Application of navel orange zinc finger protein transcription factor CsZAT12 in regulation and control of fruit penicilliosis resistance

Abstract

The invention discloses application of a navel orange zinc finger protein transcription factor CsZAT12 in regulating and controlling fruit penicilliosis resistance, and belongs to the technical field of molecular biology and genetic engineering. Overexpression and silence are instantaneously transferred into Neuron navel orange peel through an agrobacterium infection method by an injection method, the silent CsZAT12 gene can remarkably control or reduce the occurrence probability of navel orange fruit penicilliosis, meanwhile, the increase of the titratable acid content in the peel is inhibited, and the verification result of the overexpression CsZAT12 gene is contrary to that of the navel orange fruit penicilliosis. Therefore, it is proved that the CsZAT12 gene participates in regulation of navel orange fruit penicilliosis resistance, research results are applied to navel orange molecular breeding, and candidate genes and a new method are provided for creation or improvement of navel orange disease-resistant new germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and genetic engineering, and in particular to application of navel orange zinc finger protein transcription factor CsZAT12 in regulating fruit resistance to penicillium mold disease. Background Art

[0002] Navel orange (Citrussinensis Osbeck) is a perennial evergreen fruit tree of the sweet orange family of the genus Citrus in the Rutaceae family. When the fruit is ripe, it has a beautiful shape, bright orange-red color, tender flesh, and rich flavor, which is deeply loved by consumers. In my country, the harvest period of most citrus fruits is mainly concentrated in October to November, and they are mainly sold fresh. During the post-harvest storage, transportation and sales process, they are very susceptible to infection by pathogenic fungi, causing the fruit to rot and deteriorate. Post-harvest penicillium disease of navel oranges caused by infection with Penicillium italicum has caused inestimable economic losses to the main navel orange producing areas in Jiangxi Province. Therefore, breeding disease-resistant varieties is one of the main goals of the citrus planting industry.

[0003] When pathogenic fungi infect plant tissues, they may secrete organic acids or ammonia that regulate pH to acidify or alkalinize the host environment, thereby enhancing their ability to infect. Based on this mechanism of action, pathogenic fungi can be divided into two categories: acidic fungi and alkaline fungi. Penicillium italicum is 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 be the following two points: (1) lowering the pH value of the host environment and increasing the activity of cell wall degrading enzymes (such as pectin esterase, polygalacturonase, β-galactosidase, cellulase, etc.) to weaken the function of the host cell wall; (2) chelating Ca 2+ Reduces host cell intercellular Ca 2+ activity, disrupting its mineral balance and leading to host cell death.

[0004] Transcription factors (TFs) are a class of proteins that regulate gene expression by recognizing cis-acting elements in gene promoters. Current studies have identified several TFs families related to plant stress response, including APETALA2 / ethylene response factor (AP2 / ERF), MYB, WRKY, bZIP, and C2H2 (Amorim et al, 2017). The C2H2-type zinc finger protein (ZFPs) TF family is divided into multiple subfamilies, of which the C1 family is one of the largest subfamilies. C1 family members have different numbers of dispersed zinc finger structures and are divided 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 and belongs to the C1-2i subclass of the C2H2-ZFP transcription factor family. Most ZAT family members also have highly conserved QALGGH motifs and EAR motifs at the C-terminus (Ding et al, 2023). At present, ZAT genes are widely involved in the response or resistance of plants to abiotic stresses such as drought, high salt, cold damage, and oxidative stress, but there are few studies on the disease resistance function of plant transcription factor ZAT in response to P. italicum infection. Summary of the invention

[0005] The purpose of the present invention is to provide an application of navel orange zinc finger protein transcription factor CsZAT12 in regulating fruit resistance to penicillium mold, 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 navel orange zinc finger protein transcription factor CsZAT12 in regulating fruit resistance to penicillium mold disease.

[0008] The second technical solution of the present invention is the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CsZAT12 gene in regulating plant resistance to penicillium disease.

[0009] The third technical solution of the present invention is a method for regulating plant resistance to penicillium disease, silencing or knocking out the CsZAT12 gene to improve the plant's resistance to penicillium disease; overexpressing the CsZAT12 gene to reduce the plant's resistance to penicillium disease.

[0010] A fourth technical solution of the present invention is the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CsZAT12 gene in breeding new plant varieties resistant to Penicillium disease.

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

[0012] The sixth technical solution of the present invention is the application of CsZAT12 gene or the protein encoded by it 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. The present invention screened and identified a transcription factor CsZAT12 that plays an important regulatory role in the accumulation of organic acids during the occurrence of postharvest Penicillium disease in 'Newhall' navel orange through transcriptome sequencing and weighted gene co-expression network analysis. The nucleotide sequence, amino acid sequence, and related vectors of the transcription factor were published for the first time, which is of great significance for clarifying the disease resistance function of the navel orange transcription factor ZAT in response to P. italicum infection and the breeding of disease-resistant varieties.

[0015] 2. The present invention, for the first time, uses molecular biological methods to confirm that CsZAT12 has a nuclear localization signal. Silencing the CsZAT12 gene can significantly enhance the fruit's resistance to Penicillium, effectively inhibit the increase in the titratable acid content in the peel, and effectively delay the adverse effects of P. italicum infection, thereby controlling the sourness process of the peel tissue. 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 a picture of the disease development of 'Newhall' navel orange after inoculation with Penicillium italicum. A is the fruit lesion phenotype, B is the measurement result of lesion diameter and disease index, and C is the measurement result of TA content and pH value.

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

[0019] Figure 3 This is the subcellular localization map of CsZAT12 in Nicotiana benthamiana leaves.

[0020] Figure 4Figure 1 is the effect of transient expression of CsZAT12 recombinant vector in navel orange peel on fruit. A is the effect of transient expression of CsZAT12 recombinant vector in navel orange peel on fruit phenotype, B is the effect of transient expression of CsZAT12 recombinant vector in navel orange peel on fruit hardness, C is the effect of transient expression of CsZAT12 recombinant vector in navel orange peel on fruit lesion diameter, and D is the effect of transient expression of CsZAT12 recombinant vector in navel orange peel on fruit disease index.

[0021] Figure 5 The figure shows the effect of transient expression of the recombinant vector in navel orange peel on the expression level of CsZAT12, TA content and pH value. A shows the effect of transient expression of the recombinant vector in navel orange peel on the expression level of CsZAT12, B shows the effect of transient expression of the recombinant vector in navel orange peel on the TA content, and C shows the effect of transient expression of the recombinant vector in navel orange peel on the pH value. 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 application of navel orange zinc finger protein transcription factor CsZAT12 in regulating the resistance of fruit to Penicillium mold disease.

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

[0030] In some specific embodiments, the penicillium is a plant disease caused by Penicillium italica; and the fruit comprises navel orange fruit.

[0031] In some specific embodiments, silencing or knocking out the CsZAT12 gene increases the plant's resistance to Penicillium wilt; overexpressing the CsZAT12 gene reduces the plant's resistance to Penicillium wilt.

[0032] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CsZAT12 gene in regulating plant resistance to Penicillium wilt.

[0033] In some specific embodiments, the starting vectors of the recombinant vector are PBI121 and TRV2, and the gene insertion sites are between Xba I and Bam HI and between Xba I and Sam I.

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

[0035] The embodiment of the present invention also provides a method for regulating plant resistance to penicillium disease, silencing or knocking out the CsZAT12 gene to improve the plant's resistance to penicillium disease; overexpressing the CsZAT12 gene to reduce the plant's resistance to penicillium disease.

[0036] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CsZAT12 gene in cultivating new plant varieties resistant to Penicillium disease.

[0037] The embodiment of the present invention also provides a method for breeding new plant varieties resistant to Penicillium disease, using a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the CsZAT12 gene to regulate the expression of the CsZAT12 gene in the plant.

[0038] The embodiments of the present invention also provide the use of CsZAT12 gene or the protein encoded by it in regulating the synthesis of plant organic acids.

[0039] In some specific embodiments, 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] The present invention clones a transcription factor CsZAT12 whose translation is induced by Penicillium italicum from Newhall navel orange, and constructs vectors such as subcellular localization, silencing and overexpression of ZAT12. Subcellular localization finds that the transcription factor is consistent with the nuclear marker localization and has a nuclear localization signal. Overexpression and silencing are transiently transferred into the peel of Newhall navel orange by injection through Agrobacterium infection. Silencing the CsZAT12 gene can significantly control or reduce the incidence of penicillium disease in navel orange fruit, while inhibiting the increase of titratable acid content in the peel. The verification result of overexpressing the CsZAT12 gene is the opposite. This proves that the CsZAT12 gene is involved in regulating the resistance of navel orange fruit to penicillium disease. The research results are applied to navel orange molecular breeding, providing candidate genes and new methods for the creation or improvement of new germplasm with disease resistance in navel orange.

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

[0042] The present invention provides an important C2H2 transcription factor CsZAT12, and studies the regulatory mechanism of organic acid metabolism during the occurrence of postharvest Penicillium in navel orange fruit, which is of great significance for analyzing the occurrence mechanism of postharvest resistance to Penicillium in navel orange. By performing weighted co-expression network analysis on the expression level of CsZAT12 in navel orange peel at different storage time points after inoculation, it is proved that there is a certain positive regulatory relationship between it and organic acid synthesis; at the same time, with the help of molecular biological methods and genetic transformation technology, it is further verified that silencing or knocking out CsZAT12 can inhibit organic acid synthesis and accumulation, and improve the defense ability of navel orange fruit against Italian Penicillium infection.

[0043] The fruit material involved in the present invention is 'Newhall' navel orange (Citrus sinensis L.cv'Newhall'), which was collected from the navel orange planting standard garden of Junping Fruit Industry in Nankang District, Ganzhou City, Jiangxi Province.

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

[0045] Example 1

[0046] Determination of Phenotypic Characteristic Indexes Related to Penicillium Disease in Postharvest Navel Orange

[0047] Select healthy fruits with the same size and maturity (single fruit weight: 300-350g, fruit shape index: 105-110%, solid acid ratio: 14.6-16.1), uniform coloring (citrus color difference index: 5.45-6.10), no mechanical damage, and no pests and diseases. Wash the navel orange fruit with clean water and drain it, soak it in 1.0% sodium hypochlorite for 2 minutes, rinse it with running water to ensure that there is no disinfectant water residue, place it on a sterilized clean bench for ventilation and dry it, use a sterilized needle to make a hole (3mm in diameter, 3mm in depth) at the equator of the navel orange fruit, wait for the wound to dry completely, then add 15μL of Italian Penicillium spore suspension, put it in a box after drying, and then pack it in a polyethylene plastic bag, and place it in a constant temperature box (temperature is 27±1℃, relative humidity is between 90% and 95%) for 4 days. At 0h, 24h, 48h, 72h and 96h after inoculation, random samples were taken to observe changes in fruit appearance, the diameter of the lesions was measured, and peel tissue samples were taken from 10-20mm outside the fruit wound, chopped, quick-frozen in liquid nitrogen, and stored at -80℃.

[0048] 1. Determination of lesion diameter and disease index

[0049] Lesion diameter: The diameter of navel orange lesions was determined by the cross method.

[0050] Disease index: When the diameter of the lesion is greater than 3mm, the fruit is defined as diseased fruit. The disease index uses the following scale:

[0051] Grade 0: lesion diameter = 0 mm (no decay);

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

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

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

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

[0056] The disease severity calculation formula is as follows:

[0057]

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

[0059] The TA content was determined by NaOH neutralization titration method: 2.0 g of 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 NaOH consumption, and the result was expressed in %.

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

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

[0062] Example 2

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

[0064] (I) Extraction of RNA from navel orange peel

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

[0066] (1) DEPC water (2 L): Take 2 mL of diethyl pyrocarbonate in a fume hood, dilute to 2 L with ultrapure water, and sterilize by high pressure overnight.

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

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

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

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

[0071] (6) Extraction Buffer (500 mL): 47.264 g of guanidine thiocyanate, 15.224 g of ammonium thiocyanate, 0.5 g of 8-hydroxyquinoline, 190 mL of water-saturated phenol, 25 mL of glycerol, and 16.7 mL of 3 M pH 5.2 sodium acetate (NaAC). Add DEPC water and stir thoroughly until completely dissolved. Make up to 500 mL and sterilize by high pressure.

[0072] (7) TESAR (100 mL): 1 M Tris-HCl (pH 7.6) 1 mL, 10% sodium lauryl sarcosine 10 mL, 0.5 M EDTA solution 200 μL, DEPC water to 100 mL, and sterilize by high pressure.

[0073] (8) Bu / CTAB and Aq / CTAB: Add 125 mL of DEPC water and 125 mL of n-butanol to a clean, RNAse-free separatory funnel. Let stand overnight and then separate the aqueous and organic phases (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 hexadecyltrimethylammonium bromide (CTAB), and then add 100 mL of the aqueous phase. Mix well, let stand, and sterilize under high pressure. The upper layer is Bu / CTAB and the lower layer is Aq / CTAB.

[0074] The detailed steps of RNA extraction are as follows: (1) Weigh 0.5g of peel sample powder into a 10mL centrifuge tube, add 5mL of buffer, immediately vortex for 2min to mix thoroughly, and let stand for 10min. (2) After centrifugation at 4℃ and 12000rpm for 20min, transfer the supernatant to a new centrifuge tube, add 5mL of chloroform, immediately vortex for 2min to mix thoroughly, and let stand for 10min. (3) After centrifugation at 4℃ and 12000rpm for 20min, transfer the supernatant to a new centrifuge tube, add an equal volume of ice-cold isopropanol, gently invert to mix the two liquids thoroughly, and let stand at room temperature for 10min. (4) After centrifugation at 4℃ and 12000rpm for 20min, discard the supernatant, collect the debris precipitate, soak it in 3mL of pre-cooled 75% ethanol, and store it at -20℃ for 2h. (5) After centrifugation at 4°C and 12000rpm for 15min, discard the ethanol and air-dry, add 800μL TESAR, fully dissolve the precipitate, then add 800μL Bu / CTAB and Aq / CTAB respectively, and vortex vigorously for 2-5min. (6) After centrifugation at 4°C and 12000rpm for 20min, transfer the supernatant to a new 1.5mL centrifuge tube, add 350μL 3M sodium chloride, mix and vortex for 1min. (7) After centrifugation at 4°C and 8000rpm for 6min, transfer the lower layer liquid to a new 1.5mL centrifuge tube, add 50μL 3M NaAc (pH5.2) and 1mL anhydrous ethanol, mix thoroughly, and place at -20°C overnight. (8) After centrifugation at 4°C and 12000rpm for 10min, dissolve the precipitate in 50μL DEPC water, store at 4°C for 1h to obtain the navel orange RNA solution, and freeze it in a -80°C refrigerator.

[0075] (ii) Reverse transcription to synthesize cDNA

[0076] After the concentration of RNA obtained from different samples was determined, st cDNA was synthesized by reverse transcription according to the instructions of the Strand cDNA SynthesisSuperMix for qPCR reverse transcription kit (Shanghai Yisheng Biotechnology Co., Ltd.).

[0077] (III) Cloning of CsZAT12 gene

[0078] The reference sequence of the CsZAT12 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:

[0079] Overexpression primers:

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

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

[0082] Silencing Primers:

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

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

[0085] The cDNA in the peel of 'Newhall' navel orange was used as a template to amplify the full length of the target gene CDS by PCR. 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 CsZAT12 gene

[0087]

[0088]

[0089] 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. The sample was then spotted after solidification, and DL 2000 was used as a marker for verification, resulting in 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 Company for sequencing in order to obtain the accurate target gene sequence.

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

[0091] According to the sequencing results, q-PCR primers were designed online on NCBI Primer-BLAST. The sequences of the real-time fluorescence quantitative primer pairs 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 uses a fluorescent quantitative PCR instrument (T100 Thermal Cycle) to verify the relevant genes:

[0095] The total volume of the reaction system was 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 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.

[0097] The relative gene expression was calculated using the -2 △△Ct Law.

[0098] q-PCR results Figure 2 As shown, the expression level of CsZAT12 is positively correlated with the incidence of postharvest Penicillium disease in navel oranges. The expression level increases with the occurrence of Penicillium disease, indicating that CsZAT12 may be a key gene promoting postharvest Penicillium disease in navel oranges. According to the sequencing results, the cDNA sequence of the CsZAT12 gene is 923 bp in length, including a 480 bp open reading frame, the nucleotide sequence of which is shown in SEQ ID NO.1, and can encode a protein of 159 amino acid residues, the amino acid sequence of which 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 CsZAT12 gene in navel orange

[0103] (I) Construction of recombinant vector

[0104] Using PRI101-eGFP as the starting vector, the insertion site was between NdeΙ and BamHI, and the 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, 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, 10 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.

[0108] (II) Agrobacterium transformation

[0109] Take 3 μL of CsZAT12-PRI101-eGFP plasmid DNA with correct sequence confirmation and mix it with 50 μL of GV1301 Agrocybe stoloniferum competent cells, place it on ice for 5 minutes, place it in liquid nitrogen for 5 minutes, place it in a 37°C water bath for 5 minutes, and place it in an ice bath for 5 minutes. Resuspend it in 700 μL of 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 -80°C refrigerator for bacteria preservation.

[0110] (III) Tobacco leaf infection

[0111] 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 CsERF017-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.

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

[0113] Example 4

[0114] Transient Overexpression of CsZAT12 Gene in Navel Orange

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

[0116] PBI121 was used as the recombinant vector, the insertion site was between Xba Ι and Bam HI, and the amplification primer pair was:

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

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

[0119] According to the designed primers, PCR amplification was performed, and the recovered product was connected to pMD19 and 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 50μL system. The components were: XbaΙ1.5μL, Bam HI l.5μL, 10×M Buffer 5μL, plasmid DNA 20μL, H2O 22μL. After adding the system to a 200μL centrifuge tube, it was gently shaken and mixed and then centrifuged briefly, and then warmed at 37°C for 3h. The 50μL digestion product after the reaction was detected by agarose gel electrophoresis, and the remaining digestion product was recovered after successful detection. The recovered product was then connected to the PBI121 vector.

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

[0121] TRV2 was used as the recombinant vector, the insertion site was between XbaI and SamI, and the amplification primer pair was:

[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 according to the designed primers, and the recovered product was ligated to pMD19 and transformed into Escherichia coli. After plasmid DNA was extracted, the plasmid was digested by restriction endonucleases XbaΙ and SamI as the endonucleases for double digestion test. The digestion system is shown in Table 3.

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

[0126]

[0127] After adding the system to a 200 μL centrifuge tube, the tube was incubated at 37° C. for 3 h. After the reaction was completed, 50 μL of the enzyme cleavage product was detected by agarose gel electrophoresis. After successful detection, the remaining enzyme cleavage product was recovered and connected to the TRV2 vector.

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

[0129] (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.

[0130] (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.

[0131] (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.

[0132] (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.

[0133] (5) Preparation of infection fluid: Use Agrobacterium culture fluid successfully obtained in the previous transformation experiment, including the culture fluid of successfully transformed CsZAT12-PBI121 and empty vector PBI121, as well as CsZAT12-TRV2, empty vector TRV2 and empty vector TRV1, and add them to LB liquid culture medium containing kanamycin and rifampicin. Shake on a shaker at 28°C and 200 rpm until the OD value of the culture fluid at a wavelength of 600nm is between 0.6-0.8, when the activity of the culture fluid is the best. Transfer the activated culture fluid to a 50mL centrifuge tube and centrifuge 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 equal volumes of MMA solution. CsZAT12-TRV2 and empty vector TRV2 are mixed with equal volumes of TRV1 respectively. The mixture of empty vector PBI121 and empty vector TRV2 with TRV1 is used as the control of this experiment.

[0134] (6) Peel injection

[0135] After washing and draining the navel orange fruit with clean water, soak it in 1.0% sodium hypochlorite for 2 minutes, rinse it with running clean water to ensure that there is no disinfectant residue, and place it on a sterilized clean bench for ventilation and drying. Use a sterilized needle to make a hole at the equator of the navel orange fruit (3mm in diameter and 3mm in depth), use a syringe without a needle to draw 0.5mL of the Agrobacterium infection solution and inject it into the hole. Let it stand for 2 hours until the infection solution completely penetrates the peel, then make another hole 1cm to the right of each hole and inoculate 15μL of 1×10 5 CFU / mL of Penicillium italicum spore suspension, air-dried.

[0136] (7) Storage and sampling

[0137] After the above treatment, the navel orange fruits were packed in boxes (10 / box), then packed with polyethylene plastic bags, and placed in a light-proof constant temperature box (temperature of 27±1°C, relative humidity between 90% and 95%) for cultivation. Random samples were taken every other day after inoculation for 3 consecutive days. At the same time, the appearance changes of the fruits were observed for 1 to 5 days, the diameter of the lesions was measured, and the peel tissue samples were taken from 10 to 20 mm outside the wound of the fruit, chopped, quickly frozen in liquid nitrogen, and stored at -80°C for the determination of subsequent indicators.

[0138] (III) GUS staining of transient overexpressed juice cells

[0139] 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. Take 1 cm of the wound from the peel that has been stored for 1 day and soak it in the GUS staining solution. Keep it in a 28°C incubator overnight. The blue dots that appear in the juice cells are the GUS expression sites ( Figure 4 (A).

[0140] (IV) Determination of lesion diameter, disease index and hardness

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

[0142] (V) TA content and pH value determination

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

[0144] The experimental results showed that after successful transformation of CsZAT12, overexpression of CsZAT12 accelerated the occurrence of postharvest Penicillium disease in navel oranges, while silencing of CsZAT12 inhibited the occurrence of Penicillium disease ( Figure 4 ), the difference was the largest at 2d. Samples taken 2d after inoculation were selected for subsequent fluorescence quantitative analysis and TA content determination. The results showed that PBI121-CsZAT12 (overexpression) increased the TA content in the peel, while CsZAT12-TRV2 (silence) had the opposite effect ( Figure 5 ), indicating that CsZAT12 may reduce the resistance of navel orange fruit to Penicillium wilt by promoting and regulating the synthesis of organic acids in the peel of navel orange.

[0145] Based on Examples 1, 2, 3, and 4, in the process of navel orange fruit responding to Penicillium italicum 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 navel orange CsZAT12 transcription factor has a positive regulatory effect on the synthesis and accumulation of organic acids in the fruit; the transcription factor was subcellularly localized in Nicotiana benthamiana, and it was found that it had a nuclear localization signal; after the recombinant vector of CsZAT12-PBI121 was transiently overexpressed in the peel of Newhall navel orange, the severity of the disease was aggravated, the TA content increased, and the hardness 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 regulation on the occurrence of Penicillium disease.

[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 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. Application of navel orange zinc finger protein transcription factor CsZAT12 in regulating fruit resistance to Penicillium wilt.

2. The use according to claim 1, 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.

3. The use according to claim 1, characterized in that: The penicillium disease is a plant disease caused by Penicillium italicum; and the fruit includes navel orange fruit.

4. The use according to claim 1, characterized in that: Silencing or knocking out the CsZAT12 gene improves the plant's resistance to Penicillium; overexpressing the CsZAT12 gene reduces the plant's resistance to Penicillium.

5. Use of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsZAT12 gene in regulating plant resistance to Penicillium wilt.

6. A method for regulating plant resistance to Penicillium spp., characterized in that: Silencing or knocking out the CsZAT12 gene improves the plant's resistance to Penicillium; overexpressing the CsZAT12 gene reduces the plant's resistance to Penicillium.

7. The use of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsZAT12 gene in breeding new plant varieties resistant to Penicillium disease.

8. A method for breeding new plant varieties resistant to Penicillium disease, characterized in that: The expression of the CsZAT12 gene in the plant is regulated by using a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CsZAT12 gene.

9. Application of CsZAT12 gene or the protein it encodes in regulating plant organic acid synthesis.

10. The use according to claim 9, characterized in that: Silencing or knocking out the CsZAT12 gene promotes the synthesis of organic acids in plants; overexpressing the CsZAT12 gene inhibits the synthesis of organic acids in plants.

Images