Cloning of Blakeslea trispora carRA gene by PCR-driven overlap extension and construction of an activity detection system / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 990-997, 2011.
Artigo
em Chinês
| WPRIM
| ID: wpr-324511
ABSTRACT
Blakeslea trispora CarRA has both lycopene cyclase and phytoene synthase activity. In order to analyze the double functional activity of CarRA proteins and to detect the active sites of lycopene cyclase, we constructed two detection systems in Escherichia coli by color complementary. Through PCR-driven overlap extension we got carRA gene cDNA, then constructed prokaryotes expression vector pET28a-carRA. pET28a-carRA with plasmid pAC-LYC carrying crtl/crtB/crtE gene clusters were co-transformed to BL21(DE3) to validate lycopene cyclase activity. We constructed the plasmid pAC-LYC delta (crtB) carrying crtl/crtE gene clusters, then co-transtormed them with pET28a-carRA to BL21(DE3) to validate phytoene synthase activity. Based on color complementary, and HPLC analysis of metabolites, we confirmed that the CarRA protein activity detection system was reliable. Our study provides a screening model for specific mutation of lycopene cyclase without affecting phytoene synthase activity.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Proteínas Fúngicas
/
Carotenoides
/
Reação em Cadeia da Polimerase
/
Clonagem Molecular
/
DNA Complementar
/
Liases Intramoleculares
/
Alquil e Aril Transferases
/
Escherichia coli
/
Geranil-Geranildifosfato Geranil-Geraniltransferase
/
Vetores Genéticos
Tipo de estudo:
Estudo diagnóstico
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2011
Tipo de documento:
Artigo
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