赤潮藻的分子标识及荧光原位杂（FISH）技术在检测赤潮藻中的应用
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摘要
赤潮(Red tide)现象在近 20 年中的发生频率、强度以及地理分布都在逐渐增加,它对国民经济、人类健康以及海洋生态环境所造成的巨大影响使得赤潮现象已成为全球共同关注的环境问题之一。
研究赤潮,进而预防和控制赤潮,最根本的问题是要明确引发赤潮的物种。最近几年采用分子生物学技术鉴定海洋浮游植物,特别是对赤潮藻的研究证实它们已成为微型藻类形态特征观察研究的重要补充手段。
本论文通过对一些原甲藻株和硅藻藻株的 ITS 序列、5.8S rDNA 或 LSU rDNA进行 PCR 扩增、克隆和序列测定。在 NCBI 数据库中进行同源性检索,序列的比对及相似性分析采用软件 Bioedit、DNAStar、Phylip3.5、Mega 2.0 等,系统进化关系分析采用 NJ(Neighbor Joining)法构建系统进化树。核酸序列的获得一方面是希望通过分子生物学的方法对一些通过形态鉴定的赤潮藻的命名进行验证,更重要的是找出引发赤潮的主要物种的特异性序列或区域,为下一步定性定量检测方法,主要是 FISH 技术中设计种特异性探针提供更多的选择区域。研究结果如下:
1. 采用 ITS 和 5.8S rDNA 序列研究 2 株海洋原甲藻和 3 株海洋硅藻
(1)海洋原甲藻 APBM(Prorocentrum micans APBM)的 ITS 和 5.8S rDNA 的长度为 631bp,而东海原甲藻(P. donghaiense)的相应部位的碱基长度为 552bp;东海原甲藻与从 GenBank 中获得的微小原甲藻相似程度较高为 88%,与甲藻属其它原甲藻相似程度较低(约 60-70%);本文研究的海洋原甲藻 APBM 的 ITS 序列与其它原甲藻相似程度都较低(约 30%左右)。用 ITS1 或 ITS2 序列构建的系统树与用 ITS+5.8S rDNA 序列构建的系统树反映的结果基本一致,系统进化树反映的结果与序列相似性的结果基本一致。5.8S 区因过于保守似乎不适于构建系统树反映种下的亲缘关系。
(2)尖刺拟菱形藻的 ITS 和 5.8S rDNA 全长为 693bp,SK-1(分离自东海赤潮爆发区)测序得到715bp;SK-2(分离自青岛养殖场) 的ITS和5.8S长度为331bp。
During the last two decades, serious economic and public health impacts caused by Harmful Algal Blooms (HABs)have increased in intensity, frequency and geographic distribution. It had great effect on national economy, public health, and oceanic ecosystem as well as. Thus, HABs is receiving increased international attention。 Studying HAB events , then predict and govern HAB, The basic biological concern of HAB was identify and distinguish its species. The speedy progress of molecular techniques offers a new way to identify phytoplankton. At present More and more studies using LSU/SSU rRNA gene or internal transcribed spacers(ITS) as molecular criteria to study dinoflagellate phylogeny have been proved to be powerful tools.
PCR amplification, cloning and sequencing the ITS and 5.8S rDNA or LSU rDNA from some strains of dinoflagellates and diatoms. Blast searches of these sequences had been done on NCBI(the website: http:// www.ncbi.nlm.nih.gov). Softwares such as Mega 2.0, DNAStar,BioEdit and Phlip3.5 were used to compare the three sequences studied in this paper with other ITS sequences of diatoms retrieved from GenBank. The Phylogenetic tree was constructed with NJ method. The aims of this paper were hoping that verify their phylogeneny by molecular data , and more nucleotide sequences obtained could be offer more choice for designing species-specific probes as well as. The results were as follows:
1.Studing 2 strains of dinoflagellate and 3 strains of diatoms according to ITS and 5.8S rDNA sequences
(1) The ITS length of P.micans APBM was 631 base pairs, and P. donghaiense’s ITS sequence was 552 bp. In the ITS sequence, P.donghaiense has very high level of similarity with P.minimum (showing 88% identity), but low level similarity with other species of P.triestinum and P.micans (about 60%-70%), while P.micans APBM show
the lowest level similarity with other P.micans according to ITS sequence, it was about 30% only. The results from phylogenetic tree of ITS2 sequence was the same as that showed by phylogenetic tree of ITS and 5.8S sequence. The results from phylogenetic tree constructed with ITS and 5.8S sequence accorded with that of sequence similarity analysis. The results of the phylogenetic tree constructed with 5.8S sequence were inaccurate; therefore, the use of 5.8S rDNA as a classification standard between species is not reasonable. (2) The ITS sequence of P. pungens was 693 bp, and the ITS sequence of SK-1 was 715 bp, while the ITS sequence of SK-2 was 331bp. Suquence similarity and phylogenetic tree showed that biogeographic groups of P.pungens have little difference in the ITS sequence, showing 100% identity, and classifications of SK-2 and SK-1 based on morphological features maybe incongruous. SK-2 seems to belong to species of S.pseudocostatum. (3) To overcome the shortage of fresh materials demanded by molecular biological experiments, and to get complete target sequence when sequencing as well as, an improved cloning method was applied. This cloning method is simple, feasible and efficient able to meet the need for regular cloning in lab. This study can provide target DNA on call. 2.Obtaining more nucleotide sequences could be offer more choice for designing species-specific probes (1)Complete LSU rDNA sequence of two strains of dinoflagellate were obtained by PCR amplifying three DNA fragments. The complete LSU rDNA sequence of P.micans was 3332 bp,and that of P.donghaiense was 3376 bp. The D1-D2 region of P.micans was 659 bp while the same region of P.donghaiense was713 bp. The D9-D10 region of these species were both 330bp。 (2)analyzing the sequences of complete LSU rDNA, D1-D2 and D9-D10 regions showed that the D1-D2 of P.micans was hypervariable between this strain of P.micans and other species of dinoflagellate, but the same region of p.donghaiense was relatively conserve compared with that of other species of dinoflagellate, especially with P.minimun. The D1-D2 region and D9-D10 region between
p.donghaiense and P.minimun all have above 96% identity, so was the complete LSU rDNA sequ
研究赤潮,进而预防和控制赤潮,最根本的问题是要明确引发赤潮的物种。最近几年采用分子生物学技术鉴定海洋浮游植物,特别是对赤潮藻的研究证实它们已成为微型藻类形态特征观察研究的重要补充手段。
本论文通过对一些原甲藻株和硅藻藻株的 ITS 序列、5.8S rDNA 或 LSU rDNA进行 PCR 扩增、克隆和序列测定。在 NCBI 数据库中进行同源性检索,序列的比对及相似性分析采用软件 Bioedit、DNAStar、Phylip3.5、Mega 2.0 等,系统进化关系分析采用 NJ(Neighbor Joining)法构建系统进化树。核酸序列的获得一方面是希望通过分子生物学的方法对一些通过形态鉴定的赤潮藻的命名进行验证,更重要的是找出引发赤潮的主要物种的特异性序列或区域,为下一步定性定量检测方法,主要是 FISH 技术中设计种特异性探针提供更多的选择区域。研究结果如下:
1. 采用 ITS 和 5.8S rDNA 序列研究 2 株海洋原甲藻和 3 株海洋硅藻
(1)海洋原甲藻 APBM(Prorocentrum micans APBM)的 ITS 和 5.8S rDNA 的长度为 631bp,而东海原甲藻(P. donghaiense)的相应部位的碱基长度为 552bp;东海原甲藻与从 GenBank 中获得的微小原甲藻相似程度较高为 88%,与甲藻属其它原甲藻相似程度较低(约 60-70%);本文研究的海洋原甲藻 APBM 的 ITS 序列与其它原甲藻相似程度都较低(约 30%左右)。用 ITS1 或 ITS2 序列构建的系统树与用 ITS+5.8S rDNA 序列构建的系统树反映的结果基本一致,系统进化树反映的结果与序列相似性的结果基本一致。5.8S 区因过于保守似乎不适于构建系统树反映种下的亲缘关系。
(2)尖刺拟菱形藻的 ITS 和 5.8S rDNA 全长为 693bp,SK-1(分离自东海赤潮爆发区)测序得到715bp;SK-2(分离自青岛养殖场) 的ITS和5.8S长度为331bp。
During the last two decades, serious economic and public health impacts caused by Harmful Algal Blooms (HABs)have increased in intensity, frequency and geographic distribution. It had great effect on national economy, public health, and oceanic ecosystem as well as. Thus, HABs is receiving increased international attention。 Studying HAB events , then predict and govern HAB, The basic biological concern of HAB was identify and distinguish its species. The speedy progress of molecular techniques offers a new way to identify phytoplankton. At present More and more studies using LSU/SSU rRNA gene or internal transcribed spacers(ITS) as molecular criteria to study dinoflagellate phylogeny have been proved to be powerful tools.
PCR amplification, cloning and sequencing the ITS and 5.8S rDNA or LSU rDNA from some strains of dinoflagellates and diatoms. Blast searches of these sequences had been done on NCBI(the website: http:// www.ncbi.nlm.nih.gov). Softwares such as Mega 2.0, DNAStar,BioEdit and Phlip3.5 were used to compare the three sequences studied in this paper with other ITS sequences of diatoms retrieved from GenBank. The Phylogenetic tree was constructed with NJ method. The aims of this paper were hoping that verify their phylogeneny by molecular data , and more nucleotide sequences obtained could be offer more choice for designing species-specific probes as well as. The results were as follows:
1.Studing 2 strains of dinoflagellate and 3 strains of diatoms according to ITS and 5.8S rDNA sequences
(1) The ITS length of P.micans APBM was 631 base pairs, and P. donghaiense’s ITS sequence was 552 bp. In the ITS sequence, P.donghaiense has very high level of similarity with P.minimum (showing 88% identity), but low level similarity with other species of P.triestinum and P.micans (about 60%-70%), while P.micans APBM show
the lowest level similarity with other P.micans according to ITS sequence, it was about 30% only. The results from phylogenetic tree of ITS2 sequence was the same as that showed by phylogenetic tree of ITS and 5.8S sequence. The results from phylogenetic tree constructed with ITS and 5.8S sequence accorded with that of sequence similarity analysis. The results of the phylogenetic tree constructed with 5.8S sequence were inaccurate; therefore, the use of 5.8S rDNA as a classification standard between species is not reasonable. (2) The ITS sequence of P. pungens was 693 bp, and the ITS sequence of SK-1 was 715 bp, while the ITS sequence of SK-2 was 331bp. Suquence similarity and phylogenetic tree showed that biogeographic groups of P.pungens have little difference in the ITS sequence, showing 100% identity, and classifications of SK-2 and SK-1 based on morphological features maybe incongruous. SK-2 seems to belong to species of S.pseudocostatum. (3) To overcome the shortage of fresh materials demanded by molecular biological experiments, and to get complete target sequence when sequencing as well as, an improved cloning method was applied. This cloning method is simple, feasible and efficient able to meet the need for regular cloning in lab. This study can provide target DNA on call. 2.Obtaining more nucleotide sequences could be offer more choice for designing species-specific probes (1)Complete LSU rDNA sequence of two strains of dinoflagellate were obtained by PCR amplifying three DNA fragments. The complete LSU rDNA sequence of P.micans was 3332 bp,and that of P.donghaiense was 3376 bp. The D1-D2 region of P.micans was 659 bp while the same region of P.donghaiense was713 bp. The D9-D10 region of these species were both 330bp。 (2)analyzing the sequences of complete LSU rDNA, D1-D2 and D9-D10 regions showed that the D1-D2 of P.micans was hypervariable between this strain of P.micans and other species of dinoflagellate, but the same region of p.donghaiense was relatively conserve compared with that of other species of dinoflagellate, especially with P.minimun. The D1-D2 region and D9-D10 region between
p.donghaiense and P.minimun all have above 96% identity, so was the complete LSU rDNA sequ
引文
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