CN115786293A - Borneol dehydrogenase gene, expression vector and application of borneol dehydrogenase gene in preparation of camphor - Google Patents

Abstract

The invention relates to a protein, which comprises the following components 1) to 3): 1) An amino acid sequence shown as SEQ ID NO. 1; 2) A sequence having the same function as the sequence shown in SEQ ID NO.1 and obtained by substituting deletion or addition of one or more amino acids; 3) Fusion protein which has the same function with the protein shown in SEQ ID NO.1 and is obtained by connecting a label at the N end or the C end; the AarBDH5 gene is obtained by cloning from artemisia argyi slices, and the AarBDH5 gene is a key enzyme gene for biosynthesis of monoterpene compounds camphor, which is obtained from artemisia argyi for the first time. Experiments prove that: the AarBDH5 protein can catalyze Borneol (Borneol) to form Camphor (Camphor), and has important theoretical and practical significance for the synthesis research of Camphor components in folium artemisiae argyi, the quality improvement of folium artemisiae argyi medicinal materials, and the regulation and production of plant monoterpene compounds.

Description

Borneol dehydrogenase gene, expression vector and application of borneol dehydrogenase gene in preparation of camphor

Technical Field

The invention relates to the technical field of medicinal plant molecular biology and genetic engineering, in particular to a Ai Bingpian dehydrogenase AarBDH5 protein, an encoding gene and application.

Background

The Folium Artemisiae Argyi leaf is a dry leaf of Artemisia Argyi L.et Vant. of Artemisia of Compositae, is pungent, warm and bitter in taste, enters liver, spleen and kidney channels, has the effects of warming channels to stop bleeding, dispelling cold and relieving pain, is mainly used for treating hematemesis, epistaxis, metrorrhagia, menorrhagia, fetal leakage and bleeding, lower abdomen psychroalgia, irregular menstruation, cold womb infertility and other diseases, and can eliminate dampness and relieve itching when externally used. The mugwort leaves are dried in the sun and smashed to obtain mugwort floss which can be used for making mugwort sticks for moxibustion. Modern researches find that the volatile oil is the main drug effect substance basis of the folium artemisiae argyi, has the capabilities of broad-spectrum antibacterial property, antioxidation, anti-inflammation, antitumor, pain relief, asthma relief, immunoregulation and the like, and mainly comprises monoterpene compounds, sesquiterpene compounds and derivatives thereof.

The monoterpene compound camphor is one of the important components of the volatile oil of the folium artemisiae argyi, is white or transparent solid at room temperature, is easy to sublimate, and has unique fragrance. The Camphora has effects of dredging orifices, promoting qi stagnation, removing filth, killing parasites, relieving itching, and relieving swelling and pain, and can be used for treating scabies, pruritus, traumatic injury, pain, toothache, etc. Most of the camphor used in the market is obtained by chemical synthesis, but the chemical synthesis of camphor in special application in medicine and the like is difficult to completely replace the natural camphor. The chemical synthesis of camphor or the extraction of natural camphor from plants has the problems of difficult acquisition of materials, complicated steps, environmental pollution and the like, so the method for synthesizing natural camphor by a microorganism factory by utilizing a bioengineering technology has great application prospect.

Previous studies reported that bornyl dehydrogenase BDH, which belongs to the short-chain dehydrogenases/reductases (SDR) family, has the function of catalyzing borneol to camphor. BDH is cloned in a few species such as artemisia annua, camphor tree, blumea balsamifera and the like, but no relevant research that key enzyme genes with camphor synthesizing function can be obtained from the artemisia. The cloning of BDH in the folium artemisiae argyi and the research on the function of the BDH are beneficial to providing a theoretical basis for the formation of the quality of the folium artemisiae argyi medicinal material, and meanwhile, the BDH brings a wide application space for producing natural camphor by utilizing a genetic engineering technology.

Disclosure of Invention

The invention aims to provide a Ai Bingpian dehydrogenase AarBDH5 protein for efficiently synthesizing camphor, an encoding gene and application.

The invention firstly provides a protein which comprises the following components 1) to 3):

1) An amino acid sequence shown as SEQ ID NO. 1;

2) A sequence having the same function as the sequence shown in SEQ ID NO.1 and obtained by substituting deletion or addition of one or more amino acids;

3) Has the same function with the protein shown in SEQ ID NO.1, and the N end or the C end is connected with a label to obtain the fusion protein.

The protein and the protein tag refer to a polypeptide or a protein which is fused and expressed with a target protein by using a DNA in vitro recombination technology so as to be convenient for expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

Also provided is a gene encoding the protein of claim 1, wherein the coding gene is at least one of:

1) A nucleotide sequence shown as SEQ ID NO. 2;

2) A DNA molecule which hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the protein of claim 1;

3) A nucleotide sequence having 90% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the protein of claim 1;

4) A sequence obtained by substituting, deleting or adding nucleotides to the sequence shown by SEQ ID NO. 2;

5) Different transcripts or homologous gene sequences were generated from the nucleotide sequence shown in SEQ ID NO.2.

Also provides a recombinant vector, an expression cassette, a recombinant bacterium, a recombinant cell and a transgenic plant containing the coding gene.

Based on Ai Quan genome and different organ/tissue transcriptome differential expression analysis and phylogenetic evolution tree analysis constructed by the BDH protein and borneol dehydrogenase BDH with known function, a nucleotide sequence shown in SEQ ID NO.2 of the BDH gene possibly participating in camphor synthesis is screened out, or the nucleotide sequence is recombined with an expression vector and then is introduced into a receptor microorganism, the expression vector is specifically pET-28a, the receptor microorganism is specifically Escherichia coli BL21 (DE 3), a recombinant microorganism expressing AarBDH5 protein or protein with the same function as the AarBDH5 protein is obtained, the recombinant microorganism is cultured, and the protein is obtained by expression.

Also provides the application of the coding gene in catalyzing and generating camphor by taking borneol as a substrate.

Also provides the application of the recombinant vector, the expression cassette, the recombinant bacterium, the recombinant cell and the transgenic plant in catalyzing the camphor growth of the borneol.

Also provides a primer for amplifying the coding gene, wherein the primer is shown as SEQ ID NO. 3-SE Q ID NO. 4.

Also provided is a method for preparing the 1) -3) protein, the method comprising: recombining the nucleotide sequence of the coding gene with an expression vector, introducing the recombined gene into a receptor microorganism to obtain a recombined strain, culturing the recombined strain, and expressing to obtain the protein.

Also provided is a method for preparing camphor, which comprises catalyzing borneol to generate camphor by using the proteins from 1) -3) to generate camphor, wherein the camphor comprises levo-camphor or dextro-camphor. By passing

Purifying AarBDH5 protein by Ni NTA resin, performing AarBDH5 protein in-vitro enzymatic reaction in a buffer solution, and detecting the AarBDH5 gene in the buffer solution by a solid-phase microextraction technology and a gas chromatography-mass spectrometry (GC-MS) technologyAnd (3) performing in-vitro catalysis on a prokaryotic expression system to obtain a catalysis product camphor.

The AarBDH5 namely Ai Bingpian dehydrogenase is cloned from the moxa and is a key enzyme gene for synthesizing an important index component monoterpene compound borneol from the moxa for the first time. Experiments prove that the AarBDH5 can catalyze borneol to form camphor, has an important effect on further analyzing the synthesis of terpenoid in the artemisia argyi, and has important significance on further improving the camphor yield and enhancing the medicine quality.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a phylogenetic clade of the Ai Houxuan BDH gene and other identified BDH genes;

FIG. 2 is a diagram showing an analysis of the expression level of a candidate BDH gene in different tissue sites;

FIG. 3 is a graph showing the catalytic products detected by GC-MS in an in vitro expression system;

FIG. 4 is a mass spectrum of AarBDH5 catalytic product, with the upper end of the abscissa axis being the standard ion peak and the lower end of the abscissa axis being the product ion peak.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1AarBDH screening

1) AarBDH with potential camphor synthesis function is screened based on moxa genome information and known BDH gene sequences, 8 genes in total possibly have important functions of potential borneol synthesis, and the length of the amino acid sequence is 791-882.

2) The identified functional AaBDH (KT 070864), aaADH2 (GU 253890), liB DH (AFV 30207), ZSD1 (AB 480831), ccBDH3 (MN 551096) and 8 candidate genes of the AiBDH are selected, and a phylogenetic evolutionary tree is established by using MEGA software. As shown in fig. 1.

3) And (3) performing expression profile analysis on candidate BDH genes in different organs such as moxa roots, stems, leaves and flowers by adopting an RNA-seq technology. As shown in fig. 2.

Example 2 cloning of AarBDH5 Gene and protein sequence encoding the same

Designing a primer according to the AarBDH5 sequence in the moxa genome, and amplifying by using the moxa leaf sheet cDNA as a template to obtain a nucleotide sequence with the length of 821bp, such as SEQ ID NO.1. The amino acid sequence encoded by AarBDH5 is obtained after translation based on the full-length cDNA sequence, as shown in SEQ ID NO.2.

Example 3 in vitro catalytic reaction and product detection of AarBDH5

1) Selecting BamH I/EcoR I as enzyme cutting sites, carrying out enzyme cutting on a pET-28a vector, connecting a target gene fragment AarBDH5 into the enzyme cutting vector by using a seamless cloning kit, and constructing a pET-28a-Aa rBDH5 gene expression vector;

2) pET-28a empty vector (control) and pET-28a-AarBDH5 were transformed into BL21 competent cells, respectively, and the transformed cells were plated on a plate containing 50mg/L Kana (kanamycin) to select positive clones. Selecting a single colony, inoculating the single colony in an LB liquid culture medium containing corresponding antibiotics, culturing overnight, transferring according to the ratio of 1 ₆₀₀ When the concentration reaches 0.6, adding 0.5mM IPTG, and inducing in a shaking table at 16 ℃ for 16h in a dark place at the rotating speed of 110r/min;

3) And (3) freezing and centrifuging the induced bacterial liquid and pET-28a empty carrier bacterial liquid at 7000rpm/min for 10min to obtain bacterial precipitation, re-suspending the bacterial by using 5ml of cell lysate and 2 mu L of 50mg/ml protease inhibitor, ultrasonically crushing the bacterial at low temperature, and freezing and centrifuging at 7000rpm/min for 3min to obtain sample supernatant protein and control supernatant protein.

4) The sample supernatant was diluted with binding buffer (20 mM Tis-HCl pH =8.0, 10mM Imid azole,0.5M NaCl) and suspended in

On Ni NTA; after eluting the hybrid protein with the binding buffer, the target protein is eluted with a gradient of elution buffer containing 50, 150, 200, 300 and 500mM Imidazole, and the purified protein AarBDH5 at the optimal Imidazole elution concentration is obtained by SDS-PAGE electrophoresis. Purified protein concentration was determined using BCA assay kit.

5) AarBDH5 in vitro enzyme activity assay containing 10 g purified protease activity assay in 500 u l buffer, the buffer containing 10mM NAD +, 10 u g enzyme and 5 u g D-borneol as substrate, at 30 degrees C were incubated for 1 hours. Volatile products were detected by GC-MS method.

6) GC-MS detection results: the Aa rBDH5 gave a single product, d-camphor, in an in vitro enzymatic reaction with d-borneol as substrate, as shown in fig. 3 and the mass spectrum in fig. 4, relative to the control protein (pET-28 a empty vector supernatant). The results show that AarBDH5 has substrate specificity and product specificity in a prokaryotic expression system, and that AarBDH5 is expected to be developed into functional protein for efficiently and specifically synthesizing camphor.

In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. A protein, which is characterized by comprising the following components 1) to 3):

1) An amino acid sequence shown as SEQ ID NO. 1;

2) A sequence having the same function as the sequence shown in SEQ ID No.1 and obtained by substituting deletion or addition of one or more amino acids;

3) Has the same function with the protein shown in SEQ ID NO.1, and the N end or the C end is connected with a label to obtain the fusion protein.

2. A gene encoding the protein of claim 1.

3. The encoding gene according to claim 2, characterized in that: the coding gene is at least one of the following:

1) A nucleotide sequence shown as SEQ ID NO. 2;

2) A DNA molecule which hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the protein of claim 1;

3) A nucleotide sequence having 90% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the protein of claim 1;

4) A sequence obtained by substituting, deleting or adding nucleotides to the sequence shown by SEQ ID NO. 2;

5) Different transcripts or homologous gene sequences were generated from the nucleotide sequence shown in SEQ ID NO.2.

4. A recombinant vector, an expression cassette, a recombinant bacterium, a recombinant cell, a transgenic plant containing the coding gene of claim 2 or 3.

5. The use of the coding gene of claim 2 or 3 for catalyzing the production of camphor with borneol as substrate.

6. The recombinant vector, expression cassette, recombinant bacterium, recombinant cell and transgenic plant of claim 4, which can be used for catalyzing the growth of borneol and camphor.

7. A method of producing the protein of claim 1, comprising: recombining the nucleotide sequence of claim 2 or 3 with an expression vector, introducing the recombinant vector into a recipient microorganism to obtain a recombinant strain, culturing the recombinant strain, and expressing the recombinant strain to obtain the protein of claim 1.

8. A process for preparing camphor, characterized by: the method comprises catalyzing camphol production with the protein of claim 1.

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