实验海洋生物学重点实验室知识库

摘 要

浒苔是生长在潮间带的一种常见大型绿藻。近年来由于浒苔在我国青岛海域持续暴发生长引起绿潮而受到广泛关注。绿潮暴发对我国近海海域生态系统造成了严重的破坏，造成了巨大的经济损失。浒苔之所以能够暴发式生长与其强大的抗逆能力密切相关。由于长期生活在潮间带，浒苔受潮汐和陆源河流的影响，会时常遭受高盐、干出、高光以及光质等多种环境因子的影响。经过长期适应，浒苔拥有了极强的抗高光、抗高盐能力。

现已证实青岛海域的浒苔暴发与我国苏北浅滩的紫菜养殖业密切相关。在紫菜筏架上定生的浒苔会进入外海呈漂浮生长引起绿潮暴发。本文首先对绿潮暴发海域的浒苔光合活性进行了测定。结果表明，浒苔从定生到漂浮后其光合活性虽然有所不同，但仍然维持相对较高的活性，并且浒苔暴发海域从南至北浒苔光合活性逐渐降低。跟踪统计了暴发海域浒苔孢子囊形成情况，发现孢子囊形成迅速，并观察到了浒苔的原位萌发。以上结果表明了浒苔在暴发过程中光合活性变化明显，由定生到漂浮生长后孢子囊形成快速，这为其快速繁殖提供了基础。浒苔从定生到漂浮后，由于长时间漂浮在海面，在阳光照射下会持续受到高光和高盐胁迫，并且由于浒苔生物量巨大，会在海水表面形成藻席，部分浒苔沉入海水里面。此外，由于涨潮和退潮影响，定生浒苔会处于不同的水深中，由于不同波长的光在海水中穿透能力不同，海水中的光质会发生变化，浒苔会接收到不同波长的光。在这些情况下浒苔依然能够保持较高的光合活性和快速的生长速率，说明浒苔必然存在高效的生理机制应对不良环境。我们的工作重点关注了高光、光质及高盐三种胁迫因子下浒苔的非光化学淬灭（NPQ）响应机制。

在实验室设置高光条件处理浒苔，测定其光合活性并分析蛋白组成。结果表明，浒苔在高光下NPQ显著升高，伴随着玉米黄质含量升高，但是与NPQ发生相关的关键蛋白PsbS和LhcSR并没有上调，在浒苔中PsbS和LhcSR在高光下不能被诱导表达，与莱茵衣藻和小立碗藓显著不同。分析了浒苔内周天线蛋白，发现浒苔中只有蛋白CP26和CP29，没有CP24，与高等植物明显不同。我们推测，正是CP24的缺失导致浒苔及相近的大型潮间带绿藻缺少快相的NPQ发生机制。通过比较色素组成发现，浒苔存在一定本底水平的玉米黄质，不同于莱茵衣藻和小立碗藓的诱导型表达。定生浒苔长期受到周期性的高光胁迫，浒苔藻体内储存着NPQ发生必须的物质基础——PsbS、LhcSR蛋白以及玉米黄质，以便能快速启动NPQ机制进行光保护。这也是浒苔长期适应潮间带环境的结果。

在光质胁迫实验中，我们对浒苔进行8种波长不同的光进行照射处理。结果表明，经过长波长的远红光处理2天后浒苔NPQ显著升高，并且玉米黄质含量升高，但是光合生理活性降低；短波长的紫外光和蓝光下浒苔NPQ与白光相比并无显著差异。通过免疫印迹实验（WB）发现，LhcSR蛋白显著上调，而PsbS无显著差异，实时荧光定量PCR结果也证明了基因LhcSR的上调表达。以上结果表明，远红光能通过诱导LhcSR蛋白的表达和玉米黄质的合成促进NPQ的升高。作为对比，我们发现小立碗藓在远红光下并不能诱导LhcSR的表达，NPQ也没有升高，充分说明了浒苔存在响应远红光的通路。对处理后藻体的类囊体膜蛋白进行超速离心分离并对光合复合体成分的鉴定发现，远红光诱导上调表达的LhcSR结合在光系统II（PSII）捕光天线复合体（LHCII）三聚体和内周天线蛋白单体上，并不能与光系统I（PSI）复合体结合。此外，对PSI天线系统（LHCI）和LHCII进行WB分析发现，远红光条件下LHCII变大，并且PSI-LHCI复合体上叶绿素b与叶绿素a的比值下降。以上结果表明，远红光能诱导浒苔LhcSR的升高，并与PSII的结合，但不能诱导PsbS的表达，证明了在光保护中LhcSR不同于PsbS的关键作用，为LhcSR蛋白功能的研究提供了新的方向。通过转录组学方法对实验结果进行了验证，并且发现钙离子相关代谢通路在FR信号转导中起到重要作用。在转录组数据中发现远红光受体基因的存在，并据此构建了相对完整的远红光信号通路。

最后，在高盐胁迫条件下，我们研究了浒苔在高盐胁迫下的NPQ发生机制，并且以单细胞绿藻盐藻和陆生植物小立碗藓作为对比。我们发现浒苔和盐藻在响应高盐胁迫时光合生理参数YI和YII有不同的变化趋势。盐藻通过促进磷酸戊糖途径为甘油循环提供还原力和能量，促进甘油循环的高效进行，从而适应高盐环境，而磷酸戊糖途径在浒苔中作用与盐藻中有明显不同。在浒苔中，适当的高盐度处理条件（9% NaCl）能诱导浒苔NPQ的升高。而PsbS和LhcSR蛋白表达并没有升高，此外玉米黄质含量升高，这与小立碗藓在高盐胁迫下NPQ升高的机制类似，被认为是一种独特的诱导途径，还有待于更加深入的研究证实。

比较三种条件下浒苔NPQ的发生机制我们认为，高盐胁迫与高光及远红光条件下的NPQ的升高均需要玉米黄质的参与，但三种条件下NPQ的发生也有明显不同。由于潮间带环境因子变化多样且迅速，浒苔进化出多种NPQ机制共同帮助浒苔抵抗胁迫，适应潮间带的环境。

ABSTRACT

Ulva prolifera is a familiar macro-alga lived in intertidal zone. It has attracted more attentions in recent years due to the green tide occurred persistently along Qingdao bay. The green tide destroyed the ecosystem of the sea and has cased huge economic losses. The outbreak of U. prolifera is closely related to its high ability against high light, high salinity and other stress factors.

It has been proved that the green tide occurred in Qingdao is much related to the Porphyra yezoensis culture in Jiangsu province of China. After fall off from the raft, U.prolifera is floatted to the open sea. In this work, we first measured the physiological activities of U. prolifera in the green tide occurring area in situ. The results show that the physiological activities of floating thalli kept higher although decreased a little compared with adhering thalli. The photosynthetic activities varied in different ocean areas. In total, the photosynthetic activities decreased gradually from south to north area. In addition, we calculated the percentages of sporangium and suggested that it formed quickly when floating on the sea. These all contributed to the occurrence of green tide. The floating thalli would subjected to high light and high salinity stresses. Moreover, light qualities under seawater were different from that in atmosphere. These were all stress to U. prolifera. There must exist efficient pathway of U. prolifera to against adversity stress. In the following work, we mainly focused on high light, high salinity and light quality stress.

We first treated U. prolifera with high light in laboratory and analyzed the photosynthetic activities and protein compositions of thalli. The result show that NPQ value increased obviously, accompany by the increased Zeaxanthin content. However, neither PsbS nor LhcSR was up-regulated. We suggested that these two key protein for NPQ in U. prolifera were quite different from Physcomitrella patens and Chlamydomonas reinhardtii. Besides, we founded that the composition of the minor LHC proteins in U. prolifera is CP26 and CP29, but without CP24, which is different from higher plants. We deduced that, it was only the lacking of CP24 that resulting in the defect in fast NPQ induction. In conclusion, in order to active the NPQ mechanism quickly, U. prolifera keep enough PsbS, LhcSR and Zeaxanthin within the cells. This is a result of U. prolifera adapting to intertidal zone.

We further treated thalli with different light qualities for 2 days. Our results show that thalli treated with long wavelength light, far red light (FR), show high NPQ value and Zeaxanthin content compared with white light and short wavelength light. However, the photosynthetic activity of thalli under FR decreased, indicating FR as a stress factor. Our WB experiments indicated that LhcSR was up-regulated while PsbS was not when U. prolifera exposed to FR condition. This was further conformed by RT-PCR analysis. This was to say FR induced the synthesis of Zeaxanthin and the up-regulation of LhcSR to promote NPQ. We compared this in Physcomitrella patens and no similar phenomenon was found. This indicated a special NPQ mechanism existing in U. prolifera. Next, we separate the photosynthetic complexes with sucrose density gradient and identified that LhcSR was bonded to light harvesting complex II （LHCII） trimer and minor LHC but not PSI-LHCI complex. Moreover, under FR the LHCII trimer became larger and the ratio of chlorophyll b/a in PSI-LHCI. Our results declared the special role of LhcSR compared with PsbS in NPQ induction and put forward a new direction of LhcSR study. We further verified these results with transcriptomic analysis, and found calcium signal pathway play important role in U.prolifera responding to far red light. Phytochrome encoding gene was explored and we finally built a complete far red signal transduction pathway in U.prolifera.

Finally, we explored the salt stress upon U. prolifera and Dunaliella salina.We found different YI and YII changes when subjected to salt stress in U. prolifera and Dunaliella salina. The pentose phosphate pathway may play different roles in these two algae. In Dunaliella salina, the pentose phosphate pathway provides NADPH and ATP for glycerol synthesis to modulate intracellular osmotic stress. Besides, it was shown that 9% NaCl induced the increasement of NPQ and Zeaxanthin content. However, neither PsbS nor LhcSR was proved up-regulated. This was much similar with Physcomitrella patens when subjected to salt stress. This was considered as a special pathway to induce NPQ and respond to salt stress, which deserved more studies. In total, NPQ inductions under these three stress were quite different, although there exist same similarities. This was consider the result of U. prolifera to survive the complex environment in intertidal zone.