Transcriptomic Response Of Karenia Mikimotoi To Ambient Inorganic Nitrogen And Temperature Changes

Karenia mikimotoi is a typical harmful dinoflagellate species in coastal waters of China,which can produce toxin and form bloom.K.mikimotoi bloom not only seriously threatens marine ecosystem stability,development of marine aquaculture,but also ultimately influences human health through the food chain.It’s known that ambient nitrogen availability and temperature are two key factors regulating bloom formation of K.mikimotoi.Although most studies have been devoted to the effects of these two factors on K.mikimotoi in the view of physiology and ecology,the molecular mechanism behind the bloom formation is poorly known.In this thesis,we integrated physioecological and transcriptomic approaches to investigate global gene expression profiles of K.mikimotoi under different nitrogen and temperature conditions,to identify the key genes and biological processes involved in nitrogen uptake and utilization,as well as their response to nitrogen and temperature variations.Combined with the physiological parameter analysis,the present study aimed to elucidate a mechanistic understanding about the response of K.mikimotoi to ambient nitrogen and temperature variation,and to explore their roles in the bloom formation of K.mikimotoi.The main findings are as follows:(1)Under nitrogen-deficient condition,the growth of K.mikimotoi was significantly inhibited,and the Fv/Fm and chlorophyll contents were also significantly decreased.Notably,the Fv/Fm and chlorophyll content recovered rapidly after resupply of either nitrate or ammonium to the nitrogen-deficient cultures.A total of 142,479 Unigenes were assembled from the transcriptome of K.mikimotoi cells under different nitrogen conditions.Under nitrogen-deficient cells,1,885 genes were significantly upregulated and 1,677 genes were significantly down-regulated when compared with the nitrate-replete cells,while it generates 461 up-regulated genes and 600 down-regulated in contrast to the ammonium-replete cells.In the nitrate-resupplied cells,370 genes were significantly up-regulated and 1,338 genes were significantly down-regulated;while in the ammonium-resupplied cells,107 genes were up-regulated and 300 genes were down-regulated.The differentially expressed genes were mainly involved in nitrogen metabolism,photosynthesis,chlorophyll and purine metabolism,amino acid metabolism,lipid metabolism,cell growth and movement.(2)The up-regulated genes related to transport and utilization of inorganic nitrogen,organic nitrogen and cysteine synthesis under nitrogen deficiency indicated that K.mikimotoi enhanced utilization of inorganic and organic nitrogen sources from the environment.It also ensured the resistance via efficiently conversing other amino acids to cysteine in cells.Additionally,the genes related to cell growth and movement were down-regulated,and the genes related to glycolysis and lipid synthesis and accumulation were significantly up-regulated,supporting that K.mikimotoi reduced the energy for cell division and movement,redistributed intracellular carbon source,and synthesized more lipid as energy storage in order to preferentially maintain intracellular homeostasis.Compared with the nitrogen-deficient group,the number of significantly different genes in the ammonium-resupplied group was less than that of the nitrateresupplied group,indicating that fewer biological processes were influenced by ammonium resupplement.Comparison between the two nitrogen-resupplied groups showed that the amino acid metabolism,cellular immunity and antioxidant transcriptional regulation of algal cells were more active after ammonium resupply,and thus the ammonium resupply rapidly enhanced cellular immunity and antioxidant capacity of K.mikimotoi under nitrogen-deficiency.(3)In the low temperature condition(16℃),the maximum cell density of K.mikimotoi,and the values of Fv/Fm and rETRmax decreased significantly.After quick temperature rising from 16℃to 20℃,the cells responded rapidly at the physiological level as both Fv/Fm and rETRmax increased significantly.After a short adaption period,K.mikimotoi cells recovered rapid growth.A total of 137,804 Uni genes were assembled from the transcriptome of K.mikimotoi cells under different temperature conditions.Compared with the temperature control group(20℃),303 genes were significantly upregulated and 340 genes were significantly down-regulated in the low temperature group(16℃).After quick temperature rising from16℃to 20℃,855 genes were upregulated and 846 genes were down-regulated.Compared with the temperature control group(20℃),the total number of differentially expressed genes was 4,772 in the quick temperature rising group,among which 2,502 genes were up-regulated and 2,270 genes were down-regulated.The differentially expressed genes mainly participated in photosynthesis,carbon fixation,cell defense,cell movement and growth and protein metabolism.(4)Compared with the temperature control group(20℃),the genes associated with cell defense and movement,tubulin and heat shock protein were significantly down-regulated in the low temperature group(16℃),indicating that the probability of protein misfolding was increased,the cytoskeleton tended to be unstable,and the cellular immunity and motor ability were also compromised.After quick temperature rising(16℃→20℃),Rubisco,a key carbon immobilization enzyme,and the genes involved in photosynthesis were up-regulated,while the genes involved in cell division were significantly down-regulated,demonstrating that energy metabolism and organic matter accumulation were prioritized after thermal stress to support rapid growth.At the physiological level,K.mikimotoi cells could resume high division rate,and thus K.mikimotoi had a strong phenotypic plasticity to rapid thermal stress,which might support the bloom formation in marine environment.