Ribosomal protein PpRPL12 from Pyrus truncatula and its application in improving plant disease resistance

Abstract

The present invention discloses a ribosomal protein PpRPL12 in sand pear and its application in improving the disease resistance of plants. The present invention screens out a ribosomal protein PpRPL12 from sand pear that has a positive effect on resistance to pear black spot. The present invention uses an overexpression vector and a virus silencing vector constructed with the coding gene of the ribosomal protein PpRPL12 to verify the function of plant resistance to pear black spot. The results show that the resistance of pear callus and positive Arabidopsis plants with the coding gene to pear black spot is significantly higher than that of wild-type callus and wild-type Arabidopsis plants. PpRPL12 After silencing, it was found that the wild-type Pyrus betulus seedlings had better resistance to pear black spot than the silenced Pyrus betulus seedlings, which proved that the ribosomal protein PpRPL12 and its encoding gene of the present invention have important application value in the research of improving the resistance of plants to pear black spot.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a ribosomal protein PpRPL12 in sand pear and an application thereof in improving the disease resistance of plants. Background Art

[0002] Pear is a temperate fruit. As the third largest main fruit tree in China, it has a variety of varieties and rich nutritional value. In the process of the pear industry developing towards the modern cluster stage, the country has increased its research efforts on post-harvest preservation, storage and transportation of pears, pear browning, pear diseases and pests, and has achieved a series of important results. However, there are still some major problems to be solved in the pear industry. The impact of pear diseases on the yield, quality and economic benefits of pears cannot be underestimated. Among them, pear diseases seriously affect the growth and development of trees, causing withering and death, and some cause fruit rot and early drop, resulting in reduced production. It also causes spots and rot in the fruit during growth and storage, reducing the commercial value of the fruit. A lot of manpower, material and financial resources are consumed in the process of disease prevention and control. Nowadays, the most common prevention and control methods are chemical prevention and control, such as pesticides and fungicides, but this not only increases production costs but also may cause environmental pollution. Physical methods such as light insect trapping are environmentally friendly but have limited efficiency and cannot completely replace chemical prevention and control. Therefore, whether excellent disease-resistant new varieties can be cultivated is particularly important for the development of the pear industry. Faced with genetic complexity and limitations of traditional breeding, breeders have used molecular marker-assisted selection and gene editing technology, combined with plant tissue culture methods, to accelerate the improvement of disease-resistant genes and the selection of new varieties, demonstrating the application potential and development prospects of disease-resistant breeding.

[0003] Pear black spot is a common pear tree disease that seriously affects the growth of pear trees and the quality of fruit. It is mainly caused by Alternaria spp. Alternaria ) fungi, especially Alternaria tenuissima ( A. tenuissima )、Alternaria alternata A. alternata ) A. arborescens ), Alternaria pyrifolia ( A. gaisen )、Alternaria japonica A. gossypina ) and Alternaria longipes ( A. longipes ) and other species (Wang Wenqing et al., 2020). These pathogens cause damage to pear tree leaves, new shoots, fruits, etc. through different pathways. The disease is common in pear producing areas across the country, especially in the southern region where the disease is more severe. The disease mainly affects the leaves, fruits and new shoots of pear trees, causing brown or dark brown circular spots on the leaves and black circular spots on the fruits. In severe cases, it causes fruit cracking, softening and early fruit drop, resulting in significant economic losses. The main Alternaria species of pear black spot disease in my country is Alternaria tenuissima ( Alternaria tenuissima ) and common Alternaria ( Alternaria alternata), the pathogenicity of common Alternaria is relatively strong, and its infection patterns, pathways and methods have been systematically studied through common pathology, tissue pathology and pathophysiology (Li Yongcai, 2000). When plants are infected with common Alternaria, they will cause a series of disease resistance reactions, such as inducing lipid peroxidation and accumulating hydrogen peroxide (H 2 O 2 ) and cell death (ChungK-R, 2012), by regulating the antioxidant enzyme system (such as superoxide dismutase SOD, catalase CAT, peroxidase POD) to resist the cell stress caused by reactive oxygen species (ROS). Pear black spot stress can also trigger changes in the expression of multiple genes in the plant to activate the plant's defense state, and many physiological indicators regulated by disease resistance genes change. For example, chlorophyll content decreases, and total soluble sugars (reducing and non-reducing sugars) and protein content decrease. In addition, changes in specific gene expression patterns related to plant disease resistance also involve two independent hormone-dependent defense response pathways: salicylic acid (SA) and jasmonic acid (JA) pathways.

[0004] The salicylic acid (SA) and jasmonic acid (JA) pathways play a crucial role in the complex immune response of plants. The SA pathway plays a role in plant recognition of biogenic pathogens, such as bacteria and fungi. Its signal recognition and response mechanism involves the accumulation of SA in infected tissues, thereby activating local and systemic defense responses. The accumulation of SA further regulates the expression of defense-related genes such as pathogenesis-related proteins (PR proteins), and these gene products are directly involved in enhancing plant resistance to pathogens. In addition, SA-induced systemic acquired resistance (SAR) provides plants with a long-term and broad-spectrum resistance state, giving them enhanced defense capabilities against a variety of potential reinfections. The JA pathway plays a role in plant response to physical damage or attack by specific abiotic pathogens, and is involved in regulating plant wound responses and defense mechanisms. JA-induced gene expression includes plant defensins and protease inhibitors, which can inhibit the growth of pathogens and thus play a key role in plant defense. In addition, there is an ethylene pathway, which is often associated with the JA pathway. It is worth noting that the SA pathway and JA are antagonistic to each other when participating in the activation of plant defense responses, but sometimes they work synergistically. Many enzymes play a key role in these hormone-dependent defense response pathways. Phenylalanine ammonia lyase (PAL) may participate in the biosynthesis pathway of SA, affecting the synthesis of downstream secondary metabolites, thereby enhancing the disease resistance of plants. At the same time, PAL is also the first key enzyme in the phenylpropanoid pathway. It catalyzes the conversion of phenylalanine into cinnamic acid, and then synthesizes a variety of phenolic compounds, which have antioxidant and anti-pathogen properties. Polyphenol oxidase (PPO) participates in the oxidative polymerization of phenolic substances to form structural substances such as lignin, providing a physical barrier for plants. In terms of antioxidants, phenolic compounds produced by the phenylpropanoid pathway can scavenge reactive oxygen species (ROS) and reduce oxidative damage caused by pathogens. At the same time, these phenolic substances can also regulate the antioxidant enzyme system (such as SOD, POD) in plants, reducing the oxidative damage caused by plants under disease stress. In addition, chitinase and β-1,3-glucanase (β-1,3-GA) can directly act on pathogens. Chitin is a polysaccharide, and its covalent cross-linking with β-1,3-glucan and mannan together constitutes the fungal cell wall. Chitinase and β-1,3-GA can decompose the exoskeleton of pathogens and are also involved in the SAR signal transduction pathway induced by SA. Therefore, the increase in chitinase and β-1,3-GA activity and the change of allozymes are also considered to be a factor in enhancing plant disease resistance. The combined action of the above enzymes enhances plant resistance to diseases such as pear black spot.

[0005] Plant ribosomal proteins (RP) participate in the transcription, translation and intracellular biosynthesis of plant genetic information, and play an important role in plant growth and development. The ribosome is composed of two subunits, the large and small ribosomal proteins, which form a complex that can read mRNA and guide the arrangement of amino acid sequences to form polypeptide chains through specific interactions. In addition to being indispensable in the transmission of plant genetic information, the plant ribosomal protein (RP) gene family is also believed to have additional ribosomal functions, such as the occurrence of stress signals, which are speculated to be related to disease resistance (Wool, IG, 1996; Warner, JR& McIntosh, KB, 2009). A study extracted the RPL3 protein from yeast and transferred it into tobacco, which was confirmed to enhance the disease resistance of tobacco plants (Di, R.&Tumer, NE, 2005). In recent years, many studies have found that RP genes are related to plant disease resistance. For example, the peanut RPL29-1 gene has resistance to peanut bacterial wilt, the cotton RPS6 gene family has resistance to cotton Verticillium wilt, and the potato RPL28-1 gene may have resistance to potato bacterial wilt (Cao et al., 2016; Zhu Dandan, 2021; Wei Hongping et al., 2019).

[0006] Ribosomal Protein Large Subunit (RPL) is mainly responsible for catalyzing the formation of peptide bonds, which is a key step in protein biosynthesis. In the plant disease resistance mechanism, the function of the large subunit may be related to its role in rapid response to external stimuli and regulation of plant growth and development. For example, when pathogens invade, the large subunit may enhance the plant's defense ability by adjusting its activity or interacting with other disease resistance-related proteins. The interaction between RPL19, RPL14, RPL3 and rRNA large and small subunit elements may enhance the stability of the intersubunit bridge (Harms, J. et al., 2001). RPL12 protein is usually an L7 / L12 dimer and is the only multi-copy ribosomal protein involved in the regulation of protein synthesis. In immunology, RPL7 and RPL12 show cross-reaction. RPL7 and RPL12 are transcribed from the same gene. The difference between them lies in the aminoacetylation of the N-terminal serine residue of the RPL7 protein (Bailey-Serres, J.et al., 1997; Grebenyuk, E.et al., 2009). Gong et al. found that GaRPL18 has an anti-dahlia bacterium effect in cotton and Arabidopsis, and is mainly regulated by the SA signaling pathway (Gong et al., 2017). Satish Nagaraj et al. studied the resistance of RPL12 and RPL19 to non-host diseases in Nicotiana benthamiana and Arabidopsis, confirming that the RPL gene family has disease resistance function (Satish Nagaraj et al., 2016). In addition, Zheng Xiaofeng's research group found that in addition to participating in ribosome composition and protein translation, RPL6 also has a new function of regulating DNA damage response (Yang Chuanzhen & Zang Weicheng et al., 2019). Mazahar Moin et al. found that more than 50% of RP genes were upregulated in rice shoots and roots under drought stress. RPL23A The overexpression line has significant salt and drought resistance (Mazahar Moin, et al., 2017). RPL6 Overexpression of rice significantly improved salt tolerance (Mazahar Moin, et al., 2021). In addition to being involved in the root development process of Arabidopsis, RPL10 is also involved in the disease resistance response to Geminivirus (Carvalho et al., 2008; Rocha et al., 2008). In summary, the RPL gene family plays an important role in plant growth and development, stress and disease response, and the role of the RPL gene family in plant resistance to pear black spot disease needs further study.

[0007] Sand pear is the most widely planted pear cultivar and has very rich genetic diversity. The genetic diversity of sand pear varieties includes not only wild resources, but also local resources and newly bred varieties, which provides an important basis for the study of germplasm resources. The study of sand pear germplasm resources is crucial to understanding the domestication and improvement process of pears. Pear black spot disease has a huge impact on the yield of sand pears, so cloning the genes related to sand pear resistance to pear black spot disease is the key and basis of disease resistance genetic engineering. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a ribosomal protein PpRPL12 in sand pear and its encoding gene and its application in improving the ability of plants to resist pear black spot disease. The PpRPL12 has the function of regulating plant disease resistance and effectively improves the ability of transgenic plants to resist pear black spot disease.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] In the first aspect, the present invention seeks to protect at least one application of the ribosomal protein PpRPL12 in Pyrus pyrifolia with an amino acid sequence as shown in SEQ ID NO.1 or a biological material related to the protein in the following (a1)-(a13):

[0011] (a1) Application in improving the peroxidase activity of plants stressed by pear black spot disease;

[0012] (a2) Application in the preparation of products for increasing the peroxidase activity of plants stressed by pear black spot disease;

[0013] (a3) Application in improving the activity of phenylalanine ammonia lyase in plants stressed by pear black spot disease;

[0014] (a4) Use in the preparation of a product for increasing the activity of phenylalanine ammonia lyase in plants stressed by pear black spot;

[0015] (a5) Application in improving the activity of polyphenol oxidase in plants stressed by pear black spot disease;

[0016] (a6) Use in the preparation of a product for increasing the activity of polyphenol oxidase in plants stressed by pear black spot disease;

[0017] (a7) Application in improving chitinase activity in plants stressed by pear black spot disease;

[0018] (a8) Application in the preparation of a product for increasing chitinase activity in plants stressed by pear black spot disease;

[0019] (a9) Application in improving the activity of β-1,3-GA enzyme in plants stressed by pear black spot;

[0020] (a10) Application in the preparation of a method for improving the activity of β-1,3-GA enzyme in plants stressed by pear black spot disease;

[0021] (a11) Application in improving the resistance of plants to pear black spot disease;

[0022] (a12) Use in the preparation of products for improving the resistance of plants to pear black spot disease;

[0023] (a13) Application in genetic breeding of new plant germplasm with improved resistance to pear black spot.

[0024] Furthermore, the biological material related to the protein is at least one of the following (b1)-(b11):

[0025] (b1) a gene encoding the protein whose nucleotide sequence is shown in SEQ ID NO.2;

[0026] (b2) a primer pair having nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 for cloning the gene described in (b1);

[0027] (b3) an expression cassette containing the gene described in (b1);

[0028] (b4) a recombinant vector containing the gene described in (b1), or a recombinant vector containing the expression cassette described in (b3);

[0029] (b5) a recombinant microorganism containing the gene described in (b1), or a recombinant microorganism containing the expression cassette described in (b3), or a recombinant microorganism containing the recombinant vector described in (b4);

[0030] (b6) a transgenic plant cell line containing the gene described in (b1), or a transgenic plant cell line containing the expression cassette described in (b3), or a transgenic plant cell line containing the recombinant vector described in (b4);

[0031] (b7) transgenic plant tissue containing the gene described in (b1), or transgenic plant tissue containing the expression cassette described in (b3), or transgenic plant tissue containing the recombinant vector described in (b4);

[0032] (b8) a transgenic plant organ containing the gene described in (b1), or a transgenic plant organ containing the expression cassette described in (b3), or a transgenic plant organ containing the recombinant vector described in (b4);

[0033] (b9) a transgenic plant containing the gene described in (b1), or a transgenic plant containing the expression cassette described in (b3), or a transgenic plant containing the recombinant vector described in (b4);

[0034] (b10) Tissue culture produced from regenerable cells of the transgenic plant described in (b9);

[0035] (b11) Protoplasts produced from the tissue culture described in (b10).

[0036] Furthermore, the above application is to increase the activity and / or content of the ribosomal protein PpRPL12 in the target plant, or to overexpress the gene encoding the ribosomal protein PpRPL12.

[0037] In a specific embodiment of the present invention, the above application specifically comprises the following steps: recombinantly expressing the ribosomal protein PpRPL12 or the gene encoding the ribosomal protein PpRPL12 or the gene cloned using the primer pair in a target plant, and the obtained recombinant plant has disease resistance.

[0038] Furthermore, the cloning procedure is 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and 72°C extension for 10 min after the cycle is completed.

[0039] In the second aspect, the present invention claims a method for improving the resistance of plants to pear black spot disease, which comprises increasing the activity and / or content of the ribosomal protein PpRPL12 having an amino acid sequence as shown in SEQ ID NO.1 in a target plant, or overexpressing a gene encoding the ribosomal protein PpRPL12 having a nucleotide sequence as shown in SEQ ID NO.2 to improve the resistance of the target plant to pear black spot disease.

[0040] In the third aspect, the present invention claims a method for cultivating transgenic plants with improved resistance to pear black spot disease, overexpressing a gene encoding the ribosomal protein PpRPL12 as shown in SEQ ID NO.2 in a target plant to obtain transgenic plants with improved resistance to pear black spot disease, and cultivating the obtained transgenic plants.

[0041] The plant described in the present invention is pear or Arabidopsis thaliana.

[0042] The pathogen of the pear black spot disease of the present invention is Alternaria ( Alternaria ) Pathogenic fungi, such as Alternaria tenuissima ( Alternaria tenuissima ), Alternaria alternata ( Alternaria alternata ) Alternaria arborescens ), Alternaria pyrifolia ( Alternaria gaisen )、Alternaria japonica Alternaria gossypina ) and Alternaria longipes ( Alternaria longipes ); especially Alternaria alternata ( Alternaria alternata ).

[0043] In a specific embodiment of the present invention, when conducting a pear black spot disease stress experiment, the inoculation method of the pear black spot disease pathogen includes spraying a spore suspension and contacting a solid culture medium bacterial cake.

[0044] The present invention provides a coding gene for ribosomal protein PpRPL12 in sand pear, having a nucleotide sequence as shown in SEQ ID NO.2 in the sequence table. Leaves of two-week-old sand pear seedlings were inoculated with a 5 mm diameter pear black spot disease cake, samples were taken at corresponding time points, and the relative expression of the coding gene provided by the present invention was analyzed by real-time quantitative PCR. The results showed that within 192 hours, as the pear black spot disease treatment experiment was extended, the relative expression of the coding gene gradually increased except for 48 hours, which indicated that the coding gene responded to the pear black spot disease stress treatment and had a regulating disease resistance function.

[0045] Beneficial effects of the present invention:

[0046] The present invention provides the use of ribosomal protein PpRPL12 in sand pear and its coding gene or the coding gene cloned by using the primer pair in improving the ability of plants to resist pear black spot disease. By constructing a pear callus transformation vector, an Arabidopsis transformation vector and a gene-silencing pear seedling transformation vector respectively, positive callus tissue or plant seedlings are obtained for pathogen inoculation treatment, and the results show that the lesion area of ​​transgenic pear callus tissue is significantly reduced compared with the wild type (WT), the conductivity and hydrogen peroxide content are significantly reduced compared with the wild type, and the POD, PAL, PPO and chitinase activities are significantly increased compared with the wild type, indicating that the positive callus tissue has stronger disease resistance than the wild type. The leaf length and leaf area of ​​the transgenic Arabidopsis positive plant seedlings are significantly increased compared with the wild type, the lesion area of ​​the leaf inoculated with bacterial cake is significantly reduced compared with the wild type, and the POD and PAL enzyme activities are significantly increased compared with the wild type, indicating that the positive plant seedlings have stronger disease resistance than the wild type. The lesion area and hydrogen peroxide content in virus-silenced pear seedlings were higher than those in the wild type, and the POD and PAL enzyme activities were significantly lower than those in the wild type, indicating that the plants were more damaged by the pathogen. These results indicate that overexpression PpRPL12 The gene can effectively change the activity of disease-resistant enzymes to induce the disease resistance of plants and enhance the ability to degrade fungal cell walls, so that plants can better cope with the stress of pear black spot. PpRPL12 The silencing of the gene reduces the ability of the plant to stimulate changes in disease-resistant enzymes, thereby weakening the disease resistance of the plant. The ribosomal protein PpRPL12 and its encoding gene provided by the present invention have important application value in the research of improving the ability of plants to resist pear black spot disease. The present invention will have broad application space and market prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1It is a schematic diagram of the process of the technical solution of the present invention.

[0048] Figure 2 This is a schematic diagram of the expression of the coding gene of PpRPL12 in Example 2 under the stress of pear black spot and the treatment of SA, MeJA, and Eth plant hormones;

[0049] Among them, A is a detached leaf of a two-week-old sand pear seedling (not genetically modified) inoculated with a bacterial cake, and samples were taken at the corresponding time points, and the relative expression of the gene of the present invention was analyzed by real-time quantitative PCR; B is the expression pattern of the sand pear seedlings at different time points after being transplanted into the MS medium containing 100 μM SA; C is the expression pattern of the sand pear seedlings at different time points after being transplanted into the MS medium containing 100 μM MeJA; D is the expression pattern of the sand pear seedlings at different time points after being transplanted into the MS medium containing 100 μM Eth.

[0050] Figure 3 The subcellular localization of the gene encoding PpRPL12 in Example 3;

[0051] Among them, A is the imaging of GFP gene (control) in bright field (middle) and GFP field (left), and the figure (right) is the superposition of the two images; B is PpRPL12 The image of the gene under bright field (middle) and GFP field (left). The image (right) is the superposition of the two.

[0052] Figure 4 The invention is the genetic transformation and identification of the callus of eggplant and pear in Example 4;

[0053] Among them, A is the vector construction map; B is the gel electrophoresis diagram of RNA extracted from wild-type and transgenic overexpression callus samples; C is the overexpression lines of transgenic eggplant pear callus identified by qRT-PCR, OE3, OE5 and OE6 are three overexpression lines.

[0054] Figure 5 For Example 5 PpRPL12 Identification of positive seedlings of transgenic Arabidopsis;

[0055] Among them, A is the identification of transgenic Arabidopsis thaliana by PCR ("M" is Marker; "+" is recombinant plasmid; "-" is wild-type Arabidopsis thaliana; "1-9" is transgenic strain); B is the identification of transgenic Arabidopsis thaliana T1 generation by RT-PCR, OE1, OE2 and OE9 are three transgenic lines.

[0056] Figure 6 For Example 6 PpRPL12 Identification of positive seedlings of Pyrus betulaeformis seedlings transiently transformed with virus silencing;

[0057] Among them, A is the gel electrophoresis diagram of RNA extracted from the pear seedling samples of the control group and the experimental group after instantaneous injection; B is the gene expression level of gene silencing positive plants detected by qRT-PCR using gene-specific primers and internal reference primer UBQ, pTRV5, pTRV8 and pTRV9 are three silencing strains.

[0058] Figure 7 For Example 7 PpRPL12 Phenotypic and physiological parameters of overexpression lines and wild-type pear callus after inoculation with Alternaria alternata ;

[0059] Among them, A is PpRPL12 Phenotypes of overexpression and wild-type pear callus at 0, 10, and 20 days after inoculation with Alternaria; B is the lesion diameter and changes of overexpression and wild-type pear callus at 0-20 days after inoculation; C is the conductivity of overexpression and wild-type pear callus 6 days after inoculation; D is the hydrogen peroxide content of overexpression and wild-type pear callus before and 6 days after inoculation; E is the POD enzyme activity of overexpression and wild-type pear callus before and 6 days after inoculation; F is the PAL enzyme activity of overexpression and wild-type pear callus before and 6 days after inoculation; G is the PPO enzyme activity of overexpression and wild-type pear callus before and 6 days after inoculation; H is the β-1,3-GA enzyme activity of overexpression and wild-type pear callus before and 6 days after inoculation; I is the chitinase activity of overexpression and wild-type pear callus before and 6 days after inoculation.

[0060] Figure 8 In Example 7 PpRPL12 Phenotypic and physiological parameters of transgenic lines and wild-type Arabidopsis were measured before and after Alternaria inoculation;

[0061] Among them, A is the phenotype of transgenic lines and wild-type Arabidopsis before and 10 days after spraying spore suspension; B is the phenotype of transgenic lines and wild-type Arabidopsis leaves before and 3 days after inoculation with Alternaria; C is a micrograph of Alternaria hyphae penetrating the epidermal cell wall of Arabidopsis; D is a micrograph of transgenic lines Arabidopsis 0-3 days after inoculation PpRPL12 Relative expression of genes (relative to uninoculated wild-type Arabidopsis); E is the POD enzyme activity of leaves of transgenic lines and wild-type Arabidopsis before and 3 days after inoculation with Alternaria; F is the PAL enzyme activity of leaves of transgenic lines and wild-type Arabidopsis before and 3 days after inoculation with Alternaria.

[0062] Fig. 9 In Example 8, the wild type and PpRPL12 Phenotypic and physiological indexes of virus-silencing positive Pyrus betula seedlings before and after inoculation with Alternaria;

[0063] Among them, A is the wild type and PpRPL12Phenotypes of virus-silencing positive Pyrus betula seedling leaves before and 6 days after inoculation with Alternaria; B is the wild type and PpRPL12 Diameter of lesions on leaves of virus-silenced Pyrus betulaeformis seedlings 6 days after inoculation with Alternaria; C is the diameter of lesions on leaves of wild type and PpRPL12 Hydrogen peroxide content in the leaves of virus-silencing positive Pyrus betulaeformis seedlings 6 days after inoculation with Alternaria alternata. D is the difference between wild type and PpRPL12 POD enzyme activity in virus-silencing positive Pyrus betula seedling leaves 6 days after inoculation with Alternaria alternata; E is the wild type and PpRPL12 PAL enzyme activity in leaves of virus-silencing positive Pyrus betula seedlings 6 days after inoculation with Alternaria alternata. DETAILED DESCRIPTION

[0064] The present invention provides a ribosomal protein PpRPL12 in Pear sphaerocarpa, having an amino acid sequence as shown in SEQ ID NO.1 in the sequence table, comprising 167 amino acids, an isoelectric point of 9.02, and a molecular weight of 17.77 KDa. The PpRPL12 participates in the plant JA-mediated defense response, stimulates the disease resistance of the plant, and can also increase the chitinase activity to degrade the fungal cell wall, thereby indicating that this gene has a disease resistance function. In the present invention, the PpRPL12 is obtained by recombinantly expressing the coding gene of the PpRPL12. The method of the recombinant expression is not particularly limited, and the recombinant expression method known in the art can be used.

[0065] The present invention provides a gene encoding the ribosomal protein PpRPL12 in Pear sutchuenensis, which has a nucleotide sequence as shown in SEQ ID NO. 2 in the sequence table. The encoding gene is preferably obtained by cloning.

[0066] The present invention provides a primer pair for cloning the coding gene cDNA sequence, comprising a forward primer and a reaction primer, wherein the forward primer has a nucleotide sequence as shown in SEQ ID NO.3 in the sequence list; and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.4 in the sequence list. The source of the primer pair is not particularly limited, and it can be synthesized by a biosynthesis company known in the art. In the embodiment of the present invention, the primer pair is commissioned to be synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0067] The invention provides the use of the ribosomal protein PpRPL12 in sand pear, the coding gene or the coding gene cloned by using the primer pair in improving the ability of plants to resist pear black spot disease.

[0068] In the present invention, the use preferably comprises the following steps: recombinantly expressing the ribosomal protein PpRPL12 in the sand pear or the coding gene or the coding gene cloned by the primer pair in a plant, and the obtained recombinant plant has the ability to resist pear black spot disease (see Figure 1 ); and silencing the ribosomal protein PpRPL12 in the sand pear or the coding gene or the coding gene cloned by the primer pair in the plant, so that the resistance of the plant to pear black spot is weakened (see Figure 1 ).

[0069] In the present invention, the cloning procedure is preferably 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and 72°C extension for 10 min after the cycle is completed. The coding gene is preferably transformed into plants in the form of GFP-PpRPL12 recombinant vectors and silenced in plants in the form of pTRV2-PpRPL12 recombinant vectors. The present invention has no special restrictions on the construction method of the GFP-PpRPL12 and pTRV2-PpRPL12 recombinant vectors, and the construction method known in the art can be used.

[0070] The method for recombinant expression and silencing of the coding gene or the coding gene cloned by the primer pair in plants preferably adopts the method of Agrobacterium-mediated genetic transformation. The present invention has no particular limitation on the method of Agrobacterium-mediated genetic transformation, and conventional methods well known in the art can be used. The plants preferably include callus tissue of Pyrus oleraceus, Arabidopsis thaliana and Pyrus betulaeformis.

[0071] In the present invention, before testing the ability of the recombinant plants and gene silenced plants to resist pear black spot disease, it is preferred to also include screening of positive plants. The screening method for the positive transgenic plants is preferably carried out by PCR amplification. During the screening, the forward primer of the PCR amplification primer has a nucleotide sequence as shown in SEQ ID NO.3 in the sequence table; the reverse primer of the PCR amplification primer has a nucleotide sequence as shown in SEQ ID NO.4 in the sequence table. After the PCR amplification is completed, if the plant lines to be tested can amplify the expected size of the fragment (501bp), it indicates that they are positive transgenic lines.

[0072] The screening method for the positive overexpression plants and gene silenced plants is preferably carried out by using a qRT-PCR method to test the gene expression amount. During the screening, the forward primer of the primers used in the qRT-PCR has a nucleotide sequence as shown in SEQ ID NO.5 in the sequence list, and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.6 in the sequence list; the internal reference primer UBQ has a nucleotide sequence as shown in SEQ ID NO.7 and SEQ ID NO.8 in the list.

[0073] After the real-time quantitative PCR amplification is completed, if the gene expression level amplified by the tested plant line is significantly higher than the expression level of the gene in the wild-type plant, it indicates that they are positive overexpression lines. If the gene expression level amplified by the tested plant line is significantly lower than the expression level of the gene in the wild-type plant, it indicates that they are positive silenced lines.

[0074] In the present invention, the pear black spot disease strain activation method is to place the Alternaria strains in potato dextrose agar (PDA) culture medium for activation, culture them in a 28°C incubator for 4 days, punch holes at the edge of the colony with a cork puncher, and then place the bacterial cake in a new culture medium, and repeat each treatment twice. Culture them in a 28°C incubator for 4 days, calculate the average daily growth rate of the black spot disease, and screen the black spot disease strains with strong pathogenicity.

[0075] In the present invention, the artificial inoculation of the pear black spot disease adopts different methods in different plant materials. The bacterial cake inoculation method is adopted on the pear callus, and a hole is punched at the edge of the activated colony with a puncher, and the bacterial cake is placed in the middle of the culture medium with the callus tissue spread flat, and the sterile environment is sealed and placed in a 28°C incubator for dark culture for a certain period of time before sampling. The callus tissue is taken about 1 cm from the junction of the callus tissue and the alternating spore spot disease to determine the physiological indicators. The live spore suspension spraying method can be adopted on Arabidopsis, and the activated black spot disease strain is picked, and the potato glucose medium (PotatoDextrose Broth, PDB) is shaken, and the spore suspension is placed in a 28°C shaker for 48h, and filtered with gauze to obtain a spore suspension. Select a 45-day-old robust Arabidopsis plant, spray the spore suspension on the leaves every day, and place it in a 28°C incubator for dark culture. After a certain period of time, the Arabidopsis leaves are picked to determine the indicators. The bacterial cake method can also be used to inoculate Arabidopsis leaves with injuries. Pick robust Arabidopsis leaves, sterilize the detached leaves with 0.2% NaClO for 30 seconds, rinse with sterile water 5 times, and place them on a porcelain plate with sterilized moist gauze. Wrap the petiole with cotton wool dipped in sterile water, cover with plastic wrap to keep moist, and recover for 12 hours. Use a needle to puncture the center surface of the leaf (avoid the veins), place the bacterial cake on the needle-punctured hole of the leaf, and culture it in a 28℃ incubator. After 2 days, remove the bacterial cake and continue to culture at 28℃ until sampling. Pear seedling leaves are inoculated with wounds using the bacterial cake method, and the specific steps are the same as Arabidopsis.

[0076] In the present invention, the obtained positive transgenic strains and gene silenced strains are subjected to pear black spot stress treatment, and the phenotypes and physiological indicators of the positive transgenic strains and gene silenced strains obtained after the treatment are measured to verify their anti-pear black spot function. In the determination of the lesion diameter, because the black spot lesions are irregular and nearly circular, the determination method is preferably to measure the black spot diameter once every 45 degrees in the obvious black spot area with a ruler, take the average of 3 times, and repeat 3 groups. The state, leaf length and leaf area of ​​Arabidopsis plants can be identified by naked eye observation. The measurement results show that the wild-type callus has larger lesions than the transgenic line, the wild-type Arabidopsis plant leaves have obvious lesions, and the transgenic plant leaves have no obvious lesions, but the leaves are yellow. After spraying the Arabidopsis plants with spore suspension for 10 days, the wild-type Arabidopsis plants have poor growth status compared with the transgenic lines, shorter leaf length, smaller leaf area, and obvious withered and rotten leaves. This indicates that the positive transgenic lines can better defend against the infection of pear black spot disease, maintain the growth of the plants, have better external barriers and better endogenous physiological regulation capabilities when facing the stress of pear black spot disease, and better avoid the damage of pear black spot disease to plants.

[0077] In the present invention, the conductivity determination method is not particularly limited, and the extraction and determination methods known in the art can be used. Analysis of the conductivity determination results showed that the conductivity of the positive overexpression callus after inoculation was significantly lower than that of the wild type (WT), which was about 1 / 3 of the wild type, indicating that the positive overexpression strain may have better cell integrity, stability and ROS resistance than the wild type, indicating that the positive overexpression strain has a stronger resistance to pear black spot than the wild type.

[0078] The hydrogen peroxide content, PAL, POD, PPO and chitinase activity were determined using kits from Nanjing Jiancheng Company, and the β-1,3-GA activity was determined using a Solebo kit. The results showed that the POD, PAL, PPO and other enzyme activities of the overexpression callus increased after inoculation and were significantly higher than those of the wild type, indicating that PpRPL12 The overexpression line has stronger antioxidant capacity, and the damage of free radicals after infection is reduced. The disease resistance pathway mediated by PpRPL12 may be related to the phenylpropanoid metabolic pathway, which resists pathogens by affecting intracellular signal transduction. In addition, the chitinase activity of the overexpression line increased by 36.2% compared with the wild type before and after infection, and the β-1,3-GA enzyme activity was significantly improved, indicating that PpRPL12 The overexpression line has a stronger ability to degrade pathogens. The activities of POD, PAL and other enzymes in the transgenic Arabidopsis thaliana increased after inoculation and were significantly higher than those in the wild type, indicating that PpRPL12 The gene also activated the resistance of Arabidopsis plants to Alternaria alternata. The activities of POD and PAL enzymes were significantly reduced in the silenced Pyrus betula seedlings, and the content of hydrogen peroxide, which is oxidatively destructive, increased significantly. This indicates that the virus is silenced. PpRPL12 The role of phenylpropanoid pathways and other disease resistance pathways in the transgenic plants was weakened. 2 O 2 ) may cause more serious damage to the cell membrane, ultimately leading to a decrease in the plant's resistance to pear black spot.

[0079] In the present invention, the resistance of the plant to pear black spot disease is expressed by the degree of plant disease, such as the size of the diseased spots and the growth status of the plant after the disease. The pear black spot disease is caused by the pathogenic fungus of the genus Alternaria, Alternaria ( Alternaria alternata ). The plant includes Pyrus oleifera callus, Arabidopsis thaliana or Pyrus betulaeformis.

[0080] The ribosomal protein PpRPL12 in the sand pear provided by the present invention and its application in improving the resistance of plants to pear black spot disease are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0081] In the present invention, the resistance of the plant to pear black spot disease is expressed by the degree of plant disease, such as the size of the diseased spots and the growth status of the plant after the disease. The pear black spot disease is caused by the pathogenic fungus of the genus Alternaria, Alternaria ( Alternaria alternata ). The plant includes Pyrus oleifera callus, Arabidopsis thaliana or Pyrus betulaeformis.

[0082] The ribosomal protein PpRPL12 in the sand pear provided by the present invention and its application in improving the ability of plants to resist pear black spot disease are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0083] Embodiment 1 sand pear PpRPL12 Cloning of full-length cDNA of gene

[0084] A ribosomal protein PpRPL12 was screened from Pyrus pyrifolia. PpRPL12 The gene sequence and primers were designed using primerprimier5.0. The primer specificity was verified on the Pear Center website. The full length of the cDNA was amplified from Pear oleraceus by RT-PCR. The detailed steps are as follows: The synthesis of the first-chain cDNA was carried out according to the operating instructions of the reverse transcription kit of Nanjing Novezan Company. The first-chain cDNA was used for PpRPL12Amplification of genes. The total volume of PCR reaction was 50 μl, including 1 μl of Pyrus cDNA, 2 μl of upstream and downstream primers (SEQ ID NO.3 and SEQ ID NO.4), 1 μl of enzyme, 25 μl of 2×Buffer, and 19 μl of sterilized dd water. PCR was completed according to the following procedures: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and 72℃ extension for 10 min after the cycle was completed. After amplification, a single band of PCR products was generated. After 1% agarose gel electrophoresis, the target band was cut out and the specific target band was recovered according to the instructions of the gel recovery kit.

[0085] The purified product was recovered and connected to the pEASY-Blunt Zero vector. The connection method was carried out according to the operating instructions of the Nanjing Novizan recombinase kit. The molar ratio of gene to vector in the connection system was 3:1. The total reaction volume was 10μl, including 1 μl of PCR purified product, 2 μl of vector, 2 μl of 5× buffer, 1 μl of DNA recombinase, and 4 μl of sterile dd water. The connection was carried out at 37℃ for 30min, and the heat shock method was used to transform into Escherichia coli competent DH5α. The target gene sequence primers were used for PCR verification and sequencing (completed by Shanghai Shenggong Biotechnology Co., Ltd.).

[0086] Example 2 Pear black spot stress treatment and different plant hormone treatments PpRPL12 qRT-PCR analysis of genes

[0087] In order to analyze PpRPL12 The response patterns of genes to pear black spot, SA, MeJA, and Eth treatments were analyzed using real-time PCR technology. PpRPL12 The expression pattern of genes was analyzed. RNA was extracted using the FastPurePlant Total RNA lsolation Kit from Nanjing Novezan Company, and the synthesis of the first strand of DNA was carried out according to the operation manual of the reverse transcription kit from Nanjing Novezan Company. In the 10 µl reaction system, there were: 3.6 μl 2 × SYBR-Green PCR MasterMix, 500 ng cDNA, 0.2 µl primers (forward and reverse primers were SEQ ID NO.5 and SEQ ID NO.6, and UBQ was used as the internal reference primer (SEQ ID NO.7 and SEQ ID NO.8)), and deionized water was added to 10 µl. The program and reaction system of real-time quantitative PCR are shown in Tables 1 and 2.

[0088] Table 1 Real-time quantitative PCR program

[0089] .

[0090] Table 2 Real-time quantitative PCR reaction system

[0091] .

[0092] Results Figure 2 . Figure 2 It is a schematic diagram of the expression of the PpRPL12 encoding gene of the present invention under the stress of pear black spot and the treatment of SA, MeJA and Eth plant hormones; Figure 2 Middle A is a detached leaf of a two-week-old sand pear seedling (not genetically modified) inoculated with bacterial cake, samples were taken at corresponding time points, and the relative expression of the gene of the present invention was analyzed by real-time quantitative PCR; Figure 2 Middle B is the expression pattern at different time points after the sand pear seedlings were transplanted into MS medium containing 100 μM SA; Figure 2 Center C shows the expression pattern of Pyrus communis seedlings at different time points after being transplanted into MS medium containing 100 μM MeJA. Figure 2 Figure D shows the expression pattern of Pyrus ovata seedlings at different time points after transplanting into MS medium containing 100 μM Eth. Figure 2 As can be seen from Figure A, with the extension of the pear black spot disease stress time, the expression level of the coding gene showed an increasing trend in 0h-36h and 48h-144h, which indicates that the coding gene is affected by the pear black spot disease stress, thereby responding to the disease resistance mechanism and enabling the sand pear to achieve the pear black spot disease resistance function. Figure 2 From B to D, it can be seen that after exogenous application of SA, MeJA, and Eth, PpRPL12 Gene expression levels were generally upregulated. PpRPL12 The genes may be related to salicylic acid, jasmonic acid and ethylene resistance pathways, and the correlation with the salicylic acid pathway is the highest.

[0093] Example 3 Subcellular localization of the gene encoding PpRPL12

[0094] According to the nucleotide sequence of the coding gene of PpRPL12 and the map of the pCAMBIA1300-GFP vector, XbaI and BamHI restriction sites were added before and after the gene sequence, respectively. The target gene extraction plasmid with the correct sequencing result was used as a template and amplified with primers (SEQ ID NO.9 and SEQ ID NO.10) with added restriction sites. The PCR program used was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min. The termination codon TAG was removed from the 3′ of the gene in order to allow the gene to fuse with GFP. After 1% agarose gel electrophoresis, the PCR product was recovered using a gel kit. The pCAMBIA1300-GFP vector plasmid was digested with XbaI and BamHI restriction endonucleases and purified and recovered after 4 hours of digestion at 37℃. The pCAMBIA1300-GFP vector after digestion was recovered from the gel PpRPL12 The fragments were connected by recombinant ligase, connected at 37°C for 30 minutes, and transformed into E. coli competent DH5α. The transformed bacterial solution was tested by PCR, and the bacterial solution with positive PCR identification was sent to the company for sequencing, and the plasmid of the bacterial solution with correct sequencing results was extracted, and the obtained recombinant vector was named GFP-PpRPL12.

[0095] Agrobacterium-mediated transient transformation of tobacco leaf cells: The recombinant GFP-PpRPL12 vector plasmid was transformed into Agrobacterium competent GV3101, and the activated Agrobacterium containing the recombinant plasmid was propagated in LB liquid medium containing 50 mg / mL kanamycin and 50 mg / mL rifampicin, in a constant temperature shaker at 250 rpm and 28°C. The cells were centrifuged at 6000 rpm for 10 min and inoculated with infection solution (100 mL: 10 mL 100 mM MgCl 2 , 10 mL 100 mM MES, 75 µL 200mM AS, ddH 2 O 80 mL) to OD600 of 0.8. Incubate at room temperature for 3-4 h and then inject into young tobacco leaves. Infected tobacco plants were cultured in the dark for 24 h and in normal light for 48 h, then stained with DAPI to mark the cell nucleus and prepared. Photographs were taken using a laser scanning confocal microscope (Zeiss LSM 410).

[0096] Results Figure 3 . Figure 3 The subcellular localization of the gene encoding PpRPL12; Figure 3 Middle A, the imaging of GFP gene (control) under bright field (middle) and GFP field (left), and the image (right) is the superposition of the two; Figure 3 Middle B, PpRPL12The image of the gene in bright field (middle) and GFP field (left), and the image (right) is the superposition of the two. According to the cell localization map, PpRPL12 is located in the cytoplasm.

[0097] Example 4 Genetic transformation of eggplant callus

[0098] 1. Plant transformation vector construction

[0099] According to the multiple cloning site of PCAMBIA1300-GFP vector and PpRPL12 The coding region sequence of the gene was prepared, and restriction sites XbaI and BamHI were added. The upstream and downstream PCR primers (SEQ ID NO.9 and SEQ ID NO.10) were designed using primer primier5.0 software according to the general principles of primer design. PpRPL12 The clone of the gene was used as a template for PCR amplification. The annealing temperature of PCR amplification was 59 °C, and the PCR reaction system and amplification procedure were the same as those in PpRPL12 The gene clones were the same. After amplification, gel purification was performed. The volume of the PCMBIA1300-GFP vector double restriction enzyme digestion reaction was 50 µl, including: 1000 ng of the PCMBIA1300-GFP vector plasmid, 5 µl of 10×M buffer, 1 µl of XbaI and BamHI, and double distilled water to make up to 50 µl. Place at 37℃ for 3-4 hours and then purify and recover. Add to the ligation reaction system PpRPL12 The molar ratio of gene to vector pCMBIA1300 is 2:1, and the total reaction volume is 10 µl. It contains: 2 µl 5× buffer, 1 µl DNA recombinase, PpRPL12 1 ul of gene, 2 µl of PCMBIA1300-GFP vector product recovered by double enzyme digestion, 4 µl of double distilled water, reacted at 37°C for 30 min to obtain the ligation product. The ligation product was transformed into Escherichia coli DH5α and cultured on LB solid plates containing 50 mg / L kanamycin for 16 h. The positive clones screened were picked and shaken, and the plasmids were extracted for PCR identification. Sequencing confirmed that there were no coding frame mutations, and the recombinant clone containing the inserted target fragment was obtained, which was named GFP-PpRPL12 recombinant vector. The recombinant vector GFP-PpRPL12 was introduced into Agrobacterium GV3101 by freeze-thaw method.

[0100] 2. The steps of Agrobacterium-mediated genetic transformation of eggplant and pear callus are as follows:

[0101] (1) Agrobacterium culture: Take the Agrobacterium tumefaciens culture liquid stored in the ultra-low temperature refrigerator, streak it on a plate containing LB supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin, and culture it in a 28 °C incubator for 36-48 hours. Select a spot and shake it in LB liquid culture medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin for 36-48 hours. Centrifuge it at 5000 r for 5 min, remove the supernatant and re-spin it with double distilled water in an ultra-clean workbench (the sterility of the infection liquid must also be ensured in the subsequent steps). Measure and adjust the OD to 0.6, add 100 µl of AS, and culture it in a shaker at 30 °C and 120 r for 45 min-1 h for infection.

[0102] Dip staining: In a clean bench, crush the wild-type eggplant pear callus with tweezers and pour it into a conical flask containing the dip staining solution. Incubate at 30°C and 120 r in a shaker for 15 min. Filter out the callus with a colander, rinse with water appropriately, and then dry it with filter paper.

[0103] Culture: The callus tissue was spread 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 / L AS, pH = 5.8) solid culture medium with AS, and cultured at 28°C for two days.

[0104] 3. Screening of overexpression positive callus

[0105] The callus obtained by the above method was transferred to MS (2.37g / LMS+50g / L sucrose+7g / L agar+0.5mg / L 6-BA+1mg / L 2,4-D+10mg / L hygromycin+150mg / L cephalosporin, pH=5.8) solid medium for screening overexpression callus. The callus was cultivated according to the conventional method, and white tender cell clumps grew in about 3-4 weeks. Small clumps of transgenic callus were picked and transferred to new screening solid medium. Each clump was PpRPL12 An overexpression line of overexpressing callus tissue was continued to be subcultured according to the conventional method.

[0106] 3.1 RNA extraction from overexpression callus

[0107] RNA was extracted using the RNA extraction kit from Nanjing Novezan Company, and the extraction method was referred to the kit instructions.

[0108] 3.2 Detection of positive overexpression plants

[0109] Each line of pear callus obtained by the above method was sampled independently, and RNA from wild-type and transgenic callus samples was extracted. The structure was tested by running gel to check its integrity. The concentration was determined by Nanodrop (the concentration was 200-1000 ng / ul). The total amount of RNA was adjusted to 3 ug and then reverse transcribed into cDNA, and then amplified using UBQ as an internal reference. The nucleotide sequence of the UBQ primer is:

[0110] UBQ forward primer: 5'-GCACAAGAAGGTGAAGCTCG-3' (SEQ ID NO.7)

[0111] UBQ reverse primer: 5'-ACTCAGCATTGGGGCACTC -3' (SEQ ID NO.8)

[0112] The brightness of the bands amplified by UBQ was consistent, indicating that the concentration of the reverse transcribed cDNA was the same. Then, qRT-PCR was performed using PpRPL12 specific primers and pear internal reference primer UBQ to analyze the expression level of the strain to be tested. The nucleotide sequence of the PpRPL12 specific primer is:

[0113] Forward primer: 5'-TGCCTCCCAAATTCGACCC-3' (SEQ ID NO.5)

[0114] Reverse primer: 5'-GGCAGTCTCCTTAGCGATGT-3' (SEQ ID NO. 6).

[0115] according to PpRPL12 The expression levels of the genes were determined, and three plants with higher expression levels were selected as overexpression positive strains and named OE3, OE5 and OE6.

[0116] PpRPL12 was expressed in callus of pear plant through Agrobacterium-mediated transgenic method. Molecular genetic analysis identified transgenic pear callus stably expressing PpRPL12, so the phenotypic traits can be stably inherited. PpRPL12 The phenotypic changes of the overexpression material are not caused by the transgenic operation affecting other genes. Therefore, the overexpression material provides material guarantee for the research of this project. Figure 4 Middle A, Figure 4 Middle B is the gel electrophoresis diagram of RNA extracted from wild-type and transgenic overexpressing callus samples; Figure 4 Center C shows the overexpression lines of transgenic eggplant and pear callus identified by qRT-PCR, and OE3, OE5 and OE6 were selected as overexpression positive lines.

[0117] Example 5 Genetic transformation of Arabidopsis thaliana

[0118] 1. Plant transformation vector construction

[0119] Same as Example 4.

[0120] 2. The steps of Agrobacterium-mediated genetic transformation of Arabidopsis thaliana are as follows:

[0121] (1) Agrobacterium culture: Take the Agrobacterium tumefaciens culture liquid stored in the ultra-low temperature refrigerator, streak it on a plate containing LB supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin, and culture it in a 28°C incubator for 36-48 hours. Scrape the streaked plaque and add it to liquid MS (2.37 g / L MS + 50 g / L sucrose + 0.1 mg / L IBA, pH = 5.8) culture medium. Incubate it at 28°C with shaking for 30 min. When the concentration of the culture liquid reaches OD = 0.8-1.0, add 200 μl / L of surfactant sweet77 for infection.

[0122] (2) Infection: Take a wild-type Arabidopsis plant with a stem height of about 10 cm, remove all seed pods, and then invert it into a glass bottle containing the prepared Agrobacterium tumefaciens bacterial solution. Vacuum it and maintain a pressure of 0.05 MPa for 5 minutes. Place it on its side away from light for 24 hours.

[0123] (3) Cultivation: Cultivate the plants until they are fruitful according to conventional methods, and harvest mature seeds (T0 generation).

[0124] 3. Screening of transgenic positive seedlings

[0125] According to the above method, the PpRPL12 Genetic Arabidopsis T0 seeds were surface sterilized and evenly spread on MS selection medium containing 50 mg / L hygromycin and 50 mg / L timentin, cultured at 22°C with 16 h / d of light, and after one week of growth, the plants with fast growth and long roots were selected and transplanted into sterilized nutrient soil to grow for a period of time.

[0126] 3.1 DNA extraction from transgenic Arabidopsis

[0127] According to the above method, the PpRPL12 Gene Arabidopsis, extract DNA from each line of Arabidopsis, design primers and intragenic primers for PCR amplification to identify positive Arabidopsis strains.

[0128] (1) Take an appropriate amount of Arabidopsis leaves and grind them 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, fully dissolved in a 65°C water bath for later use) and 10 µl of β-mercaptoethanol and mix well.

[0129] (2) Heat in a 65°C water bath for 30 min, taking out the tube and gently inverting it every 10 min to mix it evenly; centrifuge at 10,000 g for 10 min at room temperature; take the supernatant, add 500 µl of chloroform / isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1), and mix it evenly by inversion.

[0130] (3) Centrifuge at 10,000 g for 10 min. Transfer 450 µl of the supernatant to a new 1.5 ml centrifuge tube. Add 450 µl of isopropanol and mix by inverting.

[0131] (4) Centrifuge at 10,000 g for 10 min, discard the supernatant, rinse twice with 1 mL of 75% ethanol, and centrifuge at 10,000 g for 10 min to completely remove the ethanol. Place the tube on a clean bench for ventilation and drying until the DNA becomes colorless and transparent.

[0132] (5) Add 50 µl of ultrapure water and place in a 65°C incubator to dissolve for 40 min before performing gel testing.

[0133] 3.2 Detection of positive transgenic plants

[0134] PCR amplification was performed using primer gene specific primers. The reaction procedures and systems are shown in Table 3 and Table 4, respectively. PCR was performed using gene upstream primers and vector downstream primers (SEQ ID NO.3 and SEQ ID NO.4). If fragments of the expected size could be amplified in the selected transgenic lines, it indicated that they were positive transgenic lines.

[0135] Table 3 PCR reaction procedure

[0136] .

[0137] Table 4 PCR reaction system

[0138] .

[0139] Agrobacterium-mediated expression in Arabidopsis thaliana PpRPL12 Through molecular genetic analysis, it was identified that the stable expression PpRPL12 The transgenic Arabidopsis thaliana was stably inherited. PpRPL12 The phenotypic changes of transgenic materials are not caused by the transgenic operation affecting other genes. Therefore, the transgenic materials provide material guarantee for the research of this project. Figure 4 A, PCR was used to identify transgenic Arabidopsis thaliana. Figure 5A ("M" is a marker; "+" is a recombinant plasmid; "-" is a wild-type Arabidopsis; "1-9" is a transgenic line), RT-PCR was used to identify the transgenic Arabidopsis T1 generation. Figure 5 In B, OE1, OE2, and OE9 are three transgenic lines.

[0140] Example 6 Transient transformation of Pyrus betulaeformis seedlings

[0141] 1. Construction of virus-induced gene silencing vector

[0142] The double restriction sites of the viral silencing vector pTRV2 are XbaI and SmaI. According to the general principles of primer design, the upstream and downstream primers (SEQ ID NO.11 and SEQ ID NO.12) were designed using primerprimier5.0 software. PpRPL12 The gene was amplified and inserted between the two restriction sites on the vector to obtain the recombinant vector pTRV2-PpRPL12, which was then transformed into Agrobacterium GV3101 competent cells.

[0143] 2. Virus-induced gene silencing in Pyrus betulaeformis seedlings

[0144] (1) Agrobacterium culture: Take the Agrobacterium tumefaciens culture liquid stored in the ultra-low temperature refrigerator and culture it in LB liquid medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin at 28°C and 220 rpm for 12 h. The cultured culture liquid was centrifuged at 6000g for 10 min to collect the cells and then inoculated with infection solution (10 mM MgCl 2 , 10 mM MES, 200 mM acetosyringone, pH 5.6) to resuspend the pellet to a concentration of OD = 0.8;

[0145] (2) Induction of bacterial solution: Place the bacterial solution with adjusted OD value in the dark and induce it at room temperature at 100 rpm for 4 h;

[0146] (3) Injection of pear seedlings: pTRV1 and pTRV2 bacterial solutions were mixed in a volume ratio of 1:1 as the control group, and pTRV1 and pTRV2-PpRPL12 bacterial solutions were mixed in a volume ratio of 1:1 as the experimental group. The two groups were injected into pear seedlings that were 45 days old, had consistent growth conditions and were in good health.

[0147] 3. Identification of virus-induced gene silencing-inhibition positive seedlings

[0148] After the injection, the pear seedlings were placed in the dark at room temperature for 12 h, and then returned to normal culture for 5 days. Each strain was independently sampled, and RNA from the pear seedling samples of the control group and the experimental group was extracted. The integrity of the structure was detected by running gel. Figure 6In A, after the concentration was measured by Nanodrop (the concentration was between 200-1000 ng / ul), the total amount of RNA was adjusted to 3 ug and then reverse transcribed into cDNA, and then amplified using UBQ as an internal reference. The nucleotide sequence of the UBQ primer is:

[0149] UBQ forward primer: 5'-GCACAAGAAGGTGAAGCTCG-3' (SEQ ID NO.7)

[0150] UBQ reverse primer: 5'-ACTCAGCATTGGGGCACTC -3' (SEQ ID NO.8)

[0151] The brightness of the bands amplified by UBQ was consistent, indicating that the concentration of reverse transcribed cDNA was the same. PpRPL12 qRT-PCR detection was performed using specific primers and pear internal reference primer UBQ to analyze the expression level of the strains to be tested. PpRPL12 The nucleotide sequences of the specific primers are:

[0152] Forward primer: 5'-TGCCTCCCAAATTCGACCC-3' (SEQ ID NO.5)

[0153] Reverse primer: 5'-GGCAGTCTCCTTAGCGATGT-3' (SEQ ID NO. 6).

[0154] according to PpRPL12 The expression level of the gene was determined, and three plants with lower expression levels were selected as virus silencing positive strains and named pTRV5, pTRV8 and pTRV9.

[0155] Results Figure 6 shown. Figure 6 A in the middle is the gel electrophoresis diagram of RNA extracted from pear seedling samples of the control group and the experimental group after instantaneous injection; Figure 6 In step B, gene-specific primers and internal reference primer UBQ were used to perform qRT-PCR to detect the gene expression level of gene silencing positive plants. Figure 6 The results show that the virus-silenced Pyrus betulaeformis seedling-positive lines (pTRV5, pTRV8 and pTRV9) PpRPL12 The gene is silenced.

[0156] Example 7 Identification of resistance of PpRPL12 transgenic disease-resistant plants

[0157] 1. Analysis of resistance of pear black spot in callus overexpressing the gene

[0158] For identification PpRPL12To determine whether the overexpression of pear callus was related to resistance to pear black spot, the control line and the transgenic line were inoculated with Alternaria alternata cake. PpRPL12 The callus tissues of overexpression lines (OE3, OE5 and OE6) and wild type (WT) callus tissues were flattened and densely spread on MS screening medium and commonly used MS non-antibiotic medium, respectively, to form a circular callus thin layer, and the amount of callus tissue on each medium was controlled to be equal. The callus tissue was inoculated with pear black spot pathogen cake, sealed in a sterile environment and placed in a 28°C incubator for dark culture. The phenotype after treatment was observed at 5d, 10d, 15d, and 20d, and the diameter of the lesions was measured. The conductivity and hydrogen peroxide content were measured 6d after inoculation, and the enzyme activities of PAL, POD, PPO, β-1,3-GA, chitinase, etc. were measured. The overexpression callus tissue was sampled and mixed to measure physiological indicators.

[0159] The methods for determining hydrogen peroxide content PAL, POD, PPO, and chitinase activity refer to the instructions for the kit provided by Nanjing Jiancheng Company, and the method for determining β-1,3-GA enzyme activity refer to the instructions for the kit provided by Solebold.

[0160] Figure 7 for PpRPL12 Phenotypic and physiological parameters of overexpression lines and wild-type pear callus after inoculation with Alternaria alternata ; Figure 7 A is PpRPL12 Phenotypes of overexpression lines and wild-type pear callus at 0, 10, and 20 days after Alternaria inoculation; Figure 7 Middle B is the lesion diameter and changes of overexpression line and wild-type pear callus 0-20 days after inoculation; Figure 7 Middle C is the conductivity of the overexpression line and wild-type pear callus 6 days after inoculation; Figure 7 D in the middle is the hydrogen peroxide content of the overexpression line and wild-type pear callus before inoculation and 6 days after inoculation; Figure 7 Middle E is the POD enzyme activity of overexpression line and wild-type pear callus before inoculation and 6 days after inoculation; Figure 7 F in the middle is the PAL enzyme activity of the overexpression line and wild-type pear callus before inoculation and 6 days after inoculation; Figure 7 Middle G is the PPO enzyme activity of overexpression lines and wild-type pear callus before inoculation and 6 days after inoculation; Figure 7 Middle H is the β-1,3-GA enzyme activity of overexpression lines and wild-type pear callus before inoculation and 6 days after inoculation; Figure 7 I is the chitinase activity of the overexpression line and wild-type pear callus before and 6 days after inoculation; the determination of the above indicators is an important measure to evaluate its resistance to pear black spot disease. In terms of phenotype, the lesion area and PpRPL12 The overexpression line is significantly larger, and the diameter of the lesion is more than twice that of the overexpression line. PpRPL12The overexpression line is more resistant to infection and spread of Alternaria alternata. The conductivity of wild-type callus is nearly 3 times that of the overexpression line, indicating that PpRPL12 The overexpression lines have better cell integrity and stability under Alternaria stress, and may also have stronger resistance to ROS. The enzyme activities of POD, PAL, PPO, etc. in the overexpression lines increased after inoculation and were significantly higher than those in the wild type, indicating that PpRPL12 The overexpression line has stronger antioxidant capacity, and the damage of free radicals after infection is reduced. The disease resistance pathway mediated by PpRPL12 may be related to the phenylpropanoid metabolic pathway, which resists pathogens by affecting intracellular signal transduction. In addition, the chitinase activity of the overexpression line increased by 36.2% compared with the wild type before and after infection, and the β-1,3-GA enzyme activity was significantly improved, indicating that PpRPL12 The overexpression line has a stronger ability to degrade pathogens. Figure 7 can be obtained PpRPL12 The gene enhanced the resistance of overexpressing callus to pear black spot.

[0161] 2. Analysis of Transgenic Arabidopsis Plants' Resistance to Alternaria alternata

[0162] For identification PpRPL12 To determine whether transgenic Arabidopsis thaliana is resistant to pear black spot, the control and transgenic lines were treated with pear black spot by spraying the whole plant with spore suspension and inoculating leaves with bacterial cake. PpRPL12 Transgenic lines (OE1, OE2, OE9) and wild-type (WT) Arabidopsis were sprayed with spore suspension on leaves every day and then cultured in a 28°C incubator in the dark. The phenotypes were observed after 10 days. PpRPL12 Transgenic lines (OE1, OE2, OE9) and wild-type (WT) Arabidopsis leaves were disinfected and rinsed. The petioles were wrapped with cotton wool dipped in sterile water and covered with plastic wrap to keep them moist. They were allowed to recover for 12 hours. The leaves were punctured (avoiding the veins) and inoculated with bacterial cakes. The leaves were cultured in a 28°C incubator and the bacterial cakes were removed after 2 days. Arabidopsis leaves were taken 1-3 days after inoculation for determination. PpRPL12 The relative expression of genes was measured by taking Arabidopsis leaves before inoculation and 3 days after inoculation to determine the POD and PAL enzyme activities. The phenotype was observed and microscopically photographed 5 days later.

[0163] Figure 8 for PpRPL12 Phenotypic and physiological parameters of transgenic lines and wild-type Arabidopsis were measured before and after Alternaria inoculation; Figure 8 Center A shows the phenotypes of the transgenic lines and wild-type Arabidopsis before and 10 days after spraying with spore suspension; Figure 8 Middle B is the phenotype of leaves of transgenic lines and wild-type Arabidopsis before and 3 days after inoculation with Alternaria; Figure 8Center C is a micrograph of Alternaria hyphae penetrating the epidermal cell wall of Arabidopsis thaliana; Figure 8 -D is the transgenic Arabidopsis line 0-3 days after inoculation PpRPL12 Relative expression of genes; Figure 8 Middle E is the POD enzyme activity of leaves of transgenic lines and wild-type Arabidopsis before and 3 days after inoculation with Alternaria; Figure 8 F in the middle is the PAL enzyme activity of the leaves of the transgenic line and wild-type Arabidopsis before and 3 days after inoculation with Alternaria. Phenotypically, the leaf length and leaf area of ​​the transgenic line Arabidopsis after spraying the spore suspension were significantly larger than those of the wild-type Arabidopsis. The wild-type Arabidopsis wilted severely, while the transgenic line wilted slightly and grew. The fungal spots on the leaves of the transgenic line after inoculation of the fungus cake were not obvious compared with the wild type. PpRPL12 Transgenic Arabidopsis has stronger resistance to Alternaria. Xing Caihua et al. found conidia of Alternaria in the intercellular space when inoculating Arabidopsis with Alternaria (Xing Caihua et al., 2023), proving that Alternaria can cause disease in Arabidopsis. The microscopic photographs of Alternaria hyphae passing through the epidermal cell wall of Arabidopsis in this study also proved this conclusion. In addition, the activities of enzymes such as POD and PAL in the transgenic lines increased after inoculation, and were significantly higher than those in the wild type, indicating that PpRPL12 The gene also activated resistance of Arabidopsis plants to Alternaria alternata. Figure 8 can be obtained PpRPL12 Genes enhance the ability of transgenic Arabidopsis to resist Alternaria alternata.

[0164] Example 8 Identification of resistance of PpRPL12 virus-silenced plants

[0165] 1. Disease resistance analysis of virus-silenced Pyrus betulaeformis seedlings

[0166] In order to identify whether the gene encoded by PpRPL12 is closely related to the resistance to black spot disease of pear, wild-type and virus-silenced pear seedlings were subjected to pear black spot disease stress for a certain period of time. Wild-type and virus-silenced pear seedlings (pTRV5, pTRV 8 and pTRV 9) with the same age and good growth condition were inoculated with activated Alternaria alternata cakes and cultured in a 28℃ incubator. The cakes were removed after 2 days. The phenotypes of wild-type and virus-silenced pear seedlings were observed 6 days after inoculation, and the lesion diameter, POD, PAL enzyme activity and hydrogen peroxide content were measured.

[0167] Fig. 9 For wild type and PpRPL12 Phenotypic and physiological indexes of virus-silencing positive Pyrus betula seedlings before and after inoculation with Alternaria; Fig. 9 A is the wild type and PpRPL12 The phenotypes of virus-silencing positive Pyrus betula seedling leaves before and 6 days after inoculation with Alternaria; Fig. 9B is the wild type and PpRPL12 The diameter of the lesions on the leaves of virus-silenced Pyrus betulaeformis seedlings 6 days after inoculation with Alternaria; Fig. 9 C is the wild type and PpRPL12 Hydrogen peroxide content in leaves of virus-silencing positive Pyrus betula seedlings 6 days after inoculation with Alternaria alternata. Fig. 9 D is the wild type and PpRPL12 POD enzyme activity in the leaves of virus-silencing positive Pyrus betulaeformis seedlings 6 days after inoculation with Alternaria alternata; Fig. 9 E is the wild type and PpRPL12 PAL enzyme activity in virus-silenced Pyrus betula seedling leaves 6 days after inoculation with Alternaria alternata; Phenotypically, the diameter of the lesions on the leaves of the silenced strains after inoculation was significantly larger than that of the wild type, the activity of antioxidant enzymes and the activity of phenylalanine ammonia lyase, a key enzyme in the phenylpropanoid pathway, were significantly reduced in the silenced plants, and the content of oxidatively destructive hydrogen peroxide increased significantly. This indicates that the virus is silenced PpRPL12 The role of phenylpropanoid pathways and other disease resistance pathways in the transgenic plants was weakened. 2 O 2 ) may cause more serious damage to the cell membrane, ultimately leading to a decrease in the plant's resistance to pear black spot.

[0168] 2. Comprehensive analysis shows that PpRPL12 After the transgenic callus was overexpressed, its function was identified, and it was found that the overexpression line had a greatly improved resistance to pear black spot compared with the wild type. When pear black spot stress occurred, the phenotype was observed, and the conductivity, hydrogen peroxide content, and enzyme activities such as PAL, POD, PPO, β-1,3-GA and chitinase were measured, indicating that the growth state of the transgenic callus was better than that of the wild type callus. PpRPL12 The overexpression lines had smaller lesion areas, lower electrical conductivity, and higher antioxidant enzyme activities and β-1,3-GA enzyme activities, indicating that they had stronger resistance. PpRPL12 After the gene was transferred into Arabidopsis, its function was identified, and the phenotypic changes of transgenic plants and wild-type Arabidopsis after inoculation were observed. PpRPL12 The results showed that the transgenic Arabidopsis thaliana had better growth than the wild type after inoculation and had stronger resistance. PpRPL12 After gene silencing, the phenotypes of wild-type and gene-silenced Pyrus betulae seedlings were observed after pear black spot stress, and their POD, PAL enzyme activities and hydrogen peroxide content were measured. It was found that the wild-type Pyrus betulae seedlings had better resistance to pear black spot than the silenced Pyrus betulae seedlings, indicating that PpRPL12 Gene silencing weakens the disease resistance of Pyrus betulata seedlings.

[0169] The analysis of the above results shows that PpRPL12The gene is closely related to the plant's resistance to Alternaria alternata, and the overexpressed PpRPL12 The gene can effectively enhance the antioxidant capacity of transgenic plants, increase the content of disease-resistant signaling factors in plants, promote the effects of disease-resistant pathways such as the phenylpropanoid pathway, maintain homeostasis of the intracellular environment, and enhance the ability of plants to directly destroy or inhibit pathogens, thereby improving the disease resistance of plants.

[0170] SEQ ID NO.1 amino acid sequence

[0171] MPPKFDPSQVVDVYVRVTGGEVGAASSLAPKIGPLGLSPKKIGEDIAKETAKDWKGLRVTVKLTVQNRQAKVSVVPSAAALVIKALKEPERDRKKTKNIKHSGNISLDDVIEIAKVMRNRSMAKELAGTVKEILGTCVSVGCTVDGKDPKDLQQEIADGDVEIPLD*

[0172] SEQ ID NO.2 Nucleotide sequence

[0173] ATGCCTCCCAAATTCGACCCCTCTCAGGTCGTCGATGTCTACGTCCGAGTCACCGGAGGCGAGGTCGGCGCCGCCAGTTCGCTCGCTCCCAAGATCGGTCCTTTGGGTCTCCCCCAAAAAGATCGGAGAAGACATCGCTAAGGAGACTGCCAAGGACTGGAAGGGCCTGAGAGTCACCGTCAAGCTCACCGTGCAGAATCGCCAGGCCAAGGTCTCCGTCGTCCCTCCCGCCGCAGCGCTCGTCATCA AGGCGTTGAAGGAGCCGGAGCGCGACAGGAAGAAGACTAAGAACATCAAGCACAGTGGGAACATTTCGTTGGACGACGTGATCGAGATTGCTAAGGTGATGAGGAACAGGTCCATGGCGAAGGAGTTGGCCGGGACCGTGAAGGAGATTCTGGGCACTTGCGTTTCGGTAGGGTGCACCGTAGATGGCAAGGACCCGAAGGATCTTCAGCAGGAGATTGCCGACGGTGATGTCGAAATTCCCTTGGACTGA

[0174] Cloning primers and PCR identification primers

[0175] SEQ ID NO.3 ATGCCTCCCAAATTCGACCCCTC

[0176] SEQ ID NO.4 ATTTCCCGGGAAGCTGCTAATGT

[0177] qPCR primers

[0178] SEQ ID NO.5 TGCCTCCCAAATTCGACC

[0179] SEQ ID NO.6 GGCAGTCTCCTTAGCGATGT

[0180] Internal reference primer

[0181] SEQ ID NO.7 GCACAAGAAGGTGAAGCTCG

[0182] SEQ ID NO.8 ACTCAGCATTGGGGCACTC

[0183] Subcellular localization expression vector primers

[0184] SEQ ID NO.9 gagaacacgggggactctagaATGCCTCCCAAATTCGACCCCTC

[0185] SEQ ID NO.10 gcccttgctcaccatggatccATTTCCCGGGAAGCTGCTAATGT

[0186] Gene silencing primers

[0187] SEQ ID NO.11 TCAAGCTCACCGTGCAGAATCGCC

[0188] SEQ ID NO. 12 GTCCAAGGGAATTTCGACATCACC.

Claims

1. Use of a protein with an amino acid sequence as shown in SEQ ID NO.1 or a biological material related to the protein in at least one of the following (a1) to (a13): (a1) Application in improving the peroxidase activity of plants stressed by pear black spot disease; (a2) Application in the preparation of products for increasing the peroxidase activity of plants stressed by pear black spot disease; (a3) Application in improving the activity of phenylalanine ammonia lyase in plants stressed by pear black spot disease; (a4) Use in the preparation of a product for increasing the activity of phenylalanine ammonia lyase in plants stressed by pear black spot; (a5) Application in improving the activity of polyphenol oxidase in plants stressed by pear black spot disease; (a6) Use in the preparation of a product for increasing the activity of polyphenol oxidase in plants stressed by pear black spot disease; (a7) Application in improving chitinase activity in plants stressed by pear black spot disease; (a8) Application in the preparation of a product for increasing chitinase activity in plants stressed by pear black spot disease; (a9) Application in improving the activity of β-1,3-GA enzyme in plants stressed by pear black spot; (a10) Application in the preparation of a method for improving the activity of β-1,3-GA enzyme in plants stressed by pear black spot disease; (a11) Application in improving the resistance of plants to pear black spot disease; (a12) Use in the preparation of products for improving the resistance of plants to pear black spot disease; (a13) Application in genetic breeding of new plant germplasm with improved resistance to pear black spot; The plant is pear or Arabidopsis thaliana; the pathogen of pear black spot disease is a pathogenic fungus of the genus Alternaria.

2. The use according to claim 1, characterized in that: The biological material related to the protein is at least one of the following (b1)-(b11): (b1) a gene encoding the protein whose nucleotide sequence is shown in SEQ ID NO.2; (b2) a primer pair having nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 for cloning the gene described in (b1); (b3) an expression cassette containing the gene described in (b1); (b4) a recombinant vector containing the gene described in (b1), or a recombinant vector containing the expression cassette described in (b3); (b5) a recombinant microorganism containing the gene described in (b1), or a recombinant microorganism containing the expression cassette described in (b3), or a recombinant microorganism containing the recombinant vector described in (b4); (b6) a transgenic plant cell line containing the gene described in (b1), or a transgenic plant cell line containing the expression cassette described in (b3), or a transgenic plant cell line containing the recombinant vector described in (b4); (b7) transgenic plant tissue containing the gene described in (b1), or transgenic plant tissue containing the expression cassette described in (b3), or transgenic plant tissue containing the recombinant vector described in (b4); (b8) a transgenic plant organ containing the gene described in (b1), or a transgenic plant organ containing the expression cassette described in (b3), or a transgenic plant organ containing the recombinant vector described in (b4); (b9) a transgenic plant containing the gene described in (b1), or a transgenic plant containing the expression cassette described in (b3), or a transgenic plant containing the recombinant vector described in (b4); (b10) Tissue culture produced from regenerable cells of the transgenic plant described in (b9); (b11) Protoplasts produced from the tissue culture described in (b10).

3. The use according to claim 1, characterized in that: The activity and / or content of the protein is increased in the target plant, or the gene encoding the protein is overexpressed.

4. A method for improving the resistance of plants to pear black spot disease, characterized in that: The activity and / or content of a protein having an amino acid sequence as shown in SEQ ID NO.1 is increased in a target plant, or a gene encoding the protein having a nucleotide sequence as shown in SEQ ID NO.2 is overexpressed to improve the resistance of the target plant to pear black spot disease; the plant is pear or Arabidopsis thaliana; and the pathogen of pear black spot disease is a pathogenic fungus of the genus Alternaria.

5. A method for cultivating transgenic plants with improved resistance to pear black spot disease, characterized in that: A gene with a nucleotide sequence as shown in SEQ ID NO.2 is overexpressed in a target plant to obtain a transgenic plant with improved resistance to pear black spot disease, and the obtained transgenic plant is cultivated; the plant is pear or Arabidopsis thaliana; and the pathogen of pear black spot disease is a pathogenic fungus of the genus Alternaria.

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