Study On The Function Of Chitinase From Aeromonas Salmonicida

Chitin is the second most abundant natural polysaccharide in nature,and its application in medicine has received increasing attention.Chitinase is a glycosyl hydrolase that can efficiently degrade chitin into oligosaccharides or even monosaccharides.Most bacteria in the Vibrio family are capable of producing chitinase,and Chitin is ubiquitous in the fungal cell wall and Diatom cell wall.Based on this,this study explores whether bacteria can secret chitinase to inhibit related algae,fungi by Means of gene knockout and reducing sugar detection,etc.Aeromonas salmonicida belongs to the family Vibrio in marine bacteria and is capable of producing chitinase.In this study,we will study the function of chitinase in chitin cycle by comparing the degradation of chitin and the inhibition to diatom and fungus with the knockout strain and the wild type strainIn this study,we test the ability of Aeromonas salmonicida and knockout strain degrading chitin by DNS reductive sugar detection method in first.The Aeromonas salmonicida OD value at 12 hours was 0.125,the OD value at 24 hours was 0.209,and the OD value at 48 hours was 0.283.The OD value of knockout strain was 0.000 for 12 hours,0.002 for 24 hours and 0.007 for 48 hours.The chitinase-deficient strain was lost to the ability to degrade chitin.Since bacteria and diatoms are not symbiotic in the ocean,we speculate that bacteria can show chemotaxis to diatoms containing chitinous shells,so we verified this conjecture by applying a plate method to diatom droplets.Since bacteria and diatoms are not symbiotic in the ocean,we speculate that bacteria can show chemotaxis to diatoms containing chitinous shells.We verified this conjecture by dropping the diatom on agar plates containing bacterial cells by coating the plate method.After 12 hours of observation,it was found that Aeromonas salmonicum did indeed accumulate in the direction of diatoms,confirming that the bacteria had chemotaxis.Next,we examined the algaecicidal activity of the bacteria by cocultivating the wild strain and the knockout strain with diatom for 24 hours,respectively,and then detecting the algae fluorescence in the culture using a microplate reader.The results showed that the average fluorescene intensity of the control diatom was 274,the bacterial concentration of 0.3% was 270,the bacterial concentration of 0.5% was 273,the bacterial concentration of 1.0% was 261,the bacterial concentration of 2.0% was243,and the bacterial concentration of 5.0% was 201.The results showed that the diatoms treated by the wild strain bacteria had a significant decrease in fluorescence intensity when the bacterial concentration reached 1.0%,and the higher the bacterial concentration is,the stronger the algicidal activity is,so 2.0% was selected as the subsequent experimental concentration.The diatom were treated with wild type strains and the knockout strains for 24 hours,and fluorescence was detected.The average fluorescence intensity of the diatoms in the control group was 403,the fluorescence intensity of the diatoms treated by the wild type strains was 366,and the fluorescence intensity of the diatoms treated by the knockout strain was 403,which was no change in the fluorescence intensity of diatoms relative to the control group.Therefore,the knockout strain bacteria lost the algicidal activity,demonstrating that chitinase plays a key role in the bacterial algicidal process.Finally,we co-cultured the wild strains and the knockout strains with diatom and visualized it under scanning electron microscope.It was found that the wild strains of Aeromonas salmonicum could adhere to the surface of diatom and lyse the chitin shell,killing the diatoms;the knockout strains can only adhere to the surface of the diatom,while there is no fragmentation of the diatom.In the experiment of bacterial infection of fungi,this study verified the antibacterial effect by Oxford Cup method.The results showed that the growth of the fungus in the Oxford Cup with the bacteria of the wild strains was significantly inhibited,while there was no significant inhibition curve in the Oxford Cup with the bacteria of the missing strain.This proves that the wild strains have an inhibitory effect on the growth of fungi,while the knockout strains hava no inhibitory effect on the growth of fungi.Scanning electron microscopy showed that both the wild and knockout strains of Aeromonas salmonicida could adhere to the fungal cell wall.However,since the chitin contained in the fungal cell wall was located inside the cell wall,no cell wall lysis was observed.In order to further understand the effect of chitin on Vibrio,this study used the plate method to prepare Vibrio cultured in different medium,and found that when the environment lacks carbon sources,vibrio(Vibrio Parahemolyticus and Vibrio Anguillarum)can survive with chitin as a carbon sourse,and that when the environment is sufficient,chitin will inhibit the growth of Vibrio bacteria.By experimenting with the infection of diatoms and fungi by wild strains of Aeromonas salmonicida,knockout strains,this study finally concluded that chitinase plays a key role in the process of bacteria inhibiting diatoms and fungi,which is great value to the development and protection of algae and the prevention and control of fungal pathogens.