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

  • 摘要: 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。

     

    Abstract: With rapid economic development and increasing pollution, some Chinese coastal waters have become neuritic,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 morph logically-similar species and differentiate non- toxic from toxic algae.Some new molecular taxonomy methods and related technology bismuth be developed and applied to different afternoon-ox IC from ox IC algae and to Mon it or the develop Kent of algal blooms in coastal waters.Application of the ribosome al DNA ( DNA) sequences to identification and classification harmful algal bloom species are important complements for the traditionally morphology tical indent infliction, and can give us more exact taxonomy IC in formations and valuable results some tmies.Moreover, analyst is of phylogenic and relationships among harmful algal bloom species using sequences of the rDNA can provide a bas is on molecular probes design and can detect target algae quant itatively and rapidly.Molecular taxonomy has proved particularly useful to separate closely related species and has being the most common focuses on phytoplanktonicecology. In the present study, the gene fragments of.8S, 8S rDNA and ITS ( Internal transcribed spacer) of several representative harmful algal b loom species from X iam en harbor w ere amplified by polym erase chain react ion andcloned, and then their sequencesw eremensurated and analyzed by software.The variable reg ions o f.8S rDNA w ere am pl-if ied by specific premier PCR and then analyzed by denaturing grad ient gel electrophores is.The genom ic d ifferences o f theseharm ful algal bloom speciesw ere analyzed byAP-PCR.The different levels of molecular taxonom y were described by theabove-mentioned m ethods, which w ere used to analyze the mo lecular taxonomy relationships in these harmful a lgal bloomspecies from X iam en harbor.The resultswere as follow s:.8S rDNA, detected by denaturing grad ient gel electrophoresisw astoo conservative and its precision to mo lecular taxonomy was not preferable.M olecular taxonom ic method with AP-PCRbased on the analysis of genom ic differences was m ore accurate and integrated than DGGE.T he analysis of rDNA sequences,especially the length and whole sequences analysis of IT S and variable dom ains D. and D ( about 7. bp) in8S rDNA, could be more reliable and could prov ide more exact taxonomic informations than other methods.These taxonomic informations cou ld offer a base and elem ents for the des ign ofm olecular probe which could detect, identify and classifythese harm fulalgal bloom species.Tw o phylogenetic trees were constructed us ing ITS and part ia l 8S rDNA of threerepresentative harm fu l algal bloom spec ies isolated from X iamen harbor and its correlat ive species.Phylogenet ic tree constructedusing IT S sequence of 3 species showed thatT akayama pulchella (AY764.79) was closely related toK arlod iniumm icrum, and these genuses ofA kashiw o, K arenia, Gyrod inium,K arlod inium andT akayam a could also be separated fromGymnod inium genus approxmi ately.Phylogenet ic tree constructed us ing 8S rDNA ( partial LSU, D. and D reg ion) sequenceo f. species could separateKarlodinium andKarenia from T akayama, but could not separate Gyrodinium instriatumfrom A kashiwo and Gymnodinium species clearly.

     

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