Honeysuckle LjC3H1 gene tissue-specific promoter and its application

Abstract

This invention discloses a tissue-specific promoter for the LjC3H1 gene in honeysuckle, relating to the field of biotechnology. The nucleotide sequence of the specific promoter is shown in SEQ ID NO:1. This invention also provides an expression cassette containing the LjC3H1 gene tissue-specific promoter, a recombinant vector, and a host cell. It has the function of driving or regulating the expression of the LjC3H1 gene to further increase chlorogenic acid production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the tissue-specific promoter of the LjC3H1 gene in honeysuckle and its application. Background Technology

[0002] Honeysuckle (Lonicera japonica Thunb.), also known as Lonicera japonica, is a plant belonging to the genus Lonicera in the family Caprifoliaceae. It is a widely used medicinal plant in Asian countries. Honeysuckle has antibacterial, antiviral, antipyretic, hemostatic, antioxidant, lipid-lowering, and immunomodulatory effects. It is commonly used to treat carbuncles, boils, sore throat, erysipelas, dysentery caused by heat and toxins, colds due to wind-heat, and fever caused by epidemic diseases. Currently, more than 200 compounds have been discovered in honeysuckle. Chlorogenic acids are the main active medicinal components of honeysuckle, possessing antioxidant, anti-inflammatory, antibacterial, antiviral, and antitumor activities. The 2020 edition of the Chinese Pharmacopoeia stipulates that honeysuckle is the dried flower buds or newly opened flowers of Lonicera japonica, and the chlorogenic acid content must not be less than 1.5%. Honeysuckle exhibits significant variations in chlorogenic acid content not only due to varietal differences but also because environmental factors can cause substantial variations in chlorogenic acid content within the same variety at different times and in different regions, or even within the same region in different years. Authentic honeysuckle is often easily mistaken for counterfeit products. Therefore, effectively increasing and stabilizing chlorogenic acid content is a major challenge in honeysuckle breeding and cultivation.

[0003] Chlorogenic acid is a phenylpropanoid compound produced by plants via the shikimic acid pathway during aerobic respiration. Phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), and 4-hydroxycinnamoyl-CoA ligase (4CL) are the key enzymes in the initial three steps of the reaction. Under the action of these three enzymes, phenylalanine is converted to cinnamic acid, which is then converted to coumaroyl-CoA. Afterward, there are two main pathways: the first is the coumaroyl-quinic acid pathway, where coumaroyl-quinic acid is converted to chlorogenic acid by coumaric acid hydroxylase (C3H); the second is the coumaroyl-shikimic acid pathway, where coumaroyl-shikimic acid is converted to caffeic acid shikimic acid by C3H, and then esterified to chlorogenic acid by hydroxylated cinnamoyl-CoA / quinic acid hydroxycinnamoyltransferase (LJC3H1). It can be seen that C3H plays an important role in both pathways.

[0004] Promoters are key components regulating gene transcription and expression; their precise binding to RNA polymerase determines the initiation of gene expression. Promoters contain transcription factor-binding elements, working in concert with transcription factors to regulate gene expression and playing a crucial regulatory role in plant adaptation to the environment and growth and development. Based on their regulatory mechanisms and expression sites, plant promoters can be classified into three types: constitutive promoters, tissue-specific promoters, and inducible promoters. Significant differences exist between different species and between different types of promoters. With the continuous improvement of genome analysis technology, it has become possible to increase crop yield, the content of active ingredients, and enhance resistance to adversity through genetic engineering. In honeysuckle breeding, although significant breakthroughs have been achieved in transgenic technology, the scarcity of discovered superior gene promoters severely limits the research and application of gene expression regulation related to the synthesis of active ingredients.

[0005] Therefore, discovering the honeysuckle promoter and studying its function has important economic and application value in regulating the expression of genes related to the synthesis of honeysuckle's effective components. Summary of the Invention

[0006] In view of this, the present invention provides a promoter upstream of the LjC3H1 gene of honeysuckle and performs functional analysis on it, which can provide an important promoter sequence resource for breeding practices to improve the chlorogenic acid content of honeysuckle.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A tissue-specific promoter for the LjC3H1 gene of honeysuckle, the nucleotide sequence of which is as follows:

[0009] (1) The nucleotide sequence shown in SEQ ID NO:1;

[0010] (2) The complementary sequence of the nucleotide sequence shown in SEQ ID NO:1;

[0011] (3) The nucleotide sequence shown in SEQ ID NO:1 with the addition, substitution, insertion or deletion of one or more nucleotides and having the same function, or its alleles and their derived nucleotide sequences.

[0012] The present invention also provides an expression cassette containing the above-mentioned honeysuckle LjC3H1 gene tissue-specific promoter.

[0013] The present invention also provides a recombinant vector containing the tissue-specific promoter of the honeysuckle LjC3H1 gene described above.

[0014] The present invention also provides a host cell containing the above-mentioned tissue-specific promoter, expression cassette, or recombinant vector, wherein the host cell is selected from bacterial, algal, and fungal host cells.

[0015] The present invention also provides a method for driving or regulating the expression of the LjC3H1 gene in honeysuckle, comprising effectively linking the LjC3H1 gene to the tissue-specific promoter described above, or introducing the expression cassette or the recombinant vector described above into honeysuckle plants.

[0016] The present invention also provides a method for driving or regulating chlorogenic acid metabolism in honeysuckle, including effectively linking the LjC3H1 gene to the tissue-specific promoter mentioned above, or introducing the expression cassette or the recombinant vector mentioned above into honeysuckle plants.

[0017] The present invention also provides a method for producing transgenic honeysuckle with high chlorogenic acid production, comprising effectively linking the LjC3H1 gene to the tissue-specific promoter mentioned above, or introducing the expression cassette or the recombinant vector mentioned above into honeysuckle plants.

[0018] This invention also provides the application of the above-mentioned honeysuckle LjC3H1 gene tissue-specific promoter in honeysuckle breeding. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 Map of the constructed pLjC3H1::GUS expression vector;

[0021] Figure 2 Screening for Basta resistance in transgenic Arabidopsis thaliana;

[0022] Figure 3 GUS testing for transgenic Arabidopsis thaliana;

[0023] Figure 4 GUS testing for genetically modified rice;

[0024] Figure 5 Protein concentrations in different tissues of transgenic Arabidopsis thaliana;

[0025] Figure 6 Quantitative determination of GUS activity in different tissues of transgenic Arabidopsis thaliana. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention discloses a tissue-specific promoter for the LjC3H1 gene of honeysuckle and its application. Unless otherwise specified, the reagents or methods involved are conventional reagents or methods and are not specifically limited thereto.

[0028] Example 1

[0029] 1. Obtaining the promoter of the LjC3H1 gene from honeysuckle

[0030] The promoter sequence of the honeysuckle LjC3H1 gene was cloned using chromosome walking technology.

[0031] (1) Constructing a GenomeWalker library with connector sequences

[0032] Genomic DNA was extracted from honeysuckle using the CTAB method and purified. The DNA was then digested with four restriction endonucleases with adapters: Genome Walker Adaptor BamHI, EcoRI, NdeI, and XhoI.

[0033] (2) PCR amplification

[0034] Nested PCR was used for amplification. The first PCR used the outer adapter primer (AP1: 5'-AAATGTAGACCGTGCTGT-3', SEQ ID NO.2) and the honeysuckle outer gene-specific primer (GSP1: 5'-ACCGCCTCCATCAAGCTGCACAC CG-3', SEQ ID NO.3). The PCR product was diluted and used as a template for the second PCR. The second PCR used (AP2: 5'-TAATACGGTAAGGTGTACTAG TTCA-3', SEQ ID NO.4) and (GSP2: 5'-ATACGCTTCTCTAATCG CCGTGACC-3', SEQ ID NO.5) for amplification. The LjC3H1 promoter sequence was obtained through cloning, sequencing, and sequence assembly. Finally, using honeysuckle genomic DNA as a template, PCR amplification was performed using primers (P3F: 5'-TAATGTATCAATTGTAGAGTATATT-3', SEQ ID NO.6) and (P3R: 5'-TGAGACCATCCCTTGGGAGATTTTG-3', SEQ ID NO.7).

[0035] (3) PCR product recovery and sequencing

[0036] After electrophoresis analysis of the PCR products, they were recovered using a kit from Tiangen Biotech Co., Ltd., and ligated into the pMD18-T Vector using the pMD18-T Vector ligation kit from Takara Bio Inc. The target DNA fragment was ligated into the pMD18-T vector and transformed into E. coli DH5α strain. After blue-white screening, positive clones were selected, plasmids were extracted, and enzyme digestion was performed for identification. The positive clones were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to obtain the LjC3H1 gene promoter sequence, which is 2340 bp in length. The nucleotide sequence of the promoter is shown in SEQ ID No. 1.

[0037] 2. Analysis of the LjC3H1 promoter sequence of honeysuckle

[0038] The PLACE (http: / / www.dna.affrc.go.jp / PLACE / ) and PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) online analysis systems were used to analyze the possible cis-regulatory elements in the HLjC3H1 promoter sequence. Most of the cis-regulatory elements in the LjC3H1 promoter of honeysuckle are involved in flavonoid synthesis, pollen development, petal expansion, and microspore release. The results indicate that this promoter contains abundant cis-regulatory elements, which may be regulated by various transcription factors and may be involved in plant flavonoid synthesis and flower opening-related responses.

[0039] Example 2: Functional Analysis of LjC3H1 Promoter

[0040] 1. Construction of GUS expression vector

[0041] The 35S promoter was excised from the modified pCAMBIA1301 vector using a double enzyme digestion method (SpeI and SacI), and the pCAMBIA1301 vector fragment without the 35S promoter was recovered. Primers containing the SpeI restriction site (ACT AGT) and the SacI restriction site (GAGCTT) were designed based on the honeysuckle LjC3H1 promoter sequence (primer pairs: 5'-TAATGTATCAATTGTAGAGTATATT-3', SEQ ID NO. 8 and 5'-ACTCA AGTGTTCTACTATGTTTGTA-3', SEQ ID NO. 9). After PCR product recovery, restriction endonucleases were used for digestion, and the digested products were ligated into the pCAMBIA1301 vector fragment without the 35S promoter, thus constructing the LjC3H1 promoter reporter gene GUS expression vector pLjC3H1::GUS (see Appendix). Figure 1 ).

[0042] 2. Preparation of Agrobacterium competent cells

[0043] Pick EHA105 Agrobacterium strain stored at -80℃ and inoculate it onto rifampicin-resistant YEB agar (containing 1 mg / ml rifampicin) and incubate at 28℃ with shaking at 200 rpm for 2-3 days. Pick a single colony and inoculate it into 20 ml of rifampicin-resistant YEB liquid medium and incubate at 28℃ with shaking at 200 rpm for 16 hours. Take 2 ml of the bacterial culture and inoculate it into 200 ml of (activated rifampicin (1 mg / ml) resistant YEB liquid medium at a 1:100 ratio) and incubate at 28℃ with shaking at 200 rpm for 3-5 hours until the OD600 is approximately 0.5. Place the bacterial culture on ice for 10 min, centrifuge at 5000 rpm for 5 min at 4℃ to collect the bacterial cells. Gently resuspend the bacterial cells in 10 ml of pre-chilled 10% glycerol. Incubate on ice for 10 min, centrifuge at 5000 rpm for 5 min at 4℃, discard the supernatant, and repeat once. Add 5 ml of pre-cooled 10% glycerol to resuspend the bacterial cells, dispense into 100 μl tubes, flash freeze in liquid nitrogen, and store at -80°C.

[0044] 3. Expression vector pLjC3H1::GUS was transformed into Agrobacterium.

[0045] The pLjC3H1::GUS vector was transformed into Agrobacterium EHA105 cells using plasmid electroporation. Competent Agrobacterium cells were removed from a -80°C freezer and thawed on ice. 30 μl of competent cells were taken, and 0.5 μl of plasmid DNA was added and mixed thoroughly. The mixture was transferred to a pre-cooled, clean electroporation vessel and electroporated. The electroporated mixture was then rapidly transferred to a 1.5 ml centrifuge tube containing 900 μl of YEB liquid medium. The cells were incubated at 28°C with shaking at 5000 rpm for 2–3 hours, followed by centrifugation at 8000 rpm for 2 min. The bacterial cells were collected and plated onto YEB solid medium containing both rifampicin (100 μg / ml) and kanamycin (50 μg / ml) resistance. PCR detection using gene-specific primers was performed to identify positive clones.

[0046] 4. Transformation of plants using expression vector pLjC3H1::GUS

[0047] (1) Arabidopsis thaliana was transformed with expression vector pLjC3H1::GUS.

[0048] Agrobacterium was prepared and used to infect Arabidopsis thaliana using the flower infection method. Agrobacterium containing the expression vector plasmid was streaked onto YEB agar plates and cultured for 2-3 days. Then, 100 mL of AAM transformation medium was prepared, and the cultured Agrobacterium was inoculated into AAM liquid medium using an inoculation needle loop and cultured at 150 rpm with shaking for 30 min. Finally, vigorous Arabidopsis thaliana plants that were beginning to flower were selected for transformation. The unopened flower buds were soaked in AAM transformation medium, shaking 2-3 times during the process. After soaking, the buds were blotted dry with sterile filter paper and cultured in the dark at 28℃ for 3 days. Transgenic Arabidopsis thaliana plants were obtained through two consecutive screenings using the Basta resistance method (see Appendix). Figure 2 T0 generation seeds were harvested. The harvested T0 generation seeds were surface-sterilized and then evenly spread on 1 / 2 MS plates containing the antibiotic hygromycin. After vernalization for 3 days, they were transferred to an artificial climate chamber for growth. After germination for 8 days, plants with dark green cotyledons were identified as transgenic plants, while those with light green or even yellow cotyledons were identified as non-transgenic plants. The transgenic plants were then transferred to soil for further growth until T1 generation transgenic seeds were harvested. Individual T1 generation plants were harvested, and the seeds from each plant were further screened with antibiotics (hygromycin). Positive plants with a segregation ratio of 3:1 (positive:negative) were transplanted and grown until T2 generation transgenic seeds were harvested. After individual harvesting, the seeds from each plant were screened with antibiotics (hygromycin) to obtain pure-line T3 generation transgenic seeds.

[0049] (2) Rice transformed with expression vector pLjC3H1::GUS

[0050] Callus induction: NB basic medium was used as the induction medium + 2,4-D (2.5 mg / L). The culture conditions were the same as those used by Nishimura et al.

[135] (2007): 32℃ high temperature, continuous light, and loose and swollen callus tissue could be obtained in about 7 days, which is just right for transformation.

[0051] Transformation: First, Agrobacterium with the detected expression vector plasmid was streaked on YEB solid plates and cultured for 2-3 days. Then, 30 mL of AAM transformation solution was prepared, and Agrobacterium was picked up with an inoculation needle loop and inoculated into AAM liquid medium with shaking at 150 rpm for 30 min. Finally, callus tissue with good growth viability was selected for transformation. The transformation method of Ozawa et al.

[136] (2009) was followed: the callus tissue was soaked in AAM transformation solution for 10 min, and shaken 2-3 times during the process. The soaked callus tissue was then blotted dry with sterile filter paper and cultured on MS medium at 25℃ for 3 days.

[0052] Sterilization and screening: After co-culturing the callus with Agrobacterium, rinse it 4-5 times with sterile water, then sterilize it with 500 mg / L carbenicillin (shake at 200 rpm for 15 min), and finally blot the liquid on the surface of the callus with sterile filter paper. Then place it on hygromycin (30 mg / L) screening medium for about 20 days, changing the medium every 10 days during this period.

[0053] Differentiation: Transfer the resistant callus to differentiation medium and differentiate for about 20 days, changing the medium every 10 days.

[0054] Rooting: The differentiated seedlings are transferred to a rooting medium for rooting induction, and roots will grow in about 7 days.

[0055] Hardening off seedlings: Open the bottle cap indoors, put a plastic bag over the seedlings, and harden off for about 7 days.

[0056] Transplanting to the field: Transgenic seedlings are transferred to the field and managed according to conventional methods, and herbicide resistance is screened.

[0057] PCR identification of transgenic plants yielded T0 generation seeds.

[0058] Genomic DNA was extracted from transgenic and wild-type rice using the CTAB method, and PCR detection was performed using AsRed gene-specific primers. The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 34 cycles; and a final extension at 72℃ for 5 min. AsRed-F 5'-CCCTTCGCCTTC CACATCCT-3', SEQ ID NO.10, AsRed-R 5'-TGTAGCACTTGCC CTTCTCCAC-3', SEQ ID NO.11.

[0059] 5. GUS staining of transgenic plant materials

[0060] GUS staining solution preparation: First, prepare a 50 mg / ml X-Gluc stock solution using DMSO. Then, prepare a GUS wash solution: a mixture of sodium phosphate, potassium ferrocyanide, and potassium ferricyanide, with concentrations of 50 mM sodium phosphate buffer, 0.5 mM potassium ferrocyanide K4Fe(CN)6 (pH 7.2), and 0.5 mM potassium ferricyanide K3Fe(CN)6, respectively. Finally, prepare the staining solution: 5 ml wash solution + 100 μl X-Gluc stock solution.

[0061] GUS staining:

[0062] Small pieces of tissues, including leaves, filaments, ovaries, petals, terminal buds, and axillary buds, were cut and placed in 1.5 ml centrifuge tubes. Pre-chilled 90% acetone was added, and the tissues were rinsed for 10 minutes. The tubes were then placed on ice for 25 minutes to remove some chlorophyll. The tissues were then completely covered with prepared GUS staining working solution. A vacuum pump was used to remove air until the material settled to the bottom of the centrifuge tube. The tubes were then covered with aluminum foil to prevent light exposure overnight. Gradient elution was performed with 50%, 75%, and 95% ethanol, with gentle shaking for 5 minutes each time. Microscopic observation and photography were performed (see appendix). Figure 3 and attached Figure 4 ).

[0063] GUS staining analysis of different pLjC3H1::GUS Arabidopsis pure lines showed that the tissue-specific expression sites of GUS were basically consistent among different lines of the transgenic Arabidopsis T3 generation, with T3-6 showing the highest expression level. Therefore, T3-6 was used as the material for subsequent experiments. Transgenic Arabidopsis T3-6 was planted on 1 / 2 MS medium, and GUS staining was performed on Arabidopsis at the seedling and flowering stages to observe its tissue-specific expression. The results showed that GUS driven by the LJC3H1 promoter was specifically and highly expressed in Arabidopsis petals, styles, filaments, terminal buds, and axillary buds; expression was observed in the terminal buds of transgenic rice, but not in the roots.

[0064] 6. Detection of GUS activity in transgenic plant materials

[0065] (1) Preparation of GUS quantitative detection solution

[0066] 0.1M phosphate buffer (pH 7.0): Take 5.77 ml of 1 mol / L Na2HPO4 and 4.23 ml of 1 mol / L NaH2PO4, and make up to 100 ml (1 mol / L Na2HPO4 solution: 35.814 g Na2HPO4 dissolved in 100 ml water; 1 mol / L NaH2PO4 solution: 15.601 g NaH2PO4 dissolved in 100 ml water).

[0067] 10% SDS solution: Heat 90ml of water slightly, add 10g of SDS, stir to dissolve, add a few drops of concentrated hydrochloric acid to adjust the pH to 7.2, and then add water to make up to 100ml.

[0068] 0.5MEDTA (pH 8.0): Add 18.61g Na2EDTA·2H2O to 80ml of water, adjust the pH to 8.0 with NaOH (about 2g of solid NaOH is needed), dissolve and then bring the volume to 100ml.

[0069] GUS enzyme extraction solution: 50 ml of 0.1 M phosphate buffer (pH 7.0); 1 ml of 10% SDS; 2 ml of 0.5 M EDTA (pH 8.0); 100 μl of Triton X-100; 100 μl of β-mercaptoethanol; and water to a final volume of 100 ml.

[0070] MUG substrate: Weigh 8.8 mg MUG and dissolve it in 10 ml GUS enzyme extract to prepare a working concentration of 2 mmol / L.

[0071] Reaction termination solution (0.2 mol / L Na2CO3): Weigh 2.12 Na2CO3 and dilute to 100 ml with water.

[0072] Coomassie Brilliant Blue G250 solution: 10 mg Coomassie Brilliant Blue G250, 5 ml 95% ethanol, 10 ml H3PO4, bring to a final volume of 100 ml, filter, and store at 4°C.

[0073] 1 mg / ml BSA: 20 mg BSA, diluted to 20 ml with GUS extraction buffer.

[0074] (2) Protein extraction and concentration determination

[0075] Take approximately 100 mg of different Arabidopsis thaliana tissue samples and grind them thoroughly into powder in liquid nitrogen. Transfer the powder to a 1.5 mL centrifuge tube, add 0.5 mL of GUS extraction buffer, and vortex to mix. Centrifuge at 11,000 rpm for 8 min at 4 °C, and store the supernatant at 4 °C.

[0076] Protein concentration determination: Pipette 0 μl, 1 μl, 3 μl, 5 μl, 10 μl, and 15 μl of BSA standard solution into 1.5 mL centrifuge tubes, respectively, and bring the volume to 20 μl with water. Add 980 μl of Coomassie Brilliant Blue G250 solution, mix thoroughly, incubate on ice for 10 min, and measure the absorbance at 595 nm. Construct a standard curve of protein concentration versus BSA 595 absorbance. Alternatively, take 15 μl of the protein sample to be tested, add water to 20 μl, add 980 μl of Coomassie Brilliant Blue G250 solution, vortex mix, and incubate on ice for 10 min. Measure the absorbance at 595 nm using a spectrophotometer. Calculate the protein concentration of the sample; the results are shown in the appendix. Figure 5 It can be seen that the expression level was high in Arabidopsis flowers and terminal buds, and low in leaves and roots, with the differences reaching a significant level.

[0077] (3) GUS quantitative detection steps

[0078] Preparation of standard curve: Prepare a standard solution of MU with a concentration range of 0-10 μM using the reaction termination solution, and measure their fluorescence intensity to plot a standard curve.

[0079] 100 μl of protein samples from different tissues were added to 700 μl of GUS enzyme extraction buffer and incubated at 37℃ for 10 min. Then, 200 μl of 5 MUG was added, and the mixture was thoroughly mixed. 200 μl of this solution was then quickly added to 800 μl of stop solution to terminate the reaction, and the 0-point mark of the enzymatic reaction was recorded. 200 μl of the reaction solution was taken after each reaction at 37℃ for 10 min, 15 min, 20 min, 35 min, 30 min, and 45 min, and 800 μl of stop solution was added to terminate the reaction. The solutions were then stored in aluminum foil protected from light. Fluorescence spectrophotometry was used to measure the fluorescence values ​​at different reaction time points at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. The results are attached. Figure 6 As shown, GUS activity varied significantly across different tissues, which is consistent with the results of strong chemical staining. GUS activity was high in petals and filaments, decreased in terminal buds and leaves, and extremely low in stems and roots. These results indicate that the LjC3H1 promoter is a highly expressed, specifically expressed promoter in flowers.

[0080] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0081] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not 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.

Claims

1. A Lonicera japonica LjC3H1 gene tissue-specific promoter, characterized in that, The nucleotide sequence of the specific promoter is the nucleotide sequence shown in SEQ ID NO:

1.

2. An expression cassette comprising, The LjC3H1 gene tissue-specific promoter of Lonicera japonica according to claim 1.

3. A recombinant vector, characterized in that, The LjC3H1 gene tissue-specific promoter of Lonicera japonica according to claim 1.

4. A host cell comprising the tissue-specific promoter of claim 1 or the expression cassette of claim 2 or the recombinant vector of claim 3. The host cell is selected from bacterial, algal and fungal host cells.

5. The LjC3H1 gene tissue-specific promoter of Lonicera japonica according to claim 1 is applied in the breeding of Lonicera japonica.

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