番茄miR398启动子参与低温响应的调控机理分析

MicroRNAs (miRNAs) are the important regulatory factors which play versatile roles in plant growth, development and stress responses, while the miRNAs promoters are closely related to the specific expression of miRNAs involved in these progresses. Chilling stress is one common abiotic stress for tomato cultivation which seriously affects the growth, development, fruit yield and quality of tomato. Our prophase research has proved that miR398 targeted CSD1 (Superoxide dismutase [Cu-Zn] 1) increased on chilling-tolerant tomato (Solanum habrochaites L.) under the chilling stress, and similar response was found on other species. However, the regulation mechanism of miR398 promoter in the chilling response is still unclear. Therefore, based on the information of miR398 precursor, the miR398 promoter will be cloned from Solanum habrochaites tomato and then for bioinformatics analysis. Expression vectors of promoter fragments with different length will be constructed and transformed into tomato. Then, the chilling stress responsive promoter fragments will be tested and selected. Meanwhile, Yeast one-hybrid system will be applied to analysis the transcription factors combining to the promoter fragment of miR398, which will reveal the transcriptional regulation mechanism of tomato miR398 in the chilling response. This project would not only enrich the theory of miRNA regulating chilling response, but also provide gene resources for tomato germplasm resources innovation with chilling tolerance.

MicroRNAs（miRNAs）是调控植物生长发育和逆境胁迫响应的重要调控因子，而miRNAs启动子又与miRNAs在该过程的特异表达密切相关。低温是番茄栽培生产常见的非生物胁迫，严重影响了番茄生长发育和产量品质。前期研究证实多毛番茄miR398靶向CSD1（Superoxide dismutase [Cu-Zn] 1）在低温胁迫下上调表达，其他植物miR398在低温下也存在类似响应。然而miR398启动子在该响应过程的调控机理尚不清晰。为此，本项目拟在获得miR398前体序列的基础上，克隆多毛番茄miR398启动子并进行生物信息学分析，构建不同长度启动子片段表达载体并转化番茄，筛选响应低温的启动子片段，并利用酵母单杂交分析与该启动子片段结合的转录因子，揭示番茄miR398启动子在低温响应过程中的调控机理。本项目丰富了miRNAs参与低温调控理论，并为番茄耐低温种质创新提供基因资源。

前期研究发现多毛番茄miR398靶向CSD1（Superoxide dismutase [Cu-Zn] 1）在低温胁迫下上调表达，为进一步揭示miR398调控机制，本项目首先对番茄miR398启动子进行分析，之后克隆并分析多毛番茄的miR398启动子，随后对多毛番茄miR398前体进行克隆及序列分析，构建miR398前体过量表达载体，获得转基因株系，并以其为研究对象，分析mRNA及抗氧化酶活性变化。研究结果发现，生物信息学预测到miR398启动子区域存在脱落酸响应元件、厌氧诱导必需元件、MYB结合位点等元件，克隆获得了多毛番茄miR398启动子片段，其上存在厌氧诱导必需元件和光响应元件；多毛番茄miR398前体序列获得后分析发现其成熟体具有一定保守性，前体与葡萄、大豆等植物同源性较高；随后获得了miR398前体过量表达株系，其植株地上部矮小，且miR398表达量显著增加；经比较分析发现转基因株系与未转基因植株之间存在大量差异表达显著的基因，主要是一些转录因子、蛋白激酶等基因，而抗氧化酶活性分析发现SOD酶活性显著降低。本研究初步阐明了miR398调控机制，为miRNA研究提供了理论依据。