Optimization Of Genomic DNA Extraction Methods And Preliminary Study On Genome Of Pasteuria Penetrans

Root knot nematodes(Meloidogyne spp.)are wide spread and highly specialized omnivorous plant pathogen,which parasitize,feed and reproduce in the roots of plants.They caused huge damages to agriculture,forestry,horticulture,and so on.For a long time,the chemical pesticides were used for preventing root knot nematodes.With the spread of green growing environmental awareness,biological control is replacing the chemical control and to be an important method to control root knot nematodes.Recently,Pasteuria penetrans is being researched as an ideal biological control strain.Pasteuria penetrans,an endospore-forming and gram-positive bacterium,is an obligate parasite of root-knot nematodes and becoming the most potential biocontrol agent.Nevertheless,the fastidious growing conditions of P.penetrans,has proven to be challenge for culturing this organism in vitro and wide application.Studying on the whole genome probably bring new breakthrough on property and evolution of P.penetrans,and also may lay the foundation for culturing in vitro.In this study,we explored the methods of endospores collection,decontamination,breaking-up and optimizing the genomic DNA extraction process,and then established an effective method to extract genomic DNA of P.penetrans.The genome sequence of P.penetrans was sequenced,comparative genomic analyses were processed among P.penetrans and Bacillus subtilis.Enterobacter cloacae.The results showed that the best condition of endospores collection was centrifugation at 13000 r/min for 20 min.The lysozyme,protease K,SDS reagents were used to decontaminate the endospores,and the decontamination efficiency was 100%.Based on FastPrep instrument,0.1 mm glass beads was used to break the endospores under the condition of 6 m/s for 40 seconds.Among the five used DNA extraction methods,the optimizated BacieriaGen DNA kit was the most suitable one for P.penetrans genomic DNA extraction.P.penetrans genomic DNA(PPG)and whole genomic amplification DNA(PPMDA)were sequenced by Illumina sequecing technology.P.penetrans genomic data were assembled by SOAP de novo.Nearly 2.34 Mb P.penetrans genome.46.55%G+C content were obtained and 3228 coding genes,45 tRNA and 3 rRNA operons were predicted,including 134 interspersed repeats and 495 tandem repeats.Based on the COG.GO and KEGG databases,genome annotation showed that a large number of genes were associated with energy production,energy conversion,catalytic activity and metabolic process in PPG genome,which might be related to nutrients ingestion and energy coversion process when P.penetrans grew in root knot nematodes.The PPG genome contained five histidine kinase and the genes encoding master response regular protein SpoOA,collagen-like protein,chitinase and serine protease.Phylogenetic analysis of multilocus sequences of 7 hosukeeping genes and 5 sporulation genes revealed that P.penetrans was clustered into the Clostridium-Bacillus branch of the endospore-forming bacteria,and more related to the Bacillus spp.Comparative genomic analysis showed that 49 core genes were obtained when compared with B.subtilis and E.cloacae.When compared with B.subtilis genome,147 core genes were obtained,including sporulation genes,such as spoOA,spoOF and spoIIAB.