Diversity Of Cyanobacteria And Cyanophages In Daqing Wetland Water

Cyanobacteria are a kind of photosynthetic autotrophic prokaryotes and primary producers of ecosystems.Cyanophages are a kind of viruses that specifically infect cyanobacteria.Both are widely distributed in various habitats on the earth.In order to explore the genetic diversity of cyanophages and cyanobacteria in Daqing wetland water,PCR-cloning sequencing technology was used to study the diversity of g20 gene encoding T4 cyanophage capsid assembly protein and psb A gene encoding D1 subunit of reaction center protein in cyanophage photosynthetic system II.The genetic diversity of cyanobacteria in Daqing wetland water was analyzed by high-throughput sequencing technology.The cyanobacteria and cyanophages in Daqing wetland water were obtained by traditional separation and purification methods.The biological characteristics of cyanophages are to be clarified,and the germplasm resources of cyanobacteria and cyanophages in Daqing wetland water are to be excavated,the main results are as follows:(1)The degenerate primers CPS1.1/CPS8.1 were used to amplify the DNA of virus components in Daqing wetland water by PCR amplification-clone sequencing.A total of 130 cyanophage g20 gene sequences were obtained,and the homologous sequence similarity was between 63.74% and 100 %.The g20 gene sequence obtained in this study was phylogenetically analyzed with the g20 gene of cyanomyovirus in different habitats(marine,freshwater lakes and paddy fields),found that the g20 gene sequence of Daqing wetland formed four unique groups(g20-DWW-Ⅰ～DWW-Ⅳ).Nonmetric multidimensional scaling(NMDS)analysis showed that there were regional and temporal differences in the g20 gene assemblages of cyanophages in Daqing wetland water.The g20 gene cluster of cyanophages in Liming Lake was closely related to that in freshwater lakes and paddy fields,and far from that in the ocean.The g20 gene clusters of cyanophages in Wanbao Lake and Zhalong Wetland were far from those in other habitats.(2)The degenerate primers psbA-F/psbA-R were used to amplify the DNA of virus components in Daqing wetland water by PCR amplification-clone sequencing.A total of 86 psb A gene sequences were obtained.Combined with GC content,tripeptide elements and phylogenetic analysis,68 psbA sequences were confirmed to be cyanophage origin,and there were two unique groups(psb A-DWW-I and psb A-DWW-II).Non-metric multidimensional scaling(NMDS)analysis showed that the psb A gene cluster in Daqing wetland water was more similar to the psb A gene cluster in paddy field,and was relatively far from the psb A gene cluster in the ocean compared with the psb A gene cluster in the lake.(3)The diversity of cyanobacteria in Daqing wetland water was investigated by highthroughput sequencing technology.It was found that the diversity of cyanobacteria in Zhalong wetland was significantly higher than that in Liming Lake and Wanbao Lake.The community diversity of ZLW-Jun was the highest and that of LWB-Jun was the lowest.Cyanobium PCC-6307 was the dominant genus in the other five samples except LWB-Jun.The composition of cyanobacteria community varies with time and sampling sites.(4)Four cyanobacterial strains were isolated by enrichment and separation method.By microscopic observation and identification of 16 S r RNA sequence,three strains were identified as Geitlerinema(99.70% similarity)and one strain was identified as Phormidium(96.84% similarity).(5)Four purified cyanophages were obtained from Daqing wetland water by double-layer plate method using Anabaena sp.strain as host,and all of them belonged to podoviridae through electron microscope observation.The study of their biological characteristics showed that the cyanophages were inactivated when the temperature was higher than 50 °C.The virus activity was the highest at p H weak alkali(7-8)conditions;more than 90 % of its activity was lost after 6 min UV irradiation;after preservation at different temperatures(4 °C,-20 °C and-80 °C)with protective agent,the activity of cyanophage remained unchanged in the short term.In summary,there are new cyanophages and cyanobacteria in Daqing wetland water.It is still necessary to continue to explore the germplasm resources of cyanobacteria and cyanophages in wetland water,and provide basic data for the ecological relationship between cyanobacteria and cyanophages.