【作者】 赵丽媛；

【导师】 于志刚；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【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G₁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。更多还原

【Abstract】 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 G₁ 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.更多还原