Cloning Of The Bacteriostat Related Gene And Identification Of Antifungal Substances From Bacillus Subtilis S-16

Sunflower sclerotinia is a devastating disease infected by Sclerotinia sclerotiorum, at present, there is no effective prevention and control measures. Chemical control、 agricultural control and multiresistance variety selective breeding did not achieve good control effect in the process of control sunflower sclerotinia. So, this experiment is focus on biological control, and series of sunflower sclerotinia. This experiment use Bacillus subtilisS-16as material, the Bacillus subtilisS-16is isolated in my lab, and researches the physicochemical properties of antimicrobial substances which were produced by bacillus subtilisS-16. And then, the experiment studies antibacterial gas composition and the culture conditions influences on sclerotinia sclerotiorum growth. PHV1249(contain mini-Tn10) was used for plasmid, which was transferred into strain. Finally, the transposon library of S-16is constructed successfully. Further, the experiment studies ability of biofilm formation. The purpose is to finding bacteriostat related gene, and as to provide theoretical basis for alteration of gene engineering strain of Bacillus subtilris S-16. The specific research results are as follows:1. Built3000mutants in library, and3mutants with defective of bacteriostasis were selected. Select1mutant with defective of ability of ntibacterial gas composition and6mutants with defective of ability of biofilm formation, which is selected by200mutants. The200mutants were selected randomly.2. Physicochemical properties of Antimicrobial substances were researched.40mL (100mL flask) of the fluid volume and beef extract as nitrogen source that all better for antibacterial substance, the activity of antibacterial substance is the highest. In the medium with glucose as carbon source, the activity of antibacterial substance is the highest too. Antibacterial substance is high thermostability and resistant to acid but alkali. Protease has no effect on antimicrobial substances. Ultraviolet irradiation treatment does not have effect on activity of antibacterial substance. According to the results of the above, Preliminary concluded that the antibacterial substance of bacillus subtilisS-16is the main component of lipid peptides.3. To study antibacterial gas composition shows that1-Butanamine. N-methyl-2-Hexynoic acid、3-Butenamide、Cyclobutanol、2-Octanamine and Benzothiazole.2-methyl-has ability to Inhibit the growth of Sclerotinia sclerotiorum hyphae. Formamide, N-ethyl-N-phenyl-and Piperazine,2-methyl-cause the hyphae yellowing but do not affect the formation of sclerotia. Ethyne, fluoro-and3,3-Dimethyl-4-methylamino-butan-2-one has smaller influence on sclerotium and hyphae, it Just cause hyphae sparse and do not affect the formation of sclerotia. Experimental results show that, antibacterial gas has some multiples which can inhibits fungal hyphae and sclerotium forming.4. In the experiment of the culture conditions influences on sclerotinia sclerotiorum growth, its optimum culture medium is PDA, The optimum carbon source is sucrose and optimum nitrogen source is ammonium nitrate. When culture in ammonium carbonate, its Mycelial growth was poor, and sclerotinia sclerotiorum does not produce sclerotium and sclerotium. Growth situation of sclerotinia sclerotiorum grow with the increase in volume of medium and the sugar content. Acid condition is better for sclerotinia sclerotiorum growthing and sclerotium forming.The most optimum pH is pH6.