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Hedychium coronarium sesquiterpene synthetase gene HcTPS14 and application thereof

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

Active Publication Date: 2019-05-07
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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  • Hedychium coronarium sesquiterpene synthetase gene HcTPS14 and application thereof
  • Hedychium coronarium sesquiterpene synthetase gene HcTPS14 and application thereof
  • Hedychium coronarium sesquiterpene synthetase gene HcTPS14 and application thereof

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 hedychium coronarium sesquiterpene synthetase gene HcTPS14 and application thereof. 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; and the coded amino acid sequence is shown in SEQ ID NO: 3. The HcTPS14 gene has certain expression in both leaf tissues and rhizomes; however, the HcTPS14 gene is hardly expressed in floral organs. Moreover, the expression level of the HcTPS14 gene is regulated by leaf development. Being used for catalyzing substrates, exogenous recombinant proteins with the HcTPS14gene allow generation of a medicinal sesquiterpene component, namely alpha-caryophyllene; and thus, the HcTPS14 gene can be used for preparing alpha-caryophyllene, and further into essential oil, essence and pharmaceuticals. Being connected with plant transformation vectors, and then introduced into cells of hedychium coronarium or other plants, the HcTPS14 gene allows obtaining of transgenic plants expressing the HcTPS14 gene. Moreover, specific molecular markers can be produced on basis of the gene sequence information disclosed by the invention so as to be used for identifying sesquiterpene-alpha-caryophyllene synthetase genes of hedychium coronarium and other plants, thereby realizing molecular-marker-assisted selected breeding so as to improve selection efficiency of breeding. Thus,relatively great application prospects are ensured.

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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IPC IPC(8): C12N15/60C12N9/88C12N15/11C12N15/82C12P5/00C12Q1/6895A01H5/00A01H5/10
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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