【作者】 王健；

【导师】 茅云翔；

【作者基本信息】 中国海洋大学 ， 遗传学， 2010， 硕士

【摘要】 绿潮是近几十年来在国内外频繁发生的大型海洋绿藻暴发性生长、并从原有的固着生长转变为飘浮生长的有害生态异常现象,主要发生在河口、内湾、渴湖和城市密集的海岸,其暴发的规模和种类因地而异。形成绿潮的海藻主要是石莼科(Ulvaceae)绿藻,以石莼属(Ulva)海藻为主。2008年,黄海中南部暴发的漂浮浒苔绿潮引起了国家对海洋绿潮灾害的关注；2009年,青岛沿海地区又发生了多次石莼属漂浮绿藻大量堆积的情况,对海洋生态坏境造成了负面的影响。为了寻找绿潮发生的原因,并进一步达到预防、治理绿潮的目的,首先需要做的是对引发绿潮的物种进行准确的鉴定。本研究的工作主要是对青岛沿海地区漂浮生长的绿藻进行种类鉴定,并确定它们的来源,为后续的分析和治理提供最根本的依据。本研究运用了地高辛标记分子探针技术,建立了黄海绿潮浒苔(Ulva prolifera)的快速鉴定技术,该技术通过DNA提取、扩增待测样本特定序列、双探针杂交最终达到快速准确地鉴定浒苔的目的,整个鉴定工作可在24h-36h内完成。本研究通过扩增并分析18S-28S ITS序列(internal transcribed spacer、5S核糖体RNA串联重复序列(5S rDNA spacer),成功鉴定了2009年5月至7月青岛内湾形成漂浮绿藻藻华的主要物种为长石莼(Ulva linza),并估算了生物量。与正常固着生长的长石莼比较发现,漂浮长石莼的形态发生了显著的变化。通过扩增并分析rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit)序列,发现自2009年7月开始青岛内湾漂浮生长的绿藻主要为孔石莼(Ulva pertusa)和硬石莼(Ulva rigida),以孔石莼占大多数。同时通过现场调查、以及对三种绿藻形态上特点的分析认为,三种绿藻都不具备随水流远程运输的形态条件。所以,2009年青岛海区大量漂浮生长的长石莼、孔石莼和硬石莼均为青岛本地生长的物种,而不是从外海漂来的。显微结构观察的结果表明,漂浮生长的绿藻有细胞壁加厚的生长特征,推测这与其为了适应变化的生存环境而发生的变化。针对目前研究和应用较多的一种规范化物种分类技术——DNA条形码技术(DNA barcoding),本研究对ITS序列作为石莼科藻类DNA条形码的潜在可能做了分析,发现ITS序列在一些物种的种内遗传距离大于它们在物种间的种间遗传距离,所以ITS序列还尚未达到作为DNA条形码序列的要求,但从所建立的系统发生树可以看出,ITS序列可以为石莼科物种分类提供很好的参考依据。更多还原

【Abstract】 "Green tides" are harmful phenomena refering to fast growth and vast accumulated biomass of green macroalgae, which often occur in estuaries, inner bays, lagoons and on urbanized seacoasts in different scales. This kind of phenomena has increased over the past four decades all over the world. Species forming green tides are mainly Ulvaceae, especially Ulva. They often become abnormally free-floating instead of normally attached during the occurence of green tides. Our government has raise concerns about the Yellow sea green tides since the year of 2008 when Ulva prolifera overgrowth occurred. During the year of 2009, green tides formed by Ulva occured for many times on the coasts of Qingdao as well. They all had negative impacts on the marine ecological environment. Our work is to identify these free-floating green algae and find out their orgin, in order to provide theoretical basis for subsequent analyzations and controls of green tides.With digoxin labeled DNA probes technology, we set up a rapid method of identifying Ulva prolifera, which is the yellow sea green tides forming algae. By means of DNA extraction, amplification of determinands’ DNA sequences and detection with two DNA probes, precise identifation of Ulva prolifera is done. The entire process is very simple and convenient, which could be accomplished within 24-36h.From May to July of 2009, there were large amount of free-floating green algae assembling in the inner bays of Qingdao. By amplifying and analyzing ITS DNA sequences(internal transcribed spacer) and 5SS DNA sequences(5S rDNA spacer), we managed to estimate the biomass of this green algae bloom and identify the species forming the bloom, which refered to Ulva linza. The thalli of free-floating Ulva linza were quite different from thalli of the attached ones in shape. Since late July of 2009, Ulva linza disappeared gradually; other free-floating Ulva species became dominant simultaneously.By amplifying and analyzing rbcL DNA sequances (ribulose-1,5- bisphosphate carboxylase/oxygenase large subunit), we managed to make out the species forming the new algae blooming, which were majority of Ulva pertusa and minority of Ulva rigida. According to our outdoor survey and. common traits of the three species, we conclude that Ulva linza, Ulva pertusa and Ulva rigida don’t have the morphological characteristic by which they can drift for long distance with the help of seawater current, so the three species are all locally originated and not from other regions. A phenomenon of cell wall thickening of free-floating Ulva specimens can be observed microscopically, which may be explained by their adaptions to the change of their living environment.DNA barcoding is a species identification technology based on the analysis of sequence diversity in small segments of DNA. We choose ITS DNA sequences as candidates to test whether it could be eligible for DNA barcode of Ulvaceae. The results told us that the minimum interspecific genitic distance of ITS DNA sequences involving a few species of Ulvaceae was less than their maximum intraspecific distance, so ITS DNA sequences haven’t made the grade to be DNA barcodes. But it can be learned from the molecular phylogenetic tree that data of ITS DNA sequences are still good references for Ulvaceae taxonomy study.更多还原