tailieunhanh - Biểu hiện gen mã hóa Flavonol synthase phân lập từ chè Trung Du Xanh (Camellia sinensis var. macrophylla) trong vi khuẩn E. coli
Các flavonol phổ biến ở thực vật bao gồm quercetin, kaempferol và myricetin, được tổng hợp từ các dihydroflavonols (dihydroquercetin-DHQ, dihydrokaempferol-DHK và dihydromyricetin - DHM) bởi flavonol synthase (FLS). Ở chè, FLS được chứng minh có hoạt tính chuyển hóa dihydroquercetin thành quercetin. Gen FLS đã được tạo dòng và xác định trình tự từ giống chè trung du xanh được trồng ở Thái Nguyên. | Biểu hiện gen mã hóa Flavonol synthase phân lập từ chè Trung Du Xanh (Camellia sinensis var. macrophylla) trong vi khuẩn E. coli TAP CHI SINH HOC 2020, 42(1): 21–29 DOI: EXPRESSION OF GENE ENCODING FLAVONOL SYNTHASE ISOLATING FROM TRUNG DU XANH TEA (Camellia sinensis var. macrophylla) IN E. coli Hoang Thi Thu Yen1,*, Vu Thi Lan1, Huynh Thi Thu Hue2 1 University of Sciences, Thai Nguyen University, Thai Nguyen, Vietnam 2 Institute of Genome Research, VAST, Vietnam Received 20 May 2019, accepted 10 January 2020 ABSTRACT Common flavonols in plants including quercetin, kaempferol and myricetin are synthesized from dihydroflavonols (dihydroquercetin-DHQ, dihydrokaempferol-DHK and dihydromyricetin- DHM) by flavonol synthase (FLS). In tea, FLS has been shown to metabolize dihydroquercetin to quercetin. The FLS gene was cloned and sequenced from the cultivated tea (Camellia sinensis var. macrophylla) in Thai Nguyen province. In this study, we presented the results of optimizing and designing an expression vector for recombinant FLS (recombinant FLS-rFLS). The FLS gene was ligated completely to the pET32a (+) vector, then expressed in E. coli Rosetta1 and Rosetta2 strain. Using 1mM IPTG to induce the expression of rFLS at 37oC, rFLS was obtained with kDa in size and existed predominantly as insoluble form. E. coli Rosetta1 pET32a (+)_FLS produces rFLS in the soluble fraction than E. coli Rosetta2 pET32a (+)_FLS. Next, E. coli Rosetta1 pET32a (+)_FLS was optimized for expression at temperatures of 30oC, 23oC and 16oC (24 and 48 hours). After being induced for expression with 1mM IPTG in 48 hours and cultured at 16oC, E. coli Rosetta1 strain containing pET32a (+) FLS produced the largest amount of rFLS in the soluble form. Keywords: Camelia sinensis, dihydroquercetin metabolism, flavonol synthase, recombinant FLS. Citation: Hoang Thi Thu Yen, Vu Thi Lan, Huynh Thi .
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