大菱鲆视蛋白基因表达与光谱调控技术研究

IOCAS-IR > 实验海洋生物学重点实验室

	大菱鲆视蛋白基因表达与光谱调控技术研究
其他题名	Study on opsin gene expression and spectral modulation technique in turbot (Scophthalmus maximus)
	王雨浓
学位类型	硕士
导师	李军
	2020-05
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	大菱鲆视蛋白视网膜变态底栖适应
摘要	摘要视觉是鱼类最重要的感觉系统之一，与其交配、躲避天敌及觅食等行为密切相关。视蛋白是视觉形成的分子基础，它与生色基团结合构成视觉形成的起始元件。视蛋白的组成与功能的多样化是鱼类视觉适应性进化的主要途径之一。然而鱼类视蛋白基因如何适应其生活史不同阶段光环境的变化目前仍不清楚。大菱鲆（Scophthalmus maximus）是我国重要的海水养殖品种之一，具有很高的经济价值，其早期发育经历重要的变态过程，可从变态前的浮游生活转变为底栖生活，其生活习性的改变伴随着栖息环境的巨大变化，尤其是光环境的转变。本文选取大菱鲆为对象，研究其早期发育阶段视网膜结构变化、视蛋白组成与表达变化、不同光谱环境下视蛋白的差异表达，及其幼鱼期对不同光谱的选择性，以期探明大菱鲆视觉器官结构及功能发育特征，并探寻鱼类视觉特征与栖息光环境的适应与关联。本文可为大菱鲆工厂化养殖中光照调控技术的提升奠定理论基础，进而推动大菱鲆产业的健康发展。相关结果如下： 1.1月龄大菱鲆视网膜外核层细胞核数（O.N.）与神经节细胞数（G.）的比值为2.22，此阶段视网膜网络会聚程度低，视敏性较高。4和9月龄大菱鲆视锥细胞和G.的数量均显著低于1月龄，O.N.数量的变化则刚好相反。变态后O.N./G.比值增大，视敏性降低。视杆细胞的数量和比例均增大，表明其在底栖生活阶段光敏性增强。据此推测大菱鲆通过提高视杆细胞数量来应对深海昏暗的光环境。 2.大菱鲆拥有全部5类视觉视蛋白基因，分别为视紫红质（RH1）、红视蛋白（LWS）、紫外视蛋白（SWS1）、蓝视蛋白（SWS2）以及绿视蛋白（RH2A1、RH2A2、RH2B1、RH2B2和RH2C）。不同于其他几类单拷贝基因，其绿视蛋白由5个旁系同源基因组成。大菱鲆与太平洋蓝鳍金枪鱼（Thunnus orientalis）一样，是目前为止发现RH2基因最多的物种之一。 3.大菱鲆在不同发育阶段，视蛋白基因表达存在一定变化。杆视蛋白RH1的表达随着生长发育逐渐增高，至9月龄达到最高值，其后维持高表达水平不变。锥视蛋白基因在0.5和18月龄表达量均较低，其中0.5月龄时LWS表达量最高，其表达比例为47.98%。随着变态和发育，RH2B1逐渐取代其成为表达占据主导地位的视蛋白基因，其最高比例为9月龄时的63.27%。18月龄时RH2B1表达比例无显著改变，但LWS表达比例略有增高。大菱鲆在发育过程中可能经历了光谱敏感性由红光到绿光的的转变，这可能是其在进化过程中保留下来应对生活史转变的一种适应策略。18月龄LWS比例增高可能是为其上浮至较浅水层繁殖做准备。 4.不同光谱LED光处理，可引起大菱鲆部分视蛋白基因表达变化。九种视蛋白基因中，只有RH2B1、SWS1和SWS2的基因表达水平在五种处理下存在显著差异。三者均为全光谱（F）、蓝光（B）、和绿光（G）处理组个体基因表达水平显著高于红光（R）和橙光（O）组个体。其中B和G组个体的SWS1、SWS2和RH2B1表达量约为O和R组个体的二倍，而F组略高于O和R组。其他六个视蛋白基因的表达量在各处理组间无显著差异。据此推测，这种视蛋白的差异表达是大菱鲆应对光环境迅速变化的一种响应。此外，与其他物种例如孔雀鱼（Poecilia reticulata）相比，大菱鲆幼鱼缺少了LWS表达的可塑性。 5.大菱鲆绿视蛋白基因亚型RH2C发生E122Q和M207L的氨基酸替换，二者均与视蛋白光谱敏感性向短波长偏移密切相关。枝位点模型分析发现二者在进化过程中均受到正选择。据此推测大菱鲆可能通过氨基酸替换获得光谱敏感峰值蓝移的绿视蛋白亚型，并因此增强了对蓝光的识别能力，利于其对底栖生活阶段蓝光环境的适应。 6.在对不同光谱的选择性实验中，大菱鲆幼鱼表现出明显的绿光偏好性，其在绿光区域出现的概率为48.00%，其次为蓝光（11.56%）和全光谱（8.18%），红光和黄光区域的出现次数最少，二者均在1%之下。本实验结果为大菱鲆变态后的绿光高敏感性提供了证据。大菱鲆对绿光的偏好可能与其自然环境中的底栖生活阶段，栖息水层以中短波长光谱为主有关。关键词：大菱鲆，视蛋白，视网膜，变态，底栖适应
其他摘要	Abstract Vision is one of the sensory systems in fish, which is closely related to behaviors such as mating, foraging and avoiding predation. Opsin is a protein which combines with chromophore to form the initial element of vision. Functional diversification of opsin plays a great role in the adaptive evolution of vision. However, related research is rarely reported on how opsin genes adapt to changes in light environment at different stages of life cycle in fish. The turbot (Scophthalmus maximus) is an important aquaculture species with great commercial value. During the development, it experiences metamorphosis and changes from planktonic to benthic life. The shift in living habits is accompanied by a huge change in its habitats, especially the light environment. This research conducted in turbot, we investigated the visual opsin repertoire, analyzed the changes in retina structure and heterochronic opsin expression during the development of turbot. We also measured the opsin differential expression in response to different spectral environments, and compared the spectral preference of juvenile turbot. This study attempted to identify the structural and functional characteristics of the visual organs in the development of turbot, and to explore the adaptability of fish visual characteristics and light environment. This study can provide a theoretical basis for the improvement of light modulation technique in the industrial culture of turbot. The relevant results showed that: 1. The ratio of nuclei of the outer nuclear layer (O.N.) to ganglion cells (G.) in the retina of 1-month-old turbot was 2.22. It indicated the low degree of the assembled meshwork and high visual acuity. The numbers of cone cells and G. in 4- and 9-month-old were significantly lower than that of 1-month-old, while the change in the number of O.N. was just the opposite. After metamorphosis, the ratio increased and visual acuity decreased. And the increased number and proportion of rod cells indicated that the photosensitivity of turbot may be enhanced. We speculated that turbot may survive in the dark environment by increasing the amount of rod cells. 2. Turbot possess five classes of visual opsin genes: 1) rhodopsin (RH1); 2) red-sensitive opsin (LWS); 3) ultraviolet-sensitive opsin (SWS1); 4) blue-sensitive opsin (SWS2); and 5) green-sensitive opsin (RH2A1, RH2A2, RH2B1, RH2B2 and RH2C). Unlike other single-copy genes, turbot have five RH2 paralogs. Along with Pacific bluefin tuna (Thunnus orientalis), tutbot is one of the species with the most RH2 genes among the sequenced fish to date. 3. Heterochronic changes in opsin gene expression were found in different developmental stages. Specifically, during the development of turbot, RH1 expression significantly increased and reached the highest at 9 months of age. In the larval stage, all cone opsin genes were expressed at a low level. LWS was the highest one with a proportional expression level of 47.98%. After metamorphosis, RH2B1 gradually replaced it as the dominant opsin gene, and the highest proportion level of RH2B1 was 63.27% at 9 months of age. The proportional level of LWS slightly increased at 18 months of age, but that of RH2B1 showed no significantly different. Turbot may undergo a spectral sensitivity transition from red to green. We speculated that heterochronic changes in opsin gene expression may be a strategy retained in the evolution of turbot to adapt to benthic life. And the increased proportion level of RH1 in 18 months of age may occur in preparation for reproduction in shallow water. 4. The treatment of LED lights with different spectral led to changes in turbot opsin gene expression. In all opsin genes, only the expression of RH2B1, SWS1, and SWS2 showed plasticity changes induced by ambient light. The expression levels of individuals under full, green, and blue spectrum light were significantly higher than those under orange and red spectrum light. And the expression levels of individuals under blue and green light were about twice that of the individuals under orange and red light, while individuals under full spectrum light were slightly higher than individuals under orange and red light. The expression levels of the other six opsin genes were not significantly different among the treatment groups. We speculated that the plasticity of opsin expression is a mechanism for turbot to react to rapid changes in light environment. In addition, turbot lacks the plasticity of LWS expression compared to other species such as guppy (Poecilia reticulata) in the juvenile stage. 5. The amino acid substitutions of E122Q and M207L were found in subtype RH2C of turbot. They are not only spectral tuning sites, but also closely related to the blue shift of opsin function. The branch-site model tests showed that they were under positive selection during evolution. We speculated that turbot may thus obtain blue-shift green opsin subtype. It may enhance the ability to discriminate blue light, which is beneficial for turbot to adapt to the bluish ocean in the benthic stage. 6.The spectral preference of juvenile turbot was investigated. It showed obvious green light preference. The frequency of its appearance in the green light region was 48.00%, followed by blue (11.56%) and full spectrum (8.18%). While the frequency of turbot found in red or orange light region was under 1%. The results of this experiment provided evidence for the high sensitivity to green light after metamorphosis. We proposed that the preference for different spectral may be related to the fact that the habitat of its benthic stage in the wild is dominated by short-to-medium wavelength spectrum. Keywords: turbot, opsin, retina, metamorphosis, benthic adaptation
学科领域	生物工程(亦称生物技术)
学科门类	工学 ; 工学::生物工程
页数	80
资助项目	National Natural Science Foundation of China[30970302] ; National Natural Science Foundation of China[30970302]
语种	中文
目录	目录第1章引言 1 1.1 鱼类视觉特性的研究进展 1 1.1.1 鱼眼的结构与功能 1 1.1.2 鱼眼的早期发育 2 1.1.3 视觉特性与环境适应 3 1.2 鱼类视蛋白的研究进展 3 1.2.1 视蛋白简介 4 1.2.2 多样的视蛋白基因库 5 1.2.3 发育过程中视蛋白的差异表达 6 1.2.4 不同环境下视蛋白表达的可塑性 7 1.2.5 视蛋白表达的调控 7 1.2.6 视蛋白与光谱敏感性 8 1.3 鱼类对不同光谱的选择性 9 1.4 选择压力的研究方法 9 1.4.1 选择压力与进化 9 1.4.2 选择压力的研究方法 10 1.5 本文的研究目的和意义 11 第2章大菱鲆视网膜结构与视蛋白表达变化分析 12 2.1 材料与方法 13 2.1.1 主要试剂 13 2.1.2 主要仪器 13 2.1.3 实验材料 13 2.1.4 石蜡切片 15 2.1.5 数据库比对与引物合成 16 2.1.6 系统发生分析 16 2.1.7 引物合成 16 2.1.8 荧光定量PCR 17 2.2 结果 20 2.2.1 视网膜组织学 20 2.2.2 系统发生和基因共线性分析 22 2.2.3 发育过程中视蛋白的表达变化 24 2.2.4 视蛋白表达的可塑性 26 2.3 讨论 27 2.3.1 视网膜发育与生态适应 27 2.3.2 视蛋白基因在生长发育中的表达变化 28 2.3.3 不同光谱环境下视蛋白表达的变化 29 2.3.4 视蛋白基因表达分析方法的比较 30 2.3.5 遗传调控vs光环境诱导 31 第3章大菱鲆视蛋白基因组成与进化研究 32 3.1 材料与方法 32 3.1.1 基因共线性分析 32 3.1.2 大菱鲆视蛋白基因结构分析及理化性质预测 33 3.1.3 绿视蛋白功能分化和基因分歧时间分析 33 3.1.4 选择压力与光谱敏感性分析 33 3.2 结果 34 3.2.1 基因共线性 34 3.2.2 视蛋白基因结构和理化性质分析 35 3.2.3 正选择与光谱峰值敏感性位点分析 37 3.2.4 绿视蛋白基因分歧与蛋白功能分化 41 3.3 讨论 42 第4章大菱鲆光谱选择行为初探 44 4.1 材料与方法 44 4.2 结果 45 4.3 讨论 46 第5章结论与展望 48 5.1 主要结论 48 5.2 展望 49 参考文献 51 附录 63 致谢 67 作者简历及攻读学位期间发表的学术论文与研究成果 68
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/164655
专题	实验海洋生物学重点实验室
推荐引用方式 GB/T 7714	王雨浓. 大菱鲆视蛋白基因表达与光谱调控技术研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.

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