A transcription factor CgWRKY40 involved in regulating the synthesis of lignin in Pomelo

Abstract

The present invention discloses a transcription factor CgWRKY40 involved in regulating the synthesis of lignin in Pomelo spp. and its application, belonging to the fields of molecular biology and fruit and vegetable preservation technology. The present invention finds that the CgWRKY40 gene is involved in regulating the synthesis and deposition of lignin in the juice cells of Pomelo spp. The discovery of this gene supplements and improves the lignin metabolism regulation mechanism of WRKY transcriptional regulation in Pomelo spp., provides a theoretical basis for analyzing the occurrence mechanism of Pomelo spp. granulation, and provides candidate genes for the molecular breeding industry to delay or inhibit the granulation of Pomelo spp. after harvest and improve fruit quality through gene editing.

Description

Technical Field

[0001] The invention relates to the technical fields of molecular biology and fruit and vegetable preservation, and in particular to a transcription factor CgWRKY40 involved in regulating the synthesis of Pomelo quinquefolia lignin and an application thereof. Background Art

[0002] Citrus grandis L.cv. Majiayou is a famous and high-quality pomelo variety in Jiangxi Province. It is native to Danan Town, Guangfeng District, Shangrao City. It is deeply loved by consumers for its beautiful shape, red flesh, high juice yield, rich lycopene, delicate taste, and moderate sweetness and sourness. At the same time, because of its thick peel, it can be stored for 2 to 5 months at room temperature, and is praised as "natural canned fruit". Pomelo is a non-respiratory climacteric fruit with no obvious ripening phenomenon. Compared with the wide-skinned citrus and orange fruits in the genus Citrus, the respiratory metabolism is slower. Since the fruit is picked from the tree, photosynthesis stagnates, and its edible quality will gradually decline with the extension of post-harvest storage time, and a series of adverse physiological disorders will occur, among which juice cell granulation is the most prominent. Its main characteristics are rough and waxy flesh mediated by the thickening of secondary cell walls, dry and hard juice cells, wrinkled or curved, turbid and opaque, which seriously affects its commercial value. Therefore, analyzing the granulation mechanism of Majiayou is particularly important for developing effective preservation technology.

[0003] In the study of post-harvest storage and preservation of pomelo fruits, a physiological disease that causes the deterioration of the quality of Majia pomelo after harvest was found - juice cell granulation / lignification, which is mainly manifested by the formation of secondary cell walls, seriously reducing the storage performance and commercial value of the fruit. The disease was first reported by Bartholomew et al. in 1934, which is completely different from the cell wall metabolism revealed by the softening process of horticultural products after harvest. The main reason for the granulation of juice cells in Majia pomelo after harvest is the accumulation of lignin and cellulose. The accumulation of lignin in juice cells (108.8%) is significantly higher than the accumulation of cellulose (31.9%), indicating that lignification is the main cause of the granulation of juice cells in Majia pomelo after harvest (Chen et al., 2021). Lignin synthesis is generated by phenylalanine through deamination, hydroxylation, methylation and redox reactions to generate three lignin monomers: p-hydroxyphenyl lignin (H-type lignin), guaiacyl lignin (G-type lignin), and syringyl lignin (S-type lignin). The above three lignin monomers are then dehydrogenated to generate phenol free radicals under the catalysis of peroxidase or laccase, and then coupled to form dimers. The dimers are polymerized to finally form lignin. At present, the known research on the post-harvest juice sac granulation of pomelo fruits mainly focuses on physiology and anatomy. The occurrence of pomelo juice sac granulation has a very significant correlation with lignin synthesis, transport, and accumulation. Clarifying the key genes for lignin synthesis in pomelo fruits and their associated mechanisms of action is an important breakthrough in studying how to inhibit or slow down the post-harvest juice sac granulation process of Majiayou.

[0004] WRKY transcription factors are one of the largest transcription factor families commonly found in plants, with typical DNA binding, transcriptional regulation, nuclear localization signals, and oligomerization sites. The biggest difference between WRKY transcription factors and other transcription factor families is that their conservative structure consists of a 60-amino acid stretch (the N-terminus is the heptapeptide amino acid residue WRKYGQK, and the C-terminus is a C2H2 / C2HC type zinc finger protein), which can specifically bind to the W-BOX (C / TTGACT / C) sequence on the promoter of the downstream structural gene, target the expression of the target gene, and participate in regulating the response of horticultural crops to adverse stress (Javed et al., 2023). According to the number of WRKY domains in the protein sequence of plant WRKY transcription factors, they can be divided into three groups: group I, II, and III. Among them, those with two WRKY conserved domains belong to group I, while sequences with only one conserved region are classified as group II or group III. Members of groups I and II have a C2-H2 (C-X4-5-C-X22-23-H-X1-H) type zinc finger structure, where X can be any amino acid, while group III WRKY proteins contain a C2-HC (C-X7-C-X23-HXC) type zinc finger structure. WRKY transcription factors in plants play an important regulatory role in responding to stress at the transcriptional level, and have become a hot topic of concern for scholars at home and abroad (Huang Xing et al., 2019; Saha et al., 2024). At present, the plant lignin biosynthesis pathway network has been basically clarified, but there are still few reports on transcriptional regulation at the molecular level. So far, there has been no report on the transcription factor CgWRKY40 promoting lignin synthesis in Majiayu juice cells. Summary of the invention

[0005] The purpose of the present invention is to provide a transcription factor CgWRKY40 involved in regulating the synthesis of lignin in Citrus grandis and an application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the application of CgWRKY40 in regulating lignin synthesis, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] The second technical solution of the present invention is the application of CgWRKY40 in regulating the lignin synthesis of Pomelo fruit, and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] The third technical solution of the present invention is the use of a recombinant vector, an expression cassette, and a transgenic strain containing CgWRKY40 in regulating the lignin synthesis of Pomelo fruit.

[0010] A fourth technical solution of the present invention is the use of the protein encoded by CgWRKY40 in regulating lignin synthesis, and its amino acid sequence is shown in SEQ ID NO.2.

[0011] A fifth technical solution of the present invention is the use of the protein encoded by CgWRKY40 in regulating the lignin synthesis of Pomelo fruit, and its amino acid sequence is shown in SEQ ID NO.2.

[0012] The sixth technical solution of the present invention is a method for improving the taste of Majiayou fruit, silencing or knocking out CgWRKY40 in Majiayou plants, and inhibiting the synthesis of lignin in Majiayou fruit.

[0013] The seventh technical solution of the present invention is the application of CgWRKY40 in improving the quality of pomelo fruit.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention identifies a transcription factor CgWRKY40 that plays an important regulatory role in the post-harvest juice sac granulation of Majiayu through transcriptome sequencing and co-expression network analysis, and finds that its expression level in granulated juice sac fruit is significantly higher than that in normal ungranulated fruit. Subcellular localization finds that the transcription factor is consistent with the nuclear marker localization. The overexpression vector is transiently injected into the juice sac of Majiayu by Agrobacterium infection, and its lignin content is significantly increased. At the same time, the autofluorescence signal in the pyrogallol staining and paraffin sections is enhanced, the cell wall is thickened, and the symptoms of juice sac granulation are aggravated. This proves that the CgWRKY40 gene is involved in regulating the synthesis and deposition of lignin in the juice sac of Majiayu fruit. The discovery of this gene supplements and improves the lignin metabolism regulation mechanism of WRKY transcriptional regulation in Majiayu, provides a theoretical basis for analyzing the occurrence mechanism of Majiayu juice sac granulation, and provides candidate genes for the molecular breeding industry to delay or inhibit the post-harvest juice sac granulation of Majiayu and improve the fruit quality through gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the effect diagram of the hardness (A) and lignin content (B) of Majiayou pulp during room temperature storage.

[0018] Figure 2 Paraffin sections (A, B, C) of Majiayu juice cells at room temperature storage stage 0d, 90d, 135d and transmission electron microscopy images (D, E, F).

[0019] Figure 3 This is the gene expression map of CgWRKY40 in the juice cells of Majiayou during room temperature storage.

[0020] Figure 4 This is the electropherogram of PCR amplification of CgWRKY40.

[0021] Figure 5 The subcellular localization map of CgWRKY40 in Nicotiana benthamiana leaves.

[0022] Figure 6 The figure shows the effect of transient expression of CgWRKY40 and PBI121 recombinant vectors in Majiayou juice cells on fruit phenotype and lignin staining. A is GUS staining, B is phloroglucinol staining, and C is paraffin section.

[0023] Figure 7 The figure shows the effect of transient overexpression of CgWRKY40 and PBI121 recombinant vectors in Majiayou juice cells on the expression pattern of lignin content and its synthesis-related genes. Among them, A is the relative expression of CgWRKY40, B is the lignin content, C is the relative expression of CgHCT1, D is the relative expression of CgC3H, E is the relative expression of Cg4CL1, F is the relative expression of CgPAL2, G is the relative expression of CgPAL1, H is the relative expression of CgCCR, I is the relative expression of CgPOD55, J is the relative expression of CgPOD3, K is the relative expression of CgPOD16, and L is the relative expression of CgPOD52. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0030] The embodiment of the present invention provides the use of CgWRKY40 in regulating lignin synthesis, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0031] The present invention cloned a transcription factor CgWRKY40 from Majiayu that plays an important role in regulating the granulation of Majiayu juice sacs after harvest, and found that its expression level in granulated juice sac fruits was significantly higher than that in normal ungranulated fruits. Subcellular localization found that the transcription factor was consistent with the nuclear marker localization. The present invention also constructed a plant transient overexpression vector, which was transiently transferred into Majiayu juice sacs by injection through Agrobacterium infection. The lignin in the juice sacs overexpressing the CgWRKY40 gene increased significantly, and at the same time, the autofluorescence signal in pyrogallol staining and paraffin sections was enhanced, the cell wall was thickened, and the symptoms of juice sac granulation were aggravated. This proves that the CgWRKY40 gene is involved in regulating the synthesis and deposition of lignin in the juice sacs of Majiayu fruit. The research results provide candidate genes for improving the quality of Majiayu, which is beneficial to provide an important theoretical basis for the molecular breeding and quality maintenance of Majiayu.

[0032] The CgWRKY40 gene belongs to the WRKY family member, and its nucleotide sequence is shown in SEQ ID NO.1. It contains an open reading frame of 966 bp and encodes 321 amino acids. The amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0033] The present invention performs weighted co-expression network analysis on the expression levels of WRKY transcription factors and the contents of lignin and its synthetic intermediates in pomelo fruits at different storage time points, and proves that there is a certain positive regulatory relationship between CgWRKY40 and fruit lignin synthesis. With the help of molecular biology methods and genetic transformation technology, it is further verified by transient injection of pomelo juice cells that CgWRKY40 can promote the synthesis of lignin in pomelo.

[0034] In some specific embodiments, overexpression of CgWRKY40 promotes the synthesis of lignin, and silencing or knocking out CgWRKY40 inhibits the synthesis of lignin.

[0035] The embodiment of the present invention also provides the use of CgWRKY40 in regulating the lignin synthesis of Pomelo fruit, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0036] In some specific embodiments, overexpression of CgWRKY40 promotes the synthesis of lignin in Pomelo fruit, and silencing or knocking out CgWRKY40 inhibits the synthesis of lignin in Pomelo fruit.

[0037] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, and a transgenic strain containing CgWRKY40 in regulating the synthesis of lignin in pomelo fruit.

[0038] The embodiment of the present invention also provides the use of the protein encoded by CgWRKY40 in regulating lignin synthesis, and its amino acid sequence is shown in SEQ ID NO.2.

[0039] The embodiment of the present invention also provides the use of the protein encoded by CgWRKY40 in regulating the lignin synthesis of Pomelo fruit, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0040] The embodiment of the present invention also provides a method for improving the taste of Majiayou fruit, silencing or knocking out CgWRKY40 in Majiayou plants, and inhibiting the synthesis of lignin in Majiayou fruit.

[0041] The embodiments of the present invention also provide the application of CgWRKY40 in improving the quality of pomelo fruit.

[0042] The fruit material involved in the present invention is selected from the main famous and excellent pomelo variety "Ma Jia You" in Guangfeng District, Shangrao City, Jiangxi Province, and is collected from the Xitan Ma Jia You standard orchard in the "Wanqi Xingwancun" demonstration base in Guangfeng District, Shangrao City 185 days after full flowering (about 8 mature).

[0043] Strains: The overexpression vector PBI121 was preserved in this experiment; Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0044] Example 1

[0045] Determination of phenotypic characteristic indices related to juice sac granulation in postharvest Majia pomelo

[0046] Select healthy fruits with uniform shape / color and no mechanical damage / pests and diseases and pre-store them for 4 days. After the fruits are cleaned and dried, pack them individually in polyethylene film fresh-keeping bags and store them at room temperature (20±2℃, RH: 70%~85%) for 5 months. Take samples every 30d and 15d in the first 3 months and the last 2 months respectively and keep them in a -80℃ refrigerator.

[0047] (I) Determination of pulp hardness: Use a handheld fruit hardness tester GY-4 with a 4mm probe to puncture the fruit juice cells, and the force (N) required to puncture the pulp indicates the pulp hardness.

[0048] (ii) Determination of lignin content: Accurately weigh 0.2 g of juice cell powder sample, grind it into a homogenate with 95% ethanol, and dilute to 5 mL; centrifuge at 12000 rpm for 2 min, discard the supernatant, wash the precipitate with 95% ethanol 3 times, and then wash it with ethanol:n-hexane (1:2) 3 times, and place it in a fume hood to dry; add 2 mL of 25% acetyl bromide acetic acid solution, and add 0.9 mL of 2 mol / L sodium hydroxide solution after 70°C water bath for 30 min; after the reaction is terminated, add 5 mL of acetic acid and 0.1 mL of 7.5 mol / L hydroxylamine chloride solution, dilute to 10 mL with acetic acid, and measure the absorbance at 280 nm. Finally, prepare a standard curve using a lignin standard sample to obtain the lignin content.

[0049] (III) Preparation and staining of paraffin sections

[0050] The specific process of making paraffin sections is as follows: (1) Fixation and sampling: Cut fresh fruit pulp and immediately put it into tissue fixative or special fixative corresponding to the tissue for more than 24 hours. Take the tissue out of the fixative and trim the target tissue with a scalpel in the fume hood, and put the trimmed tissue and the corresponding label in the embedding frame. (2) Dehydration and wax immersion: Put the dehydration box into the dehydrator and dehydrate it in gradient alcohol. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, melt paraffin I at 65℃ for 1 hour, melt paraffin II at 65℃ for 1 hour, melt paraffin III at 65℃ for 1 hour. (3) Paraffin embedding: Embed the wax-soaked tissue in the embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim it. (4) Paraffin sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4μm. Float the slices on the 40℃ warm water of the spreader to flatten the tissue, pick up the tissue with a slide, and bake it in a 60℃ oven. After the wax is baked dry, take it out and store it at room temperature for later use.

[0051] The observation was conducted by toluidine blue staining, and the specific steps are as follows: (1) Dewaxing paraffin sections to water: sequentially place the sections into environmentally friendly dewaxing transparent liquid I for 20 minutes, environmentally friendly dewaxing transparent liquid II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and then wash with tap water. (2) Toluidine blue staining: After the plant tissue sections are placed in the staining solution for about 2 minutes, they are washed with water and examined under a microscope. Depending on the depth of tissue staining, appropriate differentiation or non-differentiation is performed. After washing with tap water, the sections are placed in a 60°C oven to dry. (3) Transparent sealing: The sections are placed in clean xylene for 5 minutes and sealed with neutral gum. (4) Microscopic examination, image acquisition and analysis.

[0052] (IV) Transmission electron microscopy preparation and observation

[0053] The specific steps of preparing the transmission electron microscope slides are as follows: (1) Sample fixation: After peeling the fresh fruit pulp, immediately put it into a culture dish filled with electron microscope fixative and cut it into 1 mm pieces in the fixative in the culture dish with a scalpel. 3The cut small tissue blocks were transferred to EP tubes containing new electron microscope fixative for further fixation, vacuum dried, placed at room temperature for 2 hours, and then stored at 4°C overnight. Rinse with 0.1mol / L phosphate buffer (pH 7.4) 3 times, 15 minutes each time. (2) Post-fixation: Fix with 1% osmium acid prepared in 0.1mol / L phosphate buffer at room temperature in the dark for 7 hours, and then rinse with 0.1mol / L phosphate buffer 3 times, 15 minutes each time. (3) Dehydration at room temperature: Dehydrate the tissue in 30%, 50%, 70%, 80%, 95%, and 100% ethanol in turn, 1 hour each time. Soak in anhydrous ethanol: acetone (3:1) for 0.5 hour, anhydrous ethanol: acetone (1:1) for 0.5 hour, anhydrous ethanol: acetone (1:3) for 0.5 hour, and acetone for 1 hour. (4) Infiltration embedding: acetone:812 embedding agent (3:1) soak at 37℃ for 2-4h, acetone:812 embedding agent (1:1) permeate overnight at 37℃, acetone:812 embedding agent (1:3) soak at 37℃ for 2-4h, pure 812 embedding agent soak at 37℃ for 5-8h. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate and bake it in a 37℃ oven overnight. (5) Polymerization: Place the embedding plate in a 60℃ oven for polymerization for 48h, remove the resin block and set aside. (6) Ultrathin sectioning: The resin block is sliced ​​at 60-80nm on an ultrathin microtome, and the slice is picked up using a 150-mesh Fanghua membrane copper mesh. (7) Staining: After the copper mesh was stained with 2% uranyl acetate saturated alcohol solution in the dark for 8 minutes, it was washed with 70% ethanol and ultrapure water three times each. After staining with 2.6% lead citrate solution in the dark for 8 minutes, it was washed with ultrapure water three times. The filter paper was slightly dried, and the copper mesh sections were placed in a copper mesh box to dry at room temperature overnight. (8) Electron microscopy observation: The copper mesh sections were carefully placed on a glass slide and observed under a transmission electron microscope (Hitachi HT7800, 2000 times), and the images were collected for analysis.

[0054] The hardness of Majiayu flesh and the content of lignin after harvest Figure 1 As shown in the figure, during the room temperature storage period, the lignin content of the juice cells of Pomelo gradually accumulated and the pulp hardness increased; the results of paraffin sections and transmission electron microscopy ( Figure 2 ) also showed that the lignin content of Majiayu juice cells increased, the cell wall thickened, and the degree of juice cell granulation deepened with the extension of storage time. It is speculated that the excessive accumulation of lignin is the main reason for the granulation of Majiayu juice cells after harvest.

[0055] Example 2

[0056] Isolation, cloning and quantitative detection of CgWRKY40 gene in Pomelo

[0057] (I) Extraction of RNA from Majiayu juice cells

[0058] The CTAB method was used to extract total RNA from fruit samples at different storage time points. The specific steps are as follows:

[0059] 1) Weigh 0.4 g of Majiayou pulp sample powder into a 2 mL centrifuge tube, add 1.7 ml of CTAB extraction solution and 80 μL of 4% mercaptoethanol, and immediately vortex for 2 min to mix thoroughly.

[0060] 2) After incubation at 65°C for 10 min, add an equal volume of chloroform:isoamyl alcohol (24:1) and vortex to mix.

[0061] 3) After centrifugation at 12000 rpm (~13400×g) for 10 min, transfer the supernatant to a new centrifuge tube and repeat the previous step.

[0062] 4) Add 1 / 5 volume of 12M lithium chloride and let stand overnight.

[0063] 5) Centrifuge at 12000 rpm (~13400×g) for 10 min, discard the supernatant, and collect the debris precipitate.

[0064] 6) Add 500 μL of 75% ethanol to wash the precipitate, centrifuge at 12000 rpm (~13400×g) for 5 min, repeat the washing once and let it stand at room temperature.

[0065] 7) After dissolving with 100 μL DEPC, add 2 / 3 volume of 3M NaAc (pH 5.2) and 5 volumes of anhydrous ethanol, and place at -20°C for 15 min.

[0066] 8) Centrifuge at 12000 rpm (~13400×g) for 10 min, discard the supernatant and wash the precipitate twice with 75% ethanol.

[0067] 9) After standing at room temperature for 30 min, centrifuge at 12000 rpm (~13400×g) for 5 min, dissolve the precipitate in 30 μL DEPC water to obtain the pomelo RNA solution, and freeze it in a -80°C refrigerator.

[0068] (ii) Reverse transcription to synthesize cDNA

[0069] After the concentration of RNA obtained from different samples was determined, reverse transcription was performed to synthesize cDNA according to the instructions of the HifairⅢ1st Strand cDNA SynthesisSuperMix for qPCR reverse transcription kit (Shanghai Yisheng Biotechnology Co., Ltd.).

[0070] (III) Cloning of CgWRKY40 gene

[0071] The reference sequence of the CgWRKY40 gene was obtained by transcriptome sequencing, and the primers for cloning were designed using Premier 5.0 software. The primer pairs for gene amplification were:

[0072] CgWRKY40-F1 (SEQ ID NO.3): 5'-ATGGATTCAACGTGGGTGGAC-3';

[0073] CgWRKY40-R1 (SEQ ID NO. 4): 5'-GGTTGACCATCTTTGTGTTCGG-3'.

[0074] PCR amplification was performed using cDNA from Majiayou juice cells as a template. A 50 μL reaction system included: 1 μL cDNA, 2 μL each of forward and reverse primers, 20 μL ddH2O, and 25 μL high-fidelity enzyme (2×Phanta Flash Master Mix, Novozymes).

[0075] The amplification program was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 30 s, annealing at 55°C for 5 s, extension at 72°C for 5 s, 34 thermal cycles, extension at 72°C for 1 min, and storage at 4°C.

[0076] A single and clear PCR product was generated.

[0077] After the PCR product was verified by 1% agarose gel electrophoresis, the DNA fragment was recovered using a purification recovery kit (purchased from Novazonics, operated according to the instructions provided by the kit), connected to the pMD19-T vector, and transferred into Escherichia coli competent DH5α. Positive clones were screened on LB solid plates, the plasmid was extracted and digested with enzymes, and then tested by PCR. The recombinant plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing in order to obtain the accurate target gene sequence.

[0078] (IV) Real-time fluorescence quantitative PCR (q-PCR) detection and analysis

[0079] Design q-PCR primers online on NCBI Primer-BLAST based on the sequencing results:

[0080] Forward primer CgWRKY40-F (SEQ ID NO. 5): 5′-TCAAGTGTTCTTTCGCCCC-3′;

[0081] Reverse primer CgWRKY40-R (SEQ ID NO. 6): 5'-GAGTTG CAGTAGGGCTTGC-3'.

[0082] q-PCR detection was performed using a fluorescent quantitative PCR instrument (T100 Thermal Cycle) to verify the relevant genes, and the reaction system was as follows: total volume 10 μL, including 1 μL cDNA, 0.3 μL forward and reverse primers, 3.4 μL ddH2O and 5 μL TTB Green;

[0083] The reaction conditions were pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, insulation at 95°C for 15 s repeated 39 times, annealing at 60°C for 30 s (melting curve temperature), and extension at 95°C for 5 s.

[0084] The relative gene expression was calculated using the -2 △△Ct Law.

[0085] q-PCR results Figure 3 As shown in the figure, the expression level of CgWRKY40 gene in granulated juice cells is much higher than that in normal ungranulated juice cells, and its expression level is significantly increased with the extension of storage time, indicating that CgWRKY40 may be a key gene that promotes the granulation / lignification of juice cells after harvest of Majiayu. The electrophoresis results showed that the designed primers were consistent with the length of CgWRKY40 gene amplified using Majiayu cDNA as template ( Figure 4 ), M is DL2000 Marker. According to the sequencing results, the full length of CgWRKY40 gene is 966bp, and its nucleotide sequence is shown in SEQ ID NO.1, which can encode a protein of 321 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2. In the NCBI database, WRKY genes of different plants were obtained by Blast for homology comparison. The comparison results showed that the CgWRKY40 gene in Majiayu had the highest homology with the Zanthoxylum bungeanum ZaWRKY40 gene, with a homology of 83.82%.

[0086] SEQ ID NO.1:ATGGATTCAACGTGGGTGGACACCTCTCTTGACCTCAATCTC;

[0087] SEQ ID NO.2:MDSTWVDTSLDLNLNLLNHSSEVPMREFKGDHFAEFEEIASVK QETGILVEELNRISTENKKLNEMLSILCQNYNNLRQQYMDLMNKNTENEVGISKKRKAESEDHCHTIGFNVHATESSTSTDEESCKRPKDNNTKAKVSRFYVRASDSNSTLIVKDGYQWRKYGQKVTRDNPSPRAYFKCS FAPSCPVKKKVQRSAEDPSILVATYEGEHNHPQPTDSKAELSLSPRHVATIGNPIYVSAASSMRSASPTATLDMIQPGFLFDDAKKSSVQQIEAPAIHQILVQQMASNLTKDPNFTAALAAAISGRRFADQDRTQRWSH*.

[0088] Example 3

[0089] Subcellular localization of CgWRKY40 gene in Pomelo

[0090] (I) Construction of recombinant vector

[0091] Using PRI101-eGFP as the starting vector, the insertion site was between NdeΙ and BamHI, and the amplification primer pair was designed:

[0092] F (SEQ ID NO.7): 5'-GTTCTTCACTGTTGATACATATG ATGGATTCAACGT GGGTGGAC-3';

[0093] R (SEQ ID NO. 8): 5'-CTTGCTCACCATGGATCC GTGTGACCATCTTTGTGT TCGG-3'.

[0094] CgWRKY40 with the stop codon removed was amplified using PCR technology, and the constructed DNA fragment was purified and recovered. The product was then used to prepare a recombination reaction with double-enzyme-digested PRI101-eGFP according to the instructions of the one-step cloning kit (Novozymes). Finally, 5 μL of the recombinant product was added to 50 μL of DH5α competent E. coli, placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, transformed into E. coli and plated. After inverted culture in a 37°C incubator for 16 hours, positive single colonies were picked and sent to Qingke Biotechnology for sequencing verification to obtain the accurate target gene sequence.

[0095] (II) Agrobacterium transformation

[0096] Take 3 μL of CgWRKY40-PRI101-eGFP plasmid DNA with correct sequence confirmation and mix it with 50 μL GV1301 Agrocanthus competent cells, place it on ice for 5 min, place it in liquid nitrogen for 5 min, place it in a 37°C water bath for 5 min, and place it in an ice bath for 5 min. Resuspend it in 700 μL LB without antibiotics for 2 hours, then apply it to the plate and place it in a 28°C incubator for 2 days. Pick the positive monoclonal clone, which will show a single and clear band after PCR amplification. The position of the band is consistent with the plasmid DNA, and store it in a refrigerator at 80°C.

[0097] (III) Tobacco leaf infection

[0098] Sow a number of tobacco seeds and culture them for one month under 12h light before using them in the experiment. Resuspend the Agrobacterium containing the CgWRKY40-PRI101-eGFP vector in 10mM MgCl2 (containing 120μM AS) and adjust the OD 600 When the aggregator concentration reaches about 0.6, select tobacco plants with good growth conditions, inject from the lower epidermis of tobacco leaves with a 1mL syringe without a gun tip, and mark them. Use the PRI101-eGFP empty vector as a control. Culture the injected tobacco plants under weak light for 2 days, take the labeled Agrobacterium-injected tobacco leaves, make slides, observe under a laser confocal microscope, and take pictures.

[0099] Subcellular localization results Figure 5 As shown, the PRI101-eGFP empty vector is distributed in the nucleus and cell membrane of tobacco leaf epidermal cells, and the localization of CgWRKY40-PRI101-eGFP is consistent with the localization of the nuclear marker, indicating that CgWRKY40 is a transcription factor with a nuclear localization signal.

[0100] Example 4

[0101] Transient overexpression of CgWRKY40 gene in Pomelo

[0102] (I) Construction of CgWRKY40-PBI121 transient overexpression vector

[0103] PBI121 was used as the recombinant vector, the insertion site was between Xba Ι and Bam HI, and the amplification primer pair was:

[0104] CgWRKY40 upstream (SEQ ID NO.9): 5'-AGAACACGGGGGACTCTAGAATGGATTCAACGTGGGTGGAC-3';

[0105] CgWRKY40 downstream (SEQ ID NO. 10): 5'-GACTGACCACCCGGGGATCCGTGTGACCATCTTTGTGTTCGG-3'.

[0106] According to the designed primers, PCR amplification was performed, and the recovered product was connected to pMD19 and then transformed into Escherichia coli. After plasmid DNA was extracted, the plasmid was digested, and restriction endonucleases XbaΙ and Bam HI were selected as endonucleases for double digestion test. The digestion test used a 20μL system, wherein the components were: XbaΙ1μL, Bam HI lμL, 1×M Buffer 2μL, plasmid DNA 6μL, and H2O 10μL. After the system was added to a 200μL centrifuge tube, it was gently shaken and mixed and then centrifuged briefly, then warmed for 2h at 37°C, and 10×Loading Buffer was added to terminate the digestion reaction. The 20μL digestion products after the reaction were detected by agarose gel electrophoresis. After the digestion fragments were cut on a gel exciter, gel DNA was recovered. The recovered product was then connected to the PBI121 vector.

[0107] (II) Transient transformation of Majiayu juice cells

[0108] (1) IM (500 mL) solution preparation (prepared as needed): 5.137 g 4-morpholineethanesulfonic acid (MES), 2.632 g glucose, 0.164 g sodium dihydrogen phosphate, dilute to 500 mL, adjust pH to 5.6-5.7, sterilize and cool, then add 26.316 mL 20×AB Salts.

[0109] (2) 20×AB Salts (500 mL, vortex if precipitation occurs): ammonium chloride 10 g, magnesium sulfate heptahydrate 3 g, potassium chloride 1.5 g, calcium chloride 0.1 g, ferrous sulfate heptahydrate 0.025 g.

[0110] (3) MES (10 mM MgCl2, 10 mM MES 500 mL) solution: 0.475 g magnesium chloride, 1.066 g MES, dilute to 500 mL, adjust pH to 5.5-5.6, and sterilize by high pressure.

[0111] (4) Preparation of MMA suspension: First, prepare 0.1 mol / L AS solution, 1 mol / L MgCl2 solution and 0.5 mol / L MES (methyl ester sulfonate) solution. Take 2 mL of MES solution, 1 mL of MgCl2 solution and 0.1 mL of AS solution respectively, and then make up to 100 mL with ultrapure water to prepare MMA solution. The final concentrations of MES, MgCl2 and AS are 100 mmol / L, 10 mmol / L and 10 μmol / L, respectively.

[0112] (5) Preparation of infection solution: Use the Agrobacterium culture liquid successfully obtained in the previous transformation experiment, including the culture liquid of the successfully transformed CgWRKY40-PBI121 and the empty vector PBI121, and add it to the LB liquid culture medium containing kanamycin and rifampicin. Shake it on a shaker at 28°C and 200 rpm until the OD value of the culture liquid at a wavelength of 600nm is between 0.6-0.8, when the activity of the culture liquid is the best. Transfer the activated culture liquid to a 50mL centrifuge tube and centrifuge it at 4°C for 10 min at a speed of 5000 rpm. After centrifugation, discard the supernatant and retain the bacteria, then resuspend them twice with an equal volume of MMA solution. The empty vector PBI121 is used as the control of this experiment.

[0113] (6) Juice cell injection: In a clean bench, disinfect the whole fruit with 75% alcohol, separate the peel with a scalpel, and then carefully separate the juice cells from each capsule into a large culture dish covered with sterilized filter paper. Use a sterile scalpel to cut off the juice cell stalk and place it in a sterile conical flask. Pour the infection solution of Agrobacterium into the conical flask containing the juice cells, shake it gently for 5 minutes, and then let it stand for 20 minutes. Pour out the infection solution, add sterile water to wash the juice cells twice; place the washed juice cells on sterile absorbent paper, and absorb the solution on the surface of the juice cells as much as possible.

[0114] (7) Plating and sampling: Juice cells were placed on MS solid medium containing 0.35% agar and 0.1% cephalosporin and cultured in the dark at 20°C for 5 days. Samples were taken every other day, and the juice cell samples were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0115] (III) GUS staining of transiently overexpressed juice cells

[0116] Using the GUS staining kit (Coolaber), first melt the X-Gluc solvent in a 40°C water bath, then add 1 mL of the solvent to 1 tube of X-Gluc dry powder and mix to dissolve, to obtain a 50× GUS staining concentrate. Then take 0.4 mL of the GUS staining concentrate and add it to 5 mL of the GUS staining buffer and mix. Soak the juice cells cultured in MS solid culture medium in the GUS staining solution and keep it in a 28°C incubator overnight. The blue dots that appear in the juice cells are the GUS expression sites.

[0117] (IV) Lignin staining

[0118] Weisner reagent (phloroglucinol / concentrated hydrochloric acid) was used to stain the transiently overexpressed juice cells, and normal juice cells were used as controls. After 3-5 minutes of color development, the morphological changes of the juice cells were observed; toluidine blue staining was used to stain and observe the transiently overexpressed paraffin sections, and the specific operating steps were the same as in Example 1.

[0119] (V) Determination of lignin and lignin synthesis-related genes

[0120] The extraction and content determination of lignin were carried out in the same manner as in Example 1. The q-PCR primers of lignin synthesis-related genes were designed online by NCBI Primer-BLAST. The q-PCR detection was carried out using a fluorescent quantitative PCR instrument. The system and procedure were the same as in Example 2.

[0121] The experimental results showed that after CgWRKY40 was successfully transiently transformed for 3 days, the autofluorescence signal in the juice cell phloroglucinol staining and paraffin sections was enhanced ( Figure 6 ); quantitative analysis of lignin and lignin synthesis-related genes showed that the expression of CgWRKY40 gene was upregulated by 16 times, and the expression of lignin synthesis-related genes CgC3H, Cg4CL1, CgPAL2, CgPAL1, CgCCR, CgPOD3, CgPOD16, CgPOD52, CgPOD55 and CgHCT1 was upregulated by 2-5 times, and the lignin content was significantly increased ( Figure 7 ). This indicates that CgWRKY40 can positively regulate the expression of lignin synthesis-related genes, promote the accumulation of juice sac lignin, and thus promote the juice sac granulation of Pomelo.

[0122] Table 1 Primer design for CgWRKY40 and lignin synthesis-related genes in transient overexpression

[0123]

[0124]

[0125] (VI) Silencing of the CgWRKY40 gene in Pomelo

[0126] The silencing vector of CgWRKY40 gene was constructed and transformed into Majiayou juice cells. The results showed that silencing of CgWRKY40 in juice cell granulation inhibited the synthesis of lignin, reduced the lignin content, inhibited the expression of lignin synthesis-related genes, made the cell wall thinner, and the granulation phenomenon in the fruit juice cells was not obvious.

[0127] Based on Examples 1, 2, 3, and 4, the present invention found that during room temperature storage, pomelo will experience juice sac granulation due to excessive accumulation of lignin, and CgWRKY40 is highly expressed in the fruit during juice sac granulation, indicating that the pomelo CgWRKY40 transcription factor is positively correlated with post-harvest juice sac granulation of the fruit; the transcription factor was subcellularly localized in tobacco, and it was found that it has a nuclear localization signal; after the recombinant vector of CgWRKY40-PBI121 was transiently overexpressed in the juice cells of pomelo, the lignin content increased, the expression of lignin synthesis-related genes was upregulated, the cell wall thickened, and granulation symptoms appeared in the juice cells of the fruit, indicating that CgWRKY40 can promote the synthesis of lignin in pomelo and has a positive regulatory effect on post-harvest juice sac granulation of the fruit.

[0128] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. CgWRKY40 The application of the invention in regulating the lignin synthesis of pomelo fruit is characterized in that: Its nucleotide sequence is shown in SEQ ID NO.1; The application is silencing or knocking out CgWRKY40 , inhibiting the synthesis of lignin in pomelo fruit.

2. Contains CgWRKY40 The use of a recombinant vector, an expression cassette, and a transgenic strain in regulating lignin synthesis in pomelo fruit is characterized in that: CgWRKY40 The nucleotide sequence is shown in SEQ ID NO.1; The application is silencing or knocking out CgWRKY40 , inhibiting the synthesis of lignin in pomelo fruit.

3. CgWRKY40 The use of the encoded protein in regulating the lignin synthesis of Majiayu fruit is characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.2; The application is silencing or knocking out CgWRKY40 , inhibiting the synthesis of lignin in pomelo fruit.

4. A method for improving the taste of Majiayu fruit, characterized in that: Silencing or knocking out the CgWRKY40 , inhibiting the synthesis of lignin in Majiayou fruit; CgWRKY40 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.

2.

5. CgWRKY40 The application in improving the quality of grapefruit is characterized by: Said CgWRKY40 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; The pomelo fruit is a pomelo fruit; The application is silencing or knocking out CgWRKY40 , inhibiting the synthesis of lignin in pomelo fruit.

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