Background： CYP11A1, a gene belonging to the family 11 of cytochrome P450, encodes a crucial steroidogenic enzyme that catalyzes the initial step in the production of all classes of steroids. Many studies show that C...Background： CYP11A1, a gene belonging to the family 11 of cytochrome P450, encodes a crucial steroidogenic enzyme that catalyzes the initial step in the production of all classes of steroids. Many studies show that CYP11A1 plays a role in ovary function. However, the role of CYP11A1 in goose reproductive cycle remains largely unknown.Results： In this study, full-length CYP11A1 c DNA of Zhedong goose was obtained using reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of c DNA ends（RACE）. The c DNA consisted of a 96-base pair（bp） 5′untranslated region（UTR）, a 179-bp 3′UTR and a 1509-bp open reading frame. The open reading frame encodes a putative 503 amino acid protein that shares high homology with CYP11A1 of other birds. The amino acid sequence possesses conserved domains of the P450 superfamily, which include the steroid-binding domain and the heme-binding region. Real-time quantitative polymerase chain reaction（q PCR） analysis revealed CYP11A1 mR NA was expressed ubiquitously in every Zhedong goose tissue analyzed, including the heart, liver, glandular stomach,lung, spleen, kidney, intestinum tenue, intestinum crassum, cerebrum, cerebellum, muscle, oviduct, pituitary,hypothalamus and ovary.. The relatively low levels of CYP11A1 m RNA were detected in pituitary, ovary and oviduct tissues at ovulation when compared with levels at oviposition. Interestingly, higher expression was observed in ovary and oviduct tissues during brooding. Lastly, higher m RNA expression of Yangzhou geese was detected during the ovulation period than that of Zhedong geese.Conclusions： Our findings reveal the sequence characterization and expression patterns of the CYP11A1 gene during the goose reproductive cycle, which may provides correlative evidence that CYP11A1 expression is important in reproduction activity.展开更多
Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of th...Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.展开更多
基金the earmarked fund for Modern Agro-industry Technology Research System (CARS-43-3)the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province (CX(13)2034)the Priority Academic Program Development of Jiangsu Higher Education Institutions (2011–137)
文摘Background： CYP11A1, a gene belonging to the family 11 of cytochrome P450, encodes a crucial steroidogenic enzyme that catalyzes the initial step in the production of all classes of steroids. Many studies show that CYP11A1 plays a role in ovary function. However, the role of CYP11A1 in goose reproductive cycle remains largely unknown.Results： In this study, full-length CYP11A1 c DNA of Zhedong goose was obtained using reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of c DNA ends（RACE）. The c DNA consisted of a 96-base pair（bp） 5′untranslated region（UTR）, a 179-bp 3′UTR and a 1509-bp open reading frame. The open reading frame encodes a putative 503 amino acid protein that shares high homology with CYP11A1 of other birds. The amino acid sequence possesses conserved domains of the P450 superfamily, which include the steroid-binding domain and the heme-binding region. Real-time quantitative polymerase chain reaction（q PCR） analysis revealed CYP11A1 mR NA was expressed ubiquitously in every Zhedong goose tissue analyzed, including the heart, liver, glandular stomach,lung, spleen, kidney, intestinum tenue, intestinum crassum, cerebrum, cerebellum, muscle, oviduct, pituitary,hypothalamus and ovary.. The relatively low levels of CYP11A1 m RNA were detected in pituitary, ovary and oviduct tissues at ovulation when compared with levels at oviposition. Interestingly, higher expression was observed in ovary and oviduct tissues during brooding. Lastly, higher m RNA expression of Yangzhou geese was detected during the ovulation period than that of Zhedong geese.Conclusions： Our findings reveal the sequence characterization and expression patterns of the CYP11A1 gene during the goose reproductive cycle, which may provides correlative evidence that CYP11A1 expression is important in reproduction activity.
文摘Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.
