几种典型藻华生物的分子分类学分析

MOLECULAR TAXONOMY ANALYSES OF THE RELATIONSHIPS IN SEVERAL REPRESENTATIVE HARMFUL ALGAL BLOOM SPECIES

通过PCR克隆测序、rDNA序列分析、随机PCR引物扩增结合DGGE技术等三个层次的分子分类水平对厦门海域的典型藻华生物进行分子生物学分析。结果表明,基于DGGE检测的18S rDNA序列过于保守,分类的精确度不高;AP-PCR则是基于基因组的差异进行分析,结果较为精确和全面;而rDNA序列分析相对可靠,尤其是针对ITS的长度和全序列分析以及28S rDNA的D1、D2区的序列分析,可以提供更为准确的分类信息,并可为设计检测探针提供基础。据此对分离自厦门海域的3种典型甲藻及其相关藻株建立了系统进化树。针对23株甲藻ITS序列建立的系统进化树显示Takayama pulchella(AY764179)和Karlodinium micrum的距离最近,并能够把Akashiwo属、Karenia属、Gyrodinium属、Karlodinium属、Takayama属与Gymnodinium属等区分开。用28S rDNA序列建立的系统发育树只能把Karlodinium属、Karenia属和Takayama属区分开,但不能很好地把无纹环沟藻与Akashiwo属和Gymnodinium等的藻区分开。

With rapid economic development and increasing pollution,some Chinese coastal waters have become eutrophic,which has resulted in a high frequency of harmful algal blooms.Rapid and unequivocal identification of harmful species has become a focal point of recent toxic algal research.At present,morphological criteria are the primary means to identify and classify harmful algae,but it is often difficult to distinguish morphologically-similar species and differentiate non-toxic from toxic algae.Some new molecular taxonomy methods and related technologies must be developed and applied to differentiate non-toxic from toxic algae and to monitor the development of algal blooms in coastal waters.Application of the ribosomal DNA（rDNA） sequences to identification and classification harmful algal bloom species are important complements for the traditionally morphological identification,and can give us more exact taxonomic informations and valuable results sometimes.Moreover,analysis of phylogenies and relationships among harmful algal bloom species using sequences of the rDNA can provide a basis on molecular probes design and can detect target algae quantitatively and rapidly.Molecular taxonomy has proved particularly useful to separate closely related species and has being the most common focuses on phytoplanktonic ecology.In the present study,the gene fragments of 18S,28S rDNA and ITS（Internal transcribed spacer） of several representative harmful algal bloom species from Xiamen harbor were amplified by polymerase chain reaction and cloned,and then their sequences were mensurated and analyzed by software.The variable regions of 18S rDNA were amplified by specific primer PCR and then analyzed by denaturing gradient gel electrophoresis.The genomic differences of these harmful algal bloom species were analyzed by AP-PCR.The different levels of molecular taxonomy were described by the above-mentioned methods,which were used to analyze the molecular taxonomy relationships in these harmful algal bloom species from Xiamen harbor.The results were as follows：18S rDNA,detected by denaturing gradient gel electrophoresis was too conservative and its precision to molecular taxonomy was not preferable.Molecular taxonomic method with AP-PCR based on the analysis of genomic differences was more accurate and integrated than DGGE.The analysis of rDNA sequences,especially the length and whole sequences analysis of ITS and variable domains D1 and D2（about 721 bp） in 28S rDNA,could be more reliable and could provide more exact taxonomic informations than other methods.These taxonomic informations could offer a base and elements for the design of molecular probe which could detect,identify and classify these harmful algal bloom species.Two phylogenetic trees were constructed using ITS and partial 28S rDNA of three representative harmful algal bloom species isolated from Xiamen harbor and its correlative species.Phylogenetic tree constructed using ITS sequence of 23 species showed that Takayama pulchella（AY764179） was closely related to Karlodinium micrum,and these genuses of Akashiwo,Karenia,Gyrodinium,Karlodinium and Takayama could also be separated from Gymnodinium genus approximately.Phylogenetic tree constructed using 28S rDNA（partial LSU,D1 and D2 region） sequence of 21 species could separate Karlodinium and Karenia from Takayama,but could not separate Gyrodinium instriatum from Akashiwo and Gymnodinium species clearly.