低温胁迫下转录因子SlWHY1调控Rubisco酶活的分子机制

Low temperature stress is a major factor that limits the agricultural productivity of chilling-sensitive crops in protection cultivation, such as tomato in northern China. Whirly transcription factor is a DNA-binding protein that is dual-localized in nucleus and plastid and regulates plant senescence and abiotic stress response. Our previous study found that tomato SlWHY1 plays an important role in plant low temperature response, and the mechanism is worthy of further discussion. This study plans to use the obtained SlWHY1 transgenic tomato lines to explore the molecular mechanism of SlWHY1 regulating expression of Rubisco small subunit gene under low temperature stress. Before and after low temperature treatment, the expression levels of genes encoding Rubisco large and small subunits will be determined, the content of Rubisco large and small subunits will be analyzed, and the content and enzyme activity of Rubisco holoenzyme of SlWHY1 transgenic lines will be detected. ChIP, yeast one hybrid, and EMSA will be used to identify the target gene and the binding sequence of SlWHY1. Under H2O2 treatment and low temperature plus H2O2 scavenger treatment, the expression pattern of nuclear SlWHY1 will be detected. Through the above experiments, the signal molecular that induces the expression of SlWHY1 in the nucleus under low temperature will be defined and the molecular mechanism of SlWHY1 regulating the expression of gene encoding Rubisco samll subunit will be elucidated. These studies will provide a theoretical basis for guiding the cultivation of warm vegetables and stress-resistant breeding.

低温胁迫是我国北方地区番茄等喜温蔬菜保护地生产中的主要限制因素之一。Whirly转录因子是一个核质双定位的DNA结合蛋白，调控植物的衰老和非生物胁迫响应。我们前期研究发现番茄SlWHY1在植物低温应答中具有重要作用，其作用机制值得深入探讨。本项目拟利用已获得的SlWHY1转基因番茄探究SlWHY1在低温胁迫下调控Rubisco小亚基基因表达的分子机制。通过检测低温处理前后编码Rubisco大、小亚基的基因表达，分析SlWHY1转基因株系中Rubisco大、小亚基的含量、Rubisco全酶的含量和酶活，ChIP、酵母单杂交和EMSA鉴定SlWHY1的靶基因并确定其结合序列；检测核中SlWHY1在H2O2、低温与H2O2清除剂处理下的表达模式。阐明低温胁迫下诱导核中SlWHY1表达的信号分子并解析SlWHY1调控Rubisco小亚基基因表达的分子机制，为指导喜温蔬菜栽培和抗逆育种提供理论依据。

光合作用是地球上最重要的生化反应，为几乎所有生物提供物质和能量来源。然而，光合作用也是植物体内对低温最敏感的生理过程之一。Rubisco是光合作用的限速酶，也是低温胁迫下光合作用的靶点之一。低温胁迫会降低Rubisco的含量和活性，导致植物碳同化能力下降，光能利用率减少，产生过剩光能，造成光抑制，甚至光破坏。本课题主要研究了番茄SlWHY1在低温胁迫下对光合作用碳同化过程的保护机制。低温处理后，与野生型相比，过表达SlWHY1的株系CO2同化速率更高，且Rubisco的含量和酶活也更高，而RNAi株系则恰好相反；进一步分析发现，低温胁迫下，编码Rubisco小亚基的RBCS的转录水平和蛋白水平在过表达株系中都高于野生型，而在RNAi株系中则低于野生型；RT-qPCR结果显示，编码Rubisco小亚基的RBCS基因中，只有SlRBCS1的转录水平受到低温的诱导表达；YIH、ChIP及LUC实验结果表明，SlWHY1能够与SlRBCS1启动子上的ERE及类ERE元件结合激活SlRBCS1的表达，且低温促进了SlWHY1对SlRBCS1的激活；并且，我们发现SlRBCS1的过表达株系在低温胁迫下的表型、CO2同化速率、Rubisco的含量及活性都与SlWHY1过表达株系一致；抑制所有RBCS基因表达的RNAi株系在正常条件下叶片失绿，但叶绿体结构与野生型无明显差异；将SlRBCS1的特异氨基酸位点替换后进行了番茄的转化，低温处理后，特异位点突变的转基因株系与SlRBCS1的过表达株系表型无明显差异，这表明低温胁迫下Rubisco含量和活性的维持主要依赖SlRBCS1的含量，而非特异的氨基酸残基；此外，我们还发现SA信号对SlWHY1的诱导表达最为明显，我们推测低温可能通过SA信号激活了SlWHY1的表达。本课题的研究结果阐明了低温胁迫下SlWHY1通过激活编码Rubisco小亚基的核基因SlRBCS1的表达维持了Rubisco的含量和活性，从而提高了番茄的低温抗性，完善了植物的低温信号调控网络，并为指导喜温蔬菜栽培和抗逆育种提供了理论依据。