RESUMEN
Acetyl-CoA C-acetyltransferase synthase gene (AACT) cDNA, DNA and promoter were cloned from Sanghuangporus baumii. The gene ORF (1260 bp) encoded 419 amino acids. The AACT DNA includes five exons (1-84 bp, 140-513 bp, 570-1027 bp, 1090-1282 bp, 1344-1494 bp) and four introns (85-139 bp, 514-569 bp, 1028-1089 bp, 1283-1343 bp). The molecular weight of AACT protein is 43.40 kDa, it is hydrophilic with a theoretical isoelectric point of 8.96. Furthermore, The region of the transcription start site is 1997-2047 bp of AACT promoter, and it contained promoter elements (TATA Boxs, CAAT Boxs, CAAT-box, ABRE, G-Boxs, Sp1, MSA-like, LTR). AACT recombinant protein (43.40 KDa + Tag protein 22.68 KDa) was subjected in SDS-PAGE. AACT the transcription levels of in different development stages were investigated. The expression of AACT in primordia (2.4-fold) and 15 d mycelia (2.3- fold) were significantly higher than 9 d mycelia (contral). The expression level of the AACT downstream genes and triterpenoids content were determined at different developmental stages. Triterpenoid content reached its peak on day 15(7.21 mg/g).
Asunto(s)
Acetilcoenzima A/química , Acetil-CoA C-Acetiltransferasa/química , Basidiomycota/enzimología , Cuerpos Fructíferos de los Hongos/enzimología , Proteínas Fúngicas/química , Micelio/enzimología , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Basidiomycota/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Cuerpos Fructíferos de los Hongos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Intrones , Punto Isoeléctrico , Modelos Moleculares , Peso Molecular , Micelio/química , Sistemas de Lectura Abierta , Filogenia , Regiones Promotoras Genéticas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triterpenos/aislamiento & purificación , Triterpenos/metabolismoRESUMEN
The bamboo shoot of Pleioblastus amarus (Keng) Keng f. is a medicinal and edible plant product in China. In this study, the chemical composition of the total alkaloids from bamboo shoots and bamboo shoot shells of P. amarus (Keng) Keng f. (ABSP and ABSSP, respectively) were separated and investigated by UHPLC/QTOF-MS/MS. The results showed that a total of 32 alkaloids were extracted, with 15 common to both ABSP and ABSSP and 10 and 7 alkaloids distinct to ABSP and ABSSP, respectively. ABSP and ABSSP both decreased the lipopolysaccharide (LPS, 0.5 µg/mL)-induced nitric oxide (NO) production in RAW264.7 murine macrophages with half maximal inhibitory concentration (IC50) values of 78 and 55 µg/mL, respectively. We also found that ABSP and ABSSP (100 µg/mL) could decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both mRNA and protein levels in LPS-exposed RAW264.7 cells. Moreover, 100 µg/mL of ABSP and ABSSP also significantly inhibited LPS-induced mRNA expression of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α). Additionally, ABSP and ABSSP (100 µg/mL) decreased the phosphorylation of extracellular regulated protein kinase (ERK) in LPS-stimulated RAW264.7 cells. Collectively, the total alkaloids from the bamboo shoots and shells of P. amarus exhibit anti-inflammatory effects in LPS-activated RAW264.7 cells through the inhibition of ERK signaling. This result can provide support for the medicinal use and further study of P. amarus.
Asunto(s)
Alcaloides/farmacología , Antiinflamatorios/farmacología , Extractos Vegetales/farmacología , Brotes de la Planta/química , Sasa/química , Alcaloides/análisis , Alcaloides/química , Animales , Antiinflamatorios/análisis , Antiinflamatorios/química , Citocinas/genética , Citocinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Estructura Molecular , Óxido Nítrico/metabolismo , Extractos Vegetales/análisis , Extractos Vegetales/química , Células RAW 264.7 , Análisis EspectralRESUMEN
A farnesyl diphosphate synthase (FPS) cDNA and promoter region was cloned from Sanghuangporus baumii. The gene contains a 150-bp 5'-untranslated region (UTR), a 154-bp 3'-UTR, and a 1062-bp open reading frame (ORF) encoding a 354 amino acid polypeptide. The FPS-DNA includes three exons (nucleotides 1 -123, 184-321, and 505-1305) and two introns (nucleotides 124-183 and 322-504). The FPS protein has a molecular weight of 40.73 kDa, it is hydrophilic with a theoretical isoelectric point of 5.13, and the secondary and three-dimensional structure were analysed. There is a transcription start site at nucleotides 1318-1368 of the promoter, which includes typical eukaryotic promoter elements (TATA Box, CAAT Box, ARBE, AT-rich element, G-box, MBS, Sp1, LTR). FPS was expressed in Escherichia coli BL21, and the recombinant protein (63.41 kDa) was subjected to dodecyl sulphate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). FPS transcription was measured during different developmental stages, and expression in 11 and 13 days mycelia was upregulated 49.3-fold and 125.4-fold, respectively, compared with 9 days mycelia controls. Through analysing, S. baumii triterpenoid content was correlated with the transcription level of FPS during different development stages, and the triterpenoid content peaked at day 15 (7.21 mg/g).