Distribution Characteristics Of Cyanobacteria And Cyanophages In Daqing Wetland Waters

Cyanophage is a kind of double-stranded DNA virus infecting cyanobacteria.It is widely distributed in different aquatic environments.It plays an important role in regulating the density,diversity and biogeochemical cycle of cyanobacteria in water bodies.Recently,studies on the genetic diversity and biological characteristics of cyanophage have been focus on ecological research.However,there are limited reports on the gene diversity of cyanophage in Daqing wetland waters.In this study,the DNA polymerase pol gene and the phosphate auxiliary metabolism gene phoH gene in bacteriophage were used as molecular markers,and PCR amplification-cloning-Sanger sequencing technology was used to study the genetic diversity and community distribution of cyanobacteria in Daqing Wetland waters.The diversity of phage DNA pol gene and phoH gene in Daqing wetland water body were revealed,the cyanophages and cyanobacteria strains in Daqing wetland waters were obtained by separation and purification method,the morphology of cyanophages were observed by electron microscope Morphology and identified taxonomic status combined morphological method with molecular biology methods,the DNA pol gene of cultivable cyanobacteria in Daqing wetland waters were cloned and sequenced,which explored congruent relationship the cultivable cyanophage in Daqing wetlands waters and clones of environmental samples.The high-throughput sequencing technology were used to investigate the diversity of cyanobacteria in Daqing wetland waters,the results mentioned above provided materials for the future study on the relationship between cyanobacteria and cyanophage diversity.The main results are as follows:1.the pol gene encoding DNA polymerase in the cyanophage was used as a molecular marker to investigate the community distribution of the cyanophage in Daqing wetland waters.Using degenerate primers CP-DNAP-349 E and CP-DNAP-533Ra/Rb,PCR amplification was performed on the cyanophage pol gene in Daqing wetland waters.59 DNA pol gene sequences from cyanophage were obtained from three Daqing wetland water samples,the BLASTp alignment in the NCBI database found that the similarity of these sequences is between 71%-99%.Phylogenetic analysis revealed that these sequences were divided into five clusters(Groups 1-5),and the distribution of cyanopodophage communities in Lake Liming,Longfeng Wetland water and Zhalong Wetland water was different.Through phylogenetic analysis of the DNA pol gene obtained in this study and the DNA pol gene of cyanopodophage in different environments(ocean,paddy field,and wetland sediments),a new DNA pol gene cluster was found in t Daqing wetland waters.Nonmetric multidimensional scale(NMDS)analysis found that the cyanopodophage DNA pol gene assemblages in Daqing wetland waters are closely related to those of freshwater lakes and paddy fields,and are far from those from the ocean and The DNA pol gene clusters of cyanopodophage in coastal estuaries and wetland sediments.The phosphate auxiliary metabolism gene(AMG)phoH gene was used as a molecular marker to explore the community distribution of cyanophage in Daqing wetland.Using degenerate primers vPhoHf/vPhoHr,73 viral phoH gene sequences were obtained from four Daqing wetland water samples.Phylogenetic analysis found that five new subgroups(Groups 3 f,Groups 3 g and Groups 6 f,Groups 6 g,Groups 6 h)of viral phoH gene sequences from Daqing wetland water were specifically formed,indicating that there are new viralphoH gene clusters in Daqing wetland waters.Based on nonmetric multidimensional scale(NMDS)analysis,it was found that the viral phoH gene assemblages in Daqing wetland waters is far from those from the ocean and rice fields,and is closer to those from the wetland sediment.2.Fourteen strains of cultivable phagophages with Anabaena PCC7120 as the host were isolated and purified from four Daqing Wetland water samples by a double-layer plate method.Biological characteristics of 12 isolated cultivated cyanophages found that the activity of the four strains of cyanophage almost disappeared beyond 50?of temperature;the activity was the highest between pH 7-8,and the activity almost disappeared after being irradiated under ultraviolet light for more than 3min.Four isolated cyanophages were cyanopodophage observed by electron microscopy.Three DNA pol gene sequences from isolated cyanophages of Lake Liming infecting Anabaena PCC7120 were obtained and their phylogenetic status were clustered with the viral DNA pol gene sequence from Lake Liming waters together.3.Three cyanobacteria strains from Daqing wetland water were isolated by plate streaking method,and were identified based on 16 s rRNA and psbA gene by phylogenetic analysis combined with their morphological characteristics,the results found that three strains of cyanobacteria belong to the order Oscillatoria,Oscillatoria,Leptolyngbya.4.High-throughput sequencing technology was used to investigate the diversity of cyanobacteria in the Daqing wetland waters,LLM-Jun,LLM-Sep and ZLW-Jun respectively represent the water samples of Liming Lake in June,the water samples of Liming Lake in September and the water samples of Zhalong Wetland in June.The results found that the diversity richness chao1 index,diversity Shannon index and Simpson index richness of cyanobacteria in Zhalong wetland waters were higher than those in Liming Lake;There is no difference in the richness of cyanobacterial communities in the water bodies of Liming Lake in different periods;and the cyanobacterial diversity Shannon index and Simpson index found that the water body of Lake Liming in June was lower than that of Lake Liming in September;Cyanobium PCC-6307 is the dominant genus for all three samples,and the cyanobacterial diversity index of Lake Liming waters were higher than those of Zhalong wetland waters;Richness of Cyanobium PCC-6307 in Lake Liming in September is higher than that in June;Cyanobacterial composition at different locations in Daqing wetland waters at the same time is more different than thtat at the same location in different sampling time.In summary,DNA pol gene and phoH gene were used to as molecular marker genes,a novel DNA pol gene assemblage and five new phoH gene subgroups were found in Daqing wetland waters.DNA pol gene assemblages in Daqing wetland waters are closely related to the paddy floodwater,and far from those of wetland sediment and marine waters,phoH gene assemblages in Daqing wetland waters are closely related to those of wetland sediments,and far from those of marine waters and paddy floodwaters.Cyanobacterial community in Daqing wetland waters varied significantly with sampling time and location.Three DNA pol gene sequences from isolated and purified cyanophages infecting Anabaena PCC7120 were obtained,their phylogenetic status distributes special DNA pol gene clade from Lake Liming.The results mentioned above could provide theotical basis for follow-up investigating congruent relationship between cyanophage and cyanobacteria.