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Paclitaxel synthase TcTS2, coding nucleotide sequence and application thereof

A nucleotide sequence and taxadiene technology, which is applied in the field of plant genetic engineering, can solve the problems of supply, dependence and the like that cannot be completely solved, and achieve the effects of improving efficiency, increasing yield and reducing production costs.

Active Publication Date: 2020-10-09
AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main production method of paclitaxel is the semi-synthetic method, that is, firstly by extracting the natural precursors baccatin III (baccatin III) and 10-deacetylbaccatin III (10-deacetylbaccatin III, 10-DAB), and then chemically synthesizing paclitaxel ( Li et al., 2015; Liu et al., 2016), but the precursor substances used in this method still rely on plant extraction, limited by plant or cell resources, and cannot completely solve the supply problem

Method used

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  • Paclitaxel synthase TcTS2, coding nucleotide sequence and application thereof
  • Paclitaxel synthase TcTS2, coding nucleotide sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Cloning, expression and purification of taxadiene synthase TcTSs gene

[0068] 1. Cloning and sequence analysis of taxadiene synthase TcTSs gene

[0069] With the known function of Taxus brevifolia taxadiene synthase TbTS (GeneBank NO.U48796) as the target sequence, the genome of Taxus chinensis var.mairei was searched and two taxadiene synthase genes were found. , Respectively named TcTS1 and TcTS2. Among them, after sequence comparison analysis, it is found that the amino acid sequence of TcTS1 reading frame has 98% homology with TbTS, and TcTS2 is the newly discovered taxadiene synthase gene, and the genomic DNA sequence of TcTS2 is shown in SEQ ID NO.2. As shown, the nucleotide similarity to TbTS is 42%; the nucleotide sequence and amino acid sequence of the TcTS2 reading frame are shown in SEQ ID NO. 3 and SEQ ID NO. 1, respectively, and the nucleotide similarity and amino acid similarity to TbTS The origin is 79%.

[0070] Based on the nucleotide sequences o...

Embodiment 2

[0081] Example 2 Biochemical function analysis of taxadiene synthase TcTSs

[0082] 1. TcTSs-His6 in vitro activity

[0083] The in vitro enzymatic reaction system is 500μL, containing 100μg purified protein, 100μM GGPP (Sigma-Aldrich) and enzyme reaction buffer (25mM, pH8.5, 10% glycerol, 5mM DTT, 5mM ascorbate, 5mM sodium metabisulfite and 1mM MgCl 2 ), the reaction mixture was covered with 500 μL pentane (Macklin, GC-MS grade) in a 32°C water bath for 2 hours, vortexed for 2 minutes, centrifuged at 5000 rpm for 10 minutes, the pentane layer was taken out and placed in 2 mL into the sample, and concentrated at low temperature using a nitrogen blower The product was analyzed by gas chromatography mass spectrometry (GC-MS) instrument.

[0084] The control group is the reaction product of purified recombinant protein TcTSs-His6 after boiling at 100°C for 10 minutes. The GC-MS instrument is Agilent 7890B / 7000C (Agilent Technologies, Waldbronn, USA), mass spectrometer parameters: 70eV,...

Embodiment 3

[0090] Example 3 Correlation analysis of taxadiene synthase TcTSs gene expression and paclitaxel under methyl jasmonate stress

[0091] 1. Methyl jasmonate treatment of Taxus chinensis cell line 211

[0092] The Taxus chinensis 211 cell line was divided into two groups: the experimental group (MeJA+) was soaked with 100 μM methyl jasmonate; the control group (MeJA-) was soaked with 0.05% ethanol solution. The two groups were carried out at the same time. Samples were collected after 0h, 2h, 3h and 4h respectively, and used for the analysis of the expression pattern of TcTSs gene and the changes of baccatin III and paclitaxel content. Three biological replicates.

[0093] 2. Analysis of the changes in the content of Bakatine III and Paclitaxel in different treatment times

[0094] Metabolites were extracted using a modified Wolfender method. In short, 100 mg of freeze-dried cell powder was weighed and placed in a 2 ml centrifuge tube, and 1.5 mL of extraction buffer (methanol: water = ...

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Abstract

The invention provides a paclitaxel synthase TcTS2, a coding nucleotide sequence and application thereof, and relates to the technical field of plant genetic engineering. The amino acid sequence of the TcTS2 comprises or consists of the following sequences: a) an amino acid sequence shown as SEQ ID NO.1; or b) a functional homologous sequence having at least 80% sequence similarity with the aminoacid sequence shown as SEQ ID NO.1; or c) an amino acid sequence which is obtained by adding, deleting and replacing one or more amino acids in the amino acid sequence shown as SEQ ID NO.1 and has TcTS2 activity. TcTS2 and the nucleotide sequence for encoding TcTS2 provide a new gene resource for increasing the yield of paclitaxel, can be used for modifying chassis hosts, producing paclitaxel andintermediates thereof and the like in plant genetic engineering and metabolic engineering strategies, and have remarkable economic and social values.

Description

Technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a taxadiene synthase TcTS2, a coding nucleotide sequence and its application in the synthesis of baccatin III or paclitaxel. Background technique [0002] Paclitaxel is a diterpene alkaloid that was first isolated from the bark of Taxus brevifolia. It is a star anti-cancer drug and is widely used in the clinical treatment of a variety of cancers. Cancer is one of the top ten causes of death in humans. At present, the annual incidence of cancer in the world is still more than 10 million (WHO). Paclitaxel and its preparations are important first-line anticancer drugs. At present, the main production method of paclitaxel is a semi-synthetic method, that is, first by extracting the natural precursors baccatin Ⅲ (baccatin Ⅲ) and 10-deacetylbaccatin Ⅲ (10-deacetylbaccatin Ⅲ, 10-DAB), and then chemically synthesizing paclitaxel ( Li et al., 2015; Liu et al., 2016), but the...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/82C12P17/02
CPCC12N9/88C12Y402/03017C12N15/8257C12P17/02C12N15/52C12N15/8243C07K14/415
Inventor 闫建斌苟君波廖庆刚梁海菲李冲杜然熊兴耀黄三文
Owner AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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