【作者】 陈家琪；

【导师】 王嫣； 王道儒；

【作者基本信息】 海南大学 ， 渔业发展， 2021， 硕士

【摘要】 目前全球气候变化引起的海水表面温度升高、海洋酸化和增强的紫外线辐射对热带浅海的珊瑚礁造成严重的胁迫,导致其大面积白化和死亡。构成珊瑚礁基础结构的造礁珊瑚是一个复杂的共生功能体,其细胞中共生的单细胞虫黄藻(群落)不但为其提供大部分的有机物营养,而且部分塑造其环境适应特性。本研究以海南岛岸礁代表性造礁珊瑚丛生盔形珊瑚(Galaxea fascicularis)为研究对象,分离纯化和建立了其两个内共生虫黄藻的单克隆株系,研究其生理和生态特性,及其对白化丛生盔形珊瑚恢复的影响。主要研究结果如下:1、虫黄藻分离、纯化和单克隆株系的建立及分子鉴定从丛生盔形珊瑚触手(其共生虫黄藻经18S-RFLP鉴定为C系与D系),分离虫黄藻,用改良的L1培养液进行培养,水滴法分离和软平板藻落形成单克隆株系并扩大培养。藻细胞DNA经ITS1和ITS2区段测序验证,分别为共生藻科Symbiodiniaceae之Cladocopium ITS-C1和Durusdinium ITS-D1亚系群,分别命名为GF19C1和GF20D1。二者均成功分离于2018年11月,GF19C1至2019年7月已经基本适应体外环境,实现扩大培养,而GF20D1直到2020年1月才基本适应体外环境。2、GF19C1与GF20D1相对于其共生状态超微结构的改变大多数GF19C1和GF20D1细胞处于裸甲藻(gymnodinioid dinoflagellate)的球形期(coccoid stage),其细胞壁比同源内共生虫黄藻增厚大约2倍,说明它们发育为营养包囊,兼具有性生殖和无性生殖的潜力。此外,其分裂率高于内共生虫黄藻,细胞直径则明显减小。超微结构的测量结果表明,,GF19C1(0.10±0.02%)和GF20D1(0.16±0.04%)的叶绿体体积密度(Chloroplast volume density)均显著(P<0.01)低于共生C系虫黄藻(0.55±0.11%)或共生D系虫黄藻(0.50±0.07%)。而叶绿体中类囊体片层的表面积密度(Surface density of thylakoid lamellae),GF20D1(8.8±1.4)显著(P<0.05)低于D系共生虫黄藻(10.8±1.7),但GF19C1(10.3±0.9)相比于C系共生虫黄藻(11.1±1.1)仅略有减小。至于液泡体积,GF19C1相对于C系共生藻增加了近5倍,但GF20D1(0.07±0.02%)相对于D系共生藻(0.07±0.04%)仅略有增加,离体和共生态虫黄藻的线粒体体积和累积体体积则没有变化。3、GF19C1与GF20D1的体外驯化及其细胞ROS测定光温正交实验表明,在50或70μmol.m~2/s光强下,GF19C1和GF20D1在分离培养过程中所处温度为25±1°C(培养时保持该温度)时生长速度高于在28±1°C,表明其具有温度适应惯性。GF19C1与GF20D1的最适盐度相近,均在28-35之间。通过人工加温至35°C,探究在加温3h和24h后GF19C1、GF20D1与C、D系共生虫黄藻产生活性氧(ROS,Reactive Oxygen Species)的差异。实验表明产生ROS的细胞比例随着加温时间的增多而增多,说明温度胁迫可以促使虫黄藻产生更多的ROS。在常温和加温下,共生虫黄藻产生ROS的细胞比例均显著(P<0.01)大于GF19C1和GF20D1,这可能是GF19C1、GF20D1的叶绿体体积密度低于共生虫黄藻而导致的。4、体外驯化的共生藻株系在白化珊瑚的共生重建中的作用本研究通过四组实验来探究体外驯化的虫黄藻在白化珊瑚共生重建中所起的作用:(1)给藻组(Z组),给白化丛生盔形珊瑚注射GF19C1和GF20D1混合藻液,(2)共处组(D组),将白化珊瑚与健康珊瑚放在一起培养,(3)投喂虾肉组(F组),(4)单独培养组(SD组)。经九周实验后,Z组的恢复率最高(90%),D组次之(87%),SD组则显著(P<0.05)低于其他组。根据计算各组每周恢复率做出趋势线,发现Z组呈现出胞饮吸纳虫黄藻的直线恢复,而D组3-6周时呈直线恢复(但斜率即恢复速度显著低于给藻组),6-9周则呈现出以虫黄藻自分裂为主要特征的指数曲线恢复。F组和SD组一直为指数曲线恢复。说明外源性虫黄藻(包括健康珊瑚来源)对白化珊瑚的恢复速度有明显的促进作用。而投喂虾肉组恢复率高于单独培养组,说明外源性营养对珊瑚白化的恢复也有促进作用。本研究建立了丛生盔形珊瑚共生虫黄藻的单克隆株系GF19C1和GF20D1,这将为进一步研究造礁珊瑚共生体的形成和生理机制奠定基础。更多还原

【Abstract】 In recent years,the increase in sea surface temperature,ocean acidification and enhanced ultraviolet radiation caused by global climate change has caused severe stress on coral reefs in shallow tropical seas,leading to large areas of bleaching and death.The reef-building coral that constitutes the basic structure of the coral reef is a complex symbiotic function.The symbiotic unicellular zooxanthellae(community)in its cells not only provides most of the organic nutrients for it,but also partially shapes its environmental adaptation characteristics.In this study,Galaxea fascicularis,a representative reef-building coral in the coastal reefs of Hainan Island,was used as the research object.Two monoclonal strains of endosymbiotic zooxanthellae were isolated,purified and established to study their physiological and ecological characteristics.And its influence on the recovery of albino clump coral corals.The main findings are as follows:1.The isolation and purification of zooxanthellae and the establishment and molecular identification of monoclonal strainsIsolate the zooxanthellae from the tentacles of the clump coral coral(the symbiotic zooxanthellae were identified as the C and D series by 18S-RFLP),cultured with the improved L1 medium,separated by the water drop method and formed a monoclonal strain by the soft flat algae colony And expand the training.The DNA of algal cells was verified by sequencing of ITS1 and ITS2 segments,and they were Cladocopium ITS-C1 and Durusdinium ITS-D1 subgroups of Symbiodiniaceae,named GF19C1 and GF20D1,respectively.Both were successfully isolated in November 2018.GF19C1 has basically adapted to the in vitro environment and realized expansion of culture from July 2019,while GF20D1 did not basically adapt to the in vitro environment until January 2020.2.Changes in the ultrastructure of GF19C1 and GF20D1 relative to their symbiotic stateMost GF19C1 and GF20D1 cells are in the coccoid stage of gymnodinioid dinoflagellate,and their cell walls are approximately 2 times thicker than the homogenous endosymbiotic zooxanthellae,indicating that they develop into vegetative cysts with both sexual reproduction and The potential for asexual reproduction.In addition,its division rate is higher than that of endosymbiotic zooxanthellae,and the cell diameter is significantly reduced.The ultrastructure measurement results showed that the chloroplast volume density of GF19C1(0.10±0.02%)and GF20D1(0.16±0.04%)were significantly(P<0.01)lower than that of symbiotic C-type zooxanthellae(0.55±0.11%)or symbiotic D-type zooxanthellae(0.50±0.07%).The surface density of thylakoid lamellae in chloroplasts,GF20D1(8.8±1.4)was significantly(P<0.05)lower than that of the D-type symbiotic zooxanthellae(10.8±1.7),but GF19C1(10.3±1.4)0.9)Compared to the C-line symbiotic zooxanthellae(11.1±1.1),it is only slightly reduced.As for the vacuole volume,GF19C1 increased nearly 5 times compared to C-type symbiotic algae,but GF20D1(0.07±0.02%)only slightly increased compared to D-type symbiotic algae(0.07±0.04%),in vitro and symbiotic zooxanthellae The mitochondrial volume and cumulative volume of mitochondria remained unchanged.3.In vitro domestication of GF19C1 and GF20D1 and determination of cellular ROSOrthogonal experiment of light temperature showed that under the light intensity of50 or 70μmol.m~2/s,the growth rate of GF19C1 and GF20D1 during the separation and culture process was 25±1°C(maintain the temperature during culture).28±1°C,indicating that it has temperature adaptation inertia.The optimal salinity of GF19C1 and GF20D1 is similar,both are between 28-35.By artificially heating to 35°C,the difference in reactive oxygen species(ROS)produced by GF19C1 and GF20D1 and C and D symbiotic zooxanthellae after heating for 3h and 24h was explored.Experiments show that the proportion of ROS-producing cells increases with increasing heating time,indicating that temperature stress can promote zooxanthellae to produce more ROS.Under normal temperature and heating,the proportion of ROS-producing cells in symbiotic zooxanthellae was significantly higher(P<0.01)than that of GF19C1 and GF20D1.This may be caused by the lower volume density of chloroplasts of GF19C1 and GF20D1 than symbiotic zooxanthellae.4.The role of in vitro domesticated symbiotic algal lines in the symbiotic reconstr uction of bleached coralThis study explored the role of zooxanthellae domesticated in vitro in the symbiosis reconstruction of albino corals through four sets of experiments:(1)The algae group(group Z)was injected with a mixture of GF19C1 and GF20D1 into the albino clump corals.(2)Coexistence group(D group),culture bleached corals and healthy corals together,(3)Shrimp feeding group(F group),(4)Individual culture group(SD group).After nine weeks of experiment,the recovery rate of group Z was the highest(90%),followed by group D(87%),and the SD group was significantly(P<0.05)lower than the other groups.Based on the calculation of the weekly recovery rate of each group,a trend line was drawn,and it was found that the Z group showed a linear recovery of pinocytosis absorbing zooxanthellae,while the D group showed a linear recovery at 3-6 weeks(but the slope,that is,the recovery speed was significantly lower than that of the algae.Group),after 6-9 weeks,it showed an exponential curve recovery characterized by zooxanthellae self-division.The F group and SD group have been recovering the exponential curve.It shows that exogenous zooxanthellae(including healthy coral sources)can obviously promote the recovery speed of bleached corals.The recovery rate of the shrimp fed group was higher than that of the independent culture group,indicating that exogenous nutrition can also promote the recovery of coral bleaching.In this study,we established the monoclonal strains GF19C1 and GF20D1 of the symbiotic zooxanthellae of the clumping galea coral,which will lay the foundation for further research on the formation and physiological mechanism of the reef-building coral symbiont.更多还原