Application of CsZAT12 in regulating resistance of navel orange to penicillium expansum

Abstract

This invention discloses the application of the zinc finger protein transcription factor CsZAT12 in regulating resistance to Penicillium mold in navel oranges, belonging to the fields of molecular biology and genetic engineering. Overexpression and silencing of the CsZAT12 gene were transiently transferred into the peel of Newhall navel oranges via injection using Agrobacterium infection. Silencing the CsZAT12 gene significantly controlled or reduced the incidence of Penicillium mold in navel oranges, while simultaneously inhibiting the increase in titratable acid content in the peel. The results of overexpression of the CsZAT12 gene showed the opposite. This demonstrates that the CsZAT12 gene participates in regulating resistance to Penicillium mold in navel oranges. The research findings can be applied to molecular breeding of navel oranges, providing candidate genes and new methods for the creation or improvement of new disease-resistant germplasm in navel oranges.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic engineering, and in particular to the application of the navel orange zinc finger protein transcription factor CsZAT12 in regulating resistance to Penicillium rot in fruit. Background Technology

[0002] Navel orange (Citrus sinensis Osbeck) is a perennial evergreen fruit tree belonging to the genus Citrus in the Rutaceae family. When ripe, the fruit is beautifully shaped, bright orange-red, with tender, juicy flesh and a rich aroma, making it very popular with consumers. In my country, the harvest season for most citrus fruits is concentrated in October and November, primarily for fresh sale. During post-harvest storage, transportation, and sales, the fruit is highly susceptible to infection by pathogenic fungi, leading to rot and spoilage. Postharvest Penicillium italicum infection in navel oranges has caused incalculable economic losses to the main navel orange producing areas of Jiangxi Province. Therefore, breeding disease-resistant varieties is one of the main objectives of citrus cultivation.

[0003] When pathogenic fungi infect plant tissues, they may secrete pH-regulating organic acids or ammonia to acidify or alkalize the host environment, thereby enhancing their infectivity. Based on this mechanism, pathogenic fungi can be divided into two main categories: acidic fungi and alkaline fungi. *Penicillium italicum* has been identified as an acidic fungus (Jiao et al., 2021; Alkan et al., 2015). The secretion of organic acids plays an important role in the infection and pathogenesis of acidic fungi. It can create an acidic environment suitable for fungal colonization and growth, thereby enhancing its pathogenicity. Its mechanism of action may include the following two points: (1) lowering the pH value of the host environment and increasing the activity of cell wall degrading enzymes (such as pectinase, polygalacturonase, β-galactosidase, cellulase, etc.) to weaken the host cell wall function; (2) chelating Ca 2+ Reduce intercellular calcium in host cells 2+ It can disrupt the host cell's mineral balance, leading to cell death.

[0004] Transcription factors (TFs) are a class of proteins that regulate gene expression by recognizing cis-acting elements in gene promoters. Current research has identified several TF families associated with plant stress responses, including APETALA2 / ethylene response factor (AP2 / ERF), MYB, WRKY, bZIP, and C2H2 (Amorim et al., 2017). The C2H2-type zinc finger protein (ZFP) TF family is divided into several subfamilies, with the C1 family being one of the largest. C1 family members have varying numbers of dispersed zinc finger structures, classified into five subclasses: C1-1i, C1-2i, C1-3i, C1-4i, and C1-5i. The protein encoded by the ZAT gene contains two dispersed zinc finger structures, belonging to the C1-2i subclass of the C2H2-ZFP transcription factor family. Most ZAT family members also possess highly conserved QALGGH and EAR motifs at the C-terminus (Ding et al., 2023). Currently, ZAT genes are widely involved in plant responses to or resistance to abiotic stresses such as drought, high salinity, cold damage, and oxidative stress. However, there is very little research on the disease resistance function of plant transcription factor ZAT in response to P. italicum infection. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the navel orange zinc finger protein transcription factor CsZAT12 in regulating the resistance of fruit to Penicillium rot, so as to solve the problems existing in the prior art.

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

[0007] One of the technical solutions of this invention is the application of the navel orange zinc finger protein transcription factor CsZAT12 in regulating the resistance of fruit to Penicillium rot.

[0008] The second technical solution of this invention is the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsZAT12 gene in regulating plant resistance to Penicillium wilt.

[0009] The third technical solution of this invention is a method for regulating plant resistance to Penicillium spores by silencing or knocking out the CsZAT12 gene to improve plant resistance to Penicillium spores, and by overexpressing the CsZAT12 gene to reduce plant resistance to Penicillium spores.

[0010] The fourth technical solution of this invention is the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsZAT12 gene in the cultivation of new plant varieties resistant to Penicillium blight.

[0011] The fifth technical solution of the present invention is a method for cultivating new plant varieties resistant to Penicillium wilt, which utilizes a recombinant vector containing the CsZAT12 gene, an expression cassette, a transgenic cell line or recombinant bacteria to regulate the expression of the CsZAT12 gene in plants.

[0012] The sixth technical solution of this invention is the application of the CsZAT12 gene or its encoded protein in regulating the synthesis of organic acids in plants.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] 1. This invention screened and identified a transcription factor CsZAT12, which plays an important regulatory role in the accumulation of organic acids during the occurrence of postharvest Penicillium wilt in 'Newhall' navel oranges, through transcriptome sequencing and weighted gene co-expression network analysis. The nucleotide sequence, amino acid sequence, and related vector of this transcription factor are published for the first time. This is of great significance for elucidating the disease resistance function of the navel orange transcription factor ZAT in response to P. italicum infection and for the breeding of disease-resistant varieties.

[0015] 2. This invention is the first to demonstrate, through molecular biology methods, that CsZAT12 has a nuclear localization signal. Silencing the CsZAT12 gene can significantly enhance the fruit's resistance to Penicillium rot, effectively inhibit the increase of titratable acid content in the peel, and effectively delay the adverse effects of P. italicum infection, thereby controlling the rancidity process of the peel tissue. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The diagram shows the disease development of 'Newhall' navel oranges after inoculation with Penicillium italicum. In the diagram, A shows the phenotype of the fruit lesions, B shows the results of lesion diameter and disease index measurements, and C shows the results of TA content and pH value measurements.

[0018] Figure 2 The images show the expression level and PCR amplification electrophoresis diagram of the transcription factor CsZAT12 in navel orange. In the diagram, A represents the relative expression level of CsZAT12, and B is the PCR amplification electrophoresis diagram of CsZAT12.

[0019] Figure 3 Subcellular localization map of CsZAT12 in tobacco leaves.

[0020] Figure 4The graphs show the effects of transient expression of the CsZAT12 recombinant vector in navel orange peel on the fruit. Specifically, A represents the effect of transient expression of the CsZAT12 recombinant vector in navel orange peel on fruit phenotype; B represents the effect of transient expression of the CsZAT12 recombinant vector in navel orange peel on fruit firmness; C represents the effect of transient expression of the CsZAT12 recombinant vector in navel orange peel on fruit lesion diameter; and D represents the effect of transient expression of the CsZAT12 recombinant vector in navel orange peel on fruit disease index.

[0021] Figure 5 This diagram shows the effects of transient expression of the recombinant vector in navel orange peel on CsZAT12 expression level, TA content, and pH value. Specifically, A represents the effect of transient expression of the recombinant vector in navel orange peel on CsZAT12 expression level, B represents the effect of transient expression of the recombinant vector in navel orange peel on TA content, and C represents the effect of transient expression of the recombinant vector in navel orange peel on pH value. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

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

[0027] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0028] This invention provides the application of the navel orange zinc finger protein transcription factor CsZAT12 in regulating the resistance of fruit to Penicillium rot.

[0029] In some specific embodiments, the amino acid sequence of the navel orange zinc finger protein transcription factor CsZAT12 is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1.

[0030] In some specific implementations, the Penicillium wilt is a plant disease caused by Penicillium italicum; the fruit includes navel orange fruit.

[0031] In some specific implementation schemes, silencing or knocking out the CsZAT12 gene can increase plant resistance to Penicillium wilt; overexpressing the CsZAT12 gene can decrease plant resistance to Penicillium wilt.

[0032] This invention also provides the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsZAT12 gene in regulating plant resistance to Penicillium wilt.

[0033] In some specific implementations, the recombinant vector originates from PBI121 and TRV2, and the gene insertion site is between Xba I and Bam HI, and between Xba I and Sam I.

[0034] In some specific implementations, the transgenic cell line or recombinant bacteria is Agrobacterium GV3101.

[0035] This invention also provides a method for regulating plant resistance to Penicillium spores: silencing or knocking out the CsZAT12 gene to improve plant resistance to Penicillium spores; and overexpressing the CsZAT12 gene to reduce plant resistance to Penicillium spores.

[0036] This invention also provides the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsZAT12 gene in the cultivation of new plant varieties resistant to Penicillium blight.

[0037] This invention also provides a method for cultivating new plant varieties resistant to Penicillium spp., which utilizes a recombinant vector containing the CsZAT12 gene, an expression cassette, a transgenic cell line, or a recombinant bacterium to regulate the expression of the CsZAT12 gene in plants.

[0038] This invention also provides the application of the CsZAT12 gene or the protein it encodes in regulating the synthesis of organic acids in plants.

[0039] In some specific implementation schemes, silencing or knocking out the CsZAT12 gene promotes the synthesis of plant organic acids; overexpressing the CsZAT12 gene inhibits the synthesis of plant organic acids.

[0040] This invention cloned a transcription factor CsZAT12, induced by *Penicillium italicum*, from Newhall navel oranges and constructed vectors for subcellular localization, silencing, and overexpression of CsZAT12. Subcellular localization revealed that the transcription factor is consistent with nuclear marker localization, exhibiting nuclear localization signals. Overexpression and silencing were transiently transferred into the peel of Newhall navel oranges via injection using *Agrobacterium* infection. Silencing the CsZAT12 gene significantly controlled or reduced the incidence of *Penicillium italicum* in navel oranges and inhibited the increase in titratable acid content in the peel. The results of overexpression of the CsZAT12 gene were the opposite. This demonstrates that the CsZAT12 gene participates in regulating resistance to *Penicillium italicum* in navel oranges. The research findings can be applied to molecular breeding of navel oranges, providing candidate genes and new methods for the creation or improvement of new disease-resistant germplasm in navel oranges.

[0041] This invention is the first to discover a transcription factor CsZAT12 whose translation is induced by Penicillium italicum, and whose expression level is significantly positively correlated with the disease index and organic acid content of navel orange fruit.

[0042] This invention provides an important C2H2 transcription factor, CsZAT12, to investigate the regulatory mechanism of organic acid metabolism during postharvest Penicillium rot in navel orange fruit. This is of great significance for elucidating the mechanism of postharvest resistance to Penicillium rot in navel orange. Weighted co-expression network analysis of CsZAT12 expression levels in navel orange peel at different storage time points after inoculation demonstrated a positive regulatory relationship between CsZAT12 and organic acid synthesis. Furthermore, using molecular biology techniques and genetic transformation technology, it was verified that silencing or knocking out CsZAT12 can inhibit organic acid synthesis and accumulation, thereby enhancing the defense ability of navel orange fruit against Penicillium rot infection.

[0043] The fruit material used in this invention is the 'Newhall' navel orange (Citrus sinensis L.cv 'Newhall'), which was collected from the Junping Fruit Industry Navel Orange Planting Standard Orchard in Nankang District, Ganzhou City, Jiangxi Province.

[0044] Strains: Overexpression vector PBI121 and silencing vectors TRV1 and TRV2 were preserved in this laboratory; Escherichia coli DH5α and Agrobacterium GV3101 were purchased from Beijing Qingke Biotechnology Co., Ltd. *Penicillium italicum* was independently isolated and identified by our research group from *Penicillium*-infected navel oranges and preserved in the Jiangxi Provincial Key Laboratory of Fruit and Vegetable Composition and Preservation, Jiangxi Agricultural University.

[0045] Example 1

[0046] Determination of phenotypic characteristics related to postharvest Penicillium rot in navel oranges

[0047] Select healthy fruits of uniform size and maturity (single fruit weight: 300-350g, fruit shape index: 105-110%, sugar-acid ratio: 14.6-16.1), uniform coloring (citrus color difference index: 5.45-6.10), free from mechanical damage and pests. After rinsing and draining the navel oranges with clean water, soak them in 1.0% sodium hypochlorite solution for 2 minutes, rinse thoroughly with running water to ensure no disinfectant residue remains, and air dry in a sterilized laminar flow hood. Then, use a sterilized needle to make a hole (3mm in diameter, 3mm deep) at the equator of the navel orange fruit. After the wound is completely dry, add 15μL of *Penicillium italicum* spore suspension, air dry, pack into boxes, and then package in polyethylene plastic bags. Incubate in a constant temperature incubator (temperature 27±1℃, relative humidity 90%–95%) for 4 days. Fruit appearance changes were observed at random times of 0h, 24h, 48h, 72h and 96h after inoculation. The diameter of the lesions was measured, and fruit peel tissue samples were taken from within 10-20mm of the wound on the fruit. The samples were chopped, flash-frozen in liquid nitrogen and stored at -80℃.

[0048] (a) Determination of lesion diameter and disease index

[0049] Diameter of lesions: The diameter of lesions in navel oranges was determined by the cross-cross method.

[0050] Disease index: Fruits with lesions larger than 3 mm in diameter are defined as diseased. The disease index is calculated using the following scale:

[0051] Grade 0: Lesion diameter = 0mm (no rot);

[0052] Grade 1: 1mm ≤ lesion diameter ≤ 10mm;

[0053] Grade 2: 10mm < lesion diameter ≤ 20mm;

[0054] Grade 3: 20mm < lesion diameter ≤ 40mm;

[0055] Grade 4: Lesion diameter > 40 mm.

[0056] The formula for calculating the severity of disease is as follows:

[0057]

[0058] (II) Determination of TA (titerizable acid) content and pH value

[0059] The TA content was determined by NaOH neutralization titration. 2.0 g of fruit peel tissue and two drops of 1% phenolphthalein were added to 40 mL of distilled water, and then titrated with 0.1 M NaOH solution. The TA content was calculated based on the amount of NaOH consumed, and the result was expressed as a percentage.

[0060] pH values ​​were measured using an Ohaus laboratory pH meter (model: ST3100 / B, New Jersey, USA). Each reading was calibrated with deionized water before each measurement, and the results are expressed as readings.

[0061] Changes in lesion diameter and disease index in Newhall navel oranges inoculated with Penicillium italicum from 0 to 96 hours are as follows: Figure 1 As shown, the disease began to appear 48 hours after inoculation. With the extension of storage time, the diameter of fruit lesions and the disease index gradually increased. The results of TA content and pH value in the peel showed that as the infection process of Penicillium italicum progressed, the TA content in the peel increased and the pH value decreased. The above results indicate that the infection of Penicillium italicum directly affects the synthesis and accumulation of organic acids in the peel.

[0062] Example 2

[0063] Isolation, cloning, and quantitative analysis of the CsZAT12 gene in navel orange

[0064] (I) Extraction of RNA from Navel Orange Peel

[0065] RNA was extracted from citrus peel using a modified Trizol method. The solution was prepared as follows:

[0066] (1) DEPC water (2L): Take 2mL of diethyl pyrocarbonate in a fume hood, make up to 2L with ultrapure water, and autoclave overnight.

[0067] (2) 0.2M sodium chloride (100mL): Weigh 1.169g of sodium chloride, add DEPC water to make up to 100mL, and autoclave.

[0068] (3) 3M sodium acetate (NaAC, 100mL): 24.609g of anhydrous sodium acetate was added to 70mL of DEPC water and stirred. The pH was adjusted to 5.2 with glacial acetic acid and the volume was brought to 100mL. The mixture was then autoclaved.

[0069] (4) 1M Tris-HCl (pH 7.6, 100mL): Weigh 12.14g of tris(hydroxymethyl)aminomethane (Tris), add 80mL of DEPC water, adjust the pH to 7.6 with concentrated hydrochloric acid, and bring the volume to 100mL.

[0070] (5) 10% sodium dodecyl sarcosinate (25mL): Weigh 2.5g sodium dodecyl sarcosinate and add DEPC water to make up to 25mL.

[0071] (6) Extraction Buffer (500mL): 47.264g guanidine isothiocyanate, 15.224g ammonium thiocyanate, 0.5g 8-hydroxyquinoline, 190mL water-saturated phenol, 25mL glycerol, 16.7mL 3M pH 5.2 sodium acetate (NaAC), add DEPC water and stir thoroughly until completely dissolved, bring the volume to 500mL, and autoclave.

[0072] (7) TESAR (100mL): 1mL of 1M Tris-HCl (pH 7.6), 10mL of 10% sodium dodecyl sarcosinate, 200μL of 0.5M EDTA solution, and DEPC water to a final volume of 100mL. Autoclave.

[0073] (8) Bu / CTAB and Aq / CTAB: Add 125 mL of DEPC water and 125 mL of n-butanol to a clean, RNAase-free separatory funnel. After standing overnight, separate the aqueous phase and the organic phase (the upper layer is the organic phase and the lower layer is the aqueous phase). Take 100 mL of the organic phase, add 3.68 g of cetyltrimethylammonium bromide (CTAB), and then add 100 mL of the aqueous phase. Mix well and let stand. Autoclave. The upper layer is Bu / CTAB and the lower layer is Aq / CTAB.

[0074] The detailed steps for RNA extraction are as follows: (1) Weigh 0.5g of fruit peel sample powder into a 10mL centrifuge tube, add 5mL of buffer, vortex for 2min to mix thoroughly, and let stand for 10min. (2) Centrifuge at 4℃ and 12000rpm for 20min, transfer the supernatant to a new centrifuge tube, add 5mL of chloroform, vortex for 2min to mix thoroughly, and let stand for 10min. (3) Centrifuge at 4℃ and 12000rpm for 20min, transfer the supernatant to a new centrifuge tube, add an equal volume of frozen isopropanol, gently invert to mix the two liquids thoroughly, and let stand at room temperature for 10min. (4) Centrifuge at 4℃ and 12000rpm for 20min, discard the supernatant, collect the fragment precipitate, soak it in 3mL of pre-cooled 75% ethanol, and store at -20℃ for 2h. (5) After centrifuging at 12000 rpm for 15 min at 4℃, discard the ethanol and air dry. Add 800 μL of TESAR to fully dissolve the precipitate, then add 800 μL of Bu / CTAB and Aq / CTAB respectively, and vortex vigorously for 2-5 min. (6) After centrifuging at 12000 rpm for 20 min at 4℃, transfer the supernatant to a new 1.5 mL centrifuge tube, add 350 μL of 3M sodium chloride, mix well and vortex for 1 min. (7) After centrifuging at 8000 rpm for 6 min at 4℃, transfer the lower layer liquid to a new 1.5 mL centrifuge tube, add 50 μL of 3M NaAc (pH 5.2) and 1 mL of anhydrous ethanol, mix well, and incubate at -20℃ overnight. (8) After centrifuging at 12000 rpm for 10 min at 4℃, dissolve the precipitate in 50 μL of LEPC water, store at 4℃ for 1 h to obtain the navel orange RNA solution, and freeze at -80℃.

[0075] (II) Reverse transcription to synthesize cDNA

[0076] After determining the concentration of RNA obtained from different samples, follow Hifair III 1 st The Strand cDNA SynthesisSuperMix for qPCR (Shanghai Yisheng Biotechnology Co., Ltd.) reverse transcription kit is used to synthesize cDNA via reverse transcription.

[0077] (III) Cloning of the CsZAT12 gene

[0078] The reference sequence of the CsZAT12 gene was obtained through transcriptome sequencing. Primers for cloning were designed using Premier 5.0 software. The primer pairs for gene amplification are as follows:

[0079] Overexpression primers:

[0080] CsZAT12-F1 (SEQ ID NO.3): 5'-ATGAAGAGAGATAGAGAAATGGCAG-3',

[0081] CsZAT12-R1 (SEQ ID NO.4): 5'-AGCGACCATAACTTCAAATC-3',

[0082] Silent primer:

[0083] CsZAT12-F2 (SEQ ID NO.5): 5'-GCTTTAGGCGGCCACAGAG-3',

[0084] CsZAT12-R2 (SEQ ID NO. 6): 5'-TTGGATTTCTTCAGAACGGGGAG-3'.

[0085] The full-length CDS of the target gene was amplified by PCR using cDNA from the peel of 'Newhall' navel oranges as a template. The PCR reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0086] Table 1. PCR reaction system for amplifying the CsZAT12 gene.

[0087]

[0088]

[0089] 0.2 g of agarose powder was added to 20 mL of 1×TAE gel, dissolved in a microwave oven, and cooled to a temperature that was not hot to the touch. 1.5 μL of nucleic acid dye was then added, poured into a gel plate, and allowed to solidify before spotting. DL 2000 was used as a marker for verification, resulting in a single, clear PCR band. The PCR product was verified by 1% agarose gel electrophoresis. The DNA fragment was then recovered using a purification and recovery kit (purchased from Novizan, following the instructions provided). After ligation with the pMD19-T vector, the fragment was transformed into competent DH5α cells of *E. coli*. Positive clones were screened on LB agar plates, and plasmids were extracted, digested with enzymes, and detected by PCR. The recombinant plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain the accurate target gene sequence.

[0090] (IV) Real-time quantitative PCR (q-PCR) detection and analysis

[0091] q-PCR primers were designed online using NCBI Primer-BLAST based on sequencing results. The real-time quantitative primer pair sequences are as follows:

[0092] Forward primer CsZAT12-qF (SEQ ID NO.7): 5'-TGACACCGCAAATTGCTTGA-3',

[0093] Reverse primer CsZAT12-qR (SEQ ID NO.8): 5'-GCTTGGAACGATGGGAACTC-3'.

[0094] q-PCR detection was performed using a quantitative real-time PCR instrument (T100 Thermal Cycle) to validate the relevant genes.

[0095] The reaction system had a total volume of 10 μL, including 1 μL cDNA, 0.3 μL forward primer, 0.3 μL reverse primer, 3.4 μL ddH2O and 5 μL TB Green;

[0096] The reaction conditions were: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 30s, 95℃ holding for 15s, repeated 39 times, 60℃ annealing for 30s (melting curve temperature), and 95℃ extension for 5s.

[0097] The method for calculating relative gene expression levels uses -2 △△Ct Law.

[0098] q-PCR results are as follows Figure 2 As shown, the expression level of CsZAT12 is positively correlated with the severity of postharvest Penicillium rot in navel oranges; the expression level increases with the occurrence of Penicillium rot, indicating that CsZAT12 may be a key gene promoting postharvest Penicillium rot in navel oranges. The sequencing results revealed that the full-length cDNA sequence of the CsZAT12 gene is 923 bp, containing a 480 bp open reading frame, and its nucleotide sequence is shown in SEQ ID NO.1. It encodes a protein of 159 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2.

[0099] SEQ ID NO.1: ATGAAGAGAGATAGAGAAATGGCAGCCATTGACACCGCAAA TTGCTTGATGCTGCTCTCAAAAGTTGGCGAAACAGACCAGGGAAAGCGTGTATTTGCATGCAAAACATGCAACAAAGAGTTCCCATCGTTCCAAGCTTTAGGCGGCCACAGAGCAAGTCACAAGAAGCCGAAGCTGATGACGATGGCTTCATCAGGGGAAGATTTTGATCAAGCCCAGATGCCACCAGCTTCCCCCAAGAAGCCTAAAACGCACGAGTGT TCGATTTGTGGTCTTGAGTTTGCTATTGGGCAAGCGCTCGGCGGTCACATGAGGAGACACAGGGCAGCAGCTGCCATGGGCGCCGCCGCCGATGGATTGGTGACTCGTCCTTTTGCAGCTCCCCGTTCTGAAGAAATCCAATAGTTGTAAAAGAGCTTTGTGCTTGGATTTGAACTTGATGCCTACTGGGGATGATTTGAAGTTATGGGTCGCTTAG;

[0100] SEQ ID NO.2: MKRDREMAAIDTANCLMLLSKVGETDQGKRVFACKTCNKEFP SFQALGGHRASHKKPKLMTMASSGEDFDQAQMPPASPKKPKTHECSICGLEFAIGQA LGGHMRRHRAAAAMGAAADGLVTRPLLQLPVLKKSSNSCKRALCLDLNLMPTGDD LKLWVA*.

[0101] Example 3

[0102] Subcellular localization of the CsZAT12 gene in navel orange

[0103] (I) Construction of Recombinant Carrier

[0104] Using PRI101-eGFP as the starting vector, with the insertion site between NdeI and BamHI, amplification primer pair was designed:

[0105] F (SEQ ID NO.9): 5'-GTTCTTCACTGTTGATACATATGAATGAAGAGAGATAG AGAAATGGCAG-3';

[0106] R (SEQ ID NO. 10): 5'-CTTGCTCACCATGGATCCAGCGACCCATAACTTCAA ATC-3'.

[0107] CsZAT12, with the stop codon removed, was amplified using PCR technology. The constructed DNA fragment was then purified and recovered. Following the instructions of the one-step cloning kit (Novizan), the product was mixed with double-digested PRI101-eGFP to prepare a recombination reaction. Finally, 10 μL of the recombinant product was added to 50 μL of DH5α competent E. coli cells, placed on ice for 30 min, heat-shocked at 42°C for 45 s, transformed into E. coli, plated, and incubated upside down at 37°C for 16 h. Positive single colonies were picked and sent to Kexin Biotechnology Co., Ltd. for sequencing verification to obtain the accurate target gene sequence.

[0108] (II) Agrobacterium-mediated transformation

[0109] Take 3 μL of the correctly sequenced CsZAT12-PRI101-eGFP plasmid DNA and mix it with 50 μL of GV1301 competent Agrobacterium cells. Incubate the mixture sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min. Resuspend the mixture in 700 μL of antibiotic-free LB agar for 2 h, then plate it and incubate at 28°C for 2 days. Select positive single clones, amplify them by PCR, and observe a single, clear band positioned consistent with the plasmid DNA. Store the culture at -80°C.

[0110] (III) Tobacco Leaf Infection

[0111] A quantity of tobacco seeds were sown and cultured under 12-hour light for one month before being used in experiments. Agrobacterium containing the CsERF017-PRI101-eGFP vector was resuspended in 10 mM MgCl2 (containing 120 μM MgSO4) suspension, and the OD was adjusted. 600 To achieve an antigen level of approximately 0.6, select healthy tobacco plants and inject the agrobacterium-injected bacteria into the lower epidermis of the leaves using a 1mL syringe (without the syringe tip). Label the injection site. Use the empty PRI101-eGFP vector as a control. Culture the injected tobacco plants under low light for 2 days. Collect the labeled Agrobacterium-injected tobacco leaves, prepare slides, observe under a laser confocal microscope, and photograph them.

[0112] Subcellular localization results as follows Figure 3 As shown, the empty vector PRI101-eGFP is distributed in both the nucleus and cell membrane of tobacco leaf epidermal cells. The localization of CsZAT12-PRI101-eGFP is consistent with the localization of the nuclear marker, indicating that CsZAT12 is a transcription factor with nuclear localization signal.

[0113] Example 4

[0114] Transient overexpression of the CsZAT12 gene in navel orange

[0115] (I) Construction of CsZAT12-PBI121 transient overexpression vector

[0116] Using PBI121 as the recombinant vector, with the insertion site between Xba1 and Bam HI, the amplification primer pair is as follows:

[0117] CsZAT12 upstream (SEQ ID NO.11): 5'-AGAACACGGGGGACTCTAGAATGAAGAGAGATAGAGAAATGGCAG-3',

[0118] CsZAT12 downstream (SEQ ID NO. 12): 5'-GACTGACCACCCGGGGATCCAGCGACCCATAACTTCAAATC-3'.

[0119] PCR amplification was performed using the designed primers. The recovered product was ligated into pMD19 and transformed into *E. coli*. Plasmid DNA was extracted and then digested with restriction enzymes. XbaI and BamHI were selected as the restriction enzymes for the double digestion assay, using a 50 μL digestion system. The components were: XbaI 1.5 μL, BamHI 1.5 μL, 10×M Buffer 5 μL, plasmid DNA 20 μL, and H2O 22 μL. This system was added to a 200 μL centrifuge tube, gently vortexed to mix, briefly centrifuged, and then incubated at 37°C for 3 h. The 50 μL digestion product was then analyzed by agarose gel electrophoresis. After successful detection, the remaining digestion product was recovered. The recovered product was then ligated into the PBI121 vector.

[0120] (II) Construction of CsZAT12-TRV2 transient overexpression vector

[0121] Using TRV2 as the recombinant vector, with the insertion site between XbaI and SamI, the amplification primer pair is as follows:

[0122] CsZAT12 upstream (SEQ ID NO.13): 5'-TGAGTAAGGTTACCGAATTCTCTAGAGCTTTAGGCGGCCACAGAG-3',

[0123] CsZAT12 downstream (SEQ ID NO. 14): 5'-TTTAATGTCTTCGGGACATGCCCGGGTTGGATTTCTTCAGAACGGGGAG-3'.

[0124] PCR amplification was performed using the designed primers. The recovered product was ligated into pMD19 and transformed into E. coli. After extracting plasmid DNA, the plasmid was digested with restriction enzymes. Restriction endonucleases XbaI and SamI were selected as the endonucleases for the double digestion experiment. The digestion system is shown in Table 3.

[0125] Table 3. PBI121 double enzyme digestion reaction system

[0126]

[0127] The system was added to a 200 μL centrifuge tube and incubated at 37 °C for 3 h. After the reaction was complete, 50 μL of the enzyme digestion product was detected by agarose gel electrophoresis. If the detection was successful, the remaining enzyme digestion product was recovered and ligated into the TRV2 vector.

[0128] (III) Instantaneous transformation of navel orange peel

[0129] (1) Preparation of IM (500mL) solution (prepare fresh for use): 5.137g of 4-morpholinoethanesulfonic acid (MES), 2.632g of glucose, and 0.164g of sodium dihydrogen phosphate, bring the volume to 500ml, adjust the pH to 5.6-5.7, sterilize and cool, then add 26.316ml of 20×AB Salts.

[0130] (2) 20×AB Salts (500mL, vortexing is required if precipitation occurs): 10g ammonium chloride, 3g magnesium sulfate heptahydrate, 1.5g potassium chloride, 0.1g calcium chloride, 0.025g ferrous sulfate heptahydrate.

[0131] (3) MES (10mM MgCl2, 10mM MES 500mL) solution: 0.475g magnesium chloride, 1.066g MES, bring to a final volume of 500mL, adjust pH to 5.5-5.6, and autoclave.

[0132] (4) Preparation of MMA suspension: First, prepare 0.1 mol / L LAS solution, 1 mol / L MgCl2 solution and 0.5 mol / L MES (fatty acid methyl ester sulfonate) solution. Take 2 mL of the prepared 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, so that the final concentrations of MES, MgCl2 and AS are 100 mmol / L, 10 mmol / L and 10 μmol / L respectively.

[0133] (5) Preparation of infection solution: Agrobacterium bacterial suspensions successfully obtained through previous transformation experiments were used, including successfully transformed CsZAT12-PBI121 and empty vector PBI121 bacterial suspensions, as well as CsZAT12-TRV2, empty vector TRV2, and empty vector TRV1. These were added to LB liquid culture medium containing kanamycin and rifampin and shaken on a shaker at 28°C and 200 rpm until the bacterial suspension had the best activity when the OD value at 600 nm wavelength was between 0.6 and 0.8. The activated bacterial suspension was then transferred to a 50 mL centrifuge tube and centrifuged at 4°C for 10 min at 5000 rpm. After centrifugation, the supernatant was discarded, and the bacterial cells were retained. The suspensions were then resuspended twice with an equal volume of MMA solution. CsZAT12-TRV2 and empty vector TRV2 were mixed with TRV1 in equal volumes, and the mixture of empty vector PBI121 and empty vector TRV2 with TRV1 served as the control for this experiment.

[0134] (6) Fruit peel injection

[0135] After rinsing and draining the navel oranges, soak them in 1.0% sodium hypochlorite solution for 2 minutes, rinse thoroughly with running water to ensure no disinfectant residue remains, and then air dry them in a sterilized laminar flow hood. Using a sterile needle, make a hole (3mm in diameter, 3mm deep) at the equator of each navel orange. Using a syringe without a needle, inject 0.5mL of Agrobacterium infection solution into the hole. Let it stand for 2 hours until the infection solution has completely penetrated close to the peel. Then, make another hole 1cm to the right of each previous hole and inoculate with 15μL of 1×10⁻⁶ Agrobacterium tumefaciens. 5 A suspension of Penicillium spores at CFU / mL was dried.

[0136] (7) Storage and Sampling

[0137] After the above treatment, the navel oranges were boxed (10 oranges per box), then packaged in polyethylene plastic bags and placed in a light-proof constant temperature incubator (temperature 27±1℃, relative humidity 90%~95%) for cultivation. Samples were randomly taken every other day for 3 consecutive days after inoculation. Simultaneously, the appearance of the fruit was observed for 1–5 days, and the diameter of lesions was measured. Peel tissue samples were also taken from within 10–20 mm of the wound on the fruit, chopped, flash-frozen in liquid nitrogen, and stored at -80℃ for subsequent indicator determination.

[0138] (III) Transient overexpression of GUS staining in juice cells

[0139] Using a GUS staining kit (Coolaber), X-Gluc solvent was first melted in a 40°C water bath. 1 mL of this solvent was then added to one tube of X-Gluc powder and mixed thoroughly to obtain a 50× GUS staining concentrate. 0.4 mL of this concentrate was then added to 5 mL of GUS staining buffer and mixed well. A 1 cm section of the wound from a 1-day-old fruit peel was immersed in the GUS staining solution and incubated overnight at 28°C. The appearance of small blue dots in the juice cells indicated GUS expression sites. Figure 4 (A)

[0140] (iv) Measurement of lesion diameter, disease index and hardness

[0141] The specific operating steps are the same as in Example 1; the hardness is measured using a handheld hardness tester.

[0142] (V) Determination of TA content and pH value

[0143] The specific operating steps are the same as in Example 1.

[0144] Experimental results showed that successful transformation of CsZAT12 accelerated the occurrence of postharvest Penicillium mold in navel oranges, while silencing CsZAT12 inhibited its occurrence. Figure 4 The difference was greatest at 2 days. Samples taken 2 days after inoculation were selected for subsequent quantitative fluorescence analysis and TA content determination. The results showed that PBI121-CsZAT12 (overexpression) increased the TA content in the pericarp, while CsZAT12-TRV2 (silence) had the opposite effect. Figure 5 The results suggest that CsZAT12 may reduce the resistance of navel oranges to Penicillium mold by promoting the regulation of organic acid synthesis in the peel.

[0145] Based on the descriptions in Examples 1, 2, 3, and 4, during the response of navel orange fruit to Penicillium infection, the TA content in the peel increased with the increase of the fruit disease index, and the expression level of CsZAT12 also increased accordingly, indicating that the CsZAT12 transcription factor in navel orange has a positive regulatory effect on the synthesis and accumulation of organic acids in the fruit. Subcellular localization of this transcription factor in Nicotiana benthamiana revealed that it has a nuclear localization signal. After transient overexpression of the recombinant vector CsZAT12-PBI121 in the peel of Newhall navel oranges, the severity of the disease increased, the TA content increased, and the firmness and pH value decreased. The results of CsZAT12-TRV2 were the opposite, indicating that CsZAT12 can promote the synthesis of organic acids and has a positive regulatory effect on the occurrence of Penicillium infection.

[0146] 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 of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of silencing or knocking out the zinc finger protein transcription factor CsZAT12 in navel orange in improving resistance to Penicillium rot in navel orange fruit, characterized in that, The amino acid sequence of the navel orange zinc finger protein transcription factor CsZAT12 is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The aforementioned Penicillium rot is a disease of navel orange fruit caused by Penicillium italicum.

3. Silence or knockout CsZAT12 The application of recombinant gene vectors, expression cassettes, transgenic cell lines, or recombinant bacteria in improving resistance to Penicillium rot in navel oranges is characterized by, The CsZAT12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for improving the resistance of navel orange fruit to Penicillium mold, characterized in that, Silence or Knockout CsZAT12 Genes that enhance the resistance of navel oranges to Penicillium mold; The CsZAT12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. Silence or knockout CsZAT12 The application of recombinant gene vectors, expression cassettes, transgenic cell lines, or recombinant bacteria in the cultivation of new navel orange varieties resistant to Penicillium mold is characterized by, The CsZAT12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. A method for cultivating a new variety of navel orange fruit resistant to Penicillium mold, characterized in that, Using silence or knockout CsZAT12 Recombinant gene vectors, expression cassettes, transgenic cell lines, or recombinant bacteria regulate the expression of genes in navel oranges. CsZAT12 Gene expression; The CsZAT12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. Silence or Knockout CsZAT12 The application of a gene or its encoded protein in enhancing the synthesis of organic acids in navel orange fruit, characterized by: The CsZAT12 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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