[Objective] The aim was to clone H6H gene from Atropa belladonna and construct an efficient plant expression vector.[Method] The coding sequence of H6H（Hyoscyamine 6β-hydroxylase）was cloned from Atropa belladonna w...[Objective] The aim was to clone H6H gene from Atropa belladonna and construct an efficient plant expression vector.[Method] The coding sequence of H6H（Hyoscyamine 6β-hydroxylase）was cloned from Atropa belladonna with RT-PCR.Then,the sequence was subcloned into the reconstructed plant binary expression vector p2301 to construct the recombinant vector p2301-H6H,which was then introduced into Agrobacterium tumefaciens strain LBA4404 and Agrobacterium rhizogenes strain C58C1,respectively.[Result] The engineering bacteria p2301-H6H-LBA4404 and p2301-H6H-C58C1 which could be directly used in genetic improvement were obtained.[Conclusion] The present research provided basis for the increasing of alkaloid content of Atropa belladonna by plant genetic engineering technology.展开更多
Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no eff...Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as antioxidant, antiviral, and liver-protective properties. Here, we report the isolation and characterization of two CYP450 enzymes, SbCYP82D1.1 and SbCYP82D2, which function as the flavone 6-hydroxylase （F6H） and flavone 8-hydroxylase （F8H）, respectively, in S. baicalensis. SbCYP82D1.1 has broad substrate speci- ficity for flavones such as chrysin and apigenin and is responsible for biosynthesis of baicalein and scutel- larein in roots and aerial parts of S. baicalensis, respectively. When the expression of SbCYP82D1.1 is knocked down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. SbCYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, although minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that SbCYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed by neofunctionalization, whereby the ancestral F6H activity is partially retained in the derived SbCYP82D2.展开更多
基金Supported by Natural Science Foundation of Chongqing~~
文摘[Objective] The aim was to clone H6H gene from Atropa belladonna and construct an efficient plant expression vector.[Method] The coding sequence of H6H（Hyoscyamine 6β-hydroxylase）was cloned from Atropa belladonna with RT-PCR.Then,the sequence was subcloned into the reconstructed plant binary expression vector p2301 to construct the recombinant vector p2301-H6H,which was then introduced into Agrobacterium tumefaciens strain LBA4404 and Agrobacterium rhizogenes strain C58C1,respectively.[Result] The engineering bacteria p2301-H6H-LBA4404 and p2301-H6H-C58C1 which could be directly used in genetic improvement were obtained.[Conclusion] The present research provided basis for the increasing of alkaloid content of Atropa belladonna by plant genetic engineering technology.
文摘Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as antioxidant, antiviral, and liver-protective properties. Here, we report the isolation and characterization of two CYP450 enzymes, SbCYP82D1.1 and SbCYP82D2, which function as the flavone 6-hydroxylase （F6H） and flavone 8-hydroxylase （F8H）, respectively, in S. baicalensis. SbCYP82D1.1 has broad substrate speci- ficity for flavones such as chrysin and apigenin and is responsible for biosynthesis of baicalein and scutel- larein in roots and aerial parts of S. baicalensis, respectively. When the expression of SbCYP82D1.1 is knocked down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. SbCYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, although minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that SbCYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed by neofunctionalization, whereby the ancestral F6H activity is partially retained in the derived SbCYP82D2.
