Taxadiene synthase tcts2, encoding nucleotide sequence and application thereof

A taxadiene and nucleic acid sequence technology, applied in the field of plant genetic engineering, can solve problems such as dependence and inability to completely solve supply problems, and achieve the effects of improving efficiency, significant economic and social value, and solving expensive prices

Active Publication Date: 2021-12-03
AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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  • 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

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  • Taxadiene synthase tcts2, encoding nucleotide sequence and application thereof
  • Taxadiene synthase tcts2, encoding nucleotide sequence and application thereof

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Experimental program
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Embodiment 1

[0067] Cloning and expression purification of the taxadiene synthase TcTSs gene of embodiment 1

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

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

[0070] Based on the nucleotide sequences of TcTS1 an...

Embodiment 2

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

[0082] 1. In vitro activity of TcTSs-His6

[0083] The in vitro enzymatic reaction system was 500 μL, containing 100 μg purified protein, 100 μM GGPP (Sigma-Aldrich) and enzyme reaction buffer (25 mM, pH 8.5, 10% glycerol, 5 mM DTT, 5 mM ascorbate, 5 mM sodium metabisulfite and 1 mM MgCl 2 ), the reaction mixture was covered with 500 μL pentane (Macklin, GC-MS grade) and reacted 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 put into 2 mL of the sample, and concentrated using a nitrogen blower at low temperature , the product was analyzed by gas chromatography-mass spectrometry (GC-MS) instrument.

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

Embodiment 3

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

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

[0092] The Southern Taxus 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, for the expression pattern of TcTSs gene and the change analysis of baccatin Ⅲ and paclitaxel content, with three biological repetitions.

[0093] 2. Analysis of changes in the content of baccatin III and paclitaxel at different treatment times of MeJA

[0094] The metabolites were extracted using the improved Wolfender method. In short, weigh 100mg of freeze-dried cell powder and place it in a 2ml centrifuge tube, add 1.5mL of extraction buffer (methanol:...

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Abstract

The invention provides a taxadiene synthase TcTS2, encoding nucleotide sequence and application thereof, and relates to the technical field of plant genetic engineering. The amino acid sequence of TcTS2 comprises or consists of the following sequence: a) the amino acid sequence shown in SEQ ID NO.1; or, b) a functional identity having at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO.1 source sequence; or c) an amino acid sequence that adds, deletes, or replaces one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 and has TcTS2 activity. TcTS2 and the nucleotide sequence encoding TcTS2 provide new genetic resources for increasing the yield of paclitaxel, which can be used in plant genetic engineering and metabolic engineering strategies to transform chassis hosts, produce paclitaxel and its intermediates, etc., and have significant economic and social value.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a taxadiene synthase TcTS2, its coding nucleotide sequence and its application in synthesizing baccatin III or paclitaxel. Background technique [0002] Paclitaxel is a diterpene alkaloid, which was first isolated from the bark of Taxus brevifolia. It is a star anticancer drug and is widely used in the clinical treatment of various cancers. Cancer is one of the top ten causes of death for 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 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., 20...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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