Genome Sequencing Of Streptomyces Sampsonii And Expression Analysis Of Several Cloned Chitinase Genes

Streptomyces sampsonii KJ40 is an actinomycete with functions of disease prevention and promoting plant growth,it has the potential as a biological pesticide.However,there is no relevant study in the genome of S.sampsonii until now,limiting the research of its functional genes,metabolites synthesis pathway and comparative genomics etc.To analyze the genome information of the S.sampsonii KJ40 strain,and to research the mechanism of functions of the strain,and its secondary metabolite genes.The whole genome of KJ40 strain was sequenced on the Illumina Hiseq platform,and then sequenced Reads were assembled,the genes were predicted,the gene annotation,secondary metabolite biosynthesis and synteny were also analyzed with softwares.To clone and prokaryotic expression of the chitinase genes of S.sampsonii KJ40,then purify recombinant protein and investigating antibacterial characters.The mainly results were as follows:1 One linear genome of 7 070 328 bp was obtained.The average GC content was73.38%.S.sampsonii KJ40 is deposited at Gen Bank under the accession CP016824.The genome contained 6 340 genes,which constituted 87.96 % of the genome,981 tandem repeat sequences,784 minisatellite DNAs,66 microsatellite DNAs,21 r RNAs,65 t RNAs,and 17 s RNAs.2 Functional annotation and metabolic pathway analyses were achieved on GO databases,KEGG databases,COG databases,Swiss Prot databases,NR databases and CAZy databases.4 503,3 600,3 026,5 964 and 2 018 genes were able to annotate in COG,GO,KEGG,NR and Swiss-Prot databases respectively.157 proteins had CAZy orthologs,application of the CAZy databases,uncovered 65 genes encoding glycoside hydrolases(GHs),17 genes encoding glycosyl transferases(GTs),16 genes encoding carbohydrate esterases(CEs),1 genes encoding polysaccharide lyases(PLs),55 genes encoding carbohydrate-binding modules(CBMs).25 secondary metabolite biosynthetic gene clusters were also obtained.Of the 25 clusters,12 for polyketides or non-ribosomal peptides,5 were estimated for terpene biosynthesis,2 were estimated for bacteriocin biosynthesis,1 were estimated for ectoine biosynthesis,2 were estimated for siderophore biosynthesis,2 were estimated for lantipeptide biosynthesis,1 were estimated for butyrolactone biosynthesis.3 In the comparative genomics analysis of S.sampsonii KJ40,the genome sequences of Streptomyces albus J1074,Streptomyces sp.FR-008 and Streptomyces albus SM254 were obtained as references.Synteny was very conserved among the four genomes.There were deletion mutations and insert mutations among the four genomes.Futhermore,pan-genome of the four genomes was constructed and analyzed.The results showed that the total pan-genome contained 7 482 genes,core-genome contained 5 465 genes,dispensable-genome contained 2 017 genes.The genome of S.sampsonii KJ40 contained279 specific genes.Specific genes of S.sampsonii KJ40 were annotated as genes that code for hypothetical proteins and genes that code for polyketone cyclase,dehydratase family proteins and transposase,involving in transport of polyketone cyclase,dehydratase and lipid.4 Firstly we cloned the 6 chitinase genes from S.sampsonii KJ40 by PCR amplification,Chi KJ40654,Chi KJ402040,Chi KJ404958,Chi KJ405296,Chi KJ406136,Chi KJ40 were deposited at Gen Bank under the accession MG323506、MG323507、MG323508、MG323509、MG323510、MF434484.Then we ligated Chi KJ406136 and Chi KJ40 into vector p ET-32 a and expressed in Escherichia coli BL21(DE3),48 k Da and42 k Da of the recombinant chitinases were obtained.There are no significant differences in production of protein using different concentrations of IPTG at 37 °C inducing 3 h.0.2mmol/L IPTG inducing 16 °C overnight,recombinant chitinase mainly existed in the form of soluble supernatant,small existed in inclusions precipitation.The chitinase activity of Chi KJ406136 purified protein was 0.033 U/m L,the specific activity of Chi KJ406136 purified protein was 0.471 U/mg,the purification ratio was 21.41,the rate of 73.33%.The chitinase activity of Chi KJ40 purified protein was 0.046 U/m L,the specific activity of Chi KJ40 purified protein was 0.115 U/mg,the purification ratio was 2.8,the rate of 57.5%.After treating with the purified protein Chi KJ406136,mycelium cells of Cylindrocladium scoparium,Cryphonectria parasitica and Fusarium oxysporum were segmented with inflating the mycelia,and myceliums of Neofusicoccum parvum were broken.After treating with the purified protein Chi KJ40,mycelium cells of C.scoparium,C.parasitica,Alternaria alternata were segmented with inflating the mycelia,and myceliums of Rhizoctonia violacea were broken.Our results show the genome information of KJ40 strain,it gives evidence that KJ40 is linked to disease prevention and promoting plant growth,and provided reference for understanding secondary metabolic synthesis pathway of Streptomyces,which is of great significance to the future research of S.sampsonii.This study of the chitinase genes provides biocontrol background of S.sampsonii and finds a new source for the chitinase genes,and lays a theoretical foundation for its application.