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Tripterygium wilfordii diterpene synthase TwGES1 and encoding gene and application thereof

A technology of diterpene synthase and encoding, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as restricted development, slow plant growth, and low content

Active Publication Date: 2017-03-22
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is a very potential way to develop new drugs from the active ingredients in traditional Chinese medicine. However, due to the slow growth of plants and the low content of these active ingredients in plants, its development is greatly limited.

Method used

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  • Tripterygium wilfordii diterpene synthase TwGES1 and encoding gene and application thereof
  • Tripterygium wilfordii diterpene synthase TwGES1 and encoding gene and application thereof
  • Tripterygium wilfordii diterpene synthase TwGES1 and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1, the cloning of tripterygium wilfordii TwGES1 full-length cDNA sequence

[0087] 1. Cloning of the 3' end sequence and the 5' end sequence of the Twges1 gene

[0088] 1. Extraction of total RNA

[0089] Using improved CTAB method (CTAB Buffer: 2% CTAB (W / V); 100mmol L -1 Tris-HCl (pH 8.0); 25mmol L -1 EDTA; 2.0mol L -1 NaCl; 0.5g L -1 spermidine) to extract the total RNA of Tripterygium wilfordii suspension cells.

[0090] 2. Cloning of the 5' end sequence of Twges1 gene

[0091] (1) Using the RNA obtained in step 1 as a template, using SMARTer TM The 5'-CDS primer in the RACE cDNA Amplification Kit kit was reverse-transcribed to obtain 5'-RACE-Ready cDNA.

[0092] (2) Use the 5'-RACE-Ready cDNA obtained by reverse transcription as a template, and use UPM and TwGES1-GSP1 as primers to perform PCR amplification to obtain the 5' end of the cDNA of the Twges1 gene. The target product is 2135bp, and the sequence contains Twges1 The complete sequence...

Embodiment 2

[0112] Embodiment 2, Twges1 gene expression analysis after alamethicin treatment

[0113] 1. Treatment of experimental materials

[0114] 1. Put the suspension cells of Tripterygium wilfordii in the culture medium A, shake culture at 25±1°C and 120rpm in the dark for 10 days, and obtain the suspension cells of Tripterygium wilfordii treated with alamethicin;

[0115] Suspension cells of Tripterygium wilfordii were cultured in culture medium B at 25±1°C and 120 rpm for 30 h in the dark to obtain control suspension cells of Tripterygium wilfordii;

[0116] Culture solution A is obtained by mixing alamethicin (Aladdin, A132913), ethanol solution and MS liquid medium (+0.1mg / LKT+0.5mg / L IBA+0.5mg / L 2,4-D) culture solution, wherein the final concentration of alamethicin is 100ng / L, and the volume fraction of ethanol solution is 0.1%.

[0117] Culture medium B is the culture medium obtained by mixing ethanol solution and MS liquid medium (+0.1mg / L KT+0.5mg / L IBA+0.5mg / L 2,4-D), wh...

Embodiment 3

[0127] Example 3. Obtaining and functional analysis of Tripterygium wilfordii TwGES1 protein

[0128] 1. Obtaining TwGES1 protein of Tripterygium wilfordii

[0129] 1. Construction of recombinant vector

[0130] Replace the DNA fragment shown in position 62-2608 of Sequence 1 with the fragment between the BamHI and SalI restriction sites of the vector pMAL-c2X (New England Biolabs, catalog number E8000S), and keep the other sequences of the pMAL-c2X vector unchanged , to obtain the recombinant plasmid pMAL-TwGES1.

[0131] 2. Acquisition of recombinant bacteria

[0132] The recombinant plasmid pMAL-TwGES1 was transformed into the E. coli expression strain Transetta (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the pMAL-TwGES1 recombinant strain; at the same time, the pMAL-c2X empty vector without the target gene was used to transform the large intestine Bacillus expression strain Transetta (DE3) was used as control bacteria.

[0133] 3. Obtain...

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Abstract

The invention discloses a tripterygium wilfordii diterpene synthase TwGES1 and an encoding gene and application thereof. Twges1 genes are obtained through cloning from tripterygium wilfordii suspension cells; and the genes are key enzyme genes obtained through synthesis of diterpene components from tripterygium wilfordii. Experiments prove that TwGES1 proteins can catalyze GGPP to form geranyl linalool ((E, E)-geranyl linalool), can catalyze FPP to form nerolidol ((E)-nerolidol), not only have important significance on biological synthesis of such diterpene compounds as triptolide in the tripterygium wilfordii and but also have important theoretical and practical significance on adjustment and production of plant diterpene compounds and cultivation of high-quality tripterygium wilfordii.

Description

technical field [0001] The invention belongs to the field of genetic engineering of medicinal plants, and specifically relates to tripterygium wilfordii diterpene synthase TwGES1 and its coding gene and application. Background technique [0002] The medicinal plant Tripterygium wilfordii Hook.f. is a Chinese herbal medicine widely used in the treatment of rheumatoid arthritis and inflammation (Raphaela G M, Mildred W, Roy F, et al. Comparison of Tripterygium wilfordii Hook F Versus Sulfasalazine in the Treatment of Rheumatoid Arthritis: A Randomized Trial[J]. Annals of Internal Medicine, 2009, 151(4): 229-240; Tao X L, Lipsky P E. The Chinese anti-inflammatory and immunosuppressive herbal remedy Tripterygium wilfordii Hook F.[ J]. Rheumatic Disease Clinics of North America, 2000, 26(1):29-50.). Terpenes are the main active ingredients of Tripterygium wilfordii, including triptolide, triptophenolide and celastrol. It is a very potential way to develop new drugs from the act...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12P7/04
CPCC12N9/90C12P7/04
Inventor 高伟黄璐琦苏平周家伟胡添源
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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