硅酸钠提高马铃薯黑痣病抗性相关基因功能分析

Stem canker and black scurf by Rhizoctonia solani is one of the most severe disease affecting potato production worldwide. It is serious in the growing areas of potato in Inner Mongolia.Farmers are still lack of effective measures to suppress the disease. Resistance of plant was enhanced by silicon and silicate has been confirmed in many diseases.In our previous research in field and laboratory, the resistance of underground stem and tuber of potato to Rhizoctonia solani were enhanced by sodium silicate,the yield and quality of potato were also increased. In this project, molecular mechanisms of resistance enhanced by sodium silicate will be researched mainly. The new transcriptome Illumina sequencing will be used and bioinformatics will be analysised to screen resistance-related genes. Real-Time PCR will be used to understand the spatial and temporal expression and function prediction of resistance-related genes. The whole length cDNA of the resistance-related genes will be cloned and the expression product will be analysised to determine the functional genes by RACE technique.Then the functions of these genes will be tested and validated in transformed potato. The same time, the using methods of sodium silicate will be determined. This research can find a new idea for controlling stem canker and black scurf of potato, and provide the theoretical basis for this new control measures that can protect environment, improve efficiency, increase production and is safe to human bings.

马铃薯黑痣病（Rhizoctonia solani）是世界马铃薯生产上的一种重要病害。在内蒙古马铃薯种植区危害严重，目前缺乏有效的防治措施。硅及硅酸盐类能提高植物抗病性已在很多病害中得到证实。前期经室内和田间试验研究，硅酸钠能提高马铃薯地下茎、块茎对黑痣病的抗性，且能增加产量并改善品质。本课题重点研究硅酸钠提高马铃薯黑痣病抗性的分子机制，即通过新一代高通量Illumina测序及生物信息学分析，筛选出硅酸钠诱导的马铃薯抗病相关基因，再利用Real-Time PCR技术分析其时空表达和进行功能预测，并用RACE技术克隆抗病相关基因的全长cDNA进行表达产物分析，确定目标功能基因，再通过转基因超量表达进行候选基因的功能验证。同时，明确硅酸钠提高马铃薯黑痣病抗性的应用方法。为马铃薯黑痣病提供环保、高效、安全、增产的新型防治措施，并深刻揭示其理论依据，具有理论和实践双重价值。

马铃薯黑痣病是一种世界性病害，发生严重，目前无有效的防治方法。硅酸钠能增强马铃薯黑痣病抗性。增强原原种抗性的方法为在MS培养基中加入0.604 mg/L硅酸钠，或按3.02g/L浓度将硅酸钠加入到MS培养基中直接浇灌马铃薯幼苗，能增强马铃薯幼苗的长势和提高抗性。增强马铃薯商品薯抗性的方法为，硅酸钠叶面喷施70～100mmol/L和单株沟施3～5g对马铃薯安全，防效高，增加产量和改善品质，对匍匐茎防效最好的是硅酸钠喷施70mmol/L接菌、不接菌处理，防效分别为61.96%和59.72%，对块茎防效最好的是硅酸钠喷施100mmol/L不接菌处理为83.43%，对地下茎防效最好的是沟施5g不接菌处理，防效为63.30%。.在硅酸钠处理4~6d可诱导马铃薯幼苗POD活性升高，减缓PPO活性降低，对PAL活性没有影响，处理第10d可诱导CAT活性升高和增加木质素含量，处理14~16d时能提高SOD活性，处理第2d可以诱导内几丁质酶和β-1，3-葡聚糖酶活性升高。硅酸钠能通过提高其体内防御性关键酶活性增强自身防卫机制。.分子抗性机制研究结果显示，马铃薯地下茎经硅酸钠处理不同时间基因数与表达水平均存在差异，接菌施硅酸钠和不施硅酸钠处理4d中有差异基因35698个，其中上调表达的有263个，下调表达的有303个；8d有差异基因35785个，其中上调表达的有1204个，下调表达的有2445个；12d有差异基因35677个，其中上调表达的有658个，下调表达的有101个。经RT-qPCR对差异基因进行验证，初步选取4个在不同时期相对表达量较高的基因，几丁质酶（CHIT）、抗病蛋白（RGA4）、胰蛋白酶和蛋白酶抑制剂（CYRI10）和苯丙氨酸裂解酶（PAL6），进行cDNA的全长克隆，并构建表达及农杆菌转化，并验证其抗性功能。.本项目明确了硅酸钠提高马铃薯黑痣病抗性的应用方法，为马铃薯黑痣病提供安全、高效、环保、增产的新型防治措施。并揭示了其抗性提高的生理生化和分子机制，具有理论和实践意义，具有广阔的应用前景。