RESUMEN
OBJECTIVE: To screen and characterize the aptamer of Escherichia coli outer member protein tolC. METHODS: By using the recombinant E.coli outer member protein tolC for the screening target, oligonucleotides which were capable of specifically binding to the protein were screened from a random oligonucleotide library through the stematic evolution of ligand by exponential enrichment (SELEX) technique. The binding capacity of ssDNA to the targeted protein from each round was detected by the FITC fluorescence labeling technique.The ssDNA from the last cycle was cloned and sequenced,and the second structure was further analyzed by the DNAMan program. RESULTS: After 12 cycles of selection, 40 clones were selected randomly and sequenced. Although a unique conserved sequence was not obtained among the 23 obtained aptamers by the primary structure analysis,three pairs of aptamers and two pairs of aptamers were found to be identical.Analysis of the secondary structure revealed that the stem-loop and bulge loop were the main motifs,indicating that they might play a key role in the binding of aptamers to the target protein. According to the characteristic of the second structure,23 aptamers were divided into four families,and aptamer 20 bore the greatest affinity. CONCLUSION: Aptamers against E.coli outer member protein tolC were successfully identified by the SELEX method. The results laid a foundation for the investigation of the interference to the drug resistance of E. coli and the underlying mechanisms.
Asunto(s)
Aptámeros de Nucleótidos/genética , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli , Proteínas de Transporte de Membrana/química , ADN de Cadena Simple , Biblioteca de Genes , Proteínas Recombinantes/química , Técnica SELEX de Producción de AptámerosRESUMEN
We collected evapotranspiration data of Dajiuhu peatland in Shennongjia from 2016 to 2017 with eddy covariance method and estimated the value of crop coefficient (Kc) using FAO56 Penman-Monteith equation and the linear relationship between actual evapotranspiration (ETa) and referenced evapotranspiration (ET0). We analyzed the characteristics of referenced evapotranspiration and its main influencing factors and calculated the crop coefficient of the wetland dominated by Sphagnum. The results showed that the daily averaged ETa were 1.63 and 1.38 mm·d-1 in 2016 and 2017, the daily averaged ET0 were 1.61 and 1.23 mm·d-1 in 2016 and 2017. Environmental factors influencing ET0 included net radiation, air temperature, vapor pressure deficit, wind speed, and relative humidity. The Kc values for the growing seasons of 2016, 2017, and 2016-2017 were 0.95 (R2 of linear regression between ETa and ET0 was 0.96), 1.03 (R2=0.95), and 0.98 (R2=0.95). The Kc values in 2016, 2017, and 2016-2017 were 0.92 (R2=0.94), 0.95 (R2=0.89), and 0.93 (R2=0.92). Kc was effective in the range of 0.92-1.03 for the wetland dominated by Sphagnum. The identified parameters could be widely used in studies on climate change, ecosystem services, and water management in peatlands.