RNAi介导的转S1基因大豆对SMV广谱抗性启动机制的解析

Soybean mosaic virus (SMV) is the most prevalent viral pathogen of soybean and there are twenty-two SMV strains, which are controlled by the corresponding resistance genes in China. To pyramid all these resistance genes into one breeding line is difficult, and SMV appeared to break the original resistance by high-speed variation. It is an effective solution to explore the RNA interference (RNAi) mediated broad-spectrum resistance genes, but the initiated mechanisms for RNAi-mediated broad-spectrum resistance to SMV in transgenic soybean is not clear at present. We have found the transient expression of the RNAi gene S1 improved the resistance to SMV in tobacco. Meanwhile, S1-transgenic soybean were immune following inoculation with five SMV strains separately. Furthermore, sRNA sequencing suggested that different DCL genes maybe involved in the RNAi-mediated resistance to SMV in soybean. Here, we will verify the broad-spectrum resistance of S1-transgenic soybean to all twenty-two SMV strains. Furthermore, to identify the key DCL genes in RNAi-mediated resistance to SMV, we will knock out each DCL gene of S1-transgenic soybean by CRISPR/Cas9 system, and then evaluate the SMV resistance for DCL mutants. In addition, small RNA sequencing will be performed in S1-transgenic soybean and its DCL gene mutants to reveal the characteristics of siRNAs, which are derived from specific hpRNAs. In this study we expect to generate transgenic soybean conferring broad-spectrum resistance to SMV, and then elucidate the synthetic mechanism of siRNAs derived from SMV, which are mediated by soybean DCL. This study will provide a theoretical basis for improvement soybean antiviral ability by RNAi mechanism.

SMV是我国大豆全国性重大病害，共有22个株系，分别由相应抗性基因控制。育种中要聚合多个抗病基因于一体难度大，且SMV本身多变。基于RNAi介导的广谱抗性基因的发掘是有效解决途径，但目前RNAi介导的转基因大豆对SMV广谱抗性启动机制知之甚少。我们前期研究发现瞬时表达干扰基因S1可使烟草抗SMV，转S1基因大豆初步可兼抗5个SMV株系，sRNA分析启示不同DCL基因参与大豆体内RNAi介导的抗SMV通路。据此，本项目拟鉴定转S1大豆对22个SMV株系的广谱抗性；应用CRISPR/Cas9敲除转S1大豆DCL基因并评估其抗性，挖掘转S1大豆抗SMV关键DCL基因；对转S1大豆及其DCL突变体sRNA测序分析，揭示DCL切割hpRNA形成的siRNAs的特征。本项目有望获得广谱抗SMV转基因大豆材料，阐明大豆DCL介导SMV来源的siRNAs合成机制，为利用RNAi改良大豆抗病毒提供理论依据。

大豆花叶病毒（SMV）病是世界大豆产区重大病毒病害，导致大豆产量损失和种子质量降低，严重影响大豆的生产和安全。基于10个鉴别能力强、反应稳定的鉴别寄主，我国大豆产区25个省(市)的SMV被划分为22个株系。相应地，针对各SMV株系的多个抗性位点被定位，但对抗SMV基因的克隆和功能鉴定报道较少。另外，抗病基因聚合方法可以提高大豆抗病性，然而聚合周期长且会影响大豆产量和品质。鉴于此，结合转基因手段的RNAi介导的抗性策略是发展广谱抗SMV大豆的潜在方法，但对RNAi介导的转基因大豆对SMV广谱抗性启动机制却知之甚少。为回答上述科学问题，主要研究内容包括：1、对转S1基因大豆对SMV的广谱抗性进行评估；2、应用CRISPR/Cas9系统敲除转S1基因大豆DCL基因，评估敲除突变体对SMV的抗性，鉴定关键DCL基因；3、对转S1基因大豆及其DCL突变体sRNA测序分析，揭示DCL切割hpRNA形成的siRNAs的特征。重要成果及关键数据包括：1、开发了对11个SMV株系具有广谱抗性、RNAi介导的转S1基因大豆材料；2、转S1基因大豆DCL基因敲除试验显示，敲除dcl1a/1b和dcl4a/4b突变体发育异常，种子没活力，无法正常生长，揭示DCL1a/1b和DCL4a/4b在大豆生长发育过程中具有重要作用；敲除DCL3转基因事件后代未鉴定到dcl3敲除突变体；对转S1基因大豆及其dcl2a/2b突变体接种SMV，突变体植株未发病但病毒含量显著增加，表明DCL2a/2b在抗SMV过程中扮演正向调节作用，同时推测其它DCL类基因可能协同DCL2a/2b在抵抗SMV侵染过程中起作用；3、对转S1基因大豆及其dcl2a/2b突变体sRNA测序分析显示，sRNA丰度在21nt和22nt长度的居多。其中，转S1基因大豆及其dcl2a/2b突变体的21nt sRNA丰度无差异，而22nt sRNA丰度在dcl2a/2b突变体中出现显著下降，说明DCL2a/2b在调节22nt sRNA生产中扮演重要作用。本项目基于RNAi介导的植物抗病毒机制，结合转基因手段培育了SMV抗性大豆新品种，对SMV病害的广谱抗性研究具有重要的现实意义。此外，探索了RNAi介导转基因大豆对SMV广谱抗性机制，为RNAi介导的双子叶作物抗病毒机制提供理论意义。