黄瓜花叶病毒作为基因沉默载体用于玉米基因功能研究的创建和优化

Virus induced gene silencing (VIGS), being widely used in plant genes function study, is an important research area in plant virology. Construction and optimization of a virus vector is critical to obtain efficient and stable silencing. Up to now, totally 38 virus- and satellite- based VIGS vectors have been developed. However, only Brome mosaic virus-based VIGS vector could be used on several unpopular inbred lines of maize, and it induces severe symptom resulting in unsuitable for extensive application. Our previous work characterized a Cucumber mosaic virus isolate ZMBJ naturally infecting maize in China, and it was developed to be a VIGS vector by displacing partial region of 2b with a multiple cloning site. The CMV-ZMBJ-2bN81 vector had been used successfully to silence PDS and ZmZb7 in maize. This vector has outstanding advantages, including inducing very mild symptom, long-term silencing, and working on maize model inbred line B73. In this study, a series of assays will be performed as following, (a) to optimize the inoculation method on maize to get high infection efficiency, (b) to develop different vectors with variously truncated 2b, then to obtain the best VIGS vector, (c) to optimize the temperature and insert length for efficient silencing, (d) to try to use the vector on important inbred lines of maize and other monocots, such as sorghum, millet, etc.; (e) to develop a high-throughput VIGS vector and its preliminary application. This proposal focuses on one of the hottest issues in modern plant viology, and works for maize one of the most important crops. The results will provide a novel set of VIGS vectors with no obvious symptom, suitable for most maize inbred lines, and with high efficiency, for maize gene function research.

病毒诱导的基因沉默（VIGS）广泛用于植物基因功能研究，是植物病毒研究的重要方向。构建和优化沉默高效、稳定的病毒载体是关键。已有38个病毒/病毒卫星作为VIGS载体，但仅雀麦花叶病毒用于玉米的极少数不常用自交系，且症状严重。前期研究中，将自然侵染玉米的黄瓜花叶病毒北京分离物（CMV-ZMBJ）RNA2 2b部分区域改造构建了CMV-ZMBJ-2bN81载体，成功沉默了玉米PDS和ZmZb7基因，具有病毒症状轻微、沉默持续时间长、侵染模式自交系B73的显著优点，潜力极大。基于此，拟开展如下系统研究：①优化接种玉米方法，获得高侵染率；②缺失2b不同长度，确定最佳载体；③优化温度、外源片段长度范围；④尝试用于玉米重要自交系及高粱、谷子等重要作物；⑤构建高通量沉默载体和初步利用。预期结果将为研究玉米基因功能提供一套无明显病毒症状、适应自交系广、高效、新颖的VIGS载体，以期促进相关领域快速发展。

病毒诱导的基因沉默（VIGS）广泛用于植物基因功能研究，是植物病毒研究的重要方向。构建和优化沉默高效、稳定的病毒载体是关键。已有38个病毒/病毒卫星作为VIGS载体，但仅雀麦花叶病毒用于玉米的极少数不常用自交系，且症状严重。前期研究中，将自然侵染玉米的黄瓜花叶病毒北京分离物（CMV-ZMBJ）RNA2 2b部分区域改造构建了CMV-ZMBJ-2bN81载体，成功沉默了玉米PDS和ZmZb7基因，具有病毒症状轻微、沉默持续时间长、侵染模式自交系B73的显著优点，潜力极大。在此基础上，完成了以下研究：.建立了一套完整详细的可高效沉默玉米基因的ZMBJ-CMV VIGS系统。确定了以根癌土壤杆菌浸润接种的本生烟叶片汁液为毒源针刺接种玉米种胚的接种方法，VIGS载体可承载的插入片段长度为100~300 bp；接种后的玉米在16 h/20oC光照~8 h/18oC黑暗的条件下沉默效率最高（沉默72.3%）。对玉米内源基因的沉默表明，ZMBJ-CMV-2bN81能够高效沉默IPP/DMAPP合成酶（ZmIspH）基因， ZmIspH的沉默导致了玉米叶片中叶绿素和类胡萝卜素含量降低，叶绿体类囊体片层减少、排列疏松且不规则。ZMBJ-CMV-2bN81诱导ZmIspH沉默至少持续至接种后60天，在新长出的上部叶片中ZmIspH仍被沉默。首次在单子叶植物中证明了自噬途径参与淀粉代谢。ZMBJ-CMV VIGS载体分别成功沉默了自噬相关基因ZmATG3和ZmATG8a，阐明了ZmATG3、ZmATG8a参与调控玉米叶片中间淀粉的降解。对20种玉米育种和研究中重要的自交系的沉默实验表明ZMBJ-CMV-2bN81载体适用于17个玉米自交系：Oh43、8112、PH207、w22、Mo17、LH196、K22、178、F42、Q319、Zheng58、Shen5005、5003、Lv28、HuangC、Huangzao4和Q381。并可以侵染高粱、谷子等重要作物；构建了高通量沉默载体，在玉米上进行了初步应用。这些进展为研究玉米基因功能提供了一套无明显病毒症状、适应自交系广、高效的VIGS载体。