红藻糖苷异构体作为化学指示物在坛紫菜中的逆境响应机制研究

Pyropia hatianensis as one of the most important economically algae, in the intertidal zone are periodically exposed to the complex environment, which affect the healthy development of Porphyra haitanensis cultivation. Thus, it is necessary to find a chemical indicator to reflect stress resistance. Now, three isomers including floridioside, D-isofloridoside and L-isofloridoside were focused according to the clear structures and abundance activities. And it also has been reported that the levels of floridosides would change when they are responding to environmental stresses. But the response pattern and functions of each floridoside isomers has not been elucidated. Thus, a hypothesis is proposed that the three floridosides isomers are playing different roles in defense system, or different functions to response to complex environmental stress. In this study, a chiral high performance liquid chromatography coupled with mass spectrometry was developed as key technology for separation of three floridosides isomers in p. haitanensis. Combining with the changes of synthetase of floridosides, the function of each floridosides will be revealed by the relations between the changes of three floridosides isomers and floridosides phosphate synthetase under environmental stresses including wild culture environment and artificially simulated environment stresses. The different functions of three floridosides isomers in defense system will help for elucidating stress resistance mechanism of p. haitanensis, and direct the genetic breeding of Pyropia variations which have high anti-stress capacity.

坛紫菜是我国主要的潮间带大型经济海藻之一，在养殖周期内会经历剧烈复杂的环境变化，这些胁迫因素严重影响坛紫菜栽培业的健康发展，因此希望寻找到一种参与胁迫响应的小分子化学指示物快速反应胁迫应答过程。目前已发现红藻糖苷具有结构清晰、简单、生理活性丰富的特点，只有三种简单的异构体（floridioside、D-isofloridoside和L-isofloridoside），且随着环境条件改变而发生含量迅速变化，但是它们的响应模式和抗逆角色并不清晰。为此提出如下假说：为适应环境的变化，红藻糖苷三个异构体各自的角色是如何，它们与不同的红藻糖苷合成酶之间的相关性又如何，即它们在坛紫菜的逆境响应机制。由此明确不同环境胁迫下红藻糖苷作为化学指示物的防御模式，并且掌握红藻糖苷三个异构体各自的响应模式，有助于理解坛紫菜在海水栽培中的抗逆防御机制，指导坛紫菜的健康栽培和抗逆品种培育。

为明确不同环境胁迫下红藻糖苷作为化学指示物的防御模式，并且掌握红藻糖苷三个异构体各自的响应模式，本研究以不同胁迫环境下的坛紫菜作为研究对象，选择红藻糖苷三个异构体作为化学指示物，分析逆境下红藻糖苷合成酶基因和红藻糖苷异构体之间的对应关系，阐述红藻糖苷异构体的逆境响应机制。.采用离子通道为m/z 253→m/z 119、m/z 253→m/z 89的选择反应离子模式(SRM)作为定量扫描模式，结合液相色谱质谱联用和核磁共振的氢谱技术，建立起红藻糖苷、D-异红藻糖苷，L-异红藻糖苷的三种异构体的定性定量方法。基于此发现不同日龄、不同地点的坛紫菜中红藻糖苷含量相对稳定，异红藻糖苷的含量敏感于海水温度波动，故可初步认为红藻糖苷异构体能够作为坛紫菜选育过程中的一个指标物。坛紫菜两个世代体-叶状体和丝状体中红藻糖苷异构体含量在盐度、琼胶寡糖和温度的胁迫响应方式不尽相同：红藻糖苷异构体对于胁迫的响应程度为：盐度>琼胶寡糖>温度；且叶状体中红藻糖苷异构体的响应强度和速度显著高于丝状体，整体呈现上调现象，以异红藻糖苷含量为主，但是响应倍率和速度却为红藻糖苷>D-异红藻糖苷>L-异红藻糖苷。克隆并鉴定了四个可能的海藻糖-6-磷酸合酶基因PhTPS1~4，通过功能验证发现PhTPS1，4参与了红藻糖苷和异红藻糖苷的生物合成，异红藻糖苷是主要产物，PhTPS3是异红藻糖苷磷酸合酶，而PhTPS2没有活性。当受到盐度、温度等胁迫时，PhTPS3和PhTPS4的响应达到20倍以上，对应着红藻糖苷和异红藻糖苷的合成，且以异红藻糖苷合成为主。红藻糖苷异构体的前体物质甘油-3 -磷酸及其相关合成基因Phnho1和Phgpdh的表达量在胁迫后整体呈现上调现象，从而促进红藻糖苷异构体的合成。.由此可知，红藻糖苷可作为紫菜选育过程中的一个化学指标物，为坛紫菜健康养殖情况和抗逆品种培育提供指导作用。