Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system

Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance.

in LB plates containing kanamycin (10 µg/ml). Single colonies were picked and shaken in LB containing kanamycin, and glycerol was added to save the strains at a -80 ℃ refrigerator.

SEM
The empty vector and AtR472 strains stored in the -80 ℃ refrigerator were taken out and streaked on LB plates containing kanamycin, and cultured at 37 ℃ for 12 h. Single colonies were picked and shaken at 180 rpm for 36 h in LB containing kanamycin. The 25 ml of the culture solution were centrifuged in a 50 ml centrifuge tube at 5000 rpm for 6 min. The supernatant was decanted, washed three times with PBS, and finally transferred to a 2 ml EP tube. The supernatant was removed by centrifugation, fixed with 2.5% glutaraldehyde, shaken, and allowed to stand at room temperature for 2 h. Wash with 1 ml of a concentration of 30%, 50%, 70%, and 90% ethanol, shake well, 8000 rpm, 3-5 min centrifugation. Finally, it was dehydrated with 1 ml of 100% ethanol, and dried by a freeze dryer. After coated with gold on the sample, a JSM-7001F (JEOL Japan) Scanning Electron Microscope was used to observe and save the image.

Protein extraction from B. subtilis expression system
The strains stored in the -80 ℃ refrigerator were taken out and streaked on 6 LB plates containing kanamycin, and cultured at 37 ℃ for 12 h. Single colonies were picked and shaken at 180 rpm for 60 h in LB containing kanamycin. The 15 ml of the culture solution was centrifuged in a 50 ml centrifuge tube at 4 ℃, 10000 rpm for 25 min. The supernatant was filled in a pre-cooled beaker and placed on ice, then, a saturated ammonium sulfate solution was continuously added to the beaker while stirring the liquid in the beaker until the liquid became cloudy. The beaker was placed in a 4 ℃ refrigerator overnight to precipitate the protein, and the fluffy protein was aspirated in a 50 ml centrifuge tube and centrifuged at 4 ℃, 10,000 rpm for 25 min. The supernatant was decanted and the protein was dissolved with 1 ml pre-cooled PBS solution. Each protein concentration was measured by a spectrophotometer, and each protein concentration (including the control protein) was adjusted with a PBS solution, and the adjusted protein was stored in a 4 ℃ refrigerator for use.

Hemolysis analysis
The hemolytic activity was tested with porcine erythrocyte, which was washed

Liquid fermentation and spray drying
The strains stored in the -80 ℃ refrigerator were taken out and streaked on the NA plate containing kanamycin, and cultured at 37 ℃ for 12 h. Single When the temperature is lowered to 40 ℃, the alcohol is burned in the sample port, and the B. subtilis culture solution is quickly added to the fermenter and fermented for 60 h.
After the fermentation was finished, the fermentation liquid is taken out, the spray drying device is turned on, and the spray drying machine is Triowin's SD-1500. The inlet air temperature and the rotation speed are set so that the obtained powder cannot be too wet or too dry, and the rotation speed is adjusted at any time to stabilize the inlet air temperature. The B. subtilis powder is finally obtained.

Preparation of B. cinerea conidial suspension
The B. cinerea was activated on the PDA, and the activated B. cinerea was cultured on a PDA plate for one week. The B. cinerea conidia on the PDA plate were washed with 2% glucose and filtered to obtain a conidial suspension. The conidial suspension was adjusted to a concentration of 1 × 10 6 conidia/ml. The conidial suspension was mixed 1:1 with the diluted B. subtilis powder to observe the conidial germination. The picture was taken by microscope NIKON ECLIPSE 55i (NIKON, Japan).

Western blot
Purified protein using Ni-Agarose Resin from CWBIO. The purified protein was isolated by CWBIO's Tricine-SDS-PAGE Gel kit using the pre-stained marker.
The isolated protein was transferred to a polyvinylidene difluoride (PVDF) membrane at 21 V and 185 mA for 21 min. The PVDF membrane was shaken with 5% skim milk for 2.5 h at room temperature to block non-specific binding.