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Novel sesquiterpene synthetase and application thereof

An amino acid and sequence technology, applied in the direction of application, lyase, and introduction of foreign genetic material using vectors, can solve unseen problems

Inactive Publication Date: 2013-08-14
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current prior art, the production of α-bisabolol is mainly carried out by the means of cultivating plant extraction, and there are no reports of production using biotechnology methods

Method used

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  • Novel sesquiterpene synthetase and application thereof
  • Novel sesquiterpene synthetase and application thereof
  • Novel sesquiterpene synthetase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1, extraction of Artemisia annua total RNA, PCR amplification of target gene QHS3

[0090] A. Extraction of Artemisia annua total RNA

[0091] The Artemisia annua plant material (about 100 mg) was thoroughly ground in liquid nitrogen. Transfer to a 1.5ml centrifuge tube, add 1mL Trizol (Invitrogen, Cat. 15596-018), mix well, and stand at room temperature for 5min. Centrifuge at 12,000rpm for 10min and discard the precipitate. Add 200 μL of chloroform to the supernatant, mix well, and centrifuge at 12,000 rpm for 10 min. Take the supernatant and add 500 μL of isopropanol to precipitate the RNA. Centrifuge at 12,000rpm for 10min, wash the pellet with 70% ethanol, dry in vacuum, dissolve in 20-50μL H 2 O (RNase free). The RNA was properly diluted with 10 mM Tris-HCl (pH 7.5), and the UV absorption value at a wavelength of 200-300 nm was measured. RNA concentration = 40 μg / mL × A 260 ×Dilution factor. A 260 / A 280 Should be between 1.9 and 2.1. PolyAmRNA ...

Embodiment 2

[0111] Embodiment 2, PCR amplification target gene QHS3

[0112] The high-fidelity enzyme KOD-plus DNA polymerase (ToYoBo) was used to amplify the full-length gene sequence of QHS3 (SEQ ID NO: 2) (1641bp). The primer sequences are as follows:

[0113] pETQHS 3-S: 5'-TTTCCATGGCTATGTCTCTTACAGAAGAAAAACCTA-3' (SEQ ID NO: 14),

[0114] pETQHS3-AS: 5'-TTTGGATCCTCATATACTCATAGGATAAACGAGT-3' (SEQ ID NO: 15).

[0115] The PCR reaction conditions were: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 68°C for 120 s, amplification for 30 to 35 cycles; incubation at 68°C for 10 min. Keep warm at 4°C.

Embodiment 3

[0116] Example 3, Domain substitution and site-directed mutagenesis of Artemisia annua QHS3

[0117] Using overlap extension PCR technology (Aiyar et al., 1996), multiple domain substitution mutants, single point mutants, double mutants, triple mutants and multiple mutants were successfully constructed. The DNA sequences of these mutants were determined.

[0118] Mutant information and primer sequences are shown in Table 1 (5'-3').

[0119] Table 1

[0120]

[0121]

[0122]

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PUM

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Abstract

The invention relates to a novel sesquiterpene synthetase and an application thereof. The novel sesquiterpene synthetase is obtained by separation and can cyclize farnesyl pyrophosphate to produce alpha-bisabol. Product specific sites of the sesquiterpene synthetase are obtained by structural domain replacement and point mutation experiments. The sesquiterpene synthetase has good enzymatic activity and can be applied in industrial production of the alpha-bisabol.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a new sesquiterpene synthase and its application. Background technique [0002] The plant Artemisia annua L. belongs to Compositae and is an annual herb with good medicinal value. Artemisia annua contains a variety of chemical components, such as sesquiterpenoids (including Qinghaosu, Artemisinin, Artemisinol, Artemisic acid, etc.); also contains volatile components, such as camphor Camphene, β-Camphene, Isoartemisia ketone, 1-Camphor, β-Caryophyllene, etc.; also contain flavonoids, such as: Kaemp-ferol, Quercetin, Luteolin, etc.; also contains coumarins, β-galactosidase, β-glucosidase (β-Glucosidase), β-Sitosterol (β-Sitosterol), stigmasterol (Stigmasterol) and palmitic acid (Palmitic acid), etc. Various components have exerted antimalarial effect, anti-schistosome effect, antimicrobial effect and so on. Among them, artemisinin, which is a sesquiterpen...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21C12N1/19C12N5/10C12P7/02
Inventor 陈晓亚王凌健李建戌
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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