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A kind of ginger flower sesquiterpene synthase gene hctps14 and its application

A sesquiterpene synthase and gene technology, applied in the field of genetic engineering, can solve problems such as few reports

Active Publication Date: 2021-03-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current domestic research on terpene synthases in ginger mainly focuses on monoterpene synthase genes, but there are few reports on sesquiterpene synthase genes in ginger flowers.

Method used

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  • A kind of ginger flower sesquiterpene synthase gene hctps14 and its application
  • A kind of ginger flower sesquiterpene synthase gene hctps14 and its application
  • A kind of ginger flower sesquiterpene synthase gene hctps14 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Obtaining of full-length cDNA of HcTPS14 gene

[0030]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.

[0031] 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 annota...

Embodiment 2

[0033] Example 2 Expression analysis of HcTPS14 gene

[0034] 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'-ATCCAACAGCAGTAGCTCGAC-3' (as shown in SEQ ID NO:6). P2: 5'-GGTTCAACCAGCACCAAAGA-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

[0037] Example 3 Prokaryotic expression of HcTPS14 gene

[0038] S1. Vector construction: according to the coding region of the obtained HcTPS14 gene, use the specific primer F: 5'-GATCTG comprising KpnI and EcoRI restriction sites GGTACC ATGATAAAGAGAGTGGAAGA-3' (as shown in SEQ ID NO: 10); R: 5'-GAGCTC GAATTC TATAGGAATGGGTTCAACCAGCA-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 ...

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Abstract

The invention discloses a gingerflower sesquiterpene synthase gene HcTPS14 and its application. The full-length cDNA sequence of the HcTPS14 gene is shown in SEQ ID NO:1; the coding sequence is shown in SEQ ID NO:2; the encoded amino acid sequence is shown in SEQ ID NO:3. The HcTPS14 gene is expressed to a certain extent in leaf tissue and rhizome, but hardly expressed in flower organs, and its expression level is regulated by leaf development. The exogenous recombinant protein of HcTPS14, after catalyzing the substrate, can generate α-caryophyllene, a medicinal ingredient of sesquiterpene, which can be used to prepare α-caryophyllene and further prepare essential oils, essences and drugs; connect HcTPS14 to the plant transformation vector, and then introduce In Ginger Flower or other plant cells, transgenic plants expressing the gene can be obtained; specific molecular markers can also be produced according to the gene sequence information of the present invention, for identifying the sesquiterpene α-Caryophyllum of Ginger Flower or other plants The alkene synthase gene is used for molecular marker-assisted selection breeding, thereby improving the selection efficiency of breeding, and has great application prospects.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, more specifically, to a ginger flower sesquiterpene synthase gene HcTPS14 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 Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/11C12N15/82C12P5/00C12Q1/6895A01H5/00A01H5/10A01H6/00
Inventor 范燕萍熊美新李昕悦余让才岳跃冲玉云祎罗文平
Owner SOUTH CHINA AGRI UNIV
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