| As an important biocontrol resource, Bacillus subtilis can secrete lipopeptide antibiotic to inhibit fungal pathogens, and is used in various biological control of plant diseases. In recent years, the important role of bacterial biofilms in control of plant diseases is gradually realized, its sophisticated regulatory network becomes clear, and its multi-cell behaviors are gradually being studied. To further elaborate molecular mechanism of biofilms of B. subtilis Bs916 against rice sheath blight and its important regulatory mechanism in the process of disease prevention and control, under the detailed map of the whole genome of B. subtilis Bs916, the following researches are included in this study:1. The random inserted mutant library of Bs916 was constructed by introducing ransposon.Screening mutants which to appear enhanced or decreased apparently in the inhibition zone to Xanthomonas oryzae pv. oryzicola (Xooc), and to obtain the genes which may be related to anti-bacterial activities changed by cloning the DNA sequence around the transposon inserted sites. Biofilm formation of Bs916 is used as a control to screen mutants with obvious biofilm formation defects from these mutants with obvious anti-bacterial capacity changes. The results by the PCR and Southern Blot showed that the random inserted mutant library of B.subtilis 916 was constructed successfully, 85% mutants were inserted by a single copy; 30 mutants whose anti-bacterial activities changed against Xooc strongly enhanced or sharply decreased were screened; Twenty-one genes around the insertion site were cloned from the mutants above and sequenced; the bioinformatic analysis results showed that these genes were related to competence development, flagellar motility and secondary metabolites synthesis; 10 mutants with biofilm formation defects from 30 mutants with obvious anti-bacterial capacity changes were screened, 3 mutants were further screened by biofilm formation with serious defects and named AgltB,?fliZ, and AserA.2. Production of three lipopeptide antibiotic (LPs) in Bs916 and its single knockout mutants (?bac,Asrf, and Afen) were detected. Rhizoctonia solani and Bs916 were used as the target strain and the control strain for inhibition test respectively to identify the antibacterial activity of three LPs against R.solani. The results showed that three LPs bacillomycin L, surfactin, and fengycin were identified in Bs916; antibacterial activity of Abac mutant significantly reduced without bacillomycin L, antibacterial activity of ?srf mutant was completely lost without bacillomycin L and surfactin at the same,antibacterial activity of Afen was some remaining without fengycin. This study confirmed three LPs had an important role against R. solani, in which bacillomycin L was the most important. Loss of bacillomycin L and surfactin was caused by missing expression of srfAA in Bs916.3. Single knockout mutants of three genes gltB, fliZ, and serA associated with biofilms were constructed. Biofilm formation of Bs916 was used as comparison to detect biofilm formation of three mutants. Biocontrol efficiency of three mutants and Bs916 against rice sheath blight were detected. The results showed that biofilm formation of three mutant had serious flaws, only a flat two-dimensional structure, however, biofilm formation of Bs916 was a volumetric three-dimensional structure; biocontrol efficiency of three mutants against rice sheath blight appeared a significant decline compared to Bs916.In addition, there was a significant cluster effect appeared in the wound sites by R. solani in Bs916, and significant green fluorescence. No significant cluster effect and enough cells appeared in three mutants.The results showed that three genes associated with biofilm had a significant regulation for multicellular behavior of Bs916.4. Production of three LPs bacillomycin L, surfactin, and fengycin were detected in Bs916 and its three mutants with biofilm defect. The results showed that no bacillomycin L and fengycin were detected in AgltB, and production of surfactin increased fivefold compared to Bs916. No significant changes of three LPs appeared in ?fliZ and ?serA. This study preliminary viewed that lack of bacillomycin L and fengycin, severely reduced colonization caused by bad biofilm formation and together resulted in a significant decrease in biocontrol efficiency against R. solani. Severely reduced colonization caused by bad biofilm formation in AfliZ and AserA resulted in a significant decrease in biocontrol efficiency against R. solani.5. Biofilm regulatory mechanisms of AgltB mutant was investigated. Based RT-PCR data analysis with five times lower of the capB (ywsC) gene in transcript level, which was considered to be necessary for polyglutamate biosynthesis. Detecting glutamate and polyglutamate content of EM medium used for biofilm formation in Bs916 and AgltB mutant. The results showed that the ability to use glutamate to compose polyglutamate was found severely reduced in Ag1tB mutant compared to Bs916. By exogenously adding polyglutamate to restore the biofilm of AgltB mutant, the results showed that part of biofilm restored by adding 10 g/L polyglutamate, most of biofilm restored by adding 20 g/L polyglutamate. Biofilm formation of WT Bs916 was enhanced by adding 10 g/L and 20 g/L polyglutamate. By adding 30 g/L polyglutamate in WT Bs916 and AgltB mutant, due to the high concentration, both were severely inhibited. By training next to WT Bs916 by ?gltB mutant in a EM solid plate, this study demonstrated that polyglutamate produced by Bs916 restored biofilm formation of?gltB mutant. To form poly-glutamic acid by regulating glutamate, gltB regulated biofilm formation of Bs916.6. By transcriptome sequencing in AgltB, ?fliZ, and AserA and Bs916, differentially expressed genes and metabolic pathway analysis were analyzed. The results of different expressed genes analysis showed that the number of different expressed genes in Bs916 and its three mutants ?gltB, AfliZ, and AserA was significantly different, In which proportion of up-regulated and down-regulated genes of?fliZ had the most significant difference. The results of GO enrichment analysis of different expressed genes showed that changes of two subdirectories classification protein in three mutants compared with WT Bs916 mainly included cellular process, cell, and cell part. The results of KEGG enrichment.analysis of different expressed genes showed that process changes in three mutants compared with WT Bs916 mainly included amino acid metabolism, carbohydrate metabolism, and membrane transport,were closely related to energy metabolism and material transport in biofilm formation of Bs916. Further to enrich these main genes in three common processes, the results showed that yfmT, yhfS, and alsS genes were also participate in two important metabolic processes, and expressing trend were significantly different in the same process caused by different controlling genes, illustrated that the three genes played an important regulatory role in biofilm regulatory network of Bs916, and participated in multiple metabolic processes. |
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