| As the water eutrophication and cyanobacteria bloom in the water environment are more and more frequent,they have become an urgent ecological problem in China and other countries.Cyanobacteria and its secondary metabolites pose a great threat to the health and safety of aquatic animals and human beings.The prevention and control technology of cyanobacteria bloom has become one of the focuses in current research.Copper ion is one of the most classic algaecide,however,the mechanism of algae killing and the response of cyanobacteria to copper ion stress should be further studied.Although the copper algaecide has the characteristics of high algae removal efficiency and low price,the secondary pollution caused by copper ions in the process of algae-removal has aroused concern.Thus,it is very important to find an algaecide,which is efficient and environmentally friendly enough,to control cyanobacteria blooms.In this research,the killing mechanism of copper ion to Microcystis aeruginosa was studied by transcriptome sequencing,and the effects of copper stress conditions on toxicity-producing function of M.aeruginosa were also deeply explored.We also tried to find a more efficient and safer way to remove algae,and then the inhibition and killing mechanism of vitamin C to M.aeruginosa was studied.The main findings of this paper are as follows:1.M.aeruginosa PCC 7806 was exposed to different concentrations of copper ions to determine a minimum inhibitory concentration and EC50 for 96 h.The values were 0.5?M and 3 ?M,respectively.The gene regulation of M.aeruginosa treated with these two copper ions for 96 h was investigated by transcriptome sequencing technology.More significant differentially expressed genes were found in the 3 ?M copper ion treated group compared to the 0.5 ?M copper ion treated group.There were 1306 differentially expressed genes in the samples treated with 3 ?M copper ions,and 52 differentially expressed genes in the samples treated with 0.5 ?M copper ions.Metal transporter-related genes,iron-sulfur cluster-related genes,heat shock protein(HSPs)genes and microcystins(Microcystins,MCs)synthetase genes were differentially expressed in the two copper ion treatments.Gene Ontology consortium(GO)and KEGG were used to annotate the differentially expressed genes.In GO analysis,the differentially expressed genes were enriched in macromolecule complexes,membrane protein complexes,photosynthesis,thylakoid,nitrogen metabolism and so on.KEGG pathway enrichment analysis showed that there were significant differences in glycolysis and gluconeogenesis,synthesis of secondary metabolites,synthesis of non-ribosomal peptide,sulfur metabolism,propionic acid metabolism,fatty acid biosynthesis and metabolism,carbon fixation of photosynthetic organisms,cysteine and methionine metabolism between the two copper ion treatments.The acquisition of differentially expressed genes laid a foundation for further study on the mechanism of copper ions inhibiting Microcystis aeruginosa.2.M.aeruginosa PCC 7806 exposed to 0.5 and 3 ?M copper ions was used to study the relationship between the level of microcystins(MCs)and the concentration of copper ions.The results showed that the contents of MCs in M.aeruginosa cells treated with 0.5?M and 3 ?M copper ions increased significantly.The content of copper ions and iron ions in M.aeruginosa cells of different treatment groups determined by inductively coupled plasma optical emission spectrometry(ICP).At 0.5 ?M and 3 ?M copper,the content of copper and iron ions in M.aeruginosa cells increased significantly.And transcriptome data showed that the transcriptional levels of transport iron and sulfur-related genes in the 3 ?M copper ion treatment group were significantly affected.The Quantitative Real-time PCR data showed that the transcription levels of the fur A gene and MCs synthase gene were significantly changed in the 0.5 and 3 ?M copper ion treatment group compared with the control group sample.What is more,we analyzed high-throughput RNA sequencing datas.Through electrophoretic mobility shift assay,it was found that Fur A,which regulates iron ion absorption,binds to the promoter region of the MC synthesis gene mcy D,thereby regulating the synthesis of MCs.We speculate that copper ions indirectly lead to an increase in MCs content by regulating Fur A in cells of M.aeruginosa.M.aeruginosa produces more MCs in a copper-dependent manner,which seems to be regulated by Fur A,as copper stress induces genes for the formation of iron-sulfur clusters and cluster target genes.3.M.aeruginosa FACHB 905 was treated with different concentrations of vitamin C(0,0.12,0.6,3 and 6 m M)and it was found that the biomass of M.aeruginosa cells gradually decreased with the increase of vitamin C concentration.And the minimum inhibitory concentration of vitamin C against M.aeruginosa is 0.6 m M.0.6 m M of vitamin C was used to treat M.aeruginosa and the amount of intracellular vitamin C at different time points was determined.The content of iron ions in the above samples was determined by ICP,and it was found that the intracellular iron ion content gradually increased as the treatment time of vitamin C against M.aeruginosa increased.The content of ferrous ions in M.aeruginosa cells treated with 0.6 m M vitamin C at different time points was also examined.The results showed that the ferrous ion content in the cells of M.aeruginosa samples also increased significantly from 0.8 to 4 Times.The concentration of reactive oxygen species(ROS)in the 0.6 m M vitamin C-treated M.aeruginosa cells increased significantly by 1.43,3.62 and 3.15 times at 6,12 and 24 h,respectively.This indicates that vitamin C enters the M.aeruginosa cells to catalyze the Fenton reaction,which produces a large amount of ROS.Compared with the control group,the MDA content and caspase-3 protein activity in M.aeruginosa cells were significantly increased in the 0.6 m M vitamin C treatment group.TEM images of samples at different time points show that M.aeruginosa cell morphology is more severely disrupted as exposure time increases.It was shown that excessive ROS induced increased caspase-3 protein activity,lipid peroxidation,membrane deformation and leading to the death of M.aeruginosa.In the 0.6 m M vitamin C treatment group,the content of MCs decreased with the increase of exposure time.This suggests that vitamin C catalyzes the large amount of ROS produced by the Fenton reaction in M.aeruginosa cells,does not cause M.aeruginosa to produce and release more MCs. |
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