马铃薯半胱氨酸蛋白酶抑制剂CPI的转录调控机理研究

Browning is one of the major bottlenecks restricting the development of fresh-cut potato industry. To date，none consistent conclusion about the browning mechanism of fresh-cut potato has been put forward. Identification of the key genes involved in potato browning and understanding their transcriptional regulation mechanisms is the core and prerequisite to solve this problem. Potato cysteine protease inhibitor gene (CPI) was proven to play important roles in alleviating potato browning. However, the transcriptional regulation mechanisms of CPI is not clear at present. By cloning the full-length and different deletion fragments of CPI promoter, and subsequent GUS staining and GUS activity examination in transgenic potatoes, this project aims to explore the expression patterns of CPI and identify the core cis-element within its promoter. In addition, yeast one-hybrid assay will be employed to identify the upstream transcription factors of CPI from a cDNA library prepared from potato tubers. The interactions between these transcription factors and the cis-elements within the promoter of CPI will be further confirmed by EMSA and CHIP assays. Furthermore, by determining the expression patterns of upstream transcription factors, a comprehensive picture of the transcriptional regulation mechanisms of CPI in modulating fresh-cut potato browning is achieved, which will lay a solid theoretical foundation for variety improving and developing new technology to inhibit fresh-cut potato browning.

褐变是制约鲜切马铃薯产业化发展的主要瓶颈之一，但影响褐变的关键因素及分子机理国内外尚无定论，因此鉴定马铃薯抗褐变关键基因并全面解析其作用机理是解决该问题的重要前提。申请人前期研究表明马铃薯半胱氨酸蛋白酶抑制剂基因CPI在调控马铃薯采后鲜切褐变中发挥重要作用，但其转录调控机制仍不清楚。在此基础上，本项目通过对CPI基因启动子进行克隆及片段缺失，构建启动子全长及缺失片段的表达载体并转化马铃薯，通过GUS染色及活性测定，鉴定启动子的核心顺式作用元件；利用酵母单杂交从马铃薯cDNA文库中筛选CPI基因上游的转录调控因子，并通过EMSA和CHIP等实验进一步验证转录因子与启动子的结合。通过全面分析CPI基因转录因子的表达模式，阐明在马铃薯鲜切褐变过程中CPI基因转录调控的分子机制，为马铃薯品种改良及研发控制褐变新技术奠定理论基础和基因资源。

褐变是制约鲜切马铃薯产业化发展的主要瓶颈之一，但影响褐变的关键因素及分子机理国内外尚无定论，因此鉴定马铃薯抗褐变关键基因并全面解析其作用机理是解决该问题的重要前提。本项目通过在二倍体马铃薯“MDS”中超表达StCPI基因探讨了该基因对马铃薯褐变及耐盐性的影响，探讨了外源脯氨酸处理抑制鲜切马铃薯褐变的机制，通过CRISPR-Cas9敲除“Desiree”马铃薯中StCPI获得了突变体植株，并探讨了该基因的缺失对马铃薯酶促褐变的影响机制。主要研究结果如下:（1）超表达StCPI显著提高了马铃薯块茎的总抗氧化能力，降低了总游离氨基酸的含量，降低了褐变主要底物酪氨酸的含量，结果表明超表达StCPI可通过提高马铃薯抗氧化能力和减少总游离氨基酸含量和酪氨酸含量来减轻褐变。超表达StCPI减缓了马铃薯植株叶绿素分解，能维持较高的光合作用，提高了植株抗氧化胁迫和渗透胁迫的能力，进而显著提高了转基因马铃薯植株抗盐性。（2）外源脯氨酸处理可显著减低鲜切马铃薯褐变，最佳处理条件下可延长鲜切马铃薯货架期至4d。外源脯氨酸处理降低了鲜切马铃薯PPO活性、总酚含量，提高了抗氧化酶SOD、CAT活力，增加了DPPH清除率、ABTS清除率及铁离子清除能力并降低了酪氨酸等游离氨基酸含量，增加了脯氨酸、组氨酸及缬氨酸等氨基酸含量，进而显著抑制鲜切马铃薯褐变。（3）利用CRISPR/Cas9技术构建了StCPI的基因编辑载体，通过农杆菌转化法侵染马铃薯茎段，第一批获得了3株第1代杂合突变阳性植株，突变类型均为两个等位基因的单碱基缺失和单碱基替换，未发现纯合突变体，3株阳性株的块茎褐变与WT无显著性差异；第二批获得了17株第1代杂合突变阳性植株，其中块茎浆液及鲜切马铃薯褐变显著高于野生型。通过全面分析并阐明在马铃薯鲜切褐变过程中CPI基因作用的分子机制，将为马铃薯品种改良及研发控制褐变新技术奠定理论基础和基因资源。