Antimicrobial peptide sm-985 from Teosinte and its application
A technology of antimicrobial peptides and teosinte, applied in the biological field, can solve problems such as shortage, adverse environmental impact, and difficult management
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Embodiment 1
[0034] Construction of Zea mexicana (Schrad.) Kuntze) cDNA library:
[0035] Teosinte cDNA library construction: total RNA extraction, mRNA purification, cDNA synthesis, cDNA integration into vector pBE-S, transformation into Escherichia coli competent cell HST08, and transformation into Bacillus subtilis competent cell SCK6. Colonies were randomly selected from the library. The PCR protocol was 95°C for 5 minutes; 95°C for 30s, 55°C for 30s, 72°C for 50s (28 cycles) and 72°C for 10 minutes. The quality of the cDNA library was determined by gel electrophoresis.
[0036] The cDNA insert from Teosinte was cloned into Bacillus subtilis SCK6, and the cDNA library consisted of 2500 single colonies. Due to the cDNA insertion, most of the randomly selected colonies had a band size of more than 500 bp (the empty vector band size), which indicated the high quality of the cDNA library. Colony PCR results for the entire cDNA library showed that more than 2000 colonies had cDNA inserts...
Embodiment 2
[0041] Determination of antibacterial spectrum and antibacterial effect of SM-985:
[0042] 1) The indicator bacteria used in this embodiment are as follows:
[0043] Gram-positive bacteria: wheat seedling blight (Clavibacterfangii.), tomato bacterial canker (Clavibacte rmichiganesis subsp.michiganesis.) and Bacillus subtilis (Bacillus subtilis) 168;
[0044] Gram-negative bacteria: Pseudomonas syringaepv.tomato DC3000., Ralstonia solanacearum., Xanthomonas campestris pathogenic species (Xanth omonas campestris pv.holcicola.), Xanthomonasoryza epv.oryzae and Escherichia coli BL21.
[0045] Luria-Bertani (LB) medium was used to cultivate Xanthomonas, King's B (KB) medium was used to cultivate Pseudomonas, and Nutrient agar (NA) medium was used to cultivate the remaining bacteria. Non-pathogenic indicator bacteria (Bacillus subtilis 168 and Escherichia coli BL21) were cultured at 37°C, while pathogenic indicator bacteria were cultured at 28°C.
[0046] 2) Determination of min...
Embodiment 3
[0059] Antibacterial mechanism of SM-985:
[0060] 1) SM-985 inhibits bacteria by increasing the permeability of bacterial cell membrane:
[0061] According to Van de Velde et al., plasma membrane damage can be analyzed by uptake of propidium iodide (PI) (Van De Velde et al., 2010). For each indicator bacteria, use 10mM sodium phosphate buffer (pH 7.0) to prepare a concentration of 1×10 7 CFU / ml of bacterial suspension. The bacterial suspension was treated with 10 μM of SM-985, and the bacterial suspension with the same volume was treated with sterile water as a control. Both the treatment and the control were cultured at 28°C and 37°C for 4 hours (inverted gently every 1 hour) to distinguish between pathogenic and non-pathogenic indicator bacteria. After incubation, PI dye was added to the treatment and control centrifuge tubes at a final concentration of 10 μg / ml and incubated for 15 minutes in the dark. Bacterial cells were centrifuged twice with 10 mM sodium phosphate bu...
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