CRISPR/Cas9单碱基编辑技术创制非转基因抗除草剂西瓜新种质

Weeds have caused serious damage to watermelon production, which are the main obstacles to achieve labor-saving and simple cultivation. Breeding new varieties of herbicide-resistant watermelon is one primary approach to improve the competitiveness of watermelon industry, however, classical breeding techniques and transgenic technology are inherent in technical or safety limitations. CRISPR/Cas9-mediated gene editing provides the possibility for creating non-GM herbicide-resistant watermelon efficiently. We first reported the efficient CRISPR/Cas9 gene-knockout system of watermelon, and successfully obtained herbicide resistant Arabidopsis using CRISPR/Cas9-mediated technique. Based on the above achievements , the base editing vector was proposed to target the known resistance sites of the watermelon ClALS gene and ClEPSPS gene (encoding the ALS inhibitor and the glyphosate substrate, respectively), the efficiency of which will be evaluated by protoplast transformation system, further optimizing the vector. Afterwards, transgenic plants will be obtained through the stable and efficient tissue culture method. To get the non-GM high ALS inhibitor-resistant and glyphosate-resistant watermelon, all offsprings will be genotype identified and evaluated by herbicide-resistance. Our project will provide technical support for cultivating watermelon varieties in a more efficient and simplified manner, which will greatly benefit the watermelon industry.

草害严重危害西瓜生产，是实现省工轻简化栽培的主要障碍，培育抗除草剂西瓜新品种是提高西瓜产业竞争力的主要途径之一，然而经典育种技术及转基因技术均存在技术或安全局限性。CRISPR/Cas9基因编辑技术为高效创制非转基因抗除草剂西瓜新种质提供了可能。本项目课题组率先报道了西瓜高效的CRISPR/Cas9基因敲除系统，并利用CRISPR/Cas9单碱基编辑技术成功获得抗ALS抑制剂的拟南芥。在此基础上，拟采用单碱基编辑载体，以栽培西瓜ClALS基因和ClEPSPS基因（分别编码ALS抑制剂与草甘膦的作用底物）的已知抗性位点为靶标，通过原生质体瞬时转化系统评估打靶效率，优化载体，再通过稳定高效的组培转化获得转基因植株，后代经分离鉴定与除草剂抗性评价，获得非转基因高抗ALS抑制剂与抗草甘膦的西瓜新种质，为培育适应西瓜高效轻简化生产的西瓜品种提供技术手段，进而带动西瓜产业的省工节本增效。

西瓜是我国重要的经济作物，然而草害严重影响了西瓜的生产，目前尚无高效的防除杂草的方案，培育抗除草剂西瓜新品种可以解决这一难题。本项目利用CRISPR/Cas北地区9碱基编辑技术，精准突变了籽用西瓜ALS基因，成功创制世界首例抗ALS除草剂籽瓜。通过自交分离后，获无外源基因插入的抗除草剂籽瓜新种质京籽190S。针对京籽190S，本项目进行除草剂的交互抗性水平研究，并进一步开发利用“抗除草剂籽瓜品种+除草剂”组合来有效防除籽瓜田间杂草的方案。京籽190S对播后土壤处理的 “精异丙甲草胺+唑嘧磺草胺”表现出良好的抗性，有望成为籽瓜田杂草防控的新工具。此外，与野生型相比，京籽190S无不良生长表型。利用京籽190S作为父本配制杂交组合，其中组合“京籽1号” 长势强，产量高，坐瓜数多，抗病性强，苗期对除草剂的抗性强，适宜田间化学锄草。本研究为籽瓜田杂草的防除提供了有力的解决方案。