rDNA序列在几种浮游植物的分类和中肋骨条藻定量检测中的应用
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摘要
利用核糖体DNA(rDNA)序列对浮游植物进行亲缘关系分析和系统学研究往往会获得许多有价值的研究成果，已成为传统形态学鉴定的重要补充手段。同时，以rDNA序列多样性为基础建立浮游植物的定量检测方法，是目前浮游植物生态学研究的热点之一。
本论文首先对分离自胶州湾的十一种浮游植物的rDNA序列进行了PCR扩增、克隆、序列测定、序列相似性分析，并利用测得的小亚基(SSU)rDNA序列分析了这些浮游植物的系统进化关系。结果发现三株角毛藻的rDNA序列相似性非常高(全序列的相似性均大于99％)，进化距离也非常接近；而两株裸甲藻(一株裸甲藻未鉴定种(Gymnodinium sp．，编号为GYN-15)、一株米氏裸甲藻(Gymnodinium mikimotoi))虽然进化距离接近，但它们的rDNA序列相似性却很低，全长序列的相似性仅为54.1％。
在上述工作的基础上，本论文从以下几方面作了进一步的研究：
1．初步建立了定量检测中肋骨条藻的新方法——实时荧光定量PCR(Realtime Fluorescent Quantitative PCR，RFQ-PCR)。
首先以中肋骨条藻的rDNA序列为设计种特异性引物的靶区域，共设计出7对适合RFQ-PCR的引物(依次命名为Primer 1(F／R)～Primer 7(F／R))，并用常规PCR实验初步证明其中有4对引物(Primer 1(F／R)、Primer 2(F／R)、Primer 5(F／R)、Primer 6(F／R))可作为中肋骨条藻特异性引物的候选者；然后测定了以Primer6(F／R)为引物的PCR产物的序列，序列分析表明，中肋骨条藻的PCR产物序列与其他藻的PCR产物序列差别较大，从中可设计出满足RFQ-PCR需要的TaqMan探针(命名为TaqMan 6)；进一步的核酸杂交实验表明，TaqMan 6只与中肋骨条藻的PCR产物杂交，不与其他藻的PCR产物杂交。最后以Primer 6
rDNA序列在几种浮游植物的分类及中肋骨条藻定量检测中的应用
(F/R)和TaqMan6探针建立了定量检测中肋骨条藻的RFQ一PCR。以RFQ一PCR中
实际细胞数的常用对数值为横座标，以测得的CT值为纵座标，绘制出了定量检
测的标准曲线，该曲线的回归方程为:y=一3.3427x+43.443，其相关系数是:
RZ二0.9788，CT值的标准估计误差为Sy’x二0.6741。在蚌1 .3212的区域，可保
证95%的准确率。该标准曲线的CT值检测范围为20.17士1.3212一33.54上1.3212，
对应的细胞数为106乡623士0·39，2一xoZ·962，土03952个。用己知细胞数的样品验证表明，
该曲线是准确可靠的。
2.对裸甲藻属未鉴定种(GYN一15)进行了初步鉴定。
该藻的roNA序列全长2658 bp，涵盖了sSU roNA基因3’端1747 bp、
ITsl一5.85 rDNA一ITSZ全长、LSU rDNA基因5’端337bp。以此序列作BLAsTn
分析发现GYN一巧与共生甲藻属(今mbi口dinium)中的两株藻(一株是
砂功biodinium eal如rnsum，共生于海葵体内;一株是Gymnodinium varians，自
由生活)的相应序列具有很高的相似性。进一步的序列比较表明，这三株藻的各
段rDNA序列的相似性均大于99%。以SSU rDNA序列中的三个可变区
(Vz+vZ+V3)和邻接法困eighbor一Joining Method，NJ法)构建共生甲藻属的系统
进化树表明，GYN一巧与5. cal扣rnium和G.varian:在共生甲藻属内构成一个独
立的、自检支持百分率为100%的子类群(clade)，根据这些结果可将GYN一巧初
步鉴定为属于共生甲藻属。另外，鉴于GYN一巧所在的子类群与其它五个己报
道过的子类群(c ladeA一E)明显分开，因此建议将其命名为cladeF。
3.对三株角毛藻的亲缘关系和遗传差异进行了研究。
所测定的旋链角毛藻、柔弱角毛藻和纤细角毛藻的rDNA序列全长分别为
287obp、2867bp、2868bP。以ssU rDNA序列和NJ法构建的系统进化树表明三
种角毛藻与其它几种角毛藻的进化距离相对较远，而它们之间的进化距离却很
接近;三种角毛藻的SSU rDNA序列相似性)99%，明显高于它们与其它角毛
藻SSU:DNA的序列相似性(82.9%一93.1%):三种角毛藻各段rDNA的序列相似
性非常高(98%一100%);三种角毛藻的任意引物PCR(AP一PcR)指纹图谱并不完
全相同。这些结果说明三种角毛藻既具有很近的亲缘关系，又存在一定的遗传
rDNA序列在几种浮游植物的分类及中肋骨条藻定量检测中的应用
差异。
通过上述这些研究，本论文发现裸甲藻未鉴定种与共生甲藻属
(今形biodinium)的两个种具有很近的亲缘关系和进化距离，初步将该藻鉴定为属
于共生甲藻属;命名了共生甲藻属中一个新的子类群—CladeF;首次发现
了不同种角毛藻之间具有很近的亲缘关系;初步建立了定量检测中肋骨条藻的
新方法—RFQ一PCR。
Analysis of phytogenies and relationships among phytoplankton using sequences of the ribosomal DNA (rDNA) sequences are important complements for the traditionally morphological identification, and can sometimes give researchers valuable results. On the other hand, studies on the quantitatively rapid detection using the divergent sequences of rDNA are one of the most common focuses on phytoplanktonic ecology.
In this dissertation, sequences of rDNA from eleven important phytoplanktonic strains collected at Jiaozhou Bay are amplified, cloned, sequenced and compared, and the phylogenetic relationships among them are analyzed using the SSU rDNA sequences. It is found that sequences of rDNA from three Chaetoceros strains show high level of the total sequence similarities (all above 99%), and show close relationships. In contrast, sequences from two strains Gymnodinium are divergent (the sequence similarities of the small subunit (SSU) rDNA, the ITS1, the 5.8S rDNA, the ITS2 and the large subunit (LSU) rDNA are 76.5%, 25.4%, 54.2%, 23.2%, 56.5%, respectively), though their phylogenetic positions are relatively close.
On the basis of the researches above, further studies are carried out as following:
1. Development of a quantitative method for the rapid detection of Skeletonema costatum --Real-time fluorescent quantitative PCR (RFQ-PCR) .
The divergent sequences of rDNA from S. costatum are used to design primers meeting the requirements of the RFQ-PCR. Seven pairs of primers are designed and designated as Primer 1 (F/R) ~ Primer 7 (F/R), respectively, among which primer 1 (F/R), primer 2 (F/R), primer 5 (F/R), primer 6 (F/R) showed high level of specificities to S. costatum. Then, the PCR products primed by primer6 (F/R) are sequenced. Sequences comparisons show that the sequence amplified from S. costatum is very different to that from other microalga. Thus, the sequence from S.
costatum is used to design TaqMan 6 for RFQ-PCR. Experiments of nucleic acid hybridization show that TaqMan 6 only hybridizes to the PCR products amplified from S. costatum, but not to products from other microalga. These results indicate that Primer6 (F/R) and TaqMan6 can be used to establish the RFQ-PCR method for the quantitative detection of S. costatum.
Thereafter, the RFQ-PCR method for the detection and enumeration of S. costatum cells is established with Primer6 (F/R) and TaqMan6. The regression curve for enumeration is delineated according to the development of the fluorescent densities in the RFQ-PCR with the increasing number of S. costatum cells. The regression equation is y = -3.3427x + 43.443, in which x indicates the log10 of cell number, and y indicates the CT values, with R2 of 0.9788. The standard estimated error of the CT values (Sy.x) is 0.6741, thus 95% of the accurate CT values are included in the range of y 1.3212. The range of the CT values that can be detected accurately is 20.17 1.3212 ~ 33.54 1.3212, corresponding to 106-9623 0.3952 ~ 102.9625 0.3952 of cells. This equation is proved to be credible by the RFQ-PCR using the samples whose cell number is known.
2. The primary classification of a Gymnodinium sp. (GYN-15), whose species name was undetermined.
The total rDNA sequence from GYN-15 is of 2658 bp length, spanning 1748 bp of the 3' end of the SSU rDNA, the complete sequence of the ITS1-5.8S rDNA-ITS2 region, and 336 bp of the 5' end of the LSU rDNA. The BLASTn results show that GYN-15 is closely related to a symbiont of anemones, S. californium, and the free-living strain, Gymnodinium varians. Sequence comparison show that the similarities among each part of the sequences from these three strains are all above 99%. Phylogenetic reconstruction with Neighbor-joining (NJ) method using sequences of variable regions (V1+V2+V3) of SSU rDNA indicated that GYN-15, S. californium and G. varians form a new clade with 100% bootstrap support. Based on these results, it is believed that GYN-15 may belong to the genus Symbiodinium. In addition, the branch formed by GYN-15 and other two Symbiodinium strains
本论文首先对分离自胶州湾的十一种浮游植物的rDNA序列进行了PCR扩增、克隆、序列测定、序列相似性分析，并利用测得的小亚基(SSU)rDNA序列分析了这些浮游植物的系统进化关系。结果发现三株角毛藻的rDNA序列相似性非常高(全序列的相似性均大于99％)，进化距离也非常接近；而两株裸甲藻(一株裸甲藻未鉴定种(Gymnodinium sp．，编号为GYN-15)、一株米氏裸甲藻(Gymnodinium mikimotoi))虽然进化距离接近，但它们的rDNA序列相似性却很低，全长序列的相似性仅为54.1％。
在上述工作的基础上，本论文从以下几方面作了进一步的研究：
1．初步建立了定量检测中肋骨条藻的新方法——实时荧光定量PCR(Realtime Fluorescent Quantitative PCR，RFQ-PCR)。
首先以中肋骨条藻的rDNA序列为设计种特异性引物的靶区域，共设计出7对适合RFQ-PCR的引物(依次命名为Primer 1(F／R)～Primer 7(F／R))，并用常规PCR实验初步证明其中有4对引物(Primer 1(F／R)、Primer 2(F／R)、Primer 5(F／R)、Primer 6(F／R))可作为中肋骨条藻特异性引物的候选者；然后测定了以Primer6(F／R)为引物的PCR产物的序列，序列分析表明，中肋骨条藻的PCR产物序列与其他藻的PCR产物序列差别较大，从中可设计出满足RFQ-PCR需要的TaqMan探针(命名为TaqMan 6)；进一步的核酸杂交实验表明，TaqMan 6只与中肋骨条藻的PCR产物杂交，不与其他藻的PCR产物杂交。最后以Primer 6
rDNA序列在几种浮游植物的分类及中肋骨条藻定量检测中的应用
(F/R)和TaqMan6探针建立了定量检测中肋骨条藻的RFQ一PCR。以RFQ一PCR中
实际细胞数的常用对数值为横座标，以测得的CT值为纵座标，绘制出了定量检
测的标准曲线，该曲线的回归方程为:y=一3.3427x+43.443，其相关系数是:
RZ二0.9788，CT值的标准估计误差为Sy’x二0.6741。在蚌1 .3212的区域，可保
证95%的准确率。该标准曲线的CT值检测范围为20.17士1.3212一33.54上1.3212，
对应的细胞数为106乡623士0·39，2一xoZ·962，土03952个。用己知细胞数的样品验证表明，
该曲线是准确可靠的。
2.对裸甲藻属未鉴定种(GYN一15)进行了初步鉴定。
该藻的roNA序列全长2658 bp，涵盖了sSU roNA基因3’端1747 bp、
ITsl一5.85 rDNA一ITSZ全长、LSU rDNA基因5’端337bp。以此序列作BLAsTn
分析发现GYN一巧与共生甲藻属(今mbi口dinium)中的两株藻(一株是
砂功biodinium eal如rnsum，共生于海葵体内;一株是Gymnodinium varians，自
由生活)的相应序列具有很高的相似性。进一步的序列比较表明，这三株藻的各
段rDNA序列的相似性均大于99%。以SSU rDNA序列中的三个可变区
(Vz+vZ+V3)和邻接法困eighbor一Joining Method，NJ法)构建共生甲藻属的系统
进化树表明，GYN一巧与5. cal扣rnium和G.varian:在共生甲藻属内构成一个独
立的、自检支持百分率为100%的子类群(clade)，根据这些结果可将GYN一巧初
步鉴定为属于共生甲藻属。另外，鉴于GYN一巧所在的子类群与其它五个己报
道过的子类群(c ladeA一E)明显分开，因此建议将其命名为cladeF。
3.对三株角毛藻的亲缘关系和遗传差异进行了研究。
所测定的旋链角毛藻、柔弱角毛藻和纤细角毛藻的rDNA序列全长分别为
287obp、2867bp、2868bP。以ssU rDNA序列和NJ法构建的系统进化树表明三
种角毛藻与其它几种角毛藻的进化距离相对较远，而它们之间的进化距离却很
接近;三种角毛藻的SSU rDNA序列相似性)99%，明显高于它们与其它角毛
藻SSU:DNA的序列相似性(82.9%一93.1%):三种角毛藻各段rDNA的序列相似
性非常高(98%一100%);三种角毛藻的任意引物PCR(AP一PcR)指纹图谱并不完
全相同。这些结果说明三种角毛藻既具有很近的亲缘关系，又存在一定的遗传
rDNA序列在几种浮游植物的分类及中肋骨条藻定量检测中的应用
差异。
通过上述这些研究，本论文发现裸甲藻未鉴定种与共生甲藻属
(今形biodinium)的两个种具有很近的亲缘关系和进化距离，初步将该藻鉴定为属
于共生甲藻属;命名了共生甲藻属中一个新的子类群—CladeF;首次发现
了不同种角毛藻之间具有很近的亲缘关系;初步建立了定量检测中肋骨条藻的
新方法—RFQ一PCR。
Analysis of phytogenies and relationships among phytoplankton using sequences of the ribosomal DNA (rDNA) sequences are important complements for the traditionally morphological identification, and can sometimes give researchers valuable results. On the other hand, studies on the quantitatively rapid detection using the divergent sequences of rDNA are one of the most common focuses on phytoplanktonic ecology.
In this dissertation, sequences of rDNA from eleven important phytoplanktonic strains collected at Jiaozhou Bay are amplified, cloned, sequenced and compared, and the phylogenetic relationships among them are analyzed using the SSU rDNA sequences. It is found that sequences of rDNA from three Chaetoceros strains show high level of the total sequence similarities (all above 99%), and show close relationships. In contrast, sequences from two strains Gymnodinium are divergent (the sequence similarities of the small subunit (SSU) rDNA, the ITS1, the 5.8S rDNA, the ITS2 and the large subunit (LSU) rDNA are 76.5%, 25.4%, 54.2%, 23.2%, 56.5%, respectively), though their phylogenetic positions are relatively close.
On the basis of the researches above, further studies are carried out as following:
1. Development of a quantitative method for the rapid detection of Skeletonema costatum --Real-time fluorescent quantitative PCR (RFQ-PCR) .
The divergent sequences of rDNA from S. costatum are used to design primers meeting the requirements of the RFQ-PCR. Seven pairs of primers are designed and designated as Primer 1 (F/R) ~ Primer 7 (F/R), respectively, among which primer 1 (F/R), primer 2 (F/R), primer 5 (F/R), primer 6 (F/R) showed high level of specificities to S. costatum. Then, the PCR products primed by primer6 (F/R) are sequenced. Sequences comparisons show that the sequence amplified from S. costatum is very different to that from other microalga. Thus, the sequence from S.
costatum is used to design TaqMan 6 for RFQ-PCR. Experiments of nucleic acid hybridization show that TaqMan 6 only hybridizes to the PCR products amplified from S. costatum, but not to products from other microalga. These results indicate that Primer6 (F/R) and TaqMan6 can be used to establish the RFQ-PCR method for the quantitative detection of S. costatum.
Thereafter, the RFQ-PCR method for the detection and enumeration of S. costatum cells is established with Primer6 (F/R) and TaqMan6. The regression curve for enumeration is delineated according to the development of the fluorescent densities in the RFQ-PCR with the increasing number of S. costatum cells. The regression equation is y = -3.3427x + 43.443, in which x indicates the log10 of cell number, and y indicates the CT values, with R2 of 0.9788. The standard estimated error of the CT values (Sy.x) is 0.6741, thus 95% of the accurate CT values are included in the range of y 1.3212. The range of the CT values that can be detected accurately is 20.17 1.3212 ~ 33.54 1.3212, corresponding to 106-9623 0.3952 ~ 102.9625 0.3952 of cells. This equation is proved to be credible by the RFQ-PCR using the samples whose cell number is known.
2. The primary classification of a Gymnodinium sp. (GYN-15), whose species name was undetermined.
The total rDNA sequence from GYN-15 is of 2658 bp length, spanning 1748 bp of the 3' end of the SSU rDNA, the complete sequence of the ITS1-5.8S rDNA-ITS2 region, and 336 bp of the 5' end of the LSU rDNA. The BLASTn results show that GYN-15 is closely related to a symbiont of anemones, S. californium, and the free-living strain, Gymnodinium varians. Sequence comparison show that the similarities among each part of the sequences from these three strains are all above 99%. Phylogenetic reconstruction with Neighbor-joining (NJ) method using sequences of variable regions (V1+V2+V3) of SSU rDNA indicated that GYN-15, S. californium and G. varians form a new clade with 100% bootstrap support. Based on these results, it is believed that GYN-15 may belong to the genus Symbiodinium. In addition, the branch formed by GYN-15 and other two Symbiodinium strains
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26．廉双喜．赤潮监测和预报的构想．海洋技术．2002．21(2)：74-77．
27．林昱．甲藻赤潮与水体中营养盐的关系初探．台湾海峡，2001．20(1)：77-79．
28．刘令梅．赤潮的监测技术和防治措施．海洋技术．1998．17(3)：58-64．
29. SOHMA A, SATO T, NAKATA K. New Numerical Model Study on a Tidal Flat System - Seasonal, Daily and Tidal Variations. Spill Science & Technology Bulletin. 2000, 6 (2): 173～185.
30．刘玉等．湛江海域浮游藻类生态及其多样性．海洋环境科学．1994．13(3)：7～12．
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32．齐雨藻，黄长江．南海大鹏湾海洋卡盾藻赤潮发生的环境背景．海洋与湖沼．1997．28(4)：337～342．
33. Bu-Olayan A H, Al-Hassan R, Thomas B V, Subrahmanyam M N V. Impact of trace metals and nutrients levels on phytoplankton from the Kuwait Coast. Environment International. 2001, 26: 199～203.
34. Wang W X, Dei R C H. Metal uptake in a coastal diatom influenced by major nutrients (N, P, AND Si). Wat. Res. 2001, 35(1): 315～321.
35．钱宏林，粱松，齐雨藻．广东沿海赤潮和特点及成因研究．生态科学．2000．19(3)：8～16．
36. WU R S S. Eutrophication, Water Borne Pathogens and Xenobiotic Compounds: Environmental Risks and Challenges. Marine Pollution Bulletin. 1999, 39 (1～12): 11～22.
37．林荣根．海水富营养化水平评价方法浅析．海洋环境科学．1996．15(2)：28～31．
38．孙军，刘东艳．中国海区常见浮游植物种名更改初步意见．海洋与湖沼，2002，33(3)：271～286．
39．王团老，林均民．硅藻分子生物学研究的一些进展．植物生理学通讯．1998．34(2)：140～148．
40．王正方．张庆．卢勇等．氮、磷、维生素和微量金属对赤潮生物海洋原甲藻的增殖效应．东海海洋科学．1996．6：33—38．
41. Adachi M, Sako Y & Ishida Y. Analyses of Alexandrium (Dinophyceae) species using sequences of the 5.8S ribosomal DNA and internal transcribed spacer regions. J. Phycol. 1996a, 32: 424～432.
42. Adachi M, Sako Y & Ishida Y. Identification of the toxic dinoflagellates Alexandrium catenella and A. tamerense (Dinophyceae) using DNA probes and whole-cell hybridization. J. Phycol. 1996b, 32: 1049～1052.
43. Adachi M, Sako Y, Ishida Y, Anderson D M & Reguera B. Cross-reactivity of five monoclonal antibodies to various isolates of Alexandrium as determined by an indirect immunofluorescence method. Nippon Suisan Gakkaishi. 1993, 59: 1807.
44. Aguilera A, Gonzalez-Gil S., Keafer B A & Anderson D M. Immunomagnetic separation of cells of the toxic dinoflagellate Alexandrium fundyense from natural plankton samples. Mar. Ecol. Prog. Ser. 1996, 143: 255～269.
45. Anderson D M, Kulis D M, Keafer B A, Berdalet E. Detection of the toxic dinoflagellate Alexandrium fundyense (Dinophyceae) with oligonucleotide and antibody probes: variability in labeling intensity with physiological condition. J. Phycot. 1999, 35: 870～883.
46. Anderson D M, Keafer B A, Kulis DM, Waters R M & Nuzzi R. An Immunofluorescent survey of the brown tide chrysophyte Aureococcus anophagefferens along the northeast coast of the United states. J. Plankton Res. 1993, 15: 563～580.
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48． Vrieling E G, Anderson D M, Immunofluorescence in phytoplankton research: Applications and potential. J. Phycol. 1996． 32,1-16
49． 陈月琴，邱小忠，屈良鹄，曾陇梅，齐雨藻，郑磊．南海有毒塔玛亚历山大藻的分子地 理标记分析．海洋与湖沼1999． 30(1) ：45-51．
50． 陈月琴，屈良鹄，邱小忠，曾陇梅，齐雨藻．甲藻单个细胞DNA的制备及在赤潮藻类 分子鉴定中的应用．中山大学学报(自然科学版)．1997． 36(4) ：66-69．
51． Bates S S, Leger C, Keafer B A & Anderson D M. Discrimination between domoic-acid-producing and nontoxic forms of the diatom Pseudonitzschia pungens using immunofluorescence. Mar. Ecol. Prog. Ser. 1993． 100:185-195．
52． Beutler M. FluoroProbe-a new tool in algal differentiation. http://www.bbe-moldaenke.de
53． Campbell L. Carfemter. E K & lacono V J. Identification and enumeration of marine chroococcoid cyanobacteria by immunofluorescence. Appl. Environ. Microbiol. 1983． 46: 553-559．
54． Carlos A A, Baillie B K, Kawachi, M. Phylogenetic position of Symbiodinium (Dinophyceae) Isolates from Tridacnids (Bivalvia), Cardiids (Bivalvia), a sponge (Porifera), a Soft Coral (Anthozoa), and a Free-Living Strain. J. Phycol. 1999． 35: 1054-1062．
55． Daugbjerg N, Hansen G, Larsen J & Moestrup (?). Phylogeny of some of the major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including the erection of three new genera of naked dinoflagellates. Phycologia, 2000． 39: 302-317．
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