RESUMEN
Although the palynology and sporophyte stage of Isöetes sinensis have been well studied, the biology of its gametophyte and embryo is less well understood. To date, the functions of several genes of I. sinensis and the molecular mechanisms of enzymes encoded by them remain to be studied. In the present study, the Fe-SOD gene of I. sinensis was successfully cloned using RT-PCR and rapid amplification of cDNA ends (RACE), and termed IsFeSOD. IsFeSOD has certain reference value in the classification of system evolution. The study also accumulated data for further research on the SOD gene. Bioinformatic analysis was employed to compare IsFeSOD with gene sequences obtained from other plants present in the GenBank. Furthermore, the recombinant pET32-FeSOD plasmids were transformed into Escherichia coli BL21 for expression. IsFeSOD was observed to have 1469 nucleotides that were predicted to encode 247 amino acids. The bioinformatic analysis revealed that IsFeSOD contained conserved TGGGA sequences, similar to eight other species, in addition to five other conserved sequences. The recombinant protein was about 43 kDa. Recombinant FeSOD was expressed, purified, and confirmed by western blotting. Alignment of complete Fe-SOD mRNA sequences from 9 species revealed several conserved sequences. A phylogenetic tree was constructed using MEGA4.1 and ClustalX multiple-sequence alignment programs. This study could be helpful in further characterization of SOD genes and for classification of system evolution status.
Asunto(s)
Briófitas/enzimología , Briófitas/genética , Regulación de la Expresión Génica de las Plantas , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica , Filogenia , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismoRESUMEN
The main aim of this study was to understand the relationship between the drug-resistant characteristics of Klebsiella pneumoniae and CTX-M-type extended spectrum ß-lactamases (ESBLs), and to detect the distributions of CTX-M-type ESBLs in clinically isolated strains. CTX-M ESBL genes isolated from the clinical samples were amplified by polymerase chain reaction and identified by sequence analysis; the antibiotic susceptibility of the samples was determined using the Kirby-Bauer disc-diffusion method. One hundred and five strains among the 246 isolated strains of K. pneumoniae tested positive for ESBL production (42.68%); 92 of these produced CTX-M ESBLs. Of the 92 CTX-M ESBL strains, 81 produced CTX-M-1 ESBLs and 11 produced CTX-M-25 ESBLs. Fifty-seven of the CTX-M-1 ESBL- and six of the CTX-M-25 ESBL-producing bacteria had CTX-M ESBL genes that coexisted in the plasmid and chromosome. The Kirby-Bauer antibiotic susceptibility method revealed that CTX-M ESBL-positive strains showed a higher rate of resistance to cefazolin, cefoxitin, cefuroxime, ceftazidime, cefotaxime, aztreonam, levofloxacin, and cotrimoxazole, compared to the CTX-M ESBL-negative strains (P < 0.05). The CTX-M ESBL genes were commonly observed in the K. pneumoniae isolated from respiratory tract samples; these were significantly associated with the drug-resistant characteristics of K. pneumoniae to ß-lactam antibiotics.