Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Common threewingnut root sesquiterpene synthase TwNES as well as coding gene and application thereof

A sesquiterpene, coding technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of slow growth, low content, and restricted development of plants

Active Publication Date: 2017-03-29
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
View PDF0 Cites 7 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Common threewingnut root sesquiterpene synthase TwNES as well as coding gene and application thereof
  • Common threewingnut root sesquiterpene synthase TwNES as well as coding gene and application thereof
  • Common threewingnut root sesquiterpene synthase TwNES as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, the cloning of Tripterygium wilfordii Twnes full-length cDNA sequence

[0085] 1. Acquisition of cDNA

[0086] The total RNA of tripterygium wilfordii suspension cells was extracted by the improved CTAB method; using SMARTer TM The RNA obtained from the primer 5'-CDS primer in the RACE cDNA Amplification Kit kit was used as a template, and reverse transcription was performed to obtain 5'-RACE-Ready cDNA;

[0087] 2. PCR amplification

[0088] Using the 5'-RACE-Ready cDNA obtained in step 1 as a template, PCR amplification was carried out using TwNES-F and TwNES-R primers (the underlined sequence is the enzyme recognition site) to obtain PCR amplification products. And the PCR amplification products were sequenced.

[0089] TwNES-F: 5'-CGC GGATCC ATGGCCTTCTTTGGTTCCTCTCGC-3'

[0090] TwNES-R: 5'-CGC GTC GAC TTACTTAATCAGGATGGATTTAAGG-3'

[0091] PCR reaction program: 98°C pre-denaturation for 3 minutes; 98°C for 10s, 60°C for 15s, 68°C for 2min, 35 ...

Embodiment 2

[0093] Embodiment 2, Twnes gene expression analysis after alamethicin treatment

[0094] 1. Treatment of experimental materials

[0095] 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;

[0096] 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;

[0097] 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%.

[0098] 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), whe...

Embodiment 3

[0108] Example 3, Obtaining and Functional Analysis of Tripterygium wilfordii TwNES Protein

[0109] 1. Obtaining TwNES protein of Tripterygium wilfordii

[0110] 1. Construction of recombinant vector

[0111] Replace the DNA fragment shown in the 65-1723bp position 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, The recombinant plasmid pMAL-TwNES was obtained.

[0112] 2. Acquisition of recombinant bacteria

[0113] The recombinant plasmid pMAL-TwNES was transformed into the E. coli expression strain Transetta (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the pMAL-TwNES 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.

[0114] 3. Obt...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses common threewingnut root sesquiterpene synthase TwNES as well as a coding gene and application thereof. According to the common threewingnut root sesquiterpene synthase TwNES as well as the coding gene and application thereof disclosed by the invention, a Twnes gene is obtained by cloning common threewingnut root suspension cells, and this gene is a key enzyme gene which is acquired from common threewingnut root for the first time and is synthesized by sesquiterpenoids components. Proved by experiments, TwNES protein disclosed by the invention not only can catalyze FPP to form nerolidol ((E)-nerolidol), but also can catalyze GGPP to form geranyl linalool ((E, E)-geranyllinalool), plays an important role in the synthesis of sesquiterpenoids alkaloids in common threewingnut root, and also has important theoretical and practical significance in the adjustment and production of plant sesquiterpenoids compounds and in the cultivation of high-quality common threewingnut root.

Description

technical field [0001] The invention belongs to the field of genetic engineering of medicinal plants, and specifically relates to tripterygium wilfordii sesquiterpene synthase TwNES 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 ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/88C12N15/60C12N5/10C12N1/21C12N1/15C12N1/19C12P7/04
CPCC07K2319/21C07K2319/43C12N9/88C12P7/04
Inventor 高伟黄璐琦苏平周家伟胡添源
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com