CN110408618B - Middle caragana derived promoter CiNAC071 and application thereof - Google Patents

Abstract

The invention relates to the field of biological gene engineering, in particular to an inducible promoter CiNAC071 and application thereof. The use of the inducible promoter is more beneficial to the research of gene functions.

Description

Middle caragana derived promoter CiNAC071 and application thereof

Technical Field

The invention relates to an inducible promoter CiNAC071 and application thereof, in particular to activity induction of the promoter CiNAC071 under ABA treatment and GA treatment. The CiNAC071 promoter is induced by ABA and GA, and can be used for constructing a transgenic expression vector. The use of the inducible promoter is more beneficial to the research of gene functions.

Background

The growth and development of plants are the process of orderly expression and synergistic action of different genes in time and space. The switch, expression mode, expression abundance and the like of the gene in the whole growth process are accurately regulated and controlled. The expression regulation of plant genes can be divided into 5 different levels, namely, pre-transcription, post-transcription, translation, post-translation and the like, wherein the regulation on the transcription level is the most main and most key link. The regulation of the gene at the transcription level is mainly realized by regulating the combination of a transcription regulatory factor and a promoter, strength change and the like. General transgenic plants are obtained by inserting T-DNA into plant genome, if the transferred gene is closely related to plant growth and development, the transgenic plant growth and development will be changed, so that transgenic homozygote seeds are difficult to obtain, and phenotype analysis cannot be carried out. Thus, there is a need for inducible promoters, which do not express the gene in the screening of transgenic plants, and which are induced by an inducer when studying gene function. Meanwhile, the condition that the expression quantity of the exogenous gene in the transgenic plant is too low to facilitate the research of gene function exists, and the research of gene function is facilitated by utilizing the inducible promoter. Therefore, inducible promoters are of great significance in genetic engineering.

NAC transcription factor is one of the largest families of transcription factors that are plant-specific. The NAC transcription factor family consists of NAM (No. application meristm) originally found in morning glory, ATAF (Arabidopsis transcription activation factor) of Arabidopsis thaliana, and CUC (cup-shaped cottledon). The N-terminal of the family protein has a highly conserved structural domain, which is composed of about 150 amino acid sequences and is used as a DNA binding structural domain; the C terminal is relatively variable and can activate or inhibit transcription as a transcription regulation region; part of the NAC transcription factor contains a transmembrane domain. The DNA binding domain of the NAC transcription factor consists of five subdomains A-E, of which subdomain A is likely to be involved in dimer formation, subdomains C and D are positively charged for DNA binding, and subdomains B and D determine the diversity of the NAC transcription factor. The upstream promoter sequence of NAC transcription factor contains many cis-elements that regulate plant metabolism, growth and development, and stress response, but the regulation mechanism is not completely clear. In recent years, the existence of NAC transcription factor family genes has been found in many species, for example 117 NAC genes in Arabidopsis thaliana (Arabidopsis thaliana), 151 NAC genes in rice (Oryza sativa), 152 NAC genes in soybean (Glycine max), and 97 NAC genes in alfalfa (Medicago truncatula).

The research of the arabidopsis thaliana NAC transcription factor family gene is more detailed, partial genes are renamed according to the known structure and function of the NAC gene, and NAC transcription factors named by NST (NAC secondary wall stimulating factor) and SND (secondary wall-associated NAC domain protein) participate in the secondary wall growth of arabidopsis thaliana, such as AT3G61910, AT2G46770, AT1G28740, AT4G28500 and the like; NAC transcription factors named as NTL (NAC with transmembrane motif 1-like) and NTM (NAC with transmembrane motif) have transmembrane domain motifs such as AT1G01010, AT2G27300, AT3G10500, AT3G49530, AT4G01550, AT4G35580, AT4G01540 and the like; NAC transcription factors named SGS (promoter of gene silence) are gene silencing repressors, such as AT3G10490 and AT3G 10490; NAC transcription factors named NARS (NAC-regulated seed morphology) are associated with the seed morphology of Arabidopsis thaliana, such as AT3G 15510; the NAC transcription factor named CBNAC (calmodulin-binding NAC protein) AT4G35580 encodes a calmodulin-binding NAC protein, and has the function of regulating calmodulin transcription repression; NAC transcription factors named VND (transformed NAC domain) and VNI (VND-interacting) are involved in the formation of the vasculature of Arabidopsis thaliana, such as AT5G66300, AT5G62380, AT4G36160, AT2G18060, AT1G71930, AT1G12260, and AT5G13180, among others; the Arabidopsis thaliana gene AT2G43000, designated as JUB1 (JUNGBRUNNEN 1), retards senescence after overexpression and enhances tolerance to abiotic stress.

NAC transcription factor is involved in various stages of plant growth and development, including seed germination, root growth, secondary wall formation, plant hormone signaling pathways, leaf senescence, and the like. The existing research shows that NTL8 regulates and controls GA mediated salt signal response way and regulates seed germination; arabidopsis NAC1 overexpressing plants had larger leaves, more lateral roots and increased fresh weight compared to control plants; SND1 and NST1 transcription in arabidopsis activate the formation of secondary walls in fibroblasts; under the condition of abiotic stress, arabidopsis RD26 (dehydration-induced NAC protein 26) is used as a transcriptional activator of ABA inducible genes; overexpression of CiNAC1 and CiNAC4 of caragana intermedia promotes the elongation of main roots and the increase of the number of lateral roots of Arabidopsis; the middle caragana CiNAC1 gene also promotes the senescence of transgenic arabidopsis leaves; researches such as Mahmood and the like show that ANAC032 can promote the senescence of Arabidopsis thaliana and inhibit the synthesis of Arabidopsis thaliana anthocyanin under stress conditions such as sucrose, salt and oxidation; arabidopsis ANAC046 can promote Arabidopsis chlorophyll degradation and leaf senescence.

In addition, the NAC transcription factor also participates in the response of plants to abiotic stress such as drought, salt, cold, heat, mechanical damage and the like and biotic stress such as diseases, insect pests and the like, and the overexpression of NAC1 in the rice enhances the drought resistance and salt resistance of the rice; the over-expression of OsNAC6 in rice can not only enhance the tolerance capability of dehydration resistance and salt stress resistance, but also enhance the disease resistance capability; the middle caragana CiNAC3 and CiNAC4 improve the tolerance of the transgenic arabidopsis thaliana to salt stress; the arabidopsis ATAF1 regulates the expression of drought stress response genes; banana MusasnAC1 can eliminate H of guard cells ₂ O ₂ Leading to the closure of stomata and enhancing the drought resistance of the bananas; overexpression of the solanum lycopersicum ShNAC1 enhances plant sensitivity to cold, drought, and salt, and also promotes dark and salt-induced leaf senescence. All these studies indicate that NAC transcription factor plays an important role in plant growth and development and stress tolerance control.

Expression of eukaryotic genes is regulated by a promoter located upstream of the transcription start site, which is the binding site for RNA polymerase II. Promoters regulate the initiation and accuracy of transcription of genes and are central to transcriptional regulation. The comparative analysis of various promoter sequences shows that the basic structural characteristics of the promoters are that TATA sequences exist at the positions from-20 bp to-30 bp of the sequences, and CAAT regions exist at the positions from-70 bp to-78 bp of the sequences. Also, some promoters contain specific cis-acting elements that differ from other promoters. The promoters are mainly divided into: constitutive, tissue-specific and inducible promoters. Constitutive promoters, also known as non-specific promoters, express in different tissue parts at various stages of plant growth and have no time and temporal specificity, such as the 35S promoter of cauliflower mosaic virus (CaMV); the tissue-specific promoter can regulate the expression of genes in specific tissue organs of plants, for example, soybean SbPRPI gene promoter is mainly a promoter which is activated by signal stimulation and is expressed and induced in a root development system, thereby driving the expression of downstream genes, and can be divided into an abiotic stress inducible promoter, a biotic stress inducible promoter and a hormone inducible promoter according to the difference of induction factors. Analysis of the promoter elements of a gene is useful for understanding the expression pattern of the gene.

Promoters can be classified into: constitutive promoters, tissue specific promoters and inducible promoters. Inducible promoters, inducers can rapidly initiate the "on" and "off" of gene expression. When inducer exists, the gene expression can be started, and after the inducer is removed, the gene expression is closed quickly, so that the gene expression can be controlled manually, accurately and quickly.

Caragana intermedia (Caragana inermedia) belongs to the family leguminosae, caragana, perennial shrubs. The root system is developed, the underground root is nearly 5 meters, the stem is upright or obliquely upward, and the plant height is 1-2 meters; feathery compound leaves, 12-16 small leaves, inverted oval or ellipse, and most of hair; the middle caragana begins to grow in 4 middle of the month, the flowering phase is 5 middle of the month, the corolla is butterfly-shaped and yellow, the fruit phase is 6 months, the seeds are kidney-shaped, and the caragana carinata has two colors of light green brown and yellow brown. The plant is widely distributed in regions such as inner Mongolia, shanxi, ningxia and Shanxi in China, has the characteristics of drought resistance, high temperature resistance, salt and alkali resistance, cold resistance and the like, and is an excellent plant for preventing wind, fixing sand and maintaining water and soil in arid and desert regions; in addition, the middle caragana can also be used as raw materials of feed, fuel, paper making, fertilizer and plates, and also has medicinal value. The inducible promoter CiNAC071 cloned from the middle caragana can inhibit the activity of the promoter under ABA treatment and promote the activity of the promoter under GA treatment. The CiNAC071 promoter is induced by ABA and GA, and can be used for constructing a transgenic expression vector. The use of the inducible promoter is more beneficial to the research of gene functions.

Disclosure of Invention

The invention aims to provide a caragana intermedia inducible promoter CiNAC071 and an application thereof, namely a novel promoter CiNAC071 capable of being induced in plant tissues.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention provides a nucleotide CiNAC071, the sequence of which is shown in SEQ ID No. 1; the sequence can be synthesized by an artificial synthesis method, and can also be cloned from the genome of the middle caragana by a PCR (polymerase chain reaction) and enzyme digestion method.

In a second aspect, the invention provides a set of primer pairs for amplifying the nucleotide CiNAC071, wherein the sequences of the primers are shown as SEQ ID No.2 and SEQ ID No. 3.

In a third aspect, the invention provides a recombinant vector comprising said nucleotide CiNAC071, such as the pCAMBIA1305.2 vector of the examples, to which nucleotide CiNAC071 is ligated.

In a fourth aspect, the invention provides a transformant comprising nucleotide CiNAC071, wherein the host used by the transformant is Agrobacterium.

In a fifth aspect, the invention provides the use of nucleotide CiNAC071 as a promoter, ciNAC071 promoter induced by ABA and GA.

Preferably, the application is specifically that nucleotide CiNAC071 is used as an inducible promoter to drive the expression of a target gene in a plant, downstream gene activity is inhibited under ABA treatment, and downstream gene activity is promoted under GA treatment; the plants include caragana intermedia and arabidopsis thaliana.

Preferably, the application of the nucleotide CiNAC071 as a promoter in driving expression of a target gene in a plant is to transfect plant tissue by soaking the recombinant vector or directly transfect the plant tissue by a transformant.

Preferably, the use is in particular the use of nucleotide CiNAC071 as promoter in plant gene function.

In a sixth aspect, the invention provides a preparation method of the nucleotide CiNAC071, which comprises an artificial synthesis method or a biological cloning method;

preferably, the biological cloning method specifically comprises:

step 1, extracting caragana intermedia DNA;

step 2, using the DNA as a template, and sequentially using the outer primer and the inner primer for amplification;

and 3, carrying out agarose gel electrophoresis detection on the amplification product to obtain nucleotide CiNAC071.

The CiNAC071 promoter of the invention has the following characteristics: a) The expression conditions of caragana intermedia and arabidopsis tissues are very clear; b) The promoter activity was inhibited by ABA treatment and promoted by GA treatment.

The invention adopts a molecular cloning method for the first time, obtains a new CiNAC071 promoter by separating from a caragana intermedia genome, and proves that the CiNAC071 promoter is an inducible promoter through a transgenic experiment, can be used for constructing a transgenic expression vector because the CiNAC071 promoter is induced by ABA and GA, and is more beneficial to the research of gene functions by utilizing the inducible promoter, thereby achieving the aim of the invention.

Drawings

FIG. 1: cloning of CiNAC071 promoter and enzyme digestion verification of expression vector: FIG. A: chromosome walking method first amplification of CiNAC071 promoter, FIG. B: amplifying the promoter of the CiNAC071 gene for the second time by a chromosome walking method, wherein lanes 1, 2 and 3 respectively represent a first round of PCR, a second round of PCR and a third round of PCR; and (C) in a drawing: cloning CiNAC071 gene promoter, 1, 2 respectively representing the same PCR product; FIG. D: enzyme digestion verification Pro _CiNAC071 GUS, lane D: plasmid control; m1: DL5 000bp DNA marker; m: DL2 000bp DNA marker.

FIG. 2: cis-element analysis of CiNAC071 promoter.

FIG. 3: pro _CiNAC071 Histochemical staining of GUS transgenic lines and wild-type plants: to Pro _CiNAC071 GUS transformationHistochemical staining of the gene strain and the wild plant in different growth stages, wherein a-h is histochemical staining in a seedling stage, and i-r is histochemical staining in a mature stage of 40 days of plant growth.

FIG. 4: pro under MeJA, ABA and GA treatment _CiNAC071 Histochemical staining analysis of GUS transgenic plants.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of CiNAC071 promoter

Cloning by using a Genome Walking kit to obtain a promoter of the CiNAC071 gene. Based on the gene ORF sequence, primers SP1, SP2 and SP3 specific to the first round promoter clone were designed and obtained, see Table 1. The primers and four universal primers in the kit take middle caragana gDNA as a template to carry out thermal asymmetric PCR reaction, and gene flanking sequences are obtained by three times of nested PCR. And recovering a clear and specific amplification band after the third PCR amplification, connecting the pEASY-T1Simple cloning vector, transforming the recombinant plasmid into escherichia coli, and sequencing. And (3) analyzing a sequencing result to ensure that the sequence can be spliced with the open reading frame sequence of the gene without errors, and then considering the sequence as the promoter of the gene. If the first round of amplification does not result in a sequence of the desired length, a second round of amplification can be performed.

And (3) primers for the second round of promoter amplification are designed on the basis of the sequence obtained in the first round. The three homodromous specific primers 2nd-SP1, 2nd-SP2 and 2nd-SP3 are still used, and the target band of the second promoter clone is obtained by taking the middle caragana gDNA as a template. After sequencing analysis, the sequence is correctly spliced with the sequence obtained in the first round of amplification, and the sequence obtained by the two rounds of amplification is spliced to be the promoter sequence of the gene.

Meanwhile, primers p071-sense and p071-anti for amplifying the full length of the promoter are designed according to the splicing result, and the promoter sequence is amplified by taking the gDNA of the middle caragana as a template.

PCR procedure

After PCR amplification, the fragment of interest was recovered using a TIANGEN agarose gel DNA recovery kit.

TABLE 1 primer List

Example 2 ligation of the middle Caragana CiNAC071 promoter to the pCAMBIA1305.2 expression vector

According to enzyme cutting sites HindIII and Nco I contained In the specific primers, an empty vector pCAMBIA1305.2-GUS is subjected to enzyme cutting treatment and then is connected with a target fragment In an In-fusion mode. The linking system is as follows:

the substances are mixed evenly and centrifuged, and then are bathed in water at 37 ℃ for 15min and 50 ℃ for 15min. Transformation to escherichia coli competent DH5 α was verified using double digestion and colony PCR. Pro _CiNAC071 After the GUS recombinant plasmid expression vector is successfully constructed, transforming escherichia coli, extracting recombinant plasmid, and verifying by using double enzyme digestion and colony PCR. The recombinant plasmid was extracted from E.coli using a plasmid miniprep kit and electrically transformed into Agrobacterium tumefaciens GV3101. The wild arabidopsis is transfected by using a dip-dyeing method, and hygromycin is screened to obtain an over-expression plant.

Example 3 cloning of CiNAC071 promoter of Caragana intermedia and construction of GUS reporter gene expression vector driven by promoter

Obtaining the ORF full-length sequence of CiNAC071 gene from the middle caragana transcriptome database, designing three reverse primers 071-SP1, 071-SP2 and 071-SP3 in the known sequence, using middle caragana gDNA as template, carrying out three rounds of PCR reaction, amplifying to obtain the promoter of CiNAC071 gene (figure 1A), and recovering two specific sequences of the third round of PCR product. Sequencing analysis shows that the length of the sequence is between 1000bp and 1500bp, the sequence with brighter electrophoretic bands is the promoter of the CiNAC071 gene, the length of the sequence is 1246bp, and the length of the sequence is not enough for analyzing all cis-elements of the promoter of the CiNAC071 gene, so that the second promoter amplification is continuously carried out.

On the basis of the sequence generated after the first PCR sequence is connected with the full-length sequence of ORF of CiNAC071 gene, three reverse primers 071-2nd-SP1, 071-2nd-SP2 and 071-2nd-SP3 are designed. And taking the gDNA of the middle caragana as a template, and carrying out three rounds of PCR reactions to obtain a product of the second amplification of the CiNAC071 gene promoter (figure 1B). The amplification also has two specific bands, and the sequence result analysis shows that the brighter sequence with the length between 750bp and 1000bp can be spliced with the first amplification sequence, and the sequence is supposed to be a partial sequence of the CiNAC071 gene promoter.

The sequences obtained by the amplification of the two promoters are spliced to form the promoter sequence of CiNAC071 gene, which is 2107bp in total. Specific cloning primers p071-sense and p071-anti are designed according to the sequence of CiNAC071 gene promoter. Using middle caragana gDNA as template, under the catalysis of PrimeSTAR enzyme, cloning to obtain CiNAC071 gene promoter (FIG. 1C). After the pCAMBIA1305.2 vector was linearized with HindIII and NcoI endonucleases, it was ligated with the PCR product under an In-fusion system to construct Pro _CiNAC071 GUS expression vector. HindIII and Nco I double digestion identification scheme see FIG. 1D, after digestion, the target gene fragment and linearized vector are generated, indicating Pro _CiNAC071 The GUS expression vector is successfully constructed.

The sequence of the gene promoter is analyzed on line by PlantCARE to predict various response elements contained in the promoter, thereby deducing the regulation function of the gene in each signal path. Analysis of the homeopathic elements of the promoter of this sequence: eukaryotic promoter common transcription elements CAAT-box and TATA-box; plant light response elements ATC-motif, ACE, ATCT-motif, G-Box and the like; meristematic expression of CAT-box; plant hormones, namely a jasmonate-methyl response element CGTCA-motif, a gibberellin response element P-box, a TATC-box and an abscisic acid response element ABRE; stress response element TC-rich repeats, low temperature stress response element LTR; a zein metabolism control element O2-site and the like. It is speculated from these elements that CiNAC071 gene may be involved in physiological and biochemical reactions such as hormone regulation and stress response.

Example 4, pro _CiNAC071 Histochemical staining of GUS transgenic plants

Successful Pro will be constructed using the floral dip method _CiNAC071 GUS plasmid is used for transfecting Arabidopsis thaliana wild plants, and hygromycin is screened to obtain transgenic positive plants. And observing histochemical staining conditions of the T2 generation plants in different growth periods by taking wild type plants as a control. Four time points were selected for staining, the first time seed germination 48h, with staining mainly concentrated in cotyledons and hypocycloid (FIGS. 3a and e); the second time is seed germination 5d, staining was mainly concentrated at cotyledon, hypocotyl, petiole (fig. 3b and f); the third time is seed germination 10d, and staining is mainly concentrated at cotyledon vein, new true leaf blade, hypocotyl and few roots (fig. 3c, fig. 3d, fig. 3g and fig. 3 h). The fourth time is the plant growth of 40d, and the rosette leaves, flowers, stems and mature fruit pods of the plants are stained and analyzed, the staining is mainly concentrated at the veins, calyces, stigmas and the tops of the fruit pods, and the stems are not stained at all. Wild type plants were not stained at different stages of growth and in different tissues (FIGS. 3 i-r). Research shows that GUS reporter gene driven by CiNAC071 promoter is mainly expressed in plant leaf, hypocotyl, calyx and other tissues and organs.

Example 5, pro _CiNAC071 GUS transgenic plant hormone treatment histochemical staining

Among the more frequently occurring cis-elements of the CiNAC071 promoter are 5 ABA responsive elements, 4 MeJA responsive elements and 2 GA responsive elements. To analyze the response of these elements to plant hormones, pro was treated with the hormones described above _CiNAC071 GUS transgenic plants were then histochemically stained. Pro as shown in FIG. 4 _CiNAC071 GUS transgenic plants after MeJA treatmentThere was no significant change from the pretreatment. After ABA treatment, however, staining signals decreased with increasing ABA concentration but increased with increasing GA concentration, indicating that CiNAC071 promoter is able to respond to ABA and GA, an inducible promoter.

SEQUENCE LISTING

<110> Applicant

<120> caragana intermedia inducible promoter CiNAC071 and application thereof

<130> caragana intermedia inducible promoter CiNAC071 and application thereof

<160> 3

<170> PatentIn version 3.3

<210> 1

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<212> DNA

<213> Caragana inermedia

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tagggcgaat tgaagctgcc ctttgtcgac agatatgaac agagtcacgt gggccttgct 60

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agtactacct atatgttttt agaatatttt attccaaata aaagatgagc atatctattc 180

cttgaagcat gaatcacata gggggtaacg tgtctgactt gccaaaaaaa gcaaatgcgt 240

tgcatctcag aaaagatatt tcaagagaaa tttatttgtt cagtcataaa aaaaaaagag 300

agatttattt gtctttttca attactaata ttaaatagac aattatttat taaaaaaagt 360

attagtacgt ttatatataa aatccaacaa aaaaaaaatg ttaaatgaat acgtgtgcat 420

ctcacactat ctcttgcctc ggctgcttca caccttcacc attctcgatc taacgcagac 480

catcttctct gaagggccta tttgcatttt ttttcatctt cctccatcgc cgaccacttc 540

ttttggccga agttgctgtc aaccgtttcc cttcatcttc ccttcctcct ttctctgttt 600

ttttttctca tctttaccaa ctatggttga tgcttctgcc cctgcaactt gatcagcaac 660

gtgcggctcc ccctctgtac agcactccgg cgccgccatt tgcttcacct ctggtggcat 720

tgagcttacc cgggcaaatc cgtccgatcg gaaccgtcac ccgaccaaac cgtcaccggc 780

caaaccgtgc aagaaaacgg acggaattgg gccacatatt acagcccacg gtttgggccg 840

gattagagtt ttttggcccg aaaccgatcc ggcccgaacc atgtccagcc ctactcatca 900

tcatcatcat tcatgatgac gctttgataa ttgaaaggca atttaacgaa aggtcatact 960

aacaagtgct ttaaagacat tgtttaagga ttctaaataa ggtaaatact catttaaaaa 1020

ccatgtttat ctctttccaa cgtattaaat acacaacttc tcataaaaac ttactttttt 1080

ggacttctta aacaatactc ttagggcact cattagtatt ttccttaatg aaattagagg 1140

catataatcc tctgcttttg ttagtgattg aaggcaagct aatgttgacg tccattcttt 1200

aactgagatg caaaggaaga agctaatgtt aattaagaac ccaaaacgca tgaaataaat 1260

aagccataaa ataacggtcg caaattattg gaattggcgg gaagtaaact aaactttgta 1320

ggtgtgagct ggttggagtg gtgggtgggc cattttgctt tggaattttt ggtgcactcg 1380

tgacaagtaa tctcaaactt tcggttcagt gtgctcaatt ttggactgtt ggttcccttc 1440

cccaaatgct ataaatacca caccatcact gacctatgtt tttactctat agtctcttgt 1500

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gataaaattt ctgatttgtt atttttgttt tgtatatttg ggggtcaaaa gttggtaatt 1680

taataattgg taacttctga gtattactaa gttaatatca attctttttg ttccagcttt 1740

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ttagtagtag ccatggcccc tcccatatta tgttgctatg ta 42

Claims (6)

1. Nucleotide

Characterised in that the sequences are e.g. </> based on>

As shown.

2. A set of nucleic acids for amplification according to claim 1

The primer pair according to (4), characterized in that the sequence of the primer pair is e.g. < >>

、/>

As shown.

3. A recombinant vector comprising the nucleotide according to claim 1

。

4. The nucleotide according to claim 1

Use as promoter, in particular in conjunction with nucleotide(s) < >>

Use as an inducible promoter for driving expression of a gene of interest in plants in->

Inhibits downstream gene activity under treatment>

Promoting the activity of downstream genes under the treatment; the plants are caragana intermedia and arabidopsis thaliana.

5. The nucleotide according to claim 1

The method for producing (1) is characterized by comprising a synthetic method or a biological cloning method.

6. The nucleotide according to claim 5

The preparation method is characterized in that the biological cloning method specifically comprises the following steps:

step 1, extracting caragana intermedia

；

Step 2, using the above

Sequentially amplifying by using the outer primer and the inner primer as a template;

step 3, carrying out agarose gel electrophoresis detection on the amplification product to obtain nucleotide

。/>

Images