基于真菌毒素和病原细菌的SELEX发展植物病害诊断的新方法

Fungi and bacteria are the main pathogenic microorganisms that cause plant diseases, and the time of prevention and control is extremely important for both fungal and bacterial diseases. It is an important means to reduce the loss of agricultural economy, decrease the use of pesticides as well as pesticide residues in agricultural products, through the identification of pathogens in the early stage of disease and further timely prophylactico-therapeutic measures. Recently, false smut and bacterial blight are major fungal and bacterial diseases in rice production in China, the pathogens of which are Ustilaginoidea virens (Cooke) Tak and Xanthomonas oryzae pv.oryzae (Xoo in brief), respectively. In this project, we will take the mycotoxin of Ustilaginoidea virens (Cooke) Tak, Ustiloxin A, and the crude extracellular mixture or intracellular mixture of Xoo as targets, respectively, and use traditional or improved SELEX technology to screen out the aptamer of Ustiloxin A as well as the DNAzyme of Xoo. Then, we’ll do further research to build them into nucleic acid biosensors with high sensitivity, specificity and convenience, which could be applied to detect the pathogens in seeds, soils and plants without significant symptoms, and subsequently to detect false smut and bacterial blight diseases in rice as new methods. In summary, this project aims to establish SELEX platform based on mycotoxins and bacteria, and screen out the functional nucleic acids with specificity, then construct nucleic acid biosensors to detect the pathogenic fungi and bacteria, and finally develop new technology for early diagnosis of plant diseases.

真菌和细菌是引起植物病害的主要病原微生物，真菌病害和细菌病害的防控时机非常重要。在病害发生早期检测出病原菌并及时采取防治措施，是降低农业经济损失、减小农药使用量以及减少农产品农药残留的重要手段。稻曲病和白叶枯病是我国近年来水稻生产上主要的真菌性和细菌性病害，致病菌分别为稻绿核菌和白叶枯病菌。本项目拟分别以稻绿核菌毒素Ustiloxin A，白叶枯病菌代谢至胞外的粗提物或该病菌胞内粗提物为靶标，运用传统和改良的SELEX技术，筛选出Ustiloxin A的适配子和白叶枯病菌的核酸酶，并进一步构建成灵敏度高、特异性强、方便快捷的核酸生物传感器，用于稻种、土壤、田间未显症状植株材料中病原菌的鉴定，开发新的检测水稻稻曲病和白叶枯病的方法。综上，本项目旨在搭建基于真菌毒素和病原细菌的SELEX平台，筛选出与其特异性结合的功能核酸，并构建成核酸生物传感器检测病原菌，从而发展植物病害早期诊断的新方法。

水稻稻曲病和白叶枯病是我国近年来水稻生产上主要的真菌性和细菌性病害，致病菌分别为稻绿核菌和白叶枯病菌，对羟基苯基丙酮酸双加氧酶（HPPD）是重要的农药靶标。在本项目中，我们分别以稻绿核菌毒素Ustiloxi n A，白叶枯病菌代谢至胞外的粗提物以及HPPD为靶标，运用传统和改良的SELEX技术，筛选其适配子或核酸酶，并进一步构建成灵敏度高、特异性强、方便快捷的核酸生物传感器。在本项目资助下，我们筛选出Ustiloxin A倾向性结合核酸序列，以及HPPD的适配子。同时构建了三个核酸探针体系，分别是：基于滚环扩增技术（RCA）反应的可视化适配子传感器；基于CRISPR-Cas12a 信号放大系统的新型核酸生物传感器；以及基于HPPD适配子和氧化石墨烯（GO）材料的HPPD探针。另外，我们合成了三个新型过氧化氢酶（CAT）抑制剂BT-1、BT-Br和C-6，可分别诱导去势抵抗性前列腺癌（CRPC）细胞DU145，三阴性乳腺癌细胞MDA223，以及耐药性肺癌细胞A549发生凋亡或铁死亡。且动物实验结果显示，BT-1和BT-Br化合物均可有效抑制移植型前列腺实体瘤的生长。此外，我们还研究BT-Br诱导拟南芥细胞产生铁死亡压力的机制和效果，旨在进一步开发基于铁死亡通路的除草剂。综上，本项目基本按照计划执行，研究成果已发表1篇SCI论文，另外3篇正在审稿和准备投稿中。项目执行期间，共培养了5名硕士毕业生，和2名在读硕士研究生。

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