are widely used for controlling brown rot, the pathogen has developed resistance to DMI fungicides and caused the management failure. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp element (Mona), and the MfCYP51 gene is constitutively over-expressed in DMI resistant isolates but not in sensitive isolates.In this project, the biological function of Mona element will be proved by site-directed knockout of Mona element and then recovered by transforming the normal Mona element (Mona+MfCYP51) into transformants or sensitive isolates. At the same time, we are going to carry out the yeast one-hybrid to get the corresponding transcription factor using the Mona element as bait. The molecular mechanism of how Mona control the over-expression of MfCYP51 gene may be demonstrated based on the activities of recognition and interaction between the transcription factor and Mona element. Finally, the transcription factor and Mona will be recombinated into same expression vector and transformed into yeast.This project is not only scientifically significant,the resulted engineering strains are valuable in production of functional genes and demonstration of new gene functions.

桃褐腐病是严重危害桃生产的重要病害,在采前及采后均能造成严重经济损失。由于DMI杀菌剂的长年使用，桃褐腐病菌Monilinia fructicola产生了对DMI杀菌剂的抗性并导致重大经济损失。研究发现抗性菌株MfCYP51基因的上游调控区都存在一个65bp的DNA插入片断,称为Mona元件。实时荧光定量PCR分析表明DMI抗性菌株中MfCYP51基因都会超量表达。本项目拟从抗性菌株中定点敲除Mona元件，然后再用Mona元件回复敲除子或转化敏感菌株以验证其生物学功能。同时运用酵母单杂交技术调取与Mona元件识别、互作的转录因子，阐明Mona元件调控下游基因超量表达的分子机理。最后把转录因子及Mona元件整合到同一表达载体上，构建高效表达工程菌株。该项目的实施，不仅能从分子水平阐明桃褐腐菌对DMI杀菌剂抗性的机理,工程菌株在重要功能基因产物生产及病原真菌基因功能分析等方面有重大利用价值。

本研究通过实验证明了桃褐腐病菌Monilinia fructicola中Mona元件具有启动子活性，同时证明了其活性区域为TAG TTA TAT ATA CTC CTC CT。通过从抗性菌株中敲出Mona元件及将Mona元件定点转化进敏感菌株中，证明了Mona元件通过调控下游的MfCYP51基因上调表达而产生对DMI杀菌剂的抗性。根据Mona元件及其两端的序列，设计开发了一个基于LAMP扩增的褐腐病菌抗DMI杀菌剂的检测试剂盒。利用该试剂盒，基层农技人员或种植户可简单利用水浴锅即可在两小时内检测褐腐病菌对DMI杀菌剂的抗性，该技术有望在生产中得到广泛利用。另外，在该基金及其它相关基金的资助下，本研究组也对其它核果类果树如李、杏、樱桃等的褐腐病菌种群组成进行了较系统的分析研究，取得了很好的研究结果。研究结果得到了同行专家的广泛认可，受邀在2016年中国植物病理学会学术年会上做大会报告，同时在《植物病理学报》上对我国的褐腐病研究做特邀专题评述。