RESUMEN
A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70 degrees C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28 degrees C (pH 6.0) in flasks stirred at 150 x g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization.
Asunto(s)
Coriolaceae/aislamiento & purificación , Coriolaceae/metabolismo , Lignina/metabolismo , Biocombustibles , Celulasa/metabolismo , China , Coriolaceae/genética , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Filogenia , Madera/microbiologíaRESUMEN
Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger(4)Ser)(3) and short-linker (Ger(4)Ser) were established to contain 1.04 x 10(7) or 6.07 x 10(6) transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.
Asunto(s)
Ésteres/química , Región Variable de Inmunoglobulina/inmunología , Biblioteca de Péptidos , Piretrinas/química , Piretrinas/inmunología , Secuencia de Aminoácidos , Animales , Bovinos , Sueros Inmunes/inmunología , Inmunización , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
In this study, the pitted peel and non-pitted peel of 'Fengjie' navel orange fruits were used as experimental materials to construct and screen the peel pitting related genes by suppression subtractive hybridization (SSH). The results showed that suppression subtractive hybridization was very effective. A cDNA library of differentially expressed genes was constructed. The library included about 200 clones with an average insert size of around 300 bp. Part of the positive clones were picked up randomly and sequenced. Six of the 50 clones had no homologous sequences being found and three had unknown functions in GenBank. According to the analysis of the homology, four homologous (Ca2+ binding protein, cysteine proteinase, NAC-domain protein and expansin) genes were chosen to examine their expressions through semi-quantitative RT-PCR analysis in pitted and non-pitted navel orange fruits. The expression of four genes were all higher in pitted peel than that in non-pitted peel. It suggests that these genes in the SSH cDNA library may be involved with peel pitting and can be subject of future investigation to explore the molecular biological mechanism of the pitting of citrus fruit.
Asunto(s)
Citrus sinensis/genética , Citrus/genética , Hibridación de Ácido Nucleico/métodos , Secuencia de Bases , ADN de Plantas/análisis , Perfilación de la Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , Técnica de SustracciónRESUMEN
Citrus fruit is prone to develop peel pitting during development and storage, which greatly decreases its fresh market value because of the deterioration of the peel. In the present study, we have examined the effect of different temperatures (15 degrees C and 4 degrees C), waxing and mechanical damage on the changes in the activity of phenylalanine ammonia-lyase (PAL) and the incidence of peel pitting in 'Fengjie' navel orange (Citrus sinensis Osbeck) fruits. The expression levels of PAL2, PAL6 genes in the peel during the development of peel pitting have been investigated through semi-quantitative PCR method. The incidence of peel pitting was greatly enhanced by waxing and mechanical damage and was decreased in lower temperature storage (4 degrees C) (Fig.1). Waxing and mechanical damage might be the important factors inducing peel pitting and suitable low temperature could decrease the incidence of this disease. The PAL activity increased during the whole storage period in accordance with the development of this pitting (Fig.2). The expression levels of PAL2 and PAL6 genes in damaged peel were higher than those in healthy peel and the expression of PAL2 is much more higher than that of PAL6 (Figs.4 and 5). The results suggested that the enzyme activity of PAL, along with the expression of PAL2 gene is highly related to this peel pitting occurred on 'Fengjie' navel orange fruits.
