| Dinoflagellates are the major causative organisms of marine Harmful Algal Blooms(HABs)and investigating the life history of dinoflagellates is therefore one of the most important research area in order to understand the population dynamics,fundamental biological mechanisms of bloom formation,development,decline,and other ecological aspects of dinoflagellates.For many dinoflagellates,resting cyst is a key stage of their life history due to its vital roles in surviving unfavorable conditions and lengthened dormancy in marine sediments.The molecular processes associated with the formation,dormancy,and germination of resting cysts,however,have virtually not been investigated.Hence,the present research aimed to initiate our understanding about the molecular and biochemical processes during the formation,dormancy,and germination of resting cyst at the gene transcriptional level,and consequently to better understand the mechanisms of recurrences and geographic expansion of dinoflagellate HABs.In order to identify the differentially expressed genes(DEGs)between vegetative cells and resting cysts,particularly those relevant to the molecular and biochemical processes regulating cyst dormancy at different conditions in dinoflagellate,via using the common HABs-forming and cyst production-prone species,Scrippsiella trochoidea(Stein)Loeblich III,as a model organism,the following investigations were conducted:(1)Establishment of a method for massive production of resting cysts.By adding solid particles(sterilized sands)into the culture of S.trochoidea at vegetative growth,both the final yields and production rates of resting cysts in sands-treated groups were significantly higher than that in the control,with the highest mean cyst yield in sandtreated group being 107 folds higher than that of the control.This method effectively made possible the massive production of resting cysts and satisfied the requirement for a great quantity of cysts in the following investigations.Meanwhile,the enhancement effect of sand addition was also significant in Biecheleria brevisulcata,and Levanderina fissa,while,if the culture or species does not produce cysts under normal conditions,or does produce cysts with a low yield,such as Cochlodinium polykrikoides Margalef,Akashiwo sanguinea(Hirasaki)G.Hansen & Moestrup,and Pheopolykrikos hartmannii,the enhancement of sand addition would be minimal or less obvious.(2)Establishment of a cDNA subtractive library via suppression subtractive hybridization(SSH)technology.After converting the total mRNA of both vegetative cells and resting cysts into cDNA,we assigned the resting cysts cDNA that contained specific differentially expressed transcripts as tester,and the cDNA of vegetative cells cDNA as driver.The cDNA subtractive libraries of the two different types of cells in the life history of S.trochoidea were successfully constructed using SSH.In total,1,247 positive clones were selected from 1,314 strains,with the positive rate of 94.9%.The lengths of sequencesgenerally ranged from 250 to 1000 bp.Consequently,1,025 significantly differentially expressed genes were screened based on the results of dot blotting hybridization,with a selection efficiency of 86.2%.(3)Analyses and verification of the obtained cDNA subtraction library.A total of 925 sequences,ranging from 102 to 1,806 bp,were analyzed after removing the primers and those sequences less than 100 bp and 280 contigs or sequences were obtained after assembling,with 74.3% of the contigs or sequences annotated by at least one of the databases NR(NCBI non-redundant protein sequences),KOG(euKaryotic Ortholog Groups),KO(KEGG Ortholog),KEGG(Kyoto Encyclopedia of Genes and Genomes),GO(Gene Ontology)and SWISS-PROT.Except for some sequences that have particular short lengths and thus have insufficient information for annotation,the most likely explanation for failing to annotate 25.7 % of the 280 contigs/sequences may be that they are genes specifically existing in dinoflagellates and having not been characterized yet.Among the contigs/sequences with annotations,187 were annotated to be involved in biological processes,of which 55 were involved in metabolic processes;142 were annotated to be associated with molecular functions.Meanwhile,57 sequences were annotated to be involved in metabolic pathways,accounting for 70% of the KEGG metabolism pathway.Part of these sequences have been verified to be from S.trochoidea by PCR amplication,cloning,and sequencing using the dinoflagellates-specific spliced leader(SL)sequence as the forward primer and particularly designed gene-specific reverse primers.(4)Screening and validating the reference genes for the following experiments investigating gene expressions.The two genes,Cyclophilin(CYC)and Phosphoenolpyruvate carboxykinase(PEPCK)from 15 candidate housekeeping genes were determined to be suitable as reference genes for the gene expression analyses in S.trochoidea by using real-time fluorescence quantitative PCR and three widely used statistical softwares-Ge Norm,Normfinder,and Bestkeeper.(5)Investigations on the expressions of genes associated with energy metabolism in vegetative cells and resting cysts under different conditions.Based on the annotation information in(3),three key genes regulating the energy metabolism and translation process were selected to investigate the transcriptions of these genes in resting cysts in response to varied ambient conditions of dormancy.cDNA cloning and expression analyses of the genes(ATP synthase beta subunit,ATP_synt?,cytochrome c oxidase,CO1;and a translation elongation factor,EF)in vegetative cells and resting cysts under darkness,lowed temperature(4?),hypoxia/anoxia,and different durations of dormancy were performed.The results demonstrated that the expressions of these three genes in resting cysts were generally much lower than that in vegetative cells and,in resting cysts under darkness,lowered temperature,hypoxia/anoxia,and extended duration of dormancy were significantly lower than that in cysts under normal culture-maintaining conditions(light,21?,aerobic,and newly harvested).These results together suggest that resting cysts survive an elongated dormancy in marine sediments via reducing energy metabolism under conditions such as darkness,lowered temperature and hypoxia/anoxia.We believe these results provide a preliminary but solid basis for further understanding the molecular processes and mechanisms of resting cysts dormancy. |
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