RESUMO
Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.
Assuntos
Agrobacterium , Southern Blotting , Digestão , Genoma , Genômica , Métodos , Microscopia de Fluorescência , Oxirredutases , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Ferimentos e LesõesRESUMO
Se realizó un estudio descriptivo y transversal de 75 pacientes con diabetes mellitus, quienes presentaron candidiasis bucal, tratados en la consulta estomatológica del Policlínico de Especialidades del Hospital Provincial Docente Clinicoquirúrgico "Saturnino Lora Torres" de Santiago de Cuba, desde marzo de 2013 hasta igual mes de 2015, a fin de evaluar los resultados del diagnóstico clínico e histopatológico de la citada afección. Las muestras para biopsias se fijaron y procesaron con la técnica clásica de inclusión en parafina; se colorearon con una tinción especial de ácido peryódico de Schiff, lo cual confirmó la existencia de cambios celulares. Predominaron el sexo masculino, el grupo etario de 60 años y más; la ausencia de manifestaciones clínicas, el paladar duro y la comisura labial como los sitios de mayor prevalencia y la candidiasis eritematosa como la forma clínica más común. La presencia de hifas patógenas y células levaduriformes fueron los hallazgos histopatológicos más sobresalientes.
A descriptive and cross-sectional study of 75 patients with diabetes mellitus who presented oral candidiasis, treated in the stomatological service of the Specialties Polyclinic of "Saturnino Lora Torres" Teaching Provincial Clinical Surgical Hospital in Santiago de Cuba was carried out, from March, 2013 to the same month in 2015, in order to evaluate the results of the clinical and histopathologic diagnosis of the mentioned affection. The samples for biopsies were fixed and processed with the classic technique of inclusion in paraffin; they were colored with a special stain of Schiff periodic acid, which confirmed the existence of cellular changes. There was a prevalence of male sex, the 60 years and over age group; the absence of clinical symptoms, the hard palate and the corner of the mouth as the sites of greater prevalence and the erythematous candidiasis as the most common clinical form. The presence of pathogen hyphae and yeast-like cells were the most outstanding histopathologic findings.