RESUMEN
This study assessed the effect of supplementation of novel transgenic phytase on growth performance and bone mineralization in Korean native broiler chickens. The experiment was designed using four dietary groups: those with a diet supplemented with (A) recombinant phytase, (B) transgenic phytase from the plant Lemna minor, (C) or wild-type L. minor as well as (D) a control group that was supplemented with commercially available feed. Three hundred 1-day-old Korean native broiler chicks were used and divided into these four dietary treatment groups having three replicates of 25 birds each (n = 75). The results showed increases in growth performance and bone mineralization in Groups B and C; compared with Groups A and D. Hematological analyses revealed notable contrasts in erythrocyte sedimentation rate, red blood cell count, and hemoglobin levels among the experimental groups, whereas no impacts of dietary treatment were observed on total eosinophil, lymphocyte, heterophil, monocyte, and basophil levels. The relative expression profiling of candidate genes showed that the genes involved in growth response, meat quality, and P-Ca metabolism were significantly highly expressed in the phytase-supplemented groups. Hence, it is suggested that dietary supplementation with transgenic phytase plant L. minor for enhancing growth performance is a promising new approach in the broiler feed industry. To the best of our knowledge, we report here the most comprehensive analysis using a broiler model that provides a workable platform for further research on the cost-effective production of feed with different compositions that might be beneficial in the livestock feed industry.
Asunto(s)
6-Fitasa/genética , Alimentación Animal , Araceae/genética , Plantas Comestibles/genética , 6-Fitasa/química , Animales , Araceae/química , Calcificación Fisiológica/genética , Pollos/crecimiento & desarrollo , Suplementos Dietéticos , Plantas Modificadas Genéticamente/genéticaRESUMEN
A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.
Asunto(s)
6-Fitasa/metabolismo , Aspergillus nidulans/enzimología , Proteínas Fúngicas/metabolismo , Nicotiana/metabolismo , 6-Fitasa/genética , 6-Fitasa/farmacocinética , Animales , Aspergillus nidulans/genética , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacocinética , Glicosilación , Eliminación Intestinal , Masculino , Fósforo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Especificidad por Sustrato , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
Ginseng ESTs allowed us to identify an unknown transcript which is highly abundant in rhizomes and seeds. We called the cDNA ginseng-specific abundant protein (GSAP), and identified three homologues, GSAP1, GSAP2, and GSAP3. GSAP cDNAs encode a small polypeptide consisting of 121 or 117 amino acids, and GSAP3 shows 87.6% amino acid sequence homology with GSAP1. GSAP transcripts were detected in most plant tissues, but GSAP3 is highly expressed in seeds, and is up-regulated under stressed conditions, water deficit. GSAP3-GFP fusion protein is located in the cell wall when expressed in onion epidermis cells. The transgenic Arabidopsis seedlings which over-expressed GSAP3 grew faster than those of the wild-type plant on the medium containing 300 mM mannitol and 100 mM NaCl. GSAP3 may play a role in altering the characteristics of the cell wall to allow for more tolerance of water deficit stress under abiotic stress conditions.