Overexpression of antisense phosphatase 2C affords cold resistance in hybrid Populus davidiana × Populus bolleana.

BACKGROUND: Overexpression of the abiotic and biotic stress-resistance genes of the plant signaling pathway is well known for its significant role in the regulation of plant growth and enhancement of the productivity of agricultural land under changing climatic conditions. OBJECTIVES: This research aimed to clone Populus davidiana × Populus bolleana PP2C (PdPP2C) gene and analyze its structure and function, and downregulate PdPP2C by overexpression of its antisense PdPP2C (AS-PdPP2C) gene for enhancing cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. METHODS: PdPP2C was cloned and transformed to identify its function, and its antisense was overexpressed via downregulation to increase the cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. RESULTS: Antisense inhibition of protein phosphatase 2C accelerates the cold acclimation of Poplar (P. davidiana × P. bolleana) gene in terms of antifreeze. CONCLUSION: PdPP2C was expressed in the roots, stems, and leaves of P. davidiana × P. bolleana, and the expression was higher in the leaves. The expression of PdPP2C was also significantly downregulated at low-temperature (0 °C and 4 °C) stress. The relative conductivity and malondialdehyde content of non-transgenic lines were higher than those of AS-PdPP2C lines after 2 days of cold treatment at - 1 °C. The leaves of the transgenic lines were not wilted and showed no chlorosis compared with those of the non-transgenic lines. The AS-PdPP2C transgenic lines also showed higher freezing resistance than the non-transgenic lines. AS-PdPP2C participated in the regulation of freezing resistance.

Analysis of drought and salt-alkali tolerance in tobacco by overexpressing WRKY39 gene from Populus trichocarpa.

WRKY is one of the largest families of transcription factors in plants. It not only regulates plant growth and development but also participates in the regulation of plant defense against biological and abiotic stresses. In this study, research was aimed to overexpress WRKY39 gene of P. trichocarpa (PtWRKY39) and to identify its important role played in drought and saline-alkali tolerance in tobacco model plant. Under the control of CaMV35S promoter, the overexpression of PtWRKY39 gene was increased to more than 10 times in T3 generation of transgenic tobacco plant. The drought resistance and saline-alkali tolerance were evidenced in overexpressed PtWRKY39 transgenic lines at germination/seedling stage. The overall germination rate, fresh weight, and chlorophyll contents of overexpressed lines were significantly higher while the level of malondialdehyde was significantly lower in PtWRKY39 transgenic lines than that of wild type (WT) lines. The content of H2O2 in leaves was detected by the 3, 3-Diaminobenzidine method showed that the overexpression of PtWRKY39 gene could reduce the accumulation of ROS (mainly H2O2) and enhance salt-alkali tolerance. Phenotypic analysis at 7-leaf pot transgenic seedlings stage treated with the saline-alkali soil extract and salt NaCl under root irrigation stress, revealed growth of the transgenic line was significantly higher than that of WT. This work concludes that overexpression of PtWRKY39 gene can improve the regulation of drought resistance and saline-alkali tolerance of transgenic plants during seed germination and vegetative growth.

Overexpression of transcription factor SlWRKY28 improved the tolerance of Populus davidiana × P. bolleana to alkaline salt stress.

BACKGROUND: WRKY transcription factors (TFs) have been suggested to play crucial roles in the response to biotic and abiotic stresses. This study is the first to report the alkaline salt regulation of the WRKY gene. RESULTS: In this study, we cloned a WRKY gene (SlWRKY28) from the Salix linearistipularis and then transferred to the Populus davidiana × P. bolleana for expression. Sequence analysis on the transcriptome of Salix linearistipular showed the significant up-regulation of WRKY gene expression in response to salt-alkali stress in seedlings. Our data showed that SlWRKY28 localized to the nucleus. Furthermore, the expression of the SlWRKY28 from female plants increased with saline-alkali stress according to the northern blot analysis results. The results of 3,3'-Diaminobenzidine (DAB) staining showed that hydrogen peroxide (H2O2) concentration was lower under stress, but ascorbate peroxidase (APX) enzyme activity was significantly higher in the overexpressed plants than that in non-transgenic (NT) plants. CONCLUSIONS: We found out the SlWRKY28 induced regulation of the enzyme gene in the reactive oxygen species (ROS) scavenging pathway is a potential mechanism for transgenic lines to improve their resistance to alkaline salt. This study shows theoretical and practical significance in determining SlWRKY28 transcription factors involved in the regulation of alkaline salt tolerance.

Caracterización genotípica de Staphylococcus aureus resistente a la meticilina prevalente en hospitales de Pakistán / Genotypic characterization of prevalent methicillin resistant Staphylococcus aureus in Pakistani community hospitals / CaracterizaþÒo de Staphylococcus aureus resistente O meticilina prevalecente em hospitais da comunidade no PaquistÒo

El gen de la proteína A se usó como marcador genético para la caracterización de aislados de Staphylococcus aureus resistentes a la meticilina (SAMR). De un total de 130 aislados de Staphylococcus aureus, 90 fueron identificados como SAMR y 81 de éstos se pudieron caracterizar por tipificación spa. Todos estos aislados fueron obtenidos de cinco Hospitales Nacionales de la Comunidad. Se utilizaron dos juegos diferentes de cebadores para amplificar la región-X del gen de la proteína A en las cepas de SAMR. Un conjunto de cebadores, spa-F/spa-R ha identificado tres tipos de repeticiones diferentes, a saber, 7 repeticiones (spa 2), 8 repeticiones (spa 3) y 10 repeticiones (spa 4) y otro conjunto de cebadores, spa-1113F/spa-1514R ha identificado 4 tipos de repeticiones diferentes, a saber, 6 repeticiones (spa 1), 15 repeticiones (spa 6), y 17 repeticiones (spa 7) y 19 repeticiones (spa 8). Se identificó la repetición 11 (spa 5) con ambos conjuntos de cebadores. Los tipos de SAMR esporádicos que portaban las repeticiones 6, 7, 10, 17 y 19 fueron poco prevalentes mientras que los SAMR epidémicos con 8, 11, y 15 repeticiones fueron más prevalentes y se los consideró involucrados en la transmisión entre los pacientes dentro de los diferentes hospitales. Este trabajo concluye que la técnica spa es lo suficientemente eficiente como para diferenciar las cepas epidémicas, esporádicas y aquéllas que se transforman lentamente de esporádicas a epidémicas.(AU)

Protein A gene was used as a genetic marker for the characterization of Pakistani methicillin resistant Staphylococcus aureus (MRSA) isolates. Out of a total of 130 Staphylococcus aureus isolates, 90 were identified as MRSA and of these 90 MRSA, 81 MRSA isolates were characterized by spa typing. All of these isolates were collected from five National Community Hospitals. Two different sets of primers were used to amplify the X-region of Protein A gene in MRSA strains. One set of primers i.e, spa-F/spa-R identified three types of different repeats viz., 7 repeats (spa 2), 8 repeats (spa 3) and 10 repeats (spa 4) and another set of primers i.e. spa-1113F/spa-1514R identified 4 types of different repeats viz., 6 repeats (spa 1), 15 repeats (spa 6), and 17 repeats (spa 7) and 19 repeats (spa 8). Repeat 11 (spa 5) was identified with both sets of primers. Sporadic MRSA types carrying 6, 7, 10, 17 and 19 repeats were less prevalent, while the epidemic MRSA with 8, 11 and 15 repeats were more prevalent and considered to be involved in transmission among the patients within different hospitals. Research work concludes that spa technique is efficient enough to differentiate spa strains carrying variations in general and those slowly transforming from sporadic to epidemic outbreak in particular.(AU)

O gene da proteína A foi usado como marcador genético para a caracterizaþÒo de isolados de Staphylo¡coccus aureus resistentes O meticilina (SAMR). De um total de 130 isolados de Staphylococcus aureus, 90 foram identificados como SAMR e 81 destes puderam se caracterizar por tipificaþÒo spa. Todos estes isolados foram obtidos de cinco Hospitais Nacionais da Comunidade. Utilizaram-se dois jogos diferentes de cevadores para amplificar a regiÒo-X do gene da proteína A nas cepas de SAMRUn conjunto de ce¡vadores, spa-F/spa-R tem identificado trÛs tipos de repetiþ§es diferentes, a saber, 7 repetiþ§es (spa 2), 8 repetiþ§es (spa 3) e 10 repetiþ§es (spa 4) e outro conjunto de cevadores, spa-1113F/spa-1514R tem identificado 4 tipos de repetiþ§es diferentes, a saber, 6 repetiþ§es (spa 1), 15 repetiþ§es (spa 6), e 17 repetiþ§es (spa 7) e 19 repetiþ§es (spa 8). Foi identificada a repetiþÒo 11 (spa 5) com ambos os conjuntos de cevadores. Os tipos de SAMR esporádicos que tinham as repetiþ§es 6, 7, 10, 17 e 19 foram pouco prevalecentes enquanto que os SAMR epidÛmicos com 8, 11, e 15 repetiþ§es foram mais prevalecentes e sÒo considerados envolvidos na transmissÒo entre os pacientes dentro dos diferentes hospitais. Este trabalho conclui que a técnica spa é o suficientemente eficiente como para diferenciar as cepas epidÛmicas, esporádicas e aquelas que se transformam lentamente de esporádicas em epidÛmicas.(AU)

Caracterización genotípica de Staphylococcus aureus resistente a la meticilina prevalente en hospitales de Pakistán / Genotypic characterization of prevalent methicillin resistant Staphylococcus aureus in Pakistani community hospitals / Caracterização de Staphylococcus aureus resistente à meticilina prevalecente em hospitais da comunidade no Paquistão

El gen de la proteína A se usó como marcador genético para la caracterización de aislados de Staphylococcus aureus resistentes a la meticilina (SAMR). De un total de 130 aislados de Staphylococcus aureus, 90 fueron identificados como SAMR y 81 de éstos se pudieron caracterizar por tipificación spa. Todos estos aislados fueron obtenidos de cinco Hospitales Nacionales de la Comunidad. Se utilizaron dos juegos diferentes de cebadores para amplificar la región-X del gen de la proteína A en las cepas de SAMR. Un conjunto de cebadores, spa-F/spa-R ha identificado tres tipos de repeticiones diferentes, a saber, 7 repeticiones (spa 2), 8 repeticiones (spa 3) y 10 repeticiones (spa 4) y otro conjunto de cebadores, spa-1113F/spa-1514R ha identificado 4 tipos de repeticiones diferentes, a saber, 6 repeticiones (spa 1), 15 repeticiones (spa 6), y 17 repeticiones (spa 7) y 19 repeticiones (spa 8). Se identificó la repetición 11 (spa 5) con ambos conjuntos de cebadores. Los tipos de SAMR esporádicos que portaban las repeticiones 6, 7, 10, 17 y 19 fueron poco prevalentes mientras que los SAMR epidémicos con 8, 11, y 15 repeticiones fueron más prevalentes y se los consideró involucrados en la transmisión entre los pacientes dentro de los diferentes hospitales. Este trabajo concluye que la técnica spa es lo suficientemente eficiente como para diferenciar las cepas epidémicas, esporádicas y aquéllas que se transforman lentamente de esporádicas a epidémicas.

Protein A gene was used as a genetic marker for the characterization of Pakistani methicillin resistant Staphylococcus aureus (MRSA) isolates. Out of a total of 130 Staphylococcus aureus isolates, 90 were identified as MRSA and of these 90 MRSA, 81 MRSA isolates were characterized by spa typing. All of these isolates were collected from five National Community Hospitals. Two different sets of primers were used to amplify the X-region of Protein A gene in MRSA strains. One set of primers i.e, spa-F/spa-R identified three types of different repeats viz., 7 repeats (spa 2), 8 repeats (spa 3) and 10 repeats (spa 4) and another set of primers i.e. spa-1113F/spa-1514R identified 4 types of different repeats viz., 6 repeats (spa 1), 15 repeats (spa 6), and 17 repeats (spa 7) and 19 repeats (spa 8). Repeat 11 (spa 5) was identified with both sets of primers. Sporadic MRSA types carrying 6, 7, 10, 17 and 19 repeats were less prevalent, while the epidemic MRSA with 8, 11 and 15 repeats were more prevalent and considered to be involved in transmission among the patients within different hospitals. Research work concludes that spa technique is efficient enough to differentiate spa strains carrying variations in general and those slowly transforming from sporadic to epidemic outbreak in particular.

O gene da proteína A foi usado como marcador genético para a caracterização de isolados de Staphylo­coccus aureus resistentes à meticilina (SAMR). De um total de 130 isolados de Staphylococcus aureus, 90 foram identificados como SAMR e 81 destes puderam se caracterizar por tipificação spa. Todos estes isolados foram obtidos de cinco Hospitais Nacionais da Comunidade. Utilizaram-se dois jogos diferentes de cevadores para amplificar a região-X do gene da proteína A nas cepas de SAMRUn conjunto de ce­vadores, spa-F/spa-R tem identificado três tipos de repetições diferentes, a saber, 7 repetições (spa 2), 8 repetições (spa 3) e 10 repetições (spa 4) e outro conjunto de cevadores, spa-1113F/spa-1514R tem identificado 4 tipos de repetições diferentes, a saber, 6 repetições (spa 1), 15 repetições (spa 6), e 17 repetições (spa 7) e 19 repetições (spa 8). Foi identificada a repetição 11 (spa 5) com ambos os conjuntos de cevadores. Os tipos de SAMR esporádicos que tinham as repetições 6, 7, 10, 17 e 19 foram pouco prevalecentes enquanto que os SAMR epidêmicos com 8, 11, e 15 repetições foram mais prevalecentes e são considerados envolvidos na transmissão entre os pacientes dentro dos diferentes hospitais. Este trabalho conclui que a técnica spa é o suficientemente eficiente como para diferenciar as cepas epidêmicas, esporádicas e aquelas que se transformam lentamente de esporádicas em epidêmicas.

Evaluation of trace metals in tobacco of local and imported cigarette brands used in Pakistan by spectrophotometer through microwave digestion.

Uncontrolled exposure of active and passive smokers to trace metals causes increase in health risks. The primary objective of this study was to determine whether local and imported cigarette brands used in Pakistan, have elevated levels of metals or not. Six metals manganese (Mn), cobalt (Co), copper (Cu), cadmium (Cd), lead (Pb) and zinc (Zn) were determined in tobacco of twenty cigarette brands (local and imported) used in Pakistan by flame atomic absorption Spectrophotometry. To overcome contamination chances and for complete digestion of analytes a microwave digester was used. The analytical results showed highest concentration of Mn (84.78 microg/g dry weight), Cd (0.525 microg/g dry weight) and Zn (14.34 microg/g dry weight) metals in imported brands in relation to counterparts from the local brands. Certain elevated levels were observed for Co (3.344 microg/g dry weight), Pb (14.16 microg/g dry weight) and Cu (7.889 microg/g dry weight) metals in local brands. The inter-metal relationships in the tobacco of local and imported cigarette brands showed some integrated variation in the selected metal levels. In view of health risk associated with the above metals, there should be a strict quality control over monitoring of heavy metals during growing, processing and smoking of tobacco. Therefore, it is prudent to minimize exposure to toxic substances whenever possible because smoking and exposure to cigarette smoke is a confounder to be taken into account when carrying out epidemiological studies on human exposure to metals.