| The mensuration of the physiological and ecological parameters such as the insitu growth rate is the basic of understanding the regulation of phytoplanktondynamics, constructing the ecological dynamic models and even forecasting thehappening of red tide. In the past,the research in our lab shows that,the relativeexpression leavel of proliferating cell nuclear antigen(PCNA) gene can be used as agood indicator to the phytoplankton growth rate,moreover, established a formulabetween the relative expression level of PCNA gene and the growth rate μ(d-1) aboutSkeletonema costatum and Prorocentrum donghaiense. It paves a way for monitorand predicting the red tide.In this study,based on the preliminary research results,we pay a attention to usingthis methods to measure the in situ growth rate of the filed samples.We do someresearch on these fields such as the collection methods, the methods of extracting theRNA, improving the specificity of the primers about the reference gene andoptimizing the reaction system of RFQ-PCR.In order to determine the in situ growthrate of the samples from the Prorocentrum donghaiense red tide area in the EastChina Sea,we use the established method that measure the relative expression levelof PCNA gene to get growth rate,The method was validated and applied,enumeratingthe red tide monitor accurately using this methods.The main results are as follows:(1) We research on the efficiency of RNA extraction including the number ofcells,grinding methods,membrane specifications and extraction reagents.To determinethe lowest number of samples is2×106cells.To cocllect the samples by filtering thewater with cellulose acetate membrane which diameter is50mm,pore is10μm.Dealing with the samples,can grind the membrane with liquid nitrogen, or grind the microalgae scraped off with liquid nitrogen.depending on the samples. In theextraction reagent,we choose TRIZOL on extraction firstly.(2) The primers were designed based on Cyt b sequences alignment. Thespecies-specificity of the RFQ-PCR primers for Cyt b amplifying was tested by theother14algal species. The reaction system was optimized when treat the filedsamples. the suitable qualities of RNA for reverse transcription were50ng~200ngRNA in the20μL system of the reverse transcription.10-fold diluting templates oradding the bovine serum albumin (BSA) with a final concentration of0.2μg/μL inthe RFQ-PCR system could bring down the depressant effect of the inhibitors in thefiled samples so as to reduce the interference. The standard curves were constructed todetect the Cyt b gene by SYBR Green I systems, respectively. The equation fordetecting P. donghaiense Cyt b was defined as lg[copies]=-3.44Ct+36.53(3) In the field study, we enumerated the in situ growth rate by the methodmeasuring the relative expression level of PCNA gene and the method of trainingtraditional culture method.It found that,the growth rates were consistent.The both canreflect the in sisu groth rate of the redtide phytoplankton in the water.We measuredthe in situ growth rate of P. donghaiense obtained from the red tide areas in the coastof Changjiang River Estuary and the sea off Zhoushan islands during the2011springby the method that measuring the relative expression level of PCNA gene.It foundthat,the in sisu growth rate of P. donghaiense in many sites were nagtive valuereflecting the red tide had entered the death phase.This study enumerated the P. donghaiense obtained from P. donghaiense redtide area in situ growth rate with the established PCNA relative expression method，while reflecting the growth status and the trend of this red tide very well. It paved away for using molecular biology technology to monitor and forecast the red tide. |
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