坛紫菜环境适应机制中的脂质氧化代谢途径的研究

This study aimed to elucidate the response mechanisms of lipid peroxidation pathway (oxylipin pathway) of the macro-algae living in seashore zone in defending against environmental stresses. It was found previously by other investigators that plant oxylipins with highly complex structures were present in red algae. However, to date, there have been no studies on the genes encoding the enzymes involved in oxylipins pathway and the function of this pathway in algae. In our preliminary study, we found that there are several genes that encodes the key enzyme, lipoxygenase (LOX) involved in oxylipin pathways, was present in Pyropia haitanensis and that its corresponding LOX enzymes possessed the characteristics of multiple functions and flexibility. Based on these backgrounds, we hypothesized that Pyropia haitanensis employed the enzymes catalyzing in the key steps of oxylipin pathways, and the multiple LOX genes encoding LOX isoforms, using the “the differential expression and their cooperative team work” defense mode, as well as the multiple functions and the flexibility of LOXs, to achieve different combinations of the oxylipin metabolites and their quantitative changes for adapting and defending against the complex environmental stresses. In this study, we selected Pyropia haitanensis, which has evolved at low level, has a small genomic size and can be easily obtained, as the study model to conduct a systemic investigation on signal transduction cascade of the lipid metabolism pathways within the oxylipin pathway, including the genes encoding the key enzymes of these pathway → enzyme proteins → down-stream components, for elucidating the responsive mechanisms of the proteins and metabolites involved in this pathway present in Pyropia haitanensis to environmental stresses. This investigation attempted to answer the question raised by the scientific circle regarding the origins of the structural diversity of oxyipins, to elucidate the origin and evolution/development of the unique abundant oxylipins present in red algae and to gain insights into the ecological response mechanisms of Pyropia haitanensis to different environmental stresses from the aspect of lipid oxidation defense. Moreover, this study will provide a reasonable study model for further elucidating the immune system present in macro-algae.

为阐明红藻中的脂氧化代谢（oxylipin）途径在生态适应中的响应机制，本研究基于前人发现的红藻中存在结构复杂的oxylipins，但却没有相关酶基因和功能报道的事实；及前期研究发现的坛紫菜中有多条oxylipin途径关键酶—脂氧合酶（LOX）基因，且相应蛋白存在多功能、多灵活性的特点，提出如下假说：坛紫菜利用oxylipin途径中多条酶基因和蛋白的“差异表达和分工”以及该酶的多功能和灵活性，实现防御物质的不同组合的变化，来抵御复杂的环境胁迫。因此本研究将选择坛紫菜为研究对象，对oxylipin途径中的脂类物质代谢通路；关键酶的基因→酶蛋白→下游化合物的信息传递通路，及受胁迫时路中的蛋白和化学组分的响应方式进行系统研究，进而回答科学界关于红藻中的大量结构多样的oxyipins来源的疑问；阐明红藻特殊oxylipin途径的进化；并从脂氧化代谢防御角度理解坛紫菜对潮间带生态适应的响应机制。

许多藻类同时具备C18-和C20-多聚不饱和脂肪酸（PUFAs）氧化衍生物（oxylipins），参与环境胁迫的化学防御和信号转导。脂氧合酶（LOXs）是oxylipins合成中关键的起始酶，但至今关于藻类LOXs家族的系统性研究却几乎没有。.本研究借助坛紫菜基因组测序的基础，从基因组中共发现6条NhLOX基因，发现全部NhLOX基因都来自横向基因转移，红藻LOXs在进化的早期与高等植物和动物LOX进化枝的祖先分离。其中NhLOX3-6起源自蓝细菌，而NhLOX1,2则起源自海洋细菌。并且以NhLOX1为例，发现存在基因存在可变剪切现象。发现NhLOX1,2这两个酶都具有多功能的特性，比如NhLOX1在单结构域中同时具有LOX、AOS以及HPL的功能，而NhLOX2同时具有LOX和氢过氧化物酶两种活性。NhLOX1表现出异常高的HPL活性，这说明了所观察到的红藻的oxylipin物质的多样性。NhLOX的底物柔韧性，位置柔韧性和功能多样性代表了低等生物的典型特性。酶-底物相互作用的模型表明，Gln777和Asn575残基可能已经参与了NhLOX的AOS活性。提出了两步反应来解释双双加氧产物的形成，C20脂肪酸的尾对头方向和C18：3的头对尾插入。研究表明，某些红藻的氧脂蛋白生物合成基因的起源和氧脂蛋白生物合成模式是独特的，这可能解释了这些红藻的oxylipin途径的进化以及红藻的致密基因组的原因。发现坛紫菜在不同的环境胁迫和不同的发育时期，会运用多条LOX基因和不同剪切体形式的变化，以一种“差异分工”或“组合分工”的方式，并结合酶蛋白的多功能性和灵活性，来实现不同组合的防御产物来应对复杂、多样的环境胁迫。这可能解释了这些红藻如何利用致密的基因组来应对复杂潮间带环境的具体的化学防御策略。还发现NhLOX1具备一步催化形成C8-C9类挥发性物质的能力。包括催化ARA生成2E-壬烯醛，EPA生成2E,6Z-壬二烯醛。NhLOX1也可以以不同的油料水解物为底物，将葵花籽油、玉米油和鱼油分别转化生成2E,4E-戊二烯醛、2E-辛醛和2E,4E-辛二烯醛。表明了NhLOX1具备新型生物催化剂开发的潜力。