黄渤海沿岸钵水母种间遗传变异及LAMP检测技术研究

	黄渤海沿岸钵水母种间遗传变异及LAMP检测技术研究
	刘中援 1,2
学位类型	硕士
导师	刘东艳 ; 王培亮 ; 董志军
	2014-11
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	钵水母海蜇白色霞水母种间遗传变异 Lamp检测
摘要	近年来，水母暴发已成为我国黄渤海近岸海域面临的突出生态问题，给当地的渔业资源、社会生产及生态环境造成严重影响。由于不同生活史时期的水母形态差异较大，传统形态鉴定分析方法难以分辨不同种类的水母幼体，给种群动态观测与暴发机制研究带来困难。分子生物学技术在物种鉴定中不受生活史阶段和物种发育状态的影响，可有效解决不同生长阶段物种鉴定尤其是幼体鉴定的难题。本研究选取黄渤海近岸常见的钵水母海月水母、海蜇、沙海蜇和白色霞水母为研究对象，基于种间的遗传变异研究结果，筛选出适合白色霞水母（灾害水母）和海蜇（资源水母）物种鉴定的特异性序列，分别建立了两种水母的环介导等温核酸扩增（LAMP）检测方法。四种不同钵水母的线粒体细胞色素c氧化酶I基因（COΙ）序列分析表明，COΙ序列具有合适的变异速率，四种钵水母种内最大遗传距离为0.015，而种间遗传距离为0.197~0.270，种内差异明显小于种间差异，COΙ基因可以作为检测黄渤海海域钵水母类的可靠靶序列。根据白色霞水母COΙ基因，设计并筛选了白色霞水母LAMP反应的引物序列，优化确立了白色霞水母LAMP检测的最佳反应体系和反应条件，并对该方法的特异性、灵敏性和重复性进行了验证。实验结果表明，本研究设计的白色霞水母LAMP引物具有良好的特异性和重复性，所有的白色霞水母样品都扩增出特异性条带，而非目的水母均没有特异性条带出现；LAMP方法的最低检测限为0.1 ng DNA，比传统的PCR方法的灵敏度高100倍。根据海蜇COΙ基因，设计并筛选了海蜇LAMP反应引物，优化确立了海蜇LAMP检测的最佳反应体系和反应条件，对本方法的特异性、灵敏性和重复性进行了验证，并利用建立的LAMP方法对海蜇无性世代的螅状体进行检测。实验结果表明，本研究设计的海蜇LAMP引物具有良好的特异性，不同地理种群的海蜇样品均能扩增出特异性条带，而非目的水母均没有特异性条带出现；海蜇LAMP方法的最低检测限为0.1 ng DNA，与白色霞水母检测限相同。所有的海蜇螅状体都能通过LAMP方法检测出来，表明海蜇LAMP检测可适用于螅状幼体的分子鉴定。本研究根据白色霞水母和海蜇线粒体细胞色素c氧化酶I基因建立了两种水母的LAMP快速检测方法，具有良好的特异性、灵敏性和重复性，将来可应用于两种水母不同生活史阶段的快速检测，可为灾害性水母白色霞水母的预测预警和海蜇的资源调查工作提供技术支持。
其他摘要	In recent years, blooms of the harmful scyphomedusae, which were severe nuisance to local fisheries, social production, and ecological environment, has become a prominent ecological problem in coastal waters of Bohai Sea and Yellow Sea. It is difficult to distinguish the scyphomedusae larvae by traditional classification methods, due to the complex morphological characteristics of different stages of their life cycle. This also restricts people to study the mechanism of its outbreak. Molecular biology techniques are not affected by different life stages and species status in species identification, which can effectively solve the problem in species identification, especially larvae identification problem. This study selected the common jellyfish included Aurelia aurita, Rhopilema esculentum, Nemopilema nomurai, Cyanea nozakii as research object, and find specific conserved sequences for molecular identification of Rhopilema esculentum and Cyanea nozakii, through analyzing the population genetic variation, and each LAMP detection method was established according to their conserved sequence. The analysis of mitochondrial cytochrome c oxidase I gene (COΙ) sequences shows, COΙ gene has right mutation rate, the largest intraspecific genetic distance is 0.015, while the interspecific genetic distance is about 0.197~0.270, significantly greater than the intraspecific genetic distance. The COΙ gene can be used as a reliable target sequence for detecting Cyanea nozakii and Rhopilema esculentum. LAMP primers of Cyanea nozakii were designed based on its COI gene. Change reaction conditions to optimize each reaction system. The specificity, sensitivity and repeatability of the method were tested. Experimental results showed that the primers had excellent specificity and repeatability, all samples of Cyanea nozakii produced positive results, while the other non-target scyphomedusae and hydrozoan species produced negative results. The detection limit of LAMP method was 0.1 ng DNA, displayed a higher amplification efficiency than traditional PCR. LAMP primers of Rhopilema esculentum were designed based on its COI gene. Change reaction conditions to optimize each reaction system. The specificity, sensitivity and repeatability of the method were tested. In addition, the test of detecting scyphistoma in asexual generation was conducted by established LAMP method. Experimental results show that the primers had excellent specificity and repeatability, samples from different geographic populations of Rhopilema esculentum produced positive results, while the other non-target scyphomedusae and hydrozoan species produced negative results. The detection limit of LAMP method was 0.1 ng DNA, the same with Cyanea nozakii. All hydroid of Rhopilema esculentum could be detected by LAMP method, which indicated LAMP method could be applied to molecular identification of hydroid. This study established a LAMP method for detecting Cyanea nozakii and Rhopilema esculentum respectively, according to their mitochondrial cytochrome c oxidase I gene. The method can be used to detect both jellyfish at different life stages, with good specificity, sensitivity and repeatability. Therefore, it may provide early warning of harmful jellyfish Cyanea nozakii and forecasting the resource of edible jellyfish Rhopilema esculentum.
语种	英语
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/7922
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	1.中国科学院烟台海岸带研究所 2.中国科学院大学
第一作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	刘中援. 黄渤海沿岸钵水母种间遗传变异及LAMP检测技术研究[D]. 北京. 中国科学院研究生院,2014.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
黄渤海沿岸钵水母种间遗传变异及LAMP检（2488KB）	学位论文		开放获取	CC BY-ND	浏览请求全文