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CN-117737092-B - Intelligence-promoting Shan Tie synthase AoTPS1 gene and encoding protein and application thereof

CN117737092BCN 117737092 BCN117737092 BCN 117737092BCN-117737092-B

Abstract

The invention discloses a eucalyptol synthase AoTPS gene with intelligence improving effect, and a coding protein and application thereof. The invention provides a gene AoTPS for regulating and controlling the biosynthesis of a monoterpene compound eucalyptol, the nucleotide sequence is shown as SEQ ID NO.1, the total length of the sequence is 1785bp, 594 amino acids are coded, the molecular weight is 69.03kDa, and the coded amino acid sequence is shown as SEQ ID NO. 2. The alpinia AoTPS protein has the function of synthesizing eucalyptol in vitro by catalysis, and meanwhile, the content of eucalyptol in the monoterpene volatile oil compound can be obviously increased by adopting a genetic transformation technology to overexpress the alpinia AoTPS gene. Therefore, the AoTPS gene is a key gene for regulating and controlling the synthesis of the nootropic essential oil, can obviously promote the biosynthesis of eucalyptol in the monoterpene essential oil, and improves the medicinal value of the nootropic essential oil.

Inventors

  • ZHAO JIAYING
  • YU ZHENMING
  • Zeng Zhouting
  • LI XIAOXIAN
  • CHEN JUNYU
  • Ying Yuxin

Assignees

  • 浙江中医药大学

Dates

Publication Date
20260508
Application Date
20231007

Claims (8)

  1. 1. A kind of intelligence Shan Tie synthase AoTPS1 gene, characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.
  2. 2. The protein encoded by the monoterpene synthase AoTPS gene of claim 1.
  3. 3. A recombinant vector comprising the monoterpene synthase AoTPS gene of claim 1.
  4. 4. The recombinant vector according to claim 3, wherein the recombinant vector is pET28-AoTPS1 or pCAMBIA1301-AoTPS1.
  5. 5. An engineered bacterium comprising the recombinant vector of claim 3 or 4.
  6. 6. The engineering bacterium according to claim 5, wherein the engineering bacterium is E.coli BL21 containing pET28-AoTPS1 recombinant vector or Agrobacterium GV3101 (pSoup-p 19) containing pCAMBIA 1301-AoTPS.
  7. 7. Use of the monoterpene synthase AoTPS as defined in claim 2, for promoting eucalyptol biosynthesis in nootropic agents.
  8. 8. Use of the monoterpene synthase AoTPS gene according to claim 1 in improved intellectual development breeding, said use being the use of the monoterpene synthase AoTPS gene in genetic engineering breeding to promote eucalyptol biosynthesis in intellectual development.

Description

Intelligence-promoting Shan Tie synthase AoTPS1 gene and encoding protein and application thereof Technical field: the invention belongs to the technical field of medicinal plant genetic engineering, and particularly relates to an application of a educational Shan Tie synthase AoTPS1 gene and a coding protein thereof in regulation and control of eucalyptol biosynthesis. The background technology is as follows: Intelligence development is a dried mature fruit of the perennial plant alpinia (Alpinia oxyphylla) of the family zingiberaceae, has the effects of warming kidney, controlling nocturnal emission, reducing urination, warming spleen, stopping diarrhea and taking saliva, and is mainly distributed in Hainan and Guangdong parts and the like together with betel nut (Areca catechu), fructus amomi (Amomum villosum) and morinda officinalis (Morinda officinalis) and called as four southern medicine. The quality control of traditional Chinese medicine for improving intelligence mainly takes the content of volatile oil as standard, and is rich in compounds (Van HT,Thang TD,Luu TN,Doan VD.An overview of the chemical composition and biological activities ofessential oils fromAlpinia genus(Zingiberaceae).RSCAdv.2021,11:37767-37783), such as eucalyptol (1, 8-cineole), beta-pinene (beta-pinene), alpha-pinene (alpha-pinene), beta-myrcene (beta-myrcene), camphor (camphor), gamma-terpinene (gamma-terpinene) and the like, and the content of the eucalyptol is highest. The eucalyptol, also known as eucalyptol, 1, 8-eucalyptol, colorless liquid, has flavor Xin Leng similar to camphor smell, and researches show that eucalyptol has the effects of resisting bacteria, resisting inflammation, relieving asthma, resisting oxidation, easing pain and relieving spasm (WangY,Zhen D,Fu D,FuY,Zhang X,Gong G,Wei C.1,8-cineole attenuates cardiac hypertrophy in heart failure by inhibiting the miR-206-3p/SERP1pathway.Phytomedicine.2021,91:153672). on diseases such as influenza, common cold, other respiratory tract infections, rhinitis, nasosinusitis and the like, so that the excavation and identification of key enzyme genes for encoding biosynthesis of eucalyptol have important medicinal and economic values. Eucalyptol is a bicyclic monoterpene compound, and the biosynthetic pathway in plants is relatively conserved, mainly in chloroplasts/plastids. Firstly, isopentenyl pyrophosphate (isopentenyl pyrophosphate, IPP) and dimethylallyl pyrophosphate (DIMETHYLALLYL PYROPHOSPHATE, DMAPP) are condensed under the catalysis of geranyl pyrophosphate synthase (geranyl diphosphate synthase, GPPS) to form geranyl pyrophosphate (geranyl pyrophosphate, GPP), and GPP is cyclized by eucalyptol synthase (1, 8-cineole synthase, CINS) to form eucalyptol (Karuppiah V,Ranaghan KE,Leferink NGH,Johannissen LO,Shanmugam M,NíCheallaighA,Bennett NJ,Kearsey LJ,Takano E,Gardiner JM,van der Kamp MW,Hay S,MulhollandAJ,Leys D,Scrutton NS.Structural basis ofcatalysis in the bacterial monoterpene synthases linalool synthase and 1,8-cineole synthase.ACS Catal.2017,7(9):6268-6282;Ali M,Alshehri D,Alkhaibari AM,Elhalem NA,Darwish DBE.Cloning and characterization of 1,8-cineole synthase(SgCINS)gene from the leaves of Salvia guaranitica plant.Front Plant Sci.2022,13:869432)., so that eucalyptol synthase is a key enzyme for eucalyptol biosynthesis and belongs to one of the monoterpene synthase family members. The eucalyptol synthase genes that have been developed and identified at present are mainly concentrated in plants such as bacteria, fungi and arabidopsis, however, less research has been conducted in eucalyptus, laurel and alpinia plants with higher eucalyptol content. Therefore, the monoterpene synthase or eucalyptol synthase is excavated from the traditional Chinese medicine alpinia oxyphylla, so that not only can the gene resource of the eucalyptol synthase be enlarged, but also a high-efficiency protease source is provided for constructing the microorganism engineering bacteria for high-yield eucalyptol volatile oil. The invention comprises the following steps: The invention aims to provide a gene of a educational Shan Tie synthase AoTPS and a coded protein, wherein the monoterpene synthase AoTPS can promote the biosynthesis of monoterpene volatile oil-eucalyptol. In order to achieve the above object, the present invention provides a monoterpene synthase AoTPS for controlling the biosynthesis of monoterpene volatile oil-eucalyptol, wherein AoTPS has a nucleotide sequence shown as SEQ ID NO. 1. The invention provides a protein encoded by the intelligence-promoting Shan Tie synthase AoTPS1, which has an amino acid sequence shown as SEQ ID NO. 2. The invention also provides a recombinant vector containing AoTPS1, wherein the recombinant vector is pET28-AoTPS1 and pCAMBIA1301-AoTPS1. The invention also provides engineering bacteria containing the recombinant vector, wherein the engineering bacteria are escherichia coli BL21 containing pET28-AoTPS1 recombinant vector and ag