Study On Physiological Changes And Molecular Mechanism Of Phaeodactylum Tricornutum Adaptating To Warming Seawater

As an important primary producer of the ocean and an important part of the food chain,diatoms play a very important role in the marine biogeochemical cycle.With the warming of the ocean,how diatoms adapt to high-temperature environment has become scientific and popular interest in recent years.The research on diatoms adapting to ocean warming is currently mainly focused on the physiological level(such as growth rate,photosynthetic capacity,and cell composition).However,there are few reports on the regulation of gene expression in diatoms adapting to ocean warming.In this study,the model diatom Phaeodactylum tricornutum was used as the research material to conduct a heat selection experiment(about 160 generations).The dynamic changes of the cell composition and metabolism of P.tricornutum were analyzed by the determination of different physiological and biochemical parameters under wild type(20°C)and high temperature(25°C).Furthermore,the molecular mechanism of diatoms adapting to ocean warming was also explored through the analysis of gene transcription regulation and DNA methylation changes.Here,the growth rate and Fv/Fm of P.tricornutum during the heat selection period were measured.Groups “Control”,“Heat shock”(approximate 7 generations)and“Heat Selection”(approximate 160 generations)were set to determine the growth rate,Fv/Fm,photosynthetic pigment content,soluble protein content,nutrient element percentage,cell morphology and size.Our results showed that the growth rate and Fv/Fm of P.tricornutum were lower at 25°C compared to that at 20°C.As the high temperature continued,the spindle-shaped cells were gradually widened,and the photosynthetic pigment content,soluble protein content and the weight percentage content of nitrogen in the cells were significantly increased(P<0.05).These results indicated that no matter short-term or long-term treatment,high temperature(25°C)inhibited the growth,reproduction and photosynthesis of P.tricornutum,but promoted the accumulation of cellular nutrients.In addition,thermal performance assay and nitrogen limitation tests on P.tricornutum in the thermal selection group were also analyzed,and found that heat selection can help alga increase the survival rate under extreme high temperatures and tolerance under nitrogen limited environment.In order to further explore the dynamic changes of gene transcription levels during the heat selection process of P.tricornutum,ONT full-length sequencing technology was used for RNA sequencing to analyze the changes in gene transcription levels of groups “Control”,“Heat shock”(approximate 7 generations)and “Heat Selection”(approximate 160 generations).Through the screening of transcriptional differential genes and enrichment analysis,it was found that the transcription of multiple genes of P.tricornutum showed dynamic changes as the high temperature continued.After heat selection,the transcriptions of genes encoding nitrate transporter,nitrate reductase,nitrite reductase and glutamine synthetase in the nitrogen metabolism pathway of P.tricornutum were up-regulated,indicating that the nitrogen absorption and assimilation capacity of cells were activated.Heat selection induced the downregulation of multiple genes encoding Lhcf-type and Lhcr-type light-harvesting protein,while heat shock only resulted in the downregulation of the Lhcr10 protein-coding gene.However,the transcription of genes encoding Lhcx-type proteins involved in photoprotection were promoted in both experimental groups.At high temperature,the conversion efficiency of light energy was inhibited.During heat selection,light absorption was inhibited through gene expression regulation,while the non-photochemical quenching of chlorophyll(promoting the expression of Lhcx protein and increasing the carotenoid content)was promoted to avoid the generation of free radicals caused by the uncoordinated energy absorption and conversion efficiency.These results indicate that diatoms coordinate energy metabolism by improving nutrient absorption and metabolism and inhibiting photosynthetic efficiency,thereby adapting to ocean warming.Epigenetic mechanisms are involved in the regulation of gene transcription.In order to better understand the regulation of DNA methylation mechanism on gene expression,and explore the role of DNA methylation mechanism in diatoms adapting to ocean warming,Single-Molecule Nanopore DNA Sequencing was used to detect DNA methylation modifications,and the 6mA methylation profile of P.tricornutum was sequenced for the first time.It was shown that types of DNA methylation modifications were widely distributed in P.tricornutum genome.The methylation levels of 6mA,CpG,CHG and CHH on the whole genomic DNA of P.tricornutum were approximately 15.6%,3.5%,14.4% and 15.7%,respectively.The DNA methylation modification of P.tricornutum varies widely among different experimental groups,and about 90% of the differential methylation sites(DML)are in the promoter region.A joint analysis of the methylome and the transcriptome showed that 6mA methylation is associated with the high abundance of gene transcription,and CpG methylation is involved in gene transcription inhibition.For example,the downregulation of light-harvesting protein encoding genes is associated with 6mA demethylation.The transcription of the PTD15 encoding gene in the unsaturated fatty acid synthesis pathway is significantly negatively correlated with CG methylation in the promoter region.These results indicated that DNA methylation is involved in the regulation of gene expression in P.tricornutum at high temperature.