Genetic Diversity Of Cyanophage In Paddy Floodwater In Northeast China And Isolation, Identification Of Cyanobacteria And Their Associated Bacteria

Cyanophage is virus infecting cyanobacteria and a kind of innegligible and strategicbioresource. It plays an important role in regulating abuandance and diversity of plankton,sucession of community structure and biogeochemical cycle. Recently, the reseach on geneticdiversity of cyanophage was a hotspot in viral ecosystem. However, genetic diversity ofcyanophage in paddy field ecosystem in northeast China has not been reported. Therefore, targetingg20gene encoded capsid assembly protein and the psbA gene encoded D1subunit of photosyntheticreaction center protein, the genetic diversity of cyanophage from paddy floodwater in northeastChina was investigated with PCR, Cloning and DGGE of molecular technique. Meanwhile, thecyanobacteria from paddy field and cyanobacterial associated bacteria were isolated and identified,so as to provide the material for isolation of cyanophage in paddy field. The results are as follows:54different g20sequences were obtained from5different paddy floodwaters in northeastChina using degenerated primers CPS1and CPS8.These sequences are different from those inpaddy field in Japan. Phylogenetic analysis showed that these sequences distributed the Cluster α, β,γ and ε and formed3specific subclusters (PFW-VII-PFW-IX) in paddy floodwater in northeastChina. UniFrac analysis based on PCoA (principal coordinate analysis) showed that cyanophagecommunity in paddy field differed from those of marine and lake. Even though in similarenvironment, the cyanophage community in paddy field in northeast China differed from that inJapan.Seventeen different psbA sequences were obtained from3different paddy floodwaters innortheast China using degenerated primer psbA-F and psbA-R. These psbAsequences are deducedto be the origin of cyanophage through analyzing the GC content of gene sequence and signaturemotif of deduced amino acid. Phylogenetic analysis showed that the genetic composition ofcyanophage in paddy floodwater in northeast China differed from that in Japan. There was aspecific Cluster (PFW-CN) in the phylogenetic tree. Cyanophage community based on psbA gene was significantly different from those in marine and lake. The psbA assemblages in paddyfloodwater were different in between Japan and northeast China (P<0.01).The cyanobacteria frompaddy floodwaters in northeast China were isolated using platestreaking. Among isolated cyanobacteria,9cyanobacterial strains were identified. Based onphylogenetic analysis of16S rRNA gene and psbA gene isolated from cyanobacteria, combiningwith the morphological feature of cyanobacteria,2Anabaena strains,4Nostoc strains wereidentified, meanwhile3novel cyanobacterial strains were discovered in paddy floodwater innortheast China, the highest identity of16S rRNA and psbA in3novel strainswere89%-91%and85%-86%, repectively. These cyanobacteria will provide the host materials for the isolation ofcyanophage in paddy floodwater.The cyanobacterial associated bacteria were identified during isolation of cyanobacteria frompaddy field using molecular technique.25sequences of16S rRNA in associated bacteria wereobtained. The associated bacteria of model strain Anabaena PCC7120in different soils wereinvestigated with molecular technique,45sequences of16S rRNA gene were obtained.Phylogenetic analysis showed that these bacteria distributed in Proteobacteria, Bacteroidetes,Firmicutes andActinomycetes. Some bacteria with potentially degrading cyanobacteria excretionwere foundby sequences analysis of associated bacteria, for example, Pseudomonas,Flavobacterium andRhodococcus.In conclusion, there were specific gene assemblages of cyanophage using the g20and psbAgene, which were different from those in marine and laked, cyanobacteria and their associatedbacteria in paddy floodwater in Northeast China. The results will provide host materials forinvestigating the genetic diversity of cyanophage in paddy floodwaters with traditional methods andlay the theoretical basis on investigating the relationship among cyanobacteria, their associatedbacteria and cyanophage in water environment.