Application of PbrRPS29 gene in improving resistance of pear to Alternaria black spot

Abstract

The application discloses application of a pear PbrRPS29 gene in improving pear resistance to black spot disease. A ribosome small subunit protein is isolated and cloned from a pistil of Pyrus bretschneideri Rehd. var. candicans, and the applicant names the ribosome small subunit protein as PbrRPS29. The amino acid sequence of the ribosome small subunit protein is shown as SEQ ID NO: 2. The gene is constructed into a gene silencing (TRV-VIGS) vector and an overexpression vector respectively, and is introduced into Pyrus bretschneideri Rehd. var. candicans seedlings and Solanum lycopersicum calli through an agrobacterium-mediated genetic transformation method. TRV-mediated silencing strains and stably expressed overexpression strains are obtained respectively and are used for function analysis of the gene resistance to black spot disease. The results show that PbrRPS29 can significantly improve the resistance of pear to Alternaria alternata. The mining of the polypeptide has important significance for cultivating pear varieties resistant to black spot disease and exploring black spot disease-resistant antibacterial peptide drugs.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to the application of the pear PbrRPS29 gene in improving pear resistance to black spot disease. Background Technology

[0002] Plants have the characteristic of sessile growth and cannot escape predators or pathogens by moving like animals. In order to cope with this survival challenge, plants have evolved complex defense systems to resist the infection of pathogens, forming a defense system of "multi-level defense and synergistic regulation".

[0003] Pear (Pyrus L.) is a perennial fruit tree belonging to the genus Pyrus in the family Rosaceae. It is one of the three major fruits in China and is widely cultivated worldwide, possessing significant economic value. However, the frequent occurrence of pests and diseases during pear development severely restricts the development of the pear industry. In recent years, fungal diseases such as black spot, ring rot, anthracnose, and fire blight have seriously damaged the yield and quality of pear fruits. Pear black spot is a disease caused by the pathogenic fungus *Alternaria*, primarily affecting the leaves and fruits of pears, leading to weakened tree vigor, fruit cracking, and premature fruit drop. Furthermore, pear black spot is a typical latent infectious disease; its main pathogen, *Alternaria*, can invade and colonize the style during pear flowering, then gradually enter the calyx tube and intercalyx tissue of the fruit, causing severe damage. However, the mechanisms by which Rosaceae plants, especially pear trees, defend against *Alternaria* infection remain poorly understood.

[0004] Plant ribosomes are core organelles in plant cells responsible for protein synthesis, composed of ribosomal RNA (rRNA) and ribosomal proteins (RPs). Besides their classic translation function, ribosomes play crucial roles in plant growth and development, hormone responses, stress adaptation, and pathogen interactions. Plant ribosomal proteins are highly conserved in structure and possess plant-specific characteristics; their structural composition and subcellular localization together determine the diversity of their functions.

[0005] Currently, there are no reports on the role of the PbrRPS29 gene in pear's resistance to black spot disease. Given the important role of the ribosomal protein family in plant immunity, studying the characteristics and function of the PbrRPS29 gene in pear using molecular biology methods is of great significance and practical application value for regulating pear's resistance to black spot disease and improving pear yield and quality. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the pear PbrRPS29 gene in improving pear resistance to black spot disease. The protein encoded by this pear PbrRPS29 gene is the pear ribosomal small subunit protein PbrRPS29. Studies have shown that the pear PbrRPS29 gene has a positive regulatory function on pear disease resistance, providing an effective technical means for transgenic plants to resist black spot disease.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention seeks protection for the use of the pear PbrRPS29 gene, whose nucleotide sequence is shown in SEQ ID NO:1, in at least one of the following (a1) to (a2):

[0009] (a1) Application in improving pear resistance to black spot disease;

[0010] (a2) Application in the development of transgenic pears with enhanced resistance to black spot disease.

[0011] Secondly, the present invention seeks protection for the use of the PbrRPS29 protein encoded by the pear PbrRPS29 gene, whose nucleotide sequence is shown in SEQ ID NO:1, in at least one of the following (a1) to (a2):

[0012] (a1) Application in improving pear resistance to black spot disease;

[0013] (a2) Application in the development of transgenic pears with enhanced resistance to black spot disease.

[0014] Thirdly, the present invention seeks protection for the use of biological material comprising the pear PbrRPS29 gene with a nucleotide sequence as shown in SEQ ID NO:1 in at least one of the following (a1) to (a2):

[0015] (a1) Application in improving pear resistance to black spot disease;

[0016] (a2) Application in the development of transgenic pears with enhanced resistance to black spot disease;

[0017] The biomaterial is at least one of the following (c1) to (c9):

[0018] (c1) An expression cassette containing the PbrRPS29 gene;

[0019] (c2) A recombinant vector containing the PbrRPS29 gene, or a recombinant vector containing the expression cassette (c1);

[0020] (c3) A recombinant microorganism containing the PbrRPS29 gene, or a recombinant microorganism containing the expression cassette of (c1), or a recombinant microorganism containing the recombinant vector of (c2);

[0021] (c4) A transgenic plant cell line containing the PbrRPS29 gene, or a transgenic plant cell line containing the expression cassette of (c1), or a transgenic plant cell line containing the recombinant vector of (c2);

[0022] (c5) Transgenic plant tissue containing the PbrRPS29 gene, or transgenic plant tissue containing the expression cassette of (c1), or transgenic plant tissue containing the recombinant vector of (c2);

[0023] (c6) A transgenic plant organ containing the PbrRPS29 gene, or a transgenic plant organ containing the expression cassette of (c1), or a transgenic plant organ containing the recombinant vector of (c2);

[0024] (c7) A transgenic plant containing the PbrRPS29 gene, or a transgenic plant containing the expression cassette of (c1), or a transgenic plant containing the recombinant vector of (c2);

[0025] (c8) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c7);

[0026] (c9) Propagation material of the transgenic plants as described in (c7).

[0027] Furthermore, the above applications include improving the resistance of pears to black spot disease or breeding transgenic pears with enhanced resistance to black spot disease by overexpressing the PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 or increasing the activity and / or content of the PbrRPS29 protein encoded by the PbrRPS29 gene in the target plant.

[0028] Fourthly, the present invention claims protection for a method for improving the resistance of pears to black spot disease, by overexpressing the PbrRPS29 gene with a nucleotide sequence as shown in SEQ ID NO:1 in the target plant or increasing the activity and / or content of the PbrRPS29 protein encoded by the PbrRPS29 gene.

[0029] Fifthly, the present invention claims protection for a method for breeding transgenic pears with enhanced resistance to black spot disease, which involves overexpressing the PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 or increasing the activity and / or content of the PbrRPS29 protein encoded by the PbrRPS29 gene in the target plant to breed transgenic pears with enhanced resistance to black spot disease and applying them in production practice.

[0030] In the technical solution of this invention, the PbrRPS29 protein is the protein shown in (b1) or (b2) below:

[0031] (b1) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0032] (b2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

[0033] In the technical solution of this invention, the pear black spot disease is caused by the pathogenic fungus Alternaria alternata.

[0034] In the technical solution of this invention, the primer pair used for cloning the PbrRPS29 gene cDNA sequence includes a forward primer and a reverse primer. The forward primer has a nucleotide sequence as shown in SEQ ID NO:3 in the sequence listing; the reverse primer has a nucleotide sequence as shown in SEQ ID NO:4 in the sequence listing.

[0035] During the study, leaves of two-week-old Dangshan crisp pear seedlings were inoculated with pear black spot fungus cakes with a diameter of 5 mm. Real-time quantitative PCR was used to analyze the relative expression level of the PbrRPS29 gene described in this invention in the leaves at different time points after inoculation with black spot fungus. The results showed that after inoculation with black spot fungus, the relative expression level of the PbrRPS29 gene gradually increased and reached its peak expression level after 4 days. This indicates that the PbrRPS29 gene responds to pear black spot stress treatment and has a regulatory function for disease resistance.

[0036] The beneficial effects of this invention are:

[0037] This invention provides the application of the PbrRPS29 gene and its encoded protein in enhancing the resistance of plants to pear black spot disease. By constructing pear callus transformation vectors and gene-silenced pear seedling transformation vectors respectively, positive callus tissues or seedlings were obtained and inoculated with pathogens. The results showed that the mycelial cake area of ​​transgenic pear callus tissue was significantly smaller than that of wild type (WT), indicating that the positive callus tissue had stronger disease resistance than the wild type. The lesion area in virus-silenced Dangshan crisp pear seedlings was significantly increased compared with the control plants, indicating that the plants suffered greater damage from pathogens. The results indicate that overexpression of the PbrRPS29 gene can effectively enhance the disease resistance of plants, enabling them to better cope with the stress of pear black spot disease, while silencing the PbrRPS29 gene weakens the plant's disease resistance. The discovery of the PbrRPS29 gene is of great significance for breeding transgenic pears with improved resistance to black spot disease and for exploring the development of antimicrobial peptide drugs against black spot disease. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the technical process of the present invention.

[0039] Figure 2 Analysis of the expression pattern of the PbrRPS29 gene in leaves of Dangshan crisp pear infected with Alternaria pearis.

[0040] Figure 3 Analysis of the expression pattern of the PbrRPS29 gene in the flower column of Dangshan pear infected with Alternaria pearis.

[0041] Figure 4 Subcellular localization of PbrRPS29 protein; Bar = 20 μm.

[0042] Figure 5 This study aimed to identify positive seedlings of Dangshan pear seedlings transiently transformed by PbrRPS29 virus silencing. A represents the phenotype of wild-type and PbrRPS29 virus-silencing positive pear seedlings after 6 days of inoculation with *Alternaria alternata* (Bar = 1 cm). B represents the gene expression level of gene-silencing positive plants detected by qRT-PCR using gene-specific primers and the internal control primer UBQ; three silencing lines were identified. C represents the colony area measurement of wild-type and PbrRPS29 virus-silencing positive pear seedlings 6 days after inoculation with *Alternaria alternata*.

[0043] Figure 6Genetic transformation and identification of PbrRPS29 callus from pear; A shows the phenotype of wild-type and PbrRPS29 overexpression lines 7 days after inoculation with Alternaria alternata, Bar = 1 cm; B shows the gene expression levels of wild-type (WT) and gene overexpression lines (OE) detected by qRT-PCR using gene-specific primers and internal control primer UBQ, with a total of three overexpression lines; C shows the area of ​​the mycelial cake 7 days after inoculation of wild-type and PbrRPS29 overexpression lines with Alternaria alternata. Detailed Implementation

[0044] This invention screened a PbrRPS29 gene from Dangshan pear. The nucleotide sequence of this gene is shown in SEQ ID NO:1, encoding 55 amino acids with a molecular weight of 6.23 kDa. Its amino acid sequence is shown in SEQ ID NO:2. The PbrRPS29 gene provided by this invention plays a positive regulatory role in the stress of black spot disease in pear plants.

[0045] This invention provides a primer pair for cloning the cDNA sequence of the PbrRPS29 gene, comprising a forward primer and a reverse primer. The forward primer has the nucleotide sequence shown in SEQ ID NO:3 (ATGGGACACTCGAACGTATGGAAC); the reverse primer has the nucleotide sequence shown in SEQ ID NO:4 (CTAGCACTTGATGAAGCCAATCTCC). This invention does not impose any particular limitations on the design method of the primers; they can be synthesized by a biosynthetic company well-known in the art. The primer pair was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0046] In this invention, a PbrRPS29 gene silencing vector was constructed and transiently transformed into Dangshan pear seedlings. A PbrRPS29 gene overexpression vector was then constructed and introduced into pear callus tissue. After genetic transformation was completed, *Alternaria pyrifolia* inoculation was performed, and disease resistance was assessed (e.g.,...). Figure 1 (As shown in the image). The results showed that overexpression of the PbrRPS29 gene enhanced the plant's resistance to pear black spot disease. Conversely, silencing the PbrRPS29 gene weakened the disease resistance of the lines.

[0047] This invention provides a recombinant expression vector containing the PbrRPS29 gene and a silencing vector (pTRV2) for silencing the PbrRPS29 gene. The PbrRPS29 gene is preferably overexpressed or silenced in plants in the form of PbrRPS29-GFP and pTRV2-PbrRPS29 recombinant vectors. This invention does not impose any particular limitation on the construction method of the PbrRPS29-GFP and pTRV2-PbrRPS29 recombinant vectors; any construction method well known in the art can be used.

[0048] 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.

[0049] In this invention, the screening method for positive overexpression plants and gene-silenced plants preferably employs qRT-PCR to examine gene expression levels. The forward primer used in the qRT-PCR experiment has the nucleotide sequence shown in SEQ ID NO:5 (CACTCGAACGTATGGAACTC); the reverse primer has the nucleotide sequence shown in SEQ ID NO:6 (CTAGCACTTGATGAAGCCAATC); and the primers for the internal reference gene PbrUBQ have the nucleotide sequences shown in forward primer SEQ ID NO:7 (GCACAAGAAGGTGAAGCTCG) and reverse primer SEQ ID NO:8 (ACTCAGCATTGGGGCACTC). After the qRT-PCR amplification, if the expression level of the PbrRPS29 gene in the candidate transgenic lines is significantly higher than that in wild-type plants, this indicates that they are positive overexpression lines. If the expression level of the PbrRPS29 gene amplified in the tested plant lines is significantly lower than that in wild-type plants, this indicates that they are positive silenced lines.

[0050] In this invention, the pear black spot disease Alternaria strain was activated and cultured in potato dextroseagar (PDA) medium, and then cultured at a constant temperature of 28°C for 8 days before being used for inoculation experiments.

[0051] In this invention, the plant materials preferably include pear callus, Dangshan pear seedlings, and Dangshan pear styles. This invention uses Dangshan pear as a representative plant to verify the application of the gene PbrRPS29 in regulating plant resistance to pear black spot disease. Experiments show that when the gene PbrRPS29 is highly expressed in plants, the plant's resistance to pear black spot disease is enhanced; when the gene PbrRPS29 is silenced in plants, the plant's resistance to pear black spot disease is weakened. Therefore, by regulating the expression level of the gene PbrRPS29, the resistance and disease resistance of plants to pear black spot disease can be regulated (e.g., Figure 1 (Schematic diagram of the technical process of this invention).

[0052] In this invention, different inoculation methods are used for different plant materials to inoculate the pear black spot pathogen. For pear callus and pear leaves, the mycelial cake inoculation method is used. A hole punch is used to make holes at the edge of the black spot pathogen colony, and mycelial cakes of uniform size are placed in the center of the pear leaf and pear callus, respectively. The pear pistil tissue is treated with a conidial suspension spray method. ddH2O is added to the appropriate-aged black spot pathogen culture medium. After filtering the suspension containing Alternaria alternata conidia through gauze (to wash away the mycelia), the concentration of the conidial suspension is adjusted to 5 × 10⁻⁶ using ddH2O. 5 per mL.

[0053] In this invention, the plant's resistance to pear black spot disease is expressed by the degree of disease on the plant, such as the size of the lesions.

[0054] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention without departing from its spirit and scope to make it suitable for various uses and conditions.

[0055] Example 1: Cloning of PbrRPS29, a small ribosomal subunit protein family gene, from Dangshan pear.

[0056] A gene, PbrRPS29, was screened from the flower column of Dangshan pear. Primers were designed based on its CDS sequence, and its full length was cloned from Dangshan pear using RT-PCR. Detailed steps are as follows: The synthesis of the first strand of cDNA was performed according to the instructions of the reverse transcription kit from Nanjing Novizan Pharmaceutical Co., Ltd. The obtained first-strand cDNA was used for the amplification of PbrRPS29. The total volume of the PCR reaction was 50 µl, including 2 µl of Dangshan pear cDNA, 2 µl each of forward and reverse primers (SEQ ID NO:3 and SEQ ID NO:4), 1 µl of enzyme, 25 µl of 2×Buffer, and 18 µl of sterile ddH2O. The PCR reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min. After amplification, a single-band PCR product is generated. After electrophoresis on a 1% agarose gel, the target band is excised and the DNA is recovered.

[0057] The purified product from the gel was ligated into the pEASY-BluntZero vector (TransGen Biotech) to obtain the pEASY-Blunt Zero:PbrRPS29 plasmid. The total reaction volume was 5 µl, containing 1 µl of the pEASY-Blunt Zero Cloning Vector. After ligation at 37°C for 30 min, the plasmid was transformed into competent E. coli DH5α cells using the heat shock method. PCR verification was performed using primers for the target gene sequence, followed by sequencing (sequencing was performed by Shanghai Sangon Biotech Co., Ltd.).

[0058] Example 2: qRT-PCR analysis of the PbrRPS29 gene in leaves of Dangshan crisp pear under pear black spot disease stress treatment.

[0059] To analyze the response pattern of the PbrRPS29 gene in Dangshan pear leaves to pear black spot disease treatment, leaves of two-week-old Dangshan pear seedlings were inoculated with 5 mm diameter black spot fungus cakes. The expression pattern of the PbrRPS29 gene in the leaves at different time points after inoculation was analyzed using qRT-PCR. RNA was extracted from Dangshan pear leaves using the Nanjing Novizan FastPurePlant Total RNA Isolation Kit, and the synthesis of the first strand of cDNA was performed according to the instructions of the Nanjing Novizan Reverse Transcription Kit.

[0060] The forward primer used in the qRT-PCR reaction has the nucleotide sequence shown in SEQ ID NO:5 (CACTCGAACGTATGGAACTC); the reverse primer has the nucleotide sequence shown in SEQ ID NO:6 (CTAGCACTTGATGAAGCCAATC); and the primers for the internal reference gene PbrUBQ have the nucleotide sequences shown in the forward primer SEQ ID NO:7 (GCACAAGAAGGTGAAGCTCG) and the reverse primer SEQ ID NO:8 (ACTCAGCATTGGGGCACTC).

[0061] The qRT-PCR reaction system was as follows: 10 μl 2×SYBR-Green PCR Master Mix, 2 μl cDNA, 0.4 μl each of forward and reverse primers, and 7.2 μl deionized water. The PbrUBQ gene was used as an internal control. The qRT-PCR reaction procedure is shown in Table 1.

[0062] Table 1 qRT-PCR reaction procedure

[0063]

[0064] See results Figure 2Seedlings of Dangshan crisp pear were treated with pear black spot pathogen, and samples were taken at 0, 0.5, 1, 2, 3, 4, and 5 days post-treatment. qRT-PCR analysis revealed that the expression level of the PbrRPS29 gene increased after inoculation with pear black spot pathogen, reaching a peak at 4 days post-inoculation.

[0065] Example 3: qRT-PCR analysis of the PbrRPS29 gene in the flower style of Dangshan pear under stress from *Pyrus pyrifolia* var. *pear*.

[0066] To analyze the response pattern of the PbrRPS29 gene in the flower column of Dangshan pear to pear black spot disease treatment, the expression pattern of the PbrRPS29 gene was analyzed using qRT-PCR technology. RNA was extracted from the flower column of Dangshan pear using the FastPure Plant Total RNA Isolation Kit, and the synthesis of the first strand of cDNA was performed according to the instructions of the Nanjing Novizan reverse transcription kit. The qRT-PCR reaction system and reaction procedure were as described in Example 2.

[0067] See results Figure 3 Branches of Dangshan crisp pear were cultured indoors. After removing the male parts of the pear flowers at the large bud stage, they were sprayed with a suspension of pear black spot disease conidia. Samples were taken at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h. Figure 3 It can be seen that the expression level of the PbrRPS29 gene is highest in untreated pistils during the large bud stage, and the expression level in subsequent stages is lower than that during the large bud stage. In samples subjected to pathogen stress, the expression level of the PbrRPS29 gene is higher than that during the large bud stage, and significantly higher than that of untreated pistils at 12 h, and the high expression can be maintained for a long time. This indicates that after treatment of pear pistils with pear black spot pathogen, the PbrRPS29 gene can respond rapidly and resist pathogen invasion by enhancing its expression.

[0068] Example 4 Subcellular localization of PbrRPS29 protein

[0069] The PbrRPS29 gene was ligated into the pCAMBIA1300-35s-GFP vector (Tang C, Wang P, Zhu X, et al. Acetylation of inorganic pyropHospHatase by S-RNase signaling induces pollen tube tip swelling by repressing pectin methylesterase[J]. Plant Cell, 2023, 35 (9):3544-3565.), with double restriction sites of XbaI and BamHI. Using the pEASY-BluntZero:PbrRPS29 plasmid constructed in Example 1 as a template, the forward primer had the nucleotide sequence shown in SEQ ID NO:9 (gagaacacgggggactctagaATGGGACACTCGAACGTATGGAAC), and the reverse primer had the nucleotide sequence shown in SEQ ID NO:10 (gcccttgctcaccatggatccGCACTTGATGAAGCCAATCTCC). The PCR reaction system and procedure were as described in Example 1. The pCAMBIA1300-35s-GFP vector plasmid was double-digested with XbaI and BamHI restriction endonucleases, and purified after 2 hours at 37°C. The digested pCAMBIA1300-35s-GFP vector was then ligated with the gel-recovered PbrRPS29 fragment using recombinant ligase, and the ligation was performed at 37°C for 30 minutes. The ligation was then transformed into competent DH5α cells of *E. coli*. Subsequently, the bacterial cultures that tested positive by PCR were sequenced, and the plasmid from the correctly sequenced cultures was extracted. The resulting recombinant vector was named PbrRPS29-GFP.

[0070] Agrobacterium-mediated transient transformation of tobacco leaf cells: The PbrRPS29-GFP recombinant plasmid was transformed into Agrobacterium competent cells GV3101. The activated Agrobacterium containing the recombinant plasmid was propagated in LB broth containing 50 mg / L kanamycin and 50 mg / L rifampin in a shaker at 250 rpm and 28℃. After centrifugation at 6000 rpm for 10 min, the cells were resuspended in infection buffer (each 100 mL contained: 10 mL 100 mM MgCl2, 10 mL 100 mM MES, 75 µL 200 mM AS, and 80 mL ddH2O) to an OD600 of 0.8. After incubation at room temperature (25±5℃) for 3–4 h, the cells were injected into young tobacco leaves. The cells were cultured in the dark for 24 h, followed by culture under normal light for 48 h. Fluorescence detection and image acquisition were performed using a laser confocal microscope (Zeiss LSM900).

[0071] See results Figure 4 Imaging of the GFP gene (control) and the PbrRPS29 gene in GFP field, bright field, and superimposed fields; subcellular localization maps show that PbrRPS29 is located in the cytoplasm and nucleus.

[0072] Example 5: Instantaneous transformation of Dangshan crisp pear seedlings

[0073] 1. Construction of virus-induced gene silencing vectors

[0074] Using the pEASY-Blunt Zero:PbrRPS29 plasmid constructed in Example 1 as a template, the PbrRPS29 gene was amplified using the forward and reverse primers shown in SEQ ID NO:11 and SEQ ID NO:12, and inserted into the middle of the XbaI and SmaI restriction sites of the viral silencing vector (pTRV2) (Xing CH, Chen QM, Qiao QH, et al. PbrWRKY70 increases pear (Pyrusbretschneideri Rehd) black spot disease tolerance by negatively regulating ethylene synthesis via PbrERF1B-2[J]. Plant Sci, 2023, 334:111773.), resulting in the recombinant vector pTRV2-PbrRPS29, which was then transformed into Agrobacterium GV3101 competent cells.

[0075] Forward primer: aaggttaccgaattctctagaATGGGACACTCGAACGTATGGAAC (SEQ ID NO:11)

[0076] Reverse primer: tgtcttcgggacatgcccgggCTAGCACTTGATGAAGCCAATCTCC (SEQ ID NO:12).

[0077] 2. Virus-induced gene silencing in Dangshan pear seedlings

[0078] Agrobacterium culture: Agrobacterium tumefaciens culture stored at -80℃ was cultured in LB liquid medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampin for 12 h with shaking (28℃, 220 rpm). The cultured bacterial solution was centrifuged at 6000 rpm for 10 min to collect the bacterial cells, and the precipitate was resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 mM acetylsylcholine, pH 5.6) to a concentration of OD600=0.8. Induction: The bacterial solution was placed in the dark and induced at room temperature at 100 rpm for 4 h. Injection into pear seedlings: pTRV1 and pTRV2 bacterial solutions were mixed at a 1:1 ratio as the control group, and pTRV1 and pTRV2-PbrRPS29 bacterial solutions were mixed at a 1:1 ratio as the experimental group. The control group was injected into Dangshan crisp pear seedlings with uniform growth and good health at 45 days of age.

[0079] 3. Identification of virus-induced gene silencing positive vaccines and pathogen stress

[0080] After injection, pear seedlings were treated in darkness for 24 hours, followed by normal light treatment for 14 days. RNA was independently sampled from each line, and samples from both the control and experimental groups were extracted. After reverse transcription, qRT-PCR was performed to identify whether the gene-silenced lines were positive. Figure 5 In section B, the lines with significantly reduced gene expression were named pTRV-PbrRPS29-1, pTRV-PbrRPS29-2, and pTRV-PbrRPS29-3, respectively. The results indicate that the PbrRPS29 gene was silenced in virus-silenced pear seedling positive lines.

[0081] Example 6 Genetic transformation of pear and eggplant callus

[0082] 1. Construction of plant genetic transformation vectors

[0083] The recombinant vector PbrRPS29-GFP constructed in Example 4 was introduced into Agrobacterium GV3101.

[0084] 2. Agrobacterium-mediated genetic transformation of pear and eggplant callus

[0085] The specific steps are as follows: Agrobacterium culture: Take Agrobacterium tumefaciens culture stored in a -80℃ refrigerator, streak it on an LB agar plate with 50 mg / L kanamycin and 50 mg / L rifampin, and incubate it in a 28℃ incubator for 36-48 h. Pick single colonies and shake them in LB liquid medium with 50 mg / L kanamycin and 50 mg / L rifampin for 36-48 h. Centrifuge at 5000 rpm for 5 min, remove the supernatant and resuspend it in double-distilled water in a laminar flow hood (all subsequent steps are performed under aseptic conditions). Measure and adjust OD600 to 0.6, add 100 µl of 200 mM AS, and incubate at 30℃ and 120 rpm for 45 min. Inoculation: In a laminar flow hood, crush the wild-type pear callus tissue with tweezers and pour it into an Erlenmeyer flask containing the inoculation solution. Incubate at 30℃ and 120 rpm for 15 min. Use a strainer to filter out the callus tissue, rinse with sterile water as needed, and then blot dry with filter paper; culture: spread the callus tissue evenly on MS (2.37 g / L MS + 50 g / L sucrose + 7 g / L agar + 0.5 mg / L 6-BA + 1 mg / L 2,4-D + 100 µM AS, pH=5.8) solid medium containing AS, and culture at 28℃ for two days.

[0086] 3. Screening of pear-overexpressing positive callus tissue

[0087] Callus cultured for 2 days was transferred to MS (2.37 g / L MS + 50 g / L sucrose + 7 g / L agar + 0.5 mg / L 6-BA + 1 mg / L 2,4-D + 10 mg / L hygromycin + 150 mg / L cephalosporin, pH 5.8) solid medium for screening overexpressing callus. Callus was cultured using standard methods, and white, immature cell clumps appeared in approximately 3-4 weeks. Small clumps of transgenic callus were picked and transferred to a new screening solid medium; each clump represented an overexpression line of PbrRPS29 gene-overexpressing callus. The cultures were then subcultured for 3 generations according to the respective lines.

[0088] 4. Extraction of RNA from overexpressed callus tissue

[0089] RNA extraction was performed using an RNA extraction kit from Nanjing Novizan Biotech Co., Ltd., following the kit's instructions.

[0090] 5. Detection of positive overexpression plants

[0091] Each pear callus line obtained by subculture using the above method was independently sampled, and RNA was extracted from wild-type and transgenic callus samples. The RNA was reverse transcribed into cDNA and then identified by qRT-PCR. Based on the expression level of the PbrRPS29 gene, three plants with high expression levels were selected as overexpression-positive lines, named PbrRPS29-OE-1, PbrRPS29-OE-2, and PbrRPS29-OE-3. Figure 6 As shown in B, the results indicate that the PbrRPS29 gene is overexpressed in pear callus-positive lines.

[0092] Example 7: Identification of disease resistance in PbrRPS29 transgenic disease-resistant plants

[0093] 1. Disease resistance analysis of virus-silenced pear seedlings

[0094] To determine whether the PbrRPS29 gene is involved in pear resistance to black spot disease, wild-type and virus-silenced positive seedlings were subjected to pear black spot stress treatment. Wild-type and virus-silenced positive seedlings of uniform age and good growth were inoculated with activated Alternaria alternata mycelium cakes and cultured in an incubator at 28°C. After 6 days, the phenotypes of wild-type and virus-silenced positive pear seedlings were observed, and the area of ​​lesions was measured.

[0095] Figure 5 In the figure, A represents the phenotype of wild-type and pTRV-PbrRPS29 virus-silenced pear seedlings after 6 days of leaf inoculation with Alternaria. Figure 5 The bar chart with 'C' representing the area of ​​the lesions shows that the expression of the silenced gene PbrRPS29 leads to a decrease in the plant's resistance to pear black spot disease.

[0096] 2. Analysis of resistance to pear black spot disease by overexpression of pear callus

[0097] To determine whether the PbrRPS29 overexpression line in pear callus affects pear resistance to black spot disease, wild-type and transgenic lines were inoculated with Alternaria alternata fungal discs. The identified PbrRPS29 overexpression line and wild-type (WT) callus were spread evenly and densely on MS selection medium and commonly used MS antibiotic-free medium, respectively. Black spot fungal discs of similar size and growth were inoculated onto the callus, sealed under aseptic conditions, and incubated in the dark at 28°C. Phenotypic characteristics were observed and the area of ​​the fungal discs was measured after 7 days.

[0098] Figure 6 In the figure, A represents the phenotype of wild-type and PbrRPS29 overexpression line callus after 7 days of inoculation with Alternaria. Figure 6 The bar chart with 'C' representing the area of ​​the mycelial cake is shown. The results indicate that overexpression of the gene PbrRPS29 enhances the plant's resistance to pear black spot disease.

[0099] In conclusion, the PbrRPS29 gene is closely related to the plant's resistance to Alternaria alternata, the causal agent of pear black spot.

[0100] 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.

[0101] sequence list

[0102] PbrRPS29 gene (SEQ ID NO:1)

[0103] ATGGGACACTCGAACGTATGGAACTCTCACCCCAAGAACTACGGCCCCGGTTCCCGCACTTGCCGAGTGTGTGGAAACCCTCCATGGGTTGATCCGGAAGTATGCTCTGATGTGCTGCAGGCAGTGCTTCCGCAGCAATGCCAAGGAGATTGGCTTCATCAAGTGCTAG

[0104] PbrRPS29 protein (SEQ ID NO:2)

[0105] MGHSNVWNSHPKNYGPGSRTCRVCGNPHGLIRKYALMCCRQCFRSNAKEIGFIKC*.

Claims

1. The pear PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 is used in at least one of the following (a1) to (a2): (a1) Application in improving pear resistance to black spot disease; (a2) Application in the development of transgenic pears with enhanced resistance to black spot disease.

2. The PbrRPS29 protein encoded by the pear PbrRPS29 gene, whose nucleotide sequence is shown in SEQ ID NO:1, is used in at least one of the following (a1) to (a2): (a1) Application in improving pear resistance to black spot disease; (a2) Application in the development of transgenic pears with enhanced resistance to black spot disease.

3. The application according to claim 2, characterized in that, The PbrRPS29 protein is as shown in (b1) or (b2) below: (b1) A protein with the amino acid sequence shown in SEQ ID NO:2; (b2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

4. The use of biological materials containing the pear PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 in at least one of the following (a1) to (a2): (a1) Application in improving pear resistance to black spot disease; (a2) Application in the development of transgenic pears with enhanced resistance to black spot disease; The biomaterial is at least one of the following (c1) to (c9): (c1) An expression cassette containing the PbrRPS29 gene; (c2) A recombinant vector containing the PbrRPS29 gene, or a recombinant vector containing the expression cassette (c1); (c3) A recombinant microorganism containing the PbrRPS29 gene, or a recombinant microorganism containing the expression cassette of (c1), or a recombinant microorganism containing the recombinant vector of (c2); (c4) A transgenic plant cell line containing the PbrRPS29 gene, or a transgenic plant cell line containing the expression cassette of (c1), or a transgenic plant cell line containing the recombinant vector of (c2); (c5) Transgenic plant tissue containing the PbrRPS29 gene, or transgenic plant tissue containing the expression cassette of (c1), or transgenic plant tissue containing the recombinant vector of (c2); (c6) A transgenic plant organ containing the PbrRPS29 gene, or a transgenic plant organ containing the expression cassette of (c1), or a transgenic plant organ containing the recombinant vector of (c2); (c7) A transgenic plant containing the PbrRPS29 gene, or a transgenic plant containing the expression cassette of (c1), or a transgenic plant containing the recombinant vector of (c2); (c8) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c7); (c9) Propagation material of the transgenic plants as described in (c7).

5. The application according to claim 1, characterized in that, By overexpressing the PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 in the target plant or increasing the activity and / or content of the PbrRPS29 protein encoded by the PbrRPS29 gene, the resistance of pear to black spot disease can be improved or transgenic pears with improved resistance to black spot disease can be bred.

6. A method for improving the resistance of pears to black spot disease, characterized in that, By overexpressing the PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 or increasing the activity and / or content of the PbrRPS29 protein encoded by the PbrRPS29 gene in the target plant, the resistance of pear to black spot disease can be improved.

7. A method for cultivating transgenic pears with enhanced resistance to black spot disease, characterized in that, Transgenic pears with enhanced resistance to black spot disease were bred and applied in production practice by overexpressing the PbrRPS29 gene with the nucleotide sequence shown in SEQ ID NO:1 or increasing the activity and / or content of the PbrRPS29 protein encoded by the PbrRPS29 gene in the target plant.

8. The method according to claim 6 or 7, characterized in that, The PbrRPS29 protein is as shown in (b1) or (b2) below: (b1) A protein with the amino acid sequence shown in SEQ ID NO:2; (b2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

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