东海原甲藻增殖细胞核抗原基因表达量与生长关系的研究
|
摘要
浮游藻是海洋食物链的重要一环，在能量转移和营养盐循环等方面起着重要作用。对浮游藻现场生长率等基本生理生态学参数的测定，是估算浮游藻生产力、研究浮游藻种群变迁、构建生态动力学模型乃至赤潮预测的基础，特别是现场测定单种浮游藻的生长率显得尤为重要。但目前尚缺乏一种准确估算单一种类浮游藻现场生长率的方法。近年来，国际上对细胞周期标志物的研究显示，增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)可能在浮游藻生长率研究中有巨大的潜在应用价值，这为藻类生长率的估算提供了新的研究方向。
本研究以我国东海海域常见的赤潮藻——东海原甲藻(Prorocentrumdonghaiense)为研究对象，首次克隆得到了东海原甲藻的PCNA基因和其细胞色素b基因的cDNA全长序列，分别设计了符合实时荧光定量PCR检测要求的引物和TaqMan探针，建立了检测东海原甲藻PCNA与Cyt b基因的实时荧光定量PCR方法，系统研究了东海原甲藻PCNA基因表达量在不同细胞周期中的变化及其与生长率之间的关系，为运用分子生物学技术实现东海原甲藻现场生长率的估算奠定了基础。
主要研究结果如下：
(1)通过反转录PCR(RT-PCR)和cDNA末端快速扩增技术(RACE)，首次克隆得到了东海原甲藻长为1057bp的PCNA基因的cDNA全长序列和长为1124bp的细胞色素b(cytochrome b，Cyt b)基因的cDNA全长序列，并分别对PCNA和Cyt b进行了序列比对和系统进化树分析。
(2)根据得到的PCNA与Cytb基因序列，分别设计了符合实时荧光定量PCR(RFQ-PCR)检测要求的引物和TaqMan探针，并以12种藻为参照藻，对所设计PCNA基因的RFQ-PCR引物的特异性进行了分析。采用SYBR Green I染料和TaqMan探针两种荧光体系建立了检测东海原甲藻PCNA与Cytb基因的实时荧光定量PCR的标准曲线。采用SYBR Green I染料体系得到的曲线方程分别为：对PCNA，y=-3．403x+40．048，(r=0．999，其中x为细胞数对数值，y为C_T值，r为相关系数)；对Cyt b，y=-3．501x+39．351，(r=0．999)；采用TaqMan探针体系得到的曲线方程分别为：对PCNA，y=-3．211x+39．302，(r=0．999)；对Cyt b，y=-3．340x+41．041，(r=0．997)。通过对标准样品的检测，验证了所建立标准曲线的准确性。
(3)运用建立的定量PCR方法，系统研究了东海原甲藻PCNA基因表达量在不同细胞周期中的变化及其与生长率之间的关系。结果表明，Cyt b基因表达稳定，平均单细胞表达量为(45．4±4．7)拷贝，在不同的生长阶段表达量变化很小，是一个潜在的良好的内参基因。与此不同，平均单细胞中PCNA表达量在培养的不同阶段变化较大，指数期的含量比平台期高4倍左右，说明PCNA表达量与细胞分裂密切相关。以Cytb基因为内参基因，PCNA基因为目的基因，进一步研究了不同培养条件下PCNA基因的相对表达量(REL)与生长率之间的关系，结果发现，REL与生长率呈正相关性。
(4)以流式细胞术分析了同步培养的东海原甲藻的细胞周期，以RFQ-PCR测定了PCNA基因表达量，研究了不同细胞周期中PCNA基因表达的变化。结果表明，同其他物种的PCNA类似，东海原甲藻PCNA表达与细胞周期相关，表达量从G_1后期开始升高，在S期表达量最高。
本研究为建立PCNA相对表达量与东海原甲藻生长率之间的关系，进而运用分子生物学技术实现东海原甲藻现场生长率的估算奠定了基础。
Phytoplankton is a significant composition of the marine food chain,and plays animportant role in energy transfer and nutrient cycle.The mensuration of thephysiological and ecological parameters such as the in situ growth rate is the basic ofunderstanding the regulation of phytoplankton dynamics,constructing the ecologicaldynamic models and even forecasting the happening of red tide.Therefore,themeasurement of the in situ growth rate of single species seems to be especiallyimportant.While a correctly estimating method of the in situ growth rate is still lack.In recent years,the studies on the molecular markers of cell cycle have shown that theproliferating cell nuclear antigen (PCNA) is potential to be used to study thephytoplankton growth rate,which provides a new orientation of studying the algalgrowth rate.
In this study,Prorocentrum donghaiense,one dominant species of red tide algaein East China Sea area,was taken as the object.The full length cDNA sequences ofPCNA gene and cytochrome b (Cyt b) gene of P.donghaiense were obtained for thefirst time.The primers and Taqman probes were designed based on PCNA and Cyt bsequences.Furthermore,the RFQ-PCR methods were constructed to detect the twogenes.The above methods were applied to the study of the variation of expressionlevel of PCNA gene in different cell cycle phases for P.donghaiense,and therelationship between its expression level and the growth rateμ(d~(-1)).It paves a way forusing molecular biological methods to enumerate P.donghaiense in situ growth rateaccurately.
The main results are as follows:
(1) The 1057bp length full cDNA sequence encoding PCNA gene of P.donghaiense was cloned for the first time by RT-PCR and RACE technique.Moreover,the 1124bp length full cDNA of Cytochrome b (Cyt b) gene of P.donghaiense was obtained.We did the sequence alignments and constructedphylogenetic trees of PCNA and Cyt b respectively.
(2) The primers and Taqman probes were designed based on PCNA and Cyt bsequences.The species-specificity of the RFQ-PCR primers for amplifying PCNAwas testing by the other 12 algal species.The standard curves were constructed todetect the two genes by SYBR GreenⅠand Taqman probe systems,respectively.ForSYBR GreenⅠsystem,the equation for detecting P.donghaiense PCNA was definedas y=-3.403x+40.048(r=0.999),where x was the logarithm of the plasmid copynumber,and y was the C_T values;the equation for Cyt b was defined as y=-3.501x+39.351 (r=0.999).For Taqman probe system,the equation for detecting P.donghaiense PCNA was defined as y=-3.211x+39.302 (r=0.999);the equation forCyt b was defined as y=-3.340x + 41.041 (r=0.997).The accuracy of the standardcurves was verified by detecting the plasmid samples with the known concentration.
(3) The above methods were applied to the study of the variation of expressionlevel of PCNA gene in different cell cycle phases for P.donghaiense,and therelationship between its expression level and the growth rateμ(d~(-1)).We found that theexpression level of Cyt b was steady and had no obviously variation during differentculture stages,about (45.4±4.7) copies/cell,which suggested that the Cyt b could bea reference gene in P.donghaiense.The expression level of PCNA gene had highvariation in different culture stages,and varied about 4-fold between the exponentialand stationary phases,which suggested that the expression level of PCNA genecorrelated well with the cell division.Using PCNA gene as the objective gene and Cytb gene as the reference gene,we studied the relationship between the relativeexpression level of PCNA gene (REL) and the growth rateμ(d~(-1)) under differentconditions.It showed that,REL had a positive correlation withthe growth rateμ.
(4) We analysed the cell cycle phase of the synchronized cells of P.donghaiense by Flow Cytometer,and obtained the expression level of PCNA gene by RFQ-PCR.Furthermore,we analysed the variation of the expression level of PCNAgene in different cell cycle phases.It found that,the expression level of PCNA genewas related with the cell cycle phase liking other organism,which increased from thelate G_1 phase,and peaked in the S phase.
This research paved a way for establishing a formula between the relativeexpression level of PCNA gene and the growth rateμ(d~(-1)),and also enumerating the P.donghaiense in situ growth rate accurately using molecular biological methods.
本研究以我国东海海域常见的赤潮藻——东海原甲藻(Prorocentrumdonghaiense)为研究对象，首次克隆得到了东海原甲藻的PCNA基因和其细胞色素b基因的cDNA全长序列，分别设计了符合实时荧光定量PCR检测要求的引物和TaqMan探针，建立了检测东海原甲藻PCNA与Cyt b基因的实时荧光定量PCR方法，系统研究了东海原甲藻PCNA基因表达量在不同细胞周期中的变化及其与生长率之间的关系，为运用分子生物学技术实现东海原甲藻现场生长率的估算奠定了基础。
主要研究结果如下：
(1)通过反转录PCR(RT-PCR)和cDNA末端快速扩增技术(RACE)，首次克隆得到了东海原甲藻长为1057bp的PCNA基因的cDNA全长序列和长为1124bp的细胞色素b(cytochrome b，Cyt b)基因的cDNA全长序列，并分别对PCNA和Cyt b进行了序列比对和系统进化树分析。
(2)根据得到的PCNA与Cytb基因序列，分别设计了符合实时荧光定量PCR(RFQ-PCR)检测要求的引物和TaqMan探针，并以12种藻为参照藻，对所设计PCNA基因的RFQ-PCR引物的特异性进行了分析。采用SYBR Green I染料和TaqMan探针两种荧光体系建立了检测东海原甲藻PCNA与Cytb基因的实时荧光定量PCR的标准曲线。采用SYBR Green I染料体系得到的曲线方程分别为：对PCNA，y=-3．403x+40．048，(r=0．999，其中x为细胞数对数值，y为C_T值，r为相关系数)；对Cyt b，y=-3．501x+39．351，(r=0．999)；采用TaqMan探针体系得到的曲线方程分别为：对PCNA，y=-3．211x+39．302，(r=0．999)；对Cyt b，y=-3．340x+41．041，(r=0．997)。通过对标准样品的检测，验证了所建立标准曲线的准确性。
(3)运用建立的定量PCR方法，系统研究了东海原甲藻PCNA基因表达量在不同细胞周期中的变化及其与生长率之间的关系。结果表明，Cyt b基因表达稳定，平均单细胞表达量为(45．4±4．7)拷贝，在不同的生长阶段表达量变化很小，是一个潜在的良好的内参基因。与此不同，平均单细胞中PCNA表达量在培养的不同阶段变化较大，指数期的含量比平台期高4倍左右，说明PCNA表达量与细胞分裂密切相关。以Cytb基因为内参基因，PCNA基因为目的基因，进一步研究了不同培养条件下PCNA基因的相对表达量(REL)与生长率之间的关系，结果发现，REL与生长率呈正相关性。
(4)以流式细胞术分析了同步培养的东海原甲藻的细胞周期，以RFQ-PCR测定了PCNA基因表达量，研究了不同细胞周期中PCNA基因表达的变化。结果表明，同其他物种的PCNA类似，东海原甲藻PCNA表达与细胞周期相关，表达量从G_1后期开始升高，在S期表达量最高。
本研究为建立PCNA相对表达量与东海原甲藻生长率之间的关系，进而运用分子生物学技术实现东海原甲藻现场生长率的估算奠定了基础。
Phytoplankton is a significant composition of the marine food chain,and plays animportant role in energy transfer and nutrient cycle.The mensuration of thephysiological and ecological parameters such as the in situ growth rate is the basic ofunderstanding the regulation of phytoplankton dynamics,constructing the ecologicaldynamic models and even forecasting the happening of red tide.Therefore,themeasurement of the in situ growth rate of single species seems to be especiallyimportant.While a correctly estimating method of the in situ growth rate is still lack.In recent years,the studies on the molecular markers of cell cycle have shown that theproliferating cell nuclear antigen (PCNA) is potential to be used to study thephytoplankton growth rate,which provides a new orientation of studying the algalgrowth rate.
In this study,Prorocentrum donghaiense,one dominant species of red tide algaein East China Sea area,was taken as the object.The full length cDNA sequences ofPCNA gene and cytochrome b (Cyt b) gene of P.donghaiense were obtained for thefirst time.The primers and Taqman probes were designed based on PCNA and Cyt bsequences.Furthermore,the RFQ-PCR methods were constructed to detect the twogenes.The above methods were applied to the study of the variation of expressionlevel of PCNA gene in different cell cycle phases for P.donghaiense,and therelationship between its expression level and the growth rateμ(d~(-1)).It paves a way forusing molecular biological methods to enumerate P.donghaiense in situ growth rateaccurately.
The main results are as follows:
(1) The 1057bp length full cDNA sequence encoding PCNA gene of P.donghaiense was cloned for the first time by RT-PCR and RACE technique.Moreover,the 1124bp length full cDNA of Cytochrome b (Cyt b) gene of P.donghaiense was obtained.We did the sequence alignments and constructedphylogenetic trees of PCNA and Cyt b respectively.
(2) The primers and Taqman probes were designed based on PCNA and Cyt bsequences.The species-specificity of the RFQ-PCR primers for amplifying PCNAwas testing by the other 12 algal species.The standard curves were constructed todetect the two genes by SYBR GreenⅠand Taqman probe systems,respectively.ForSYBR GreenⅠsystem,the equation for detecting P.donghaiense PCNA was definedas y=-3.403x+40.048(r=0.999),where x was the logarithm of the plasmid copynumber,and y was the C_T values;the equation for Cyt b was defined as y=-3.501x+39.351 (r=0.999).For Taqman probe system,the equation for detecting P.donghaiense PCNA was defined as y=-3.211x+39.302 (r=0.999);the equation forCyt b was defined as y=-3.340x + 41.041 (r=0.997).The accuracy of the standardcurves was verified by detecting the plasmid samples with the known concentration.
(3) The above methods were applied to the study of the variation of expressionlevel of PCNA gene in different cell cycle phases for P.donghaiense,and therelationship between its expression level and the growth rateμ(d~(-1)).We found that theexpression level of Cyt b was steady and had no obviously variation during differentculture stages,about (45.4±4.7) copies/cell,which suggested that the Cyt b could bea reference gene in P.donghaiense.The expression level of PCNA gene had highvariation in different culture stages,and varied about 4-fold between the exponentialand stationary phases,which suggested that the expression level of PCNA genecorrelated well with the cell division.Using PCNA gene as the objective gene and Cytb gene as the reference gene,we studied the relationship between the relativeexpression level of PCNA gene (REL) and the growth rateμ(d~(-1)) under differentconditions.It showed that,REL had a positive correlation withthe growth rateμ.
(4) We analysed the cell cycle phase of the synchronized cells of P.donghaiense by Flow Cytometer,and obtained the expression level of PCNA gene by RFQ-PCR.Furthermore,we analysed the variation of the expression level of PCNAgene in different cell cycle phases.It found that,the expression level of PCNA genewas related with the cell cycle phase liking other organism,which increased from thelate G_1 phase,and peaked in the S phase.
This research paved a way for establishing a formula between the relativeexpression level of PCNA gene and the growth rateμ(d~(-1)),and also enumerating the P.donghaiense in situ growth rate accurately using molecular biological methods.
引文
1． 陈炳章，王宗灵，朱明远，李瑞香．温度、盐度对具齿原甲藻生长的影响及其与中肋骨条藻的比较．海洋科学进展，2005，23：60-64．
2． 陈翰林，吕颂辉，张传松，朱德弟．2004年东海原甲藻赤潮爆发的现场调查和分析．生态科学，2006，2：226-230．
3． 郭皓．中国近海赤潮生物图谱．北京：海洋出版社，2004．
4． 国家海洋局，中国海洋灾害公报，2001-2008．
5． 国家海洋局，中国海洋环境质量公报，2005-2008．
6． 郭沛涌，沈焕庭，张利华．流式细胞术在水体微型生物分子生物学研究中的应用．生命的化学，2001，21：520-522．
7． 何闪英．中肋骨条藻增殖细胞核抗原基因表达量与生长关系的研究[博士论文]．，中国海洋大学，2007．
8． 洪君超，黄秀清，蒋晓山，王佳兰．长江口中肋骨条藻赤潮发生过程环境要素分析—营养盐状况．海洋与湖沼，1994，25：179-184．
9． 李淑冰，李惠珍，许旭萍．贝毒素的研究现状及产生源探究．福建水产，2000，1：70-74．
10．李铁，胡立阁，史致丽．营养盐对中肋骨条藻和新月菱形藻生长及氮磷组成的影响．海洋与湖沼，2000，3 1：46-52．
11．李瑞香，朱明远，王宗灵．东海两种赤潮生物种间竞争的围隔实验．应用生态学报，2003，7：1049-1054．
12．林以安．黄、东海营养盐来源、分布和更新．见唐启升，苏纪兰等著《中国海洋生态系统动力学研究1关键科学问题与研究发展战略》北京：科学出版社，2000，1 17-122．
13．刘静雯，焦念志，蔡慧农．海洋浮游植物的细胞周期与细胞信号转导．自然科学进展，2006．16：385-392．
14．刘仁沿，马德毅，梁玉波．赤潮甲藻毒素麻痹性贝毒的生物合成研究进展．海洋环境科学，2004，23：71-75．
15．龙华，周燕，余骏，胡益峰，傅国君．2001～2007年浙江海域赤潮分析．海洋环境科学，2008，27增刊．
16．陆斗定，齐雨藻，Jeaneae Goebel，邹景忠，高亚辉．东海原甲藻修订及与相关原甲藻的分类学比较．应用生态学报，2003，14：1060-1064．
17．陆赛英，葛人峰，刘丽慧．东海陆架水域营养盐的季节变化和物理输运的规律．海洋学报(中文版)，1996，18：41-51．
18．吕颂辉，张玉宇，陈菊芳．东海具齿原甲藻的扫描电子显微结构．应用生态学报，2003，14：1070-1072．
19．吕颂辉，欧美珊．不同N源及N／P对东海原甲藻生长的影响．海洋环境科学，2006，25： 33-36．
20．欧美珊，吕颂辉．不同无机氮源对东海原甲藻生长的影响．生态科学，2006，25：28-31．
21．徐宁，吕颂辉，陈菊芳．温度和盐度对锥状斯氏藻生长的影响．海洋环境科学，2004，3：123-125．
22．王保栋．黄海和东海营养盐分布及其对浮游植物的限制．应用生态学报，2003，14：1122-1126．
23．王长友，王修林，孙百晔，苏荣国．Cu、Pb、Zn和Cd对东海原甲藻的生态毒性效应．中国环境科学，2008，28：264-268．
24．王金辉．东海赤潮生物具齿原甲藻及其特征．浙江海洋学院学报，2003，22：129-131．
25．王金辉，黄水清．具齿原甲藻的生态特征及赤潮成因浅析．应用生态学报，2003，14：1065-1069．
26．王宗灵，李瑞香，朱明远，陈炳章，郝彦菊．半连续培养下东海原甲藻和中肋骨条藻种群生长过程与种间竞争研究．海洋科学进展，2006，24：495-503．
27．卫玮，肖雯．藻毒素的类型、危害和防治．生物学通报，2004，39：21-23．
28．张秀芳．东海原甲藻基因表达特征研究[博士论文]．，中国海洋大学，2006．
29．张秀芳，杨官品，刘永建，李瑞香．东海原甲藻eDNA文库构建及尝试性EST分析．中国海洋大学学报，2006，36：36 1-364．
30．赵水东．温度、盐度和营养盐对东海原甲藻生长的影响[硕士论文]．，暨南大学，2005．
31．朱明，张学成，茅云翔，阎斌伦，滕亚娟，陆正和．温度、盐度及光照强度对海链藻(T'halassiosira sp．)生长的影响．海洋科学，2003，27：58-61．
32．朱明远，叶属峰，李瑞香，何建宗，吕宋辉，俞子修等．2002年东海区的赤潮发生[A]．何建宗，吕宋辉，俞子修等．南中国海红潮预防和管理的前沿发展[C]．香港：南中国海赤潮学会，2003，35-40．
33．邹景忠．海洋环境科学．济南：山东教育出版社，2004，273-279．
34．邹景忠，李福东，黄长江．中国沿海赤潮：赤潮生物的生理生态特征一赤潮生物的营养生理．齐雨藻等著，北京：科学出版社，2003，75-86．
35.Ahrens,C.H.,Russell,R.L.,Funk,C.J.,Evans,J.T.,Harwood,S.H.,and Rohrrnann,G.F.The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome.Virology,1997,229:381-399.
36.Almendral,J.M.,Huebsch,D.,Blundell,P.A.,Macdonald-Bravo,H.,and Bravo,R.Cloning and sequence of the human nuclear protein cyclin:homology with DNA-binding proteins.Proc.Natl.Acad.Sci.U.S.A.,1987,84:1575-1579.
37.Altschul,S.F.,Madden,T.L.,Schaffer,A.A.,Zhang,J.,Zhang,Z.,Miller,W.,and Lipman,D.J.Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Res,1997,25:3389-3402.
38.Arun,K.D.,Michelle,M.R.,and Kurt,R.K.Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry. J Virol Methods, 2002, 104: 69-82
39. Avise, J.C. Mitochondrial DNA and the evolutionary genetics of higher animals. Philos Trans R Soc Lond B Biol Sci, 1986, 312: 325-342.
40. Ball, K.L., and Lane, D.P. Human and plant proliferating-cell nuclear antigen have a highly conserved binding site for the p53-inducible gene product p21WAFl. Eur J Biochem, 1996, 237:854-861.
41. Bauer, GA., and Burgers, P.M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res, 1990, 18: 261-265.
42. Bravo, R., Frank, R., Blundell, P.A., and Macdonald-Bravo, H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature, 1987, 326: 515-517.
43. Bult, C.J., White, O., Olsen, GJ., Zhou, L., Fleischmann, R.D., Sutton, G.G, Blake, J.A., FitzGerald, L.M., Clayton, R.A., Gocayne, J.D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science, 1996, 273: 1058-1073.
44. Burgers, P.M. Saccharomyces cerevisiae replication factor C. Ⅱ. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem, 1991,266: 22698-22706.
45. Burton, R.S. Molecular tools in marine ecology. J Exp Mar Bio Eco, 1996, 200: 85-101.
46. Carpenter, E.J., Lin, S., and Chang, J., eds. Phytoplankton growth studies by cell cycle analysis. Molecular Approaches to the Study of the Ocean, ed. K.E.E. Cooksey. Chapman and Hall: London, 1998,227-245
47. Cayrol, C, Knibiehler, M., and Ducommun, B. p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene, 1998, 16: 311-320.
48. Celis, J.E., and Celis, A. Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc.Natl.Acad.Sci.U.S.A., 1985, 82: 3262-3266.
49. Celis, J.E., and Madsen, P. Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett, 1986,209:277-283.
50. Chang, J., and Carpenter, E.J. Active growth of the oceanic dinoflagellate Ceratium teres in the Caribbean and Sargasso Seas estimated by cell cycle analysis. J. Phycol., 1994, 30: 375-381.
51. Chang, J., and Dam, H.G. The influence of grazing on the estimation of phytoplankton growth rate via cell cycle analysis: modeling and experimental evidence. Limnol & Oceanogr, 1993,38:202-212.
52. Cheng, L.C., Hwang, S.P., and Chang, J. Gene sequence and expression of an analog of proliferating cell nuclear antigen (PCNA) in the alga Tetraselmis chui and detection of the encoded protein with anti-rat PCNA monoclonal antibody. Appl Environ Microbiol, 1997, 63: 4010-4014.
53. Chisholm, S.W. Temporal patterns of cell division in unicellular algae[A].In: Platt T. eds. Canadian Bulletin of Fisheries and Aquatic Sciences, Physiological Bases of Phytoplankton Ecology, Bacterial growth and division[C] Academic Press, Inc, San Diego, 1991, 210-215.
54. Chuang, L.S., Ian, H.I.,Koh, T.W., Ng, H.H., Xu, G, and Li, B.F. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21 WAF1. Science, 1997,277: 1996-2000.
55. Costas, E. and Rodas, V.L. Identification of marine dinoflagellates using fluorescent lectins. J. Phycol., 1994,30:987-990.
56. Dumonceaux, T.J., Hill, J.E., Briggs, S.A., Amoako, K.K., Hemmingsen, S.M., and Van Kessel, A.G. Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target. J Microbiol Methods, 2006, 64: 46-62.
57. Felsenstein, J.. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 1985, 39:783-791
58. Fuchs, B.M., Wallner, G, Beisker, W., Schwippl, I., Ludwig, W., and Amann, R. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol, 1998, 64: 4973-4982.
59. Furnas, M.J. In situ growth rates of marine phytoplankton: approaches to measurement, community and species growth rates. J Plankton Res, 1990, 12: 1117-1151.
60. Gibbs, E., Kelman, Z., Gulbis, J.M., O'Donnell, M., Kuriyan, J., Burgers, P.M., and Hurwitz, J. The influence of the proliferating cell nuclear antigen-interacting domain of p21(CIP1) on DNA synthesis catalyzed by the human and Saccharomyces cerevisiae polymerase delta holoenzymes. J Biol Chem, 1997,272: 2373-2381.
61. Gisselson, L.A., Graneli, E., Carlsson, P. Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate Dinophysis acuminata: drawbacks of the DNA quantification method. Mar Ecol Progr, 1999, 184: 55-62.
62. Gisselson, L.A., Carlsson, P., Graneli, E. Dinophysis blooms in the deep euphotic zone of the Baltic Sea: do they grow in the dark? Harmful Algae, 2002, 1: 401-418.
63. Gray, M.W. Origin and evolution of mitochondrial DNA. Annu Rev Cell Biol, 1989, 5: 25-50.
64. Guerini, M.N., Que, X., Reed, S.L., and White, M.W. Two genes encoding unique proliferating-cell-nuclear-antigens are expressed in Toxoplasma gondii. Mol Biochem Parasitol, 2000, 109: 121-131.
65. Guillard, R.R., and Ryther, J.H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can. J. Phys., 1962, 8: 229-239.
66. Higashi, Y., and Seki, H. Ecological adaptation and acclimatization of natural freshwater phytoplankters with a nutrient gradient. Environ Pollut, 2000, 109: 311 -320.
67. Hodgkiss, 1. J., Ho, K. C. Are changes in N: P ratios incoastal waters the key to increased red tide blooms? Hydrobiologia, 1997,352: 141-147.
68. Hodgkiss, I.J., and Yang, Z.B. New and dominant species from Sam Xing wan sai Kung during the 1998b massive fish killing red tide in Hong Kong. Harmful Algal Blooms 62-65 Paris: Intergovermental Oceanographic Commission of UNESCO, 2000
69. Horiguchi, T., et al. Prorocentrum dentatum. In: Fukuyo, Y(Ed), Red Tide Organisms in Japan The Japan Fisheries Resource Conservation Association, Tokyo, Japan, 1990, 26-27.
70. Howell, N., and Gilbert, K. Mutational analysis of the mouse mitochondrial cytochrome b gene. J Mol Biol, 1988,203: 607-618.
71. Irwin, D.M., Kocher, T.D., and Wilson, A.C. Evolution of the cytochrome b gene of mammals. J Mol Evol, 1991, 32: 128-144.
72. Jackson, C.J., Norman, J.E., Schnare, M.N., Gray, M.W., Keeling, P.J., and Waller, R.F. Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria. BMC Biol, 2007, 5: 41-51.
73. Kim, M.C., Yoshinaga, I., and Imai, I. A close relationship between algicidal bacteria and termination of Heterosigma akashiwo (Raphidophyceae) blooms in Hiroshima Bay. Mar Ecol Progr, 1998, 170:25-32.
74. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., and Wilson, A.C. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc.Natl.Acad.Sci.U.S.A., 1989, 86: 6196-6200.
75. Kodama, H., Ito, M., Ohnishi, N., Suzuka, I., and Komamine, A. Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells. Eur J Biochem, 1991, 197: 495-503.
76. Kontogianni, E., Koukoura, E., and Christopoulou, E. Squamous cell carcinoma arising in mature cystic teratoma of the ovary: a case report. Eur J Gynaecol Oncol, 2001, 22: 238-239.
77. Krishna, T.S., Kong, X.P., Gary, S., Burgers, P.M., and Kuriyan, J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell, 1994, 79: 1233-1243.
78. Lee, C.S., Redshaw, A., and Boag, G Assessment of cell proliferation in human laryngeal cancers using proliferating cell nuclear antigen and Ki-67 antigen immunostaining. Cell Vision, 1995, 2: 296-300.
79. Leveson, A.C, and Wong, J.T. PCNA-like proteins in dinoflagellates. J. Phycol., 1999, 35: 798-805.
80. Li, A.H., Forestier, E., Rosenquist, R., and Roos, G. Minimal residual disease quantification in childhood acute lymphoblastic leukemia by real-time polymerase chain reaction using the SYBR green dye. Exp Hematol, 2002, 30: 1170-1177.
81. Li, R., Waga, S., Hannon, G.J., Beach, D., and Stillman, B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature, 1994, 371: 534-537.
82. Lin, S., and Carpenter, E.J. Identification and preliminary characterization of PCNA gene in the marine phytoplankton Dunaliella tertiolecta and Isochrysis galbana. Mol Mar Biol Biotech, 1998,7:62-71.
83. Lin, S., Chang, J., and Carpenter, E.J. Detection of proliferating cell nuclear antigen analog in four species of marine phytoplankton. J. Phycol., 1994, 30: 449-456.
84. Lin, S., Chang, J., and Carpenter, E.J. Growth characteristics of phytoplankton determined by cell cycle proteins: PCNA immunostaining of Dunaliella tertiolecta (Chlorophyceae). J. Phycol., 1995,31:388-395.
85. Lin, S., and Corstjens, PL.A.M. Molecular cloning and expression of the proliferating cell nuclear antigen gene from the coccolithorid Pleurochrysis carterae (Haptophyceae). J. Phycol., 2002, 38: 164-173.
86. Lin, S., and Zhang, H. Mitogen-activated protein kinase in Pfiesteria piscicida and its growth rate-related expression. Appl Environ Microbiol, 2003, 69: 343-349.
87. Lin, S., zhang, H., and Jiao, N. Potential utility of mitochondrial cytochrome b and its mRNA editing resolving closely related Dinoflagellates: a case study of prorocentrum (Dinoflagellate). J. Phycol., 2006,42: 646-654.
88. Liu, J., Jiao, N., Hong, H., Luo, T., and Cai, H. Proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in the marine dinoflagellate Prorocentrum donghaiense Lu and the green alga Dunaliella salina Teodoresco. J. Appl. Phycol, 2005, 17: 323-330.
89. Loor, G., Zhang, S.J., Zhang, P., Toomey, N.L., and Lee, M.Y. Identification of DNA replication and cell cycle proteins that interact with PCNA. Nucleic Acids Res, 1997, 25: 5041-5046.
90. Lu, D., Goebel, J., and Qi, Y. Prorocentrum donghaiense: a high-biomass bloom-forming species in the East China Sea. IOC Newslett Toxic Algae Algal Blooms, 2002, 23: 1-5.
91. Lu, D., and Goebel, J. Five red tide species in genus Prorocentrum including the description of Prorocentrum donghaiense Lu SP. nov. from the East China Sea. Chin. J. Oceanol. Limnol, 2001,19:337-344,.
92. Lu, D., Goebel, J., Qi, Y., Zou, J., Han, X., Gao, Y, and Li.Y. Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species. Harmful Algae, 2005,4: 493-505.
93. Maga, G., and Hubscher, U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci, 2003,116: 3051-3060.
94. Markley, N.A., Bonham-Smith, P.C., and Moloney, M.M. Molecular cloning and expression of a cDNA encoding the proliferating cell nuclear antigen from Brassica napus (oilseed rape). Genome, 1992, 36: 459-466.
95. Markley, N.A., Young, D., Laquel, P., Castroviejo, M., and Moloney, M.M. Molecular genetic and biochemical analysis of Brassica napus proliferating cell nuclear antigen function. Plant Mol Biol, 1997,34:693-700.
96. Mathews, M.B., Bernstein, R.M., Franza, B.R., Jr., and Garrels, J.I. Identity of the proliferating cell nuclear antigen and cyclin. Nature, 1984, 309: 374-376.
97. Matsumoto, K., Moriuchi, T., Koji, T., and Nakane, P.K. Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin. EMBO J, 1987, 6: 637-642.
98. Matsumoto, Y., Kim, K., and Bogenhagen, D.F. Proliferating cell nuclear antigen dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol Cell Biol, 1994,14: 6187-6197.
99. McDuff, R.E., and Chisholm, S.W. The calculation of in situ growth rates of phytoplankton populations from fractions of cells undergoing mitosis: a clarification. Limnol & Oceanogr, 1982,27:783-788.
100. Moarefi, I., Jeruzalmi, D., Turner, J., O'Donnell, M., and Kuriyan, J. Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage. J Mol Biol, 2000, 296: 1215-1223.
101. Morris, GF., and Mathews, M.B. Regulation of proliferating cell nuclear antigen during the cell cycle. J Biol Chem, 1989, 264: 13856-13864.
102. Mossi, R., Jonsson, Z.O., Allen, B.L., Hardin, S.H., and Hubscher, U. Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen. J Biol Chem, 1997, 272: 1769-1776.
103. Mudie, P.J., Rochon, A., and Levac, E. Palynological records of red tide-producing species in Canada: past trends and implications for the future. Palaeogeography Palaeoclimatology Palaeoecology, 2002, 180: 159-186.
104. Nicholas, K.B., Nicholas, H.B.J., and Deerfield, D.W.I. GeneDoc: Analysis and visualization of genetic variation. EMBNET news, 1997,4:1-4.
105. O'Reilly, D.R., Crawford, A.M., and Miller, L.K. Viral proliferating cell nuclear antigen. Nature, 1989, 337: 606.
106. Paunesku, T, Mittal, S., Protic, M., Oryhon, J., Korolev, S.V., Joachimiak, A., and Woloschak, G.E. Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int J Radiat Bio, 2001,177: 1007-1021.
107. Prelich, G, Kostura, M., Marshak, D.R., Mathews, M.B., and Stillman, B. The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro. Nature, 1987, 326: 471-475.
108. Wang, R., Xiao, H., Wang, Y., Zhou, W., and Tang, X. Effects of three macroalgae, Ulva linza (Chlorophyta), Corallina pilulifera (Rhodophyta) and Sargassum thunbergii (Phaeophyta) on the growth of the red tide microalga Prorocentrum donghaiense under laboratory conditions. J Sea Res, 2007, 58:189-197.
109. Redfield, A.C. The biological control of Chemical factors in the enviroment. Am Sci, 1958, 46:561-600.
110. Saccone, C, Gissi, C, Lanave, C, Larizza, A., Pesole, G, and Reyes, A. Evolution of the mitochondrial genetic system: an overview. Gene, 2000,261: 153-159.
111. Saitou, N., and Nei, M. The neighbour-joining method: A new method for reconstructing phylogenetic trees. Mol Biochem Parasitol, 1987, 4: 406-425
112. Sasaki, K., Kurose, A., Ishida, Y., and Matsuta, M. Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA. J Histochem Cytochem, 1994, 42: 957-960.
113. Schmittgen, T.D., and Zakrajsek, B.A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods, 2000,46:69-81.
114. Schoepp, B., Breton, J., Parot, P., and Vermeglio, A. Relative orientation of the hemes of the cytochrome bc(1) complexes from Rhodobacter sphaeroides, Rhodospirillum rubrum, and beef heart mitochondria. A linear dichroism study. J Biol Chem, 2000,275: 5284-5290.
115. Simon, C, Frati, F., and Becckenbach, A. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am, 1994, 87: 651-701.
116. Swift, E., Stuart, M., and Meunier, V. The in situ growth rates of some deep living oceanic dinoflagellates Pyrocystis fusiformia Pand Pyrocystis noctiluca. Limnol & Oceanogr, 1972, 21:418-426.
117. Takahashi, H., Strutton, GM., and Parsons, P.G. Determination of proliferating fractions in malignant melanomas by anti-PCNA/cyclin monoclonal antibody. Histopathology, 1991, 18: 221-227.
118. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 1997,24: 4876-4882.
119. Trama, J.P., Mordechai, E., and Adelson, M.E. Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. Mol Cell Probes, 2005,19: 145-152.
120. Van Bleijswijk, J.D.L. and Veldhuis, M.J.W. In situ growth rates of Emiliania huxleyi in enclosures with different phosphate loadings revealed by diel changes in DNA content. Mar Ecol Prog Ser, 1995, 121: 271-277.
121. Vandesompele, J., Preter, K.D., Pattyn, F., Poppe, B., and Roy, N., De Paepe, A., and Speleman, F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002, 3: RESEARCH0034.
122. Vaulot, D., Marie, D., Olson, R.J., and Chisholm, S.W. Growth of Prochlorococus, a photosynthetic prokaryote, in the equatorial Pacific Ocean. Science, 1995, 268: 1480-1482.
123. Waga, S., Hannon, GJ., Beach, D., and Stillman, B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature, 1994, 369: 574-578.
124. Warbrick, E., Lane, D.P., Glover, D.M., and Cox, L.S. A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21 WAF1 and proliferating cell nuclear antigen. Curr Biol, 1995, 5: 275-282.
125. Wei, S.F., Hwang, S.-P.L., and Chang, J.. Influence of ultraviolet radiation on the expression of proliferating cell nuclear antigen and DNA polymerase alpha in Skeletonema costatum (Bacilariophyceae). J. Phycol., 2004,40: 655-663.
126. Weltzien, F.A., Pasqualini, C, Vernier, P., and Dufour, S. A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. Gen Comp Endocrinol, 2005, 142: 134-142.
127. Williams, J. T. Practical Considerations Relevant to Effective Evaluation. In Brown, A.H.D., Frankel, O.H., Marshall, D.R. and Williams, J.T. eds. The Use of Plant Genetic Resources Cambridge: Cambridge University Press, 1989.
128. Xiong, Y., Zhang, H., and Beach, D. D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA. Cell, 1992, 71: 505-514.
129. Wang, Y., Yu, Z., Song, X., Tang, X., and Zhang, S. Effects of macroalgae Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of four species of bloom-forming dinoflagellates. Aquatic Botany, 2007, 86: 139-147.
130. Wang, Y, Yu, Z., Song, X., and Zhang, S. Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures. J Sea Res., 2006, 56: 17-26.
131. Yoo, K., and Lee, J.B. Taxonomical studies on dinoflagellates in Masan Bay 1 .Genus Prorocentrumg Ehrenberg. J Oceanol Soc Korea, 1986, 21: 46-55.
132. Zardoya, R., and Meyer, A. Phylogenetic performance of mitochondrial protein-coding genes in resolving relationships among vertebrates. Mol Biol Evol, 1996, 13: 933-942.
133. Zhang, H., Debashish, B., and, Lin, S. Phylogeny of Dinoflagellates based on mitochondrial ctyochrome b and nuclear small subunit rDNA sequence comparisons. J. Phycol., 2005, 41: 411-420.
134. Zhang, H., Hou, Y., and Lin, S. Isolation and characterization of proliferating cell nuclear antigen from the dinoflagellate Pfiesteria piscicida. J Eukaryot Microbiol, 2006, 53: 142-150.
135. Zhang, P., Sun, Y, Hsu, H., Zhang, L., Zhang, Y, and Lee, M.Y. The interdomain connector loop of human PCNA is involved in a direct interaction with human polymerase delta. J Biol Chem, 1998,273:713-719.
136. Zhang, S.J., Zeng, X.R., Zhang, P., Toomey, N.L., Chuang, R.Y., Chang, L.S., and Lee, M.Y. A conserved region in the amino terminus of DNA polymerase delta is involved in proliferating cell nuclear antigen binding. J Biol Chem, 1995, 270: 7988-7992.
137. Zingone, A., and Enevoldsen, H.O. The diversity of harmful algal blooms: a challenge for science and management. Ocean Coast Manage, 2000, 43: 725-748.
2． 陈翰林，吕颂辉，张传松，朱德弟．2004年东海原甲藻赤潮爆发的现场调查和分析．生态科学，2006，2：226-230．
3． 郭皓．中国近海赤潮生物图谱．北京：海洋出版社，2004．
4． 国家海洋局，中国海洋灾害公报，2001-2008．
5． 国家海洋局，中国海洋环境质量公报，2005-2008．
6． 郭沛涌，沈焕庭，张利华．流式细胞术在水体微型生物分子生物学研究中的应用．生命的化学，2001，21：520-522．
7． 何闪英．中肋骨条藻增殖细胞核抗原基因表达量与生长关系的研究[博士论文]．，中国海洋大学，2007．
8． 洪君超，黄秀清，蒋晓山，王佳兰．长江口中肋骨条藻赤潮发生过程环境要素分析—营养盐状况．海洋与湖沼，1994，25：179-184．
9． 李淑冰，李惠珍，许旭萍．贝毒素的研究现状及产生源探究．福建水产，2000，1：70-74．
10．李铁，胡立阁，史致丽．营养盐对中肋骨条藻和新月菱形藻生长及氮磷组成的影响．海洋与湖沼，2000，3 1：46-52．
11．李瑞香，朱明远，王宗灵．东海两种赤潮生物种间竞争的围隔实验．应用生态学报，2003，7：1049-1054．
12．林以安．黄、东海营养盐来源、分布和更新．见唐启升，苏纪兰等著《中国海洋生态系统动力学研究1关键科学问题与研究发展战略》北京：科学出版社，2000，1 17-122．
13．刘静雯，焦念志，蔡慧农．海洋浮游植物的细胞周期与细胞信号转导．自然科学进展，2006．16：385-392．
14．刘仁沿，马德毅，梁玉波．赤潮甲藻毒素麻痹性贝毒的生物合成研究进展．海洋环境科学，2004，23：71-75．
15．龙华，周燕，余骏，胡益峰，傅国君．2001～2007年浙江海域赤潮分析．海洋环境科学，2008，27增刊．
16．陆斗定，齐雨藻，Jeaneae Goebel，邹景忠，高亚辉．东海原甲藻修订及与相关原甲藻的分类学比较．应用生态学报，2003，14：1060-1064．
17．陆赛英，葛人峰，刘丽慧．东海陆架水域营养盐的季节变化和物理输运的规律．海洋学报(中文版)，1996，18：41-51．
18．吕颂辉，张玉宇，陈菊芳．东海具齿原甲藻的扫描电子显微结构．应用生态学报，2003，14：1070-1072．
19．吕颂辉，欧美珊．不同N源及N／P对东海原甲藻生长的影响．海洋环境科学，2006，25： 33-36．
20．欧美珊，吕颂辉．不同无机氮源对东海原甲藻生长的影响．生态科学，2006，25：28-31．
21．徐宁，吕颂辉，陈菊芳．温度和盐度对锥状斯氏藻生长的影响．海洋环境科学，2004，3：123-125．
22．王保栋．黄海和东海营养盐分布及其对浮游植物的限制．应用生态学报，2003，14：1122-1126．
23．王长友，王修林，孙百晔，苏荣国．Cu、Pb、Zn和Cd对东海原甲藻的生态毒性效应．中国环境科学，2008，28：264-268．
24．王金辉．东海赤潮生物具齿原甲藻及其特征．浙江海洋学院学报，2003，22：129-131．
25．王金辉，黄水清．具齿原甲藻的生态特征及赤潮成因浅析．应用生态学报，2003，14：1065-1069．
26．王宗灵，李瑞香，朱明远，陈炳章，郝彦菊．半连续培养下东海原甲藻和中肋骨条藻种群生长过程与种间竞争研究．海洋科学进展，2006，24：495-503．
27．卫玮，肖雯．藻毒素的类型、危害和防治．生物学通报，2004，39：21-23．
28．张秀芳．东海原甲藻基因表达特征研究[博士论文]．，中国海洋大学，2006．
29．张秀芳，杨官品，刘永建，李瑞香．东海原甲藻eDNA文库构建及尝试性EST分析．中国海洋大学学报，2006，36：36 1-364．
30．赵水东．温度、盐度和营养盐对东海原甲藻生长的影响[硕士论文]．，暨南大学，2005．
31．朱明，张学成，茅云翔，阎斌伦，滕亚娟，陆正和．温度、盐度及光照强度对海链藻(T'halassiosira sp．)生长的影响．海洋科学，2003，27：58-61．
32．朱明远，叶属峰，李瑞香，何建宗，吕宋辉，俞子修等．2002年东海区的赤潮发生[A]．何建宗，吕宋辉，俞子修等．南中国海红潮预防和管理的前沿发展[C]．香港：南中国海赤潮学会，2003，35-40．
33．邹景忠．海洋环境科学．济南：山东教育出版社，2004，273-279．
34．邹景忠，李福东，黄长江．中国沿海赤潮：赤潮生物的生理生态特征一赤潮生物的营养生理．齐雨藻等著，北京：科学出版社，2003，75-86．
35.Ahrens,C.H.,Russell,R.L.,Funk,C.J.,Evans,J.T.,Harwood,S.H.,and Rohrrnann,G.F.The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome.Virology,1997,229:381-399.
36.Almendral,J.M.,Huebsch,D.,Blundell,P.A.,Macdonald-Bravo,H.,and Bravo,R.Cloning and sequence of the human nuclear protein cyclin:homology with DNA-binding proteins.Proc.Natl.Acad.Sci.U.S.A.,1987,84:1575-1579.
37.Altschul,S.F.,Madden,T.L.,Schaffer,A.A.,Zhang,J.,Zhang,Z.,Miller,W.,and Lipman,D.J.Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Res,1997,25:3389-3402.
38.Arun,K.D.,Michelle,M.R.,and Kurt,R.K.Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry. J Virol Methods, 2002, 104: 69-82
39. Avise, J.C. Mitochondrial DNA and the evolutionary genetics of higher animals. Philos Trans R Soc Lond B Biol Sci, 1986, 312: 325-342.
40. Ball, K.L., and Lane, D.P. Human and plant proliferating-cell nuclear antigen have a highly conserved binding site for the p53-inducible gene product p21WAFl. Eur J Biochem, 1996, 237:854-861.
41. Bauer, GA., and Burgers, P.M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res, 1990, 18: 261-265.
42. Bravo, R., Frank, R., Blundell, P.A., and Macdonald-Bravo, H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature, 1987, 326: 515-517.
43. Bult, C.J., White, O., Olsen, GJ., Zhou, L., Fleischmann, R.D., Sutton, G.G, Blake, J.A., FitzGerald, L.M., Clayton, R.A., Gocayne, J.D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science, 1996, 273: 1058-1073.
44. Burgers, P.M. Saccharomyces cerevisiae replication factor C. Ⅱ. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem, 1991,266: 22698-22706.
45. Burton, R.S. Molecular tools in marine ecology. J Exp Mar Bio Eco, 1996, 200: 85-101.
46. Carpenter, E.J., Lin, S., and Chang, J., eds. Phytoplankton growth studies by cell cycle analysis. Molecular Approaches to the Study of the Ocean, ed. K.E.E. Cooksey. Chapman and Hall: London, 1998,227-245
47. Cayrol, C, Knibiehler, M., and Ducommun, B. p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene, 1998, 16: 311-320.
48. Celis, J.E., and Celis, A. Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc.Natl.Acad.Sci.U.S.A., 1985, 82: 3262-3266.
49. Celis, J.E., and Madsen, P. Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett, 1986,209:277-283.
50. Chang, J., and Carpenter, E.J. Active growth of the oceanic dinoflagellate Ceratium teres in the Caribbean and Sargasso Seas estimated by cell cycle analysis. J. Phycol., 1994, 30: 375-381.
51. Chang, J., and Dam, H.G. The influence of grazing on the estimation of phytoplankton growth rate via cell cycle analysis: modeling and experimental evidence. Limnol & Oceanogr, 1993,38:202-212.
52. Cheng, L.C., Hwang, S.P., and Chang, J. Gene sequence and expression of an analog of proliferating cell nuclear antigen (PCNA) in the alga Tetraselmis chui and detection of the encoded protein with anti-rat PCNA monoclonal antibody. Appl Environ Microbiol, 1997, 63: 4010-4014.
53. Chisholm, S.W. Temporal patterns of cell division in unicellular algae[A].In: Platt T. eds. Canadian Bulletin of Fisheries and Aquatic Sciences, Physiological Bases of Phytoplankton Ecology, Bacterial growth and division[C] Academic Press, Inc, San Diego, 1991, 210-215.
54. Chuang, L.S., Ian, H.I.,Koh, T.W., Ng, H.H., Xu, G, and Li, B.F. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21 WAF1. Science, 1997,277: 1996-2000.
55. Costas, E. and Rodas, V.L. Identification of marine dinoflagellates using fluorescent lectins. J. Phycol., 1994,30:987-990.
56. Dumonceaux, T.J., Hill, J.E., Briggs, S.A., Amoako, K.K., Hemmingsen, S.M., and Van Kessel, A.G. Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target. J Microbiol Methods, 2006, 64: 46-62.
57. Felsenstein, J.. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 1985, 39:783-791
58. Fuchs, B.M., Wallner, G, Beisker, W., Schwippl, I., Ludwig, W., and Amann, R. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol, 1998, 64: 4973-4982.
59. Furnas, M.J. In situ growth rates of marine phytoplankton: approaches to measurement, community and species growth rates. J Plankton Res, 1990, 12: 1117-1151.
60. Gibbs, E., Kelman, Z., Gulbis, J.M., O'Donnell, M., Kuriyan, J., Burgers, P.M., and Hurwitz, J. The influence of the proliferating cell nuclear antigen-interacting domain of p21(CIP1) on DNA synthesis catalyzed by the human and Saccharomyces cerevisiae polymerase delta holoenzymes. J Biol Chem, 1997,272: 2373-2381.
61. Gisselson, L.A., Graneli, E., Carlsson, P. Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate Dinophysis acuminata: drawbacks of the DNA quantification method. Mar Ecol Progr, 1999, 184: 55-62.
62. Gisselson, L.A., Carlsson, P., Graneli, E. Dinophysis blooms in the deep euphotic zone of the Baltic Sea: do they grow in the dark? Harmful Algae, 2002, 1: 401-418.
63. Gray, M.W. Origin and evolution of mitochondrial DNA. Annu Rev Cell Biol, 1989, 5: 25-50.
64. Guerini, M.N., Que, X., Reed, S.L., and White, M.W. Two genes encoding unique proliferating-cell-nuclear-antigens are expressed in Toxoplasma gondii. Mol Biochem Parasitol, 2000, 109: 121-131.
65. Guillard, R.R., and Ryther, J.H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can. J. Phys., 1962, 8: 229-239.
66. Higashi, Y., and Seki, H. Ecological adaptation and acclimatization of natural freshwater phytoplankters with a nutrient gradient. Environ Pollut, 2000, 109: 311 -320.
67. Hodgkiss, 1. J., Ho, K. C. Are changes in N: P ratios incoastal waters the key to increased red tide blooms? Hydrobiologia, 1997,352: 141-147.
68. Hodgkiss, I.J., and Yang, Z.B. New and dominant species from Sam Xing wan sai Kung during the 1998b massive fish killing red tide in Hong Kong. Harmful Algal Blooms 62-65 Paris: Intergovermental Oceanographic Commission of UNESCO, 2000
69. Horiguchi, T., et al. Prorocentrum dentatum. In: Fukuyo, Y(Ed), Red Tide Organisms in Japan The Japan Fisheries Resource Conservation Association, Tokyo, Japan, 1990, 26-27.
70. Howell, N., and Gilbert, K. Mutational analysis of the mouse mitochondrial cytochrome b gene. J Mol Biol, 1988,203: 607-618.
71. Irwin, D.M., Kocher, T.D., and Wilson, A.C. Evolution of the cytochrome b gene of mammals. J Mol Evol, 1991, 32: 128-144.
72. Jackson, C.J., Norman, J.E., Schnare, M.N., Gray, M.W., Keeling, P.J., and Waller, R.F. Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria. BMC Biol, 2007, 5: 41-51.
73. Kim, M.C., Yoshinaga, I., and Imai, I. A close relationship between algicidal bacteria and termination of Heterosigma akashiwo (Raphidophyceae) blooms in Hiroshima Bay. Mar Ecol Progr, 1998, 170:25-32.
74. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., and Wilson, A.C. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc.Natl.Acad.Sci.U.S.A., 1989, 86: 6196-6200.
75. Kodama, H., Ito, M., Ohnishi, N., Suzuka, I., and Komamine, A. Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells. Eur J Biochem, 1991, 197: 495-503.
76. Kontogianni, E., Koukoura, E., and Christopoulou, E. Squamous cell carcinoma arising in mature cystic teratoma of the ovary: a case report. Eur J Gynaecol Oncol, 2001, 22: 238-239.
77. Krishna, T.S., Kong, X.P., Gary, S., Burgers, P.M., and Kuriyan, J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell, 1994, 79: 1233-1243.
78. Lee, C.S., Redshaw, A., and Boag, G Assessment of cell proliferation in human laryngeal cancers using proliferating cell nuclear antigen and Ki-67 antigen immunostaining. Cell Vision, 1995, 2: 296-300.
79. Leveson, A.C, and Wong, J.T. PCNA-like proteins in dinoflagellates. J. Phycol., 1999, 35: 798-805.
80. Li, A.H., Forestier, E., Rosenquist, R., and Roos, G. Minimal residual disease quantification in childhood acute lymphoblastic leukemia by real-time polymerase chain reaction using the SYBR green dye. Exp Hematol, 2002, 30: 1170-1177.
81. Li, R., Waga, S., Hannon, G.J., Beach, D., and Stillman, B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature, 1994, 371: 534-537.
82. Lin, S., and Carpenter, E.J. Identification and preliminary characterization of PCNA gene in the marine phytoplankton Dunaliella tertiolecta and Isochrysis galbana. Mol Mar Biol Biotech, 1998,7:62-71.
83. Lin, S., Chang, J., and Carpenter, E.J. Detection of proliferating cell nuclear antigen analog in four species of marine phytoplankton. J. Phycol., 1994, 30: 449-456.
84. Lin, S., Chang, J., and Carpenter, E.J. Growth characteristics of phytoplankton determined by cell cycle proteins: PCNA immunostaining of Dunaliella tertiolecta (Chlorophyceae). J. Phycol., 1995,31:388-395.
85. Lin, S., and Corstjens, PL.A.M. Molecular cloning and expression of the proliferating cell nuclear antigen gene from the coccolithorid Pleurochrysis carterae (Haptophyceae). J. Phycol., 2002, 38: 164-173.
86. Lin, S., and Zhang, H. Mitogen-activated protein kinase in Pfiesteria piscicida and its growth rate-related expression. Appl Environ Microbiol, 2003, 69: 343-349.
87. Lin, S., zhang, H., and Jiao, N. Potential utility of mitochondrial cytochrome b and its mRNA editing resolving closely related Dinoflagellates: a case study of prorocentrum (Dinoflagellate). J. Phycol., 2006,42: 646-654.
88. Liu, J., Jiao, N., Hong, H., Luo, T., and Cai, H. Proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in the marine dinoflagellate Prorocentrum donghaiense Lu and the green alga Dunaliella salina Teodoresco. J. Appl. Phycol, 2005, 17: 323-330.
89. Loor, G., Zhang, S.J., Zhang, P., Toomey, N.L., and Lee, M.Y. Identification of DNA replication and cell cycle proteins that interact with PCNA. Nucleic Acids Res, 1997, 25: 5041-5046.
90. Lu, D., Goebel, J., and Qi, Y. Prorocentrum donghaiense: a high-biomass bloom-forming species in the East China Sea. IOC Newslett Toxic Algae Algal Blooms, 2002, 23: 1-5.
91. Lu, D., and Goebel, J. Five red tide species in genus Prorocentrum including the description of Prorocentrum donghaiense Lu SP. nov. from the East China Sea. Chin. J. Oceanol. Limnol, 2001,19:337-344,.
92. Lu, D., Goebel, J., Qi, Y., Zou, J., Han, X., Gao, Y, and Li.Y. Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species. Harmful Algae, 2005,4: 493-505.
93. Maga, G., and Hubscher, U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci, 2003,116: 3051-3060.
94. Markley, N.A., Bonham-Smith, P.C., and Moloney, M.M. Molecular cloning and expression of a cDNA encoding the proliferating cell nuclear antigen from Brassica napus (oilseed rape). Genome, 1992, 36: 459-466.
95. Markley, N.A., Young, D., Laquel, P., Castroviejo, M., and Moloney, M.M. Molecular genetic and biochemical analysis of Brassica napus proliferating cell nuclear antigen function. Plant Mol Biol, 1997,34:693-700.
96. Mathews, M.B., Bernstein, R.M., Franza, B.R., Jr., and Garrels, J.I. Identity of the proliferating cell nuclear antigen and cyclin. Nature, 1984, 309: 374-376.
97. Matsumoto, K., Moriuchi, T., Koji, T., and Nakane, P.K. Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin. EMBO J, 1987, 6: 637-642.
98. Matsumoto, Y., Kim, K., and Bogenhagen, D.F. Proliferating cell nuclear antigen dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol Cell Biol, 1994,14: 6187-6197.
99. McDuff, R.E., and Chisholm, S.W. The calculation of in situ growth rates of phytoplankton populations from fractions of cells undergoing mitosis: a clarification. Limnol & Oceanogr, 1982,27:783-788.
100. Moarefi, I., Jeruzalmi, D., Turner, J., O'Donnell, M., and Kuriyan, J. Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage. J Mol Biol, 2000, 296: 1215-1223.
101. Morris, GF., and Mathews, M.B. Regulation of proliferating cell nuclear antigen during the cell cycle. J Biol Chem, 1989, 264: 13856-13864.
102. Mossi, R., Jonsson, Z.O., Allen, B.L., Hardin, S.H., and Hubscher, U. Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen. J Biol Chem, 1997, 272: 1769-1776.
103. Mudie, P.J., Rochon, A., and Levac, E. Palynological records of red tide-producing species in Canada: past trends and implications for the future. Palaeogeography Palaeoclimatology Palaeoecology, 2002, 180: 159-186.
104. Nicholas, K.B., Nicholas, H.B.J., and Deerfield, D.W.I. GeneDoc: Analysis and visualization of genetic variation. EMBNET news, 1997,4:1-4.
105. O'Reilly, D.R., Crawford, A.M., and Miller, L.K. Viral proliferating cell nuclear antigen. Nature, 1989, 337: 606.
106. Paunesku, T, Mittal, S., Protic, M., Oryhon, J., Korolev, S.V., Joachimiak, A., and Woloschak, G.E. Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int J Radiat Bio, 2001,177: 1007-1021.
107. Prelich, G, Kostura, M., Marshak, D.R., Mathews, M.B., and Stillman, B. The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro. Nature, 1987, 326: 471-475.
108. Wang, R., Xiao, H., Wang, Y., Zhou, W., and Tang, X. Effects of three macroalgae, Ulva linza (Chlorophyta), Corallina pilulifera (Rhodophyta) and Sargassum thunbergii (Phaeophyta) on the growth of the red tide microalga Prorocentrum donghaiense under laboratory conditions. J Sea Res, 2007, 58:189-197.
109. Redfield, A.C. The biological control of Chemical factors in the enviroment. Am Sci, 1958, 46:561-600.
110. Saccone, C, Gissi, C, Lanave, C, Larizza, A., Pesole, G, and Reyes, A. Evolution of the mitochondrial genetic system: an overview. Gene, 2000,261: 153-159.
111. Saitou, N., and Nei, M. The neighbour-joining method: A new method for reconstructing phylogenetic trees. Mol Biochem Parasitol, 1987, 4: 406-425
112. Sasaki, K., Kurose, A., Ishida, Y., and Matsuta, M. Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA. J Histochem Cytochem, 1994, 42: 957-960.
113. Schmittgen, T.D., and Zakrajsek, B.A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods, 2000,46:69-81.
114. Schoepp, B., Breton, J., Parot, P., and Vermeglio, A. Relative orientation of the hemes of the cytochrome bc(1) complexes from Rhodobacter sphaeroides, Rhodospirillum rubrum, and beef heart mitochondria. A linear dichroism study. J Biol Chem, 2000,275: 5284-5290.
115. Simon, C, Frati, F., and Becckenbach, A. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am, 1994, 87: 651-701.
116. Swift, E., Stuart, M., and Meunier, V. The in situ growth rates of some deep living oceanic dinoflagellates Pyrocystis fusiformia Pand Pyrocystis noctiluca. Limnol & Oceanogr, 1972, 21:418-426.
117. Takahashi, H., Strutton, GM., and Parsons, P.G. Determination of proliferating fractions in malignant melanomas by anti-PCNA/cyclin monoclonal antibody. Histopathology, 1991, 18: 221-227.
118. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 1997,24: 4876-4882.
119. Trama, J.P., Mordechai, E., and Adelson, M.E. Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. Mol Cell Probes, 2005,19: 145-152.
120. Van Bleijswijk, J.D.L. and Veldhuis, M.J.W. In situ growth rates of Emiliania huxleyi in enclosures with different phosphate loadings revealed by diel changes in DNA content. Mar Ecol Prog Ser, 1995, 121: 271-277.
121. Vandesompele, J., Preter, K.D., Pattyn, F., Poppe, B., and Roy, N., De Paepe, A., and Speleman, F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002, 3: RESEARCH0034.
122. Vaulot, D., Marie, D., Olson, R.J., and Chisholm, S.W. Growth of Prochlorococus, a photosynthetic prokaryote, in the equatorial Pacific Ocean. Science, 1995, 268: 1480-1482.
123. Waga, S., Hannon, GJ., Beach, D., and Stillman, B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature, 1994, 369: 574-578.
124. Warbrick, E., Lane, D.P., Glover, D.M., and Cox, L.S. A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21 WAF1 and proliferating cell nuclear antigen. Curr Biol, 1995, 5: 275-282.
125. Wei, S.F., Hwang, S.-P.L., and Chang, J.. Influence of ultraviolet radiation on the expression of proliferating cell nuclear antigen and DNA polymerase alpha in Skeletonema costatum (Bacilariophyceae). J. Phycol., 2004,40: 655-663.
126. Weltzien, F.A., Pasqualini, C, Vernier, P., and Dufour, S. A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. Gen Comp Endocrinol, 2005, 142: 134-142.
127. Williams, J. T. Practical Considerations Relevant to Effective Evaluation. In Brown, A.H.D., Frankel, O.H., Marshall, D.R. and Williams, J.T. eds. The Use of Plant Genetic Resources Cambridge: Cambridge University Press, 1989.
128. Xiong, Y., Zhang, H., and Beach, D. D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA. Cell, 1992, 71: 505-514.
129. Wang, Y., Yu, Z., Song, X., Tang, X., and Zhang, S. Effects of macroalgae Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of four species of bloom-forming dinoflagellates. Aquatic Botany, 2007, 86: 139-147.
130. Wang, Y, Yu, Z., Song, X., and Zhang, S. Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures. J Sea Res., 2006, 56: 17-26.
131. Yoo, K., and Lee, J.B. Taxonomical studies on dinoflagellates in Masan Bay 1 .Genus Prorocentrumg Ehrenberg. J Oceanol Soc Korea, 1986, 21: 46-55.
132. Zardoya, R., and Meyer, A. Phylogenetic performance of mitochondrial protein-coding genes in resolving relationships among vertebrates. Mol Biol Evol, 1996, 13: 933-942.
133. Zhang, H., Debashish, B., and, Lin, S. Phylogeny of Dinoflagellates based on mitochondrial ctyochrome b and nuclear small subunit rDNA sequence comparisons. J. Phycol., 2005, 41: 411-420.
134. Zhang, H., Hou, Y., and Lin, S. Isolation and characterization of proliferating cell nuclear antigen from the dinoflagellate Pfiesteria piscicida. J Eukaryot Microbiol, 2006, 53: 142-150.
135. Zhang, P., Sun, Y, Hsu, H., Zhang, L., Zhang, Y, and Lee, M.Y. The interdomain connector loop of human PCNA is involved in a direct interaction with human polymerase delta. J Biol Chem, 1998,273:713-719.
136. Zhang, S.J., Zeng, X.R., Zhang, P., Toomey, N.L., Chuang, R.Y., Chang, L.S., and Lee, M.Y. A conserved region in the amino terminus of DNA polymerase delta is involved in proliferating cell nuclear antigen binding. J Biol Chem, 1995, 270: 7988-7992.
137. Zingone, A., and Enevoldsen, H.O. The diversity of harmful algal blooms: a challenge for science and management. Ocean Coast Manage, 2000, 43: 725-748.