CN114774430A - Gene of liriodendron adventitious root promoting factor LhWRKY1 and application thereof - Google Patents

Abstract

The invention discloses an liriodendron adventitious root promoting factor LhWRKY1 gene, wherein the full-length nucleotide sequence of the LhWRKY1 gene is shown as SEQ ID NO.1, and the CDS sequence is shown as SEQ ID NO. 2; a WRKY gene is separated and cloned by an RACE method, and the function of the WRKY gene is verified by constructing an expression vector and simultaneously transforming the expression vector into arabidopsis, so that the growth advantage of the main root of the transgenic arabidopsis is weakened, the number of lateral roots is increased and the root length is increased. Therefore, the gene LhWRKY1 of the liriodendron manihot has good application prospects in promoting root growth and development and improving the rooting rate and survival rate of cuttage. The invention provides a foundation for the application of the liriodendron LhWRKY1 gene in molecular breeding.

Description

Gene of liriodendron adventitious root promoting factor LhWRKY1 and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an unlined root promoting factor LhWRKY1 gene of liriodendron and application thereof.

Background

The Liriodendron L is a fast-growing deciduous tree with straight trunk, excellent material, beautiful flowers and leaves, less plant diseases and insect pests, strong pollution resistance and higher ornamental value. The species of this genus exhibits a typical east Asia-North American discontinuous distribution pattern, with only Liriodendron tulipifera in east Asia and Liriodendron tulipifera in North America. By means of artificial hybridization, scientists in China successfully perform interspecific hybridization of the two species for the first time, and obtain a filial generation of Liriodendron sino-american Liriodendron, which is called hybrid Liriodendron or hybrid Liriodendron. The liriodendron has obvious hybrid vigor, has stronger growth performance and stress resistance compared with parents, is determined as a tree species for landscaping and forestation by a plurality of cities in China, and has extremely strong development and utilization prospects. In order to maintain good characters, the hybrid Chinese tulip tree can realize the large-scale production of hybrid nursery stocks by asexual propagation technologies such as cuttage, tissue culture, grafting, somatic embryogenesis and the like. However, because the tissue culture breeding technology of the liriodendron is complicated and the cost is high, cuttage is the simplest and most effective mode for quickly obtaining a large number of liriodendron seedlings at present, and clone progeny with stable inheritance can be quickly obtained.

The research of the adventitious root occurrence mechanism becomes an important frontier field of forest breeding research. However, the asexual propagation of the hybrid liriodendron has a difficult rooting problem, which hinders the asexual breeding and popularization and application steps. By continuously screening the subject group, the clonal improved variety of the south American liriodendron (GanS-SC-LC-001) with high rooting rate of cuttage is obtained (GanS-SC-LC-001) 2019). The clone with high rooting rate has been popularized and applied in more than 20 counties and cities in China, and remarkable economic, social and ecological benefits are obtained, but the mechanism of high rooting rate in cuttage is still unclear.

Transcription Factors (TF) are trans-acting factors that can regulate gene expression, and a single Transcription factor can often regulate the expression of multiple functional genes simultaneously, thereby realizing different functions. WRKY is a transcription factor found in plants at first, and has a highly conserved domain composed of 60 amino acids, the N-terminal of the domain contains 1 to 2 conserved amino acid sequences composed of WRK YGQK, and the C-terminal of the domain contains a zinc finger structure, so that the transcription factor is named as WRKY transcription factor. The WRKY transcription regulation factor can be combined with a specific DNA sequence of a target gene promoter region so as to regulate the expression of a target gene, and can be combined with various target gene promoter regions, so that the WRKY transcription regulation factor plays an important role in pathogen defense, abiotic stress and plant hormone signal transduction, and has important significance on multiple aspects of plant growth and development, metabolic regulation, physiological response and the like.

Therefore, how to clone and apply a gene of an unlined long gown adventitious root promoting factor is a problem which needs to be solved by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides an unlined long gown adventitious root promoting factor LhWRKY1 gene and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

an adventitious root promoting factor LhWRKY1 gene of liriodendron, the full-length nucleotide sequence of the gene is shown as SEQ ID NO. 1; the CDS nucleotide sequence is shown as SEQ ID NO. 2.

As the same inventive concept as the technical scheme, the invention also claims the application of the gene of the liriodendron amboinense adventitious root promoting factor LhWRKY1 in promoting the rooting of plants.

As the same inventive concept as the above technical scheme, the invention also claims a method for promoting the growth and development of plant roots and improving the rooting rate and survival rate of cuttage, which comprises the following steps: firstly, a pBWA (V) BS-LhWRKY1 plant expression vector is constructed, then the pBWA (V) BS-LhWRKY1 plant expression vector is transformed into agrobacterium, and then plant leaves are infected by agrobacterium.

As the same inventive concept as the technical scheme, the invention also claims a protein expressed by gene of the adventitious root promoting factor LhWRKY1 of the liriodendron, and the amino acid sequence of the protein is shown as SEQ ID NO. 3.

As the same inventive concept as the technical scheme, the invention also claims a plant expression vector containing an adventitious root promoting factor LhWRKY1 gene of an liriodendron, and the plant expression vector is formed by connecting a pBWA (V) BS vector and an LhWRKY1 gene.

As the same inventive concept as the technical proposal, the invention also claims a construction method of the plant expression vector containing the gene of the liriodendron adventitious root promoting factor LhWRKY1, which comprises the following steps: firstly, synthesizing a cDNA sequence of the LhWRKY1 gene, and then amplifying a coding region sequence to obtain a target sequence; and finally, connecting the target sequence with a vector to obtain a plant expression vector.

As the preferable technical scheme of the technical scheme, the primers used for amplifying the coding region sequence are as follows:

LhWRKY 1-F1: cagtCACCTGCaaaacaacatggccgttgatctgatggg, as shown in SEQ ID NO. 4;

LhWRKY 1-R1: cagtCACCTGCaaaatacattacagctggccttcctggg, as shown in SEQ ID NO. 5.

According to the technical scheme, compared with the prior art, the method provided by the invention has the advantages that by analyzing the transcriptome difference of different periods in the development process of the cloned root of the cutting high-rooting-rate liriodendron, the expressed EST expression sequence of the liriodendron expressed in a difference mode is excavated, and the full-length cDNA nucleotide sequence is obtained by utilizing RACE technology to separate and clone and is named as LhWRKY 1. Through constructing an expression vector and simultaneously transforming the expression vector into arabidopsis thaliana to verify the function of the arabidopsis thaliana, the growth advantage of the main root of a transgenic arabidopsis thaliana plant is weakened, the number of lateral roots is increased, and the length of the lateral roots is increased. The gene LhWRKY1 of the liriodendron can inhibit the top advantages of the main root and promote the increase of the number of lateral roots in the early growth and development stage of the root system, and has good application prospects in promoting the growth and development of the root, improving the cuttage rooting rate of the plant which is difficult to root and improving the cuttage survival rate.

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 embodiments or the prior art descriptions 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 drawing of a 1% agarose gel electrophoresis of total RNA of an American gown;

FIG. 2 is a 1% agarose gel electrophoresis of the full-length PCR product of LhWRKY1 gene cDNA according to the present invention;

FIG. 3 is a drawing showing an expression vector map of LhWRKY1 for constructing pBWA (V) BS in accordance with the present invention;

FIG. 4 is a diagram showing an agarose gel electrophoresis image of the PCR of the Escherichia coli liquid of the LhWRKY1 gene of the present invention;

FIG. 5 is a PCR positive verification diagram of LhWRKY1 transgenic Arabidopsis thaliana of the present invention;

FIG. 6 is a drawing showing a root system comparison between LhWRKY1 transgenic Arabidopsis thaliana and wild type seedlings transplanted for 4 days in accordance with the present invention;

FIG. 7 is a drawing showing the root system comparison between LhWRKY1 gene-transferred Arabidopsis thaliana and wild type seedlings transplanted for 8 days.

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 obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

In the embodiment, the used plant material, namely the liriodendrons, is obtained from an experiment base of the liriodendrons in yellow Maxiang, south Chang, Jiangxi, and adventitious roots which are regenerated during cutting propagation of the liriodendrons are taken in 2017 months and are preserved at the temperature of minus 80 ℃ for later use after being frozen in liquid nitrogen.

Example 1: mining and cloning of LhWRKY1 gene

1) Sequence mining and alignment

Selecting four different growth periods of cutting and rooting of the liriodendron aristata with high rooting rate, extracting RNA, constructing transcriptome sequencing data of an adventitious root cDNA library, finding out a section of EST sequence (LH-SXY-1962) differentially expressed at different development time by a bioinformatics method, finding that the similarity of the EST sequence and WRKY genes of other species is highest in NCBI database Blast, and supposing that the EST sequence may be the WRKY transcription factor gene of the liriodendron aristata.

2) Total RNA extraction of leaf of liriodendron

Taking 1g of fresh young leaves of liriodendron, quickly grinding into powder in liquid nitrogen, extracting total RNA of liriodendron according to the instruction by using a TRizol kit, dissolving in sterilized ultrapure water, detecting by using 1% agarose gel electrophoresis (figure 1), and storing in an ultra-low temperature refrigerator at-80 ℃ for later use.

3) Full-length amplification of LhWRKY1 gene

Amplification of the full length of the LhWRKY1 gene comprises amplification of a gene core sequence, amplification of a 3 'end sequence and amplification of a 5' end sequence. Wherein, we proceed gene core primer and 3'RACE and 5' RACE primer design according to EST conservative sequence, the primers are:

gene core sequence amplification primers:

LhWRKY1-CF ATGGACGACCAGATCGCG shown as SEQ ID NO. 6;

LhWRKY1-CR: ACGTGCTTCTCGCAGGGC, shown as SEQ ID NO. 7;

3' RACE amplification primers:

LhWRKY 1-31: GAATCGGACGGGTCACGCTCG, as shown in SEQ ID NO. 8;

LhWRKY 1-32: TTGATTTCACGAAGCCGAGCC, as shown in SEQ ID NO. 9;

5' RACE amplification primers:

LhWRKY 1-51: GATTCATCGTCATCTCCGTGATAGGG, as shown in SEQ ID NO. 10;

LhWRKY 1-52: CGGACTCGCCCCATTCTGATTCGGT, as shown in SEQ ID NO. 11;

LhWRKY 1-53: TGGGGCTGGTTCTGGTGGGACATCA, as shown in SEQ ID NO. 12.

The specific operation steps are carried out according to the instruction of a kit corresponding to TaKaRa. And after the amplification is finished, performing agarose electrophoresis on the PCR product obtained by amplification, and performing product recovery by using a gel recovery kit. And carrying out vector connection on the recovered product, transforming escherichia coli competent cells, picking positive monoclonals and sending the positive monoclonals to Shanghai workers for Sanger double-end sequencing. Finally, a core sequence 701bp of the LhWRKY1 gene is obtained, a sequence 703bp is obtained by 3'RACE sequencing, and a sequence 292bp is obtained by 5' RACE sequencing. And finally, carrying out sequence comparison and splicing on the 3 partial sequences by utilizing a SeqMan program under a lasergene software package to finally obtain the cDNA full length of the LhWRKY1 gene of 1142bp, wherein the cDNA full length is shown as SEQ ID NO.1, the open reading frame CDS nucleotide sequence is shown as SEQ ID NO.2, and the encoded protein amino acid sequence is shown as SEQ ID NO. 3.

Example 2: construction of liriodendron ohWRKY 1 gene plant expression vector

1) Total RNA extraction of leaves of liriodendron

Same as example 1, step 2);

2) synthesis of first Strand cDNA

Synthesis of the first Strand cDNA of the Abelia Adinandra by means of TaKaRa PrimeScript^TMII 1st Strand cDNA Synthesis Kit. The reaction system is as follows: total RNA 2. mu.L (including RNA 2. mu.g), Oligo dT Primer 1. mu.L, dNTP Mixture 1. mu.L, RNase free dH₂O6. mu.L, and the total volume of the system is 10. mu.L. The reaction conditions were 65 ℃ for 5 minutes and then quickly placed on ice. Then, according to the kit use instruction, the following reaction liquid is added: 5 Xbuffer 4 μ L, 200U/. mu.L RTase 1 μ L, 40U/. mu.L RNase Inhibitor 0.5 μ L and RNase free dH₂O4.5. mu.L, and slowly mixing. The reaction conditions are as follows: 45 minutes at 42 ℃ and 5 minutes at 95 ℃Cooling on ice, and storing in a-80 deg.C ultra-low temperature refrigerator.

3) Amplification of Gene coding region (CDS) sequence

Designing a primer containing a coding region sequence for high-fidelity RT-PCR amplification, introducing enzyme cutting sites and protective bases at two ends of the primer, and designing the primer as follows:

the upstream primer LhWRKY 1-F1: cagtCACCTGCaaaacaacatggccgttgatctgatggg, as shown in SEQ ID NO. 4;

the downstream primer LhWRKY 1-R1: cagtCACCTGCaaaatacattacagctggccttcctggg, as shown in SEQ ID NO. 5;

and 3) carrying out PCR amplification by taking the cDNA subjected to reverse transcription in the step 2) as a template. The 25. mu.L amplification system contained PfuPCRMix 12.5. mu.L, the above upstream and downstream primers 0.8. mu.L (100. mu.M), cDNA 2. mu.L, ddH₂O8.9. mu.L. The amplification conditions were: pre-denaturation at 94 ℃ for 3min, followed by denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, for 33 cycles, and finally at 72 ℃ for 8min, and holding at 4 ℃. The PCR product was electrophoresed on 1% agarose gel (FIG. 2), the objective product was excised under an ultraviolet lamp, and the objective fragment was recovered using TaKaRa kit.

4) Ligation of the enzyme to the vector

Carrying out enzyme digestion on the target fragment recovery product obtained in the step 3), wherein an enzyme digestion reaction system is as follows: mu.L of the target product, 1. mu.L of AarI enzyme, 2. mu.L of 10 XBuffer, plus ddH₂O to the system reaches 20. mu.L. The reaction conditions were 37 ℃ for 1 h.

Selecting expression vector pBWA (V) BS vector, and carrying out enzyme digestion reaction on the vector. The reaction system contained 2. mu.L of 10 Xbuffer, 1. mu.L of BsaI/Eco31I enzyme, 4. mu.L of pBWA (V) BS-ccdB vector, ddH₂O13. mu.L, totaling to 20. mu.L. The reaction conditions were 37 ℃ for 1 h.

And combining the recovered fragment enzyme digestion product with the vector enzyme digestion product, and purifying by using a PCR purification kit for the next ligation reaction. The ligation of the vector to the target fragment was performed using T4 DNA ligase. The reaction system and the operation steps are as follows: vector and target fragment digestion mixture 2.5. mu.L, 10 Xbuffer 1. mu.L, T4 ligase 1. mu.L, ddH₂O5.5. mu.L, ligation at 20 ℃ for 1 h.

5) Transformation competence and positive identification of ligation products

10 mu L of the ligation product is taken to transform the competence of the escherichia coli, the operation steps are carried out according to the conventional transformation steps, after the completion, the bacterial liquid is smeared on an LB solid culture medium containing the kanamycin, the culture is carried out for 12 hours at 37 ℃, and the monoclonal is picked for carrying out the bacterial plaque PCR identification. Selecting 3 bacterial solutions corresponding to the positive bands, taking 100 mu L of the bacterial solutions, sampling, sequencing, inoculating the rest bacterial solutions into LB culture medium containing 10mL of kanamycin resistance, and shaking the bacteria in a test tube. Finally, taking a bacterial liquid with correct corresponding sequencing, taking a tube to extract plasmids, obtaining a pBWA (V) BS (LhWRKY) expression vector containing a target fragment, and storing for later use.

Example 3: agrobacterium-mediated genetic transformation of Arabidopsis thaliana and functional analysis

1) Wild type arabidopsis seeds (columbia Col-0) were sown in nutrient soil previously watered with water (organic matter: vermiculite: covering a preservative film for 3 days when the perlite is 1:1:1), removing the film after the seeds germinate, culturing in an artificial climate chamber at the culture environment of 25 ℃ for 14h, and culturing until the flowering period.

2) Adding 1 mu L of positive plasmid containing target fragment pBWA (V) BS LhWRKY1 expression vector detected by PCR into 50 mu L EHA105 agrobacterium-infected cells stored at 80 ℃, fully mixing uniformly, then carrying out electric transformation, adding 1mL LB liquid culture medium after electric transformation, carrying out shake culture at 30 ℃ and 180rpm for 30min for activation, and placing the activated agrobacterium on an LB solid culture medium for culture. Picking single colony from a plate culture medium, inoculating the single colony into a liquid culture medium containing Basta antibiotics, culturing for 16h in a shaking incubator at 28 ℃, and performing PCR identification on bacterial liquid by using primers (LhWRKY1-F and LhWRKY1-R) with CDS sequences, wherein an electrophoresis picture is shown in figure 4; and (3) sending the positive bacteria liquid detected by the PCR to Shanghai workers for Sanger sequencing confirmation, and storing the confirmed positive clone at 4 ℃ for later use.

The upstream primer LhWRKY 1-F: ATGGCCGTTGATCTGATGGGA, as shown in SEQ ID NO. 13;

the downstream primer LhWRKY 1-R: TTACAGCTGGCCTTCCTGGGG, as shown in SEQ ID NO. 14;

selecting the positive agrobacterium with completely correct sequencing, preparing a resuspension, and culturingTo OD₆₀₀0.8. The inflorescence of the full-bloom arabidopsis is infected for 2-3s by using the suspension solution, covered by a preservative film, sealed and kept away from light for 24h, and the operation is repeated for 3 times in total, wherein the interval is 7 days. After infection is completed, the seeds are cultured to the mature stage under normal conditions, and arabidopsis T0 generation seeds are harvested.

3) Screening of transgenic plants

About 30T 0 generation seeds are randomly put in a 1.5ml centrifuge tube, 75% alcohol is added for disinfection for 5 minutes, then sterile water is used for rinsing and washing for 2 times, disinfected toothpicks are used for dibbling the seeds on 1/2MS culture medium containing hygromycin element (40 mu g/ml), the seeds are placed in a refrigerator at 4 ℃ for vernalization for 3 days and then are moved to an illumination incubator 22 ℃, 16h illumination/8 h dark culture is carried out, and after about 2 weeks, survival arabidopsis thaliana is screened out and is transplanted in mixed culture of matrix and vermiculite for normal culture.

4) PCR identification of transgenic plants

Selecting young leaves of transgenic arabidopsis thaliana, extracting DNA (deoxyribonucleic acid) by using a CTAB (cetyl trimethyl ammonium bromide) method as a template, and performing PCR (polymerase chain reaction) detection by using LhWRKY1-F and LhWRKY1-R as primers. Wild type Arabidopsis DNA was used as a negative control (-). The PCR product detection was performed on 1.5% agarose. Detecting 10 transgenic arabidopsis thaliana in total, wherein negative control shows no strip, the transgenic arabidopsis thaliana is amplified to obtain a product fragment with a target length, and the detected positive rate is 100%; the results are shown in FIG. 5;

example 4 transgenic Arabidopsis phenotypic Observation of the LhWRKY1 Gene

And continuously culturing the transgenic arabidopsis thaliana with positive PCR detection of the T1 generation to a mature period, and harvesting seeds to obtain the T2 generation. The wild type and LhWRKY1 gene are subjected to T2 generation of transgenic positive plant seeds, sterilized and cleaned by 75% ethanol, dibbled to 1/2MS culture medium containing herbicide (BASTA 15 mu g/ml) for growth, placed in a refrigerator at 4 ℃ for vernalization for 2 days, and cultured under the conditions of 26 ℃ and 16h light/8 h dark. After 5 days of growth, wild arabidopsis thaliana and transgenic arabidopsis thaliana seedlings which grow consistently are selected and transferred onto a common 1/2MS culture medium, the culture dish is sealed by a sealing film, the culture dish is vertically placed on a culture shelf, the culture is continuously carried out under the conditions of 26 ℃, 16h illumination/8 h darkness, and the difference of the root system phenotype of the transgenic plant compared with the wild type is observed, as shown in the figure 6 and the figure 7.

As shown in FIGS. 6 and 7, the transgenic Arabidopsis has a weak growth advantage of the main root, developed lateral roots, a long length and a large number. The character plays an important role in increasing the number of adventitious roots of the tulip tree after cuttage and increasing the cuttage survival rate.

The embodiments in the present description 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 device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

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.

Sequence listing

<110> institute of biological resources of academy of sciences of Jiangxi province

Gene of <120> liriodendron adventitious root promoting factor LhWRKY1 and application thereof

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Gly Asp Gly Ser Val Ser Asn Gly Lys Gln Gly Ser Ser Leu Leu Leu

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Ala Ala Val Pro Ala Val Ser Ala Gly Lys Pro Pro Leu Ser Ser Ser

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Tyr Arg Lys Lys Cys Gln Gly His Gly His Ser Asp Asp Leu Ser Gly

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Arg Gly Cys Pro Ala Arg Lys His Val Glu Arg Asp Pro Asp Asp Pro

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atggccgttg atctgatggg a 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttacagctgg ccttcctggg g 21

Claims (7)

1. An liriodendron amboinense adventitious root promoting factor LhWRKY1 gene is characterized in that the full-length nucleotide sequence of the LhWRKY1 gene is shown as SEQ ID NO. 1; the nucleotide sequence of ORF is shown in SEQ ID NO. 2.

2. The application of an adventitious root promoting factor LhWRKY1 gene of liriodendron in promoting plant rooting.

3. A method for promoting plant rooting, comprising: firstly, a pBWA (V) BS-LhWRKY1 plant expression vector is constructed, then the pBWA (V) BS-LhWRKY1 plant expression vector is transformed into agrobacterium, and then the plant leaves are infected by the agrobacterium.

4. A protein expressed by an adventitious root promoting factor LhWRKY1 gene of an liriodendron is characterized in that the amino acid sequence of the protein is shown as SEQ ID No. 3.

5. A plant expression vector containing an adventitious root promoting factor LhWRKY1 gene of liriodendron is characterized in that the plant expression vector is formed by connecting a pBWA (V) BS vector and an LhWRKY1 gene.

6. A construction method of a plant expression vector containing an liriodendron manihot adventitious root promoting factor LhWRKY1 gene is characterized by comprising the following steps: firstly, synthesizing a cDNA sequence of the LhWRKY1 gene, and then amplifying a coding region sequence to obtain a target sequence; finally, the target sequence is connected with a pBWA (V) BS vector to obtain the plant expression vector.

7. A construction method of a plant expression vector containing an adventitious root promoting factor LhWRKY1 gene of liriodendron is characterized in that primers used for amplifying a coding region sequence are as follows:

LhWRKY 1-F1: cagtCACCTGCaaaacaacatggccgttgatctgatggg, as shown in SEQ ID NO. 4;

LhWRKY 1-R1: cagtCACCTGCaaaatacattacagctggccttcctggg, as shown in SEQ ID NO. 5.

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