Aquilaria sinensis 14-3-3 protein gene AsGRF1 and application thereof

Abstract

The invention provides a 14-3-3 protein gene AsGRF1 of aquilaria sinensis and application of the 14-3-3 protein gene AsGRF1, and belongs to the field of genetics. The cDNA (complementary deoxyribonucleic acid) sequence of the aquilaria sinensis 14-3-3 protein gene AsGRF1 is as shown in SEQ ID NO: 1. A protein sequence translated by a coding region of the aquilaria sinensis 14-3-3 protein gene AsGRF1 is as shown in SEQ ID NO: 2. The 14-3-3 protein gene AsGRF1 is cloned from aquilaria sinensis calluses, RT-qPCR analysis shows that the expression quantity of the gene is remarkably increased after NaCl induction for 1 d and 5 d, the expression quantity is consistent with the FPKM value of transcriptome data, and it is shown that the gene can participate in 2-(2-phenethyl) chromone biosynthesis regulation and control; the soluble protein of the AsGRF1 is obtained by constructing a recombinant expression vector of the AsGRF1 and adopting a heterologous expression mode. Through a gene silencing technology, the AsGRF1 is proved to positively regulate the biosynthesis of the characteristic component 2-(2-phenethyl) chromone in the aquilaria sinensis.

Description

Technical Field

[0001] The invention relates to a Aquilaria sinensis 14-3-3 protein gene AsGRF1 and application thereof, and belongs to the field of genetics. Background Art

[0002] Aquilaria is a resin-containing wood that is slowly formed by plants of the genus Aquilaria (Thymelaeaceae) after being stimulated by external damage. It has important medicinal and aromatic values, and is a commonly used Chinese herbal medicine in clinical practice and an internationally famous spice. Aquilaria sinensis is the main aromatic tree species in my country and the only original plant of medicinal aquilaria listed in the Chinese Pharmacopoeia. The mechanism of aquilaria sinensis formation has not yet been elucidated, which seriously restricts the healthy and sustainable development of the aquilaria sinensis industry. 2-(2-phenylethyl) chromone is a characteristic component and main active ingredient of aquilaria sinensis, and has multiple pharmacological activities, such as anti-inflammatory, antibacterial and antioxidant. Therefore, revealing the regulatory mechanism of 2-(2-phenylethyl) chromone biosynthesis is conducive to clarifying the mechanism of aquilaria sinensis formation.

[0003] The inventors previously revealed that polyketide synthase AsPECPS (2-(2-phenylethyl)chromone precursorsynthase) is a key enzyme in the biosynthesis of agarwood 2-(2-phenylethyl)chromone, which can catalyze the synthesis of 2-(2-phenylethyl)chromone key precursor 1,5-diphenyl-1,3-pentanone, i.e., C6-C5-C6 structural skeleton.

[0004] 14-3-3 proteins are a class of extremely conservative and ubiquitous proteins in eukaryotes. Based on the differences in the conserved core regions of 14-3-3 proteins, 14-3-3 proteins can be divided into two categories: ε group and non-ε group. 14-3-3 proteins play an indispensable role in plant growth and development, hormone signal transduction, biotic and abiotic stresses, primary metabolism and other physiological processes. In recent years, the regulatory function of 14-3-3 proteins in the biosynthesis of secondary metabolites in plants has also been gradually analyzed. For example, in the food crop soybean, it was found that 14-3-3 proteins can bind to MYB transcription factors to jointly regulate isoflavone biosynthesis. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a 14-3-3 protein gene AsGRF1 of Aquilaria sinensis and its application. Studies have shown that the AsGRF1 protein of Aquilaria sinensis can be used to regulate the biosynthesis of 2-(2-phenylethyl) chromone compounds of Aquilaria sinensis and improve the quality of artificial incense making technology.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a Aquilaria sinensis 14-3-3 protein gene AsGRF1, and the cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is shown in SEQ ID NO:1.

[0008] In some embodiments of the present invention, the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is strongly expressed in Aquilaria sinensis after being induced by NaCl.

[0009] In some embodiments of the present invention, the protein sequence translated from the coding region of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is shown in SEQ ID NO:2.

[0010] In some embodiments of the present invention, the primer pair AsGRF1-F1 and AsGRF1-R1 are used to clone and obtain the 14-3-3 protein gene AsGRF1, the sequence of AsGRF1-F1 is shown in SEQ ID NO:3, and the sequence of AsGRF1-R1 is shown in SEQ ID NO:4.

[0011] In a second aspect, the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 described in the present invention.

[0012] In some embodiments of the present invention, the primer pair for amplifying the entire cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is AsGRF1-F2 and AsGRF1-R2, the sequence of AsGRF1-F2 is shown in SEQ ID NO:5, and the sequence of AsGRF1-R2 is shown in SEQ ID NO:6, wherein the lowercase bases are homology arms.

[0013] In a third aspect, the present invention provides a method for expressing the Aquilaria sinensis 14-3-3 protein gene AsGRF1, comprising the following steps:

[0014] (1) transforming Escherichia coli cells with the recombinant vector described in claim 4 to obtain a recombinant prokaryotic expression strain expressing AsGRF1;

[0015] (2) inoculating the recombinant prokaryotic expression strain into a liquid culture medium containing kanamycin and culturing the activated strain overnight;

[0016] (3) The activated recombinant prokaryotic expression strain was transferred to a liquid culture medium containing kanamycin, and after shaking culture, IPTG was added to induce the expression of the recombinant protein AsGRF1;

[0017] (4) After induction is completed, the expressed AsGRF1 recombinant protein is recovered and purified.

[0018] In a fourth aspect, the present invention provides a recombinant gene silencing vector, wherein the recombinant gene silencing vector is a partial specific cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 described in the present invention.

[0019] In some embodiments of the present invention, the primer pairs for amplifying the partial specific cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 are RNAi-F1 and RNAi-R1, RNAi-F2 and RNAi-R2; the sequence of the RNAi-F1 is shown in SEQ ID NO:7, the sequence of the RNAi-R1 is shown in SEQ ID NO:8, the sequence of the RNAi-F2 is shown in SEQ ID NO:9, and the sequence of the RNAi-R2 is shown in SEQ ID NO:10.

[0020] In a fifth aspect, the present invention provides the use of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 in the biosynthesis of 2-(2-phenylethyl) chromone compounds, a characteristic component in Aquilaria sinensis.

[0021] In some embodiments of the present invention, the Aquilaria sinensis 14-3-3 protein gene AsGRF1 positively regulates the biosynthesis of 2-(2-phenylethyl) chromone.

[0022] The beneficial effects of the present invention are as follows: the present invention cloned the 14-3-3 protein gene AsGRF1 from the callus tissue of Aquilaria sinensis, and RT-qPCR analysis showed that the expression of the gene was significantly increased after 1d and 5d of NaCl induction, which was consistent with the FPKM value of the transcriptome data, indicating that it may be involved in the regulation of 2-(2-phenylethyl) chromone biosynthesis; by constructing a recombinant expression vector of AsGRF1 and using heterologous expression to obtain the soluble protein of AsGRF1. Through gene silencing technology, it was proved that AsGRF1 positively regulated the biosynthesis of 2-(2-phenylethyl) chromone, a characteristic component in Aquilaria sinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 This is the expression diagram of the AsGRF1 gene of the present invention after being induced by NaCl for 1 day and 5 days. In the figure, * indicates the statistical comparison between the experimental group and the control group; **: P<0.01.

[0025] Figure 2 The figure is an agarose gel electrophoresis diagram of the gene coding region of AsGRF1 described in the present invention.

[0026] Figure 3 This is a phylogenetic tree of the protein encoded by AsGRF1 described in the present invention.

[0027] Figure 4 This is a multiple sequence alignment diagram of the AsGRF1 protein described in the present invention and other plant 14-3-3 proteins.

[0028] Figure 5 This is the SDS-PAGE electrophoresis diagram of the 14-3-3 protein AsGRF1 obtained by heterologous expression in the present invention, wherein M: Marker; 1-2: AsGRF1-GST proteins.

[0029] Figure 6 This is a diagram of AsGRF1 and AsPECPS gene expression in Aquilaria sinensis callus after AsGRF1 silencing described in the present invention, in which * indicates statistical comparison between the experimental group and the control group; *: P<0.05, **: P<0.01.

[0030] Figure 7 The structural formulas and TIC diagrams of eight 2-(2-phenylethyl) chromone components in Aquilaria sinensis callus quantitatively analyzed by Qtrap-MS of the present invention are shown.

[0031] Figure 8 This is a graph showing the change in 2-(2-phenylethyl) chromone content in callus tissue of Aquilaria sinensis with AsGRF1 gene silencing of the present invention, in which * indicates statistical comparison between the experimental group and the control group; *: P<0.05, **: P<0.01. DETAILED DESCRIPTION

[0032] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0033] The invention is further described below in conjunction with the accompanying drawings and specific embodiments:

[0034] The cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 of the present invention is shown as SEQ ID NO:1; the protein sequence translated from the coding region of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is shown as SEQ ID NO:2.

[0035] Example 1: Acquisition and analysis of the gene of Aquilaria sinensis 14-3-3 protein AsGRF1

[0036] 1.1 Amplification and expression analysis of AsGRF1 gene

[0037] According to the kit method (NORGEN, Total RNA Purification Kit, catalog number: 17200), RNA was extracted from Aquilaria sinensis callus tissues induced by NaCl for 1d and 5d, and Aquilaria sinensis callus tissues not induced by NaCl were used as the control group, and the RNA was reversed into cDNA (catalog number: R211) according to the Norwegian HiScript II 1st Strand cDNA Synthesis Kit, and the AsRPL gene was used as the internal reference. The expression changes of the AsGRF1 gene were analyzed by RT-qPCR method. The RT-qPCR primers are shown in Table 1. The two-step method was adopted according to the reaction system (Table 2).

[0038] Three replicates were set for each reaction, and the RT-qPCR data were statistically analyzed using Microsoft Excel software2020 and Graphpad Prism 9. -△△Cq Methods The calculation was performed by paired one-way analysis of variance (ANOVA) for Duncan's test, where P<0.05 was considered significant and P<0.01 was considered extremely significant. RT-qPCR analysis of the expression of AsGRF1 in Aquilaria sinensis induced by NaCl used primers AsRPL-F1 and AsRPL-R1, AsGRF1-qpcr-F1 and AsGRF1-qpcr-R1, and the corresponding sequences were SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0039] Table 1 Primer sequences for RT-qPCR analysis of the expression changes of AsGRF1 in Aquilaria sinensis after NaCl induction.

[0040]

[0041] Table 2 Fluorescence quantitative PCR reaction system

[0042]

[0043] The expression of AsGRF1 gene induced by NaCl is as follows Figure 1When the callus tissue of Aquilaria sinensis was treated with NaCl for 1 day, the expression level of AsGRF1 gene increased by 1.5 times compared with the control group, and after 5 days of NaCl treatment, the expression level of AsGRF1 gene further increased to 2.3 times that of the control group, which was consistent with the accumulation pattern of 2-phenylethyl chromone after NaCl induction, indicating that AsGRF1 gene can respond positively to NaCl stress and may be involved in the biosynthesis of diphenylethyl chromone.

[0044] The full length of the AsGRF1 gene coding region was amplified using AsGRF1-F1 and AsGRF1-R1, and the PCR product was electrophoresed in a 1% agarose gel (the agarose gel electrophoresis of the AsGRF1 gene coding region is shown in Figure 2 As shown), cut the fragments of the corresponding target fragment size with a knife under ultraviolet light, and recover the fragments according to the above method. Refer to the Novozyme 5min TA / Blunt-Zero Cloning Kit (Cat. No.: C601) to connect the recovered PCR product with the pCE2 TA / Blunt-Zero vector. The specific steps are as follows:

[0045] The optimal amount of inserted fragment is [0.05×fragment editing logarithm]ng

[0046] A PCR reaction was performed at 37° C. according to the following reaction system (Table 4), using the primer pair AsGRF1-F1 and AsGRF1-R1 for cloning the 14-3-3 protein gene AsGRF1. The sequence of AsGRF1-F1 is shown in SEQ ID NO: 3, and the sequence of AsGRF1-R1 is shown in SEQ ID NO: 4.

[0047] Table 3 Primer sequences for PCR amplification of AsGRF1

[0048]

[0049] Table 4 Ligation reaction system of PCR products and pCE2 TA / Blunt-Zero vector

[0050]

[0051] The ligation product was transformed into cloning competent cells DH5α and inverted for 12 hours at 37°C. Single clones were selected for colony PCR identification. Those with positive PCR identification were sent to Beijing Liuhe Huada Co., Ltd. for sequencing. The correct fragments were aligned with SnapGene and shaken to extract the pCE2TA / Blunt-Zero-AsGRF1 plasmid, which was stored at -20°C. The electrophoresis results of the agarose gel are shown in Figure 2As shown, the lanes on the left are DNA Markers, indicating DNA fragments with molecular weights of 2000, 1500, 1000, 750, 500, 250 and 100 bp from top to bottom respectively. Lane 1 is the AsGRF1 gene, and a single and obvious fragment can be seen at 500-750 bp, which is consistent with the size of the full-length sequence of the coding region of AsGRF1, indicating that the AsGRF1 gene was successfully cloned from the cDNA of Aquilaria sinensis.

[0052] 1.2 Phylogenetic tree analysis of AsGRF1 protein

[0053] All 14-3-3 proteins in Arabidopsis were downloaded from the Arabidopsis database (https: / / www.arabidopsis.org / ), and a total of 13 AtGRF1~13 were obtained. The phylogenetic evolutionary tree of AsGRF1 and Arabidopsis 14-3-3 proteins was constructed using MEGA 7 software. According to literature reports, the 14-3-3 protein family in Arabidopsis is divided into two major categories: ε subtype and non-ε subtype. The ε group includes Episilon group GFR10, GRF11 and GRF13; mu group GRF9 and lota group GRF12; non-ε group includes Kappa group GRF6 and GRF8; Psi group GRF3, GRF5, GRF7; and Omega group GRF1, GRF2 and GRF4. The phylogenetic tree of AsGRF1 protein is shown in the figure. Figure 3 As shown in the phylogenetic tree, AsGRF1 and At14-3-3 proteins are divided into three branches, among which AtGRF6 and AtGRF8 are clustered into one branch, AtGRF9-AtGRF13 are clustered into one branch, and the remaining AtGRF1-AtGRF5, AtGRF7 and AsGRF1 are clustered into one branch; AsGRF1 is closely related to AtGRF1, AtGRF2 and AtGRF4, all of which belong to the Omega group, and it is speculated that they may have similar functions.

[0054] 1.3 Multiple sequence alignment analysis of AsGRF1 protein

[0055] The amino acid sequence of AsGRF1 was compared with that of known 14-3-3 proteins in plants using DNAMAN software. The amino acid sequences of 14-3-3 proteins in other plants were directly downloaded into fasta format files according to the NCBI number. The multiple sequence alignment of AsGRF1 protein and other plant 14-3-3 proteins is shown in Figure 1. Figure 4The results of amino acid sequence comparison showed that AsGRF1 from Aquilaria sinensis had high homology with GrGRF6 from Gossypium raimondii (NCBI No.: XP_012480020.1), HuGRF6 from Malva columbia (NCBI No.: XP_021278594.1), GmSGF141 from soybean (NCBI No.: NP_001340991.1), and AtGRF1 from Arabidopsis thaliana (NCBI No.: NP_567344.1), indicating that 14-3-3 proteins are a relatively conservative class of proteins, with partial variations in their N-terminal and C-terminal sequences, while the middle sequence is relatively conservative, with only partial substitutions of amino acids. In addition, the AsGRF1 protein contains 9 conserved α-folded secondary structures (α1 to α9), which are arranged in reverse parallel to form an L-shaped structure, of which α1 to α4 are the key to forming dimers. In addition, AsGRF1 also contains a nuclear translocation signal in the ninth α-fold region, indicating that it may have related functions that affect the subcellular localization of its interacting proteins.

[0056] Example 2: Construction of AsGRF1 recombinant expression vector and heterologous expression

[0057] 2.1 Construction of AsGRF1 gene recombinant expression vector

[0058] The full length of the AsGRF1 gene was amplified using AsGRF1-F2 / R2 in Table 5, the vector pGEX-KG was double-digested with BamH I and Sal I, the PCR product with the homology arm and the vector were connected using a homologous recombinase, and transformed into Escherichia coli DH5α. After that, colony PCR and sequencing were performed for verification, the pGEX-KG-AsGRF1 recombinant plasmid was extracted and transformed into the competent Escherichia coli BL21 (DE3), and the primer pair for amplifying the entire cDNA sequence of the Aucklandia odorifera 14-3-3 protein gene AsGRF1 was AsGRF1-F2 and AsGRF1-R2. The sequence of AsGRF1-F2 is shown in SEQ ID NO: 5, and the sequence of AsGRF1-R2 is shown in SEQ ID NO: 6, wherein the lowercase bases are homology arms.

[0059] Table 5 Primer sequences for the construction of AsGRF1 prokaryotic expression vector

[0060]

[0061] 2.2 Heterologous expression of AsGRF1 gene recombinant protein

[0062] (1) The E. coli expression strain containing the pGEX-KG-AsGRF1 recombinant plasmid was inoculated into 20 mL LB liquid medium containing the corresponding resistance and cultured at 37°C and 200 rpm until the OD 600 Value: 0.6~0.8.

[0063] (2) Add IPTG (final concentration 0.8 mM) and induce expression at 37°C, 200 rpm for 4 h.

[0064] (3) Centrifuge at 5000 rpm for 10 min at 4°C to collect the cells and weigh them.

[0065] (4) Add 4 mL of lysis buffer containing lysozyme (1 mL of lysis solution plus 10 μL of 100 mg / mL lysozyme, the formula of lysis buffer is 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) per gram of bacterial pellet, place on ice for 30 min and ultrasonically disrupt and lyse the bacteria.

[0066] (5) Centrifuge at 8000 rpm at 4°C for 10 min and collect the supernatant.

[0067] (6) Take the prepared BeyoGold TM GST-tag Purification Resin, add about 4 mL of supernatant, and shake horizontally at 100 rpm at 4°C for 1 h.

[0068] (7) The above mixture was loaded into the empty affinity chromatography column tube provided in the kit (Biyuntian, product number FCL03), and the bottom cover was opened to allow the liquid to flow down by gravity.

[0069] (8) Add 0.5-1 mL of lysis buffer each time and wash the column five times.

[0070] (9) Add 0.5 mL of elution buffer (50 mM Tris-HCl, 10 mM GSH, adjusted to pH 8.0) each time, and elute the target protein 6 to 10 times. The eluate is the purified AsGRF1-GST-tagged protein sample.

[0071] The SDS-PAGE electrophoresis diagram of the 14-3-3 protein AsGRF1 obtained by heterologous expression of the present invention is as follows: Figure 5 As shown in the figure, M: protein marker; 1-2: AsGRF1-GST protein. It can be seen from the figure that a single protein band can be seen between 40 and 55 kDa in lanes 1-2, which is consistent with the size of the protein band expressed by AsGRF1 with a GST tag of 55.7 kDa, thus indicating that AsGRF1 has achieved soluble expression and purification.

[0072] Example 3: Construction of AsGRF1 gene silencing vector and its application in 2-(2-phenylethyl) chromone biosynthesis

[0073] 3.1 Construction of AsGRF1 gene silencing vector

[0074] (1) Design and synthesize the following three pairs of primers; each pair of primers is used as a 50 μL system, and an amplification reaction is performed according to the following procedure. The primer pairs for amplifying the partial specific cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 are RNAi-F1 and RNAi-R1, RNAi-F2 and RNAi-R2, loop-F and loop-R; the sequence of the RNAi-F1 is shown in SEQ ID NO:7, the sequence of the RNAi-R1 is shown in SEQ ID NO:8, the sequence of the RNAi-F2 is shown in SEQ ID NO:9, the sequence of the RNAi-R2 is shown in SEQ ID NO:10, the sequence of the loop-F is shown in SEQ ID NO:11, and the sequence of the loop-R is shown in SEQ ID NO:12.

[0075] Table 6 Primer sequences for construction of AsGRF1 gene silencing vector

[0076]

[0077] Table 7 PCR reaction program for the construction of AsGRF1 gene silencing vector

[0078]

[0079]

[0080] Table 8 PCR system for the construction of AsGRF1 gene silencing vector

[0081]

[0082] Use 1.5% agarose gel electrophoresis, 5v / cm voltage, 20 minutes, cut out the electrophoresis fragments of tgtF (201bp), loop (200bp), and tgtR (201bp) under ultraviolet light, put them in a system for sol recovery, dissolve and recover DNA with a total volume of 30uL of water (the recovered product is marked as: rDNAtlt3), and connect it to the vector after detection.

[0083] (2) Double Enzyme Digestion of Gel-Recovered Product and Vector The pBWA(V)HS-ccdB-ami vector and the gel-recovered product rDNAtlt3 were digested at 37°C for 1 h using the following enzyme system.

[0084] Table 9 Enzyme digestion system of pBWA(V)HS-ccdB-ami vector and gel-recovered product rDNAtlt3

[0085]

[0086] The vector digestion product and the recovered fragment digestion product were combined and purified using a PCR purification kit (the purified product was labeled P-rDNAtlt3) for the next ligation reaction.

[0087] (3) Ligation reaction

[0088] The purified product P-rDNAtlt3 from the above step was reacted with T4 ligase at 20°C for 1 h.

[0089] Table 10T4 ligase ligation system

[0090]

[0091] (4) Conversion

[0092] Thaw the chemical competent cells DH5α used for cloning on ice, take 5-10 μL of the recombinant product and add it to 100 μL of competent cells, flick the tube wall to mix, and let it stand on ice for 30 minutes. The transformation volume of the recombinant product should not exceed 1 / 10 of the volume of the competent cells used; after heat shock in a 42℃ water bath for 45 seconds, immediately place it on ice to cool for 2-3 minutes. Add 500 μL of LB liquid culture medium without antibiotics, and shake the bacteria at 37℃, 200-250rpm for 1 hour. Preheat the LB solid culture medium plate with the corresponding resistance in a 37℃ incubator. Centrifuge at 5000rpm for 5 minutes and discard 500 μL of supernatant. Resuspend the bacteria with the remaining culture medium and gently spread it on the plate containing the correct resistance with a sterilized pipette tip. Incubate inverted in a 37℃ incubator for 12-16 hours.

[0093] (5) Colony PCR Identification

[0094] Eight plaques were picked for simultaneous 1.5 mL EP tube inoculation and PCR identification, with identification primers 35seq: tTCATTTGGAGAGAACACGGGggac (2861 bp), NOSseq-R: caagaccggcaacaggatt caatc (3759 bp). The colony PCR system is shown in the following table:

[0095] Table 11 Colony PCR system

[0096]

[0097] The target band is a fragment of about 918 bp. Take the bacterial solution corresponding to 1-3 positive bands and send them for sequencing. After the sequencing results come out, shake the corresponding colonies with correct sequencing to extract plasmids.

[0098] 3.2 AsGRF1 positively regulates the biosynthesis of 2-(2-phenylethyl) chromone in Aquilaria sinensis callus

[0099] 3.2.1 Transformation of Agrobacterium with pBWA(V)HS-AsGRF1-RNAi-ami

[0100] Thaw the competent cells GV3101 on ice, take 0.1-1μg of the recombinant product and add it to 100μL competent cells, flick the tube wall to mix, note that the volume of the recombinant product transformation should not exceed 1 / 10 of the volume of the competent cells used, then stand on ice for 5min, liquid nitrogen for 5min, 37℃ water bath for 5min, ice bath for 5min, add 700μL LB liquid medium without antibiotics, shake and culture at 28℃ for 2-3h. Centrifuge at 6000rpm for one minute to collect the bacteria, take about 100μL of the supernatant, gently blow and resuspend the bacteria, apply it on the LB or YEB plate containing kanamycin and rifampicin antibiotics, and invert it in a 28℃ incubator for 2-3 days.

[0101] 3.2.2 Genetic transformation of Aquilaria sinensis callus by gene silencing

[0102] The fresh callus of Aquilaria sinensis was infected with Agrobacterium liquid containing the recombinant plasmid pBWA(V)HS-RNAi-AsGRF1, so as to obtain the transgenic system of Aquilaria sinensis callus.

[0103] (1) Select the callus tissue of Aquilaria sinensis with vigorous growth and loose texture, divide it into small clumps of uniform size using sterilized tweezers in a clean bench, and pre-culture it in MS solid culture medium in the dark for 24 hours.

[0104] (2) Agrobacterium GV3101 containing the pBWA(V)HS-RNAi-AsGRF1 recombinant plasmid was transferred into LB liquid medium containing Kan and Rif. First, 5 mL of LB was used for small shaking, and then the medium was transferred into 50 mL of LB liquid medium for amplification and shaking culture. The culture was cultured at 28°C and 200 rpm until the OD 600 It is 0.6~0.8.

[0105] (3) The bacterial solution was centrifuged at 4°C, 4000 rpm for 10 min, the bacteria were collected, and then resuspended in MS liquid culture medium at a volume ratio of 1:2 (bacterial solution volume: MS liquid culture medium volume) for infecting Aquilaria sinensis callus.

[0106] (4) Place the pre-cultured Aquilaria sinensis callus in the resuspended bacterial solution in a clean bench and gently shake it to ensure full contact. Then let it stand for 5 minutes. Use sterilized filter paper to absorb the bacterial solution on the callus and make it completely dry. Then transfer the infected callus to MS medium containing 50 ng / μl Hyg and 300 ng / μl Cef and culture it at 25°C in the dark for 8 days. The Aquilaria sinensis callus that has not been infected with Agrobacterium is used as the blank control group.

[0107] 3.2.3 RT-qPCR detection of the expression of related genes after gene silencing

[0108] 3.2.3.1 RNA extraction from Aquilaria sinensis callus

[0109] RNA was extracted using the NORGEN kit (Cat. No. 17200) as follows:

[0110] (1) Preliminary preparation: sterilize the mortar, pestle, imported pipette tip, and imported 1.5 mL and 2 mL EP tubes; prepare 70% ethanol using sterilized ddH2O or DEPC water.

[0111] (2) Prepare liquid nitrogen. First, pre-cool the mortar and pestle with liquid nitrogen for 2 to 3 times. After sufficient cooling, place the Aquilaria sinensis callus in the mortar and add liquid nitrogen for quick freezing.

[0112] (3) Add liquid nitrogen and grind repeatedly for 3 to 4 times until the callus tissue is completely turned into powder.

[0113] (4) Place the imported 1.5 mL EP tube in liquid nitrogen for quick freezing, then scrape the powder on the wall of the mortar, add 600 μL Buffer RL, and mix by vortexing.

[0114] (5) Centrifuge at 12,000 rpm for 2 min and transfer the supernatant to a 1.5 mL EP tube (if the supernatant contains a lot of powder, it will clog the separation column and can be centrifuged again after transfer).

[0115] (6) Add an equal volume of 70% ethanol and mix by inverting to obtain a lysate.

[0116] (7) Assemble the column and collection tube in the kit, add 600 μL of lysate, and centrifuge at 14,000 rpm for 1 min. Repeat the operation to allow all the lysate to pass through the column, and finally centrifuge the empty column at 14,000 rpm for 2 min.

[0117] (8) Add 400 μL Wash Solution A, centrifuge at 14,000 rpm for 1 min, discard the filtrate, and repeat the process 3 to 4 times.

[0118] (9) Place the column in a new collection tube, add 20 μL of Elution Buffer to the center, centrifuge at 2000 rpm for 2 min, and then centrifuge at 14000 rpm for another 1 min.

[0119] (10) The integrity of the extracted RNA was tested by 1.2% agarose gel electrophoresis, and the quality of RNA was tested by NanoDrop2000.

[0120] 3.2.3.2 Reverse transcription of RNA into cDNA

[0121] The procedure was carried out according to the instructions of the Novozymes HiScript II 1st Strand cDNA Synthesis Kit (Cat. No.: R211).

[0122] 3.2.3.3 Real-time PCR detection of related gene expression after gene silencing

[0123] The RNA of Aquilaria sinensis callus tissue after AsGRF1 gene silencing was extracted and reverse transcribed, and the expression changes of the key enzyme type III polyketide synthase gene AsPECPS and AsGRF1 gene in the synthesis of 2-(2-phenylethyl) chromone were analyzed by qPCR method. The RT-qPCR primers of AsPECPS are AsPECPS-qpcr-F1: GCAGCGTGGTGGTATA, AsPECPS-qpcr-R1: TACAACCTCTAGCAGCAGCGTTCA. The reaction conditions and data processing methods refer to Example 1.1. The expression of AsGRF1 and AsPECPS genes in Aquilaria sinensis callus tissue after AsGRF1 silencing is shown in the figure below. Figure 6 As shown in the figure, the expression level of AsGRF1 is only 0.3 of that in the control group, indicating that the AsGRF1 gene was successfully silenced; in addition, in the callus strain of Aquilaria sinensis with AsGRF1 gene silenced, the expression level of AsPECPS also dropped to half of that in the control group, indicating that the AsGRF1 gene may have a positive regulatory effect on the transcription of AsPECPS.

[0124] (4) Detection of 2-(2-phenylethyl) chromone content in callus tissue of Aquilaria sinensis after gene silencing

[0125] The callus tissues of Aquilaria sinensis infected with Agrobacterium containing the recombinant plasmid pBWA(V)HS-RNAi-AsGRF1 for 8 days and those of the control group were transferred to MS solid culture medium containing 150mM NaCl, 50ng / μL Hyg and 300ng / μL Cef and continued to be cultured for 10 days; the callus tissues were collected and naturally dried in the shade at room temperature, 0.5mL of gold-labeled methanol solution was added to 0.1g of naturally dried callus tissues, ultrasonically extracted for 1h, and the extract was centrifuged at 4°C, 13000rpm for 60min, the supernatant was aspirated, and stored at -20°C for detecting the content of 2-(2-phenylethyl) chromone.

[0126] The contents of eight 2-(2-phenylethyl) chromone components in CK and AsGRF1-RNAi single and double gene silencing callus were determined by Qtrap-MS (Table 12). Acquity HSS T3 column (2.1 mm inner diameter × 100 mm, 1.7 μm Waters Corporation, Wexford, Ireland) was used, and the LC column was maintained at 40°C in the thermostatic column chamber. Two eluents were used in the gradient program, 0.1% formic acid (A) and acetonitrile (B): 0-20 min, 40-90% (B). The flow rate was 0.3 mL / min and the injection volume was 2 μL. Qtrap-MS was equipped with an ESI ion source and collected in positive ion mode. ESI ion source parameters, nebulizer gas (GS1): 55psi, heater gas (GS2): 55psi; curtain gas (CUR): 35psi; collision activated dissociation (CAD): high; ion spray needle voltage (IS): +5500V; nebulization temperature (TEM): 550℃; deionization cluster voltage (DP): 80V; collision activated dissociation (CAD) gas: high; collision energy (CE): 30 (eV). Analyst 1.6.3 software (SCIEX, USA) was used for data processing. 1,5-diphenyl-1,3-pentanone (C6-C5-C6) was used as the internal standard, and online optimization was performed to obtain the optimal parameters.

[0127] All data were statistically analyzed using Graphpad Prism 9. All values ​​are mean ± SD of three replicates. Duncan's paired t-test was performed, and P < 0.05 was considered significant.

[0128] Table 12 Quantification of eight 2-(2-phenylethyl) chromone components in callus of Aquilaria sinensis by Qtrap-MS

[0129]

[0130] The structural formulas of eight 2-(2-phenylethyl) chromone components in callus tissue of Aquilaria sinensis quantified by Qtrap-MS and their TIC images on Qtrap-MS are shown in Figure 2 Figure 7 As shown. Among these 8 chromones, there are simple chromones without substituents (PEC-1), chromones with single hydroxyl substitution (PEC-2), chromones with dihydroxyl substitution (PEC-6, PEC-7), chromones with multiple methoxyl substitutions (PEC-3, PEC-4, PEC-5), and chromones with both methoxyl and hydroxyl substitutions (PEC-8). The above chromone structural formulas more comprehensively reflect the main chromone components in agarwood, and therefore have good representativeness.

[0131] The content of 2-(2-phenylethyl) chromone in callus of Aquilaria sinensis after AsGRF1 gene silencing Figure 8 As shown in the figure, the contents of the eight chromones were significantly reduced after AsGRF1 gene silencing; among them, PEC-4 with a higher content was only 14.1% of the control group, and PEC-5 with the second highest content was only 7.1% of the control group. Combined with the above RT-qPCR results, it is speculated that AsGRF1 may positively regulate the biosynthesis of 2-(2-phenylethyl) chromone in Aquilaria sinensis callus by affecting the transcription level of AsPECPS.

[0132] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A 14-3-3 protein gene AsGRF1 from Aquilaria sinensis, characterized in that: The cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is shown in SEQ ID NO:

1.

2. The Aquilaria sinensis 14-3-3 protein gene AsGRF1 according to claim 1, characterized in that: The Aquilaria sinensis 14-3-3 protein gene AsGRF1 is strongly expressed in Aquilaria sinensis after being induced by NaCl; the protein sequence translated from the coding region of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is shown in SEQ ID NO:

2.

3. The Aquilaria sinensis 14-3-3 protein gene AsGRF1 according to claim 1, characterized in that: The primer pair AsGRF1-F1 and AsGRF1-R1 used for cloning and obtaining the 14-3-3 protein gene AsGRF1, the sequence of the AsGRF1-F1 is shown in SEQ ID NO:3, and the sequence of the AsGRF1-R1 is shown in SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 described in claim 1.

5. The recombinant expression vector according to claim 4, characterized in that The primer pair for amplifying the entire cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 is AsGRF1-F2 and AsGRF1-R2. The sequence of AsGRF1-F2 is shown in SEQ ID NO: 5, and the sequence of AsGRF1-R2 is shown in SEQ ID NO: 6, wherein the lowercase bases are homology arms.

6. A method for expressing the Aquilaria sinensis 14-3-3 protein gene AsGRF1, characterized in that: The following steps are involved: (1) transforming Escherichia coli cells with the recombinant vector described in claim 5 to obtain a recombinant prokaryotic expression strain expressing AsGRF1; (2) inoculating the recombinant prokaryotic expression strain into a liquid culture medium containing kanamycin and culturing the activated strain overnight; (3) The activated recombinant prokaryotic expression strain was transferred to a liquid culture medium containing kanamycin, and after shaking culture, IPTG was added to induce the expression of the recombinant protein AsGRF1; (4) After induction is completed, the expressed AsGRF1 recombinant protein is recovered and purified.

7. A recombinant gene silencing vector, characterized in that: The recombinant gene silencing vector contains the specific cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 described in claim 1.

8. The recombinant gene silencing vector according to claim 8, characterized in that: The primer pairs for amplifying the partial specific cDNA sequence of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 are RNAi-F1 and RNAi-R1, RNAi-F2 and RNAi-R2; the sequence of the RNAi-F1 is shown in SEQ ID NO:7, the sequence of the RNAi-R1 is shown in SEQ ID NO:8, the sequence of the RNAi-F2 is shown in SEQ ID NO:9, and the sequence of the RNAi-R2 is shown in SEQ ID NO:

10.

9. Use of the Aquilaria sinensis 14-3-3 protein gene AsGRF1 as claimed in any one of claims 1 to 4 in the biosynthesis of 2-(2-phenylethyl) chromone compounds, a characteristic component in Aquilaria sinensis.

10. The use according to claim 9, characterized in that The white agarwood 14-3-3 protein gene AsGRF1 positively regulates the biosynthesis of 2-(2-phenylethyl) chromone.

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