浒苔（Ulva prolifera）共附生细菌群落结构分析的方法学创新与CFB类群的初步研究

发布时间：2018-08-02 16:42

【摘要】：黄海历年大规模绿潮系由浒苔(Ulva prolifera)单一生态型构成,提示其生物学特性是绿潮暴发的重要成因之一。藻类共附生细菌密切参与宿主的营养吸收、形态建成等关键过程,这为黄海绿潮的遗传溯源与暴发机制研究提供了新角度。与附生菌相比,内生菌与宿主的特异性往往更加稳定,有必要开展差异性分析,但相关研究的方法学基础尚未建立。本文首先使用16S rDNA通用引物,对漂浮与定生浒苔共附生细菌群落结构进行了比较分析,发现以下规律与问题:1)在细菌门和科的水平,漂浮与定生群体间存在明显差别;2)发现CFB、浮霉菌等功能类群,但CFB缺乏功能验证方法,浮霉菌占比与文献有显著差异,提示须对内生菌与附生菌区别研究;3)浒苔叶绿体DNA对扩增具严重影响,须开展引物优化或设计。针对上述方法学瓶颈,我们通过比较化学、物理、酶解等十几种消毒方法,发明了用特定粒径石英砂与浒苔共震荡以彻底清除附生菌的高效方法并优化了参数,DGGE结果表明,新方法对内生菌几无影响,可以用于内、附生菌群落结构的差异性分析。为了消除藻类叶绿体编码16S rDNA同源序列对内生菌群落结构分析的严重影响,共建立两种引物优化方案:一是从引物设计、热启动、退火温度三个方面优化现有799F引物,可显著降低叶绿体来源扩增产物的比例,揭示了优化原理;二是通过比较浒苔叶绿体与31个细菌门类的16S同源序列,发现了位于高度保守区的唯一双碱基差异位点,设计了新引物1525R,经测序检验,可完全排除叶绿体影响。绿潮浒苔高度分枝、利于漂浮,是区别于定生群体的显著形态差异,CFB细菌是参与浒苔形态建成(MG)的重要功能类群。本文用CFB细菌特异引物对8个漂浮与定生浒苔群体进行了分析比较,分离鉴定获得了可培养的CFB菌株,利用抗生素法建立了浒苔无菌发育实验体系,将用于菌株的MG功能验证。本文建立了附生菌选择性去除、16S引物优化与设计、无菌发育实验体系构建等关键方法,为后续开展浒苔共、附生菌群落结构差异性分析及功能类群的检验奠定了必要的方法学基础。
[Abstract]:The large green tide of Huang Hai over the years is composed of a single ecotype of Enteromorpha enteromorpha, indicating that its biological characteristics are one of the important causes of green tide outbreak. Algae co-epiphytic bacteria are closely involved in the host nutrient absorption, morphogenesis and other key processes, which provides a new perspective for the genetic traceability and outbreak mechanism of Huang Hai green tide. The specificity of endophytic bacteria and host is more stable than that of epiphytic bacteria, so it is necessary to carry out diversity analysis, but the methodological basis of related studies has not been established. In this paper, using 16s rDNA universal primers, the community structure of coepiphytic bacteria of floating and stationary Enteromorpha enteromorpha was compared and analyzed. It was found that the following laws and question: 1) were found to be at the level of phylum and family. There were significant differences between floating and metaplastic populations (2) the functional groups such as CFB and floating fungi were found, but CFB was lack of functional verification method, and the proportion of floating fungi was significantly different from that in literature. It is suggested that the difference between endophytic and epiphytic bacteria should be studied. 3) chloroplast DNA of Enteromorpha enteromorpha has a serious effect on amplification and primer optimization or design should be carried out. In view of the above methodological bottlenecks, we have developed a highly efficient method of eliminating epiphytic bacteria thoroughly by shaking together with quartz sand and enteromorpha with specific particle size by comparing a dozen disinfection methods such as chemistry, physics and enzymatic hydrolysis, and optimizing the parameters of DGGE. The new method has little effect on endophytic bacteria and can be used to analyze the difference of endophytic bacteria community structure. In order to eliminate the serious influence of the 16s rDNA homologous sequence encoded by algae chloroplast on the analysis of endophytic community structure, two primer optimization schemes were established: one was to optimize the existing 799F primers from three aspects: primer design, hot start and annealing temperature. By comparing the 16s homologous sequences of chloroplasts with 31 bacteria species, we found the only double base difference site located in highly conserved region of Enteromorpha enteromorpha by reducing the proportion of chloroplast products derived from chloroplast and revealing the principle of optimization, and comparing the 16s homologous sequences of chloroplasts with 31 bacterial families. A new primer 1525 Rwas designed and sequenced. Green tide Enteromorpha enteromorpha is highly branched and easy to float, which is a significant morphological difference from Dingsheng population. CFB bacteria are important functional groups involved in the morphogenetic (MG) of Enteromorpha enteromorpha. In this paper, CFB bacteria specific primers were used to analyze and compare 8 floating and stationary populations of Enteromorpha enteromorpha. The culturable CFB strains were isolated and identified. The aseptic development test system of Enteromorpha margin was established by using antibiotic method, which will be used to verify the MG function of the strains. In this paper, the key methods, such as the optimization and design of 16s primer for selective removal of epiphytic bacteria and the construction of aseptic development experiment system, were established for the subsequent development of Enteromorpha enteromorpha. The difference analysis of epiphytic community structure and the examination of functional groups lay the necessary methodological foundation.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X55

【相似文献】