| Diatoms,the most widely distributed microalgae due to their success in variaties of environment conditions,often form massive blooms under turbulent conditions while nutrient supplies are highly variable,this may be explained by their complicated evolutionary history and element-rich genomes.Diatoms have also received extensive attention due to their critical role in biogeochemical cycles and global climate change.However,to understanding the evolutionary status,physiological and ecological characteristics of diatoms in a more comprehensive way requires targeted research on diatom genes.Recently,the successful application of gene editing method in diatoms has opened a new gate for diatom research.In this thesis,we focus on the gene Phatr3_J55010(Pt55010)of model diatom P.tricornutum.We knock out Pt55010 by CRISPR/Cas9 and obtain the mutant,the responses of the mutant were investigated to clarify the physiological and ecological function of Pt55010 in combination with the bioinformatics,physiological methods.The main results are as follows:1.The distribution of Pt55010 homologous genes in diatoms varies among species,not all diatoms contain Pt55010 homologous genes.The homologous genes of Pt55010 are only distributed in dinoflagellates,diatoms and haptophytes in the range of all organisms,there is no homologous gene in prokaryotes.These conclusions specifically support that Pt55010 is the origin of diatom lineages.Furthermore,the protein subcellular localization of Pt55010 homologous genes were mainly in cytoplasm and endomembrane.2.Pt55010 and its homologs are widely distributed in the global ocean,and the abundance of Pt55010 is positively correlated with the concentration of nitrate,POC and phosphate in marine areas.The Pt55010 homologs are distributed from the tropics to the high latitudes,but it was only found in dinoflagellates,diatoms and haptophytes,especially in Gymnodinium.3.Pt55010 and its homologs are potential pyridoxal phosphate dependent transferase gene,Pt55010 is significantly upregulated when nitrate is deficient,and significantly downregulated in the dark or exposure to more than 6 hours light,suggesting it is mainly expressed at the start of circadian cycle.We also note that Pt55010 is broadly coregulated with the genes related to nitrate reductase,nitrite reductase and glutamate synthesis,which might be consistent with a role in N metabolism.4.We successfully obtained the Pt55010 knock-out mutant Crispr-4-7 of P.tricornutum by CRISPR/Cas9 gene editing method.We verified that the target gene of this mutant was missing 17 bases,and the expression level of Pt55010 in the mutant was significantly decreased.5.The knockout of gene Pt55010 did not significantly affect the photosynthetic activity and nutrients(nitrogen and phosphorus)uptake in P.tricornutum.However,the growth rate and maximum biomass of Crispr-4-7 were significantly decreased,while the cell size was significantly increased.Meanwhile,the lipid content of the mutant decreased and the soluble protein content per cell increased,respectively.6.The knockout of Pt55010 broadly affects the carbon and nitrogen metabolism of P.tricornutum.We note that genes involved in nitrate reduction,nitrite reduction,carboxyl phosphate synthesis,urea degradation and malate dehydrogenation were all significantly down-regulated among the mutant,which indicate that nitrogen assimilation,urea cycle and TCA cycle were all inhibited to varying degrees after Pt55010 knockout.In addition,genes related to metabolic processes such as glutamate/glutamine synthesis,lipid synthesis and glycolysis,were also downregulated in the mutant.However,the genes related to branched-chain amino acid degradation and arginine synthesis were significantly up-regulated.We also find that ribosome process and DNA replication in mutant were significantly suppressed.In conclusion,Pt55010 is important for the growth,reproduction and regulation of carbon/nitrogen metabolism in P.tricornutum.Pt55010 is an important factor in the ecological success of these microalgal. |
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