RESUMEN
OBJECTIVE: To find a natural plant essential oil (EO) with excellent antimicrobial effects on food-borne bacteria and to explore the mechanism of its antimicrobial function against Escherichia coli (E. coli). METHODS: The antimicrobial activity of seven EOs against Gram-negative E. coli ATCC 8739 and Gram-positive Staphylococcus aureus ATCC 6538 was investigated using agar disk diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each EO was determined using the broth dilution method. The chemical composition of the Trachyspermum copticum (T. copticum) EO was analyzed using gas chromatography-mass spectrometry (GC/MS). In order to explore the mechanism of the antimicrobial action, 1 MIC and 2 MIC of T. copticum EO was added to a suspension of E. coli, the growth curve and the scanning electron microscopy (SEM) analysis of E. coli, and the release of cell constituents and protein and potassium ions from the bacterial cell were measured. RESULTS: The T. copticum EO had the best antimicrobial activity against the test bacteria, and 10 compounds accounting for 94.57% of the total oil were identified, with the major components being thymol (46.22%), p-cymene (19.03%), and γ-terpinene (22.41%). The addition of 1 MIC that T. copticum EO significantly inhibited the growth of E. coli and increased the release of cell constituents and protein and potassium ions from the bacterial cells. Scanning electron micrographs showed that T. copticum EO caused most of the E. coli cell membranes to collapse and rupture, leading to cell death. CONCLUSIONS: These results indicate that T. copticum EO is a good natural antimicrobial agent for food-borne pathogens.
RESUMEN
According to the sequences of the gene nhaA coding for Na+ / H+ antiporter,a structural gene was cloned from Pseudomonas sp. cn4902 by PCR reaction with a set of primers. It was 1089 bp in length and codes for 362 amino acids sharing homology with the gene nhaA of E. coli K12 as high as 97.0%. It was inserted into plasmid pBV220 to form a high level expression reconstruction plasmid pBVA. So an overexpression 41 kD protein band could be found in the lane of transformant harbored with pBVA after SDS-PAGE electrophoresis. The detection of growth curve showed that the biomass of the transformant was 2.3 times over that of the control in the medium containing 1.0 mol/L NaCl. It was found that Na+ concentration in cytoplasm of the transformant was low to 60.4% of the control by the detection of atomic absorption spectrum. Evidence of SDS-PAGE electrophoresis of membrane proteins also showed that the NhaA was located in membrane. Purified NhaA was harvested and digested by FXa proteinase. The sequence of eight amino acids in N termination of NhaA protein was entirely identical with the polypeptide deduced from the nhaA gene. Then ten strains of transformant were continuously cultivated for 18 generations under 42 degrees C hot shock condition,all of their reconstructed plasmids were lost with the result that salt-tolerant-level went back to the original standard. In summary, all the experiments proved that the cloned gene is nhaA gene. The gene has been accepted in GenBank by the accession number AY643494.
Asunto(s)
Proteínas Bacterianas/genética , Pseudomonas/genética , Intercambiadores de Sodio-Hidrógeno/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Pseudomonas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/aislamiento & purificación , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
In order to improve the embryogenic callus induction rate and the regeneration rate of JiaHe-ZaoZhan rice, the influence of different factors were investigated, media with different hormones were used. Induction medium was supplemented with 1.5 mg/L 2,4-D + 3 mg/L NAA + 0.1 mg/L KT + 1 mg/L phytic acid + 20 mg/L PAA. Embryogenic call were treated under the condition of 25 degrees C before transferring to regeneration medium, the regeneration medium contained 0.5 mg/L 6-BA + 3 mg/L NAA + 0.5 mg/L KT + 1 mg/L phytic acid. The experiment results indicated that the hormone treatments had certain effects on the callus induction. Under the optimal medium, culture condition and the hormone treatments, the embryogenic callus induction could reach over 95%, and dry treatment of embryogenic callus had been found to increase the frequency of plant regeneration, significantly the plant regeneration rate could reach over 80%. Transplanted into pots, the young plants grew well. Then a experimental system with stability and high regenerating efficiency has been established for the mature seeds of rice (JiaHe-ZaoZhan).
