Study On DMSP Metabolism Of Antarctic Diatoma And Its Epiphytic Fungi

3-dimethylsulphoniopropionate(DMSP)is an important carrier material in the process of marine carbon-sulfur cycle.DMS,a degradation product of DMSP and its oxidation products participate in the earth climate regulation.Also,mainly produced by marine phytoplankton,DMSP plays an important regulatory role in the adaptation of phytoplankton to environmental changes.While Antarctic diatom(Nitzschia sp.ICE-H)is the dominant species of Antarctic ice algae,it is still unknown whether the Antarctic diatom can produce DMSP or not and what the synthesis mechanism is.Therefore,we chose Nitzschia sp.ICE-H as research object to study the metabolic mechanism of DMSP.Through transcriptome study of Antarctic diatoms,key genes for DMSP synthesis were identified,cloned,expressed and bioinformatically analyzed to further improve the degradation pathway of DMSP in Antarctic diatoms.At the same time,with the combination of environmental adapTableility study of DMSP synthesis,it also helps to clarify the synthesis mechanism of DMSP in Antarctic diatoms.Based on that,the degradation mechanism of DMSP in the epiphytic fungi of Antarctic diatoms was carried out.And by sequencing the genome and searching for key degradation genes,we also discussed the degradation mechanism of DMSP in Antarctic fungi.The specific research results are as follows:1.In order to explore the DMSP synthesis rules of Antarctic diatoms in extreme environments,purge and trap-gas chromatography-cold technology was used to study the regulation of DMSP synthesis in Antarctic diatoms at different temperatures and salinities.And it turned out as expected that both of them affected the DMSP production of Antarctic diatoms.The yield of DMSP in Antarctic diatom cells significantly increased at low temperature(0 ~oC)(up to 53.21 nmol/10~7cells),but not at the high one.In additon,high salinity(64‰)significantly promoted DMSP synthesis in Antarctic diatoms(up to 104.45 nmol/10~7cells);however,higher(96‰)or lower(16‰)salinity could inhibit DMSP production.According to these results,it can be seen that low temperature and high salinity promoted the synthesis of DMSP,which was related to its freezing regulation and osmotic regulation.2.According to the non-sentence transcriptome sequencing results of Nitzschia sp.ICE-H,two full length S-methyltransferase genes for DMSP sythesis were successfully obtained and cloned.Bioinformatics analysis revealed that the complete open reading frame of them were 1386 bp and 1140 bp respectively,and encoded for461 and 379 amino acids with theoretical relative molecular weight of 50.74 KDa and42.78 KDa.Besides,both of them were located in the cytoplasm and belonged to the methyltransferase family,AdoMet-MTases superfamily,with no transmembrane domain and N-terminal signal-free peptide structure.Moreover,they were hexamers and consist of six identical monomers,with low protein conservativeness and differentiation.Phylogenetic tree analysis showed that the two proteins were clustered with Sediminimonas qiaohouensis and Actinopolymorpha singaporensis,indicating that they had relatively closer evolutionary relationships.3.In low temperature induced expression test,the S-methyltransferase gene recombinant engineered strain of Nitzschia sp.ICE-H was constructed and transformed to Transetta(DE3)Chemically Competwnt Cell to express target protein with the condition of 0.5mmol/L concentration of IPTG,at 16 ~oC,160rpm for 12hours.Then,the engineering bacteria were subjected to cell breakage and SDS-PAGE electrophoresis.And the results revealed that there were a thicker band at 50 KDa and40 KDa,respectively,while the control group were not.So we hypothesized that proteins were successfully expressed by the engineering bacteria.Moreover,it turned out the target proteins existed in the cell precipitation as an inclusion body.4.Three Antarctic diatom epiphytes were successfully screened,and they were Tilletiopsis sp.ICE-01,Cladosporium sp.ICE-02 and Aspergillus sp.ICE-03,which could degrade DMSP,produce DMS,and use DMSP as sole carbon source and energy source.Furthermore,in the aspects of DMSP degradation and DMS yield,Tilletiopsis sp.ICE-01 ranked first with the rate of 57.13 nmol and 59.91 nmol respectively.Besises,the basic information of coding gene(24952761 bp),annotated gene(8494123 bp)and non-coding RNA were obtained by genomic sequencing of Tilletiopsis sp.ICE-01,and two dddD genes of DMSP lyases were also found by reference to gene function annotation of the database.These two genes belonged to the acetyl-Co-transferase familly III,and could generated DMS and 3-hydroxypropionate by degradating DMSP.