Isolation And Identification Of Antimicrobial Substance Produced From Bacillus Amyloliquefaciens HAB-2 And The Regulation Mechanism Of Key Genes

By the methods of morphology,separation technique and molecular biology,isolation and identification of the antimicrobial substance produced from strain HAB-2,lipopeptides and related-genes,and the mechanism of controlling plant pathogens and promoting plants growth were studied.The results obtained in the present investigation were summarized as below:1.The strain HAB-2 was identified as Bacillus amyloliquefaciens though the morphology observation,analysis of 16SrDNA sequence and specific sequences,and phylogenetic tree.The activity of strain HAB-2 was evaluated against 17 plant pathogens such as Colletotrichum gloeosporioides Penz,Alternaria solani(E.et M.)Jones et Grout.and Fusarium oxysporum f.sp.cubense.The inhibition rates were ranged from 42.99%-69.76%and the rate of Dothiorella dominicana Cif was the highest,reaching up to 69.76%.2.By using the methods of ammonium sulfate precipitation,hydrochloric acid precipitation and organic solvent to extract the antifungal substances from the strain HAB-2,the extraction of n-butyl alcohol and the lipopeptides extracted by acid precipitation method had strong antifungal activity.The extraction of n-butyl alcohol retained 93.64%antifungal activity under UV for 120 min,kept stable over a wide ranged pH,and the antifungal activity was still 81.12%of that of the control after treatment at 100?.There are three kinds of lipopeptides(surfactin,iturins and fengycin)in the extraction of n-butyl alcohol by analysis of MALDI-TOF-MS.3.The structure of compound H2 was identified as C48H73N9O16 with the molecular mass 1031.3,by using analyses of mass spectrometry,amino acid analysis,IR spectroscopic,UV spectroscopic and NMR.Compound H2 composed of Asp,Tyr,Pro,Ser,Gin,Thr,was a new C14 bacillomycin D,which was only one amino acid different with C14 bacillomycin D.It was named C14bacillomycin DC.The antifungal activity of compound H2 was 131 times more potent than prochloraz at the same concentration of 1 mg/100?L.4.There were 7 lipopeptides genes including srfAB?ituA?ituB?ituD?fenD?yndj?bamC,and 4 key enzyme genes in the synthesis of lipopeptides,which were ituA?lpa?mycB?fenB,amplified in genome of strain HAB-2 by PCR.But sfp was not amplified,which was the key gene in the synthesis of lipopeptides.By using the principle of homologous recombination,lpaHAB-2 was knockout in strain HAB-2 and we obtained the lpaHAB-2-deficient mutant strain which lost the antimicrobial activity.The genetically engineered strains,lpaHAB-2 transfered into B.subtilis 168 which has not antimicrobial activity,has antimicrobial ability.It demonstrated that lpaHAB-2 could regulate the synthesis of lipopeptides instead of sfp,which is the key gene in the synthesis of lipopeptides.5.The extraction of n-butyl alcohol and compound H2 control plant pathogens and the mechanism were studied.The extraction of n-butyl alcohol and compound H2 could effectively inhibited the infection of Colletotrichum gloeospo,rioides Penz.on the mango leaf and fruits,Alternaria solani on the tomato fruits,and prevent and control Oidium heveae.Analyses by optical and epifluorescent microscopy,scanning electron microscopy,and transmission electron microscopy,compound H2 caused collapsed and lysis of Xanthomonas oyzae pv.oryzae(Ishiyama)cell walls and Pantoea ananatis cell walls.It also inhibited conidia germination and caused damaged in the hyphae of Colletot,richum gloeospo,rioides Penz.6.The quantitative real-time PCR demonstrated that the application of compound H2 could strengthen the expression of HR marker gene hsr203,defense-related genes PR-1b and NPR1,and the plant growth promotion gene NtEXP2,and induced the induced systemic resistance in the tobacco challenged with TMV.The hsr203 gene in the treatment was significantly and rapidly activated during 24 h and the expression was 92.37-fold increase relative.The transcriptional expression of PR-1b was raised by 5.09-fold when treated with compound H2 upon TMV challenge during 48 h.The significantly higher expression of NPR1 following treatment from 12 h to 120 h and it was up-regulated by 85.03-fold.The accumulation of NtEXP2 gene in the tobacco leaves was inhibited from 12 h to 120 h(0.5-fold at 48 h)and this value decreased to 1.66-fold in the control group.7.After treated by the extraction of n-butyl alcohol on tomato seedlings,the content of the chlorophyll were 39.98 mg/g and the length and weight were significantly higher than those of the control group,indicate significant difference at P<0.05 level by Duncan's new multiple range test.It demonstrated that the extraction of n-butyl alcohol from strain HAB-2 promote the growth of the tomatoes.But the content of soluble sugar was 103.6 ?g/g which was low compared with the control.It might be related to protect tomato seedlings against Alternaria solani.The content of soluble protein was 11.34 ?g/g and higher than the control.It might be related to compound H2 induced the ISR in the tobacco.The goal of the tests was to study the application prospect of strain HAB-2.The test of Vigna unguiculata(Linn.)Walp by extract of HAB-2,the length and weight were significantly higher than those of the control group,indicate significant difference at P<0.05 level by Duncan's new multiple range test.Labeling strain HAB-2 with green fluorescent protein(GFP),and it has colonization ability in the tobacco seedlings root at 7 d.8.Though the static bioassay,the aquatic ecotoxicity of compound H2 in zebrafish(Danio rerio)was investigated that the 96 h-LCso value of compound H2 was 22.191 mg/L,thus showing low toxicity.