Sequence, expression and application of a borneol dehydrogenase gene for producing camphor

Abstract

The present invention relates to a protein, including the following 1)-3): 1) an amino acid sequence shown in SEQ ID NO.1; 2) a sequence having the same function as the sequence shown in SEQ ID NO.1, and obtained by replacing, missing or adding one or more amino acids; 3) a fusion protein having the same function as the protein shown in SEQ ID NO.1, and connected to a tag at the N-terminus or C-terminus; the present invention clones the AarBDH4 gene from mugwort leaves, and the AarBDH4 gene is a key enzyme gene for the biosynthesis of the monoterpene compound camphor obtained from mugwort for the first time. Experiments have proved that the AarBDH4 protein of the present invention can catalyze borneol to form camphor, which has important theoretical and practical significance for the synthesis research of camphor components in mugwort, the quality improvement of mugwort medicinal materials, and the regulation and production of plant monoterpene compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal plant molecular biology and genetic engineering, and more specifically to borneol dehydrogenase AarBDH4 Proteins, encoding genes and applications. Background Art

[0002] Artemisiae Argyi Folium is a plant of the genus Artemisia in the family Asteraceae. Artemisia argyi The dried leaves of Lévl. et Vant. are pungent, warm, bitter, and enter the liver, spleen, and kidney meridians. They have the effects of warming the meridians to stop bleeding, dispelling cold and relieving pain. They are mainly used to treat hematemesis, epistaxis, metrorrhagia, menorrhagia, fetal bleeding, lower abdominal cold pain, menstrual cold, uterine cold and infertility, etc. They can be used externally to remove dampness and relieve itching. The leaves of mugwort are dried and crushed to get "mugwort wool", which can be made into moxa sticks for moxibustion. Modern research has found that volatile oil is the main material basis of mugwort, which has broad-spectrum antibacterial, antioxidant, anti-inflammatory, anti-tumor, analgesic, anti-asthmatic, and immunomodulatory capabilities. Its main components are monoterpenes, sesquiterpenes and their derivatives.

[0003] Camphor, a monoterpene compound, is one of the important components of volatile oil of Artemisia argyi. It is a white or transparent solid at room temperature, easy to sublimate, and has a unique aroma. Camphor has the effects of clearing the meridians, promoting stagnant qi, eliminating filth, killing insects and relieving itching, reducing swelling and relieving pain. It is mainly used to treat symptoms such as scabies itching, bruises and pain, and toothache. Most of the camphor currently used on the market is obtained by chemical synthesis, but it is difficult for chemically synthesized camphor to completely replace natural camphor in special uses in medicine and other fields. Chemically synthesizing camphor or extracting natural camphor from plants faces problems such as difficulty in obtaining materials, cumbersome steps, and environmental pollution. Therefore, the use of bioengineering technology to establish a microbial factory to synthesize natural camphor has great application prospects.

[0004] Previous studies have reported that borneol dehydrogenase (BDH), which belongs to the short-chain dehydrogenases / reductases (SDR) family, has the function of catalyzing borneol into camphor. BDH has been cloned in a few species such as Artemisia annua, Cinnamomum camphora, and Blumea balsamifera, but no relevant research has been found on the key enzyme gene with the function of synthesizing camphor from mugwort. Cloning BDH in mugwort and studying its function will help provide a theoretical basis for the formation of the quality of mugwort medicinal materials, and at the same time bring broad application space for the production of natural camphor using genetic engineering technology. Summary of the invention

[0005] The purpose of the present invention is to provide an borneol dehydrogenase AarBDH4 protein for efficiently synthesizing camphor, a coding gene and an application thereof.

[0006] The present invention first provides a protein, including the following 1)-3):

[0007] 1) The amino acid sequence shown in SEQ ID NO.1;

[0008] 2) A sequence having the same function as the sequence shown in SEQ ID NO.1, and obtained by substituting, deleting or adding one or more amino acids;

[0009] 3) A fusion protein having the same function as the protein shown in SEQ ID NO.1 and having a tag connected to the N-terminus or C-terminus.

[0010] The above-mentioned protein, protein tag refers to a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and / or SUMO tag, etc.

[0011] Also provided is a coding gene encoding the above protein, wherein the coding gene is at least one of the following:

[0012] 1) The nucleotide sequence shown in SEQ ID NO.2;

[0013] 2) a DNA molecule that hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the above protein;

[0014] 3) a nucleotide sequence that has 90% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the above-mentioned protein;

[0015] 4) A sequence obtained by substituting, deleting or adding nucleotides from the sequence shown in SEQ ID NO.2;

[0016] 5) Producing different transcripts or homologous gene sequences from the nucleotide sequence shown in SEQ ID NO.2.

[0017] Also provided are a recombinant vector, an expression box, a recombinant bacterium, a recombinant cell and a transgenic plant containing the encoding gene.

[0018] Based on the differential expression analysis of the whole genome of Artemisia annua and the transcriptomes of different organs / tissues and the phylogenetic evolutionary tree analysis constructed with the known function of borneol dehydrogenase BDH protein, the nucleotide sequence shown in SEQ ID NO.2 of the BDH gene that may be involved in camphor synthesis was screened out, or the nucleotide sequence was recombined with an expression vector and introduced into a recipient microorganism, the expression vector was specifically pET-28a, and the recipient microorganism was specifically Escherichia coli BL21 (DE3), to obtain a recombinant microorganism expressing AarBDH4 protein or a protein with the same function, and the recombinant microorganism was cultured to express the protein.

[0019] Also provided is the use of the encoding gene for catalyzing the production of camphor with borneol as substrate.

[0020] Also provided is the use of the recombinant vector, expression box, recombinant bacteria, recombinant cells and transgenic plants in catalyzing borneol to grow camphor.

[0021] Also provided is a primer for amplifying the coding gene, and the primer is shown as SEQ ID NO.3 to SEQ ID NO.4.

[0022] A method for preparing the proteins 1)-3) is also provided, comprising: recombining the nucleotide sequence of the coding gene with an expression vector and introducing the vector into a recipient microorganism to obtain a recombinant strain, culturing the recombinant strain, and expressing the protein.

[0023] A method for preparing camphor is also provided, the method comprising using the 1)-3) proteins to catalyze borneol to produce camphor, the camphor comprising left-handed camphor or right-handed camphor. The AarBDH4 protein is purified by ProteinIso® Ni NTA resin, an in vitro enzymatic reaction of the AarBDH4 protein is carried out in a buffer, and the catalytic product of the AarBDH4 gene in a prokaryotic expression system in vitro is detected by solid phase microextraction technology and gas chromatography-mass spectrometry (GC-MS) technology to obtain the catalytic product camphor.

[0024] The present invention clones AarBDH4, i.e., borneol dehydrogenase, from mugwort. This gene is a key enzyme gene for the synthesis of borneol, an important indicator component monoterpenoid compound, obtained for the first time from mugwort. Experiments have shown that the AarBDH4 of the present invention can catalyze borneol to form camphor, which plays an important role in further analyzing the synthesis of terpenoid compounds in mugwort, and is of great significance for further improving the yield of camphor and enhancing the quality of medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The attached figure is a phylogenetic tree of candidate BDH genes of Ai and other identified BDH genes;

[0027] Figure 2 The attached figure is an analysis diagram of the expression of candidate BDH genes in different tissue parts;

[0028] Figure 3 The attached figure shows the catalytic product detected by GC-MS in the in vitro expression system;

[0029] Figure 4 The attached figure is a mass spectrum of the catalytic product of AarBDH4, wherein the upper end of the abscissa axis is the standard ion peak, and the lower end of the abscissa axis is the product ion peak. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 AarBDH screening

[0032] 1) Based on the genome information of Artemisia selengensis and known BDH gene sequences, AarBDH with potential camphor biosynthesis function was screened. A total of 8 genes may have important functions in borneol biosynthesis, and their amino acid sequence lengths ranged from 791 to 882.

[0033] 2) Select the identified functional AaBDH (KT070864), AaADH2 (GU253890), LiBDH (AFV30207), ZSD1 (AB480831), CcBDH3 (MN551096) and 8 candidate AiBDH genes, and use MEGA software to build a phylogenetic tree. Figure 1 shown.

[0034] 3) RNA-seq technology was used to analyze the expression profiles of candidate BDH genes in different organs such as roots, stems, leaves, and flowers. Figure 2 shown.

[0035] Example 2 Gene cloning of AarBDH4 and its encoded protein sequence

[0036] According to the Ai genome AarBDH4 Primers were designed based on the sequence, and cDNA of Artemisia argyi leaf was used as template for amplification to obtain a nucleotide sequence of 810 bp in length, as shown in SEQ ID NO. 2. AarBDH4 The encoded amino acid sequence is as shown in SEQ ID NO.1.

[0037] Example 3 In vitro catalytic reaction of AarBDH4 and product detection

[0038] 1) Select Bam H I / Eco R I was used as the restriction site to digest the pET-28a vector, and the target gene fragment was cloned using a seamless cloning kit. AarBDH4 Ligated into the restriction digest vector to construct pET-28a- AarBDH4 Gene expression vectors;

[0039] 2) pET-28a empty vector (control) and pET-28a- AarBDH4 Transform into BL21 competent cells, and spread the transformed cells on a plate containing 50 mg / L Kana (kanamycin) to screen for positive clones. Select a single colony and inoculate it in LB liquid medium containing the corresponding antibiotics. After culturing overnight, transfer at a ratio of 1:100 for expansion culture. When the OD of the bacterial solution reaches 600 When it reached 0.6, 0.5 mM IPTG was added and induced for 16 h in a shaking incubator at 16 °C in the dark at a speed of 110 r / min;

[0040] 3) The induced bacterial solution and the pET-28a empty vector bacterial solution were centrifuged at 7000 rpm / min for 10 min to obtain bacterial pellets. The bacteria were resuspended in 5 ml cell lysis buffer and 2 μL 50 mg / ml protease inhibitor, and the bacteria were ultrasonically disrupted at low temperature. The sample supernatant protein and control supernatant protein were obtained by centrifugation at 7000 rpm / min for 3 min.

[0041] 4) Dilute the supernatant protein with binding buffer (20 mM Tis-HCl pH=8.0, 10 mM Imidazole, 0.5 MNaCl) and attach it to ProteinIso®Ni NTA; after eluting the impurities with binding buffer, use the gradient elution buffer containing 50, 150, 200, 300, 500 mM Imidazole to elute the target protein, and detect it by SDS-PAGE electrophoresis to obtain the purified protein AarBDH4 at the optimal Imidazole elution concentration. Use the BCA detection kit to determine the concentration of the purified protein.

[0042] 5) Determination of in vitro enzymatic activity of AarBDH4 was performed in 500 μl of buffer containing 10 mM NAD+, 10 μg of enzyme and 5 μg of borneol as substrate at 30°C for 1 hour. Volatile products were detected by GC-MS.

[0043] 6) GC-MS test results: Compared with the control protein (pET-28a empty vector supernatant protein), AarBDH4 produced a single product, dextrorotatory camphor, in the in vitro enzymatic reaction with dextrorotatory borneol as a substrate, such as Figure 3 As shown in the mass spectrum Figure 4 This indicates that AarBDH4 has substrate specificity and product specificity in the prokaryotic expression system, indicating that AarBDH4 is expected to be developed into a functional protein for efficient and specific synthesis of camphor.

[0044] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0045] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of the AarBDH4 protein encoding gene in catalyzing the production of camphor using borneol as a substrate, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2, and the protein sequence is shown in SEQ ID NO.

1.

2. A method for preparing camphor, characterized in that: The method comprises using the AarBDH4 protein according to claim 1 to catalyze borneol to produce camphor.

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