Method for decreasing content of C-14 oxygenated taxoids in Taxus x media cells by using RNA interference technique
A mandia yew, RNA interference technology, applied in recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as reducing synthesis
Inactive Publication Date: 2010-07-21
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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Problems solved by technology
There are no reports at home and abroad on inhibiting the expression of taxane 14β-hydroxylase gene in Taxus x media cells and reducing the synthesis of C-14 oxidized taxanes in Taxus x media cells
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Embodiment 1
[0029] Embodiment 1: use the method of the present invention to effectively reduce the synthesis of C-14 oxidized taxanes in Taxus x media (Taxus x media) cells, and target taxus taxane 14β hydroxylase gene (abbreviated 14OH) fragment The constructed RNA interference vector was introduced into the cultured cells of Taxus x media. Through the optimization of the transformation method and a period of cultivation, the expression level of the 14OH gene in the Taxus chinensis cells and the three C-14 oxidized taxanes sinenxan A, sinenxan C, and Yunnanxane in the Taxus chinensis cells were improved. The total content is reduced.
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The invention relates to a method for effectively decreasing synthetic C-14 oxygenated taxoids in Taxus x media cells, which belongs to the technical field of genetic engineering. The method introduces RNA interference vectors which are constructed for Taxus taxoids 14 Beta hydroxylase gene (14OH for short) fragments into the Taxus x media culture cells by way of a binary vector transformation system mediated by Agrobacterium tumefaciens. By using the optimized transformation method, the transgenic cells are successfully obtained. The results of PCR-Southern hybridization, RT-PCR gene expression analysis and chemical ingredient detection show that: the Taxus x media cell transgenic experiment is successful and the mRNA expression level of the 14OH gene in transgenic cell lines evidently falls. The invention significantly inhibits the flow direction from the Taxus x media cells to C-14 oxygenated taxoids, which is hopeful to divert metabolic flux to the C-13 oxygenated taxoids which contain anticancer drug taxol and baccatin III which is the precursor of the taxol, thereby providing a new method for increasing the biosynthesis yield of the taxol.
Description
technical field [0001] The invention relates to a method for effectively reducing the synthesis of C-14 oxidized taxanes by Taxus x media cells. It belongs to the technical field of genetic engineering developed in the 1980s on the basis of plant cell culture. As a new technology, plant cell suspension culture technology can produce useful drugs on a large scale, and has promising prospects. Background technique [0002] Paclitaxel (taxol) is an important secondary metabolite isolated from the bark of Taxus Brevifolia for the first time by Wall et al. It can be used clinically to treat ovarian cancer, lung cancer, bladder cancer, uterine cancer and AIDS-related Karposis sarcoma, etc. It is one of the most widely used natural anticancer drugs at present. However, currently paclitaxel is mainly extracted from the yew plant, and the 2000-pound bark of 2000-3000 yew trees is required to produce 1 kg of paclitaxel. Due to over-cutting, the wild resources of yew have been serio...
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IPC IPC(8): C12N15/11C12N15/82C12Q1/68C12Q1/02G01N30/02
Inventor 邱德有李凤岚胡新玲
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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