AnGolS1提高番茄低温抗性的作用机制研究

Low-temperature stress is the main factor affecting the growth and yield of crops. The raffinose oligosaccharides with inositol galactosidase as the rate-limiting enzyme play an important role in improving the low-temperature resistance of plants. We had isolated the galactinol synthase gene (AnGolS1) from a cold-induced Suppression Subtractive Hybridization (SSH) library derived from Ammopiptanthus nanus (A. nanus) seedlings, and obtained AnGolS1 overexpression tomato plants with enhanced cold resistance. In this project, we will construct SlGolS1-4 knocked-out and overexpression tomato plant, and then to study the mechanism of AnGolS1 in confering tomato resistance against low-temperature stress. Firstly, the enzymatic properties of galactinol synthase will be analyzed by galactinol synthase crude extraction or expressed protein in prokaryotic expression system to identify the difference between AnGolS1 and SlGolS. Secondly, comparing the resistance to low temperature stress of difference transgenic materials will be implemented to reseach the effects of AnGolS1 on the growth and development of tomato under low temperature stress. Finally, most importantly, metabolomics and transcriptome will be used to analyze the effects of AnGolS1 on the synthesis, metabolism and transport of oligosaccharides and other related carbohydrates. The project will lay the foundation for the breeding of low temperature resistant tomato varieties and improvement of tomato low temperature resistance through exogenous raffinose oligosaccharides.

低温逆境是影响作物生长发育与产量的重要因子，以肌醇半乳糖苷酶为限速酶的棉子糖系列寡糖对提高植物低温抗性具有重要作用。本项目从抗寒性极强的新疆沙冬青中分离到与其抗寒性密切相关的肌醇半乳糖苷合成酶基因（AnGolS1），并获得了抗冷性增强的超表达番茄植株，在此基础上，拟构建番茄SlGolS1-4基因敲除以及番茄SlGolS超表达植株，分析基因表达产物-肌醇半乳糖苷酶的酶学性质，明确AnGolS1酶学特性；分析低温胁迫下不同转基因番茄材料的抗冷性，明确AnGolS1提高番茄不同发育阶段抗冷性的作用；通过代谢组学与转录组学分析AnGolS1对番茄不同部位棉子糖代谢、蔗糖代谢以及肌醇代谢的相关产物与基因，明确其提高植株低温抗性的作用位点，揭示其提高番茄抗低温胁迫的可能机制。为明确棉子糖系列寡糖在植物抗低温逆境胁迫的作用机制，以及培育低温抗性番茄品种或通过外源棉子糖提高番茄低温抗性等均具有重要意义。

低温逆境是影响作物生长发育与产量的重要因子，以肌醇半乳糖苷酶为限速酶的棉子糖系列寡糖对提高植物低温抗性具有重要作用。本项目获得了新疆沙冬青肌醇半乳糖苷合成酶基因（AnGolS1）超表达的番茄植株，在此基础上，研究基因表达产物-肌醇半乳糖苷酶的酶学性质，明确AnGolS1酶学特性；分析低温胁迫下不同转基因番茄材料的抗冷性，明确AnGolS1提高番茄不同发育阶段抗冷性的作用；通过代谢组学与转录组学分析AnGolS1对番茄不同部位棉子糖代谢、蔗糖代谢以及肌醇代谢的相关产物与基因，明确其提高植株低温抗性的作用位点，揭示其提高番茄抗低温胁迫的可能机制。项目主要研究结果表明，AnGolS1超表达番茄种子与幼苗的低温抗性显著增强，主要原因是AnGolS1超表达提高了肌醇半乳糖苷含量，并不同程度增加葡萄糖、果糖与蔗糖含量；AnGolS1超表达植株显著增强了叶片的抗氧化活性；低温胁迫下，AnGolS1超表达诱导番茄多个代谢通路相关基因发生差异表达，尤其是乙烯（ET）信号转导相关基因，研究表明表明肌醇半乳糖苷能够诱导ERF转录，ERF正向反馈促进ET生物合成；AnGolS1超表达能通过肌醇半乳糖苷影响次生代谢产物的生物合成，促进氨基酸类、核酸类、有机酸类及脂质类次生代谢物的生物合成，而降低黄酮类次生代谢产物的积累，提高番茄植株的抗冷性；AnGolS1能够诱导丝氨酸蛋白酶抑制因子SlSPI5的表达，SlSPI5又抑制枯草杆菌类蛋白酶SlSBT17的表达，促进丝氨酸/苏氨酸蛋白激酶Y1677的表达，Y1677蛋白将激活多种次生代谢产物的合成酶或分解酶的催化活性，从而改变次生代谢物的含量，提高低温抗性。研究结果为明确棉子糖系列寡糖在植物抗低温逆境胁迫的作用机制，以及培育低温抗性番茄品种或通过外源棉子糖提高番茄低温抗性等均具有重要意义。