肽适体抑制柱花草胶孢炭疽病的分子机理及应用

Stylosanthes anthracnose is the primary fungal disease in tropical legume forages grass. Cultivating the new species of Stylosanthesguianensis resistant to the pathogen faces great challenges because ofdisease resistance involving minor polygenes, obscurely pathogenic mechanisms invading to Stylosanthesguianensis, intractable acquirement of resistant genes. Our previous studies revealed that the selected artificial peptideapatamer interacted with calmodulin of Stylosanthes anthracnose specifically, resulting in the inhibitions of spore germination and germ tube elongation. Based on the above results, we propose to develop peptide apatamer as biological agent for the control of Stylosanthes anthracnose. In this study, antimicrobial mechanism of peptide apatamer inhibiting Stylosanthes anthracnose is explored by scanning electron microscope (SEM), flow cytometry. How the peptide aptamer penetrates into protoplasm of Stylosanthesguianensis is traced by gold nanoparticles. The effects of prevention and treatment with peptide aptamer are analyzed in the field. The key sites of reciprocity between peptide apatamer and calmodulin are identified by site-directed mutagenesis and surface plasmon resonance (SPR).Our work will also illustrates how calcium signaling plays an important role in the pathogenesis of Stylosanthesanthracnose, and seek for the new therapy targets. These are also helpful to develop the agents for the biological control, and increase the sustainable utilization of farmland, and reduce the agricultural source pollutions. It has a broad application prospect for alleviating the bear pressure of the forage grass land in north China and flourishing grass production in south China.

胶胞炭疽病是热带豆科牧草柱花草主要真菌病害之一。由于炭疽病抗病受微效多基因控制，病菌入侵柱花草分子机制尚不清楚，以及抗病靶基因获取困难等，使得柱花草抗炭疽病新品种培育步履维艰。我们前期结果表明，人工肽适体可以和炭疽病中的钙调蛋白特异互作，抑制孢子生长。基于此，我们提出用肽适体及细胞穿透肽融合表达，作为生物农药防控炭疽病的策略。本项目拟用扫描电镜、流式细胞仪等，深入剖析肽适体对炭疽病的抑菌机理；原核串联表达肽适体和细胞穿透肽，用纳米金法追踪肽适体进入柱花草抗病的最适条件，并验证田间防护效果；用定点突变、表面等离子共振等解析肽适体与钙调蛋白互作的关键位点。本工作有助于阐明钙信号传导在炭疽病致病过程的重要作用，发掘植物病原真菌新靶标；还将为开发新型肽适体药剂用于生物防治奠定基础，对增加农田可持续利用、减轻农业面源污染，有实际意义；对缓解北方饲草土地承载压力，发展华南地区牧草产业，有广阔应用前景。

胶胞炭疽病是热带豆科牧草柱花草主要真菌病害之一。由于炭疽病抗病受微效多基因控制，病菌入侵柱花草分子机制尚不清楚。抗病靶基因获取困难，使得柱花草抗炭疽病新品种培育步履维艰。基于此，我们提出用肽适体SNP-D4作为生物农药防控炭疽病的策略。首先，利用电镜、流式细胞仪等评估SNP-D4对炭疽病的抑制作用，发现SNP-D4通过破坏炭疽病孢子外膜结构，特异性抑制孢子萌发。其次，利用ICP-OES定量检测柱花草原生质体对SNP-D4-纳米金复合物的吸收，发现在柱花草原生质体和40μg/mL的SNP-D4-纳米金复合物混合培养4-6h时，柱花草原生质体内的金浓度达到饱和，说明SNP-D4-纳米金复合物已成功进入柱花草原生质体内。再次，利用PBS（阴性对照）、0.2%多菌灵（阳性对照）以及不同浓度的SNP-D4处理柱花草幼苗，发现用PBS处理的柱花草叶片发病率在90%以上，病情指数多在40以上。用浓度为8μM的SNP-D4处理后，柱花草叶片发病率和病情指数更接近阳性对照组，说明SNP-D4对柱花草炭疽病防护具有一定效果。最后，利用免疫胶体金标记SNP-D4和柱花草炭疽病的钙调蛋白互作区域，发现SNP-D4主要在细胞质中与钙调蛋白结合，且结合区域为可变肽段。本项目的研究成果在理论和实际应用中都有较强价值，一方面，发展新型肽适体抑制柱花草炭疽病菌，相较过去的化学防治，对增加农田土壤可持续利用、减轻农业面源污染具有重要意义。另一方面，从分子水平阐明肽适体抑制炭疽病的作用机理，为深入揭示炭疽病致病的原因，进一步挖掘炭疽病的治疗靶点提供理论基础。本项目还为植物真菌病害防治提供新思路，在目前急需发掘新的防控措施的情况下，无疑具有广阔的应用前景。