A transcription factor ppcbp60b affecting resistance of plants to alternaria alternata and encoding gene and application thereof

Abstract

The application discloses a transcription factor PpCBP60B affecting the resistance of plants to alternaria alternata, and an encoding gene and application thereof. The amino acid sequence of the transcription factor PpCBP60B is shown as SEQ ID NO:1, and the nucleotide sequence of the encoding gene, the transcription factor gene PpCBP60B, is shown as SEQ ID NO:2. The gene PpCBP60B is introduced into plants for overexpression, the obtained transgenic callus can effectively improve the alternaria alternata resistance of the transgenic plants compared with wild callus, and the cell damage is smaller; meanwhile, the gene is silenced, the obtained transgenic plants weaken the alternaria alternata resistance of the transgenic plants compared with wild plants, and the lesion diameter is larger. Therefore, the transcription factor PpCBP60B provided by the application has important significance for cultivating new varieties of plants resistant to alternaria alternata and improving the tolerance of plants to alternaria alternata.

Description

Technical Field

[0001] This invention belongs to the field of functional gene technology, specifically relating to a transcription factor PpCBP60B that affects plant resistance to black spot disease, its encoding gene, and its applications. Background Technology

[0002] Plants cannot actively avoid adverse environmental stresses, therefore they have evolved a series of internal response mechanisms to cope with various adverse environmental stresses. Plants have a gene regulatory network that can rapidly induce the expression of stress-related genes when exposed to external stress signals, thereby responding to various stressful environments. Transcription factors are a crucial part of this regulatory network; they respond to various external signals, thereby activating or inhibiting the expression of downstream genes, thus regulating the plant's resistance to various environmental changes.

[0003] Black spot disease is a group of diseases caused by the fungus *Heliotropium indicum*, which infects the fruits, leaves, flower spikes, and branches of plants, leading to localized necrosis of the infected tissues. It has a wide range of impacts, affecting fruit trees, vegetables, and other crops, as well as forest trees and flowers. The conidia of *Heliotropium indicum* can be spread frequently throughout the year via rain, wind, and insects. Furthermore, the fungus can infect plants at different growth stages, including leaf growth, flowering, and fruiting, directly impacting plant growth and yield. Therefore, the control of black spot disease is a significant concern in agricultural cultivation. Unfortunately, there is currently no method to regulate transcription factor expression to control black spot disease. Summary of the Invention

[0004] To address the technical problems raised in the background art, the present invention aims to provide a transcription factor PpCBP60B, the transcription factor gene PpCBP60B encoding the transcription factor PpCBP60B, and its applications. Studies have shown that overexpression of the aforementioned transcription factor gene PpCBP60B increases plant resistance to black spot disease, while knocking out or silencing the gene makes the plant susceptible to black spot disease.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention seeks protection for a transcription factor PpCBP60B, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Secondly, the present invention seeks protection for the transcription factor gene PpCBP60B encoding the transcription factor PpCBP60B, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0008] Thirdly, the present invention claims protection for primers used to amplify the transcription factor gene PpCBP60B, the primers comprising a forward primer with a nucleotide sequence as shown in SEQ IN NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ IN NO:4.

[0009] Fourthly, the present invention claims protection for a biological material containing the transcription factor gene PpCBP60B, or a biological material used to interfere with, inhibit, silence or knock out the transcription factor gene PpCBP60B, wherein the biological material is an expression cassette, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0010] Furthermore, the biological material containing the transcription factor gene PpCBP60B is at least one of (1) to (7):

[0011] (1) An expression cassette containing the transcription factor gene PpCBP60B;

[0012] (2) A recombinant expression vector containing the transcription factor gene PpCBP60B;

[0013] (3) A recombinant expression vector containing the expression cassette described in (1);

[0014] (4) Recombinant bacteria containing the transcription factor gene PpCBP60B;

[0015] (5) Recombinant bacteria containing the expression cassette described in (1);

[0016] (6) Recombinant bacteria containing the recombinant expression vector described in (2);

[0017] (7) Recombinant bacteria containing the recombinant expression vector described in (3).

[0018] Furthermore, the biological material used to interfere with, inhibit, silence, or knock out the transcription factor gene PpCBP60B is a viral silencing vector or a recombinant bacterium containing the viral silencing vector.

[0019] Fifthly, the present invention seeks protection for the use of the above-mentioned transcription factor PpCBP60B, the above-mentioned transcription factor gene PpCBP60B, or the above-mentioned biological material in (I) to (IV):

[0020] (I) Improve plant resistance to black spot disease;

[0021] (II) Downregulates plant resistance to black spot disease;

[0022] (III) Develop new plant varieties resistant to black spot disease;

[0023] (IV) Develop new plant varieties that are sensitive to black spot disease.

[0024] Furthermore, the transcription factor gene PpCBP60B as described in claim 2 is overexpressed or overexpressed in the target plant to improve plant resistance to black spot disease or to cultivate new plant varieties resistant to black spot disease; or the transcription factor gene PpCBP60B as described in claim 2 is silenced or knocked out in the target plant to downregulate plant resistance to black spot disease or to cultivate new plant varieties susceptible to black spot disease.

[0025] Sixthly, the present invention claims protection for the use of a reagent for detecting the expression level of the transcription factor gene PpCBP60B in assessing plant resistance to black spot disease and its pathogens.

[0026] In a seventh aspect, the present invention claims protection for a method for improving plant resistance to black spot disease or for breeding new plant varieties resistant to black spot disease, wherein the transcription factor gene PpCBP60B is overexpressed or overexpressed in the target plant to improve plant resistance to black spot disease or to breed new plant varieties resistant to black spot disease.

[0027] The plant described in this invention is a dicotyledonous plant, preferably a plant of the Rosaceae family, and more preferably a pear, but not limited thereto.

[0028] The beneficial effects of this invention are:

[0029] This invention screened a transcription factor, PpCBP60B, that exhibits resistance to black spot disease and provided the transcription factor gene PpCBP60B encoding this transcription factor. During the research, the transcription factor gene PpCBP60B was transferred into pear callus tissue. The resulting overexpressed transgenic plants exhibited resistance to black spot fungus. The gene PpCBP60B can serve as a marker of black spot fungus resistance, providing early warning of fungal infection in plants. This invention also knocked out or silenced the gene PpCBP60B to reduce its expression level, causing plants to exhibit resistance to black spot disease infection. Experimental results showed that the transcription level of the gene PpCBP60B gradually increased after plants were treated with black spot fungus, indicating that the transcription factor gene PpCBP60B plays an important role in the plant's resistance to black spot disease. This invention involves transferring the transcription factor gene PpCBP60B into pear callus tissue. The resulting transgenic plants exhibit overexpression of the PpCBP60B gene. Compared to wild-type callus tissue, the transgenic callus tissue effectively enhances the transgenic plants' resistance to black spot disease, resulting in less cell damage. Conversely, gene silencing of PpCBP60B reduces the transgenic plants' resistance to black spot disease compared to wild-type plants, resulting in larger lesion diameters. Therefore, the transcription factor PpCBP60B and its gene provided by this invention are of significant importance for breeding new black spot-resistant varieties and for researching plant tolerance to black spot fungus. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the technical process of the present invention.

[0031] Figure 2 The expression patterns of the transcription factor gene PpCBP60B (Pbr000282.1) in the susceptible variety 'July Red Fragrance' and the resistant variety 'Weining Fragrant Noodles' under black spot disease treatment were shown.

[0032] Figure 3 Subcellular localization of transcription factor PpCBP60B.

[0033] Figure 4 The results of identifying callus tissue overexpressing the transcription factor gene PpCBP60B.

[0034] Figure 5 The results of the identification of plants with the PpCBP60B transcription factor gene silenced;

[0035] Figure 6 Phenotypic and physiological results of callus treatment with overexpression of the transcription factor gene PpCBP60B;

[0036] Among them, A is a comparison of the growth rate of mycelium in pear callus overexpressing the PpCBP60B gene and wild-type pear callus; B is a comparison of the diameter of lesions in pear callus overexpressing the PpCBP60B gene and wild-type pear callus; C is a comparison of the hydrogen peroxide content in pear callus overexpressing the PpCBP60B gene and wild-type pear callus; D is a comparison of the phenylalanine ammonia-lyase (PAL) activity in pear callus overexpressing the PpCBP60B gene and wild-type pear callus; E is a comparison of the chitinase activity in pear callus overexpressing the PpCBP60B gene and wild-type pear callus; F is a comparison of the POD activity in pear callus overexpressing the PpCBP60B gene and wild-type pear callus.

[0037] Figure 7 Phenotypic and physiological results of plants treated with black spot disease with PpCBP60B transcription factor gene silencing.

[0038] In this image, A shows lesion photos of leaves from PpCBP60B silent plants and the control group; B shows a comparison of lesion diameters between leaves from PpCBP60B silent plants and the control group. Detailed Implementation

[0039] This invention provides a transcription factor PpCBP60B that affects plant resistance to black spot disease, the amino acid sequence of which is shown in SEQ ID NO:1.

[0040] MQTRYMERSNSMAREKRSLDSSSAEEGQPDRKRPALASVIVEALKVDSLQKLCSSLEPIL

[0041] RRVVSEEVERALAKLGPAKLTGRSSPKQIEGPDGRNLQLHFRSRLSLPLFTGGKVEGEWG

[0042] SAIPIVLIDANTKNVVTSGPESAVKLDVVVLEGDFNNEDDENWTEEEFESHVVKEREGK

[0043] RPLLTGDLQVTLKEGVGTLGELTFTDNSSWIRSRKFRLGLKVASGYCDNIRIREAKTDAF

[0044] TVKDHRGELYKKHYPPALNDEVWRLEKIGKDGSFHKRLNKAGIVTVEDFLRLVVRDSQ

[0045] RLRNILGSGMSNKMWDVLIQHAKTCLLGGKLYVYYPEDARNVGVVFNNIYELSGLITN

[0046] EQFYSADSLSEDQKVYVDGLVTKAYENWMHVMEYDGKSLLKQQKSPDVSLPEVPIASQ

[0047] DYPNSFDQQFTLPSLPASVSSEPPTMDSGLNVGGYTDGMANRFSVQSQNLNLSAPSQLD

[0048] GLSFPLQNQQPITSHQSQFQGNENMLALGPPQSSTSGFQNIGTSNPSSYRGAEDLFPEEEI

[0049] RMRSHEILENEDMQHLLRIFNMGGQGQGQGHGYGHASMNITEDNYPYSSPYMPTPQVNYSVDDQSRSSGKAVVGWLKLKAALRWGIFIRKKAAERRAQLVELDDSG (SEQ ID NO: 1).

[0050] The present invention provides a transcription factor gene PpCBP60B encoding the above-mentioned transcription factor PpCBP60B, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0051] ATGCAGACGAGGTATATGGAGAGATCGAATAGCATGGCGAGGGAGGAAGCGAAGTTTG

[0052] GATTCGTCTTCAGCTGAAGAAGGCCAGCCGGATAGGAAACGGCCTGCTCTCGCCAGT

[0053] GTAATTGTTGAAGCTCTCAAGGTGGATAGTCTGCAGAAGCTTTGCTCGTTCACTGGAG

[0054] CCGATTCTACGCAGAGTTGTTAGTGAAGAGGTGGAGCGTGCTTTAGCAAAATTAGGC

[0055] CCTGCCAAACTTACTGGAAGGTCTTCTCCTAAACAAATTGAAGGGCCCGATGGAAGA

[0056] AACTTGCAGCTGCACTTTAGGTCCAGGTTGTCCCTTCCTCTTTTTTACTGGTGGAAAAG

[0057] TAGAAGGGGGAGTGGGGTTCTGCAATCCCCTATTGTCTTGATTGATGCAAATACAAAGAA

[0058] CGTTGTGACATCAGGCCCGGAGTCAGCAGTGAAACTTGACGTTGTTGTGCTTGAAGG

[0059] TGATTTTAACAACGAGGATGATGAGAACTGGACTGAAGAAGAATTTGAGAGCCATGT

[0060] AGTAAAAGAGCGTGAAGGAAAGAGGCCACTTCTTACTGGCGATCTGCAAGTGACAC

[0061] TGAAGGAAGGTGTAGGAACACTAGGCGAATTGACATTTACCGATAATTCTAGCTGGAT

[0062] TAGGAGCAGGAAGTTTTAGGCTAGGACTGAAAGTAGCCTCTGGCTATTGCGACAACAT

[0063] TCGTATACGCGAAGCAAAAACAGATGCCTTTACTGTTAAGGATCACCGTGGGGAATTA

[0064] TACAAGAAACACTACCCTCCTGCGTTAAATGATGAGGTCTGGAGATTGGAGAAAATT

[0065] GGAAAAGATGGCTCATTCCACAAGAGACTGAATAAAGCTGGAATTGTTACAGTTGAA

[0066] GACTTCCTGCGACTTGTGGTTAGAGACTCGCAGAGATTGCGGAATATTCTTGGAAGTG

[0067] GCATGTCGAATAAGATGTGGGATGTACTCATACAGCATGCAAAAACTTGTCTTCTTGG

[0068] CGGGAAACTCTATGTCTACTATCCTGAGGATGCAAGGAACGTTGGTGTTGTTTTTAAC

[0069] AATATCTACGAGTTGAGTGGCCTAATTACCAATGAACAATTTTACTCAGCTGATTCTCT

[0070] CTCGGAAGATCAGAAGGTCTACGTGGATGGTTTGGTAACAAAGGCATATGAGAACTG

[0071] GATGCATGTTATGGAGTACGATGGAAAGTCTCTTCTTAAGCAGCAGAAGAGTCCCGAT

[0072] GTTTCTCTACCTGAGGTCCCAATAGCCTCGCAGGATTATCCAAACTCATTTGATCAGC

[0073] AGTTCACTCTACCTAGCCTGCCAGCTTCAGTTTCTTCAGAACCGCCTACTATGGATTCT

[0074] GGCCTAAATGTGGGAGGTTATACTGACGGCATGGCTAACAGATTCTCGGTACAGTCAC

[0075] AGAATCTAAATCTCAGTGCTCCCAGTCAGCTCGATGGCTTGTCATTCCCTCTACAAAA

[0076] TCAGCAGCCTATCACTTCACACCAGTCTCAGTTTCAAGGAAATGAAAACATGCTTGC

[0077] CCTTGGTCCACCACAGTCGTCCACATCTGGGTTCCAGAATATTGGTACATCCAATCCAT

[0078] CTTCTTATAGGGGAGCCGAGGACCTCTTCCCAGAGGAAGAAATTCGTATGAGGAGCC

[0079] ACGAGATTCTCGAAAATGAAGATATGCAGCATCTACTTCGTATCTTTAACATGGGCGG

[0080] TCAGGGGTCAGGGTCAAGGCCACGGTTATGGTCATGCCTCCATGAACATCACTGAAGA

[0081] CAATTATCCATATTCGTCACCATACATGCCCACTCCACAAGTGAACTACAGTGTTGATG

[0082] ATCAGAGCCGTTCATCTGGGAAAGCTGTTGTTGGCTGGCTCAAGCTCAAGGCAGCCC

[0083] TTAGATGGGGCATATTCATCAGAAAGAAGGCTGCTGAGAGACGGGCACAGCTTGTTGAGTTGGATGATTCAGGATAA (SEQ ID NO: 2).

[0084] This invention screened a transcription factor gene, PpCBP60B, from *Pyrus pyrifolia* that exhibits resistance to black spot disease. The PpCBP60B transcription factor gene provided by this invention plays a positive regulatory role in plant black spot disease stress and has an important function in combating various biotic stresses.

[0085] The present invention also provides a primer for amplifying the transcription factor gene PpCBP60B, the primer comprising a forward primer with a nucleotide sequence as shown in SEQ IN NO:3 (GAGAACACGGGGGACTCTAGA ATGCAGACGAGGTATATGG) and a reverse primer with a nucleotide sequence as shown in SEQ IN NO:4 (GCCCTTGCTCACCATGGATCC TGAATCATCCAACTCAACAAGCTGT).

[0086] This invention does not impose any special restrictions on the design method of the primers; they can be synthesized by biosynthetic companies well-known in the art. In this embodiment of the invention, the primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0087] This invention provides a recombinant expression vector containing the transcription factor gene PpCBP60B and a silencing vector for silencing the transcription factor gene PpCBP60B. In this invention, the backbone vector of the recombinant expression vector is an expression plasmid (PCMBIA1300), and the backbone vector of the silencing vector is a viral silencing vector (pTRV2). The multiple cloning sites for the PpCBP60B gene insertion in the recombinant expression vector PCMBIA1300-PpCBP60B are two restriction enzyme sites: Xbal I and BamHI. The multiple cloning sites for the PpCBP60B gene fragment sequence insertion in the silencing vector pTRV2-PpCBP60B are two restriction enzyme sites: Xbal I and SmaI. This invention does not impose any particular limitations on the preparation methods of the recombinant expression vector and the silencing vector; any method well-known in the art for constructing recombinant expression vectors and silencing vectors can be used.

[0088] The present invention provides a recombinant bacterium containing the transcription factor gene PpCBP60B or the recombinant expression vector.

[0089] In this invention, the host of the recombinant bacteria preferably includes Agrobacterium. This invention does not impose any particular limitation on the preparation method of the recombinant bacteria; any method well-known in the art for preparing recombinant bacteria may be used.

[0090] This invention provides the application of the transcription factor PpCBP60B, the transcription factor gene PpCBP60B, the recombinant expression vector, the viral silencing vector, or the recombinant bacteria in regulating plant resistance to black spot disease.

[0091] In this invention, the plant is preferably a dicotyledonous plant. The plant preferably includes callus tissue and pear (e.g., *Pyrus pyrifolia*). This invention uses *Pyrus pyrifolia* as a representative plant to illustrate the application of the gene PpCBP60B in regulating plant resistance to black spot disease. Experiments have shown that when the gene PpCBP60B is highly expressed in a plant, the plant exhibits higher resistance to black spot disease and is more susceptible to it. When the gene PpCBP60B is downregulated or silenced in a plant, the plant's resistance to black spot disease decreases, making it more susceptible to the disease. Simultaneously, experiments have shown that recombinant expression of the transcription factor PpCBP60B or its encoding gene, or the encoding gene cloned from the primer pair, in plants results in recombinant plants with resistance to black spot disease. Silencing the transcription factor PpCBP60B or its encoding gene, or the encoding gene cloned from the primer pair, in plants weakens the plant's resistance to black spot disease. Therefore, by targeting the gene PpCBP60B and regulating its expression level, it is possible to regulate plant resistance and disease tolerance to black spot disease (e.g., Figure 1 (Schematic diagram of the technical process of this invention).

[0092] Based on the relationship between the expression level of the above-mentioned gene PpCBP60B and the plant's resistance to black spot disease, this invention provides a reagent for detecting the expression level of the transcription factor gene PpCBP60B in the application of assessing the plant's resistance to black spot disease and its pathogens.

[0093] This invention provides a reagent for overexpressing the transcription factor gene PpCBP60B in improving the resistance of plants to black spot disease or for breeding plant varieties resistant to black spot disease. The reagent preferably comprises an overexpression vector (PCMBIA1300-PpCBP60B) containing the PpCBP60B gene sequence.

[0094] In this embodiment of the invention, physiological index measurements revealed that pear callus tissue overexpressing PpCBP60B was more resistant to black spot disease infection than the control, while pear leaves with silenced PpCBP60B were more susceptible to black spot disease infection than the control. This indicates that upregulating the expression of the PpCBP60B gene is beneficial for improving the plant's resistance to black spot disease. Furthermore, constructing transgenic plants overexpressing the PpCBP60B gene allows for the cultivation of a new variety resistant to black spot disease.

[0095] The room temperature described in this invention is 25±10℃.

[0096] The following detailed description, in conjunction with embodiments, illustrates a transcription factor PpCBP60B that influences plant resistance to black spot disease, its encoding gene, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0097] Example 1: Cloning of the full-length cDNA of the Pyrus pyrifolia transcription factor gene PpCBP60B

[0098] A transcription factor gene, PpCBP60B, was screened from *Pyrus pyrifolia*. Primers were designed based on the sequence of the PpCBP60B gene and Primer Premier 5.0, and its full-length cDNA was amplified from *Pyrus pyrifolia* using RT-PCR. The detailed steps are as follows: The synthesis of the first strand of cDNA was performed according to the instructions of the TIANGEN reverse transcription kit. The obtained first-strand cDNA was used for the amplification of the transcription factor gene PpCBP60B. The total volume of the PCR reaction was 50 μl, including 1 μl of *Pyrus pyrifolia* cDNA, 2.5 μl each of forward and reverse primers (SEQ ID NO:3 and SEQ ID NO:4), 1 μl of Taq enzyme, 25 μl of buffer, and 18 μl of ddH2O. PCR was performed according to the following procedure: 1. Pre-denaturation at 94℃ for 3 min; 2. Denaturation at 94℃ for 30 s; 3. Annealing at 58℃ for 30 s; 4. Extension at 72℃ for 55 s; repeat steps 2-4 for 35 cycles; 5. Extension at 72℃ for 10 min after cycling; 6. Final storage at 4℃ for 30 min. After amplification, a single-band PCR product was generated. After electrophoresis on a 1% agarose gel, the target band was excised and recovered according to the instructions of the gel extraction kit.

[0099] The purified product was ligated into the PCMBIA1300 vector at a gene-to-vector molar ratio of 2:1. The total reaction volume was 5 μl, consisting of 4.5 μl of the purified PCR product and 0.5 μl of the vector. Ligation was performed at 25°C for 30 min, followed by transformation into competent E. coli DH5α cells using the heat shock method. PCR verification and sequencing were performed using primers for the target gene sequence (performed by Shanghai Sangon Biotech Co., Ltd.).

[0100] Example 2: qRT-PCR analysis of transcription factor gene PpCBP60B under biological stress conditions

[0101] To analyze the response pattern of the PpCBP60B gene in pear to black spot disease treatment, the expression pattern of the PpCBP60B gene was analyzed using Real-time PCR. RNA was extracted using the Plant Total RNA Isolation Kit Plus from Chengdu Fuji Biotechnology Co., Ltd., and the synthesis of the first strand of DNA was performed according to the TANGEN reverse transcription kit manual. The 10 μl reaction mixture contained: 5 μl 2×SYBR Premix ExTaq, 0.1 μl cDNA, 0.4 μl primers (the qRT-PCR primers for PpCBP60B are SEQ ID NO: 5: (TCCAGGTTGTCCCTTCCTCT) and SEQ ID NO: 6: (GTTTCACTGCTGACTCCGGG); UBQ was used as an internal control primer (sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8), and 4.5 μl water.

[0102] UBQ forward primer: 5'-GCACAAGAAGGTGAAGCTCG-3' (SEQ ID NO: 7)

[0103] UBQ reverse primer: 5'-ACTCAGCATTGGGGCACTC-3' (SEQ ID NO: 8)

[0104] The procedure for real-time quantitative PCR is shown in Table 1:

[0105] Table 1 Real-time quantitative PCR program

[0106]

[0107] See results Figure 2 Samples of the sand pear 'Weining Xiangmian' and the European pear 'Qiyue Hongxiang' were taken at corresponding time points after treatment with black spot fungus cake, and the relative expression levels of the encoding genes were analyzed using real-time quantitative PCR. Figure 2 It can be seen that the PpCBP60B gene in the disease-resistant varieties has a very strong response to black spot disease, and its expression level shows an upward trend.

[0108] Example 3: Subcellular localization of transcription factor PpCBP60B protein

[0109] Based on the nucleotide sequence of the gene encoding transcription factor PpCBP60B and the PCMBIA1300-GFP vector diagram, Xba I and BamHI restriction sites were added before and after the gene sequence, respectively. The target gene plasmid with correct sequencing results was extracted and used as a template for amplification using primers with added restriction sites (SEQ IN NO:3 and SEQ IN NO:4). The PCR program used was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 10 min. The stop codon was removed from the 3′ gene to allow for gene fusion with GFP. After 1% agarose gel electrophoresis, the target band was recovered using a gel electrophoresis kit. The PCMBIA1300-GFP vector plasmid was digested with Xba I and BamHI restriction endonucleases at 37℃ for 4 hours, and then purified and recovered. The enzyme-digested PCMBIA1300-GFP vector and the gel-recovered PpCBP60B fragment were ligated using recombinant ligase at 37°C for 30 min, and then transformed into competent DH5α cells of *E. coli*. The transformed bacterial cultures were tested by PCR. Cultures showing positive PCR results were sent to the company for sequencing. Plasmids from cultures with correct sequencing results were extracted, and the resulting recombinant vector was named PCMBIA1300-PpCBP60B.

[0110] Agrobacterium-mediated transient transformation of tobacco cells: The recombinant PCMBIA1300-PpCBP60B vector plasmid was transformed into Agrobacterium competent cells GV3101. The activated Agrobacterium cells containing the recombinant plasmid were propagated in LB broth containing 50 mg / mL kanamycin and 50 mg / mL rifampin in a shaker at 250 rpm and 28 °C. After centrifugation at 6000 rpm for 10 min, the cells were resuspended in infection buffer (100 mL: 10 mL 100 mM MgCl2, 10 mL 100 mM MES, 75 μL 200 mM AS, 80 mL ddH2O) to OD200. 600 The concentration was 0.8. After incubation at room temperature for 3–4 hours, the inoculum was used to infect tobacco leaf cells. The infected tobacco cells were then cultured in the dark for 24 hours. Images were taken using an inverted laser scanning confocal microscope (Zeiss LSM 780).

[0111] See results Figure 3 . Figure 3 This refers to the subcellular localization of the transcription factor PpCBP60B. According to the cell localization map, PpCBP60B is located in the cell nucleus.

[0112] Example 4: Genetic transformation of callus

[0113] 1. Construction of plant transformation vectors

[0114] Based on the multiple cloning site of the PCMBIA1300 vector and the coding region sequence of the PpCBP60B gene, Xba I and BamHI restriction enzyme sites were added. Upstream and downstream PCR primers (SEQ IN NO:3 and SEQ IN NO:4) were designed using Primer Primer 5.0 software according to general primer design principles. PCR amplification was performed using a clone of the PpCBP60B gene as a template. The annealing temperature for PCR amplification was 58℃, and the PCR reaction system and amplification program were the same as those for the PpCBP60B gene clone. After amplification, gel purification and recovery were performed. The double digestion reaction volume of the PCMBIA1300 vector was 40 μl, containing: 10 μl of the PCMBIA1300 vector plasmid, 4 μl of 10×M buffer, 1 μl each of Xba I and BamHI, and 24 μl of double-distilled water. After digestion at 37℃ for 3-4 h, purification and recovery were performed. The ligation reaction system contained 10 μl of PpCBP60B gene and vector PCMBIA1300 at a molar ratio of 2:1. This mixture included: 1 μl of 10× buffer, 1 μl of DNA recombinase, 4 μl of double-digested and recovered PpCBP60B gene, 2 μl of double-digested and recovered PCMBIA1300 vector product, and 2 μl of double-distilled water. The reaction was carried out at 37°C for 30 min to obtain the ligation product. The ligation product was transformed into *E. coli* DH5α and cultured on LB agar plates containing 50 mg / L kanamycin for 16 h. Positive clones were picked, cultured, and plasmids were extracted for PCR identification. Sequencing confirmed the absence of coding frame mutations, yielding recombinant clones containing the inserted target fragment. These clones were named the PCMBIA1300-PpCBP60B recombinant vector and introduced into *Agrobacterium* GV3101 using the freeze-thaw method.

[0115] 2. The steps of Agrobacterium-mediated callus genetic transformation are as follows:

[0116] (1) Agrobacterium culture: Agrobacterium tumefaciens culture stored in an ultra-low temperature freezer was streaked on LB agar plates containing 50 mg / L kanamycin and 50 mg / L rifampin. The plates were incubated at 28°C for 36–48 hours. The streaked plaques were scraped off and added to liquid MS medium (2.37 g / L MS + 50 g / L sucrose + 0.1 mg / L IBA, pH = 5.8). The plates were incubated at 28°C with shaking for 30 min. When the bacterial concentration reached OD of 0.8–1.0, 200 μl / L of surfactant sweet77 was added for inoculation.

[0117] (2) Inoculation: Take wild-type callus in good condition, then soak it in Agrobacterium tumefaciens solution for 1 hour and culture it in the dark for 24 hours.

[0118] 3. Screening of transgenic positive seedlings

[0119] The PpCBP60B gene-transgenic callus obtained according to the above method was transferred to MS selective medium containing 50 mg / L hygromycin and 50 mg / L termethin and cultured at 22°C in the dark. After one month of growth, the fast-growing callus was selected and transplanted to a new medium for subculture.

[0120] 3.1 Extraction of transgenic callus DNA

[0121] The PpCBP60B gene-transformed callus was obtained using the above method. DNA was extracted from the callus, and primers were designed for PCR amplification to identify positive callus.

[0122] (1) Take an appropriate amount of callus and grind it into powder with liquid nitrogen. Then add 500 μl of CTAB (100 mmol / L Tris-HCl pH 8.0, 1.5 mmol / L NaCl, 50 mmol / L EDTA pH 8.0, 2% w / v (g / 100 ml) CTAB, and dissolve it thoroughly in a water bath at 65°C) and 10 μl of β-mercaptoethanol, and mix well.

[0123] (2) Heat in a 65℃ water bath for 30 min, and remove and gently invert every 10 min to mix; centrifuge at room temperature for 10 min at 10000g; take the supernatant, add 500μl of chloroform-isoamyl alcohol (chloroform:isoamyl alcohol volume ratio is 24:1), and invert to mix;

[0124] (3) Centrifuge at 10000g for 10 min, take 450 μl of the supernatant into a new 1.5 ml centrifuge tube, add 450 μl of isopropanol, and mix by inverting the tube.

[0125] (4) Centrifuge at 10000g for 10min, discard the supernatant, rinse twice with 1mL of 75% ethanol, centrifuge at 10000g for 10min to completely remove the ethanol, place on a clean bench and air dry until the DNA is colorless and transparent.

[0126] (5) Add 50 μl of ultrapure water, place in a 65℃ incubator for 40 min to dissolve, and then perform gel detection.

[0127] 3.2 Detection of positive transgenic callus

[0128] PCR amplification was performed using specific gene primers. The reaction procedures and systems are shown in Tables 2 and 3, respectively. PCR was performed using upstream and downstream primers (SEQ ID NO: 3 and SEQ ID NO: 4). Lines that amplified the expected size fragment were considered positive transgenic lines.

[0129] Table 2 PCR reaction procedure

[0130] step 94℃ 58℃ 72℃ 4℃ Cycle number Step 1 3min 1 Step 2 30s 30s 55s 35 Step 3 90s 1 Step 4 10min Step 5 30min

[0131] Table 3 PCR reaction system

[0132] Reaction components Dosage (μl) Template DNA 1 PCR Buffer 2 dNTD Mix (2.5 mmol / L) 1.6 Right-side forward primer 1 Left-side reverse primer 1 Taq DNA polymerase (5U) 0.2 Nuclease-free water 13.2

[0133] like Figure 4 The target band could be observed in the transgenic lines, but not in the WT lines, indicating that the obtained callus was a transgenic positive callus.

[0134] Example 5: Instantaneous transformation of pear seedlings

[0135] 1. Construction of virus-induced gene silencing vector

[0136] The viral silencing vector was constructed according to the method in Example 4. The viral silencing vector pTRV2 has two restriction enzyme sites: Xba I and Sma I. Following the general principles of primer design, upstream and downstream primers (SEQ ID NO: 9: (AAGGTTACCGAATTCTCTAGA TCCAGGTTGTCCCTTCCTCT) and SEQ ID NO: 10: (TGTCTTCGGGACATGCCCGGG CACGCTCTTTTACTACATGGCT)) were designed using Primer Primer 5.0 software to amplify the PpCBP60B gene and insert it between the two restriction enzyme sites on the vector, resulting in the recombinant vector pTRV2-PpCBP60B, which was then transformed into Agrobacterium GV3101 competent cells.

[0137] 2. Virus-induced gene silencing in pear seedlings

[0138] (1) Agrobacterium culture: Agrobacterium tumefaciens bacterial culture stored in an ultra-low temperature freezer was cultured in LB liquid medium supplemented with kanamycin 50 mg / L and rifampin 50 mg / L at 28°C and 220 rpm for 12 h. The cultured bacterial culture was centrifuged at 6000g for 10 min to collect the bacterial cells. The precipitate was resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 mM acetylsyl syringone, pH 5.6) until the concentration reached OD 600 It is 0.8-1.0;

[0139] (2) Induction of bacterial culture: Place the bacterial culture with adjusted OD value in the dark and induce at room temperature for 4 hours at 100 rpm;

[0140] (3) Pear seedling injection: pTRV1 and pTRV2 bacterial solutions were mixed in a 1:1 ratio as the control group, and pTRV1 and pTRV2-PpCBP60B bacterial solutions were mixed in a 1:1 ratio as the experimental group. The seedlings were injected with pear seedlings that were 30 days old, had the same growth status and good health.

[0141] 3. Identification of virus-induced gene silencing suppression positive vaccines

[0142] After injection, pear seedlings were treated in the dark at room temperature for 12 hours, followed by normal culture for 10 days. RNA was extracted from seedlings of each line from both the control and experimental groups. The RNA structure was verified by gel electrophoresis, and its concentration was determined using Nanodrop (200–1000 ng / µl). The total RNA amount was adjusted to 3 µg and then reverse transcribed into cDNA. UBQ primers from pear were then used as an internal control for amplification. The UBQ primer nucleotide sequences are as follows:

[0143] UBQ forward primer: 5'-GCACAAGAAGGTGAAGCTCG-3' (SEQ ID NO: 7)

[0144] UBQ reverse primer: 5'-ACTCAGCATTGGGGCACTC-3' (SEQ ID NO: 8)

[0145] like Figure 5 The bands amplified by UBQ were all of the same brightness, indicating that the concentration of reverse-transcribed cDNA was the same. Then, qRT-PCR was performed using PpCBP60B specific primers to analyze the expression level of the test lines. Based on the expression level of the PpCBP60B gene, the two plants with lower expression levels were selected as virus silencing positive lines.

[0146] See results Figure 5 As shown, qRT-PCR was used to detect gene expression levels in gene-silenced positive plants using gene-specific primers and the internal reference primer UBQ. Figure 5 This indicates that the PpCBP60B gene in the positive strain of *Pyrus pyrifolia* seedlings was silenced by the virus.

[0147] Example 6: Identification of resistance in PpCBP60B transgenic plants

[0148] To determine whether PpCBP60B transgenic callus is related to resistance to black spot disease stress, both control and transgenic lines were subjected to black spot disease stress treatment. The results showed that pear callus overexpressing the PpCBP60B gene was more resistant to black spot disease than wild-type callus, as evidenced by the significantly slower mycelial growth rate on PpCBP60B-overexpressing pear callus compared to the wild type (e.g., ...). Figure 6 A and Figure 6B), hydrogen peroxide, chitinase (CHI), phenylalanine ammonia-lyase (PAL), and peroxidase (POD) are all physiological indicators that can measure a plant's resistance to pathogens. After infection with black spot disease, the activities of H2O2, CHI, PAL, and POD in callus overexpressing the PpCBP60B gene were significantly higher than those in the control, while there were no differences before infection. Therefore, pear callus tissue overexpressing the PpCBP60B gene is less susceptible to black spot disease infection than the control. Figure 6 C- Figure 6 F).

[0149] Example 7: Identification of resistance in PpCBP60B virus-silenced plants

[0150] To investigate the function of the PpCBP60B gene in responding to black spot disease, we treated PpCBP60B-silenced plants and control plants with sterile needle puncture, then inoculated them with black spot fungal cakes. The leaves were then incubated in the dark at 25°C for 9 days. The results showed that the diameter of lesions on the leaves of PpCBP60B-silenced plants was significantly larger than that of the control. In conclusion, PpCBP60B silenced plants using VIGS are more susceptible to black spot disease infection than the control (e.g., ...). Figure 7 ).

[0151] Analysis of the above results shows that the PpCBP60B gene is closely related to plant resistance to black spot disease. Overexpression of the PpCBP60B gene can effectively enhance the reactive oxygen species scavenging ability of transgenic plants, maintain intracellular ion balance and osmotic potential homeostasis, thereby improving the plant's resistance to black spot disease.

[0152] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of overexpression of transcription factor gene PpCBP60B with nucleotide sequence as shown in SEQ ID NO:2 or increasing the activity or content of transcription factor PpCBP60B protein with amino acid sequence as shown in SEQ ID NO:1 in (I)~(II): (I) Improve resistance to pear black spot disease; (II) Develop new pear varieties resistant to black spot disease.

2. Application of biomaterials containing the transcription factor gene PpCBP60B with the nucleotide sequence shown in SEQ ID NO:2 in (I)~(II): (I) Improve resistance to pear black spot disease; (II) Develop new pear varieties resistant to black spot disease; Biological materials containing the transcription factor gene PpCBP60B are at least one of (1) to (7): (1) An expression cassette containing the transcription factor gene PpCBP60B; (2) A recombinant expression vector containing the transcription factor gene PpCBP60B; (3) A recombinant expression vector containing the expression cassette described in (1); (4) Recombinant bacteria containing the transcription factor gene PpCBP60B; (5) Recombinant bacteria containing the expression cassette described in (1); (6) Recombinant bacteria containing the recombinant expression vector described in (2); (7) Recombinant bacteria containing the recombinant expression vector described in (3).

3. The application according to claim 1 or 2, characterized in that, Overexpression or overexpression of the transcription factor gene PpCBP60B in pears can enhance resistance to pear black spot disease or breed new pear varieties resistant to black spot disease.

4. Application of reagents for detecting the expression level of transcription factor gene PpCBP60B with nucleotide sequence as shown in SEQ ID NO:2 in assessing pear resistance to black spot disease.

5. A method for improving pear resistance to black spot disease or cultivating new pear varieties resistant to black spot disease, characterized in that, Overexpression or overexpression of the transcription factor gene PpCBP60B, with the nucleotide sequence shown in SEQ ID NO:2, in pears can enhance resistance to pear black spot disease or breed new pear varieties resistant to black spot disease.

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