钙信号介导丹参丹酚酸B生物合成机制研究

Secondary metabolites is an important natural resource to screen and develop new drugs, but their utilizition is limited due to the low level in plants. Althought the biosynthesis of them can be induced by elicitors, the quantity would not meet demand if the functional mechanisms of those elicitors are not well understood..In our previous work, salicylic acid (SA) was found to significantly enhance the biosynthesis of salvianolic acid B (SAB) in Salvia miltiorrhiza cell cultures. We have found that SA elicits the Ca2+ burst in Salvia cells, and Ca2+ is involved in the signal transduction of the biosynthesis of SAB, but the mechanism is not well clarified. SA also induces the burst of H2O2, and H2O2 is also involved in the biosynthesis of SAB. It is yet unclear that how the intereaction of Ca2+ and H2O2 regulate the biosynthesis of SAB..The aim of this proposal is to reveal the mechanism of Ca2+ mediating SAB accumulation in SA-induced cells at cell level, as well as its crosstalk with H2O2. For this purpose, non-invasive micro-test (NMT) and confocal laser scanning microscope will be used to detect Ca2+ flow speed and its transportation after SA induction. .The results will be significance in explaining how signal molecules modulate the production of secondary metabolites when they respond to elicitor induction, and will lay the foundation for specifically regulate the biosynthesis through the control of the metabolic process.

次生代谢物是天然新药研发与筛选的重要资源，因在植物中含量低而使其利用受到限制。利用诱导子诱导可促进次生代谢物合成，但因不明确诱导子的作用机制而无法通过控制代谢过程定向调控其生物合成。. 我们发现水杨酸(SA)诱导可提高丹参中丹酚酸B(SAB)含量，Ca2+作为第二信使响应SA刺激介导SAB合成，其应答机制不清楚。前期我们还发现，过氧化氢也参与SA诱导SAB合成过程，不明确钙信号与过氧化氢如何协同调控SAB合成。. 本研究采用非损伤微测和胞内成像技术检测SA诱发丹参活体细胞产生的原位Ca2+流速、运动方向及浓度，从细胞层面揭示特异位点的钙信号介导SAB合成规律，进一步探讨钙信号与过氧化氢协同作用调控SAB合成的信号应答机制。. 此项目对阐明信号分子介导诱导子调控次生代谢的应答机制具有重要意义，为通过控制代谢过程定向调控次生代谢物合成奠定基础。

丹酚酸B是药用植物丹参（Salvia miltiorrhiza Bung.）的指标性成分，生产中一直无法克服其含量低且不稳定的现象。利用诱导子诱导可促进丹酚酸B合成，但因不明确诱导子的作用机制而无法通过控制代谢过程定向调控其生物合成。前期研究发现Ca2+作为第二信使响应水杨酸（SA）刺激介导丹酚酸B合成，但其应答机制仍不清楚。本项目针对钙信号介导丹参丹酚酸B生物合成机制进行了研究，取得了以下结果：. 1.确定了SA诱发丹参细胞产生Ca2+的来源及变化规律。外源SA处理能够诱发丹参细胞Ca2+信号形成，胞外Ca2+内流是丹参胞质Ca2+迸发的主要来源，水杨酸诱发Ca2+内流的瞬间伴随与H+的交换，Ca2+与H+的转运方向相反，Ca2+内流与H+外排正相关。. 2.明确了SA诱导的Ca2+促进了丹酚酸B的生物合成。SA处理诱发了Ca2+迸发，上调了丹酚酸B合成相关酶PAL、TAT、RAS基因的表达，提高了丹酚酸B合成相关酶活性，促进了丹酚酸B的生物合成。. 3.揭示了钙依赖的磷脂膜结合蛋白SmANN1在Ca2+介导丹酚酸B合成中具有重要的作用。胞质Ca2+稳态发生变化使SmANN1表达量上调。过表达SmANN1通过提高丹酚酸合成途径关键酶基因表达量提高丹酚酸B合成积累。. 4.分析了丹参MAPK基因家族及丹参中MAPK可能存在的级联反应，确定了SmMAPK3与SmMYBs和SmAREB1物理互作，初步明确了SmMAPK3可调控丹参次生代谢。. 5.发现了LPS通过Ca2+依赖方式促进丹参酮生物合成。LPS诱导促进了丹参毛状根中丹参酮累积，上调了转录因子SmWRKY1和SmWRKY2的表达，转录因子SmWRKY1和SmWRKY2激活了丹参酮合成酶基因SmCPS和SmCYP76AH1的表达，提高了丹参酮的生物合成。. 6.阐明了SmPHA4正调控丹酚酸B生物合成的机理。过表达SmPHA4引发了Ca2+信号迸发，激活了SmMYC2转录因子，启动了TAT1、RAS1的表达，促进了丹酚酸B的生物合成。. 以上研究揭示了特异位点的钙信号介导丹酚酸B合成规律及信号应答机制，对阐明信号分子介导诱导子调控次生代谢的应答机制具有重要意义，为通过控制代谢过程定向调控丹参次生代谢物的生物合成奠定了理论基础。