CN118184755B - Photinia fraseri transcription factor SgWRKY gene and application thereof - Google Patents

Abstract

The invention discloses a phoebe-nana transcription factor SgWRKY gene and application thereof, and relates to the technical field of genetic engineering. The transcription factor of the invention can be directly combined with a promoter of a terpene synthase gene SgTPS to activate SgTPS expression, thereby improving the yield of plant terpenoid. The invention provides an effective gene resource for accurately expressing a large amount of plant secondary metabolites (terpenoids) in plants through reading SgWRKY gene functions, and has important significance for high-efficiency production of important secondary metabolites.

Description

Transcription factor SgWRKY13 gene of Elaeagnus oleifera and its application

Technical Field

The invention relates to the technical field of genetic engineering, and more particularly to a oleanus chinensis transcription factor SgWRKY13 gene and an application thereof.

Background Art

Sindora glabra is a tree of the genus Sindora of the Leguminosae family. Its stem is rich in a large amount of terpene resin oil, the main components of which are copaene and caryophyllene. Copaene has important ecological value and can strongly attract agricultural pests such as the Mediterranean fruit fly. At the same time, copaene is also the main component of many high-end essential oils, with multiple functions such as bactericidal, anti-inflammatory, antioxidant, and analgesic. Caryophyllene can be used as a food flavoring. Therefore, Sindora glabra resin oil shows great application potential in medicine, food, and essential oils.

SgTPS is a key terpene synthase gene that catalyzes the synthesis of copaene, which was identified from the genome of Elaeagnus oleifera. It can catalyze the substrate farni pyrophosphate (FPP) to produce specific α-copaene and β-copaene compounds, and can be used for the mass production and synthesis of copaene.

However, the current use of metabolic engineering to produce target metabolites is mostly limited to single functional genes, and the application of transcription factors that initiate structural gene expression is rarely involved. Transcription factors can directly bind to the promoter of the target gene and precisely regulate the expression of the target gene. Moreover, transcription factors can simultaneously activate the expression of multiple structural genes in the metabolic pathway, thereby leading to the large-scale synthesis of the target product.

Therefore, discovering transcription factors that can accurately initiate the expression of target genes is an urgent problem that technicians in this field need to solve.

Summary of the invention

In view of this, the present invention provides a transcription factor for regulating the expression of the terpene synthase gene SgTPS. The transcription factor can directly bind to the SgTPS promoter to activate the expression of SgTPS, thereby providing an effective target gene resource for producing terpene compounds such as copaene by metabolic engineering.

In order to achieve the above object, the present invention adopts the following technical solution:

The amino acid sequence of the protein encoded by the SgWRKY13 gene of the oil nan transcription factor is shown in SEQ ID NO:2.

As a preferred technical solution, the nucleotide sequence of the oil nan transcription factor SgWRKY13 gene is shown in SEQ ID NO: 1.

ATGTCAACTACATCTGAGGCTATTGCGAACCACAGCTTGTTTGAGGAGAATCAGGGTGATCAGATGCATACGCAGATGGGTTTCCTTCCTTTTCCAGCAAATCTGAACCTTCCCTTTGCTTGTAACCAATCTCTGAAAGCCTTCAGCACCATGCCTCCTCCTTCTCTTGCATCAGAAGCAGGTTCAGCTACTGCAAACCTAGCCGAAACCCTACTCATTTCCGCCGCTCAAAAGCAAAGAGAAGACCTCACTTCTTGTTT GGGAGCAGCCCAACTCCTTTCCTTGCAAAGATCGAGTGCAAATCCATGGGCATGGGGAGAAGTATGCGATTTGAGCGGAAAGGGAAATGGGGGA GAAGAGCATCATCATCTGAGTGTCTCTGCAATGAAGATGAAGAAGATGAAGGCAAGAAGGAAGGTGAGGGAGCCAAGGTTTTGCTTCAAAACCATGAGCGACGTGGATGTGTTAGATGATGGCTACAAGTGGAGAAAGTACGGCCAGAAAGTGGTCAAGAACACCCAGCACCCCAGAAGCTACTACCGGTGCACACAAGATAACTGCCGCGTAAAGAAACGAGTGGAGCGCTTAGCGGAAGATCCAAGGATGGTAATAACC ACATACGAAGGGAGACATGTCCATTCTCCCTCAAATGATCTTGAAGATTCTCAATCTCCTTCCCATCACCTTAACAATTTCTTCTGGTAA, SEQ ID NO. 1.

MSTTSEAIANHSLFEENQGDQMHTQMGFLPFPANLNLPFACNQSLKAFSTMPPPSLASEAGSATANLAETLLISAAQKQREDLTSCLGAAQLLSLQRSSANPWAWGEVCDLSGKGNGGEEHHHLSVSAMKMKKMKARRKVREPRFCFKTMSDVDVLDDGYKWRKYGQKVVKNTQHPRSYYRCTQDNCRVKKRVERLAEDPRMVITTYEG RHVHSPSNDLEDSQSPSHHLNNFFW, SEQ ID NO.2.

The SgWRKY13 gene of the present invention is a transcription factor, which can directly bind to the SgTPS promoter to activate the expression of SgTPS and further synthesize terpenoids including eucalyptol, geraniol D, caryophyllene, and/or pinane.

The present invention cloned the SgWRKY13 gene using the following method:

(1) Extracting total RNA from phloem tissue of C. truncatum In the present invention, the total RNA of C. truncatum can be extracted by using the technical scheme commonly used in the art for extracting total RNA of cells. In the embodiments of the present invention, the RNA kit extraction method (TIANGEN, DP441) can be used.

(2) After extracting the total RNA of the oil nan tree, the total RNA is reverse transcribed to synthesize cDNA. In the present invention, the synthesis of the cDNA can be performed by a conventional cDNA synthesis method in the art without any other special requirements; in the specific embodiment of the present invention, the HiScript 1st Strand cDNA Synthesis Kit of Vazyme Company is used to complete the synthesis.

(3) After obtaining cDNA, PCR amplification of the SgWRKY13 gene was performed to obtain the target fragment. In the present invention, the PCR amplification of the SgWRKY13 gene can be performed by a conventional PCR amplification method in the art. Specifically, in the embodiment of the present invention, the PCR amplification of the Sg WRKY13 gene was performed using phanta max super-fidelity DNA Polymerase from Vazyme. The reaction procedure was: 95°C, 5min; 95°C, 30s; 55°C, 1min; 72°C, 1min 30s; 35 cycles; 72°C extension for 5min. The primer sequences are as follows: forward primer: 5'-GCGGGTCGACGGTACCATGTCAACTACATCTGAGGC-3', SEQ ID NO.3; reverse primer: 5'-TAGACATATGGGTACCTTACCAGAAGAAATTGTTAAGGTG-3', SEQ ID NO.4.

(4) After obtaining the target fragment through PCR amplification, the results were interpreted by electrophoresis to obtain the SgWRKY13 gene.

(5) The amplified target fragment was connected to pCambia2301-KY, introduced into E. coli DH5α competent cells, and sequenced after being verified as a positive clone by colony PCR. In the present invention, the vector linearization can be carried out using the conventional KpnI enzyme in the art; the recombination reaction can be carried out using conventional methods. Specifically, in the embodiment of the present invention, the KpnI enzyme product of Thermo or Takara was used for enzyme digestion; and the ClonExpress-IIOne Step CloningKit kit of Vazyme was used for recombination.

Another object of the present invention is to provide a transcription factor SgWRKY13 protein of the oil nan tree, whose amino acid sequence is shown in SEQ ID NO:2.

Another object of the present invention is to provide a biological material containing the above-mentioned oil nan transcription factor SgWRKY13 gene, wherein the material is an expression cassette, an expression vector, a cloning vector and/or an engineered bacterium.

Another object of the present invention is to provide an application of the above gene or the above biomaterial, wherein the application is any one of the following:

A. Application in regulating the synthesis of plant terpenoids;

B. Plant breeding;

C. Preparation of transgenic plants.

As a preferred technical solution, the terpene compounds include eucalyptol, geraniol D, caryophyllene, and/or pinane.

As a preferred technical solution, the plants include but are not limited to Arabidopsis thaliana and Elaeagnus oleifera.

Another object of the present invention is to provide a method for cultivating plants with high yield of terpenoid compounds, constructing the SgWRKY13 gene into the plant expression vector pCambia2301-KY, and transferring the oil jelly transcription factor SgWRKY13 gene into Arabidopsis plants using the pollen tube pathway method to obtain transgenic plants with overexpression of the SgWRKY13 gene.

It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a transcription factor SgWRKY13 gene for regulating the expression of the key terpene synthase SgTPS gene of oleophytum cochinchinensis and its application. The present invention provides an effective gene resource for accurately and massively expressing plant secondary metabolites (terpenoid compounds) in plants by interpreting the function of the SgWRKY13 gene, which is of great significance for the efficient production of important secondary metabolites.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is an electrophoresis diagram of the SgWRKY13 gene cloning according to an embodiment of the present invention, wherein M is DNA marker DL2000 and the arrow is the target gene SgWRKY13 fragment.

FIG. 2 is a schematic diagram of the pCambia2301-KY vector of an embodiment of the present invention.

FIG3 is a PCR electrophoresis diagram of the colonies transformed with the vector of SgWRKY13 according to an embodiment of the present invention, wherein M is DNA marker DL2000, and No. 1-8 are positive bacteria.

FIG. 4 is a diagram showing the screening of SgWRKY13 transgenic seedlings in an embodiment of the present invention.

FIG. 5 is an analysis of the expression level of SgWRKY13 in the overexpressing transgenic plants according to an embodiment of the present invention.

FIG6 is an analysis of the expression levels of terpene synthase genes in the overexpressing transgenic plants according to the embodiment of the present invention.

FIG. 7 is a schematic diagram showing changes in terpenoid content in overexpression plants.

DETAILED DESCRIPTION

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.

Unless otherwise specified, the examples of the present invention are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

Example 1

Cloning of the transcription factor SgWRKY13 gene

1. RNA was extracted from the phloem tissue of T. tiliaceus (TIANGEN, DP441), and the bands were intact by electrophoresis. The RNA was reverse transcribed using reverse transcriptase M-MLV and reversely transcribed into cDNA (using HiScript 1st Strand cDNA Synthesis Kit of Vazyme). The obtained cDNA was used as a template and the gene was cloned using primers, wherein the forward primer was: 5'-GCGGGTCGACGGTACCATGTCAACTACATCTGAGGC-3', SEQ ID NO.3, and the reverse primer was: 5'-TAGACATATGGGTACCTTACCAGA AGAAATTGTTAAGGTG-3', SEQ ID NO.4. PCR amplification was performed using phantamax super-fidelity DNA Polymerase of Vazyme; the PCR conditions were: 95℃, 5min; 95℃, 30s; 55℃, 1min; 72℃, 1min 30s; 35 cycles; 72℃ extension for 5min. The PCR product was detected by 1% agarose gel electrophoresis, and the result is shown in Figure 1, where M in Figure 1 is DNA marker DL2000, and the arrow is the target gene SgWRKY13 fragment. The electrophoresis result showed that the SgWRKY13 gene was amplified to obtain a band of the target size.

2. The vector pCambia2301-KY (see FIG. 2 ) was linearized by digesting with KpnI (using the corresponding restriction endonuclease products of Thermo or Takara).

Enzyme digestion reaction system: 10×L Buffer 2μl, vector plasmid 5μl, KpnI enzyme 2μl, ddH ₂ O to make up to 40μl.

3. Purify the enzyme-digested product and carry out a recombination reaction with the above-mentioned PCR product (the recombination reaction kit is ClonExpress-II One Step Cloning Kit from Vazyme).

Recombination ligation reaction system: 4 μl of linearized vector, 1 μl of inserted fragment, 2 μl of 5×CE II Buffer, 1 μl of ExnaseII, and ddH ₂ O to make up to 10 μl.

The reaction solution was gently pipetted and mixed, and the reaction solution was collected at the bottom of the tube by brief centrifugation. The reaction was placed at 37°C for 30 minutes, and then immediately placed on ice for cooling.

4. Transform the recombinant product into E. coli DH5α cells. Specific steps:

(1) Add 10 μL of the ligation product to 100 μL of E. coli competent DH5α cells;

(2) Ice bath for 30 min;

(3) Heat shock at 42°C for 60–90 s;

(4) Ice bath for 2 min;

(5) Add 800 μl LB liquid medium;

(6) Incubate at 37°C in a shaking incubator for 30 min;

(7) Centrifuge at 6000 rpm for 3 min, discard the supernatant, and coat the plate with Kana (50 mg/L) resistance culture medium;

(8) After inverted culture at 37°C for 12-16 h, pick out resistant colonies;

(9) In a 96-well plate, add 100 μL LB (containing Kana) liquid medium to each well;

(10) Take 4-8 colonies from each plate and expand them at 37°C, 180 rpm for 2 h;

(11) Take 1 μL of bacterial solution for PCR positive detection.

Pick the PCR-positive transformants and culture them to extract the plasmids. At the same time, the amplified products are sent for sequencing. The amplification and sequencing primers are the vector sequences inserted on both sides of the target gene, which are

35S-F: 5’-GACGCACAATCCCACTATCC-3’, SEQ ID NO.5;

2301-F: 5’-GCTTCCGGCTCGTATGTTG-3’, SEQ ID NO. 6.

The results of colony PCR are shown in Figure 3. The positive monoclonal colonies were selected, and the plasmids were extracted and sent for sequencing. After sequencing analysis, the transcription factor gene SgWRKY13 sequence obtained by clone No. 2 was correct.

Example 2

Obtaining plants with overexpression of transcription factor SgWRKY13

The correctly sequenced pCambia2301-SgWRKY13 vector was transferred into Agrobacterium, and Arabidopsis was transformed using the pollen tube channel method. When the Arabidopsis inflorescence axis grew to about 20 cm for the first time, it was cut from the base to promote branching; the frozen pCambia2301-SgWRKY13 glycerol bacteria were streaked on the LB resistance plate; a single clone was picked and cultured in 2-3 mL liquid LB for 24 hours; the bacteria were collected by centrifugation at 8000g for two minutes and resuspended with an equal amount of 5% sucrose solution. The single clone was cultured for one day and one night until the OD ₆₀₀ was about 2.4, and the final concentration of the resuspended was about 2.0; Silwet L-77 was added to a final concentration of 0.05% (0.5 microliters per milliliter of bacterial solution) and mixed thoroughly; all fertilized and developed fruit pods were cut off, leaving the newly opened and unopened flower buds, and watered enough one day before transformation. On the day of transformation, use a plastic pipette to suck up a small amount of transformation solution and drop it on all flower buds (including green unopened flower buds), and try not to leave too much liquid. After transformation, culture under normal light.

The transgenic T1 generation seeds after harvest are stored in a refrigerator at 4°C for about 1 week (vernalization). Prepare screening culture medium. Prepare MS culture medium (without sucrose) with 50 mg/L of kanamycin added for screening pressure. Take an appropriate amount of T0 generation seeds, put them in a 1.5 ml centrifuge tube, disinfect them with 75% alcohol for 5 minutes, and wash them with sterile water 5 times. In the clean bench, spread the disinfected seeds on the MS culture medium (without sucrose) with kanamycin added for screening pressure made above, and evenly disperse them with 0.05% agarose water. Dry and seal with paraffin film. Place the plate covered with seeds under the conditions of 16L/8D of light and 22-24°C to germinate the seeds and screen SgWRKY13 positive transgenic seedlings (see Figure 4). The first pair of true leaves of the transgenic seedlings to be screened unfold and take root, and then they can be transplanted. Transgenic seedlings with true leaves and roots are transplanted into a nursery soil mixed with peat, vermiculite and perlite for cultivation, and seeds are collected.

Example 3

Analysis of SgWRKY13 and TPSs gene expression in transgenic plants

Fluorescence quantitative PCR was used to amplify the SgWRKY13 gene and TPS gene of the overexpressing transgenic plants, and the changes in their expression levels were determined. The results showed that compared with the wild-type control, the expression levels of the SgWRKY13 gene and the TPS gene in the overexpressing strain were significantly increased, indicating that overexpression of SgWRKY13 promoted the expression of the terpene synthase gene TPS in the transgenic plants (see Figures 5 and 6).

The primers are as follows:

SgWRKY13-F:5’-AGCTACTGCAAACCTAGCCG-3’,SEQ ID NO.7,

SgWRKY13-R: 5’-GAGACACTCAGATGATGATGC-3’, SEQ ID NO.8;

TPS5-qF:5’-GCACTGGAGCTGTTCTAAAG-3’,SEQ ID NO.9,

TPS5-qR:5'-GGGTAGCTTTGTAGCAAAGTC-3',SEQ ID NO.10; TPS6-qF:5'-CGATCGGTATCAGGAACTGTC-3',SEQ ID NO.11,TPS6-qR:5'-GCTATCTCTGCTGATGAAGTG-3',SEQID NO .12; TPS7-qF:5'-GAGAGCAGGCCTACATTTC-3',SEQ ID NO.13,

TPS7-qR:5’-CCACCTTGTAATTTCTGCAAGC-3’, SEQ ID NO.14;

TPS9-qF:5’-TAGTGGTGGCCGCCTATGTC-3’,SEQ ID NO.15,

TPS9-qR:5’-GTGAACCCTGAGGTGTATAGC-3’, SEQ ID NO.16;

TPS10-qF:5’-GAAGCATTGCCTGCTCTATATAA-3’,SEQ ID NO.17,

TPS10-qR:5’-GTTTCCAGTAGCCATGAAAGC-3’, SEQ ID NO.18;

TPS11-qF:5’-GCAGACCATATTAGGAATGCTT-3’,SEQ ID NO.19,

TPS11-qR:5’-TTGGCAAACTTGAGTAGAGTG-3’, SEQ ID NO.20;

TPS12-qF:5’-GATTTCAGTTGGAGCACCATTGGC-3’,SEQ ID NO.21,

TPS12-qR:5’-CTCTTTTTATTTCATCCGAGGAGG-3’, SEQ ID NO.22;

TPS15-qF:5’-ATGACGTAAAGGGTTTTGATGG-3’,SEQ ID NO.23,

TPS15-qR:5’-GCTCTTGTGATAAGGATGCTTC-3’, SEQ ID NO.24;

The quantitative PCR reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

Table 1

Table 2

Example 4

Identification of the function of SgWRKY13 in regulating terpenoid biosynthesis in plants

SgWRKY13 transgenic seeds were taken and planted in MS medium. After culturing for 2 weeks, leaves of SgWRKY13 transgenic overexpressing plants were collected and terpenoid components in the leaves were extracted.

50 mg of ground leaf tissue was added with 1 ml of pentane and isobutylbenzene (0.1 mg ml-1) as an internal standard, and metabolites were extracted by inversion mixing at room temperature for 24 h. The extract was centrifuged at 1000 g for 15 min, and the pentane phase was transferred to a new GC vial for GC/MS analysis. The relative abundance of sesquiterpenoid metabolites was calculated by manually integrating the peak areas and normalizing with the internal standard and the amount of tissue used (dry weight). Four biological replicates were used for analysis, each containing two technical replicates. Sesquiterpenoid compounds extracted from leaf samples of SgWRKY13 transgenic plants were analyzed using an Agilent Technologies 7890A/5975C GC/MS system operated in electron ionization selected ion monitoring mode. Samples were analyzed on a DB-Wax fused silica column (Agilent Technologies) (30 m length, 250 μm inner diameter, 0.25 μm film thickness). The injector was operated in pulsed splitless mode and the injector temperature was maintained at 250 °C. Helium was used as carrier gas with a flow rate of 0.8 ml min ^-1 and a pulse pressure setting of 25 psi for 0.5 min. Scan range: m/z 40–500; SIM: m/z 93, 94, 105, 107, 119, 122, and 202. The dwell time was 50 ms. The oven program included 40 °C for 3 min, 10 °C min ^-1 to 130 °C, 2 °C min ^-1 to 200 °C, 50 °C min ^-1 to 250 °C, and then 250 °C for 15 min. Data acquisition and processing were performed using ChemStation software (Agilent Technologies). Compounds were identified by comparing mass spectra with the NIST/EPA/NIH Mass Spectral Library Version 2.0 (http://chemdata.nist.gov/).

The results are shown in Figure 7. The contents of terpenoids in the three transgenic strains were significantly increased, including α-Eudesmol, Germacrene D, Caryophyllene and Pinane. The content of Caryophyllene showed the greatest change, reaching up to about 3 times.

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.

The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 (7)

1. The photinia transcription factor SgWRKY gene is characterized in that the amino acid sequence of the coded protein is shown as SEQ ID NO. 2.

2. The photinia transcription factor SgWRKY gene of claim 1, wherein the nucleotide sequence is shown in SEQ ID No. 1.

3. The photinia transcription factor SgWRKY protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

4. A biological material containing the photinia transcription factor SgWRKY gene according to claim 1 or 2, characterized in that the material is an expression cassette, an expression vector, a cloning vector and/or an engineering bacterium.

5. Use of a gene according to claim 1 or 2 or a biomaterial according to claim 4, characterized in that the use is for regulating the synthesis of plant terpenoids; the plant is Arabidopsis thaliana or Photinia fraseri.

6. The use according to claim 5, wherein the terpenoid comprises eucalyptol, megaerene D, caryophyllene, and/or pinane.

7. A method for cultivating high-yield terpenoid plants is characterized in that SgWRKY genes are constructed on plant expression vectors pCambia2301-KY, a pollen tube channel method is adopted to transfer the photinia fraseri transcription factor SgWRKY genes into arabidopsis plants to obtain transgenic plants with SgWRKY genes over-expressed, and the nucleotide sequence of the SgWRKY genes is shown as SEQ ID NO. 1.