玉米耐烟嘧磺隆基因的克隆和功能分析

Maize (Zea mays L.) plays a critical role to serve as food, feed and biomass production around the world. Nicosulfuron is a commonly used herbicide which has been extensively used to control annual grass weeds，broadleaf weeds and some Cyperaceae in maize field. Nicosulfuron was widely applied in China since early 1990s, it was used as an efficient，broad-sulfonylurea maize herbicide at seedling stage. However, recent reports showing that Nicosulfuron was genotype dependent – it cause serious injuries to some maize varieties, which can result in a reduction of maize grain yield, moreover, it can also increase the risks for feeding livestock because of mycotoxins problems. Recently, some progress has been made in understanding the mechanism about the resistant system involved in maize infected by Nicosulfuron, including physiological and biochemical characteristics, genetic architecture underlying resistance traits and gene mapping, et al. However, there is limited research about the isolation and molecular characterization of resistance genes. In this study, we would use Cas9 technology and UniformMu mutants to verify a candidate gene NSFY from previous experiments, our objective is to confirm its resistance function mechanism. This study could provide important information on understanding the molecular resistance mechanism to Nicosulfuron in maize. Furthermore，it could provide important gene resource and technical support for a new generation of genetically modified herbicide resistant crops.

玉米是世界及我国最重要的粮食、饲料及工业原料。烟嘧磺隆(Nicosulfuron) 对玉米田中发生的多数一年生禾本科杂草、部分阔叶杂草及部分莎草科杂草具有较好的防治效果。我国于20世纪90年代初期开始推广烟嘧磺隆，作为一种高效、广谱的磺酰脲类除草剂，大量应用于玉米苗后的杂草防治。多年来生产实践发现，烟嘧磺隆会对部分玉米品种产生一定药害，从而造成玉米产量下降。国内外学者从该除草剂的生理生化、抗性遗传关系、基因定位等进行了相关研究，但在玉米中对该基因进行克隆和功能验证目前还没有相关报道。本项目拟在前期筛选得到的耐烟嘧磺隆候选基因NSFY基础上，利用农杆菌介导转化、CRISPER/Cas9基因敲除和UniformMu突变体等三种技术方法对NSFY基因进行功能验证，进而为玉米烟嘧磺隆抗性分子机制的研究奠定基础，为发展新一代转基因耐烟嘧磺隆农作物提供了重要的基因资源和技术基础。

烟嘧磺隆是玉米苗后专用除草剂，但不同材料之间对烟嘧磺隆的敏感性有很大差异。本研究利用前期已构建的H631和H649两套BC1群体，将烟嘧磺隆敏感基因定位于标记A1和B2之间，两标记间物理距离为10.3 Kb，该区段内仅存GRMZM2G090432一个基因。通过测序分析目的基因GRMZM2G090432全长1859 bp，包含2个外显子，1个内含子，共编码445个氨基酸。GRMZM2G090432基因属于P450家族，但与其它物种的同源性相对较远。选取63份不同抗感材料进行目的基因测序，发现敏感材料在1007-1008 bp处插入两个AA碱基，随着碱基移码突变，从而导致1117-1119 bp处提前出现了终止密码子TGA。通过激光共聚焦显微镜观察目的基因是在细胞核中表达；进一步经过表达、基因编辑和EMS突变体互补验证了目的基因的正确性。抗性材料B73和敏感材料JS188生长到六叶一心时，进行喷施80 mg/L烟嘧磺隆处理，选取0 h、2 h、4 h、8 h、12 h、24 h、48 h、72 h、96 h、192 h（其中0 h为未喷药处理的样品）等10个不同时间段的根和叶两个组织部位，对GRMZM2G090432基因进行荧光定量PCR分析，B73在处理的各个时间段表达量都明显高于JS188，这说明GRMZM2G090432基因在抗性材料中转录水平高于敏感材料。本研究成功克隆了玉米烟嘧磺隆抗性基因GRMZM2G090432，为烟嘧磺隆抗性基因GRMZM2G090432机理研究奠定了一定基础。