基于基因靶向切割的核酸扩增技术构建及其在马铃薯晚疫病检测中的应用

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight in potatoes, one of the most devastating diseases of potato worldwide. Early detection of late blight has been paid attention to by many countries and international organizations. The toxicity of Phytophthora infestans is determined by the pathogenesis-related genes, so accurate detection of gene sequence at the molecular level can effectively prevent outbreak of disease. However, the traditional detection methods are challenged due to the high rate of genetic mutation in Phytophthora infestans. Based on the previous researches of our laboratory, this project makes use of Cas9-mediated gene disruption to construct a novel self-designed nucleic acid amplification strategy for diagnosis of late blight with good specificity, high sensitivity and convenient operation. In this study, we use the gene-targeting ability of Cas9/sgRNA complex to satisfy the specificity of the detection; use the nicking endonuclease and polymerase-mediated super-exponential amplification to satisfy the sensitivity of the detection; use the nano particles to satisfy the convenience of the detection. The completion of this project will provide new technical support for the early and accurate diagnosis of potato late blight disease, and then provide reasonable suggestions for using pesticide and cultivating disease-resistant potato. And this strategy also provide a novel research techniques for exploring the epidemic trend and pathogenesis of pathogens by analyzing the genes of Phytophthora infestans.

由致病卵菌引起的马铃薯晚疫病严重威胁农业生产，晚疫病早期诊断已经为众多国家和国际组织所重视。病菌毒性强弱由致病相关基因所决定，因此在分子层面上精确检测晚疫病基因才能有效控制病害爆发。但是，由于晚疫病菌基因组突变几率高，使得传统检测方法面临挑战。研究组在前期分子诊断和纳米粒子制备的工作基础上，拟利用Cas9酶的基因靶向切割特性构建特异性好、灵敏度高和操作简便的晚疫病诊断新策略。本研究中，我们利用Cas9/sgRNA复合体靶向切割靶核酸序列能力保证了基因识别的特异性;利用基于聚合酶和缺口酶的核酸超指数扩增策略保证了检测的灵敏度;利用纳米粒子保证了操作的方便性。本项目的研究和完成，将为马铃薯晚疫病早期诊断和精确分型提供新的技术支持，进而为田间合理用药和抗病马铃薯品种培育提供合理建议。并通过对晚疫病基因分析，为探究病菌流行趋势和致病机理提供新的研究手段。

致病疫霉引起的马铃薯晚疫病会严重危害植物的叶片、叶柄、茎和块根，导致大面积减产。同时，致病疫霉的全球扩散和杂交型出现使基因结构进一步复杂化。因此，将CRISPR基因编辑系统引入到核酸检测之中，在分子层面对致病疫霉进行精准检测对晚疫病防治具有重要的意义。基于以上研究目的，我们首先构建了基于CRISPR/Cas9系统介导的纳米金比色检测方法，实现了单基因位点的精准识别，可视化检测限为5 pM。我们进一步利用切刻酶CRISPR/Cas9n系统对基因组中双基因位点进行同时检测，检出限为1.1 pM。并基于该扩增方法建立了分子逻辑门识别基因组中复杂突变关系，实现野生型和突变型疫霉的有效识别。通过引入三维金纳米枝晶（AuNDs）电化学传感器和CdS/C3N4/B-TiO2/FTO光电传感器，结合CRISPR/Cas12a系统实现了双重信号放大，大幅提高了基因检测灵敏度，检测限分别达到了39 fM和5.8 fM。因此，本项目的研究为马铃薯晚疫病检测提供了一类可视化、高保真、高灵敏度的新策略。此外，我们扩展研究中制备的纳米材料应用范围。利用纳米金的聚集诱导发光特性结合MnO2纳米片的荧光猝灭效应自组装合成了MnO2-AuNCs-SiO2纳米复合材料，实现了有机磷农药的双模态传感。合成了SiO2@Ni和Cu(OH)2SC纳米笼两种金属模拟酶，实现了微囊藻毒素的高灵敏度检测。