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Prorocentrum donghaiense Lu and Alexandrium catenella are two key harmful algal bloom(HAB) species widely spread along the coastal waters of China.In the past few years,this two HAB species have formed extensive blooms in the coast of the Southeast China Sea,and become of economic and public concern due to its impact on the marine ecosystem,aquaculture and public health.This study established the total protein preparation method for proteomic study of dinoflagellates,compared the differential protein expressions of P.donghaiense Lu and A.catenella under various growth phases and growth conditions using proteomic approach,identified and characterized physiological indicator proteins related to cell growth phase and environmental stresses.The main results were as followings:1.Established and optimized the total protein preparation method for proteomic study in HAB species.Four protein preparation methods,TritonX-100/urea extraction method,TCA-aceton precipitation method,40mM with 5%glycerol extraction method and 40mMTris extraction method were compared using 2-DE method,the results showed that TritonX-100/Urea method was the best method to prepare total proteins for proteomic studies,the high quality of 2-DE protein profile pattern and maximum protein spots(more than 1,000 spots) were achieved, meanwhile alkaline proteins and higher molecular weight proteins were extracted out using this method.Moreover,this method provides a simple and convenient protocol for preparing proteins for proteomic study in dinoflagellates;2.Comparative studies on protein expression of two Alexandrium species,A. tamarense DH01 and A.catenella DH01 grown in the same culture condition using 2-DE method together with morphology and ITS sequence results showed that,99%of protein profiles in 2-DE of two Alexandrium species were same, indicating that A.tamarense DH01 and A.catenella DH01 should be considered as conspecific.While several distinctive proteins were also found in two species respectively,protein spots AT1-5 were only observed in A.tamarense DH01 and protein spots ACl-3 only in A.catenella DH01,these proteins could be used as potential indicators for species-specific discrimination;3.The results from 2-DE proteins profiles of P.donghaiense Lu and A.catenella collected in various growth phase showed that there were some specific proteins related to growth phase.In P.donghaiense Lu,cdc2-related protein kinase 1 was found to highly express in the exponential phase and decrease in the dissipation phase,while some proteins such as spots 17-19(RubisCo) and 21,23-26,28 and 30 were almost disappeared in the dissipation phase.In A.catenella,the expressions of several proteins,spots 31-35,46-48 and 61-70 were declined significantly in the dissipation phase.These proteins could be applied as the biomarkers to indicate different growth phase of cells;4.Comparison of differential expressions of proteomics of P.donghaiense Lu and A. catenella grown in various nutrient conditions showed that,several proteins,PCl, PC3,PC5 and PC7-9 were not observed in nitrogen or phosphate-starved P. donghaiense Lu while the expression of PC2,a acidific protein was enhanced significantly,these proteins could be used as potential indicators for nitrogen or phosphate-starved conditions.However,some specific protein contents,such as PC4 and PC6 decreased significantly,or did not expressed,such asPP-13 under nitrogen-starved cells,and could be used as indicators of nitrogen-starved condition.In A.catenella,a protein related to nitrogen-starved,AN1 was found while some proteins expressions,ANl-5,AP1 and ACl were enhanced when nitrogen or phosphate added to the nutrient-starved A.catenella.These proteins could be used as potential biomarkers for nutrient un-limitation environment;5.In P.donghaiense Lu,some specific proteins,such as heat shock protein 78(spot 10) and proteins spots 13 and14 were found under continuous light and lower temperature,respectively.In A.catenella,two group proteins,groupl,spots 36-45 and group2,spots 49-51 expression were enhanced significantly in continuous light,while they were not observed in continuous dark and nitrogen-starved conditions,and their expressions were low in normal culture,phosphate-starved and lower temperature conditions.These proteins could be used as potential biomarkers of cells to environmental stress.
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