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Chamomile farnesyl diphosphatesynthase gene clone and prokaryotic expression

A technology of farnesyl pyrophosphate and chamomile, applied in genetic engineering, plant gene improvement, transferase, etc., can solve problems that have not yet been seen

Inactive Publication Date: 2012-10-03
ANHUI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

But so far, there are no research reports on key enzyme genes such as FPS gene in the metabolic synthesis pathway of chamomile terpenoids at home and abroad.

Method used

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  • Chamomile farnesyl diphosphatesynthase gene clone and prokaryotic expression
  • Chamomile farnesyl diphosphatesynthase gene clone and prokaryotic expression
  • Chamomile farnesyl diphosphatesynthase gene clone and prokaryotic expression

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Embodiment Construction

[0037] 1 Materials and methods

[0038] 1.1 Materials

[0039]Chamomile (Matricaria chamomilla L.) was provided by Xuancheng Yueping Ecological Co., Ltd. Escherichia coli DH5α, BL21(DE3), and prokaryotic expression vector pET-30a (+) were provided by the College of Life Sciences, Anhui Agricultural University. The cloning vector PMD19-T was provided by TAKARA Company. Restriction enzyme, DNA polymerase, T4 ligase, IPTG (isopropyl-β-D-thiogalactopyranoside), X-gal (5-bromo-4-chloro-3-indole- β-D galactoside), DNA gel recovery kit, plasmid small extraction kit, RNAiso plus (total RNA extraction reagent), etc. were purchased from TakaRa Company. Gene sequencing was completed by Shanghai Handsome Biotech Co., Ltd.

[0040] 1.2 Method

[0041] 1.2.1 Chamomile total RNA extraction.

[0042] (1) Grind 0.2g of chamomile petals with liquid nitrogen to powder

[0043] (2) Add 2ml of total RNA extraction reagent, cover the sample, and let it stand at room temperature for 15 minute...

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Abstract

The invention discloses a chamomile farnesyl diphosphatesynthase and a construction and expression method of a recombinant expression vector thereof, the amino acid sequence of the chamomile farnesyl diphosphatesynthase is shown as SEQ NO2, and the nucleotide sequence of the chamomile farnesyl diphosphatesynthase is shown as SEQ NO1. The ORF (open reading frame) of the chamomile FPS (frames per second) gene is 1032bp, and the chamomile FPS gene has higher homology compared with other plant sesquiterpene synthase genes through sequence comparison; and Westerning verification can be carried out by constructing a prokaryotic expression vector pET-30a(+) / FPS and inducting for expressing soluble protein to obtain a target protein with molecular weight of 38KDa, so that a foundation is established to further studies on FPS protein purification and protein structure and characteristics, and a reference is provided for improving the yield of chamomile chamazulene by a transgene method. Furthermore, the condition for induction expressing of protein is optimized in the method, the optimal induction time is six hours, and the optimal induction temperature is 37 DEG C.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chamomile farnesyl pyrophosphate synthase gene cloning, prokaryotic expression and application. Background technique [0002] Chamomile (Matricaria chamomilla L.) is a herbaceous plant of the genus Matricaria in the Asteraceae family. The whole plant is fragrant and contains volatile aromatic oils. Its volatile oil has anti-inflammatory, anti-fungal, antispasmodic, sedative, pain-relieving and improving symptoms of lung cancer patients. Its essential oil is widely used in food, beverage, tobacco, daily chemical, medicine and other fields due to its unique aroma and antioxidant effect. It is a new variety of agricultural industrial structure adjustment with great market potential, which integrates landscaping, beautification, fragrance and color. [0003] The active ingredients in chamomile are mainly concentrated in volatile oil, and the volatile oil content in flowers is general...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/66
Inventor 袁艺邰玉玲杨秀梅
Owner ANHUI AGRICULTURAL UNIVERSITY
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