寄主诱导枯萎病菌FoERG6基因沉默提高香蕉抗病性的机理研究

Fusarium wilt has become one of the most destructive diseases for banana production in the world now. So creating disease resistant germplasm and exploring its resistance mechanism has theoretical and practical significance for banana production. Our previous study has found host-induced gene silencing of the ergosterol biosynthesis pathway gene FoERG6 that give high resistance against Fusarium wilt in banana. It will be helpful to find the disease resistance strategy by exploring the disease resistance mechanism in transgenic banana. This project aims to clarify the effect of FoERG6 on growth and pathogenicity of Foc TR4 by mutation analysis. The regularity and mechanism of the germination, growth and invasion of pathogen in the target siRNA transgenic plants will be examined with GFP marked-Foc TR4, and the banana small RNA extract bacteriostatic experiment will be performed concurrently. The Results will decode the resistance mechanism of FoERG6 dsRNA expressed-banana to Fusarium wilt. Moreover, the outcome of this project will not only provide reference for disease-resistance breeding in banana using HIGS technology, but also provide theoretical foundation for developing double strands RNA fungicide to control crop fungal diseases.

枯萎病是危害香蕉产业的世界难题，创制抗病香蕉新种质并探究其抗病机理具有重要理论和现实意义。申请人前期以香蕉枯萎病菌Foc TR4麦角甾醇合成相关基因FoERG6为靶标，通过寄主诱导基因沉默技术（HIGS）在香蕉中表达FoERG6 dsRNA，显著提高了香蕉的抗病性，揭示其抗病机理有助于更好地寻找抗病策略。本项目拟利用突变体，研究FoERG6对Foc TR4生长发育及致病力的影响，明确FoERG6在Foc TR4中的生物学功能；利用GFP标记的病原菌接种抗病转基因香蕉，研究转基因植株中表达的靶标siRNA抑制病原菌萌发、生长及侵入的规律及机理，同时结合香蕉小RNA提取液体外抑菌效果评价，系统阐明表达FoERG6 dsRNA提高香蕉抗病性的机理。项目的实施将不但为利用HIGS技术进行香蕉抗病分子育种提供参考，并将为后续以病原菌基因为靶标开发dsRNA杀菌剂防控真菌病害提供理论指导。

枯萎病是危害香蕉产业的世界难题，创制抗病香蕉新种质并探究其抗病机理具有重要理论和现实意义。申请人前期以香蕉枯萎病菌Foc TR4麦角甾醇合成相关基因FoERG6为靶标，通过寄主诱导基因沉默技术（HIGS）在香蕉中表达FoERG6 dsRNA，显著提高了香蕉的抗病性，揭示其抗病机理有助于更好地寻找抗病策略。本项目利用overlap PCR方法获得Foc TR4突变体，明确了FoERG6是Foc TR4生长发育和致病力的关键基因；利用GFP标记的病原菌接种抗病转基因香蕉，通过激光共聚焦显微镜观察发现，转基因香蕉植株可显著抑制病原菌的萌发、生长及侵入；进一步对转基因/野生植株进行小RNA测序，发现FoERG6衍生的特异性siRNA在转基因植株的根系中被显著富集，其长度主要分布在18-25bp之间，这些发现进一步证实了靶基因FoERG6衍生的特异性siRNA在转基因系中的表达是决定转基因香蕉植株抗病性提高的主要因素；最后，通过提取转基因和野生型植株叶片中的小RNA进行平板抑菌实验，发现转基因植株中小RNA提取液对Foc TR4生长发育有显著的抑制作用。综上所述：FoERG6在香蕉枯萎病菌Foc TR4的致病过程中起到关键作用，利用HIGS技术沉默FoERG6可显著提高香蕉的抗枯萎病能力。上述研究成果为后续以病原菌FoERG6基因为靶标开发dsRNA杀菌剂防控真菌病害提供理论指导。