Study On The Effect Of Temperature And Nitrate Limitation On The Formation Of Phaeodactylum Tricornutum Dimethylsulfoniopropionate And The Key Genes In This Pathway

Dimethyl propionate（DMSP）is an important dissolved organic sulfur in the ocean.Its decomposition product is dimethyl sulfide（DMS）.These two substances not only have important ecological significance,such as participating in the ocean sulfur cycle and regulating global climate,but also have vital physiological functions in marine plankton.They can be used as algal antioxidant or cryoprotectant,and can also regulate osmotic pressure,therefore they play an important role in plankton adaptation to environmental changes.Phytoplankton is an important contributor to DMSP,but the mechanism of DMSP generation in algae is currently unclear.In this paper,we used model diatom Phaeodactylum tricornutum to investigate the underlying mechanism of DMSP synthesis.Through comparative genomics,four candidate genes related to this step of the reaction were detected by comparative genomics analysis.Based on protein structure and function prediction and physiological experiments under different conditions,PtMMT（Phatr3₄8704）was selected as candidate gene as putative S-adenosyl-Lmethionine-dependent methyltransferasc in DMSP synthesis for gene editing.The distribution and expression of PtMMT homologies in a global scale in field were analyzed using Tara ocean database.The results were as follows:1.In P.tricornutum,four homologous genes of Thalassiosira methyltransferase（TpMMT）:Phatr3_J48704,Phatr3_J15217,Phatr3_J33530 and Phatr3_J10824.All of which belong to the homologous superfamily of S-adenosyl-L-methioninedependent methyltransferase.These genes will be co-cxpressed with fucoxanthinChlorophyll a/c binding protein（FCP）,fatty acid,phospholipid and nucleic acid metabolism related genes.2.There is no significant change in DMSP production during different growth periods of P.tricornutum,which may be related to different culture time.3.Under 18 degree the total DMSP production（DMSPt）of P.tricornutum（Ptl strain）increased by nearly 16.98%,and the cellular DMSPt increased slightly,although there was no statistical difference.Dissolved DMSP（DMSPd）and particulate DMSP（DMSPp）also increased by nearly 62.79%and 12.88%.Algae cells may resist the effects of low temperature by accumulating intracellular DMSP（DMSPp）.It is worth noting that when the temperature decreased,only the expression of Phatr3_J48704 increased among the four candidate genes.4.Nitrate limitation inhibited the cell growth of P.tricornutum（Ptl and Pt4）,but the content of cellular DMSPt increases by more than 10 times.There may be three reasons for this:1)cells may regulate osmotic pressure by producing DMSP;2)under nitrate limitation conditions,the cell size becomes smaller and the probability of being predated increases.Algae cells may also increase the production of DMSP to resist predators;3)DMSP has an anti-oxidant effect and is used to resist the highly toxic reactive oxygen species produced when the C:N ratio is imbalanced.Due to the genetic variations between Pt4 and Ptl,which results from the differences in their original living environments,the response of Pt4 to nitrate restriction is more obvious,and the yield of DMSP is increased more.the response of Pt4 to nitrate limitation is more obvious in the context of more significant increase of DMSP content.5.Based on the above results,the putative S-adenosyl-L-methionine-dependent methyltransferase（PtMMT,encoded by Phatr3_J48704）involved in DMSP synthesisrelated in P.tricormtum was selected for further investigation.According to the analysis of Tara ocean,PtMMT homologs mainly distrubuted in coastal seawater,their expression levels are significant higher in the photic zone.Low nitrogen and low phosphorus conditions sign ificantly promoted the expression of PtMMT homologs.A small number of prokaryotes（mainly Cyanobacteria）and a large number of eukaryotic algae contain the homologous sequence of this gene.6.The knock-down and knock-out plasmids targeting PtMMT using antisense RNA and CRISPR/Cas 9 technique were constructed.The plasmids were preserved for plasmid transformation into P.tricornutum in future study.