Study On Molecular Response Mechanism Of Prorocentrum Donghaiense Lu And Karenia Mikimotoi Hansen Grown Into Nitrogen Limitation Or Phosphorus Limitation

In this study, take two dinoflagellates as research objects, that is, Prorocentrum donghaiense Lu and Karenia mikimotoi Hansen, by a combination of a variety of research tools including high-throughput transcriptomics, real time fluorescence quantitative PCR, physiology and biochemistry, the nitrogen and phosphorus metabolic networks and their regulation mechanisms of P. donghaiense under the conditions of nitrogen or phosphorus limitation was revealed, and compared with saturation condition, genes expression of relative abundance and differences of P.donghaiense related to nitrogen and phosphorus metabolism under the conditions of different phosphorus source recovery in phosphorus limitation was also illustrated.And we also clarified the nitrogen and phosphorus metabolic networks and their regulation mechanisms under the condition of nitrogen and phosphorus saturation in K. mikimotoi; Thus there provided a certain amount of molecular theoretical support for studying the change of phosphorus nutrition structures on the impact of interspecific competition of phytoplankton and developing the research on the characteristics using different phosphorus source of red tide algae.RNA-seq Technology was used to detect the differences of gene expression in different conditions in P. donghaiense, obtained the transcript gene pool of K.mikimotoi under the saturation condition and build the putative model of nitrogen and phosphorus transport and assimilation in K. mikimotoi. Differential gene expressions were verified by real-time fluorescence quantitative PCR and analyzed genes expression of relative abundance and differences of P. donghaiense related to nitrogen and phosphorus metabolism under the conditions of different phosphorus source recovery in phosphorus limitation. Finally, combined with cell density counting,photosynthetic efficiency and phosphorus nutrient measuration, bioinformatics analysis was introduced to build the putative model of nitrogen and phosphorus transport and assimilation in P.donghaiense.12 h after nitrogen limitation in P. donghaiense, RNA- seq found differentially expressed genes, including 34 up-regulated genes and 31 down-regulated genes; 12 h after Phosphorus limitation in P. donghaiense, also found differentially expressed genes, including 224 up-regulated genes and 507 down-regulated genes.Real-time fluorescence quantitative PCR confirmed the reliability of the differential expression by high flux transcriptome study. The results showed that the expression of 896 geneswas elevated or inhibited by after P deprivation. Specifically, genes involved in RNA transport, oxidative phosphorylation, photosynthesis, endocytosis, pyrimidine metabolism, glycolysis/gluconeogenesis, biosynthesis of amino acids, vitamin digestion and absorption, protein processing in endoplasmic reticulum were downregulated, while the expression of genes involved in ribosomal protein metabolism were significantly raised; There are also 896 genes expression elevated or inhibited by after N deprivation, genes involved in RNA transport, oxidative phosphorylation,photosynthesis, endocytosis, pyrimidine metabolism, biosynthesis of amino acids,nicotinate and nicotinamide metabolism, vitamin digestion and absorption, protein processing in endoplasmic reticulum were down-regulated, while genes expression of ribosomal protein metabolism were significantly up-regulated. Combined with gene annotation from high-throughput transcriptomics and KEGG analysis,we constructed the putative model of nitrogen and phosphorus transport and assimilation in P.donghaiense and K. mikimotoi, also presented the metabolic networks and their regulation mechanisms in detail.the researcher found that in addition to the existence of several common gene on the cell membrane of P. donghaiense and K. mikimotoi, there also have transport protein genes which can directly absorpt macromolecular organic matter, namely Polyamine transporter(PUT) and Oligopeptide transporter(PepT); In addition, there also exist Nucleoside transporter(ENT) on the cell membrane of P. donghaiense, and K. mikimotoi has four types of urease assitant protein UreD/F/G/H. These all make P.donghaiense and K. mikimotoi have obvious advantages over other phytoplankton species in the use of soluble organic nitrogen, interspecific competition and community succession, which may also be one of the reasons for frequently outbreaking the red tide disasters of P. donghaiense and K. mikimotoi in domestic waters. Furthermore, there not only exsit the common inorganic phosphate transporter genes on the cell membrane of P. donghaiense and K. mikimotoi, but also exsit inorganic diphosphatase/hydrogen-translocating pyrophosphatase, ATP-binding casse-tte transporter, and organic phosphonate transmembrane-transporting ATPase genes.Interestingly, P.donghaiense and K. mikimotoi also can directly absorb macromolecu-lar dissolved organic phosphorus phospholipids by Phospholipid transporter. In addit-ion, there also exsit triose-phosphate transporter genes on cell membrane of P. dong-haiense, which can directly absorpt small molecules dissolved organic phosphorus.This two algae contain alkaline phosphatase, just alkaline phosphatase gene of P.don-ghaiense is located on the cell membrane, and alkaline phosphatase gene of K. miki-motoi are located in the extracellular matrix. And two algae are mixotrophic dinoflag-ellates.Studies have shown that dissolved organic phosphorus, which has similar structures to small molecule organic phosphorus(pyrophosphate, ATP) and macromolecular organic phosphorus(phospholipids, organic phosphonate) in a certain extent, can also be the source of phosphorus for P. donghaiense and K.mikimotoi in the outbreak and maintain of red tides.