RESUMO
Resumen Este estudio tuvo como objetivo aislar, caracterizar e identificar bacterias de control biológico que poseen actividad antifúngica de amplio espectro de la filosfera de diferentes cultivos, incluidos el maíz, el trigo y la papa, así como evaluar su actividad en la promoción del crecimiento. En este estudio, 14/113 bacterias de control biológico mostraron actividad antifúngica. Los aislamientos bacterianos M11 y M33 (de maíz), del total de 113, fueron reseleccionados debido a su fuerte actividad antifúngica de amplio espectro (más del 50%) después de su evaluación contra cuatro hongos fitopatógenos que afectan cultivos de alta importancia económica, entre ellos, Alternaría alternata, Rhizoctonia solani, Fusarium oxysporum y Fusarium verticillioides. Los aislamientos se evaluaron, adicionalmente, para determinar los rasgos que promueven el crecimiento de las plantas, es decir, producción de ácido indolacético, solubilización de fosfato, producción de celulasa, compuestos orgánicos volátiles microbianos, cianuro de hidrógeno y sideróforos. Las 14 cepas aisladas mostraron resultados positivos para la producción de la hormona ácido indolacético y la enzima celulasa; 10 cepas fueron positivas para la producción de cianuro de hidrógeno. Además, se observó producción de sideróforos en el caso de 7 cepas, mientras que 5 pudieron solubilizar fosfato inorgánico. Los compuestos orgánicos volátiles microbianos solo fueron sintetizados por los aislamientos M11 y M33, que fueron identificados como Bacillus amyloliquefaciens y Bacillus subtilis, respectivamente, mediante secuenciación del gen ARNr 16S. El estudio de supervivencia mostró que las bacterias de control biológico, es decir, B. amyloliquefaciens y B. subtilis, tienen la capacidad de sobrevivir sobre un sustrato a base de melasa, por un período de tres meses.
RESUMO
Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.
Assuntos
Antifúngicos/farmacologia , Quitinases/farmacologia , Hordeum/enzimologia , Proteínas Recombinantes/metabolismo , Antifúngicos/química , Antifúngicos/isolamento & purificação , Western Blotting , Quitinases/química , Quitinases/genética , Quitinases/isolamento & purificação , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hordeum/genética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
RNA interference(RNAi)is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the eapsid protein gene of potato virus Y(CP-PVY)was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.