中文摘要
本研究于国际首次成功应用荧光免疫的方法检测生产中极易混淆的小麦三种锈菌，无论在科学领域以及实际应用价值方面都很有意义。通过该项目的研究，首先创新了一种小麦叶锈菌单克隆抗体的制备方法，并在制备的百余株杂交瘤细胞中优选（直接法和竞争法）了1株抗小麦叶锈菌PHT、THT的单克隆抗体杂交瘤细胞株，其可产生高效价的用于检测小麦三种锈菌的单克隆抗体，具有特异性好、灵敏度高、操作简便快速的优点，其与叶锈菌的孢子上清反应效果较佳，已经在中国科学院微生物研究所菌种保藏中心获得了保藏（保藏号为CGMCC No.4414）。另外，应用该单克隆抗体成功建立了区别小麦三种锈菌的荧光免疫方法的检测体系，并发现该单克隆抗体的检测浓度可达2 ng/ml。通过Western Blot，发现该单克隆抗体可在小麦三种锈菌中分别沉淀三种不同分子量大小的蛋白，说明该抗体可以特异性地识别小麦三种锈菌中各自不同的蛋白。现已申请国家发明专利2项，目前均处于授权公告阶段；发表小麦锈病诊断检测技术进展的相关综述1篇；后续的几篇主体研究论文已经整理完毕，有待发表。
英文摘要
Under the help of this fund, we firstly developed the method for monoclonal antibody of P. triticinia, and by different detection method, choose one of monoclonal antibody (CGMCC No.4414) which can have good reaction with the suspension of the spores of P. triticinia. We described a rapid and sensitive immunofluorescence method for the detection of urediniospores of the fungi Puccinia striiformis f. sp. tritici, P. triticinia, and P. graminis f. sp. tritici which are causal agents of wheat rust. The method uses monoclonal antibody against the urediniospores of P. triticinia and PE-cy3 goat anti-mouse. Antigen (urediniospores of P. triticinia or those of two species that are difficult to distinguish from P. triticinia: P. striiformis f. sp. tritici or P. graminis f. sp. tritici) was immobilized on a glass slide, and the slide was then treated with the selected monoclonal antibody. A second antibody, goat anti-mouse conjugated PE-cy3, was then added, and the slide was observed with fluoroscope. The fluorescent signal was strong with P. triticinia urediniospores,weak with P. striiformis f. sp. tritici urediniospores, and weak-to-intermediate with P. graminis f. sp. tritici urediniospores. The detection limit of this method was 2 ng/ml of the monoclonal antibody. We found that the monoclonal antibody can get 3 different proteins in the P. triticinia: P. striiformis f. sp. tritici or P. graminis f. sp. tritici by western blot. With further development, this method should be useful for on-site identification P. triticinia urediniospores and will therefore help in the selection and timing of fungicide applications for control of wheat rust. We have been applied two patents in China and published one paper in the abstract of annual meeting of China Society of Plant Protection, and other research articles are ready for submit in the near future, we will give an acknowledgement to this fund.
结题摘要
本研究于国际首次成功应用荧光免疫的方法检测生产中极易混淆的小麦三种锈菌，无论在科学领域以及实际应用价值方面都很有意义。通过该项目的研究，首先创新了一种小麦叶锈菌单克隆抗体的制备方法，并在制备的百余株杂交瘤细胞中优选（直接法和竞争法）了1株抗小麦叶锈菌PHT、THT的单克隆抗体杂交瘤细胞株，其可产生高效价的用于检测小麦三种锈菌的单克隆抗体，具有特异性好、灵敏度高、操作简便快速的优点，其与叶锈菌的孢子上清反应效果较佳，已经在中国科学院微生物研究所菌种保藏中心获得了保藏（保藏号为CGMCC No.4414）。另外，应用该单克隆抗体成功建立了区别小麦三种锈菌的荧光免疫方法的检测体系，并发现该单克隆抗体的检测浓度可达2 ng/ml。通过Western Blot，发现该单克隆抗体可在小麦三种锈菌中分别沉淀三种不同分子量大小的蛋白，说明该抗体可以特异性地识别小麦三种锈菌中各自不同的蛋白。现已申请国家发明专利2项，目前均处于授权公告阶段；发表小麦锈病诊断检测技术进展的相关综述1篇；后续的几篇主体研究论文已经整理完毕，有待发表。