Use of substances that modulate the expression level of cd lac15 in the regulation of the degree of polymerization of lignin

Abstract

The application discloses application of a substance for regulating CdLAC15 expression level in regulating lignin polymerization degree. A first aspect of an embodiment of the application provides application of a substance for regulating CdLAC15 expression level and / or activity in regulating lignin polymerization degree of a target plant. It is found in the embodiment of the application that expression of a CdLAC15 gene of Camellia chingii can regulate the polymerization degree of lignin. When the gene is overexpressed in a wild-type Arabidopsis thaliana as a model plant, the lignin polymerization degree in tissues such as pericarp, leaf and stem of the Arabidopsis thaliana is obviously increased. Therefore, the lignin polymerization degree in parts such as pericarp of a plant can be regulated by a reagent for regulating the expression level of the CdLAC15 gene, and the reagent has important application value in breeding and the like.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, and in particular to the application of substances that regulate the expression level of CdLAC15 in regulating the degree of lignin polymerization. Background Technology

[0002] Camellia oleifera is a unique woody oil crop in my country, widely distributed in the middle and lower reaches of the Yangtze River, and has a very long cultivation history. Compared to Camellia oleifera (Camellia oleifera), the southernmost variety distributed in my country, the Gaozhou Camellia oleifera has advantages such as larger fruit, higher oil content in the kernel, and richer active ingredients, making it a more suitable native Camellia oleifera species for the Guangdong region. In production, the pericarp of Camellia oleifera is often discarded or burned as waste, its economic value far less than that of the kernel. However, the pericarp of Gaozhou Camellia oleifera is significantly thicker than that of Camellia oleifera, and this thicker pericarp consumes more energy during fruit growth, restricting the accumulation of kernel and oil. Therefore, it is urgent to analyze the regulatory mechanism of pericarp thickening in Gaozhou Camellia oleifera to guide breeding work on improving pericarp thickness.

[0003] Mature camellia oleifera pericarp exhibits lignofibrosis characteristics, with its main components including lignin, cellulose, and hemicellulose, of which lignin accounts for approximately 40%. During plant growth and development, lignin continuously accumulates in the plant cell walls, gradually lignifying the cells. In this process, lignin monomers are transformed into lignin polymers under the action of various enzymes, thus completing lignin synthesis. Based on the different monomer components, lignin can be classified into syringyl lignin (S-lignin), polymerized from syringylpropane monomers; guaiacyl lignin (G-lignin), polymerized from guaiacylpropane monomers; and hydroxy-phenyl lignin (H-lignin), polymerized from p-hydroxyphenylpropane monomers. The polymerization of lignin monomers is the key step in the final formation of lignin from camellia oleifera pericarp. Therefore, it is necessary to regulate the pericarp thickness by controlling the degree of polymerization of lignin in the pericarp of Gaozhou camellia oleifera. Summary of the Invention

[0004] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes the application of a substance that regulates the expression level of CdLAC15 in regulating the degree of lignin polymerization.

[0005] A first aspect of this application provides the use of substances that regulate the expression level and / or activity of CdLAC15 in regulating the degree of lignin polymerization in target plants.

[0006] In some embodiments of this application, substances that upregulate the expression level and / or activity of CdLAC15 increase the degree of lignin polymerization in the target plant, while substances that downregulate the expression level and / or activity of CdLAC15 decrease the degree of lignin polymerization in the target plant.

[0007] In some embodiments of this application, the substance that upregulates the expression level and / or activity of CdLAC15 includes any one of A1) to A5);

[0008] A1) Substances that specifically knock in or knock up the gene encoding CdLAC15;

[0009] A2) Substances that specifically increase the mRNA level of CdLAC15;

[0010] A3) Substances that specifically increase the expression level of CdLAC15 protein;

[0011] A4) Substances that specifically enhance the activity of CdLAC15 protein;

[0012] A5) includes any one of the carriers from A1) to A4).

[0013] In some embodiments of this application, the substance that downregulates CdLAC15 expression levels and / or activity includes any one of B1) to B5):

[0014] B1) Substances that specifically knock down or eliminate the gene encoding CdLAC15;

[0015] B2) Substances that specifically inhibit the mRNA level of CdLAC15;

[0016] B3) Substances that specifically inhibit the expression level of CdLAC15 protein;

[0017] B4) Substances that specifically inhibit the activity of CdLAC15 protein;

[0018] B5) includes any one of B1) to B4) as a carrier.

[0019] In some embodiments of this application, the material that specifically knocks in or knocks up the CdLAC15 encoding gene includes at least one of the CRISPR / Cas, ZFN, TALEN, and Cre-LoxP gene editing systems.

[0020] In some embodiments of this application, the substance that specifically knocks down or eliminates the gene encoding CdLAC15 includes at least one of the CRISPR / Cas, ZFN, TALEN, and Cre-LoxP gene editing systems.

[0021] In some embodiments of this application, substances that specifically inhibit the mRNA level of CdLAC15 include at least one of antisense nucleic acid sequences, siRNA, shRNA, dsRNA, and miRNA.

[0022] In some embodiments of this application, the carrier is any one of an inorganic carrier, an organic carrier, or an inorganic-organic composite carrier.

[0023] In some embodiments of this application, the vector is at least one of lentivirus, adenovirus, and adeno-associated virus.

[0024] In some embodiments of this application, the target plant includes angiosperms.

[0025] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0026] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0027] In some embodiments of this application, the target plant includes at least one of plants from the order Ericales and the order Brassicales.

[0028] In some embodiments of this application, the target plant includes at least one plant from the Theaceae family and the Brassicaceae family.

[0029] In some embodiments of this application, the target plant includes at least one of the Camellia genus and Arabidopsis genus.

[0030] In some embodiments of this application, the target plant includes at least one of Camellia oleifera, Camellia spp., and Arabidopsis thaliana.

[0031] In some embodiments of this application, the substance regulates the degree of lignin polymerization in at least one tissue of the target plant, including the pericarp, leaves, and stem.

[0032] According to the application of the embodiments of this application, at least the following beneficial effects are achieved:

[0033] In this application, it was discovered that the expression of the CdLAC15 gene in *Camellia oleifera* can regulate the degree of lignin polymerization. When this gene was overexpressed in wild-type *Arabidopsis thaliana*, a model plant, the degree of lignin polymerization increased in the pericarp, leaves, and stems of *Arabidopsis thaliana* without significant differences in total lignin content. Therefore, the degree of lignin polymerization in plant parts such as the pericarp can be regulated by adjusting the expression level of the CdLAC15 gene, which has important application value in breeding and other fields.

[0034] A second aspect of this application provides a method for adjusting the pericarp thickness of a target plant, comprising regulating the expression level and / or activity of CdLAC15 in the target plant.

[0035] In some embodiments of this application, the expression level and / or activity of CdLAC15 are upregulated or downregulated in the target plant. Substances that upregulate CdLAC15 expression level and / or activity can increase the pericarp thickness of the target plant, while substances that downregulate CdLAC15 expression level and / or activity can decrease the pericarp thickness of the target plant.

[0036] In some embodiments of this application, the substance that upregulates the expression level and / or activity of CdLAC15 includes any one of A1) to A5);

[0037] A1) Substances that specifically knock in or knock up the gene encoding CdLAC15;

[0038] A2) Substances that specifically increase the mRNA level of CdLAC15;

[0039] A3) Substances that specifically increase the expression level of CdLAC15 protein;

[0040] A4) Substances that specifically enhance the activity of CdLAC15 protein;

[0041] A5) includes any one of the carriers from A1) to A4).

[0042] In some embodiments of this application, the substance that downregulates CdLAC15 expression levels and / or activity includes any one of B1) to B5):

[0043] B1) Substances that specifically knock down or eliminate the gene encoding CdLAC15;

[0044] B2) Substances that specifically inhibit the mRNA level of CdLAC15;

[0045] B3) Substances that specifically inhibit the expression level of CdLAC15 protein;

[0046] B4) Substances that specifically inhibit the activity of CdLAC15 protein;

[0047] B5) includes any one of B1) to B4) as a carrier.

[0048] In some embodiments of this application, the material that specifically knocks in or knocks up the CdLAC15 encoding gene includes at least one of the CRISPR / Cas, ZFN, TALEN, and Cre-LoxP gene editing systems.

[0049] In some embodiments of this application, the substance that specifically knocks down or eliminates the gene encoding CdLAC15 includes at least one of the CRISPR / Cas, ZFN, TALEN, and Cre-LoxP gene editing systems.

[0050] In some embodiments of this application, substances that specifically inhibit the mRNA level of CdLAC15 include at least one of antisense nucleic acid sequences, siRNA, shRNA, dsRNA, and miRNA.

[0051] In some embodiments of this application, substances that specifically inhibit the activity of the CdLAC15 protein include CdLAC15 antibodies.

[0052] In some embodiments of this application, the carrier is any one of an inorganic carrier, an organic carrier, or an inorganic-organic composite carrier.

[0053] In some embodiments of this application, the vector is at least one of lentivirus, adenovirus, and adeno-associated virus.

[0054] In some embodiments of this application, the target plant includes angiosperms.

[0055] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0056] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0057] In some embodiments of this application, the target plant includes at least one of plants from the order Ericales and the order Brassicales.

[0058] In some embodiments of this application, the target plant includes at least one plant from the Theaceae family and the Brassicaceae family.

[0059] In some embodiments of this application, the target plant includes at least one of the Camellia genus and Arabidopsis genus.

[0060] In some embodiments of this application, the target plant includes at least one of Camellia oleifera, Camellia spp., and Arabidopsis thaliana.

[0061] A third aspect of this application also provides a plant breeding method, including adjusting the pericarp thickness of a target plant according to the aforementioned method.

[0062] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0063] Figure 1 This is a comparison of the pericarps of Gaozhou Camellia oleifera and Camellia oleifera Abel. at different stages. The first and third rows are photos of the two sides of the pericarp of Gaozhou Camellia oleifera G255 after the fruit is cut open, while the second and fourth rows are photos of the two sides of the pericarp of Camellia oleifera YTZ13 after the fruit is cut open.

[0064] Figure 2 This table shows the total lignin content of the pericarp of *Camellia oleifera* var. *gaozhouensis* and *Camellia oleifera* var. *small-fruited* at different developmental stages. Co represents *Camellia oleifera* var. *small-fruited*, and Cd represents *Camellia oleifera* var. *gaozhouensis*. From left to right, the table compares the content at the ripening, fruitlet, and enlargement stages.

[0065] Figure 3 This represents the relative expression levels of the LAC15 gene in *Camellia oleifera* and *Camellia oleifera var. chinensis* during the maturation period.

[0066] Figure 4 This is a peak diagram of the GPC polymerization degree of lignin in the pericarps of Camellia oleifera and Camellia spp. from Gaozhou.

[0067] Figure 5 This is a comparison of the degree of polymerization of lignin in the pericarp of Camellia oleifera from Gaozhou and Camellia oleifera from small fruit. From left to right, they are the number average molecular weight (Mn) of lignin polymers of different Camellia oleifera at different times, the weight average molecular weight (Mw) of polymerized lignin polymers of different Camellia oleifera at different times, and the degree of polymerization of lignin (Mw / Mn) of different Camellia oleifera at different times.

[0068] Figure 6 This is an electrophoresis image of the CdLAC15 gene clone. The left lane M represents the marker, and the two lanes on the right represent two replicates of LAC15-2300.

[0069] Figure 7 This is a graph showing the results of CdLAC15-2300-CpYGFP Escherichia coli culture PCR detection. M represents the marker, lane 1 is the negative control, and lanes 2-8 are the electrophoresis results of LAC15-2300-CpYGFP positive transformants, with the target gene fragment size being 1710 bp.

[0070] Figure 8 This is a diagram showing the process of the CdLAC15-2300-CpYGFP transformation experiment. In the diagram, A represents the results of the T1 screening using kanamycin T0, B represents the results of positive seedling selection, C represents the results of transplanting positive seedlings, and D represents the identification of positive seedlings and seed harvesting.

[0071] Figure 9This is a graph showing the PCR identification results of the T1 positive strain of the CdLAC15-2300-CpYGFP gene. M represents the marker, lanes 1-2 are the negative and positive controls respectively, and lanes 3-11 are LAC15-2300-CpYGFP positive seedlings. The target band size is 1164 bp.

[0072] Figure 10 This is a comparison of the expression levels of the transgenic CdLAC15 gene in Arabidopsis thaliana T3 generation lines and wild-type lines, as detected by qRT-PCR.

[0073] Figure 11 This is a phenotypic comparison of the CdLAC15 gene in wild-type, mutant, and overexpressed transgenic Arabidopsis thaliana.

[0074] Figure 12 The total lignin content in the mature pericarp of wild-type Arabidopsis thaliana WT and Arabidopsis thaliana OE-LAC15 overexpressing was not significantly different (P=0.16).

[0075] Figure 13 This is a comparison of the degree of lignin polymerization at maturity between wild-type Arabidopsis thaliana WT and Arabidopsis thaliana OE-LAC15 overexpressing the species. Detailed Implementation

[0076] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0077] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0078] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number, and "approximately" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the stated number. The use of "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0079] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0080] The camellia oleifera materials selected in the following examples are Gaozhou camellia oleifera G255 and small-fruited camellia oleifera YTZ13. After the fruits of the two varieties mature, the thickness of the pericarp is significantly different. Figure 1 The fruit was planted in Xiaokeng State-owned Forest Farm in Shaoguan City and managed under normal forest land management practices. Sampling was performed by selecting three uniformly growing, fresh, and undamaged fruits, separating the pericarp from the kernel on-site, chopping the pericarp, placing it into centrifuge tubes, and immediately immersing them in liquid nitrogen for temporary storage.

[0081] The reagents and consumables used in the embodiments of this application are as follows:

[0082] Prime Script TM RT reverse transcription kit (RR037A), KpNI (1618A), SaII (1636A), and DL5000 DNA Marker (3427A) were purchased from Takara. The Hieff™ qPCR kit, SYBR Green MasterMix (low Rox), and 2× Hieff were also included. Gold PCR Master Mix high-fidelity enzyme premix was purchased from Yisheng Biotechnology Co., Ltd., 2×Easy PCR Super Mix (+dye) (AS111-11) was purchased from Beijing TransGen Biotech Co., Ltd.; gel extraction kit (DP209) and plasmid miniprep kit (DP103) were purchased from Beijing Tiangen Biotech Co., Ltd.; competent DH5α cells (DL1001) were purchased from Weidi Biotechnology Co., Ltd.; and recombinant ligation reagent Novo was used. The plus One step PCR Cloning Kit (NR005-01A) was purchased from Anolun (Beijing) Biotechnology Co., Ltd., and kanamycin (B25656) and rifampin (B25308) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0083] Other drugs: Agarose Regula (5260, TaKaRa), Star Stain Red nucleic acid dye 10,000× (E109-01, GenStar), ethanol (ACS:64-17-5), chloroform (ACS:67-66-3), methanol (ACS:67-56-1), acetone (ACS:67-64-1), acetic acid (ACS:64-19-7), sodium acetate (ACS:127-09-3), sodium azide (ACS:26628-22-8), α-amylase (ACS:9000-85-5), pullulanase (ACS:9075-68-7).

[0084] Culture medium: tryptone (LP0042, OXOID), yeast extract (LP0021, OXOID) and NaCl (Shanghai test).

[0085] Main instruments: Bio-Rad C1000 PCR amplification instrument, ViiA7 (Thermo Fisher Scientific) real-time PCR instrument, DYCP-32B agarose gel electrophoresis system (medium size) (Beijing Liuyi, Beijing), Alpha Imager HP (Protein Simple, USA) gel imaging system.

[0086] Example 1: Data collection on the fruits of Camellia oleifera and Camellia oleifera var. ...

[0087] Camellia oleifera and Camellia spp. from Gaozhou were collected, sectioned, and photographed at six different time points. The results are as follows: Figure 1 As shown, the corresponding dates are: March 30, 2021 (denoted as A), May 30, 2021 (B), July 1, 2021 (C), July 30, 2021 (D), September 12, 2021 (E), and October 10, 2021 (F).

[0088] Example 2: Determination of total lignin content in pericarps of *Camellia oleifera* and *Camellia oleifera var. chinensis*.

[0089] 1. Extraction and separation of cell walls

[0090] Weigh an appropriate amount of dried plant powder; add 1 mL of 70% ethanol, vortex thoroughly, centrifuge at 10000 rpm for 10 min, and discard the supernatant; add 1 mL of chloroform / methanol (1:1 v / v) solution, vortex thoroughly, centrifuge at 10000 rpm for 10 min, and discard the supernatant; add 1 mL of acetone, vortex thoroughly, and vacuum dry the acetone; add 1.5 mL of 0.1 M pH 5.0 sodium acetate buffer, and heat at 80 °C for 20 min; add 10 μL of 0.01% sodium azide, amylase, and pullulanase, and incubate at 37 °C overnight; heat at 100 °C to terminate the reaction, centrifuge at 10000 rpm for 10 min, discard the supernatant, and wash the precipitate with distilled water; add 1 mL of acetone, vortex thoroughly, and vacuum dry the acetone; dry the residue at 40 °C.

[0091] 2. Dissociation and determination of total lignin

[0092] Weigh approximately 2 mg of cell wall material and add 100 μL of acetyl bromide (25%) solution. Heat at 37°C for 6 h. Add 100 μL of 2M sodium hydroxide and 100 μL of 0.5M hydroxylamine hydrochloride, vortex to mix, and bring the volume to 2 mL with acetic acid. Centrifuge at 10000 rpm for 10 min, then aspirate the supernatant and measure the absorbance at 280 nm. Calculate the corresponding total lignin content (%) based on the standard curve.

[0093]

[0094] ABS: Absorbance; Coeff: Absorption coefficient, in L / (g·cm); Weight: Mass, in mg; 0.539cm: Optical path length.

[0095] The percentage lignin content value multiplied by 10 is the lignin content (μg / mg cell wall material).

[0096] Experimental results are as follows Figure 2 As shown.

[0097] Example 3: Determination of LAC15 gene expression level in pericarp tissue of *Camellia oleifera* and *Camellia oleifera var. chinensis* at maturity.

[0098] The pericarp thickness of *Camellia oleifera* G255 and *Camellia oleifera* YTZ13 differed significantly after fruit ripening. Lignin synthesis is closely related to pericarp thickening, and key genes involved in lignin synthesis showed higher expression levels in thicker pericarps. In Example 4 of this invention, *Camellia oleifera* G255 with thick pericarps and *Camellia oleifera* YTZ13 with thin pericarps were selected. RNA was extracted from the mature pericarps, and its expression level was detected using qRT-PCR. The detection results are as follows: Figure 3As shown in the figure, the results indicate that the expression level of this gene, detected by qRT-PCR, was significantly higher in the pericarp of mature Camellia oleifera from Gaozhou than in Camellia oleifera from small-fruited varieties. Therefore, this gene regulating lignin synthesis may be related to the formation of pericarp thickness in Camellia oleifera.

[0099] The specific steps for analyzing the differential expression levels of the LAC15 gene at the maturity stages of two materials, Camellia oleifera and Camellia simonii, are as follows:

[0100] 1. Grinding sample

[0101] Take about 100 mg of sample into a frozen mortar, add liquid nitrogen and grind into powder, transfer to a 1.5 mL centrifuge tube containing 1 mL of RNAisoPlus, vortex to mix, and let stand at room temperature for about 5 min.

[0102] 2. RNA extraction

[0103] 1) Add 0.2 mL of chloroform to each 1 mL of RNAiso Plus, vortex to mix for 15 seconds, let stand at room temperature for about 3 minutes, then centrifuge at 12000 rpm for 15 minutes at 4°C.

[0104] 2) Precipitation: Transfer the aqueous phase to a new 1.5 mL centrifuge tube, add 0.5 mL of isopropanol to each 1 mL of RNAiso Plus, mix well, let stand at room temperature for 10 min, then centrifuge at 12000 rpm for 10 min at 4 °C.

[0105] 3) Washing: Discard the supernatant, add 1 mL of 75% ethanol, mix well, and centrifuge at 7500 rpm for 5 min at 4°C.

[0106] 4) Dissolve: Discard the supernatant, air-dry the RNA precipitate for about 5 minutes, and add an appropriate amount of DEPC-treated water to dissolve the RNA precipitate.

[0107] 5) Determine concentration and purity: Determine RNA concentration using a spectrophotometer.

[0108] 3. Synthesis of reverse-transcribed cDNA

[0109] Prepare the RT reaction solution on ice according to the components in Table 1 below. To ensure the accuracy of the reaction solution preparation and reduce errors caused by aliquoting, prepare the reaction solution by a slightly larger volume than the actual amount needed, and finally add the RNA sample.

[0110]

[0111]

[0112] Perform reverse transcription at 37℃ for 15 minutes and 85℃ for 5 seconds, then store at -20℃ for later use.

[0113] 4. Quantitative Real-Time PCR Detection

[0114] 1) Using the NCBI online website, specific primers for the CdLAC15 gene were designed, and the Camellia oleifera GAPDH gene was used as an internal reference gene.

[0115] The primers are as follows:

[0116] LAC15-RT-F:CCACCGCTATTTCCACACCT(SEQ ID NO.1);

[0117] LAC15-RT-R: AGCATGTACGTTGGGATGG (SEQ ID NO. 2);

[0118] GAPDH-RT-F: AAGGAGGCTTCGGAGGGTAG (SEQ ID NO. 3);

[0119] GAPDH-RT-R: ATGCTGGACCTGCAATCACC (SEQ ID NO. 4).

[0120] 2) Preparation of the real-time PCR system:

[0121] Melt the mixture at 4°C, gently invert to mix, and briefly centrifuge. Prepare the reaction solution on ice according to the table below:

[0122]

[0123] Briefly centrifuge the reaction tube to ensure all reaction solution is at the bottom of the well. A three-step reaction procedure is used: 95℃ for 5 min; 95℃ for 10 sec, 60℃ for 30 sec, 40 cycles; after the above reaction is completed, melting curve analysis is performed from 60℃ to 95℃. Export the data and use 2 -ΔΔCt The method calculates the data, and Tbtools analyzes and displays the results.

[0124] Example 4: Determination of the degree of polymerization of lignin in the pericarp of Camellia oleifera and Camellia spp.

[0125] The degree of polymerization of lignin in samples from different growth stages of *Camellia oleifera* and *Camellia oleifera var. chinensis* was determined by gel permeation chromatography (GPC) using a Waters Breeze (Waters, America) system equipped with a Waters 2414 detector. The eluent was HPLC-grade THF, and the flow rate was 1 mL / min. -1 The degree of polymerization was calculated based on Mw / Mn, and the test results are as follows: Figure 4 and Figure 5 As shown.

[0126] Example 5: Bioinformatics analysis of the CdLAC15 gene in Camellia oleifera

[0127] The CDS sequence and encoded amino acid sequence of the LAC15 gene were obtained from transcriptome data. Its open reading frame is 1713 bp and encodes 570 amino acids. The gene was named CdLAC15 and its function was studied.

[0128] The open reading frame sequence of CdLAC15 is as follows:

[0129]

[0130] The amino acid sequence encoded by CdLAC15 is as follows:

[0131] MWFGKKSLILCLLGYLLFDGIGIVHCQAWIRRHRFVVKEAPYTRLCSTKKILTVNGKFPGPTLYAHKEETLIVDVYNRGKYNITIHWHGVKMPRYPWSDGPEYITQCAIKPGGKFSQKVICSAEEGTLWWHAHSDWSRATVHGAIIVYPKHGTVYPFPKPYAEVPIVLGEWWKQDVM KVLEEFVRTGGTPNDSDAFTINGQPGDLYPCSKPGTFKLTVEHGKTYLLRIINAAMNEILFLSIAKHKLTVVGADASYTKPLKTKYITISPGQTIDALFHANRHHGRYYIAARAYSAGNIPFDNTTTTAIIQYAGKKSTATSPPLFPHLPYHNDTRAMVNFTGSLRSLGSKDHPVNVPLKITTPMISTVLLNTLPCHP NRTCLGPNGTRLAASMNNLSWVNPSLDILQAYYYHIKGVFGDRFPNFPPYVFNFTADYFPMELEIPKKTRQVKILKYNSTVEVVFQGTNLVAGIDHPIHLHGHSFYVVGWGFGNYDKFKDPLTYNLVDPPLLNTVAVPRNGWITIRFKANNPGVWFLHCHFERHLMWGMETVFIVKNGKHHNATMLPPPPDMPPC(SEQ ID NO.6).

[0132] Example 6: Cloning of the CdLAC15 gene and construction of an overexpression vector

[0133] 1. Design of gene primers

[0134] Primers were designed using the NCBI online website, and PCR was used to amplify cDNA from the pericarp of *Camellia oleifera* var. *gaozhouensis*. The cDNA used was from samples of mature *Camellia oleifera* pericarp collected in the previous steps. To amplify the full-length coding region of the gene and add specific restriction enzyme sites, primers containing suitable restriction enzyme sites were designed at the start and stop codons based on the CDS sequence of CdLAC15. The vector used was PCAMBIA2300-CpYGFP, and the restriction enzyme sites used were KpNI and SaII double digestion. The upstream adapter sequence added to the PCAMBIA2300-CpYGFP backbone primers was: GAACACGGGGGACGAGCTCGGTACC (KpNI); the downstream adapter sequence added was: TTTTGAAGGTTGTCATGGTGTCGAC (SaII).

[0135] The primer sequences for the CdLAC15 vector are as follows:

[0136] LAC15-2300-CpYGFP-F: GAACACGGGGGACGAGCTCGGTACCATGTGGTTTGGGAAGAAGAGTTTGATC (SEQ ID NO. 7);

[0137] LAC15-2300-CpYGFP-R:TTTTGAAGGTTGTCATGGTGTCGACACAAGGGGGCATAT CTGGTGG (SEQ ID NO. 8).

[0138] 2. PCR reaction system and procedure for gene cloning

[0139] 1) PCR reaction system. Add all components, including primers and template, according to the table below, vortex to mix, and centrifuge briefly.

[0140]

[0141] 2) PCR reaction program: 98℃ for 3 min; 98℃ for 10 s, 68℃ for 1 min, 35 cycles; 72℃ for 5 min.

[0142] 3. Detection of PCR products

[0143] Take 2 μL of PCR product, add 3 μL of 6× Loading Buffer, mix well, and detect by 1% agarose gel electrophoresis. The results are as follows: Figure 6 As shown, the extended strip size is approximately 1713bp.

[0144] 4. Gel recovery of PCR products

[0145] The product was recovered and purified using a gel extraction kit, and the steps are as follows:

[0146] 1) After DNA electrophoresis, cut the single target DNA band from the agarose gel (remove as much excess as possible) and place it in a clean centrifuge tube, then weigh it.

[0147] 2) First, add 500 μL of equilibration solution BL to the empty adsorption column CA2 (place the adsorption column in the collection tube), centrifuge at 12000 rpm (~13400×g) for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0148] 3) Add an equal volume of PN solution to the gel block (if the gel weight is 0.1g, its volume can be considered as 100μL, then add 100μL of PN solution), and place in a 50°C water bath. During this time, gently rotate the centrifuge tube continuously to ensure that the gel block is fully dissolved. If there is still undissolved gel block, continue to let it stand for a few more minutes or add more sol solution until the gel block is completely dissolved (if the gel block is too large, it can be cut into small pieces beforehand).

[0149] 4) Add the solution obtained in the previous step to an adsorption column CA2 (place the adsorption column in the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm (~13400×g) for 30~60 s, discard the waste liquid in the collection tube, and place the adsorption column CA2 into the collection tube.

[0150] 5) Add 600 μL of washing buffer PW to the adsorption column CA2 (check that anhydrous ethanol has been added before use), let stand for 2-5 min, centrifuge at 12000 rpm (~13400×g) for 30-60 s, discard the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube.

[0151] 6) Repeat step 5.

[0152] 7) Place the CA2 adsorption column back into the collection tube and centrifuge at 12000 rpm (~13400×g) for 2 min to remove as much of the washing solution as possible. Place the CA2 adsorption column at room temperature for several minutes to dry completely to prevent residual washing solution from affecting the next step of the experiment.

[0153] 8) Place the CA2 adsorption column into a clean centrifuge tube, and add 30 μL of elution buffer EB to the center of the adsorption membrane. Incubate at room temperature for 2 min. Centrifuge at 12000 rpm (~13400×g) for 2 min to collect the DNA solution into a centrifuge tube. Store the product at -20℃.

[0154] Example 7: Construction of the LAC15-2300-CpYGFP plant expression vector

[0155] 1. Add the components to the backbone vector enzyme digestion system according to the table below, vortex to mix, and then centrifuge briefly;

[0156]

[0157]

[0158] Incubation procedure: 37℃ for 20 min; 80℃ for 15 min. After the enzyme digestion reaction is complete, electrophoresis is performed, and large fragments are recovered from the gel.

[0159] 2. Homologous recombination system:

[0160] 1) Add each component according to the table below, shake to mix, and centrifuge briefly;

[0161]

[0162] 2) Incubation procedure: 50℃ for 20 minutes.

[0163] 3) After the homologous recombination reaction is completed, the reaction solution is added to 100 μL of competent cells for heat shock transformation.

[0164] 4) Spread on kanamycin solid LB agar plates and incubate overnight at 37°C.

[0165] 3. Detection and sequencing of positive Escherichia coli bacterial culture clones by PCR

[0166] 1) Pick white colonies from the transformation plate and place them in liquid LB medium containing Kan, and incubate at 37°C in a shaker for 8 hours.

[0167] 2) For colony PCR, add each component according to the table below, vortex to mix, and then centrifuge briefly;

[0168]

[0169] 3) The PCR amplification procedure is as follows:

[0170] 95℃ for 3 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ for 5 min.

[0171] 4) After the PCR reaction is complete, electrophoresis is used to verify positive clones, and the results are as follows: Figure 7 As shown.

[0172] 5) Sequencing of the verified single clones, with three replicates for each sequence.

[0173] Example 8: Agrobacterium-mediated transformation of Arabidopsis thaliana

[0174] 1. Arabidopsis thaliana culture

[0175] Colombian wild-type Arabidopsis thaliana, transplanted from 1 / 2MS plates, was planted in an artificial climate chamber. Once it reached its peak flowering period, the formed pods were removed, and the moisture level of the nutrient soil around the roots of the Arabidopsis thaliana was maintained.

[0176] 2. Agrobacterium-mediated transformation of Arabidopsis thaliana using the flower-dipping method

[0177] 1) Activate Agrobacterium.

[0178] 2) Inoculate 100 mL of LB medium at a ratio of 1:50 and incubate at 28°C and 200 rpm until OD600 = 1.2–1.6.

[0179] 3) Centrifuge at 4000 rpm for 15 min and collect the bacterial cells.

[0180] 4) Resuspend in osmotic buffer (1 / 2 MS medium + 5% sucrose, sterilized at 121℃ for 20 min, add Silwett L-77 to a final concentration of 0.03%, and shake to mix). Use the resuspended osmotic buffer as a control and adjust OD600 to 0.4-0.6.

[0181] 5) Place Arabidopsis thaliana upside down on a container of appropriate size containing osmotic buffer and incubate for 30 seconds. Cover the entire tray with plastic film, leaving appropriate ventilation holes, and incubate under low light. After 24 hours, remove the film and continue incubating at room temperature.

[0182] 6) The genetically modified T0 generation seeds mature in about 30 days and are then harvested.

[0183] Example 9: Phenotypic identification of transgenic Arabidopsis plants

[0184] 1) Soak the harvested genetically modified seeds in Triton X-100 containing 0.2% for 10 minutes.

[0185] 2) Disinfect the surface with 10% sodium hypochlorite for 10-12 minutes.

[0186] 3) Rinse with sterile water 5-6 times, each time for about 2 minutes.

[0187] 4) Place the transgenic seeds on an MS plate containing 50 mg / L Kan and incubate in the dark at 4°C for 3 days.

[0188] 5) Seeds selected for resistance screening were cultured at 22℃ with a 16h light / 8h dark light cycle.

[0189] 6) Two weeks later, positive plants grew well on antibiotic plates, while negative plants either failed to germinate or died shortly afterward. The well-grown Arabidopsis thaliana plants from the plates were planted in the substrate. When the Arabidopsis thaliana had 7-8 true leaves, DNA was extracted from the lowest leaf for PCR detection. The results are referenced below. Figure 9The primers used for detection were LAC15-2300-CpYGFP-F and LAC15-2300-CpYGFP-R.

[0190] 7) Positive lines must be tested for each generation of plants until the T3 generation is obtained, yielding homozygous transgenic Arabidopsis lines. The T3 generation lines are then subjected to qRT-PCR detection, with the following quantitative verification process:

[0191] RNA was extracted and reverse transcribed into cDNA. Primers for CdLAC15 fluorescence quantitative quantification were used.

[0192] LAC15-RT-F:CCACCGCTATTTCCACACCT(SEQ ID NO.1);

[0193] LAC15-RT-R: AGCATGTACGTTGGGATGG (SEQ ID NO. 2).

[0194] A qRT-PCR reaction system was prepared on ice for quantitative real-time PCR. Results are as follows: Figure 10 As shown in the figure, the quantitative fluorescence verification results show that the transcription level of the CdLAC15 gene in the transgenic lines is significantly higher than that in the non-transgenic Arabidopsis.

[0195] 8) Transgenic T3 generation plants and non-transgenic plants were sterilized and cultured on 1 / 2 MS medium. After vernalization at 4°C for two days, Arabidopsis seedlings, which developed true leaves in about 10 days, were transplanted into small flowerpots for further cultivation under the same conditions. Results were as follows: Figure 11 As shown, phenotypic observation revealed morphological differences between the overexpressing transgenic Arabidopsis and the wild-type Arabidopsis.

[0196] Example 10: Determination of total lignin content and degree of lignin polymerization in Arabidopsis pericarp

[0197] The method is the same as in Example 4, and the detection results are as follows: Figure 12 and Figure 13 As shown, the total lignin content in the pericarp of wild-type Arabidopsis thaliana was 252.98±15.41 μg / mL, while the total lignin content in Arabidopsis thaliana OE_LAC15 overexpressing the LAC15 gene was 277.50±13.20 μg / mL. There was no significant difference in total lignin content between the two (P=0.16), but the degree of lignin polymerization increased by approximately two times, making the difference statistically significant. These results indicate that overexpression of CdLAC15 can increase the degree of lignin polymerization without affecting the total lignin content.

[0198] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. Furthermore, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

Claims

1. Application of CdLAC15 overexpression vector in increasing the degree of lignin polymerization in Arabidopsis pericarp without affecting the total lignin content, wherein the degree of lignin polymerization is calculated based on the weight-average molecular weight / number-average molecular weight of the lignin polymer, and the amino acid sequence encoded by CdLAC15 in the CdLAC15 overexpression vector is shown in SEQ ID NO.

6.

2. A method for increasing the degree of lignin polymerization in Arabidopsis pericarp without affecting the total lignin content, characterized in that, This includes using a CdLAC15 overexpression vector to upregulate the expression level and / or activity of CdLAC15 in the Arabidopsis thaliana, wherein the amino acid sequence encoded by CdLAC15 in the CdLAC15 overexpression vector is shown in SEQ ID NO.

6.

3. A plant breeding method, characterized in that, This includes the method according to claim 2, which increases the degree of lignin polymerization in Arabidopsis pericarp without affecting the total lignin content.

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