| Phosphorus is one of the essential nutrients for the growth of marine organisms.Its content and distribution affect the growth of marine organisms and participate in the synthesis and decomposition of nucleic acids,proteins,lipids,etc.,it plays an important role in the storage,replication and transcription of genetic information.The ecosystem composed of zooxanthellae and coral reefs has a high social,economic and cultural status,but now,due to human intervention and natural conditions change,resulting in changes in phosphorus in seawater,this in turn affects the exacerbation of coral bleaching and the separation of zooxanthellae from coral reef systems,resulting in the destruction of the Coral—zooxanthellae ecosystem.By contrast,some zooxanthellae can coexist with corals and the like,but also exist as planktonic organisms,such as the Clade E,which may result in their unique physiological processes.As an important component of the coral reef ecosystem,a great deal of research has been done on the effects of isolated and free—living zooxanthellae on the growth of corals under different phosphorus sources and phosphate concentrations,however,the physiological differences of planktonic algae caused by different phosphate concentrations and the analysis of these differences at molecular level have not been studied.In this paper,the growth and physiological characteristics of free—living zooxanthellae isolated from Zhoushan Sea area of Zhejiang Province were studied by morphological and molecular identification.Subsequently,the growth,physiological characteristics and alkaline phosphatase activity of this microalga under different concerntrations of phosphate were analyzed.Furthernore,the differentially expressed genes under high and low phosphate concentration groups and their functions and pathways were analyzed by RNA—Seq technique.The results are shown as below:1.Light micrographs(LM)and scanning electron micrographs(SEM)were used to identify the species of zooxanthellae,followed by the comfirmation with phylogenetic tree construction based on the large subunit(LSU)sequences.The results showed that this free-living zooxanthellae was one species of Clade E,which was highly supported by the bootstrap valued of 100%.Therefore,it was determined that the separated algae were Symbiodinium sp.Clade E.2.According to the current phosphate concentrations in the East China Sea and the specific cultivation condition in the laboratory,four phosphate concentration groups were set up.Symbiodinium sp.was cultured under four different DIP initial concentrations(0.15,10 20 and 35?M PO43-)for 7 days in short term test,while the algal cells were cultured under a high DIP initial concentration(35?M PO43-)for 55days in long term test.Subsequently,the growth,photosynthetic parameters and alkaline phosphatase activity(APA)of the microalga were measured.The results revealed that there were significant differences in the growth of Symbiodinium sp.under different initial concentrations of phosphate.The cell density of the high initial concentration group was significantly higher compared with the other three groups(P<0.05),indicating that the growth of free-living zooxanthellae was positively correlated with phosphate concentration.Under the high concentration of phosphate(35?mol/L),changes in the content of chlorophyll a(chl a)and in Symbiodinium sp.were consistent with the trend of algal cell density.However,the changes in cell growth and chl a showed a 5-day lag trend.Along with the consumption of phosphate,the specific growth rate(?)and effective photochemical efficiency(Fq'/Fm')of Symbiodinium sp.were decreased,suggesting that a positive correlation existed between phosphorus limitation and photosynthetic efficiency.In contrast,the APA was significantly enhanced(P<0.05)after 20 days of culture at the phosphate initial concentration of 35?M,indicating that the DIP content was negatively correlated with APA,which was also presented when Symbiodinium sp.was cultivated under the low phosphate initial concentration(0.15?M).Alkaline phosphatase catalyzes dissolved organic phosphorus to DIP,which could enable Symbiodinium sp.to keep growing under low DIP concentration..3.To understand how would Symbiodinium sp.,a species of free-living zooxanthellae,respond to the dynamic change of dissolved inorganic phosphorus(DIP),we furthermore carried out comparative analyses upon the transcriptome of this dinofllagete under high phosphate(35?M)and low phosphate(0.15?M)concentrations of phosphate in 0 d,5 d and 10 d.The de novo assembly results showed that231,642,201 Transcripts and 80,955 Unigenes were obtained.A total of 4,407(5.44%)Unigenes were co-annotated in NR,GO,KEGG,eggnog,Swiss Prot and Pfam database.In addition,27,236(33.64%)Unigenes had significant matches in NR database.All Unigenes annotated in NR database were selected for annotation in GO database,suggesting that a total of 23,998(29.64%)unigenes were mapped into three categories,including molecular function,cellular population and biological process.Among the annotation in GO database,there were 13 sub-categories of molecular functions,16sub-categories of cellular components and 19 sub-categories of biological processes.The result of egg NOG functional analysis showed that 45,387(56.06%)Unigenes were divided into 25 categories according to their functional characteristics.The largest category was post-translational modification,protein turnover and chaperone(3,704,8.16%),followed by signal transduction mechanism(3,272,7.21%),translation,ribosomal structure and biological development(2091,4.61%),extracellular structure(168,0.37%),nuclear structure(78,0.17%)and cell movement(24,0.05%).To obtain the differentially expressed genes related to phosphorus utilization,transcriptome data of five experimental groups were compared and analyzed.The result suggested that there were 9 phospholipase genes,2 alkaline phosphatase genes,4 acid phosphatase genes,3 inorganic phosphate transporter genes,2 sodium-dependent phosphate transporter genes and 3 mitochondrial phosphate transporter genes differentially expressed.According to the expression profile of the differentially expressed genes,it was hypothesized that Symbiodinium sp.could keep growing under low phosphorus stress by both enhancing the ability of transporting inorganic phosphate into cell and utilizing organic phosphorus via phosphatase.In conclusion,the results of this study suggested that like other dinoflagellates,the free-living Symbiodinium sp.could adapt to low phosphorus condition by both enhancing the ability of transporting inorganic phosphate into cell and utilizing organic phosphorus via phosphatase.This study may pave the avenue for revealing the molecular regulation mechanism of Symbiodinium sp.in response to different phosphate concentration and helps us better understand the survival mechanism of zooxanthellae in oligotrophic sea area. |
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