Use of smo gene to improve stress resistance of plants

Abstract

The application provides application of SMO genes in improving stress resistance of plants, and belongs to the technical field of biotechnology.The nucleotide sequence of the aforementioned SMO gene is compared with the sequence shown in SEQ ID NO:1, the branch support of a phylogenetic tree is at least 90%, and the encoded protein is functionally equivalent.By improving the expression amount of the SMO gene in plants, the stress resistance of the plants to plant pathogenic bacteria can be significantly improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the application of the SMO gene in improving plant stress resistance, the application of the SMO gene in improving fruit tree canker resistance, plant materials, and methods for targeted selection or identification of stress-resistant plants. Background Technology

[0002] Plants are susceptible to infection by various pathogens during their growth and development, such as *Phytophthora indica*, *Phytophthora*, and *Botrytis cinerea*. Pathogen infection can lead to a significant decrease in crop yield and quality, posing a serious threat to agricultural production and economic benefits.

[0003] In related technologies, control measures for plant diseases caused by pathogen stress include scraping off lesions and then applying pesticides. The principle is to first physically remove the rotten lesions from the infected area manually or mechanically, and then apply chemical pesticides to directly kill or inhibit the growth and reproduction of pathogens, thereby controlling the spread of the disease. However, this method not only consumes a lot of manpower and resources, increasing production costs, but also the pesticides used may remain in the plant, polluting the environment and ultimately harming human health through the food chain. Therefore, there is an urgent need for a more efficient, environmentally friendly, and safer method of plant disease control to replace traditional chemical control methods. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems in the related art. To this end, one objective of this application is to propose an application of the SMO gene in improving plant stress resistance, by significantly enhancing plant pathogen stress resistance through increasing the expression level of the SMO gene in plants.

[0005] Specifically, the technical solution of this application is as follows:

[0006] In the first aspect, the embodiments of this application propose the application of the SMO gene in improving plant stress resistance, wherein the stress resistance includes pathogen stress resistance.

[0007] In an exemplary embodiment, the SMO gene nucleotide sequence has at least 90% phylogenetic tree branch support compared to the sequence shown in SEQ ID NO: 1, and the encoded protein is functionally equivalent.

[0008] In an exemplary embodiment, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5.

[0009] In an exemplary embodiment, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

[0010] In an exemplary embodiment, the plant is selected from apple trees, pear trees, tomatoes, tobacco, Arabidopsis thaliana, or rice.

[0011] In an exemplary embodiment, the pathogens include: *Caryophyllum maculatum*, *Phytophthora*, and *Botrytis cinerea*.

[0012] Secondly, this application proposes the application of the SMO gene in improving resistance to fruit tree canker, wherein the nucleotide sequence of the SMO gene has at least 90% phylogenetic tree branch support compared with the sequence shown in SEQ ID NO: 1 and the encoded protein is functionally equivalent.

[0013] In an exemplary embodiment, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5.

[0014] In an exemplary embodiment, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

[0015] Thirdly, this application provides a plant material in which the SMO gene is upregulated by at least 2-fold compared to the control group, and the nucleotide sequence of the SMO gene has at least 90% phylogenetic tree branch support and the encoded protein is functionally equivalent to the sequence shown in SEQ ID NO: 1.

[0016] In an exemplary embodiment, the plant material is callus, plant, leaf, or protoplast; the plant is selected from apple trees, pear trees, tomatoes, tobacco, Arabidopsis thaliana, or rice.

[0017] In an exemplary embodiment, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5.

[0018] In an exemplary embodiment, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

[0019] Fourthly, embodiments of this application provide a method for targeted selection or identification of stress-resistant plants, the method comprising: identifying the expression of the SMO gene or its encoded protein in the plant to be tested, wherein the nucleotide sequence of the SMO gene has at least 90% phylogenetic branch support compared with the sequence shown in SEQ ID NO: 1 and the encoded protein is functionally equivalent.

[0020] In an exemplary embodiment, the plant is an apple tree, tobacco, or Arabidopsis thaliana.

[0021] In an exemplary embodiment, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5.

[0022] In an exemplary embodiment, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

[0023] In an exemplary embodiment, RT-PCR amplification was used to identify the expression level of the SMO gene.

[0024] Fifthly, embodiments of this application provide a method for preparing resistant plant materials, the method comprising: amplifying a plant SMO gene sequence, wherein the nucleotide sequence of the SMO gene has at least 90% phylogenetic branch support compared with the sequence shown in SEQ ID NO: 1 and the encoded protein is functionally equivalent; linking the amplified sequence to an overexpression vector to obtain an overexpression vector; converting the overexpression vector into an Agrobacterium strain capable of infecting plant materials by heat shock; and introducing the Agrobacterium strain into plant materials to obtain resistant plant materials.

[0025] In an exemplary embodiment, the plant material is selected from callus tissue, plant, leaf, or protoplast.

[0026] In an exemplary embodiment, the plant is selected from apple trees, pear trees, tomatoes, tobacco, Arabidopsis thaliana, or rice.

[0027] In an exemplary embodiment, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5.

[0028] In an exemplary embodiment, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

[0029] In an exemplary embodiment, the overexpression vector is pBIN-GFP (NTCC Typical Culture Collection Center, pBIN-GFP plant green fluorescent overexpression vector).

[0030] In an exemplary embodiment, the method further includes: screening the resistant plant material to obtain target resistant material, wherein the screening conditions include antibiotic resistance and the expression level of the SMO gene.

[0031] Based on the above solution, the embodiments of this application have the following beneficial effects:

[0032] 1. This application provides a novel resistance gene SMO. Increasing the expression level of this gene can significantly improve the plant's resistance to pathogen stress, while avoiding the high cost, environmental pollution, and food safety issues associated with chemical control methods.

[0033] 2. The resistance gene SMO in this application embodiment exhibits resistance to various pathogens in a variety of plants, including but not limited to model crops and fruit trees. By introducing this gene into plants, it is expected to solve similar disease problems encountered in plant production, thereby realizing cross-species disease-resistant breeding and having broad agricultural application prospects.

[0034] 3. This application is the first to identify and confirm that the apple-derived SMO gene (its nucleotide sequence is shown in SEQ ID NO: 1, and the encoded protein amino acid sequence is shown in SEQ ID NO: 2) is significantly upregulated in apple tissue when infected by the rot fungus (Cytospora mali). By constructing transgenic apple callus tissue stably overexpressing SMO and conducting rot fungus inoculation experiments, the results showed that SMO overexpression significantly reduced lesion size and increased the resistance of apple tissue to the rot fungus, confirming that this gene can significantly enhance the stress resistance of apple trees to the rot fungus.

[0035] 4. Verification showed that upregulating the expression of the tobacco resistance gene SMO can significantly inhibit the growth of Phytophthora tobaccoii;

[0036] This application is the first to identify and confirm that the tobacco-derived SMO gene (its nucleotide sequence is shown in SEQ ID NO: 3, and the encoded protein amino acid sequence is shown in SEQ ID NO: 4) plays a role in tobacco resistance to Phytophthora cirrhosa. By constructing transgenic tobacco plants stably overexpressing SMO and conducting Phytophthora cirrhosa inoculation experiments, the results showed that SMO overexpression significantly inhibited the growth of Phytophthora cirrhosa and increased the resistance of tobacco tissues to Phytophthora cirrhosa, confirming that this gene can significantly enhance tobacco resistance to Phytophthora cirrhosa stress.

[0037] 5. It has been verified that upregulating the expression of the Arabidopsis resistance gene SMO can significantly inhibit the growth of Arabidopsis botrytis.

[0038] This application is the first to identify and confirm that the SMO gene from Arabidopsis thaliana (its nucleotide sequence is shown in SEQ ID NO: 5, and the encoded protein amino acid sequence is shown in SEQ ID NO: 6) plays a role in Arabidopsis thaliana's resistance to Botrytiscinerea infection. By constructing transgenic Arabidopsis thaliana plants stably overexpressing SMO and conducting Botrytiscinerea inoculation experiments, the results showed that SMO overexpression significantly inhibited the growth and lesion expansion of Botrytiscinerea, and improved the resistance of Arabidopsis thaliana tissues to Botrytiscinerea, confirming that this gene can significantly enhance Arabidopsis thaliana's resistance to Botrytiscinerea stress.

[0039] 6. The embodiments of this application provide a method for preparing plant resistance materials. Compared with traditional chemical methods, this method can avoid pesticide residues and environmental hazards, and can quickly and cost-effectively obtain plant materials with durable and high pathogen resistance.

[0040] 7. Verification has shown that plant materials (such as apple callus) prepared based on the method of this application have significant resistance to pathogens (such as Cytospora mali, the causal agent of apple rot). Currently, there is a lack of effective endogenous disease resistance genes against apple rot, and this gene fills this gap.

[0041] 8. Traditional disease resistance breeding requires inoculation experiments, which are time-consuming and the results are easily affected by the environment. This application reveals a positive correlation between SMO gene expression levels and resistance. Therefore, by detecting the expression level of this gene, the disease resistance potential of plants can be predicted indirectly, rapidly, and accurately. This method allows for early screening of a large number of progeny materials, greatly improving breeding efficiency.

[0042] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram illustrating the expression level of the SMO gene in apples infected with rot fungi, as provided in one embodiment of this application.

[0045] Figure 2 A schematic diagram illustrating the conservative functional domain analysis results of an SMO provided in one embodiment of this application;

[0046] Figure 3 This is a schematic diagram illustrating the expression level of the SMO gene in a stably overexpressed callus tissue according to an embodiment of this application.

[0047] Figure 4 This is a schematic diagram illustrating the identification results of the resistance of apple callus with stable overexpression of the SMO gene to apple rot fungus in one embodiment of this application.

[0048] Figure 5 This is a schematic diagram illustrating the detection results of the NbSMO gene expression level in transgenic tobacco provided in one embodiment of this application;

[0049] Figure 6 A schematic diagram illustrating the identification results of resistance to Phytophthora in transgenic tobacco leaves provided in one embodiment of this application;

[0050] Figure 7 This is a schematic diagram illustrating the detection results of the AtSMO gene expression level in transgenic Arabidopsis thaliana provided in one embodiment of this application;

[0051] Figure 8 This is a schematic diagram illustrating the results of resistance identification of transgenic Arabidopsis thaliana leaves to Botrytis cinerea, provided in one embodiment of this application.

[0052] Figure labeling: *: indicates a statistically significant difference compared to the control group (P < 0.05); **: indicates a highly statistically significant difference compared to the control group (P < 0.01). Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0055] The term "optionally" is used for descriptive purposes only and should not be construed as indicating or implying relative importance. Therefore, a feature specified as "optionally" may explicitly or implicitly include or exclude that feature.

[0056] Before introducing the technical solution of this application, the relevant knowledge of this application will be introduced below:

[0057] In this application, "stress resistance" refers to the ability of a plant to resist adverse environmental factors, including biotic and abiotic stresses. In the embodiments of this application, stress resistance specifically includes at least pathogenic stress resistance, such as resistance to pathogens like *Carya caryopsis*, *Phytophthora*, and *Botrytis cinerea*. Resistance to *Carya caryopsis* is also referred to as resistance to apple tree rot.

[0058] In this application, "SMO gene" refers to the gene encoding squalene monooxygenase. In a specific example of this application, the gene is the MdSMO gene derived from apple (Malus domestica), whose nucleotide sequence is shown in SEQ ID NO: 1, and whose encoded protein sequence is shown in SEQ ID NO: 2.

[0059] In this application, "phylogenetic tree branch support" refers to the statistical support of a phylogenetic tree constructed based on the nucleotide sequence of a target gene when the target sequence and a reference sequence (e.g., SEQ ID NO: 1) are classified into the same evolutionary branch. In an exemplary embodiment, the branch support is preferably obtained through bootstrap analysis, i.e., the sequence data used to construct the phylogenetic tree is repeatedly resampled and the phylogenetic tree is constructed multiple times. The proportion in which the target sequence and the reference sequence are classified into the same branch in the repeatedly constructed phylogenetic trees is counted and expressed as a percentage.

[0060] In some implementations, the phylogenetic tree can be constructed based on multiple sequence alignment results using the Neighbor-Joining method, the Maximum Likelihood method, or the Maximum Parsimony method; the branch support is the support value obtained through no less than 100 times, preferably no less than 1000 times of bootstrapping iterations.

[0061] When the branch support is at least 90% (including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%), it indicates that in at least 90% of the repeated construction results, the target sequence and the reference sequence stably cluster in the same branch, thus indicating that they have high evolutionary homology. In an exemplary embodiment, through the construction and analysis of a phylogenetic tree in the early stage, it was found that the phylogenetic tree branch support of sequences SEQ ID NO: 3 and 5 is at least 90% compared with the sequence shown in SEQ ID NO: 1, and the encoded proteins are functionally equivalent.

[0062] In this application, "protein functional equivalence" refers to proteins that have substantially the same biological function, even if they differ in amino acid sequence, species of origin, three-dimensional structure, or post-translational modifications. Such equivalent proteins can achieve the same or substantially the same functional performance as the target protein in a given biological system, such as catalyzing the same biochemical reactions, binding the same substrates or ligands, activating the same signaling pathways, or producing phenotypic effects consistent with the target protein in the same experimental model.

[0063] In this application, "plant material" refers to any part of a plant, including but not limited to complete plants, plant organs (such as roots, stems, leaves, flowers, and fruits), plant tissues (such as callus, phloem, and xylem), plant cells, protoplasts, embryos, and seeds. These materials can be wild-type or genetically modified.

[0064] The sequences involved in this application are shown in Table 1.

[0065] Table 1

[0066] SEQ ID NO: Sequence (5’→3’) Description 1 ATGGTTTATGAGTATGTTCTTGCTGGTGTCCTGGTTTCTTTGTTGAGTTCTGTTTTCTTCCTCATCATAAACACCACCTACAGCGAAAAGAAGAAGGAAGACGTCGCGAATGTTTCTGAAACTGGTGTTTTTTTGGAGACGGAGATTGAGAAAAATACTGATGTTGTCATTGTTGGTGCTGGAGTTGCTGGTGCTGCTCTTGCTTACACTCTTGGGAAGGAAGGACGGCGTGTACATGTGATCGAAAGAGACTTGAACGAGCCAGACAGAATTGTTGGTGAACTATTGCAACCTGGAGGCTATCTCAAGTTGATTGAATTAGGTCTTGAAGATTGTGCTAATGAGTCCATTGATGCTCAAAAAGTGTTTGGTTATGCCCTTTACAAAAATGGCAATGACACAAAACTGTCATATCCCTTAGAAACATATAGTTCAGATATCGCCGGGAGAAGTTTCCACAATGGACGTTTCATCCAAAGAATGCGCGAAAGGGCTGCAACTCTTTCAAATGTGAAAATGGAACAAGGAACAGTGACAACACTAATTGAAGAAAATGGCATTATCAGAGGAGTGATGTACAAGAACAAGGCCGGAGAGGAGATGAGAACATATGCTCCGCTAACCATAGTATGCGATGGCTGCTTTTCAAATCTGCGCCGCTCTATCTCTACTCCCAATATTGAAAATCCATCCTGCTTTGTCGGTTTGATCTTGGAGAACTGTGAGCTTCCTCATGCAAACCATGGACATGTGATTTTGGGAGACCCTTCCCCCATCTTGTTTTATCCTATTAGTAGTACCGAGGTTCGTTGTTTGGTAGATGTACCCGGCACAAAAGTACCATCAGTAGCTAACGGT GAAATGGCTCAGTATTTGAAAACTGTGGTGGCTCCTCAGGTTCCCCATCAGCTCTTAAAGGCTTTTCTAGCAGCGGTTGATAAAGGAATCATCAGAACAATGCAAAACAAAAGCATGCCGGCTGCTCCTCAGCCCACCCCTGGCGCAATTTTATTGGGGGATGCTTTCAACATGAGACACCCTTTAACTGGAGGAGGGATGACTGTGGCTCTTTCCGACATTGTTCTTCTCAGGGATCTTTTGAGACCCCTAAGTGACTTCAACGATGCACCTGCTTTGTGCGATTATCTCGAATCATTTTACACGCTCCGCAAGCCTGTGTCATCTACTATAAACACATTGGCCGGTGCCCTTTACAAGGTGTTTTGTGCTTCGCCTGACCTCGCAAGACAGGAAATGCGTGAAGCATGTTTCGACTACTTGAGCCTTGGCGGCATCTGTTCAAATGGACCAATATCTCTACTCTCCGGTCTTAACCCTTGTCCAGTCAGCCTGTTTCTCCATTTCTTTGCTGTGGCTGTCTATGGAGTCGGCCGCTTAATGATTCCATTCCCTACACCGAAACGGATGTGGTTAGGGGCTAGATTGATCGTGAGTGCATCAGGAATCATATTCCCGATTATTAAAGCCGAAGGAGTTAGACAGATGTTCTTTCCTGCAACAATGCCAGCATATTACAGAGCTCCTCCTGTTCACAGAAGAATCGAAACAAGAACGAAGCTTAAATGTTAA Apple SMO gene nucleotide sequence 2 MVYEYVLAGVLVSLLSSVFFLIINTTYSEKKKEDVANVSETGVFLETEIEKNTDVVIVGAGVAGAALAYTLGKEGRRVHVIERDLNEPDRIVGELLQPGGYLKLIELGLEDCANESIDAQKVFGYALYKNGNDTKLSYPLETYSSDIAGRSFHNGRFIQRMRERAATLSNVKMEQGTVTTLIEENGIIRGVMYKNKAGEEMRTYAPLTIVCDGCFSNLRRSISTPNIENPSCFVGLILENCELPHANHGHVILGDPSPILFYPISSTEVRCLVDVPGTKVPSVANGEMAQYLKTVVAPQVPHQLLKAFLAAVDKGIIRTMQNKSMPAAPQPTPGAILLGDAFNMRHPLTGGGMTVALSDIVLLRDLLRPLSDFNDAPALCDYLESFYTLRKPVSSTINTLAGALYKVFCASPDLARQEMREACFDYLSLGGICSNGPISLLSGLNPCPVSLFLHFFAVAVYGVGRLMIPFPTPKRMWLGARLIVSASGIIFPIIKAEGVRQMFFPATMPAYYRAPPVHRRIETRTKLKC Amino acid sequence of the apple SMO gene 3 ATGCTACTTATAAATATCCCCAACAATCCACCTTTCTCCCTCGTCTTCTCCAAACACAATTTTCCTGTGTCAACAAACCAAACATCCCCACAATTATCTCAGCCCTCTGCTTCAAGTAGAAAGAAAAAATTAGCCTTGATTCCTTTTTCCAAATATTTGCGTAACACACAGCTTAAATTCATCTCTACCAACGAGAAAATGGTGAATTTCATGATGGATAAATATATTGTCCCCACTTTTTTTGTTTCTCTATTGGGGTTCCTTCTTCTTTATATTTTGCGACCAAGATTAAGAACCACCTATTACAAGAAAAAAGACCCTAAATCGACACAAAAATGTGGCGCCCACAACGTAATTTCTAGCAATTTTACCAATGGTGAATGCAAATTGGAAAAAGGGACCGATGCTGATATCATCATTGTTGGAGCTGGGGTTGCTGGTGCTGCTCTTGCTCATACCCTTGCCAAGGAAGGGCGAAAAGTTCTTGTAATTGAAAGGGATTTGACAGAGCCCGACCGGATTGTTGGCGAGTTGCTACAGCCTGGGGGTTATCTGAAATTGATTGAGTTGGGCCTTGAAGATTGTGTTGAGGATATTGATGCCCAGCGGGTGGTTGGATATGCTCTTTTCAAGGATGGGAAAAGCACAAACGTTTCCTATCCCTTGGAAAATTTCCATTCTGATGTTGCTGGGAGAAGCTTCCACAATGGCCGTTTCATACAAAAGATGAGAGAAAAAGCAGCTACTTTTCCCAATGTACGATTGGAGCAAGGCACTGTAACATCCCTGATTGAAGAAAATGGATCCGTTAAGGGTGTCCAGTACAAAACGAAGGCTGGTCAAGAACTTAAAGCACAT GCTCCTCTTACAGTAGTTTGTGATGGGTGCTTTTCAAACTTGCGACGCTCTCTTTGCAACCCTAAGGTTGATATCCCATCTTGCTTTGTTGGTTTGGTATTGGAACTGGAGAACGATCAACTTCCATACCCAAACCACGGGCATGTTATTCTGGCAGATCCTTCGCCCATCTTATTTTATCCTATTAGTAGCACAGAAATCCGCTGCTTGGTCGATGTACCTGGTCAAAAGCTTCCTTCTCTTGCTAATGGTGATATGGCAAATTATTTGAAGAATATGGTGGCTCCCCAGGTCCCACCTGAGCTACATGATGCTTTTATAACTGCAATTGATAAGGGGCATATCAGAACTATGCCAAATAGGAGCATGCCAGCTGCTCCGTATCCTACCCCTGGAGCTCTGTTACTTGGTGATTCTTTCAACATGCGCCATCCTTTAACTGGTGGGGGAATGACAGTCGCACTTTCAGATATTGCAGTGTTAAGGAATCTTCTTAAGCCATTGAATGACCTGAATGATGCAGATGAGTTATGTAAATATCTGGAGTCCTTTTATACTTTGCGCAAGCCTGTAGCTTCAACAATAAATACTTTGGCTGGAGCACTGTACAAGGTGTTCTGTGCTTCTCCTGATCAAGCGAGGAAGGAGATGCGAGAAGCATGTTTCGACTATTTGAGTCTTGGAGGTACTTGTTCAACAGGACCCGTAGCTCTACTCTCTGGTCTTAATCCTAGCCCGCTGAGCTTGGTACTCCATTTCTTTGCTGTGGCCATATATGGAGTTGGTCGTTTACTCGTTCCATTTCCTTCCCCAAAGAGATTGTGGATTGGAGCTAGATTAATCTCGGCTGCATCGAGT ATCATATTTCCCATTATAAAAGCAGAAGGGATCAGGCAAATGTTCTTCCCAACAACAATACCAGCATATCACAGAGCTCCTCCAGTAAACAAGGGATCAAATTAA Nucleotide sequence of tobacco SMO gene 4 MLLINIPNNPPFSLVFSKHNFPVSTNQTSPQLSQPSASSRKKKLALIPFSKYLRNTQLKFISTNEKMVNFMMDKYIVPTFFVSLLGFLLLYILRPRLRTTYYKKKDPKSTQKCGAHNVISSNFTNGECKLEKGTDADIIIVGAGVAGAALAHTLAKEGRKVLVIERDLTEPDRIVGELLQPGGYLKLIELGLEDCVEDIDAQRVVGYALFKDGKSTNVSYPLENFHSDVAGRSFHNGRFIQKMREKAATFPNVRLEQGTVTSLIEENGSVKGVQYKTKAGQELKAHAPLTVVCDGCFSNLRRSLCNPKVDIPSCFVGLVLELENDQLPYPNHGHVILADPSPILFYPISSTEIRCLVDVPGQKLPSLANGDMANYLKNMVAPQVPPELHDAFITAIDKGHIRTMPNRSMPAAPYPTPGALLLGDSFNMRHPLTGGGMTVALSDIAVLRNLLKPLNDLNDADELCKYLESFYTLRKPVASTINTLAGALYKVFCASPDQARKEMREACFDYLSLGGTCSTGPVALLSGLNPSPLSLVLHFFAVAIYGVGRLLVPFPSPKRLWIGARLISAASSIIFPIIKAEGIRQMFFPTTIPAYHRAPPVNKGSN Amino acid sequence of tobacco SMO gene 5 ATGGCTCCGACGATATTCGTTGATCACTGTATCCTCACAACAACGTTTGTCGCATCCTTGTTCGCGTTTCTGCTTCTATACGTCCTGCGTCGCCGGAGCAAGACGATTCATGGGTCTGTCAATGTCCGTAACGGAACCCTAACGGTGAAATCTGGAACAGACGTTGATATTATCATTGTCGGTGCTGGTGTCGCCGGCGCTGCCCTTGCTCATACCCTCGGCAAGGAAGGAAGAAGAGTTCACGTTATAGAAAGAGACTTAACGGAGCCTGATCGAATTGTCGGTGAATTACTTCAGCCTGGTGGTTACTTGAAGTTAATCGAACTCGGGCTTGAAGATTGTGTGAAGGATATAGATGCGCAGAGAGTTCTTGGTTATGCTCTCTTTAAAGATGGGAAACACACTAAACTCTCTTACCCGTTGGATCAGTTTGATTCCGATGTTGCGGGTCGTAGCTTTCACAATGGGAGATTTGTGCAGAGGATGCGAGAGAAAGCTTCTTTACTTCCCAATGTTCGAATGGAGCAAGGAACAGTGACATCGTTGGTGGAAGAAAACGGAATAATCAAAGGTGTTCAATACAAAACCAAAGATGGCCAAGAGCTTAAGTCATTTGCTCCTCTCACTATTGTATGTGATGGTTGTTTCTCCAACTTGCGTCGATCTCTCTGCAAACCTAAGGTGGAGGTGCCATCCAATTTCGTTGGTCTGGTATTGGAGAATTGCGAACTCCCGTTTCCGAACCACGGGCACGTTGTTCTTGGCGATCCGTCACCCATTTTATTCTATCCTATCAGCAGCTCGGAAGTCCGTTGCTTGGTAGATGTACCAGGTTCAAAACTTCCTTCAGTTGCAAGT GGTGAAATGGCTCACCATCTCAAAACAATGGTTGCACCGCAGGTACCACCTCAGATCCGTGATGCTTTCATTTCTGCAGTCGAAAAAGGTAACATAAGAACAATGCCGAACAGAAGCATGCCAGCTGATCCAATTCATACACCTGGAGCTTTGCTTTTAGGTGATGCGTTCAACATGCGCCATCCTCTTACTGGAGGTGGTATGACCGTTGCATTGTCTGATATAGTCATCCTCCGTGATCTATTGAACCCGCTCGTCGACTTAACCAACAAAGAGTCCTTATCCAAATACATAGAATCATTCTACACATTGCGGAAACCGGTTGCTTCGACTATCAATACACTCGCAGGCGCTCTTTACAAAGTCTTTTTAGCATCTCCAGACGATGCAAGAAGCGAAATGCGTCGAGCTTGCTTCGATTATCTTAGCCTCGGAGGGGTTTGCTCATCAGGACCTGTGGCTTTGCTATCTGGTTTGAACCCACGACCTATGAGCCTTGTTCTTCATTTCTTCGCAGTTGCGATTTTCGGGGTTGGTCGTTTGCTTGTACCTCTCCCGTCCGTTAAACGGTTATGGCTTGGAGCTAGACTAATCTCGAGTGCTTCAGGGATCATATTTCCAATAATAAAAGCAGAAGGTGTGAGGCAAATGTTCTTCCCTCGAACTATTCCTGCCATTTACAGAGCTCCTCCTACTCCTTCTTCTTCTTCTCCTCAATGA Arabidopsis thaliana SMO gene nucleotide sequence 6 MAPTIFVDHCILTTTFVASLFAFLLLYVLRRRSKTIHGSVNVRNGTLTVKSGTDVDIIIVGAGVAGAALAHTLGKEGRRVHVIERDLTEPDRIVGELLQPGGYLKLIELGLEDCVKDIDAQRVLGYALFKD GKHTKLSYPLDQFDSDVAGRSFHNGRFVQRMRERASLLPNVRMEQGTVTSLVEENGIIKGVQYKTKDGQELKSFAPLTIVCDGCFSNLRRSLCKPKVEVPSNFVGLVLENCELPFPNHGHVVLGDPSPILF YPISSSEVRCLVDVPGSKLPSVASGEMAHHLKTMVAPQVPPQIRDAFISAVEKGNIRTMPNRSMPADPIHTPGALLLGDAFNMRHPLTGGGMTVALSDIVILRDLLNPLVDLTNKESLSKYIESFYTLRKP VASTINTLAGALYKVFLASPDDARSEMRRACFDYLSLGGVCSSGPVALLSGLNPRPMSLVLHFFAVAIFGVGRLLVPLPSVKRLWLGARLISSASGIIFPIIKAEGVRQMFFPRTIPAIYRAPPTPSSSSPQ The amino acid sequence of the Arabidopsis thaliana SMO gene 7 atgtggaagcttaagattgcagatgATGGTTTATGAGTATGTTCTTGC upstream primer SMO-F 8 tcacgctttggaaggcaatggaaccTTAACATTTAAGCTTCGTTCTTGT Downstream primer SMO-R 9 TGACTGTGGCTCTTTCCGAC Upstream primer MdSMO-qRTF 10 GCTTGCGGAGCGTGTAAAAT Downstream primer MdSMO-qRTR 11 AACATGCGCCATCCTTTAACTG Upstream primer NbSMO-qRTF 12 CACAATCTCTTTGGGGAAGG Downstream primer NbSMO-qRTR 13 CAGCTGATCCAATTCATACACC Upstream primer AtSMO-qRTF 14 AGGAGGAGCTCTGTAAATGG Downstream primer AtSMO-qRTR

[0067] Note: Lowercase letters in the above sequences indicate restriction sites or vector homologous arm sequences;

[0068] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0069] The statistical analysis of the data in the following examples was performed using SPSS 22.0.

[0070] In the examples below, the overexpression vector is pBIN-GFP (NTCC Typical Culture Collection Center, pBIN-GFP plant green fluorescent overexpression vector).

[0071] In the following examples, the apple SMO gene is abbreviated as MdSMO.

[0072] Example 1: Expression analysis of apple callus infected with putrefactive fungi

[0073] To investigate the role of the SMO gene in the apple response to pathogen infection, this example analyzed the expression pattern of this gene in apple tissue infected with the rot fungus *Cytospora mali*. Details are as follows:

[0074] Wild-type apple callus was inoculated with *Pseudomonas aeruginosa* and cultured for 36 hours. Total RNA was then extracted. Apple callus not inoculated with *Pseudomonas aeruginosa* served as a control group. RNA was reverse transcribed into cDNA using a Quantiec reverse transcription kit (Takara) and stored at -20°C. The cDNA sample obtained from reverse transcription was then used as a template for RT-PCR using the SYBR Green dye method to determine the expression level of the MdSMO gene in apple tissue inoculated with *Pseudomonas aeruginosa* 36 hours after inoculation. Actin gene was used as an internal reference gene, and amplification was performed using primers MdSMO-qRTF (SEQ ID NO: 9) and MdSMO-qRTR (SEQ ID NO: 10).

[0075] Experimental results are as follows Figure 1 As shown, data analysis revealed that infection by *Pseudomonas canis* significantly induced the expression of the MdSMO gene in apple callus. Compared to the uninoculated control group, the expression level of the MdSMO gene in apple callus infected with *Pseudomonas canisis* was upregulated nearly 7-fold (inoculated group). This result indicates that the expression of the MdSMO gene is activated by pathogen infection and is naturally involved in the apple's defense response against *Pseudomonas canisis*.

[0076] Example 2: Preparation of transgenic apple callus

[0077] This embodiment uses the apple SMO gene as an example to prepare transgenic apple callus tissue to verify the function of this gene in improving plant stress resistance. Its nucleotide sequence is shown in SEQ ID NO: 1, and its protein sequence is shown in SEQ ID NO: 2, encoding squalene monooxygenase, containing the PLN02985 superfamily (squalene monooxygenase) domain. Figure 2 It participates in the synthesis of apple terpene metabolites. Specifically:

[0078] 1. Cloning of the apple SMO gene and construction of an overexpression vector

[0079] Based on the SMO sequence in the apple genome, specific upstream primer SMO-F (SEQ ID NO: 7) and downstream primer SMO-R (SEQ ID NO: 8) were designed. Total RNA was extracted from apple callus using the Omega Plant RNA Kit. After reverse transcription to obtain cDNA, SMO was amplified using specific primers. The PCR product was purified and recovered by agarose gel electrophoresis to obtain the full-length CDS sequence of the SMO gene. After successful sequencing, the sequence was stored at -80°C.

[0080] The SMO gene was digested with Bam HI, ligated, transformed, screened on kanamycin medium plates, and sequenced to obtain the pBIN-eGFP-SMO overexpression vector. The vector was then heat-shocked and transformed into E. coli DH5α. Positive clones were selected for verification by bacterial PCR or plasmid sequencing. After confirming that the inserted sequence was correct, positive bacteria were cultured in large quantities and plasmids were extracted to obtain the overexpression vector carrying the MdSMO gene. The vector was stored at -80℃ for later use.

[0081] 2. Obtain Agrobacterium carrying the overexpression vector pBIN-eGFP-SMO

[0082] After obtaining the overexpression vector, it is necessary to acquire Agrobacterium for plant transformation. The correctly constructed and validated overexpression vector pBIN-eGFP-MdSMO from the previous steps was transformed into competent cells of Agrobacterium tumefaciens strain GV3101, which is capable of infecting apple callus, using a heat shock method. The transformed Agrobacterium was plated on LB agar containing rifampin and kanamycin for selection. Because strain GV3101 itself contains rifampin resistance, and the pBIN-eGFP-MdSMO plasmid contains a kanamycin resistance gene, only Agrobacterium successfully transformed with the plasmid could grow on this medium. The selected single colonies were amplified by PCR and sequenced to confirm that they carried the correct pBIN-eGFP-MdSMO overexpression vector, thus obtaining the engineered strain for subsequent genetic transformation of apple callus.

[0083] 3. Preparation of apple callus transduced with SMO gene overexpression vector

[0084] Agrobacterium GV3101 carrying the overexpression vector pBIN-eGFP-MdSMO was inoculated into LB liquid medium containing rifampicin and kanamycin and cultured in a constant temperature shaker at 28°C and 200 rpm for approximately 48 hours until the optical density (OD600) of the bacterial culture reached 0.5-0.8. The cultured bacterial culture was transferred to sterile centrifuge tubes and centrifuged at 5000×g for 10 minutes at room temperature, and the supernatant was discarded. The bacterial cells were resuspended in approximately 30 mL of sterile water or infection buffer and washed, and centrifuged again under the same conditions. Finally, the bacterial cells were resuspended in approximately 40 mL of sterile water or infection buffer, and the OD600 of the bacterial culture was adjusted to 0.6-0.8 for transformation of apple callus tissue.

[0085] The overexpression vector pBIN-eGFP-MdSMO was introduced into the callus tissue of "Wanglin" apples using Agrobacterium-mediated genetic transformation. Healthy, loosely textured callus tissues were selected and placed in a resuspended Agrobacterium solution, ensuring complete submersion. The tissues were co-cultured in a constant-temperature shaker at 28°C and 180 rpm in the dark for approximately 30 minutes. Excess solution was then blotted away with sterile filter paper. The co-cultured callus tissues were transferred to MS co-culture medium and co-cultured for another 3 days at 22°C in the dark to facilitate Agrobacterium infection and T-DNA transfer. After co-culture, the infected callus tissues were collected in sterile bottles and repeatedly washed with sterile distilled water until the liquid became clear to remove any residual Agrobacterium.

[0086] 3) The cleaned callus tissue was spread evenly on MS resistance selection medium containing cephalosporin and kanamycin, and cultured in the dark at 28°C for about 20 days to perform preliminary screening of positive transformants. The preliminary positive apple transformants were further subcultured on MS selection medium containing the same antibiotics and cultured in the dark at 28°C. Through multiple generations of antibiotic resistance screening, a transgenic apple callus tissue line that can grow and proliferate stably was finally obtained.

[0087] Example 3: Identification of transgenic apple callus tissue

[0088] This embodiment uses molecular methods to identify the apple callus tissue obtained in Example 2. Details are as follows:

[0089] Total RNA was extracted from transgenic apple callus tissue that had undergone multiple generations of screening and from wild-type callus tissue used as a control. The RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit (Qiagen), strictly following the product instructions. RT-PCR reaction conditions included: incubation at 25°C for 10 minutes, incubation at 42°C for 15 minutes, and heating at 85°C for 5 seconds to terminate the reaction. The obtained cDNA samples were stored at -20°C for later use. Then, using the cDNA samples obtained from reverse transcription as templates, the relative expression level of the MdSMO gene in transgenic apple callus tissue was determined using real-time quantitative PCR (RT-PCR) based on the SYBR Green dye method. The housekeeping gene Actin was used as an internal control gene to correct for errors in sample loading and reverse transcription efficiency. The specific primer sequences used to detect MdSMO expression were SMO-qRTF (SEQ ID NO: 9) and SMO-qRTR (SEQ ID NO: 10).

[0090] Experimental results are as follows Figure 3As shown, analysis of RT-PCR data revealed that the relative expression level of the MdSMO gene was significantly upregulated in stable transgenic apple callus compared to wild-type apple callus (wild-type), with an upregulation of nearly 25-fold. These results demonstrate that transgenic apple callus stably overexpressing the MdSMO gene was successfully obtained through the aforementioned transformation and screening process.

[0091] Example 4: Verification of the anti-rot function of transgenic apple callus.

[0092] In this embodiment, apple callus tissue with stable overexpression of the SMO gene obtained in Example 3 was used as material to detect and analyze its resistance to apple rot fungus.

[0093] The fungal strain used in the experiment was *Cytospora mali*, provided by the Fungal Laboratory of the College of Plant Protection, Northwest A&F University. First, the strain was activated and cultured on potato dextrose agar (PDA) until vigorous colony growth was achieved. Using a 5mm diameter punch, uniformly sized mycelial cakes were prepared at the edge of the activated *Cytospora mali* colonies. These mycelial cakes were then inoculated onto the center of the surface of transgenic apple callus stably overexpressing the SMO gene and wild-type apple callus (as a control). The inoculated callus was cultured under suitable conditions. Three days after inoculation, the diameter of lesions caused by the *Cytospora mali* on different callus tissues was observed and measured using calipers. Lesion size was used to assess the resistance of the callus.

[0094] Experimental results are as follows Figure 4 As shown, compared with wild-type apple callus (wild-type) as a control, the diameter of canker lesions caused by *S. rot* was significantly reduced in transgenic apple callus stably overexpressing the MdSMO gene (MdSMO overexpression transgenic tissue). The results indicate that overexpression of the apple SMO gene can significantly enhance the resistance of apple callus to *S. rot*, clarifying the key function of the apple SMO gene in enhancing resistance to *S. rot* in apple tissue. This also demonstrates that constructing transgenic plant materials with stable overexpression of apple SMO is an effective way to create new apple materials with high resistance to *S. rot*, providing valuable genetic resources and technological potential for breeding new apple varieties with high resistance to *S. rot*.

[0095] Example 5: Creation and verification of disease resistance function of NbSMO-overexpressing transgenic tobacco

[0096] Agrobacterium GV3101 carrying the overexpression vector pBIN-eGFP-NbSMO was inoculated into LB liquid medium containing rifampicin and kanamycin and cultured in a constant temperature shaker at 28°C and 200 rpm for approximately 48 hours until the optical density (OD600) of the bacterial culture reached 0.5-0.8. The cultured bacterial culture was transferred to sterile centrifuge tubes and centrifuged at 5000×g for 10 minutes at room temperature, and the supernatant was discarded. The bacterial cells were resuspended in approximately 30 mL of sterile water or infection buffer and washed, and centrifuged again under the same conditions. Finally, the bacterial cells were resuspended in approximately 40 mL of infection buffer (containing MS medium basal salts, 3% sucrose, and 0.02% Silwet L-77), and the OD600 of the bacterial culture was adjusted to 0.5-0.6 for transformation of tobacco leaf discs.

[0097] The overexpression vector pBIN-eGFP-NbSMO was introduced into sterile leaves of tobacco (Nicotiana benthamiana) seedlings using Agrobacterium-mediated genetic transformation. Healthy, fully expanded sterile tobacco leaves were selected and cut into leaf discs approximately 0.5-1.0 cm in diameter using a sterile perforator or blade. These discs were then placed in a resuspended Agrobacterium solution, ensuring complete immersion, and soaked at room temperature for 10-15 minutes, gently agitating occasionally. Excess solution was then blotted away with sterile filter paper. The leaf discs, after co-culturing with Agrobacterium, were transferred to MS co-culture medium (containing MS basal salts, 3% sucrose, 0.8% agar, 2 mg / L 6-BA, and 0.2 mg / L NAA) and co-cultured at 22°C in the dark for 2-3 days to facilitate Agrobacterium infection and T-DNA transfer. After co-cultivation, remove the infected leaf discs and wash them repeatedly with sterile distilled water 3-4 times until the liquid becomes transparent to remove any remaining Agrobacterium on the surface. Then, use sterile filter paper to absorb the surface moisture.

[0098] Cleaned leaf discs were laid flat on MS selection and differentiation medium (containing MS basal salts, 3% sucrose, 0.8% agar, 2 mg / L 6-BA, and 0.2 mg / L NAA) containing cephalosporin (250 mg / L) and kanamycin (100 mg / L). The medium was cultured at 25°C under 16 hours of light / 8 hours of darkness for approximately 2-3 weeks to induce adventitious shoot differentiation and perform preliminary screening for positive transformants. When the adventitious shoots reached 1-2 cm in length, they were excised from the callus tissue and transferred to MS rooting medium (containing MS basal salts, 3% sucrose, 0.8% agar, and 0.1 mg / L NAA) containing cephalosporin (200 mg / L) and kanamycin (100 mg / L). The medium was cultured under the same conditions for approximately 2 weeks to induce rooting. Through multiple generations of antibiotic resistance screening, transgenic tobacco plants capable of stable growth and rooting were finally obtained.

[0099] Total RNA was extracted from transgenic tobacco plants that had undergone multiple generations of screening and from wild-type tobacco plants used as controls. The RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit (Qiagen), strictly following the product instructions. RT-PCR reaction conditions included: incubation at 25°C for 10 minutes, incubation at 42°C for 15 minutes, and heating at 85°C for 5 seconds to terminate the reaction. The obtained cDNA samples were stored at -20°C for later use. Then, using the reverse-transcribed cDNA samples as templates, the relative expression level of the NtSMO gene in transgenic tobacco plants was determined using real-time quantitative PCR (RT-PCR) based on the SYBR Green dye method. The housekeeping gene NbActin was used as an internal control gene to correct for errors in sample loading and reverse transcription efficiency. The specific primer sequences used to detect NbSMO expression were NbSMO-qRTF (SEQ ID NO: 11) and NbSMO-qRTR (SEQ ID NO: 12).

[0100] Experimental results are as follows Figure 5 As shown, analysis of RT-PCR data revealed that the relative expression level of the NbSMO gene was significantly upregulated in stable transgenic tobacco plants compared to wild-type tobacco plants (wild-type), with an upregulation fold of nearly 25-fold. These results demonstrate that transgenic tobacco plants stably overexpressing the NbSMO gene were successfully obtained through the aforementioned transformation and screening process.

[0101] Tobacco plants stably overexpressing the SMO gene were used as material to detect and analyze their resistance to *Phytophthora tobaccosifolia*. The *Phytophthora tobaccosifolia* strain used in the experiment was provided by the Fungal Laboratory of the College of Plant Protection, Northwest A&F University. First, the strain was activated and cultured on potato dextrose agar (PDA) until vigorous colony growth was achieved. Using a 5 mm diameter punch, uniformly sized mycelial cakes were prepared at the edge of the activated *Phytophthora tobaccosifolia* colonies. These mycelial cakes were inoculated onto the center of the surface of leaves from transgenic tobacco plants stably overexpressing the SMO gene and wild-type tobacco leaves (as a control). The inoculated leaves were cultured under suitable conditions. Three days after inoculation, the diameter of lesions caused by *Phytophthora tobaccosifolia* on different leaves was observed and measured with calipers. The size of the lesions was used to measure the resistance of the tobacco plants.

[0102] Experimental results are as follows Figure 6As shown, compared with wild-type tobacco (as a control), the diameter of lesions caused by Phytophthora in transgenic tobacco plants stably overexpressing the SMO gene was significantly reduced. The results indicate that overexpression of the tobacco SMO gene can significantly enhance the resistance of tobacco plants to Phytophthora tobacco, clarifying the key function of the tobacco SMO gene in enhancing the resistance of tobacco tissues to Phytophthora tobacco. This also demonstrates that constructing transgenic plant materials with stable overexpression of tobacco SMO is an effective way to create new tobacco materials highly resistant to Phytophthora tobacco, providing valuable genetic resources and technological potential for breeding new varieties of tobacco highly resistant to Phytophthora tobacco.

[0103] Example 6: Creation and verification of disease resistance function of AtSMO-overexpressing transgenic Arabidopsis thaliana

[0104] Agrobacterium GV3101 carrying the overexpression vector pBIN-eGFP-AtSMO was inoculated into LB liquid medium containing rifampicin and kanamycin and cultured at 28°C and 200 rpm for approximately 48 hours until the OD600 reached 0.8-1.0. The cultured bacterial solution was transferred to sterile centrifuge tubes and centrifuged at 5000×g for 10 minutes at room temperature, discarding the supernatant. The bacterial cells were resuspended in approximately 30 mL of sterile water or infection buffer and washed, then centrifuged again under the same conditions. Finally, the bacterial cells were resuspended in approximately 40 mL of infection buffer (containing 5% sucrose and 0.02% Silwet L-77), and the OD600 of the bacterial solution was adjusted to 0.8 for transformation of Arabidopsis thaliana inflorescences.

[0105] The overexpression vector pBIN-eGFP-AtSMO was introduced into wild-type Arabidopsis thaliana (Col-0 ecotype) using an Agrobacterium-mediated inflorescence immersion method. Healthy Arabidopsis plants in the early flowering stage, with open flowers and pods removed, were selected. The inflorescences were completely immersed in a resuspended Agrobacterium solution, ensuring all flower buds were wetted. The immersion time was approximately 30 seconds, during which the container was gently shaken. After immersion, the plants were laid flat and covered with a black plastic bag to maintain high humidity, and cultured at 22°C in the dark for approximately 24 hours. Subsequently, the plants were restored to normal growth conditions (22°C, 16 hours light / 8 hours dark) and cultured for approximately 4 weeks until mature T0 generation transgenic seeds were obtained.

[0106] T0 generation transgenic Arabidopsis seeds were collected, surface-sterilized, and evenly sown on 1 / 2 MS selection medium containing kanamycin (50 mg / L). The plants were cultured at 22°C under 16 hours of light / 8 hours of darkness for approximately 10-14 days, and their growth was observed. Plants exhibiting normal growth, well-developed root systems, and green true leaves were initially selected as positive transformants. These positive plants were transplanted into nutrient soil and cultured under the same environmental conditions until maturity. T1 generation seeds were then harvested from each mature plant. The T1 generation seeds were again cultured on kanamycin-containing selection medium. Lines with a resistance segregation ratio of 3:1 were selected for further planting. Through multiple generations of resistance selection, homozygous transgenic Arabidopsis lines with stable inheritance were finally obtained.

[0107] Total RNA was extracted from homozygous transgenic Arabidopsis thaliana plants selected through multiple generations of screening and from wild-type Arabidopsis thaliana plants used as controls. The RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit (Qiagen), strictly following the product instructions. RT-PCR reaction conditions included: incubation at 25°C for 10 minutes, incubation at 42°C for 15 minutes, and heating at 85°C for 5 seconds to terminate the reaction. The obtained cDNA samples were stored at -20°C for later use. Then, using the reverse-transcribed cDNA samples as templates, the relative expression level of the AtSMO gene in transgenic Arabidopsis thaliana plants was determined using real-time quantitative PCR (RT-PCR) based on the SYBR Green dye method. The housekeeping gene AtACTIN2 was used as an internal control gene to correct for errors in sample loading and reverse transcription efficiency. The specific primer sequences used to detect AtSMO expression were AtSMO-qRTF (SEQ ID NO: 13) and AtSMO-qRTR (SEQ ID NO: 14).

[0108] Experimental results are as follows Figure 7 As shown, analysis of RT-PCR data revealed that the relative expression level of the AtSMO gene was significantly upregulated in stable transgenic Arabidopsis plants compared to wild-type plants (wild-type), with an upregulation of nearly 21-fold. These results demonstrate that transgenic Arabidopsis plants stably overexpressing the AtSMO gene were successfully obtained through the aforementioned transformation and screening process.

[0109] Using Arabidopsis thaliana plants stably overexpressing the SMO gene as material, this study investigated and analyzed their resistance to *Botrytis cinerea*. The *Botrytis cinerea* strain used in the experiment was provided by the Fungal Laboratory of the College of Plant Protection, Northwest A&F University. First, the strain was activated and cultured on potato dextrose agar (PDA) until vigorous colony growth was achieved. Using a 5 mm diameter punch, uniformly sized mycelial cakes were prepared at the edge of the activated *Botrytis cinerea* colonies. These mycelial cakes were inoculated onto the center of the surface of leaves from transgenic Arabidopsis thaliana stably overexpressing the SMO gene and leaves from wild-type Arabidopsis thaliana (as a control). The inoculated leaves were cultured under suitable conditions. Three days after inoculation, the diameter of lesions caused by *Botrytis cinerea* on different callus tissues was observed and measured using calipers. The size of the lesions was used to assess the leaf resistance.

[0110] Experimental results are as follows Figure 8 As shown, compared with the wild-type Arabidopsis thaliana (wild-type) as a control, the diameter of lesions caused by Botrytis cinerea was significantly reduced in transgenic Arabidopsis thaliana plants stably overexpressing the SMO gene (transgenic). The results indicate that overexpression of the Arabidopsis thaliana SMO gene can significantly enhance the resistance of Arabidopsis thaliana plants to Botrytis cinerea, clarifying the key function of the Arabidopsis thaliana SMO gene in enhancing the resistance of Arabidopsis thaliana tissues to Botrytis cinerea. This also demonstrates that constructing transgenic plant materials with stable overexpression of Arabidopsis thaliana SMO is an effective way to create new Arabidopsis thaliana materials with high resistance to Botrytis cinerea, providing valuable genetic resources and technological potential for breeding new varieties of Arabidopsis thaliana with high resistance to Botrytis cinerea.

[0111] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0112] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application without departing from the principles and spirit of this application.

Claims

1. Application of the SMO gene in enhancing plant stress resistance, wherein the stress resistance includes: Pathogen stress resistance.

2. The application according to claim 1, characterized in that, The SMO gene nucleotide sequence has at least 90% phylogenetic tree branch support compared to the sequence shown in SEQ ID NO: 1, and the encoded protein is functionally equivalent.

3. The application according to claim 2, characterized in that, The SMO gene nucleotide sequence is shown in any one of SEQ ID NO: 1, 3, or 5; Preferably, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

4. The application according to any one of claims 1-3, characterized in that, The plants are selected from apple trees, pear trees, tomatoes, tobacco, Arabidopsis thaliana, or rice.

5. The application according to claim 4, characterized in that, The pathogens include: *Malus maculatus*, *Phytophthora*, and *Botrytis cinerea*.

6. Application of the SMO gene in improving resistance to fruit tree canker, wherein the nucleotide sequence of the SMO gene has at least 90% phylogenetic branch support compared with the sequence shown in SEQ ID NO: 1 and the encoded protein is functionally equivalent; Preferably, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5; Preferably, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

7. A plant material, characterized in that, Compared to the control group, the SMO gene in the plant material was upregulated by at least 2-fold, and the nucleotide sequence of the SMO gene had at least 90% phylogenetic branch support compared to the sequence shown in SEQ ID NO: 1, and the encoded protein was functionally equivalent.

8. The plant material according to claim 7, characterized in that, The plant material is callus, plant, leaf, or protoplast; the plant is selected from apple tree, pear tree, tomato, tobacco, Arabidopsis thaliana, or rice. Preferably, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5; Preferably, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6.

9. A method for targeted selection or identification of stress-resistant plants, characterized in that, include: To identify the expression of the SMO gene or its encoded protein in a plant, wherein the nucleotide sequence of the SMO gene has at least 90% phylogenetic branch support compared with the sequence shown in SEQ ID NO: 1 and the encoded protein is functionally equivalent; Preferably, the plant is an apple tree, tobacco, or Arabidopsis thaliana; Preferably, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5; Preferably, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6; Optionally, the expression level of the SMO gene was identified by RT-PCR amplification.

10. A method for preparing resistant plant materials, characterized in that, include: The plant SMO gene sequence was amplified, and the nucleotide sequence of the SMO gene had at least 90% phylogenetic tree branch support compared with the sequence shown in SEQ ID NO: 1 and the encoded protein was functionally equivalent. The amplified sequence is ligated into an overexpression vector to obtain the overexpression vector; The overexpression vector was converted into an Agrobacterium strain capable of infecting plant materials by a heat shock method; The Agrobacterium strain was introduced into plant material to obtain resistant plant material; Optionally, the plant material is selected from callus, plant, leaf or protoplast; Optionally, the plant is selected from apple trees, pear trees, tomatoes, tobacco, Arabidopsis thaliana, or rice; Preferably, the SMO gene nucleotide sequence is as shown in any one of SEQ ID NO: 1, 3, or 5; Preferably, the amino acid sequence of the protein encoded by the SMO gene is as shown in any one of SEQ ID NO: 2, 4, or 6; Preferably, the overexpression vector is pBIN-GFP.

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