CN109825510B - Lilium regale LrWRKY2 gene and application thereof - Google Patents

Abstract

The invention discloses Lilium regaleLrWRKY2Genes and uses thereofLrWRKY2The nucleotide sequence of the gene is shown as SEQ ID NO.1, the coded amino acid sequence is shown as SEQ ID NO.2, and the invention containsLrWRKY2The expression vector of the gene is transferred into an arabidopsis plant, and the obtained transgenic arabidopsis plant shows creeping growth, so that the original growth form of wild arabidopsis is thoroughly changed; and a plurality of rosette-shaped basal leaves grow on the stem of the arabidopsis thaliana growing in the creeping way, the edge of each leaf has a special shape with regular protrusion, and the method is very suitable for large-area cultivation and application in courtyard and garden greening.

Description

Lilium regale LrWRKY2 gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to Lilium regaleLrWRKY2Genes and applications thereof.

Background

The plant type is one of important characters of ornamental plants, and the breeding of an ideal plant type is an important target of ornamental plant breeding. The stolon-growing plants grow close to the ground due to the low plants and small branching angles, so the stolon-growing plants are more suitable for the construction of ground cover landscape and have wide application prospect in gardens. The research on the creeping transgenic plant is beneficial to expanding the application value of the creeping transgenic plant and further popularizing the application of the creeping transgenic plant in landscaping and landscaping.

WRKY is a family of transcription factors that are ubiquitous and conserved in plants. Earliest identified plantsWRKYThe gene is sweet potatoImpoea batatas）SPF1(SPF 1: SWEET POTATO FACTOR 1) gene, the gene product thereof is specific to SWEET POTATOSporaminThe SP8 sequence of the promoter region of the gene and the beta-amylase gene is identified, thereby participating in and regulating the establishment of plant sugar signal pathways. At present, the methodWRKYGenes are sequentially found in most plants, for example, 74 WRKY members are found in Arabidopsis, and 109 WRKY members are found in rice; the WRKY transcription factor includes 3 domains, namely a DNA binding domain, a nuclear localization signal, and an oligomerization site. The WRKY transcription factor is mainly structurally characterized in that the DNA binding domain of each member at least comprises one WRKY domain. The WRKY domain is a segment of about 60 amino acid residuesThe polypeptide sequence, wherein WRKYGQK is 7 highly conserved amino acid residues in all members. Except for the more conserved regions, the homology of the remaining amino acid compositions in the WRKY members is not high. In addition, the DNA binding domain of WRKY transcription factor generally contains a zinc finger structure.

The WRKY transcription factor regulates and controls a target gene on the transcription level, most of WRKY protein is positioned with a nucleus, and analysis of amino acid sequences of some WRKY protein shows that a nucleus positioning signal region (NLS) exists outside a WRKY structural domain, which means that the WRKY protein is synthesized in cytoplasm and then enters the nucleus through transmembrane transport to play a role. In vitro transient experiments also prove that some WRKY proteins with NLS are located in the cell nucleus. The WRKY transcription factor regulates the expression of a corresponding gene by combining with a W box (C/T) TGAC (T/C) nucleotide sequence of a promoter region of a target gene. Research shows that the WRKY transcription factor is a key component of a plant to pathogen defense response and plays an important role in the plant defense response. WRKY transcription factors have been shown to be involved in plant injury and senescence reactions, in plant growth and development metabolic processes, and in response to biotic and abiotic stresses in plants, such as high salinity, high temperature, drought, and cold. Overexpression of OsWRKY11, e.g., driven by the HSP101 promoter, has shown tolerance to drought by slowing the wilting rate and increasing stress survival in plants (Wu x,et al.plant Cell Rep. 2009, 28: 21-30); overexpression in Arabidopsis35S:OsWRK45And35S:OsWRK72plants acquire drought resistance by inducing ABA/stress-related genes (Qiu y.p., Yu d.q., environ. exp. bot. 2009,65: 35-47); the transcription of OsWRKY08 can be induced by PEG, NaCl or ABA, and abiotic stress related genes are up-regulatedAtCOR47AndAtRD21so as to make the transgenic arabidopsis obtain the resistance to osmotic pressure (Song Y., hanging S.J., Yu D.Q., Chin. Sci. Bull. 2009, 54: 4671-4678); patent CN 105441460A discloses Lilium regale WRKY transcription factor geneLrWRKY1And use of the same, results show overexpressionLrWRKY1The transgenic tobacco has obvious inhibition effect on the growth of four fungi including aschersonia, sclerotinia, botrytis cinerea, fusarium oxysporum and the likeThe application is as follows. But are up to now relatedWRKYFunctional studies of gene regulated plant growth and development are relatively delayed and relevantWRKYThe research on the gene is mainly focused on model plants such as arabidopsis thaliana, rice and poplar, the research on WRKY regulatory factors in other species is less, the research history is shorter, and the functional information is lacking.

Lilium regale (Lilium regale)Lilium regale Wilson) is an important wild germplasm resource in Lilium of Liliaceae, widely distributed in the Sichuan Minjiang river basin (Yangliping and Yaoya, a study on the utilization of Lilium Viridium resource in China [ M)]And Harbin: northeast forestry university press, 2018.11). Compared with the model plant, the growing and developing regulation mechanism of the Lilium regale has complexity and specificity. The previous research shows that Lilium regale has stronger resistance to lily mycosis and virosis, and a plurality of adversity stress related genes such as Lilium regale are obtained from Lilium regaleLr14-3-3、LrPR10、LrbZIP1AndLrWRKY1etc. (Li h.,et al., Sci. Hort. 2014, 168:9-16; He H., et al., Genes Genom. 2014, 36:497-507; Zhang N.N., Genes Genom. et al., 2014, 36:789-798; Han Q., et al.sci, Hort, 2016, 198: 370-378; patent No. 201610001896.4), but no report is found on the research on the growth and development of Lilium regale WRKY transcription factor gene regulated plant, and the biological function and transcription regulation mechanism are not yet elucidated.

Disclosure of Invention

Aiming at the blank existing in the prior art, the invention aims to provide Lilium regaleLrWRKY2The gene and the cDNA sequence of the gene are used for preparing a novel transgenic ground cover plant, and the research blank of lily WRKY transcription factor gene regulation and control of plant growth and development is filled.

The invention also provides a new product or a method which is suitable for large-area cultivation and application in courtyard and garden greening and has a special shape that the edge of the leaf has regular protrusions.

In order to achieve the purpose, the invention adopts the following technical scheme: lilium regaleLrWRKY2The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

As described aboveLilium regaleLrWRKY2The amino acid sequence of the gene-coded protein is shown in SEQ ID NO. 2.

A recombinant vector comprising saidLrWRKY2The nucleotide sequence of the gene.

A genetically engineered host cell comprising the recombinant vector or genome of the aboveLrWRKY2A gene.

LrWRKY2The genes or the encoded proteins thereof can be used to modulate the creeping growth of Arabidopsis thaliana.

A preparation method of creeping growth arabidopsis thaliana comprises the following steps: prepared or provided containsLrWRKY2Transforming agrobacterium with gene expression vector to obtain engineering agrobacterium strain, and infecting arabidopsis with the engineering agrobacterium strain to obtain the transgenic agrobacterium strainLrWRKY2And (4) performing gene overexpression to obtain the transgenic arabidopsis thaliana with creeping growth.

Preferably, the cauline leaves of the transgenic arabidopsis thaliana are rosette leaves and/or regular leaf edge protrusions of leaves.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention firstly clones Lilium regaleLrWRKY2The gene has a full-length sequence, and the gene is found to contain 2 typical WRKY functional domains through amino acid sequence comparison analysis; further construction ofLrWRKY2The gene and green fluorescence labeling protein GFP fusion expression vector is analyzed and found after transforming tobacco epidermal cells, and the LrWRKY2 transcription factor has the characteristic of specific expression on cell nucleus, which proves thatLrWRKY2The gene belongs to WRKY transcription factor family.

2. The invention firstly utilizes the transgenic technology to regulate and controlLrWRKY2The cDNA sequence of the gene is over-expressed in arabidopsis thaliana to obtain a transgenic model plant with creeping growth, which shows that Lilium regaleLrWRKY2The gene has the important biological function of regulating and controlling the herbaceous plant from the vertical growth to the creeping growth.

3. The invention heterologously transforms a WRKY transcription factor gene of lily in herbaceous model plantsLrWRKY2Not only thoroughly changes the growth phenotype of the plant, but also regulates and controls a plurality of rosette shapes grown on the stem of the transgenic plantThe single leaf edge of the basal leaf has a special shape of regular protrusion, is beautiful and novel, and is very suitable for cultivation and application in courtyards or green lands.

4. The invention provides a Lilium regaleLrWRKY2The method for preparing the novel ground cover plant by the gene sequence has important scientific significance for the growth and development regulation of herbaceous plants, and the obtained transgenic ground cover plant has potential application value in the fields of gardening and landscape design.

Drawings

FIG. 1 shows Lilium regaleLrWRKY2PCR amplification electrophoretogram of gene;

FIG. 2 shows the construction of recombinant expression vectorspTF101-35S::GFP-LrWRKY2-NOSA map of (a);

FIG. 3 is subcellular localization analysis of Lilium regale LrWRKY2 transcription factor;

the contrast group comprises a dark field diagram under the excitation light, a bright field diagram under the white light and a combined diagram in sequence from left to right; the experimental group comprises a dark field diagram under exciting light, a bright field diagram under white light and a combined diagram in sequence from left to right;

FIG. 4 shows the construction of recombinant expression vectorspBI121-35S::LrWRKY2-NOSA map of (a);

FIG. 5 is a schematic representation of the molecular detection of an overexpressed transgenic Arabidopsis line;

FIG. A is an electrophoretogram of kanamycin (Kan) marker gene amplified from genomic DNA of different lines of transgenic Arabidopsis thaliana using conventional PCR; FIG. B shows Lilium regaleLrWRKY2Semi-quantitative expression analysis of genes in over-expressed transgenic lines; wherein M is Marker, WT is wild type Arabidopsis plant,35S::LrWRKY2-L1、35S::LrWRKY2-L2、35S:: LrWRKY2l3 is a transgenic Arabidopsis line, respectively;

FIG. 6 is overexpressionLrWRKY2Phenotype map of transgenic Arabidopsis plants;

panel A is a wild-type Arabidopsis plant; panel B is overexpressionLrWRKY2The transgenic Arabidopsis plant of (1);

FIG. 7 is overexpressionLrWRKY2Phenotype map of transgenic Arabidopsis leaves;

FIG. A is a leaf on the stem of a wild type Arabidopsis plant; panel B is overexpressionLrWRKY2The rosette leaves growing early on the transgenic arabidopsis stolons; panel C is rosette leaves grown late in the stolons of transgenic Arabidopsis;

FIG. 8 is an overexpressionLrWRKY2Leaf edge map of transgenic Arabidopsis thaliana;

from top to bottom, the single leaf and overexpression of wild type Arabidopsis thaliana, respectivelyLrWRKY2Single leaves of transgenic Arabidopsis thaliana of (1); bars stands for actual length 1 cm.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. In the examples, the raw materials are ordinary commercial products unless otherwise specified. The experimental procedures described in the examples are not specifically described, i.e., they are carried out according to conventional molecular biological experimental procedures.

Example 1 cloning Lilium regaleLrWRKY2full-Length Gene sequences

At present, Lilium regaleLilium regaleWilson) has not been completely initiated, and the prophase of the subject group was analyzed by sequencing transcripts from different tissues to obtain information onLrWRKY2The full-length sequence of the reads sequence is obtained preliminarily through sequence splicing; in order to verify the true existence of Lilium regaleLrWRKY2Full-length gene, and specific primers (LrWRKY2-ATG-F and LrWRKY 2-Stop-F) are further designed for PCR amplification analysis.

Taking bud-stage Minjiang lily leaf tissue as a material, adopting TRIzol ™ Plus RNA Purification Kit (12183555, Invitrogen ™), extracting total RNA according to the instruction steps, removing residual trace DNA by using DNase I (18047019, Invitrogen [), and measuring the concentration of RNA by using a spectrophotometer for later use.

About 2.0. mu.g of total RNA from Lilium regale leaf was used to synthesize first strand cDNA according to PrimeScript II first-strand cDNA synthesis kit (6210A, Takara) instructions.

The PCR amplification system is high fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25 μ L, 5 XPrimeSTAR Buffer (Mg)²⁺Plus) 5. mu.L, forward primer (LrWRKY2-ATG-F, 10. mu.LM) 0.5. mu.L, reverse primer (LrWRKY2-Stop-R, 10. mu.M) 0.5. mu.L, template (DNA) 1. mu.L, dNTP (2.5mM) 2. mu.L, sterile ddH₂Make up to 25. mu.L of O.

The sequences of the forward and reverse primers are shown below:

LrWRKY2-ATG-F：TCCATGGAGAACAAAATGGGATGG

LrWRKY2-Stop-R：TCTCACCACCACCTGAAGTTGG

the PCR reaction program is: pre-denaturation at 95 deg.C for 5 min; at 95 ℃ for 30 s; 60 ℃ for 40 s; 72 ℃, 2min, 38 cycles; 72 ℃ for 10 min.

The obtained PCR product was analyzed by agarose gel electrophoresis, and as shown in FIG. 1, a specific amplified band was observed at about 2kb under UV irradiation. Purified according to the gel recovery kit (9672, Takara) for use.

Adding A into the purified DNA fragment by using a blunt end and A reagent, connecting the DNA fragment with pMD20-T vector (6019, Takara) through TA cloning, transforming a ligation product into Escherichia coli DH5a, picking 2-3 positive clones from an LB culture plate containing ampicillin (100 mg/L) for sequencing analysis, and the result shows that Lilium regale et alLrWRKY2The full-length gene sequence is shown as SEQ ID NO.1 and comprises a 930 bp open reading frame (containing a termination code). Will be provided withLrWRKY2Full-length sequences were BLAST analyzed at the NCBI database and no homologous sequences were found.

Example 2 recombinant expression vectorsp-TF101-P35S::GFP-LrWRKY2-NOSConstruction and subcellular localization analysis

(1) Construction ofp-TF101-P35S::GFP-LrWRKY2-NOSExpression vector

Lilium regaleLrWRKY2Full-length gene sequence (SEQ ID NO. 1), primers LrWRKY2-gfp-F and LrWRKY2-gfp-R are designed, and a seamless cloning (In-fusion) carrier linker sequence and an enzyme digestion site sequence are introduced into the primers. The positive cloning plasmid connected with TA in example 1 was used as a template, and LrWRKY2-gfp-F (forward primer) and LrWRKY2-gfp-R (reverse primer) were used as primers to carry out gene cloningLrWRKY2Specific amplification of (3).

The primer sequences are as follows:

LrWRKY2-gfp-F：

5’-TTCCCCGGGCTCGAGAAGCTTATGGAGAACAAAATGGGATGG-3’；

LrWRKY2-gfp-R：

5’-TTATCTAGATCCGGTGGATCCTCACCACCACCTGAAGTTGG-3’；

wherein the thickened sequence of LrWRKY2-gfp-F is Hind III restriction enzyme site, and the thickened sequence of LrWRKY2-gfp-R is Bam HI restriction enzyme site. The In-fusion cloning vector linker sequence is underlined. The LrWRKY2-gfp-F primer sequence does not containGFPThe password is terminated.

PCR reaction System: high fidelity amplification enzymePrimeSTAR HS (R010A, TaKaRa) 0.5μL，5xPrimeSTAR Buffer (Mg²⁺ Plus) 10. mu.L, forward primer (10. mu.M) 1. mu.L, reverse primer (10. mu.M) 1. mu.L, template (50-fold diluted plasmid) 1. mu.L, dNTP4 μ L (2.5mM) of sterile ddH₂O make up to 50. mu.L.

PCR reaction conditions: pre-denaturation at 95 deg.C for 5 min; at 95 ℃ for 30 s; 60 ℃ for 40 s; 72 ℃, 2min, 38 cycles; 72 ℃ for 7 min.

Will be provided withPThe CR amplification products were detected by agarose gel electrophoresis (FIG. 1). The amplified target fragment has the same size as the expected fragment, and is recovered and purified according to the instruction procedures of a gel recovery kit (9672, Takara), so that the target gene fragment is obtained.

The pTF101-GFP expression vector was digested with HindIII and BamHI. The enzyme cutting system is as follows: 5 mu L of pTF101-GFP vector; hind III 0.5. mu.L; BamHI 0.5. mu.L; buffer 10XK 2 μ L; sterile ddH₂O is complemented to 20 mu L; react at 37 ℃ for 3 h. After completion of the digestion, the pTF101-GFP vector fragment was recovered according to the Takara agarose gel recovery kit.

Construction Using the seamless Cloning technique (In-fusion HD Cloning Kit, Takara)p-TF101-P35S:: GFP-LrWRKY2-NOSA recombinant expression vector.

The recombination reaction system is as follows:

Purifed PCR fragment (recovered)LrWRKY2Fragment of interest) 50 ng; linear vector (pTF101-GFP vector) 100ng；5X In-fusion HD Enzyme P2 mu L of remix; sterile ddH₂Make up to 10. mu.L of O. Then, the recombinant reaction system was transformed into E.coli DH5a according to the molecular cloning protocol, plated on a selection plate containing spectinomycin (Spec, 100 mg/L), and sequenced by positive clones to obtain correct DNA containing spectinomycinLrWRKY2Recombinant expression vector of gene fragmentp-TF101-P35S::GFP-LrWRKY2-NOS(FIG. 2). Reporter gene in recombinant expression vectorGFPAnd a target geneLrWRKY2After fusion at the 5' end of (A), the promoter is located in a constitutive promoterP35S downstream, fusion expression is formed;LrWRKY2the 3' end of the fusion gene is assembled with an NOS terminator, which can effectively terminate the transcription of the fusion gene. The reporter gene GFP can emit green fluorescence without auxiliary factors and substrates after being excited by blue light, and can detect the expression condition of a target gene when being used as the reporter gene.

(2) Subcellular localization analysis of Lilium regale LrWRKY2

1) Agrobacterium-mediated transient transformation of tobacco

The constructed recombinant expression vector is subjected to a conventional freeze-thawing methodp-TF101-P35S::GFP-LrWRKY2-NOSTransferring the strain into an agrobacterium strain EHA105, and screening positive clones by PCR. To containpTF101-GFPThe agrobacterium strain of the plasmid is used as a positive control, and the agrobacterium injection buffer solution is prepared by the methods of hollyn and the like (agrobacterium-mediated tobacco transient expression test condition optimization, molecular plant breeding, 2016, 14(1): 80-85). The tobacco normally grown in the light incubator with 8-10 leaves fully expanded was selected for injection and the injection buffer was slowly pushed into the back of the leaves using a syringe with the needle removed. Then, the transformed plant is placed back into the incubator again, and observed after being cultured for 36h-48 h.

2) Expression and observation of GFP reporter gene in tobacco epidermal cells

Carefully shearing the transformed tobacco leaves with scissors, placing the tobacco leaves on a glass slide, adding 1 drop of distilled water, and preparing into tablets; then, the sample was placed on a fluorescence microscope and fluorescence observation was performed under blue light with an excitation light wavelength of 488-507 nm. As shown in FIG. 3, the control group (in FIG. 3) showed fluorescence expression in both the nucleus and the cell membrane of tobacco epidermal cells, whereas the experimental group (in FIG. 3) showed specific expression only in the nucleus of tobacco epidermal cells. The results show that Lilium regale LrWRKY2 is a transcription factor protein with 1 nuclear localization, and is consistent with the expression localization condition of most WRKY factors in model plants.

Example 3 recombinant expression vectorspBI121-P35S::LrWRKY2-NOSConstruction of (3) and genetic transformation of Arabidopsis

(1) Construction ofpBI121-P35S::LrWRKY2-NOSOverexpression vectors

Lilium regaleLrWRKY2The full-length gene sequence (SEQ ID NO. 1) is designed with primers LrWRKY2-inf-F and LrWRKY2-inf-R, and a seamless cloning (In-fusion) carrier linker sequence and an enzyme digestion site sequence are introduced into the primers. The positive cloning plasmid connected with TA in example 1 is used as a template, and LrWRKY2-inf-F and LrWRKY2-inf-R are used as primers to carry out geneLrWRKY2Specific amplification of (3).

The primer sequences are as follows:

LrWRKY2-inf-F:

5’-GGACTCTAGAGGATCCATGGAGAACAAAATGGGATGG-3’

LrWRKY2-inf-R:

5’-GATCGGGGAAATTCGAGCTCTCACCACCACCTGAAGTTGG-3’

wherein, the thickening sequence of the forward primer LrWRKY2-inf-F is BamHI enzyme cutting site, and the thickening sequence of the reverse primer LrWRKY2-inf-R is Sac I enzyme cutting site. The In-fusion cloning vector linker sequence is underlined.

PThe CR reaction system is as follows: high fidelity amplification enzymePrimeSTAR HS (R010A, TaKaRa) 0.5μL，5xPrimeSTAR Buffer (Mg²⁺ Plus) 10. mu.L, forward primer (10. mu.M) 1. mu.L, reverse primer (10. mu.M) 1. mu.L, template (50-fold diluted plasmid) 1. mu.L, dNTP4 μ L (2.5mM) of sterile ddH₂O make up to 50. mu.L.

PCR reaction conditions: pre-denaturation at 95 deg.C for 5 min; at 95 ℃ for 30 s; 60 ℃ for 40 s; 72 ℃, 2min, 38 cycles; 72 ℃ for 10 min.

Will be provided withPDetecting the CR amplification product by agarose gel electrophoresis. The amplified target fragment has the same size as the expected fragment, and is recovered and purified according to the instruction procedures of a gel recovery kit (9672, Takara), so that the target gene fragment is obtained.

The pBI121 plant binary expression vector was double digested with Bam HI and Sac I. The enzyme cutting system is as follows: pBI121 vector 5. mu.L; BamHI 0.5. mu.L; sac I0.5 μ L; buffer 10XK 1 uL; sterile ddH₂O is complemented to 20 mu L; react at 37 ℃ for 3 h. After the enzyme digestion, the pBI121 vector large fragment is recovered according to a Takara agarose gel recovery kit.

Construction Using the seamless Cloning technique (In-fusion HD Cloning Kit, Takara)P35S:: LrWRKY2- NOSA recombinant expression vector.

The recombination reaction system is as follows:

Purifed PCR fragment (recovered)LrWRKY2Fragment of interest) 50 ng; linear vector (pBI121) 100 ng; 5X In-fusion HD EnzymeP2 mu L of remix; sterile ddH₂Make up to 10. mu.L of O. Then transforming the recombinant reaction system into Escherichia coli DH5a according to the molecular cloning experimental instruction, coating the Escherichia coli DH5a on a screening culture plate containing kanamycin (100 mg/L), and obtaining the correct content through positive cloning screening and sequencingLrWRKY2Recombinant expression vector of gene fragmentpBI121-35S:: LrWRKY2-NOS(FIG. 4). Target gene in recombinant expression vectorLrWRKY2The 5' end of (A) is located in a constitutive promoterP35S downstream, it enablesLrWRKY2Overexpression of the gene;LrWRKY2the 3' end of the gene is assembled with an NOS terminator, which can effectively terminate the transcription of the gene. On the recombinant expression vector is assembledNPTIIThe gene can be used as a screening marker of transgenic plants, and can be used for screening the transgenic plants by using kanamycin. The assembly of LB and RB sequences on a recombinant expression vector, facilitating the assembly of an expression framework and a selectable marker gene therebetweenNPTIIIntegrated into the plant recipient chromosome.

(2) Agrobacterium-mediated genetic transformation of Arabidopsis

Genetic transformation of arabidopsis thaliana was performed by the floral dip method (Zhang x.,et al.nat Protoc. 2006, 1: 641-646). Will carry withpBI121-35S::LrWRKY2-NOSAgrobacterium of the vector was introduced into Columbia Col type Arabidopsis thaliana. Resistant Arabidopsis regenerated seedlings were selected with kanamycin (100 mg/L) and the resistance marker gene was amplified by conventional PCR methods. The PCR amplification primers were a forward primer (NPTII-F: TTGGGTGGAGAGGCTATTCGG) and a reverse primer (NPTII-R: GCCACAGTCGATGAATCCAG). The PCR detection result is shown in FIG. 5A, and could not be detected in wild type plant (WT)NPTIIThe presence of marker genes only in transgenic plants (35S::LrWRKY2-L1、35S::LrWRKY2L2 and35S::LrWRKY2amplification in L3)NPTIIMarker genes, indicating that the recombinant expression cassette has been introduced into the arabidopsis genome.

Further, the over-expression with good phenotype is obtained through the identification of semi-quantitative RT-PCRLrWRKY2The transgenic line of (4) was synthesized by using Trizol reagent (Invitrogen), extracting total RNA from Arabidopsis leaves according to the procedures described in the specification, removing residual DNA using DNase I (Invitrogen), and synthesizing first-strand cDNA using cDNA reverse transcription reagent (Takara) according to the procedures described in the specification. Using Arabidopsis thalianaβ–actinThe gene is used as an internal reference, and the transgenic line and the wild type are analyzed by a semi-quantitative RT-PCR methodLrWRKY2The level of expression of the gene.

Detection ofβ–actinThe gene primers are as follows:

β–actin-F: 5'- CACTTGCACCAAGCAGCATGAAGA -3'

β–actin-R:5'- AATGGAACCACCGATCCAGACACT -3'

the detection target gene primer is as follows:

CaMV35S-F: AGAAGACGTTCCAACCACGTCT

LrWRKY2-R: TCACCACCACCTGAAGTTGG

the results are shown in FIG. 5B, in 3 representative transgenic lines: (35S::LrWRKY2-L1、35S::LrWRKY2L2 and35S::LrWRKY2-L3) destination gene Lilium regaleLrWRKY2All up-regulate the expression, and35S::LrWRKY2the level of expression of L3 was highest and could not be detected in wild type plants (WT)LrWRKY2Is shown to indicateLrWRKY2The Arabidopsis genome has been introduced and successfully transcribed.

Example 4 phenotypic Observation and analysis of transgenic Arabidopsis

The phenotype of Arabidopsis plants growing in soil for about 45 days was identified and the results are shown in FIG. 6, with overexpressionLrWRKY2The transgenic plants of (a) exhibited creeping growth, while the wild-type control plants exhibited normal upright growth. And is overexpressedLrWRKY2The stem of the transgenic plant of (a) occurs from the lower part of the basal rosette leaves, whereas the stem of the wild type control plant occurs from the upper part of the basal rosette leaves. The most prominent phenotype was a marked change in cauline leaves, as shown in FIG. 7, where the cauline leaves of the wild type control plants appeared as single leaves at around 45 days, while the cauline leaves of the transgenic plants still appeared as rosette leaves, and FIG. 8 shows the rosette leaf morphology of the cauline leaves of the transgenic plants after 60 days. Further observation of transgenic rosette leaves (FIG. 8) revealed that the leaf edge of each single leaf in rosette leaves was also altered, showing regular leaf edge protrusion. According to the previous report (Bilsborough g.d.,et al., PNAS 2011, 108:3424-3429; Kasprzewska A., et al.plant J.2015, 83: 705-718), the occurrence of leaf and the edge bulge of the leaf are closely related to auxin signals, which indicates that Lilium regale LrWRKY2 may regulate key regulatory factors in the auxin signal pathway from upstream or directly interact with the key regulatory factors; thereby thoroughly changing the growth and development shape of the Arabidopsis plant.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

SEQUENCE LISTING

<110> Changjiang university academy;

<120> Lilium regale LrWRKY2 gene and application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 930

<212> DNA

<213> Lilium regale Wilson

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atggagaaca aaatgggatg ggagcagagc ttgatataca tgctagctca agttgaggag 60

cagacgaagc aacttgaagc ccacctcgac gccgccacac catcccagat gtgcaagcca 120

ctggcacaga agattcgctc caccatatgc acggctatca ccatggtgac ggagggttcg 180

cagctgcacg gcggatcaga ctcaccgcgg tcagccagcg atagcccacg gagtgagaac 240

tctggcagag ctttcacaga cgagcaaagg gaattgtcga agaagaggaa gaccatgcca 300

aaatggagca gccaagtacg ggttggttcg ggatcaggga ctgaggcacc aatggatgat 360

ggctatagtt ggaggaagta tgggcagaag gacattctcg gagctcacca tccaagggcc 420

tactaccggt gcacacatcg caattctcgt ggatgtccgg cgacaaagca agtgcagcga 480

tcagacgagg accctttcgt ttttgatgtc atttatagag gtgagcatat atgcgtcgaa 540

aatactcaga tgattccatg tcaacagaat cagttacagc aaccgccgcc acctgcggca 600

gctttgcaca ccgagttgat tccgggttta cagactggtt tgcacgttaa gacgcaagtt 660

atagacttag aagcccagga cccagcgtcg tctttctcct tcccatcaac accgtctagc 720

tttgctccgg cttctgtctc taacacgtct tccccgacac taggcgggat agctgatact 780

tttggtgctg gacaatcaga acttgctgag atcttcgctg ctgcaactgt ggcggaagat 840

tcgcctgtag ttgacatgaa ttttgggctt gatcaagtgg agctcgacac tgcttttcca 900

tatgacaatt ccaacttcag gtggtggtga 930

<210> 2

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<213> Lilium regale Wilson

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MENKMGWEQS LIYMLAQVEE QTKQLEAHLD AATPSQMCKP LAQKIRSTIC TAITMVTEGS 60

QLHGGSDSPR SASDSPRSEN SGRAFTDEQR ELSKKRKTMP KWSSQVRVGS GSGTEAPMDD 120

GYSWRKYGQK DILGAHHPRA YYRCTHRNSR GCPATKQVQR SDEDPFVFDV IYRGEHICVE 180

NTQMIPCQQN QLQQPPPPAA ALHTELIPGL QTGLHVKTQV IDLEAQDPAS SFSFPSTPSS 240

FAPASVSNTS SPTLGGIADT FGAGQSELAE IFAAATVAED SPVVDMNFGL DQVELDTAFP 300

YDNSNFRWW 309

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tccatggaga acaaaatggg atgg 24

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tctcaccacc acctgaagtt gg 22

<210> 5

<211> 42

<212> DNA

<213> Artificial sequence

<400> 5

ttccccgggc tcgagaagct tatggagaac aaaatgggat gg 42

<210> 6

<211> 41

<212> DNA

<213> Artificial sequence

<400> 6

ttatctagat ccggtggatc ctcaccacca cctgaagttg g 41

<210> 7

<211> 37

<212> DNA

<213> Artificial sequence

<400> 7

ggactctaga ggatccatgg agaacaaaat gggatgg 37

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence

<400> 8

gatcggggaa attcgagctc tcaccaccac ctgaagttgg 40

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<400> 9

ttgggtggag aggctattcg g 21

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

gccacagtcg atgaatccag 20

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence

<400> 11

cacttgcacc aagcagcatg aaga 24

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<400> 12

aatggaacca ccgatccaga cact 24

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence

<400> 13

agaagacgtt ccaaccacgt ct 22

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

tcaccaccac ctgaagttgg 20

Claims (6)

1. Lilium regaleLrWRKY2The gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. The Lilium regale as claimed in claim 1LrWRKY2The gene coded protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

3. A recombinant vector comprising the vector of claim 1LrWRKY2The nucleotide sequence of the gene.

4. The method of claim 1LrWRKY2Use of a gene or protein encoded thereby for modulating the creeping growth of Arabidopsis thaliana.

5. A preparation method of creeping growth arabidopsis thaliana is characterized by comprising the following steps: prepared or provided containing the compound of claim 1LrWRKY2Transforming agrobacterium with gene expression vector to obtain engineering agrobacterium strain, and infecting arabidopsis with the engineering agrobacterium strain to obtain the transgenic agrobacterium strainLrWRKY2And (4) performing gene overexpression to obtain the transgenic arabidopsis thaliana with creeping growth.

6. The method for preparing creeping growth arabidopsis thaliana according to claim 5, wherein the cauline leaves of the transgenic arabidopsis thaliana are regular leaf edge protrusions of rosette leaves and/or leaves.

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