香榧角鲨烯合成酶的转录调控机制及其对β-谷甾醇合成功能的解析

Torreya grandis‘Merrillii’ is a rare economic tree native to China. It has very high nutritional and economic value. There are a large number of beta-sitosterols in Torreya grandis seeds, which have important functions of anti-cancer and cholesterol reduction. Squalene synthase is a key enzyme in the synthesis of beta-sitosterol, which has not been reported in Torreya grandis. In this project, the enzyme kinetics of squalene synthase from Torreya grandis was analyzed by E. coli and tobacco expression system, and the function of squalene synthase was verified by transformation into Torreya grandis callus. There were significant differences in squalene synthase expression between different developmental stages and cultivars of Torreya grandis, suggesting that there may be transcriptional regulation of squalene synthase. This project aims to screen transcription factors that can regulate squalene synthase by(1)Searching for co-expressed transcription factors in transcriptome data at different developmental stages and(2)screening yeast single hybrid library, and verify their functions by transforming Torreya grandis callus. The regulation of functional transcription factors on the synthesis of beta-sitosterol will be further studied. The results will provide a clear theoretical basis for seed quality improvement of Torreya grandis.

香榧是原产于中国的稀有经济树种，具有非常高的营养价值和经济价值。香榧种实中存在大量具有抗癌及降低胆固醇等重要功能的β-谷甾醇。角鲨烯合成酶是β-谷甾醇合成途径的关键酶，目前在香榧中还未见报道。本项目拟利用大肠杆菌和烟草表达系统对香榧角鲨烯合成酶进行酶动力学分析，并转化香榧愈伤组织验证其功能。在不同发育时期和不同栽培品种的香榧种实中角鲨烯合成酶的表达量都存在显著差异，表明可能存在对角鲨烯合成酶的转录调控。本项目拟通过（1）在不同发育时期的转录组数据中查找共表达的转录因子和（2）筛选酵母单杂交文库两种方法筛选能够调控角鲨烯合成酶的转录因子，并通过转化香榧愈伤组织等方法验证其功能。对于有功能的转录因子将进一步研究其对β-谷甾醇合成途径的调控作用。研究结果将为香榧种实品质改良提供清晰的理论依据。

香榧（Torreya grandis cv.Merrillii）是一种珍贵的坚果，具有谷甾醇和类黄酮等多种生物活性化合物，具有很高的经济价值。谷甾醇不仅是含量最丰富的植物甾醇，而且具有抗菌、抗癌、抗炎、降脂、抗氧化和抗糖尿病等多种生物活性。在本研究中，对香榧鲨烯合成酶基因TgSQS进行了克隆和功能鉴定。TgSQS蛋白的原核表达能够催化法尼基二磷酸生成角鲨烯。过表达TgSQS的转基因拟南芥植株角鲨烯和β-谷甾醇的含量显著增加，而且其耐旱能力也强于野生型。香榧幼苗的转录组数据表明，干旱处理后，甾醇生物合成途径相关基因HMGS、HMGR、MK、DXS、IPPI、FPPS、SQS和DWF1的表达水平均显著提高。我们还通过酵母单杂交实验和双荧光素酶实验证明了TgWRKY3直接与TgSQS启动子区结合并上调其表达。综上，这些结果表明TgSQS在谷甾醇生物合成和防御干旱胁迫方面具有重要作用，能够作为同时改善谷固醇生物合成和植株抗旱能力的目标基因。另外我们还通过关联分析找到9个与香榧种仁抗氧化话活性显著相关的黄酮类化合物，且发现CHS、DFR和ANS与这9个黄酮类化合物的生物合成显著相关，这一结果能够香榧果实品质改良提供理论基础。