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条斑紫菜Pyropia yezoensis响应高盐胁迫机制的初步研究
余斌
学位类型硕士
导师王广策
2018-05-17
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位名称工程硕士
学位专业生物工程
关键词条斑紫菜 脱落酸 抗氧化系统 Ferredoxin: Nadp +还原酶 环式电子传递
摘要

条斑紫菜是一种常见的潮间带大型红藻,也是我国重要的经济海藻。条斑紫菜在每天低潮干出时都会经历严重的失水胁迫,是研究潮间带海藻对极端环境适应性机制的良好材料。为研究条斑紫菜对高盐失水环境的抗性机制,本论文开展了以下三方面的研究:

1. 条斑紫菜中叶黄素循环的缺乏使其更依赖于其他光保护机制,比如环式电子传递。首先我们测定了不同盐度胁迫对藻体光合参数的影响,结果表明随盐度升高,CET呈现先升后降的趋势。抑制剂实验表明条斑紫菜中至少存在三种不同类型的环式电子传递途径。为探究不同路径之间的协调方式,我们对条斑紫菜中的FNR进行克隆测序,并构建系统发育树,经分析发现条斑紫菜FNR与蓝藻FNR进化地位更相近。因此我们推测,条斑紫菜FNR通过介导NAD(P)HFd+之间的双向电子转移参与环式电子传递。FNR对不同电子供体的连接使电子能够根据代谢需求改变流向,使叶绿体中的电子转移更加灵活,更有利于条斑紫菜适应潮间带的极端环境。

2.抗氧化系统包括多种抗氧化酶和抗氧化分子,在条斑紫菜适应极端多变的环境中起着重要的作用。我们用不同盐度的海水对条斑紫菜进行胁迫,并取胁迫后不同时间的材料测定主要抗氧化酶活性,抗氧化分子的含量以及过氧化氢含量。结果表明,较低程度的盐胁迫或者短时间胁迫下条斑紫菜可依赖抗氧化酶和原有的抗氧化库,通过加快还原性物质的再生速率清除活性氧;而随着胁迫程度加深,已有的抗氧化库不足以周转,抗氧化小分子大量增加并在活性氧清除中起重要作用;谷胱甘肽在胁迫初期总量很快增加,在应对活性氧的瞬时爆发上具有重要意义。

3. ABA参与抗氧化系统的活化和逆境适应,在陆地植物中已得到较为深入的研究,但ABA在红藻中的合成途径及生理功能尚不清楚。我们对不同盐度胁迫的藻体进行ABA含量测定发现,随盐度升高,ABA含量呈现明显的上升趋势,尤其在极端高盐(150‰)胁迫下,说明ABA在胁迫响应中具有一定作用。为探索条斑紫菜中ABA的合成方式,采用AAO3抑制剂钨酸钠、NCED抑制剂萘普生和IPP合成抑制剂多效唑对藻体进行预处理,之后150‰盐度胁迫并测定ABA含量。结果表明三种抑制剂均能显著降低ABA的积累量,据此我们推测ABA在条斑紫菜中主要通过类胡萝卜素途径合成,而ABAC5前体IPP主要通过MVA途径合成。另外,外源ABA的使用能够显著降低高盐胁迫下藻体中丙二醛的积累量,说明ABA在减缓氧化损伤上具有重要作用,而ABA对条斑紫菜抗氧化系统的调控功能有待进一步研究。

其他摘要

Pyropia yezoensis (previously called Porphyra yezoensis), belongs to the genus of Pyropia, is a common rhodophyta of intertidal zone. It’s also an important economic marine algae in China. Py. yezoensis can survive the severe water loss that occurs during low tide, making it an ideal species to investigate the acclimation mechanism of intertidal seaweed to the special extreme environments. To study the mechanisms of resistances to abiotic stresses in Py. yezoensis, we carried out research in the following aspects:

1. Because of the lack of xanthophylls cycle, some other photo-protective mechanisms are much more important for Py. yezoensis, such as cyclic electron transport. Firstly, we determined the effects of high salinity on photosynthesis using increasing salinity around algal tissues. CEF increased and remained at a stable state in 50‰-120‰ salinity and decreased sharply when Py. Yezoensis were treated with 150‰ or higher salinity. Inhibitors experiments illustrated that there were three different cyclic electron transportation pathways at least. To figure out how those different CETs were coordinated, we had cloned the gene sequence (HM370553) of ferredoxin-NADP+ oxidoreductase (FNR). Then, a phylogenetic tree was constructed and the evolutionary relationship among different FNRs indicated that the Py. yezoensis FNR showed closer relation to the cyanobacterial FNR. Due to the structure-function correlation in organism, Py. yezoensis FNR was proposed to be involved in the NAD(P)H-dependent Fd+ reduction under severe salinity conditions. Thus, through the connection between different donors bridged by FNR, electrons were channeled towards distinct routes according to the different metabolic demands. FNR was also directly involved in certain CET pathways. This made the electron transfer in the chloroplasts became more flexible and contributed greatly to acclimation of Py. yezoensis to the extreme variable environments in the intertidal zone.

2. The antioxidant mechanisms which consists of a variety of antioxidases and antioxidant molecules in Py. yezoensis might play a very important role to survive the extreme variable environments. We treated Py. yezoensis with different hypersaline seawater and determined the time variation of photosynthetic parameters, antioxidases activity and the content of antioxidants. Our results showed that Py. yezoensis can cope with a short period of hypersaline or mild hypersaline with minimal disruption of their total antioxidants pools. This could be achieved by increasing the activities of antioxidases, and/or increasing the rate of antioxidants regeneration. With the increasing salinity and the extension of time, it is insufficient for the primary antioxidants pools to scavenge the generated ROS. Thus, antioxidant pools increased and play an important role in ROS scavenging. GSH and total glutathione pool increased quickly to cope with the tempory ROS burst.

3. The phytohormone abscisic acid (ABA) is involved in the adaptive stress response and regulates the activation of anti-oxidization system, which has been studied thoroughly in land plants. However, its function in marine algae remains unclear. The algal tissues were treated with increasing salinity and ABA content was determined. Our results showed that the ABA content in high salinity stressed Py. yezoensis presented a significant up-regulation compared with that of the control, especially in 150‰ salinity group. The results strongly suggested that there were some positive effects which mediated by ABA. To find out the possible ABA synthesis pathway in Py. yezoensis, thallus of the algae were treated firstly with the inhibitors which was related to the biosynthetic pathway of ABA. After that, ABA content was determined using 150‰ salinity seawater around thallus tissues. The results showed that sodium tungstate and naproxen reduced ABA synthesis significantly, which meant the synthesis of ABA in Py. yezoensis was through the carotenoid biosynthetic pathway. Paclobutrazol showed the same effect on ABA content, indicating that IPP (the precursor of ABA synthesis) was derived from MVA pathway. In addition, exogenous ABA could decrease the accumulation of MDA when Py. yezoensis was treated with increasing salinity, indicating that there might be an antioxidant pathway mediated by ABA in Py. yezoensis. Still, the effect of ABA on antioxidases activity needs to be studied further.

学科门类工学::生物工程
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154534
专题实验海洋生物学重点实验室
第一作者单位中国科学院海洋研究所;  实验海洋生物学重点实验室
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余斌. 条斑紫菜Pyropia yezoensis响应高盐胁迫机制的初步研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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