The Study Of The Relationship Between The Proliferating Cell Nuclear Antigen Gene Expression Level And The Growth Of Prorocentrum Donghaiense

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 Greenâ… and Taqman probe systems,respectively.ForSYBR Greenâ… system,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.