玉米穗粒腐病表观遗传修饰及关键调控因子的鉴定

Ear rot is a destructive disease in maize caused by Fusarium verticillioides (FV), which results in reduction of grain yield and increase of risks in raising livestock by mycotoxins production. Especially, a high incidence of ear rot occurs in the moist and humid regions of Southwest China, as well as other regions with similar longitude in other countries. In recent years, considerable progresses have been made in understanding the resistant system involved in maize ear rots infected by FV, including physiological and biochemical characteristics, resistant genetic relationship, QTL, isolation of disease resistance genes, characterization of defense responses, and elucidation of signal transduction leading to activation of defense responses. In addition, plant epigenetics research has become the new focus today according to biology development. Based on previous research, we used two maize cultivars, Bt-1 and Ye478 as completely resistant and highly susceptible to FV due to many years evaluation of field in southwest of China in this study to characterize the specific host response to FV infection. To better know the processes involved of defense in maize ear rot upon the FV infection, the DNA methylation change was investigated through Illumina Methylated DNA Immunoprecipitation Sequencing (MeDIP Sequencing) to screen out special differentially regulated genes. At the same time, the modulation of histone modifications were detected by Chromatin Immunoprecipitation (ChIP). The key transcription factors were screened out based on the association analysis from the data of upper results. The function of the key transcription factors by using TALEN targeted gene knockout technology. This study could provide important information on understanding the defense mechanisms to combat invasion in maize ear rot.

玉米穗粒腐病是世界范围里广泛发生的一种真菌性病害，特别是在我国高温高湿的西南地区该病害流行甚广，造成了玉米产量的巨大损失和品质的严重下降，国内外学者从生理生化、抗性遗传关系、QTL定位、抗病基因克隆、信号转导、抗病分子机理等方面展开了广泛而深入的研究。随着生物科学的发展，植物表观遗传学的研究已成为当今新的焦点，本项目拟在前期研究的基础上，以玉米抗（Bt-1）、感（掖478）穗粒腐病自交系为研究材料，利用高通量甲基化技术检测自交系在病原胁迫下DNA甲基化的动态变化情况，获得甲基化水平存在差异的基因片段；同时采用组蛋白检测技术解析病原胁迫后组蛋白修饰特征。通过关联分析，确定引起玉米穗粒腐病的关键调控因子，采用TALEN靶向基因敲除技术对关键调控因子进行功能验证，探明其中关键调控分子的功能、结构及相互作用网络，进而为玉米穗粒腐病抗病分子机制的研究奠定基础。

针对玉米穗腐病研究中的优质抗病种质资源缺乏、优异抗病基因难以挖掘和关键调控因子难以获得等瓶颈问题，通过本项目的实施，取得了一些重要结果。.①以人工接菌方式进行田间和室内抗病性鉴定和筛选，从玉米IBM Syn10 DH群体中获得稳定的极端抗（感）玉米穗腐病种质资源IBM81和IBM85。.②利用RNA-seq测序，在抗、感材料苞叶组织中分别得到差异表达基因495、395个，共有的差异表达基因186个；而在抗、感材料花丝组织中分别发现差异表达基因84、30 个，其中共有基因6个。分析发现众多基因参与水杨酸及茉莉酸介导信号通路、生物性刺激应答反应、新陈代谢及次生代谢物的生物合成等途径，尤其是检测到PR1家族在玉米穗腐病抗病过程中扮演重要角色。.③利用全基因组DNA甲基化测序技术对抗、感材料胁迫前后苞叶组织DNA甲基化水平进行检测，获得了玉米穗腐病表观调控的甲基化谱；在抗、感材料中分别鉴定出特异甲基化基因754、596个，其中共有特异甲基化基因 56个，并浅析了玉米穗腐病抗病分子机制。.④综合利用RNA-seq和甲基化数据，分别在抗、感材料中鉴定特异差异基因27、和10个，发现与玉米抗病密切相关特异差异基因13个，并进一步分析了特异表达差异基因在不同通路中的富集情况，从中挖掘出玉米穗腐病抗病调控关键因子基因片段4个。.⑤以IBM81（抗）和IBM85（感）为亲本进行玉米穗腐病抗病QTL初定位及精细定位，在第7染色体发现一个贡献率为33.26%的主效QTL，通过图位克隆方式挖掘出一抗病候选基因ZmLBP，并对该候选基因进行CRISPR/Cas9和过表达功能鉴定。.⑥通过项目实施，获得关键调控因子，并浅析了玉米穗粒腐病抗病分子机制，为下一步培育抗玉米穗腐病新品种奠定了理论基础；已发表论文2篇，培养博士1名、硕士2名，在读博士研究生1名、硕士研究生2名。