水稻对硝磺草酮抗性分子机制的研究

As one kind of HPPD inhibitors herbicides, mesotrione can effectively control a variety of broad-leaved weeds and gramineae weeds, with the advantages of high crop safety and good environmental compatibility. If mesotrione is used in combination with the corresponding resistant rice, it can effectively kill the malignant weeds in the rice field. Furthermore, it can solve the problem of herbicide residues in the existing resistant rice on subsequent crops and the problem of resistant weeds. However, at present, there has not been the emergence of rice varieties resistant to HPPD inhibitor herbicides. Also, there are few studies on rice OsHPPD and few reports on rice resistance to HPPD inhibitor herbicides caused by OsHPPD gene site mutation. Analysis showed that the amino acid sites on the active gate region H11 of OsHPPD protein C-terminal were critical to the affinity of substrates (or inhibitors); such mutations at these sites might lead to HPPD inhibitor resistance in rice. In addition, increasing the expression of OsHPPD may be one of the ways to enhance such resistance, since the combination of mesotrione and OsHPPD is a competitive inhibitory process. This project intends to adopt site-directed mutation, gene editing, mutagenesis screening, genome sequencing and other molecular biology methods to clarify the resistance sites of OsHPPD, the relationship between its expression level and resistance, and analyze the resistance mechanism of rice to mesotrione. This project can provide theoretical basis and data support for the development a new kind of herbicide-resistant rice.

硝磺草酮属于HPPD抑制剂类除草剂的一种，可有效防除多种阔叶杂草和禾本科杂草，具有作物安全性高与环境相容性好等优势。若将硝磺草酮与相应的抗性水稻结合使用，可有效杀灭稻田中的恶性杂草，解决现有抗性水稻的农药残留对后茬作物的影响以及抗性杂草的问题。然而，目前还未有抗HPPD抑制剂类除草剂水稻品种的问世，对水稻OsHPPD的研究也并不多见，鲜有因基因位点突变而产生抗性的报道。研究分析表明，OsHPPD蛋白C端活性门控区域H11上氨基酸位点对底物（或抑制剂）的亲和力至关重要，这些位点的突变可能导致水稻产生抗性。此外，由于硝磺草酮与OsHPPD的结合是一种竞争性的抑制过程，提高OsHPPD的表达量可能是增强抗性的途径之一。本项目拟采取定点突变，基因编辑，突变体筛选，基因组测序等方法，旨在明确OsHPPD的抗性位点，阐明其表达水平与抗性之间的关系，解析水稻对硝磺草酮的抗性机制，开发新型抗除草剂水稻。

水稻是全球（尤其是亚洲地区）最为主要的粮食作物之一，稻田中的杂草严重危害水稻的品质和产量。硝磺草酮属于HPPD抑制剂类除草剂的一种，可有效防除多种阔叶杂草和禾本科杂草，具有作物安全性高与环境相容性好等优势。若将硝磺草酮与相应的抗性水稻结合使用，可有效杀灭稻田中的恶性杂草，解决现有抗性水稻的农药残留对后茬作物的影响以及抗性杂草的问题。然而，目前还未有抗HPPD抑制剂类除草剂水稻品种的问世，对水稻OsHPPD的研究也并不多见，鲜有因基因位点突变而产生抗性的报道。此外，由于硝磺草酮与OsHPPD的结合是一种竞争性的抑制过程，提高OsHPPD的表达量可能是增强抗性的途径之一。.本项目通过转基因以及启动子编辑的途径，提高HPPD在水稻中的表达量，对不同表达量的水稻进行抗性分析，发现提高OsHPPD的表达量可以增强水稻对硝磺草酮的抗性。通过对OsHPPD进行定点突变以及突变基因体外原核表达结果表明,V224I位点突变后抗性增强,V364M位点突变后活性增强,同源建模以及分子对接结果表明,位点突变后OsHPPD蛋白酶的活性催化位点发生改变,导致OsHPPD的活性和对硝磺草酮的抗性增强。利用CRISPR/Cas9对水稻OsHPPD进行定向进化,在硝磺草酮的筛选压力下获得4株水稻苗,H11结构域测序结果表明,OsHPPD H11序列发生一系列的突变、缺失,导致H11上氨基酸残基的二级结构、亲疏水性或空间位阻发生变化导致硝磺草酮不能进入活性位点区域与Fe2+结合,从而不能竞争性地抑制底物HPPA与Fe2+结合,导致水稻对硝磺草酮具有一定的抗性。后续将继续利用突变体筛选以及基因组测序，获得抗硝磺草酮水稻材料，进一步解析水稻对硝磺草酮的抗性机制，开发新型抗除草剂水稻。