Application of CsWRKY41 gene in regulation and control of plant lignin synthesis

Abstract

The invention provides application of a CsWRKY41 gene in regulation and control of plant lignin synthesis, and belongs to the technical field of plant genetic engineering. A plant overexpression vector and a gene silencing vector of the CsWRKY41 gene are constructed and are respectively subjected to genetic transformation in arabidopsis thaliana and a tea tree, and the result proves that the lignin content of a transgenic plant can be obviously improved by overexpressing the CsWRKY41 gene; on the contrary, silencing of the gene leads to reduction of lignin content. The invention discloses a molecular mechanism of the tea tree CsWRKY41 gene for enhancing plant drought resistance by positively regulating lignin biosynthesis for the first time. The gene and the related expression vector can be used for cultivating tea trees with high lignin content and strong drought resistance and even other new forest or crop varieties, and have wide application prospects in agricultural water conservation and stress resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the CsWRKY41 gene in regulating plant lignin synthesis. Background Technology

[0002] Drought stress is one of the most significant abiotic stresses affecting global agricultural production, severely restricting crop growth, development, and yield. As an important economic crop, the tea plant (Camellia sinensis) is extremely sensitive to water conditions in terms of growth and tea quality. Therefore, identifying key drought-resistant genes in the tea plant, elucidating its molecular mechanisms of drought resistance, and using genetic engineering to cultivate new drought-resistant tea varieties are of great theoretical and practical significance for ensuring the stable development of the tea industry and conserving water resources.

[0003] Lignin is a major component of the secondary cell wall in plants, providing not only mechanical support but also playing a crucial role in plant responses to environmental stress. Studies have shown that lignin accumulation strengthens cell walls, reduces water evaporation, and enhances xylem strength to prevent drought-induced blockage, thereby improving the overall drought tolerance of plants. Therefore, regulating lignin biosynthesis pathways is an effective strategy for improving plant drought resistance. However, in tea plants, which key transcription factors directly regulate lignin synthesis and thus affect drought resistance remains a subject of in-depth research and clear reporting.

[0004] The WRKY transcription factor family is a large family of regulatory proteins in plants, widely involved in plant responses to biotic and abiotic stresses. Previous studies have reported that certain WRKY members in model plants such as Arabidopsis thaliana and rice can regulate the expression of genes related to lignin synthesis. However, in tea, although several WRKY genes have been identified, whether CsWRKY41 participates in the regulation of lignin synthesis and its direct association with drought resistance in tea plants has not been reported. Summary of the Invention

[0005] In view of this, this invention provides the application of the CsWRKY41 gene in regulating lignin synthesis in plants. Through transcriptomic analysis of tea plants under drought stress, this invention, for the first time, screened and cloned a CsWRKY41 gene significantly induced by drought. Through systematic expression pattern analysis, verification of expression induced by various stresses, and combined with gain-of-function and loss-of-function experiments in the model plant Arabidopsis thaliana and tea plants, this invention for the first time confirms that the CsWRKY41 gene in tea plants is a positive regulator of lignin biosynthesis and positively regulates drought resistance in tea plants through this pathway. This discovery provides a novel, proprietary key gene target and theoretical basis for improving the drought resistance of tea plants and related species using molecular breeding methods.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the CsWRKY41 gene in regulating plant lignin synthesis. The nucleotide sequence of the CsWRKY41 gene is shown in SEQ ID NO.1.

[0008] Preferably, overexpression of the CsWRKY41 gene promotes lignin synthesis and increases the lignin content of plants; knockout or silencing of the CsWRKY41 gene inhibits lignin synthesis and reduces the lignin content of plants.

[0009] The present invention also provides the application of a protein encoded by the CsWRKY41 gene in regulating plant lignin synthesis, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] Preferably, the plants include Arabidopsis thaliana and tea tree.

[0011] The present invention also provides a biomaterial for regulating the lignin content of plants, the biomaterial comprising: a nucleic acid fragment of the CsWRKY41 gene or an expression cassette, recombinant vector or engineered bacteria containing the nucleic acid fragment.

[0012] The present invention also provides a method for improving tea alginate synthesis, comprising the following steps:

[0013] (1) Using tea plant genomic cDNA as a template, amplify the nucleotide fragment of the CsWRKY41 gene;

[0014] (2) The super1300 vector was digested with SacI and XbaI, and the nucleotide fragment from step (1) was ligated with the digested super1300 vector to obtain the CsWRKY41-super1300 recombinant plasmid.

[0015] (3) The recombinant plasmid CsWRKY41-super1300 was transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium;

[0016] (4) Transfect tea plants with the recombinant Agrobacterium to obtain transgenic tea plants.

[0017] Preferably, the primers for amplifying the CsWRKY41 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0018] Preferably, the recombinant plasmid CsWRKY41-super1300 is transformed into Agrobacterium GV3101 by freeze-thaw method.

[0019] Preferably, the method for transfecting tea plants with recombinant Agrobacterium is as follows: the recombinant Agrobacterium is prepared into a bacterial suspension and injected into the underside of the leaves.

[0020] Preferably, the OD of the bacterial suspension 600 The value is 0.8 to 1.0.

[0021] By adopting the above technical solution, this invention has the following beneficial effects: This invention constructs plant overexpression vectors and gene silencing vectors for the CsWRKY41 gene, and performs genetic transformations in Arabidopsis thaliana and tea trees, respectively. The results demonstrate that overexpression of the CsWRKY41 gene significantly increases the lignin content of transgenic plants; conversely, silencing the gene leads to a decrease in lignin content. This invention reveals for the first time the molecular mechanism by which the CsWRKY41 gene in tea trees enhances drought resistance by positively regulating lignin biosynthesis. This gene and related expression vectors can serve as important gene resources for breeding new varieties of tea trees with high lignin content and strong drought resistance, as well as other forest trees or crops, showing broad application prospects in agricultural water-saving and stress-resistance breeding. Attached Figure Description

[0022] Figure 1 Image of CsWRKY41 gene PCR product detected by agarose gel electrophoresis.

[0023] Figure 2 Semi-quantitative analysis of three overexpressing Arabidopsis thaliana lines and WT, and quantitative analysis of overexpressing Arabidopsis thaliana lines, are presented in section (A). * indicates significant difference at the p < 0.05 level; ** indicates extremely significant difference at the p < 0.01 level; *** indicates extremely significant difference at the p < 0.005 level, and the same applies below.

[0024] Figure 3 To analyze the expression of structural genes related to lignin synthesis in Arabidopsis thaliana with overexpression of the CsWRKY41 gene.

[0025] Figure 4 The expression levels of the CsWRKY41 gene in tea plants overexpressing the CsWRKY41 gene and control tea plants were measured on days 3, 4, and 5 after transfection.

[0026] Figure 5 Figure 1 shows the lignin content determination results of tea plant lines overexpressing the CsWRKY41 gene and control tea plants.

[0027] Figure 6 The relative expression levels of the CsWRKY41 gene in tea leaves at 0, 2, 6, and 24 h after transient silencing of the CsWRKY41 gene.

[0028] Figure 7The results show the determination of lignin content in fresh tea leaves after CsWRKY41 gene silencing.

[0029] Figure 8 The results of gene expression analysis of lignin-related structures in fresh tea leaves after CsWRKY41 silencing. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1. Cloning of the CsWRKY41 gene

[0032] 1. Cloning of the CsWRKY41 gene

[0033] Using the genomic cDNA of early-maturing tea tree leaves as a template, and CsWRKY41-F and CsWRKY41-R as primers, amplification was performed using Novizan high-fidelity enzyme.

[0034] CsWRKY41-F: 5'-ATGGAAAACTCACCAGGTTGG-3' (SEQ ID NO. 3);

[0035] CsWRKY41-R: 5'-TTAGTTAATGAATCCGGGATTATTG-3' (SEQ ID NO. 4);

[0036] The amplification system consisted of 10 μL of high-fidelity enzyme, 1 μL of cDNA, 10 μL of ddH2O, and 2 μL each of CsWRKY41-F / R-terminal primers. The amplification program was as follows: (98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; storage at 4℃).

[0037] The PCR products were subjected to 1% agarose gel electrophoresis, and a single, bright target band of the correct size was observed under ultraviolet light. Figure 1 The target fragment was purified and recovered using the Novizan agarose gel DNA recovery kit, and its concentration was determined for later use.

[0038] The CDS sequence of the CsWRKY41 gene is 1095 bp, as shown in SEQ ID NO.1:

[0039]

[0040] The CsWRKY41 gene encodes 364 amino acids, and its amino acid sequence is shown in SEQ ID NO.2:

[0041] MENSPGWEQKTLINELARGKELAKQLQNNLTSSSHETCELLIHNILNSYDKALSMLKWNGSVAGEPQLTGAAMGITSESPSLCGSPHSEDSDRDLKDHEHKDGSRKRKSTPRWTQQVQVCPGTGLEGPLDDGYSWRKYGQKDILGAKYPRGYYRCTHRNVQGCLATKQVQRSDEDPTIFEITYR GRHTCTPQPPPPPEKQEQPPNTTLDNPPPPPPQQQQQSLETLLNFQTGLKVITEDLETPNNNQTLPFFYFPSTSQENCNVHLHSPSMGNFSDPSFISPATSGTSYFSLSPSPTGTGMAGFGGNQSLDGSDQSELAKIVAAAAAVTSSAATSPTAGLDFPFGSMDQFEPSFSFDNNPGFIN (SEQ IDNO.2).

[0042] Example 2. Construction and transformation of Arabidopsis thaliana using CsWRKY41 expression vector based on Gateway cloning technology

[0043] 1. Using the pEASY®-Blunt Cloning Kit, the gel-recovered product from Example 1 was ligated into the pEASY-Blunt vector. Subsequently, positive colonies were screened using Kan-resistant medium. Single colonies were streaked onto fresh LB solid medium. After confluent colonies were observed, colony PCR was performed using a standard tap enzyme. Positive colonies were selected for shaking and then sent for sequencing. After confirming correct sequencing results, plasmid was extracted from the positive bacteria to obtain the CsWRKY41-pEASY-Blunt recombinant plasmid.

[0044] 2. Using the CsWRKY41-pEASY-Blunt recombinant plasmid as a template, primers for amplifying the CsWRKY41 gene with attb adapters were designed. Attb1 and Attb2 adapters were added to the 5' and 3' ends of the CsWRKY41 gene, respectively. The primer sequences are shown below:

[0045] pDonor207-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3' (SEQ ID NO.5);

[0046] pDonor207-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3' (SEQ ID NO. 6);

[0047] After performing 1% agarose gel electrophoresis on the amplified products, the CsWRKY41 fragment with the Attb linker was obtained. The CsWRKY41 fragment with the linker was then recombined into the pDonor207 entry-level vector (Novozymes Biotechnology Co., Ltd.) using the BP reaction. The BP reaction system is shown in Table 1.

[0048] Table 1 BP reaction system

[0049] Components volume pDonor207 Entry-level carrier 1 µL (75 ng) CsWRKY41 gene with adapter 1 µL (25~50 ng) BP Clonase™ enzyme mix 0.3 µL <![CDATA[ddH2O]]> 2.7 µL

[0050] The PCR reaction was performed overnight at 25°C, and the cells were transformed into E. coli DH5α competent cells. The cells were then cultured overnight at 37°C on LB plates containing gentamicin resistance. Positive clones were screened, and after sequencing verification, the pDonor207-CsWRKY41 plasmid was extracted and stored at -20°C for later use.

[0051] The CsWRKY41 gene on the pDonor207-CsWRKY41 plasmid was recombined into the pCB2004 binary expression vector (Novizan Biotechnology Co., Ltd.) using the LR reaction. The reaction system is shown in Table 2.

[0052] Table 2 LR reaction system

[0053] Components volume overexpression vector pCB2004 1 µL (75 ng) plasmids after BP reaction 1 µL (25~50 ng) LR Clonase™ enzyme mix 0.3 µL <![CDATA[ddH2O]]> 2.7 µL

[0054] After reacting overnight at 25°C, the cells were transformed into E. coli DH5α competent cells and cultured overnight in a 37°C incubator on LB plates containing Kan resistance. Positive clones were screened and sequenced to verify their integrity. The recombinant plasmid CsWRKY41-PCB2004 was obtained.

[0055] The overexpression vector was transformed into Agrobacterium GV3101 using the freeze-thaw method, and then Arabidopsis thaliana was infected using the inflorescence infection method. The specific method is as follows:

[0056] (1) Agrobacterium tumefaciens transformed with the CsWRKY41-PCB2004 recombinant plasmid was activated and resuspended to determine the OD value between 0.8 and 1.0.

[0057] (2) Completely immerse the Arabidopsis inflorescence in the dye solution, gently shake for 20 seconds, place the plant on its side in a tray, cover with plastic wrap to keep it moist, and incubate in the dark for 24 hours. Move it back to normal growing conditions, maintain humidity, and avoid direct sunlight. Water normally, and wait for the new inflorescence to grow and the transformed siliques to gradually mature (about 4-6 weeks).

[0058] Transgenic plants were screened using glufosinate. Initial screening yielded four Arabidopsis thaliana plants overexpressing CsWRKY41 on a glufosinate-added selection medium. To further validate the positive plants, RNA was extracted from Arabidopsis thaliana and cDNA was obtained through reverse transcription. Semi-quantitative PCR analysis was performed using specific quantitative primers. The results showed that the CsWRKY41 band was brightest at lines 3 and 4, followed by line 2, with line 1 being the darkest. Figure 2 To further clarify the expression abundance of the CsWRKY41 gene in transgenic Arabidopsis thaliana, qRT-PCR analysis was performed on the transgenic materials. RNA was extracted from the test plants, and qRT-PCR analysis was performed using reverse-transcribed cDNA as a template and CsGAPDH as an internal control. The primers used are shown in SEQ ID NO.7~SEQ ID NO.10, and the reaction system is shown in Table 3. The reaction program was: 95℃ for 10 min; 94℃ for 10 s, 60℃ for 15 s, 72℃ for 12 s, for 45 cycles.

[0059] qCsWRKY41-F: 5'-GCAAGTCCAAAGATCAGACGAAG-3' (SEQ ID NO. 7);

[0060] qCsWRKY41-R: 5'-GAAGTTGAAGGGAAGTAGAAGAAAGG-3' (SEQ ID NO. 8);

[0061] CsGAPDH-F: 5'-TTGGCATCGTTGAGGGTCT-3' (SEQ ID NO. 9);

[0062] CsGAPDH-R: 5'-CAGTGGGAACACGGAAAGC-3' (SEQ ID NO. 10).

[0063] Table 3 qRT-PCR reaction system

[0064] reagents volume TB Green Fast qPCR Mix (2×) 10 μL Forward primer 0.8 μL Reverse primer 0.8 μL ddH2O 6.4 μL cDNA 2 μL

[0065] The qRT-PCR results were largely consistent with the semi-quantitative results, with line 1 upregulated by approximately 8-fold, line 2 by approximately 27-fold, line 3 by approximately 16-fold, and line 4 by approximately 30-fold. Subsequent experiments were conducted using the three lines with the highest expression levels (line 2, line 3, and line 4).

[0066] To further investigate the function of the CsWRKY41 gene, qRT-PCR analysis was performed on five lignin synthesis-related structural genes in Arabidopsis thaliana: AtCCR, AtCOMT, AtF5H, AtHCT, and AtCCoAOMT. The primers are shown in Table 4.

[0067] Table 4. Quantitative primers for lignin synthesis-related genes in Arabidopsis thaliana

[0068] Primer name Primer sequence Serial Number AtHCT-F CATTCACTCTTTCCCGCTTC SEQ ID NO.11 AtHCT-R GTTCCCATCCTCCTTGGATT SEQ ID NO.12 AtCCoAOMT-F GGCCCTGCTCTTCCCGTTC SEQ ID NO.13 AtCCoAOMT-R GGTGCATCAGGAGGAGCCA SEQ ID NO.14 AtCCR-FF TGTGGATGTTCGCGATGTC SEQ ID NO.15 AtCCR-R GAGGAGCAAGATGGCCTTTC SEQ ID NO.16 AtCOMT-F GTCATGCTCGACCGTATCCT SEQ ID NO.17 AtCOMT-R GCATCTTCGATGACATGTGG SEQ ID NO.18 AtF5H-F GGTCCGGTCGGTCTCTTGTAA SEQ ID NO.19 AtF5H-R CACGAGCCGCTTGTTTATCC SEQ ID NO.20

[0069] The results showed that the expression levels of these five genes in CsWRKY41-OE-overexpressing Arabidopsis thaliana were all higher than those in wild-type ( Figure 3 These results indicate that CsWRKY41 promotes the expression of lignin synthesis-related structural genes in Arabidopsis thaliana.

[0070] Example 2. Overexpression of the CsWRKY41 gene in tea plants

[0071] 1. Construction of overexpression vectors

[0072] The Super1300 plant binary expression vector (purchased from the BioFeng Vector Database, link: https: / / www.biofeng.com / zaiti / zhiwu / Super1300.html) was double-digested with SacI and XbaI to purify and recover the linearized backbone vector. The gel-recovered target fragment from Example 1 was then homologously recombinated with the purified backbone vector to construct the overexpression vector.

[0073] The recombinant product was transformed into Escherichia coli DH5α competent cells using the heat shock method, and then plated onto LB solid medium containing kanamycin resistance. The cells were incubated upside down at 37°C for 12 h. Single colonies were randomly picked from the plates, and colony PCR was performed after shaking. Corresponding positive clones were selected for sequencing verification. Clones with correct sequences were screened to obtain the recombinant plasmid CsWRKY41-super1300.

[0074] 2. CsWRKY41-super1300 recombinant plasmid transformed Agrobacterium

[0075] (1) The CsWRKY41-super1300 recombinant plasmid was transformed into Agrobacterium GV3101 by freeze-thaw method and cultured on LB plates with kan / rif resistance. After single clones were selected for PCR verification, single clones were selected into 12mL centrifuge tubes and 6mL LB, 6μL Kan, and 6μL Rif were added. The tubes were cultured overnight at 28℃.

[0076] (2) Inoculate the bacterial culture from step (1) overnight into 50 mL of LB medium (containing 50 μL Kan and 50 μL L Rif) and culture overnight again.

[0077] (3) Centrifuge the bacterial culture that has been cultured overnight at 5000 rpm for 5 min, collect the bacterial cells, wash twice with buffer, and resuspend the bacterial cells to adjust the OD of the bacterial suspension. 600 The value is 1.0.

[0078] 3. Agrobacterium GV3101-mediated CsWRKY41-super1300 overexpression vector transfection of tea plants

[0079] Twenty 'Zhongcha 108' cuttings of similar size and growth were selected. The bacterial solution from step three was injected into the underside of the leaves using a sterile syringe, with tea plants injected with Super1300 as a control. The tea seedlings were then transferred to a light incubator for cultivation. Samples were taken at 3 and 5 days after injection, with three biological replicates for each group. After removal, the samples were flash-frozen in liquid nitrogen and then stored at -80°C.

[0080] RNA was extracted from samples stored at -80℃ by grinding in liquid nitrogen and then reverse transcribed to obtain cDNA. The relative expression level of CsWRKY41 was then verified by qPCR. Figure 4 As shown in the figure. The results showed that the relative expression level of the CsWRKY41 gene was increased in tea plants that overexpressed the CsWRKY41 gene.

[0081] The lignin content in tea plant samples overexpressing the CsWRKY41 gene and control tea plant samples was determined using a lignin content assay kit (Soluble Technologies Co., Ltd.). The method using the lignin content assay kit is as follows:

[0082] (1) First, dry the leaf to be tested in an oven, grind it into powder with a mortar and pestle, and then screen it with a 40-mesh sieve. Take 2 mg of the screened powder, add 1.5 mL of 80% ethanol, vortex and mix well, and bathe in a 50℃ water bath for 20 min, shaking once every 3 min. After taking it out, place the test tube in a water trough and cool it with flowing water. Centrifuge at 12000 rpm for 10 min, discard the supernatant and keep the precipitate (try to retain the precipitate).

[0083] (2) Add 1 mL of 80% ethanol to the precipitate and shake to mix for 2 min. Heat in a 50℃ water bath for 20 min, shaking once every 3 min. After removing the tube, place it in a water trough and cool it with flowing water. Centrifuge at 12000 rpm for 10 min, discard the supernatant, and keep the precipitate (try to retain the precipitate). Dry the precipitate at 95℃ and set aside for later use.

[0084] (3) Preheat the microplate reader for 30 min, set the temperature to 25℃, and set the wavelength to 280 nm.

[0085] (4) Add 750 μL of reagent one to the test tube (the blank tube is an empty centrifuge tube of the same specification) (add slowly along the tube wall), mix well, and then incubate in a 70℃ water bath for 30 min (clamp the tube opening with a test tube explosion-proof clamp). Gently shake once every 15 min, and then let it cool naturally to room temperature. Continue to add 300 μL of reagent two (add slowly along the tube wall), and then add 450 μL of acetic acid. Shake well to mix thoroughly, and the total volume is 1.5 mL.

[0086] (5) Centrifuge at 5000 rpm for 5 min at room temperature, take 100 μL of supernatant into a 96-well UV plate, add 100 μL of acetic acid into the well, read the absorbance at 280 nm, and then perform calculations according to the formula in the instruction manual.

[0087] Lignin determination results as follows Figure 5 As shown, Figure 5 This indicates that, compared to the control group (CK), the lignin content of tea plants decreased significantly after overexpression of the CsWRKY41 gene.

[0088] Example 3. Transient silencing of the CsWRKY41 gene in tea plants

[0089] 1. Obtaining plants with transiently silenced CsWRKY41 gene

[0090] Antisense oligodeoxynucleotides (asODNs) for the CsWRKY41 gene were designed, and the asODNs were BLASTed on the TPIA website to obtain two asODNs as transient silencing primers for the CsWRKY41 gene in tea plants. At the same time, sense oligonucleotides (sODNs) with antisense complementary sequences of these two asODNs were designed as controls. The primers are shown in Table 5.

[0091] Table 5 Primers used for gene silencing

[0092] Primer name Primer sequence Serial Number asODN-1 ATCCGGGATTATTGTCA SEQ ID NO.21 asODN-2 AATCCGGGATTATTGTC SEQ ID NO.22 sODN-1 TGACAATAATCCCGGAT SEQ ID NO.23 sODN-2 GACAATAATCCCGGATT SEQ ID NO.24

[0093] To investigate the changes in the expression levels of the CsWRKY41 gene and related structural genes in tea leaves after transient silencing of the gene, primers for two asODN and two sODN genes were diluted to 10 μM and injected into tea leaves. The specific injection method is as follows:

[0094] Select the second mature leaf below the bud, and use a sterile syringe to inject the diluted primer mixture onto the underside of the leaf, filling it completely. Inject one healthy, mature leaf into each tea seedling. For both treatments, inject 20 'Shuchazao' cuttings of similar size and growth. The tea seedlings were then transferred to a light incubator for cultivation. Samples were taken at 0 h before injection and at 2 h, 6 h, and 24 h after injection, with three biological replicates for each group. After removal, the samples were flash-frozen in liquid nitrogen and then stored at -80°C.

[0095] Total RNA was extracted from the processed samples and reverse transcribed into cDNA for qRT-PCR to detect the expression of the CsWRKY41 gene. The results are as follows: Figure 6 As shown, at 24 h, the expression level of CsWRKY41 in tea leaves treated with asODN decreased significantly (approximately 21-fold) compared to the sODN treatment group. These results demonstrate that the present invention successfully and transiently inhibited the expression of the CsWRKY41 gene in tea leaves.

[0096] 2. CsWRKY41 gene silencing reduces lignin content and related gene expression in tea leaves.

[0097] To further investigate the function of the CsWRKY41 gene using a gene silencing assay, the lignin content of the silenced samples was determined. The results are as follows: Figure 7 As shown. Figure 7 This indicates that, compared to the control (CK), the lignin content decreased significantly after silencing. To further verify the results, qRT-PCR analysis was performed on the silencing samples to determine the expression levels of lignin synthesis-related structural genes. A total of 12 lignin synthesis-related genes were measured, and the primers are shown in Table 6.

[0098] Table 6. Quantitative primers for lignin synthesis-related genes in tea plants after silencing.

[0099] Gene name Forward sequence (5´- →3´-) Serial Number Reverse sequence (5´- →3´) Serial Number CsC4H TATTCGGCAACTGGCTCC SEQ ID NO.25 CACCATATCCTGACCCTTACC SEQ ID NO.26 Cs4CL CTTCGCTGAGGTCTGGATG SEQ ID NO.27 GGGATGAGTTGAGGAGTGGT SEQ ID NO.28 CsHCT CGACAATGGTGAAGGCGG SEQ ID NO.29 CGTCTTTTAGCACATGGGGTT SEQ ID NO.30 CsC3'H ATGGCTTTACCTCTGCTACCC SEQ ID NO.31 GCCGACGATTGGAAGTGG SEQ ID NO.32 CsCCoAOMT CAACAAACGGAGAAGGAGAA SEQ ID NO.33 GGTGGCTAGAAGAGAGTAACCA SEQ ID NO.34 CsF5H CGAAATAGCCCGACAAGTT SEQ ID NO.35 CGTAGTGAGCGAAAGCCAT SEQ ID NO.36 CsCOMT TCACGGCTCCCCAATCAC SEQ ID NO.37 TCTGCGGACCAGCAACCT SEQ ID NO.38 CsLAC-3 GTCTTGGCTCAAGGGTTTGGA SEQ ID NO.39 GGAGGCAAGGTAGTTGAAGGTG SEQ ID NO.40 CsLAC-4 TTCACTGGCATGGAGTAAGACAAC SEQ ID NO.41 TGATGGGCACTTCAGCATAAGG SEQ ID NO.42 CsLAC-6 TGAGCAGATTGTGCCATTCC SEQ ID NO.43 ATGCCACCAGAGCGTTCC SEQ ID NO.44 CsPER ATCTTGGCTTTCCTTGTTGC SEQ ID NO.45 ​ ​ ​ ​ ​ ​ ​

[0100] qRT-PCR results are as follows ​ Among them, eight structural genes (CsHTC, CsC3H, CsCCR, CsF5H, CsLAC-3, CsLAC-4, CsLAC-6, and CsPER) were significantly downregulated after silencing, indicating that CsWRKY41 is associated with lignin biosynthesis in tea plants.

[0101] As can be seen from the above embodiments, the present invention provides the application of the CsWRKY41 gene in regulating plant lignin synthesis. Overexpression of the CsWRKY41 gene can increase the lignin content of plants.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. CsWRKY41 The application of genes in regulating plant lignin synthesis is characterized by, CsWRKY41 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, overexpression CsWRKY41 Genes that promote lignin synthesis and increase the lignin content of plants; knockout or silencing. CsWRKY41 Genes inhibit lignin synthesis, thus reducing the lignin content of plants.

3. CsWRKY41 The application of gene-encoded proteins in regulating plant lignin synthesis, characterized by: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. The application according to any one of claims 1 to 3, characterized in that, The plants mentioned include Arabidopsis thaliana and tea tree.

5. A biomaterial for regulating the lignin content of plants, characterized in that, The biological materials include: CsWRKY41 Nucleic acid fragments of genes or expression cassettes, recombinant vectors, or engineered bacteria containing said nucleic acid fragments.

6. A method for improving tea arboresin synthesis, characterized in that, Includes the following steps: (1) Using tea plant genomic cDNA as a template, amplification CsWRKY41 Nucleotide fragments of a gene; (2) The super1300 vector was digested with SacI and XbaI, and the nucleotide fragment from step (1) was ligated into the digested super1300 vector to obtain CsWRKY41 -super1300 recombinant plasmid; (3) The recombinant plasmid CsWRKY41 - Super1300 was used to transform Agrobacterium GV3101 to obtain recombinant Agrobacterium; (4) The recombinant Agrobacterium was used to transfect tea plants to obtain transgenic tea plants.

7. The method according to claim 6, characterized in that, Amplification CsWRKY41 The primers for the gene are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The method according to claim 6, characterized in that, Recombinant plasmids were prepared using a freeze-thaw method. CsWRKY41 -super1300 transforms Agrobacterium GV3101.

9. The method according to claim 6, characterized in that, The method for transfecting tea plants with recombinant Agrobacterium is as follows: the recombinant Agrobacterium is prepared into a bacterial suspension and injected into the underside of the leaves.

10. The method according to claim 9, characterized in that, The OD of the bacterial suspension 600 The value is 0.8 to 1.0.

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