ABSTRACT
RESUMEN La expresión transitoria es una métodología ampliamente utilizada para el estudio de genes. Sin embargo, hasta la fecha no existe un reporte en donde se utilice esta técnica en hojas de yuca de plantas adultas. Por esta razón este trabajo se centró en la determinación de algunos parámetros críticos para la expresión transitoria del gen GUS en yuca como son: la metodología para introducir [a bacteria, [a cepa de Agrobacterium, el tiempo post-inoculación, la introducción del gen VirG y la expresión del gen GUS en algunas variedades de yuca. Los resultados indicaron niveles más altos de expresión del gen GUS entre 5-7 días postinoculación (dpi), agroinfiltrando con la cepa GV3101 y un incremento en la virulencia de esta cepa mediante la introducción del gen VirG. Por último se observaron diferentes niveles de expresión del gen GUS entre [as variedades de yuca evaluadas, [o que indica que el factor genético es clave en la eficiencia de la agroinfiltración en este cultivo.
ABSTRACT Transient expression is a common technique used for the co-expression of proteins for a wide range of experiments in molecular biology. This technique has been frequently used for gene study in plant pathogen interaction and has proved to be versatile and effective. However, there are still few reports and sparse data for transient expression in cassava leaves from adult/mature plants. For this reason, in this study we evaluated some regards of transient expression for the gene GUS in cassava: bacteria introduction methods, type of Agrobacterium strains, time post-inoculation, introduction of gene VirG and GUS and their expression into different cassava varieties. The results show that GUS expression is more efficient between 5-7 days post-inoculation with the GV3101 strain, moreover the virulence of this strain increases with the VirG gene. Finally, we observed a range of GUS expression pattern between different cassava varieties, altogether showing that the genetic factor is important for an accurate and effective agroinfiltration in cassava.
ABSTRACT
Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.
Objetivo. Evaluar la expresión transitoria del gen GUS en hojas de yuca (Manihot esculenta Crantz) por medio de infiltración con Agrobacterium tumefaciens. Materiales y métodos. Se utilizaron las cepas GV3101 y AGL1 de A. tumefaciens conteniendo el plásmido pCAMBIA1305.2, para evaluar la expresión transitoria del gen GUS. La infiltración de A. tumefaciens (agroinfiltración) se realizó tanto en hojas de plantas "in Vitro" como de plantas adultas de 1 mes. Las hojas se incubaron en tampón X-GLUC, se destiñeron y se fotografiaron para detectar la actividad de la enzima β-glucuronidasa (GUS). Resultados. Los ensayos de agroinfiltración en hoja muestraron la expresión transitoria del gen GUS en variedades cultivadas en la costa norte y en los llanos orientales, MCOL2215 (Venezolana) y CM6438-14 (Vergara) respectivamente, tanto en plantas "in Vitro" como en plantas adultas. La cepa hipervirulenta de A. tumefaciens AGL1 mostró una mayor eficiencia para la expresión transitoria en hojas de yuca. Conclusiones. Se recomienda utilizar la cepa AGL1 para evaluar la expresión transitoria de genes de interés por agroinfiltración en hojas de yuca.
Subject(s)
Agrobacterium tumefaciens , Glucuronidase , ManihotABSTRACT
OBJECTIVES: The purpose of this study was to get help in order to diagnose orthopaedic disease, measure its activity and determine treatment plan by measuring the beta-glucuronidase activity in urine, serum and joint fluid. METHODS: The beta-glucuronidase activity was determined in the serum, urine and joint fluid of the patients with degenerative arthritis, rheumatoid arthritis, osteomyelitis and osteogenic sarcoma, and some other disease to study the change of the enzyme activity. These values of each specimen were calculated by standard curve and treated by statistical analysis. RESULTS: The results obtained were summarized as follows. 1. The beta-glucuronidase activity in the serum, urine and joint fluid was increased in patients with degenerative arthritis, rheumatoid arthritis, osteomyelitis and osteogenic sarcoma etc. 2. The increased beta-glucuronidase activity in the serum and joint fluid of each disease does not show a specific finding about respective disease, but the increased beta-glucuronidase activity was statistically significant in the urine of all disease groups(male:p=0. 0041, female:p=0. 0001). CONCLUSIONS: On the basis of these results, it was suggested that beta-glucuronidase activity was affected by the orthopaedic disease and differed according to each specimen.
Subject(s)
Humans , Arthritis, Rheumatoid , Glucuronidase , Joints , Osteoarthritis , Osteomyelitis , OsteosarcomaABSTRACT
BACKGROUND: The confirmative diagnosis of pulmonary tuberculosis(Tb) can be made by the isolation of Mycobacterium Tuberculosis(MTb) in the culture of the sputum, respiratory secretions or tissues of the patients, but positive result could not always be obtained in pulmonary Tb cases. Although there are many indirect ways of the diagnosis of Tb, clinicians still experience the difficulty in the diagnosis of Tb because each method has its own limitation. Therefore development of a new diagnostic tool is clinically urgent. It was reported that silica cause some lysosomal enzymes to be released from macrophages in vitro and one of these enzymes is elevated in workers exposed to silica dust and in silicotic subjects. In pulmonary Tb, alveolar macrophages are known to be activated after ingestion of MTb. Activated macrophages can kill MTb through oxygen free radical species and digestive enzymes of lysosome. But if macrophages allow the bacilli to grow intracellularly, the macrophages will die finally and local lesion will enlarge. Then it is assumed that the lysosomal enzymes would be released from the dead macrophages. The goal of this investigation was to determine if there are differences in the plasma activities of lysosomal enzymes, beta-glucuronidase(GLU) and beta-N- acetyl glucosaminidase(NAG), among the groups of active and inactive pulmonary Tb and healthy control, and to see if there is any possibility that the plasma activity of GLU and NAG can be used as diagnostic indicies of active pulmonary Tb. METHODS: The plasma were obtained from 20 patients with bacteriologically proven active pulmonary Tb, 15 persons with inactive Tb and 20 normal controls. In 10 patients with active pulmonary Tb, serial samples after 2 months of anti-Tb medications were obtained. Plasma GLU and NAG activities were measured by the fluorometric methods using 4-methylumbelliferyl sub- strates. All data are expressed as the mean +/-the standard error of the mean. RESULTS: The activites of GLU and NAG in plasma of the patients with active Tb were 21.52 +/-3.01 and 325.4+/-23.37(nmol product/h/ ml of plasma), respectively. Those of inactive pulmonary Tb were 24.87+/-3.78, 362.36+/-33.92 and those of healthy control were 25.45 +/-4.05, 324.44+/-28.66 (nmol product/ h/ml of plasma), respectively. There were no significant differences in the plasma activities of both enzymes among 3 groups. The plasma activities of GLU at 2 months after anti-Tb medications were increased(42.18+/-5.94 nmol product/h/ ml of plasma) in the patients with active pulmonary Tb compared with that at the diagnosis of Tb(P-value <0.05). CONCLUSION: The results of the present investigation suggest that the measurement of the plasma activities of GLU and NAG in the patients with active pulmonary Tb could not be a useful method for the diagnosis of active Tb. Further investigation is necessary to define the reasons why the plasma activities of the GLU was increased in the patients with active pulmonary Tb after Tb therapy.