Application of transcription factor CgGATA8 in regulating lignin synthesis

Abstract

The present invention discloses the application of transcription factor CgGATA8 in regulating lignin synthesis, and belongs to the technical field of genetic engineering breeding and fruit and vegetable preservation. The present invention transiently transfers the transcription factor CgGATA8 into Ma Jiayou juice cells by Agrobacterium infection method, and the lignin content in the overexpressed juice cells is significantly higher than that in the empty control. At the same time, the autofluorescence signal in the pyrogallol staining and paraffin sections is enhanced, the secondary cell wall is thickened, and the symptoms of juice cell granulation are aggravated. Through molecular biological function verification, it is found that Ma Jiayou transcription factor CgGATA8 can bind to and activate CgPOD16 promoter, increase the expression level of CgPOD16 gene, and increase the accumulation of lignin synthesis in the fruit. It is proved that the CgGATA8 gene is involved in regulating the synthesis and deposition of lignin in the juice cells of Ma Jiayou fruit, and has the function of promoting the ripening and aging of the fruit after harvest.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering breeding and fruit and vegetable preservation, and in particular to the application of transcription factor CgGATA8 in regulating lignin synthesis. Background Art

[0002] Citrus is the fruit tree with the largest cultivation area and the most important economic status in southern my country, and it also occupies a leading position in global fruit production. According to the production and climate characteristics of the main citrus producing areas, my country's citrus industry can be divided into four industrial belts (including the middle and upper reaches of the Yangtze River, the sweet orange belt, the southern Jiangxi-southern Hunan-northern Guangxi, the navel orange belt, the southern Zhejiang-western Fujian-northern Guangdong, the wide-skinned orange belt and the western Hubei-western Hunan, the wide-skinned orange belt) and the two characteristic advantage bases of "Shaanxi-Hanzhong, Yunnan and Guizhou" (Deng Xiuxin, 2022). Majiayou (Citrusgrandis L.cv.Majiayou) is one of the famous and high-quality citrus varieties in Jiangxi Province. It is deeply loved by consumers for its beautiful fruit 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-climacteric fruit. Due to its huge size, it is mainly stored in piles during actual storage. It is very easy to become lignified after harvest and rots seriously. These unfavorable factors seriously restrict the healthy development of its industry. Therefore, analyzing the granulation mechanism of Majia pomelo is particularly important for developing effective preservation technology.

[0003] The post-harvest juice sac granulation of Majiayu is also known as the lignification process, which refers to the dynamic biological process in which lignin monomers are oxidatively polymerized under the catalysis of peroxidase or laccase and deposited on the secondary cell wall. During the juice sac granulation process of Majiayu, the lignification process of the secondary cell wall is regulated by external environmental factors (temperature, humidity, light, gas, etc.) and internal genetic factors (transcription factors, microRNA, photosensitive pigments, plant hormones, etc.), involving the synergistic effect of multiple transcription factors at multiple levels. Since lignin is a secondary metabolite in plant growth and development, its monomer synthesis and transport as well as terminal oxidation polymerization pathways are subject to multiple constraints of factors such as transcription factors, microRNA and hormones, thus affecting the thickening process of its secondary cell wall. Among them, during the lignification process, the research on the regulation of key genes of lignin synthesis by transcription factors and the interaction between transcription factors has laid a solid theoretical foundation for the construction of a regulatory network for lignin synthesis.

[0004] The GATA family is an important transcriptional regulatory factor in organisms. It has the ability to recognize GATA motifs and is a member of the zinc finger protein family. It can recognize and specifically bind to DNA sequences. Its family members have been found to be widely present in animals, fungi, plants and other organisms. GATA is named for its specific binding to the W-GATA-R gene sequence. It can specifically bind to the A / T and A / G sequences of the target gene promoter. Its DNA domain consists of a class IV zinc finger structure (C-X2-C-X17-20-C-X2-C) and the basic region behind it. GATA transcription factors are a type of DNA binding domain with one or two Cys2 / Cys2 type zinc finger structures. This transcription factor has the ability to recognize the DNA consensus sequence 5'-(T / A)HGATA(A / G)-3' present in the promoter region of the target gene. The first GATA transcription factor in plants, NTL1, was identified in tobacco and is related to the nitrogen cycle. Subsequently, GATA transcription factors have been studied in depth in many plants. The GATA transcription factor family is involved in many processes of plant growth and development and has been verified in multiple species. 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 CgGATA8 promoting lignin synthesis in Majiayu juice cells. Summary of the invention

[0005] The purpose of the present invention is to provide an application of transcription factor CgGATA8 in regulating lignin synthesis, 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 CgGATA8 in regulating lignin synthesis, and its nucleotide sequence is shown in SEQID NO.1.

[0008] The second technical solution of the present invention is the application of CgGATA8 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 CgGATA8 in regulating the synthesis of lignin in pomelo fruit.

[0010] A fourth technical solution of the present invention is the application of the protein encoded by CgGATA8 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 CgGATA8 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, which regulates the synthesis of lignin in Majiayou fruit by regulating CgGATA8 in Majiayou plants.

[0013] The seventh technical solution of the present invention is the application of CgGATA8 in improving the quality of pomelo fruits.

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

[0015] The present invention found that the transcription factor CgGATA8 of Majiayu can bind to and activate the promoter of CgPOD16 gene, increase its expression level, and promote the synthesis of lignin, thereby proving that the CgGATA8 gene is involved in regulating the synthesis and deposition of lignin in the juice cells of Majiayu fruit, and has the function of promoting the ripening and aging of fruits after harvest. The discovery of this gene supplements and improves the lignin metabolism regulation mechanism of zinc finger protein transcription regulation in Majiayu, provides a theoretical basis for analyzing the occurrence mechanism of Majiayu juice cell granulation, and provides candidate genes for the molecular breeding industry to delay or inhibit Majiayu juice cell granulation and improve 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 electrophoresis diagram of PCR amplification of the transcription factor CgGATA8 of Majiayou.

[0018] Figure 2 The figure shows the effect of transient expression of Majiayou transcription factor CgGATA8 and PBI121 recombinant vector in Majiayou juice cells on fruit phenotype and lignin staining. A is GUS staining, B is phloroglucinol staining, and C is paraffin section.

[0019] Figure 3The figure shows the effect of transient overexpression of Majiayou transcription factor CgGATA8 and PBI121 recombinant vector in Majiayou juice cells on lignin content and the expression pattern of genes related to its synthesis. Among them, A is the relative expression of CgGATA8, B is the relative expression of CgPOD3, C is the relative expression of CgPOD16, D is the relative expression of CgPOD55, E is the relative expression of CgPOD52, F is the relative expression of Cg4CL1, G is the relative expression of CgC3H, and H is the lignin content.

[0020] Figure 4 This is the subcellular localization map of the Majiayou transcription factor CgGATA8 in Nicotiana benthamiana leaves.

[0021] Figure 5 This is the result of yeast one-hybrid experiment of the promoters of the Majiayou transcription factors CgGATA8 and CgPOD16 genes. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] 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.

[0028] The embodiment of the present invention provides the use of CgGATA8 in regulating lignin synthesis, and its nucleotide sequence is shown in SEQ ID NO.1.

[0029] In some specific embodiments, overexpression of CgGATA8 promotes the synthesis of lignin, and silencing or knocking out CgGATA8 inhibits the synthesis of lignin.

[0030] The embodiment of the present invention also provides the use of CgGATA8 in regulating the lignin synthesis of Pomelo fruit, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0031] In some specific embodiments, overexpression of CgGATA8 promotes the synthesis of lignin, and silencing or knocking out CgGATA8 inhibits the synthesis of lignin.

[0032] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, and a transgenic strain containing CgGATA8 in regulating the synthesis of lignin in pomelo fruit.

[0033] The embodiment of the present invention also provides the use of the protein encoded by CgGATA8 in regulating lignin synthesis, and its amino acid sequence is shown in SEQ ID NO.2.

[0034] The embodiment of the present invention also provides the use of the protein encoded by CgGATA8 in regulating the lignin synthesis of Pomelo fruit, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0035] The embodiment of the present invention also provides a method for improving the taste of Majiayou fruit, by regulating CgGATA8 in Majiayou plants to regulate the synthesis of lignin in Majiayou fruit.

[0036] The embodiments of the present invention also provide the application of CgGATA8 in improving the quality of pomelo fruit.

[0037] At present, there are few reports on the research of post-harvest juice sac granulation of Majiayu. The present invention identifies a transcription factor CgGATA8 that plays an important regulatory role in post-harvest juice sac granulation of Majiayu through transcriptome sequencing and co-expression network analysis. Subcellular localization finds that the transcription factor is consistent with the nuclear marker localization. The transcription factor CgGATA8 overexpression vector is transiently transferred into Majiayu juice sac by Agrobacterium infection, and its lignin content is significantly increased. At the same time, the autofluorescence signal in pyrogallol staining and paraffin sections is enhanced, the secondary cell wall is thickened, and the symptoms of juice sac granulation are aggravated. Through yeast single hybrid experiments, it is found that Majiayu transcription factor CgGATA8 can bind to and activate the CgPOD16 gene promoter, increase its expression level, and promote the synthesis of lignin. This proves that the CgGATA8 gene is involved in regulating the synthesis and deposition of lignin in Majiayu fruit juice sacs, and has the function of promoting fruit ripening and aging after harvest. The discovery of this gene supplements and improves the lignin metabolism regulation mechanism of zinc finger protein transcriptional regulation in Majiayou, provides a theoretical basis for analyzing the occurrence mechanism of Majiayou juice sac granulation, and provides candidate genes for the molecular breeding industry to delay or inhibit Majiayou post-harvest juice sac granulation and improve fruit quality through gene editing.

[0038] The invention provides an important zinc finger protein transcription factor, and studies the regulatory mechanism of lignin synthesis in the process of juice sac granulation of pomelo fruits after harvest, which is of great significance for analyzing the occurrence mechanism of pomelo juice sac granulation and improving fruit quality. By performing weighted co-expression network analysis on the expression level of transcription factors and the content of lignin and its synthetic intermediates in pomelo fruits at different storage time points, it is proved that there is a certain positive regulatory relationship between the pomelo transcription factor CgGATA8 and the juice sac granulation of the fruit; with the help of molecular biological means and genetic transformation technology, it is further verified by transient injection of pomelo juice sacs that the pomelo CgGATA8 can promote the synthesis of lignin in the fruit and accelerate the process of juice sac granulation.

[0039] 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).

[0040] 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.

[0041] Example 1

[0042] Screening and cloning of genes related to juice sac granulation in postharvest pomelo

[0043] (I) Obtaining the target gene: Based on the transcriptome data analysis of 9 groups of juice cell samples of Majiayou fruit with different degrees of granulation (non-granulation, light granulation, and heavy granulation) during room temperature storage, a gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana. It was determined that the gene belongs to the GATA-type zinc finger protein family and was named CgGATA8 according to the location of the gene family member on the chromosome.

[0044] (II) Extraction and reverse transcription of RNA from Majiayu juice cells: The total RNA was extracted from Majiayu samples using the CTAB method. The specific steps are as follows:

[0045] (1) Weigh 0.4 g of Majiayou pulp sample powder into a 2 mL centrifuge tube, add 1.7 mL of CTAB extract and 80 μL of 4% mercaptoethanol, and immediately vortex for 2 min to mix thoroughly. (2) After 10 min in a 65°C water bath, add an equal volume of chloroform:isoamyl alcohol (24:1) and vortex to mix thoroughly. (3) After centrifugation at 12000 rpm (~13400×g) for 10 min, transfer the supernatant to a new centrifuge tube and repeat the previous step. (4) Add 1 / 5 volume of 12M lithium chloride and let stand overnight. (5) Centrifuge at 12000 rpm (~13400×g) for 10 min, discard the supernatant, and collect the debris precipitate. (6) Add 500 μL of 75% ethanol to wash the precipitate, centrifuge at 12000 rpm (~13400×g) for 5 min, repeat the wash once, and let stand at room temperature. (7) After dissolving with 100 μL DEPC, add 2 / 3 volume of 3M NaAc (pH 5.2) and 5 times volume of anhydrous ethanol, and place at -20°C for 15 minutes. (8) Centrifuge at 12000 rpm (~13400×g) for 10 minutes, discard the supernatant and wash the precipitate twice with 75% ethanol. (9) After standing at room temperature for 30 minutes, centrifuge at 12000 rpm (~13400×g) for 5 minutes, dissolve the precipitate in 30 μL DEPC water to obtain the pomelo RNA solution, and freeze it in a -80°C refrigerator. (10) Reverse transcription and cDNA synthesis: After the concentration of RNA obtained from different samples was determined, reverse transcription and cDNA synthesis were performed according to the instructions of the HifairⅢ1st Strand cDNASynthesis SuperMix for qPCR (Shanghai Yisheng Biotechnology Co., Ltd.) reverse transcription kit.

[0046] (III) Primer design: The reference sequence of the CgGATA8 gene was obtained by transcriptome sequencing, and cloning primers were designed using Premier 5.0 software. The specific primer pairs for amplifying the full-length CDS of the CgGATA8 gene are shown in Table 1. The synthesis of amplification primers was completed by Qingke Biotechnology Co., Ltd.

[0047] Table 1 Primer sequences

[0048]

[0049] (IV) Target gene amplification: PCR amplification was performed using cDNA from Majiayou juice cells as a template, and the full length of the target gene CDS was amplified with reference to the instructions of the high-fidelity enzyme (2×Phanta Flash Master Mix, Norwegian). The PCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0050] Table 2 PCR reaction system for amplifying CgGATA8 gene

[0051]

[0052] Table 3 PCR reaction program for amplifying CgGATA8 gene

[0053]

[0054] (V) Gel electrophoresis detection and gene sequencing: 0.2g agarose powder was added to 20mL 1×TAE, heated in a microwave oven to dissolve, and 1.5μL nucleic acid dye was added after cooling until it was not hot to the touch, and poured into a gel plate. After solidification, the sample was spotted and verified with DL 2000 as a marker to produce a single and clear PCR band product. After the PCR product was verified by 1% agarose gel electrophoresis, the DNA fragment was recovered using a purification recovery kit (purchased from Novozymes, 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, and the plasmid was extracted and digested by 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.

[0055] The electrophoresis results showed that the designed primers were consistent with the length of the CgGATA8 gene amplified using Majiayu cDNA as a template ( Figure 1 ), M is DL2000 Marker. According to the sequencing results, the full length of CgGATA8 gene is 1026bp, and its nucleotide sequence is shown in SEQ ID NO.1, which can encode a protein with 341 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2. In the NCBI database, Blast obtained zinc finger protein genes of different plants for homology comparison. The comparison results showed that the CgGATA8 gene in Majiayu had the highest homology with the PvGATA8 gene of pistachio and the ZjGATA8 gene of jujube, with homologies of 81.04% and 78.11%, respectively.

[0056] SEQ ID NO.1:ATGATTGGAACAAACTTCATGGACGAAATAGACTGCGGCAGCTTCTTTGATCACATAGACGATTTACTCGACTTCCCAAATGAAGATGTCGAAGCCGGGTTACCCAACGCCGATTCTAACCCGTTCCCCTCCATTTGGCCGACTCAGTCCGACTCGTTACCCGGGTCCGACTCAGTTTTCTCCAACAGCTCCACTGACCTGTCCACCCAGCTCTCTGTTCCATATGAGGACATTGTTCAGCTTGAGTGGCTTTCGAACTTTGTTGAGGATTCCTTCTCTGGAGGAAGCCTTACTATGAGCAAACAAGAGTCATCCACGATCACTAAGGACGATTCATCTCACAACCAGTTTCAGACCTCCAGCCCGGTCTCTGTTTTAGAGAGCAGCAGCTCCTGCTCGGGGGAGAAAACCGTGTTGGGAAGCCCTGAGACTACGGCCCCTGGGAGGCGCGGGCGTGCTCGTAGCAAGCGCCCTCGCCCCGCAACATTTAATCCCCGTCCTCCGGTTCAACTTGTGTCGCCAACTTCCTCAGTCACTGAAACAGAACCACAGCATCGCTTAATTGCCCCCAAGGCCTCATCTGATTCTGAGAATTTTGCAGAGTCTCGCCTTGTGATTAAAATACCAAAGCAATTCAACCCGGAGCACAAGAAGAAAAAGAAAATCAAATTGTCAGTTCCAAAAGTTTCCGATGAGACTAGTGAAGTTGGGCCAACACAAGCGGTTAGGAAATGCATGCATTGCGAGATAACGAAGACGCCTCAATGGAGAGCAGGGCCAATGGGGCCAAAAACCCTTTGCAATGCATGCGGTGTTCGCTATAAGTCTGGCAGGCTCTTCCCCGAATACAGGCCTGCTGCTAGCCCGACATTTGTCCCATCCTTGCACTCCAATTCCCACAAAAAGGTTGTTGAAATGAGAAACAAGAATTGCCAGAAGCCAATTGTGGCTGGCACTGAAACGATGATGACAGATGCACCAGAGTTGATTCCAAACAATAGCCACCTTGCACTGGAGTACGTTTGA;

[0057] SEQ ID NO.2:MIGTNFMDEIDCGSFFDHIDDLLDFPNEDVEAGLPNADSNPFPSI WPTQSDSLPGSDSVFSNSSTDLSTQLSVPYEDIVQLEWLSNFVEDSFSGGSLTMSKQESSTITKDDSSHNQFQTSSPVSVLESSSCSSGEKTVLGSPETTAPGRRGRARSKRPRPATFNPRPPVQLVSPTSSVTETEPQHRLIAPKASS DSENFAESRLVIKIPKQFNPEHKKKKKIKLSVPKVSDETSEVGPTQAVRKCMHCEITKTPQWRAGPMGPKTLCNACGVRYKSGRLFPEYRPAASPTFVPSLHSNSHKKVVEMRNKNCQKPIVAGTETMMTDAPELIPNNSHLALEYV*.

[0058] Example 2

[0059] Transient overexpression of CgGATA8 gene

[0060] (I) Construction of CgGATA8-PBI121 transient overexpression vector

[0061] PBI121 was used as the recombinant vector, the insertion site was between XbaΙ and BamHI, and the amplification primer pair was: CgGATA8-upstream: 5'-AGAACACGGGGGACTCTAGAATGATTGGAACAAACTTCATGGACG-3', CgGATA8-downstream: 5'-GACTGACCACCCGGGGATCC AACGTACTCCAGTGCAAGGTG-3'.

[0062] PCR amplification was performed according to the designed primers, and the product was purified and recovered and stored at 4° C. The next step was to perform restriction digestion of the PBI121 vector plasmid, and restriction endonucleases Xba Ι and BamHI were selected as endonucleases for double digestion test. The digestion system is shown in Table 4.

[0063] Table 4 PBI121 double restriction enzyme digestion reaction system

[0064]

[0065] Incubate at 37°C for 2 hours. After the reaction, 20 μL of the digestion product was subjected to agarose gel electrophoresis and stored at 4°C. The linearized vector and the target fragment plasmid were then prepared on ice to prepare the following reaction system, as shown in Table 5.

[0066] Table 5 Recombination reaction system

[0067]

[0068] After mixing, centrifuge briefly to collect the reaction solution to the bottom of the tube, react at 37℃ for 30min, and then cool on ice. Then add 5μL of the recombinant product to 50μL DH5α competent medium, place on ice for 30min, heat shock in 42℃ water bath for 45s, transform E. coli and plate, incubate inverted in 37℃ incubator for 16h, pick positive single colonies and send to Qingke Biotech for sequencing verification to obtain accurate target gene sequence. Take 3μL of CgGATA8-PBI121 plasmid DNA with correct sequence confirmation and mix with 50μL GV1301 competent medium, stand on ice for 5min, stand in liquid nitrogen for 5min, bath in 37℃ water bath for 5min, and bath on ice for 5min, resuspend in 700μL LB without antibiotics for 2h, plate, and place in 28℃ incubator for 2d. 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. Store in a -80℃ refrigerator to preserve bacteria.

[0069] (II) Transient transformation of Majiayu juice cells

[0070] (1) Preparation of 100 mM AAS solution: Weigh 0.1962 g of acetosyringone, dissolve it in 5 mL of DMSO (dimethyl sulfoxide), and then add water to make up to 10 mL.

[0071] (2) MES (10 mM MgCl 2 Preparation of 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.

[0072] (3) Preparation of MMA suspension: Take 2.0 mL of MES solution and MgCl 2 800 μL of solution and 300 μL of AS solution were added to make up to 200 mL with ultrapure water.

[0073] (4) Preparation of infection fluid: The Agrobacterium culture fluid successfully obtained in the previous transformation experiment, including the culture fluid of the successfully transformed CgGATA8-PBI121 and the empty vector PBI121, was added to the LB liquid medium containing kanamycin and rifampicin, and shaken on a shaker at 28°C and 200 rpm. After centrifugation and enrichment, it was suspended in the permeate to make the final concentration of the infection fluid OD 600 The value is between 0.6-0.8.

[0074] (5) Preparation of juice cell samples: In a clean bench, the whole fruit was disinfected with 75% alcohol, the peel was separated with a scalpel, and then the juice cells were carefully separated from each capsule valve into a large culture dish covered with sterilized filter paper. The juice cell stalk was removed with a sterile scalpel and placed in a sterile conical flask.

[0075] (6) Infection: Pour the infection solution of recombinant Agrobacterium into the conical flask containing juice cells, shake gently for 5 minutes, and then let it stand for 20 minutes. Pour out the infection solution and 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.

[0076] (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.

[0077] (III) GUS staining of transiently overexpressed juice cells

[0078] 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.

[0079] (IV) Lignin staining

[0080] Weisner reagent (phloroglucinol / concentrated hydrochloric acid) was used to stain transiently overexpressed juice cells, with normal juice cells as control. After 3-5 minutes of color development, the morphological changes of juice cells were observed. Toluidine blue staining was used to stain transiently overexpressed paraffin sections.

[0081] (V) Determination of lignin and lignin synthesis-related genes

[0082] Determination of lignin content: Accurately weigh 0.2g of juice cell powder sample, grind to homogenate with 95% ethanol, and dilute to 5mL; centrifuge at 12000rpm for 2min, discard the supernatant, wash the precipitate 3 times with 95% ethanol, and then wash it 3 times with ethanol: hexane (1:2), and place it in a fume hood to dry; add 2mL 25% acetyl bromide acetic acid solution, add 0.9mL 2mol / L sodium hydroxide solution after 70℃ water bath for 30min; after the reaction is terminated, add 5mL acetic acid and 0.1mL 7.5mol / L hydroxylamine chloride solution, dilute to 10mL with acetic acid, measure the absorbance at 280nm, and finally prepare a standard curve through lignin standard sample to obtain lignin content. Primer-BLAST on NCBI online design CgGATA8 gene and q-PCR primers for lignin synthesis related genes, as shown in Table 6.

[0083] Table 6 Primer design for CgGATA8 and lignin synthesis related genes in transient overexpression

[0084]

[0085] q-PCR detection was performed using a fluorescent quantitative PCR instrument (T100 Thermal Cycle) to verify the relevant genes. The reaction system was: total volume 10 μL, including 1 μL cDNA, 0.3 μL forward and reverse primers, 3.4 μL ddH 2 O and 5 μL TB Green; the reaction conditions were 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 60°C annealing for 30 s, 95°C insulation for 15 s, repeated 39 cycles, 60°C annealing for 30 s (melting curve temperature), 95°C extension for 5 s. The relative expression of genes was calculated using -2 △△Ct Law.

[0086] The experimental results showed that after CgGATA8 was successfully transiently transformed for 3 days, the autofluorescence signal in the juice cell phloroglucinol staining and paraffin sections was enhanced ( Figure 2 ); quantitative analysis of lignin and lignin synthesis-related genes showed that the expression of CgGATA8 gene was upregulated by 1.8 times, and the expression of lignin synthesis-related genes CgC3H, Cg4CL1, CgPOD3, CgPOD16, CgPOD52 and CgPOD55 was upregulated by 1.5-3.0 times, and the lignin content was significantly increased ( Figure 3 ). This indicates that CgGATA8 can positively regulate the expression of genes related to lignin synthesis, promote the accumulation of juice sac lignin, and thus promote the granulation of juice sacs in Pomelo.

[0087] (VI) Silencing of the CgGATA8 gene in Majiayou

[0088] The silencing vector of CgGATA8 gene was constructed and transformed into Majiayou juice cells. The results showed that silencing of CgGATA8 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.

[0089] Example 3

[0090] Subcellular localization

[0091] (I) Construction of recombinant vector

[0092] Using PRI101-eGFP as the starting vector, the insertion site was between NdeΙ and Bam HI, and the amplification primer pair was designed to be CgGATA8-PRI101-F and CgGATA8-PRI101-R. The primer sequences are shown in Table 1.

[0093] CgGATA8 with the stop codon removed was amplified using PCR technology, and the constructed DNA fragment was purified and recovered. The product was then prepared for a recombination reaction with double-enzyme-digested PRI101-eGFP according to the instructions of the one-step cloning kit (Novozymes). The linearized vector was then recombined with the target fragment plasmid, and Escherichia coli was transformed. The positive single colony was picked and sent for sequencing to obtain the accurate recombinant plasmid, and then transformed into Agrobacterium. The specific operation steps are the same as those in Example 2.

[0094] (II) Tobacco leaf infection

[0095] Sow a few tobacco seeds and culture them for one month under 12h light before using them for experiments. 2 (containing 120 μM AS) suspension and adjust 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.

[0096] Subcellular localization results Figure 4 As shown, the PRI101-eGFP empty vector is distributed in the nucleus and cell membrane of tobacco leaf epidermal cells, and the localization of CgGATA8-PRI101-eGFP is consistent with the localization of the nuclear marker, indicating that CgGATA8 is a transcription factor with a nuclear localization signal.

[0097] Example 4

[0098] Yeast one-hybrid assay

[0099] (I) Construction of recombinant vector

[0100] pAbai was used as the starting vector of the CgPOD16 gene promoter, and the insertion site was between Kpn I and Xho Ι. The amplification primer pairs were designed to be CgPOD16-pAbai-F and CgPOD16-pAbai-R according to the promoter 2000bp before the start codon of the CgPOD16 gene. pGADT7 was used as the starting vector of the CgGATA8 gene, and the insertion site was between Nde I and Bam HΙ. The amplification primer pairs were designed to be CgGATA8-AD-F and CgGATA8-AD-R according to the full length of the CDS of the CgGATA8 gene. The primer sequences are shown in Table 1. The CgPOD16 promoter amplification product was connected to the pAbai vector double-digested with Kpn I and Xho Ι, and the CgGATA8 gene amplification product was connected to the pGADT7 vector double-digested with Nde I and Bam HΙ. Escherichia coli was transformed, positive single colonies were identified, and plasmids were extracted. The amplification, recombination reaction and E. coli transformation methods are the same as in Example 2.

[0101] The CgPOD16 gene sequence is shown in SEQ ID NO.3.

[0102] SEQ ID NO.3:ATGGAAACTAAAAGCTTCTTCATTATTCTGTCATCTGTTGTTT TCTCTCTCATAATGACAGGTGCTTCTGCTCAACTTCGTGAAGACTTTTATAGAAGCACCTGCCCAAATGTAGAATCGCTTGTACGCTCGGCAGTCACCAAGAAGTTCACTCAGACCTTTGTCACAGCCCCAGCAACTCTTAGACTCTTTTTCCACGATTGCTTTGTCCGGGGATGTGATGCTTCAGTGCTGCTATCGTCGCCGAATAATCGGGCAGAGAAGGATCACCCAGAAGATATATCACTAGCTGGAGATGGATTTGATACTGTGGTCAAAGCTAAGGAAGCTGTTGATAGTGATCCTCAGTGCAGAAACAAAGTTTCATGTGCTGATATTTTAGCTCTTGCCACTAGAGATGTTGTTTCCTTGGCAGGGGGACCATTCTATAAAGTTGAATTGGGGAGGCGTGATGGTAGAATATCGACAATAGCAAGTGCTCAGCACAAACTTCCTCAACCTGATTTTAATTTAGACCAACTCAACAGAATGTTCAGCTCACATGGTCTTGATCAAACGGACATGATTGCATTATCAGGTGCGCATACGATAGGATTCTCTCACTGCAGCCGCTTCTCGAAAAGAATCTACAACTTCAGCCCCAGGAACAGAATCGATCCCACTTTAAATTTCAATTATGCAATGCAGCTGAGGGGCATGTGCCCTGTAAGAGTTGATCCTAGAATTGCCATTGACATGGACCCCACGACGCCTCGGATTTTCGACAATGCTTACTACAAGAATCTTCAGCAGGGAAAGGGTTTGTTTACGTCTGATCAAATTTTGTTTACTGATGGAAGATCAAGAGACACGGTTGTTCGATTTGCTTCCGACAAAGAAGCTTTTAATAGGGCTTTTATTTCTGCTATTACAAAGCTTGGACGTGTGGGAGTCAAGACAGGAAATCAAGGAGAAATTCGAAGGGATTGCGCGCTTGTGAACTAA。

[0103] (ii) Linearized CgPOD16-pAbai transformed into Y1H Gold yeast

[0104] The CgPOD16-pAbai recombinant plasmid was linearized by single restriction digestion with BstBΙ. The restriction digestion system was 1μL BstBΙ, 2μL 10× Buffer, 10μL recombinant plasmid DNA and 7μL enzyme-free ddHO. 2 O, pipette and mix well, then digest at 65℃ for 2h.

[0105] Pick a yeast monoclone on the YPDA medium and place it in a 3mL YPDA liquid medium, place it in a shaker (30°C, 200r / min) and culture it for 18h. Collect the bacterial solution in a 2mL centrifuge tube, centrifuge it at room temperature at 12000r / min for 1min, and discard the supernatant. Dilute the carrier DNA to 2mg / mL with sterile water, denature it at 100°C for 5min, and place it on ice for later use. Add 240μL 50% PEG, 34μL LiAc solution, 50μL arrier DNA and 10μL linearized pBait-AbAi plasmid to the bacteria in sequence, place it in a 42°C metal bath for 2h, centrifuge it at room temperature at 12000r / min for 1min, add 500μL ddH 2 Wash the cells with 200 μL ddHO and repeat centrifugation and washing twice. 2 Resuspend the bacteria in 5% CO, and spread 30 μL of the bacterial solution on SD-Ura solid medium and incubate at 30°C for 2-3 days.

[0106] (III) Screening the optimal AbA concentration for bait yeast strains

[0107] After the above transformation verification was successful, a fresh single colony was picked from each sample and resuspended in 1 mL of sterile aqueous solution. 600 Adjust to 0.2. Dilute with sterile water 10 times, 100 times, 1000 times (i.e. OD 600 =0.2, 0.02, 0.002, 0.0002).

[0108] Spot 3 μL on SD-Ura; SD-Ura with AbA (50ng / mL, 100ng / mL, 150ng / mL and 200ng / mL) plates respectively. Cultivate at 30℃ for 2-3 days, and observe the growth of bait yeast on plates with different AbA concentrations to determine the best AbA concentration. The best AbA concentration is when the yeast plaques are minimal or absent on plates with different AbA concentrations.

[0109] (IV) CgGATA8-AD transformation of Y1H Gold yeast

[0110] Pick the yeast monoclone on SD-Ura medium and place it in 3mL YPDA liquid medium, and culture it in a shaker (30℃, 200r / min) for 18h. Collect the bacterial solution in a 2mL centrifuge tube, centrifuge it at room temperature at 12000r / min for 1min, and discard the supernatant. Add 240μL 50% PEG, 34μL LiAc solution, 50μL arrier DNA and 10μL linearized pBait-AbAi plasmid to the bacteria in sequence, place it in a 42℃ metal bath for 2h, centrifuge it at room temperature at 12000r / min for 1min, and add 500μL ddH 2 Wash the cells with 200 μL ddHO and repeat centrifugation and washing twice. 2 Resuspend the bacteria in 5% CO, and spread 30 μL of the bacterial solution on SD-Leu solid medium and incubate at 30°C for 2-3 days.

[0111] (V) Verification of the interaction between CgPOD16-pAbai and CgGATA8-AD

[0112] After the above transformation verification was successful, a fresh single colony was picked from each sample and resuspended in 1 mL of sterile aqueous solution, and the OD600 was adjusted to 0.2. The sample was then diluted 10 times, 100 times, and 1000 times with sterile water (i.e., OD600 = 0.2, 0.02, 0.002, 0.0002).

[0113] In the order of experimental group first and control group, 3 μL was spotted on the corresponding SD / -Leu withAbA plate. Cultured at 30℃ for 2-3 days, and the growth of each group of recombinant yeast on the corresponding self-activating AbA concentration plate was observed to determine whether there was interaction.

[0114] The results showed that the combination of transcription factor CgGATA8 and CgPOD16 promoter could enable the recombinant yeast colonies to grow on leucine-deficient medium containing 150 ng / mL aureobasidin, while AD empty vector could not grow normally ( Figure 5 ). This indicates that the transcription factor CgGATA8 can directly bind to and activate the promoter of CgPOD16.

[0115] Based on Examples 1, 2, 3, and 4, it was found that during room temperature storage, pomelo would experience juice sac granulation due to excessive accumulation of lignin, and CgGATA8 was highly expressed in the fruit during juice sac granulation, indicating that the pomelo CgGATA8 transcription factor was positively correlated with the juice sac granulation of the fruit after harvest; the transcription factor was subcellularly localized in tobacco, and it was found that it had a nuclear localization signal; after the recombinant vector of CgGATA8-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 CgGATA8 can promote the synthesis of lignin in pomelo and has a positive regulatory effect on the juice sac granulation of the fruit after harvest. The results of yeast one-hybrid experiments showed that CgGATA8 could bind to and activate the CgPOD16 promoter, increase the expression level of the CgPOD16 gene, and promote the accumulation of lignin synthesis in Majiayou fruit. This proved that the CgGATA8 gene was involved in regulating the synthesis and deposition of lignin in the juice cells of Majiayou fruit, and had the function of promoting the ripening and aging of the fruit after harvest. The silencing of CgGATA8 in juice cell granulation inhibited the synthesis of lignin, reduced the lignin content, inhibited the expression of genes related to lignin synthesis, made the cell wall thinner, and the granulation phenomenon in the juice cells of the fruit was not obvious.

[0116] 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. CgGATA8 The application of the method in inhibiting the synthesis of lignin in pomelo fruit is characterized in that: CgGATA8 The nucleotide sequence is shown in SEQ ID NO.1, and the application is to silence or knock out CgGATA8 , inhibiting the synthesis of lignin.

2. A method for improving the taste of Majiayu fruit, characterized in that: By silencing or knocking out the CgGATA8 , inhibiting the synthesis of lignin in Pomelo fruit; Said CgGATA8 The nucleotide sequence is shown in SEQ ID NO.1.

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