Garland-flower sesquiterpene synthetase gene HcTPS12 and application thereof

A technology of sesquiterpene synthase and sesquiterpene, which is applied in the field of genetic engineering and can solve the problems of difficult separation and purification, low product content and the like

Active Publication Date: 2019-05-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the industrial production of bisabolene is mainly realized by plant extraction, but the extraction of bisabolene from plant tissues has the disadvantages of low product content and difficulty in separation and purification.

Method used

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  • Garland-flower sesquiterpene synthetase gene HcTPS12 and application thereof
  • Garland-flower sesquiterpene synthetase gene HcTPS12 and application thereof
  • Garland-flower sesquiterpene synthetase gene HcTPS12 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Obtaining the full-length cDNA of HcTPS12 gene

[0032] S1. Extraction of RNA from the leaves of Gingerflower: the leaves of Gingerflower preserved in ultra-low temperature refrigerators were used as materials for extracting RNA. Pipette tips and Eppendorf tubes used for RNA extraction were soaked in 0.1% DEPC overnight at 37°C and then sterilized at 121°C for 25 minutes. Glassware and mortars were wrapped in aluminum foil and then dry-heated at 180°C for 3 hours. Cooled and set aside. The total RNA of ginger flower leaves was extracted by Trizol method according to the instructions of Trizol (TaKaRa). The integrity of RNA was detected by 1% agarose gel electrophoresis, and its concentration and purity were determined by micro-spectrophotometer. Store at -80°C for later use.

[0033] S2. The total RNA of ginger flower leaves was used as a template, and the first-strand cDNA was synthesized with Sangon's M-MuLV First cDNA Synthesis Kit. According to the rele...

Embodiment 2

[0035] Example 2 Expression analysis of HcTPS12 gene

[0036] 1. Select different tissue parts and leaves of different developmental stages of ginger flower to extract RNA. Trizol method (TaKaRa) is used for RNA extraction, and SYBR green (TaRaKa) method is used for fluorescence quantitative PCR. The specific principle of dye method is shown in the instruction manual. Using Primer Premier 5.0 software to design real-time fluorescent quantitative PCR primers, according to the principles of fluorescent quantitative PCR primer design, use Primer premier 5.0 to design primers respectively, and detect whether there are mismatches or primer dimers and their amplification efficiency by fluorescent quantitative PCR. A pair of optimal primers was selected, P1: 5'-TGGAAGGCGTGGTTGTTGAT-3' (as shown in SEQ ID NO: 6). P2: 5'-AGACCGTGATTTCTGATTTGT-3' (shown in SEQ ID NO: 7). The internal reference gene RPS was designed with Primer premier 5.0 according to the design principles of Real-time...

Embodiment 3

[0039] Example 3 Prokaryotic expression of HcTPS12 gene

[0040] S1. Vector construction: according to the coding region of the obtained HcTPS12 gene, use the specific primer F: 5'-GATCTG comprising KpnI and EcoRI restriction sites GGTACC ATGGAGCTTGCTGGTACTCC-3' (as shown in SEQ ID NO: 10); R: 5'-GAGCTC GAATTC AATAGGGATAGGATGAACAA-3' (shown as SEQ ID NO: 11). Perform PCR amplification. The PCR product was recovered with a Takara recovery kit, and the recovered product was directly digested with KpnI and EcoRI restriction endonucleases, and the target fragment was recovered on 1% agarose gel. The pET-30a prokaryotic expression vector was double-digested with KpnI and EcoRI restriction enzymes to recover large fragments on 1% agarose gel. After ligation at 16°C overnight, the ligation product was transformed into Escherichia coli (E.coli) DH5α competent cells; the recombinant prokaryotic expression vector was obtained after the extracted plasmid was identified by enzyme dig...

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Abstract

The invention discloses a garland-flower sesquiterpene synthetase gene HcTPS12 and an application thereof. The full-length cDNA (complementary deoxyribonucleic acid) sequence of the gene HcTPS12 is asshown in SEQ ID NO: 1, the coding sequence of the gene is as shown in SEQ ID NO: 2, and the coded amino acid sequence of the gene is as shown in SEQ ID NO: 3. The expression quantity of the gene HcTPS12 in garland-flower leaf tissues is high, the gene is hardly expressed in organs such as rhizomes and flowers, and the expression quantity is regulated by leaf development. After a heterologous recombinant protein substrate of the gene HcTPS12 is catalyzed, bisabolene serving as a sesquiterpene medicinal component can be generated, and the gene can be used for preparing the bisabolene and further preparing essential oil, essence and medicines. The gene HcTPS12 is connected with a plant transformation vector and then led into cells of a garland-flower or other plants, and a transgenic plant expressing the gene can be acquired. Besides, a specific molecular marker is generated according to gene sequence information and used for identifying the sesquiterpene bisabolene synthetase gene of the garland-flower or other plants and molecular marker assistant selection, so that selection efficiency is improved, and the gene has a large application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, more specifically, to a gingerflower sesquiterpene synthase gene HcTPS12 and its application. Background technique [0002] Ginger flower is a perennial herb of Zingiberaceae Zingiberaceae, which is a traditional medicinal plant in India (Ray, et al., 2016). A large amount of essential oil can be extracted from the rhizome, petals and leaves of ginger flower. Studies have shown that ginger flower essential oil has medicinal properties such as antibacterial, anti-oxidation and anti-inflammatory. It is also widely used in skin care products, flavors and fragrances and other industries, and its main component is terpene compounds (Santos, et al., 2010; Dixit, 2018). Terpenes are a general term for a class of compounds composed of several isoprene (Isoprene, C5) structural units, which can be divided into monoterpene (Monoterpene, C10), sesquiterpene (Sesquiterpene , C15) and diterpene ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/11C12N15/82C12P5/00C12Q1/6895A01H5/00A01H5/10A01H6/00
Inventor 范燕萍熊美新岳跃冲余让才李昕悦玉云祎
Owner SOUTH CHINA AGRI UNIV
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