Banana gene maRBL1 for improving disease resistance of plants, method and application

Abstract

The application discloses a banana gene MaRBL1 for improving disease resistance of plants, a method and application, wherein the banana gene MaRBL1 nucleotide sequence is shown as SEQ ID NO. 1; the gene encodes cytidine diphosphate diacylglycerol synthetase, is a key enzyme for phospholipid biosynthesis, and participates in synthesis of phosphatidylinositol and derivatives phosphatidylinositol 4, 5-diphosphate; a core functional domain of the gene is a CTP_transf_1 domain, retains catalytic activity of CDP-DAG synthetase, participates in phospholipid metabolism and conduction of disease resistance signals, and the application provides a new strategy for cultivating high-resistance banana varieties.

Description

Technical Field

[0001] This invention relates to the field of gene function and plant disease resistance breeding technology, and in particular to a banana gene MaRBL1 for improving plant disease resistance, its method, and its application. Background Technology

[0002] As a vital global economic and food crop, bananas have long been severely threatened by Fusarium wilt, a fungus caused by Fusarium oxysporum f.sp.cubense (Foc). This disease can lead to yellowing of leaves, browning of pseudostems, and even death of the entire plant. Among these strains, tropical race 4 (Foc TR4) has become a major biological stressor restricting the global banana industry due to its wide range of infection and high pathogenicity.

[0003] Currently, the control of banana wilt relies on the synergy of multiple technologies, including agricultural measures, biological control, and disease-resistant breeding. However, traditional disease-resistant breeding has limitations such as long breeding cycles and low efficiency because bananas are mostly triploid, highly sterile, and parthenocarpy. Although gene editing and transgenic technologies have provided new ways to create disease-resistant germplasm, the key lies in the precise discovery and utilization of disease-resistant functional genes.

[0004] Studies have shown that lipid metabolism plays a crucial role in plant disease resistance mechanisms. Phospholipid metabolism, in particular, directly influences plant defense against pathogens by regulating cell membrane integrity and the synthesis of signaling molecules such as phosphatidylinositol derivatives. Cytidine diphosphate diglyceride synthase (CDP-DAG synthase, CDS), a key enzyme in phospholipid biosynthesis, catalyzes the reaction of phosphatidic acid (PA) and cytidine triphosphate (CTP) to produce CDP-DAG (a precursor to phosphatidylinositol). Abnormalities in CDS function can significantly impact plant disease resistance.

[0005] For example, the rice RBL1 gene encodes CDS, and its mutations confer broad-spectrum disease resistance by reducing the level of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). In bananas, the MaRBL1 gene (containing the CTP_transf_1 domain), orthologous to rice RBL1, has been identified. Its encoded protein is involved in phosphatidic acid synthesis and disease resistance signal transduction, and its expression is significantly upregulated under Foc TR4 infection, suggesting it may be a key target for banana disease resistance breeding.

[0006] However, there are no systematic reports on the functional analysis of the banana MaRBL1 gene and its application in disease-resistant breeding in the existing technology. Summary of the Invention

[0007] In view of this, the present invention provides a banana gene MaRBL1 for improving plant disease resistance, a method thereof, and its application.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A banana gene, MaRBL1, that enhances plant disease resistance, has the nucleotide sequence shown in SEQ ID NO.1.

[0010] Preferably, the recombinant vector, transgenic cell line, or recombinant bacteria containing the above-mentioned genes.

[0011] Preferably, the recombinant bacteria is a recombinant bacteria obtained by inserting the above-mentioned gene into an expression vector;

[0012] The transgenic cell lines include banana embryogenic cell lines or Arabidopsis callus cell lines.

[0013] Application of the banana gene MaRBL1, which enhances plant disease resistance, in improving plant disease resistance.

[0014] Preferably, in the aforementioned application, banana MaRBL1 encodes cytidine diphosphate diglyceride synthase, a key enzyme in phospholipid biosynthesis.

[0015] Preferably, the plant is Arabidopsis thaliana.

[0016] A method to improve plant disease resistance includes:

[0017] By introducing the banana MaRBL1 gene, which encodes CDP-DAG synthase, into target plant cells, transgenic plants with enhanced disease resistance were obtained.

[0018] Preferably, the method for obtaining the transgenic plant includes:

[0019] The expression vector containing the banana MaRBL1 gene was used to infect Arabidopsis thaliana via Agrobacterium-mediated transformation, and transgenic plants with improved disease resistance were obtained through screening and cultivation.

[0020] The present invention achieves the following technical effects compared to the prior art:

[0021] This application identified the presence of the MaRBL1 gene (containing the conserved CTP_transf_1 domain) in banana through multi-omics analysis of the banana genome. Quantitative real-time PCR data showed that the expression of the banana MaRBL1 gene was significantly upregulated after infection with Fusarium oxysporum (Foc TR4), suggesting its involvement in the banana's resistance to Fusarium wilt.

[0022] To further elucidate the function of MaRBL1, an overexpression vector was constructed and transformed into Arabidopsis thaliana. The results showed that the disease incidence of transgenic plants was significantly reduced after inoculation with Fusarium wilt, indicating that MaRBL1 gene expression can significantly enhance plant resistance to pathogens. Therefore, the application of the banana MaRBL1 gene holds promise for providing a key target for disease-resistant breeding in bananas and other crops. Attached Figure Description

[0023] Figure 1 This is a diagram illustrating the construction of the pCAMBIA1321-MaRBL1 recombinant plasmid vector of the present invention;

[0024] Note: MaRBL1 is the target fragment, and the restriction enzyme sites are SacI and XbaI;

[0025] Figure 2 This is a diagram illustrating the construction of the pCAMBIA1321-MaRBL1 plant expression vector of the present invention;

[0026] Note: Electrophoresis image of bacterial culture PCR verification of pCAMBIA1321-MaRBL1 recombinant plasmid; Marker: DL2000 DNA Marker; pCAMBIA1321-MaRBL1: target gene band of pCAMBIA1321-MaRBL1.

[0027] Figure 3 This is a diagram illustrating the genetic transformation process of pCAMBIA1321-MaRBL1 Arabidopsis thaliana in this invention.

[0028] The process includes: a) Arabidopsis thaliana cultivation; b) Arabidopsis thaliana infection; c) Arabidopsis thaliana transformation; d) Arabidopsis thaliana T0 generation seed screening; e) Arabidopsis thaliana T1 generation seedling transplantation; and f) Arabidopsis thaliana T1 generation seed harvesting.

[0029] Figure 4 This is a PCR verification diagram of the pCAMBIA132-MaRBL1 transgenic Arabidopsis thaliana T1 generation of this invention;

[0030] Note: Positive: pCAMBIA1321-MaRBL1 Agrobacterium thaliana bacterial suspension control group; M: 1000 DNA Marker; CK: wild-type Arabidopsis thaliana plant control group; W: water control group; 1-6: pCAMBIA1321-MaRBL1 Arabidopsis thaliana transgenic plants;

[0031] Figure 5 This is a graph showing the expression level analysis of MaRBL1 in T2 generation transgenic Arabidopsis thaliana according to the present invention;

[0032] Note: Different letters indicate significant differences (n=3, P<0.05, Duncan test);

[0033] Figure 6This is a diagram of the disease resistance inoculation experiment of the MaRBL1 transgenic Arabidopsis thaliana of this invention. Detailed Implementation

[0034] 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.

[0035] This invention discloses a banana gene MaRBL1 that enhances plant disease resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0036] Recombinant vectors, transgenic cell lines, or recombinant bacteria containing the above-mentioned genes.

[0037] The recombinant bacteria are those obtained by inserting the above-mentioned genes into an expression vector;

[0038] Transgenic cell lines include banana embryogenic cell lines or Arabidopsis callus cell lines.

[0039] This invention also discloses the application of the banana gene MaRBL1, which enhances plant disease resistance, in improving plant disease resistance.

[0040] Banana MaRBL1 encodes cytidine diphosphate diglyceride synthase, a key enzyme in phospholipid biosynthesis.

[0041] The plant is Arabidopsis thaliana.

[0042] This invention also discloses a method for improving plant disease resistance, comprising:

[0043] By introducing the banana MaRBL1 gene, which encodes CDP-DAG synthase, into target plant cells, transgenic plants with enhanced disease resistance were obtained.

[0044] Methods for obtaining transgenic plants include:

[0045] The expression vector containing the banana MaRBL1 gene was used to infect Arabidopsis thaliana via Agrobacterium-mediated transformation, and transgenic plants with improved disease resistance were obtained through screening and cultivation.

[0046] Example 1:

[0047] 1. Materials

[0048] 1.1. Experimental Materials

[0049] The banana variety *Musa spp. AAA* Cavendish subgroup cv'baxi' originated from the Guangzhou branch of the National Banana Improvement Center, Fruit Tree Research Institute, Guangdong Academy of Agricultural Sciences. The *Arabidopsis* seeds used were of the Columbia ecotype (Columbia-0).

[0050] 1.2. Experimental Reagents and Instruments

[0051] Experimental reagents: Conventional restriction endonucleases (BioLabs); Taq DNA polymerase, high-fidelity DNA polymerase, homologous recombinase (Nanjing Novizan); Plant RNA extraction kit (AG); Plasmid extraction kit, DNA gel recovery kit (Tiangen); cDNA reverse transcription kit (TaKaRa); DNA marker, nucleic acid dyes, gel electrophoresis agarose (Beijing Qingke). The pCAMBIA1321-Ubi-GFP vector was preserved and provided by the Banana Genetic Improvement Research Laboratory of the Fruit Tree Research Institute, Guangdong Academy of Agricultural Sciences.

[0052] Experimental instruments: clean bench, high temperature and high pressure sterilizer, high speed centrifuge, gradient PCR instrument, gel imaging analysis system, real-time PCR instrument, electronic balance, pure water system, pH meter.

[0053] 1.2. Primer synthesis and sequencing

[0054] Primer synthesis and sequencing were both completed by Shanghai Sangon Biotech Co., Ltd.

[0055] 2. Methods

[0056] 2.1. Extraction of total RNA from banana leaves

[0057] Total RNA was extracted from fresh banana leaves using the SteadyPure Plant RNA Extraction Kit from AG (Aikerui Biotechnology Co., Ltd.), following the instructions in the manufacturer's manual.

[0058] (1) Transfer the fresh banana tissue sample into a mortar pre-cooled by liquid nitrogen and grind the tissue into powder using a pestle;

[0059] (2) Take 60 mg of powder sample and place it in a 1.5 mL enzyme-free centrifuge tube containing 500 μL of lysis buffer RLS Buffer (make sure 50×DTT solution has been added). Quickly mix with a high-speed vortex or repeatedly pipette until the sample is completely lysed (no visible precipitate).

[0060] (3) After letting the lysis buffer stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 5 minutes at 4°C.

[0061] (4) Transfer the supernatant to a new 1.5 mL enzyme-free centrifuge tube;

[0062] (5) Add half a volume of anhydrous ethanol to the lysis buffer;

[0063] (6) Transfer the mixture and precipitate into a Plant RNA Mini Column, centrifuge at 12,000 rpm for 2 minutes at room temperature and discard the filtrate (if the mixture exceeds 700 μL, it needs to be added in portions);

[0064] (7) Add 500 μL of RWA Buffer to a Plant RNA Mini Column tube, centrifuge at 13,000 rpm for 3 minutes, and discard the filtrate;

[0065] (8) Add 650 μL of RWB Buffer to the tube above, centrifuge at 13,000 rpm for 3 minutes, and then discard the filtrate;

[0066] (9) Repeat step 8;

[0067] (10) Place the new 2 mL collection tube into the Plant RNA Mini Column tube and centrifuge at 13,000 rpm for 5 minutes;

[0068] (11) Place the new enzyme-free centrifuge tube into the Plant RNA Mini Column tube, add 70 μL of enzyme-free sterile water to the center of the filter membrane, place it at 25°C for 7 minutes, and then centrifuge at 13,000 rpm for 4 minutes at 25°C to elute RNA.

[0069] If not used immediately, the dissolved RNA should be stored at -80°C.

[0070] 2.2. Reverse transcription of total RNA from bananas into cDNA

[0071] The total RNA obtained in section 2.16.2 was reverse transcribed using the TakaRa PrimeScript™ II cDNA Synthesis Kit. The specific procedures were performed according to the manufacturer's instructions, as follows:

[0072] (1) Prepare the reaction mixture for the first step in centrifuge tubes. The reaction system is shown in Table 1:

[0073] Table 1:

[0074] Component Name concentration volume OligodTPrimer 50μM 1μL dNTPMixture 10nMeach 1μL template RNA Total RNA: less than 5 μg - <![CDATA[RNaseFreedH2O]]> - Up to 10 μL

[0075] (2) After holding at 65℃ for 5 minutes, cool rapidly on ice;

[0076] (3) Prepare the reverse transcription reaction solution shown in Table 2 in the centrifuge tubes above, with a total volume of 20 μL;

[0077] Table 2:

[0078] Component Name volume The above-mentioned denatured reaction solution (from step 2) 10μL 5×PrimeScriptⅡBuffer 4μL RNase inhibitor (40 U / μl) 0.5μL PrimeScript II RTase (200 U / μl) 1μL <![CDATA[RNaseFreedH2O]]> Up to 20μL

[0079] (4) Mix slowly, 30℃ for 10 min, 42℃ for 30-60 min, 95℃ for 5 min (enzyme inactivation), then cool on ice and store at -20℃ for use.

[0080] 2.3. Primer design for gene sequences

[0081] Download the banana gene MaRBL1 (Macma4_09_g27330.1) sequence from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ):

[0082]

[0083] Primers were designed using Primer Primer 5.0 software to clone the target sequence:

[0084] MaRBL1-F:

[0085] 5'-AATTACTATTTACAATTACGGATCATGCATAAAGACAGTGGCTCCAG-3',

[0086] MaRBL1-R:

[0087] 5'-CAGCTCCTCGCCCTTGCTCACCATAGATCCTCCTCCAGATCCTCCT CC-3'.

[0088] 2.4. Amplification of the target fragment of banana MarBL1

[0089] Using banana cDNA as a template, the full-length sequence of the MaRBL1 gene was amplified using 2×PhantaMax Master Mix high-fidelity enzyme and primers MaRBL1-F / MaRBL1-R. The PCR reaction system is shown in Table 3.

[0090] Table 3:

[0091] Element volume 2×PhantaMaxMasterMix 25μL MaRBL1-F (10μM) 2μL MaRBL1-R (10μM) 2μL Banana cDNA 3μL <![CDATA[ddH2O]]> 18μL Total 50μL

[0092] PCR amplification program: 95℃ for 3 min for 1 cycle; 95℃ for 15 s for 34 cycles; 65℃ for 15 s for 34 cycles; 72℃ for 1 min for 34 cycles; 72℃ for 5 min for 1 cycle.

[0093] After electrophoresis, the amplified products were purified using the Tiangen Biotech Agarose Gel DNA Recovery Kit, and the recovered DNA was stored at -20℃ for later use.

[0094] 2.5. Recombination and ligation of target gene and vector

[0095] The plasmid vector was digested with enzymes, and the recovered target fragment was recombined and ligated. The recombinant ligation product was transformed into E. coli DH5α competent cells, cultured overnight in plates containing the corresponding antibiotics, and single clones were selected for PCR identification. Positive clones were sent to the company for sequencing.

[0096] The target fragment connection system is shown in Table 4;

[0097] Table 4:

[0098] Target fragment 5×pClone007VersatileSimpleVectorMix <![CDATA[ddH2O]]> Total 1μL 1μL 3μL 5μL .

[0099] Reaction conditions: 26℃ for 5 minutes.

[0100] 2.6. Target Fragment Connectivity System Transformation

[0101] The recombinant ligation product was added to DH5α competent cells, incubated on ice for 30 min, followed by heat shock at 42℃ for 60 s and then on ice for 5 min. 700 μL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ for 200 rpm for 1 h. After centrifugation at 5000 rpm for 1 min, 100–200 μL of the bacterial culture was spread onto plates containing antibiotic LB and incubated upside down at 37℃ for 12–16 h. Single colonies were picked and inoculated into LB liquid medium containing antibiotics, and incubated at 37℃ for 200 rpm for 6–7 h. After verification by bacterial PCR and electrophoresis, the bacterial cultures containing positive bands were sent to Sangon Biotech for sequencing. If the sequencing results met expectations, the MaRBL1 plasmid could be extracted for later use.

[0102] The bacterial culture PCR system is shown in Table 5;

[0103] Table 5:

[0104]

[0105]

[0106] 2.7. Transformation of Agrobacterium with recombinant plasmids and identification of positive clones

[0107] The plasmid was transformed into EHA105 competent cells (1 μg DNA / 100 μL), incubated on ice for 5 min, followed by liquid nitrogen shock for 5 min, and then incubated in a 37°C water bath for 3 min. 700–800 μL of antibiotic-free LB medium was added, and the cells were incubated at 28°C, 200 rpm in the dark for 2 h. The cells were then plated on plates containing antibiotic LB and incubated at 28°C in the dark for 36–48 h. Single colonies were picked and expanded (containing antibiotic LB, incubated at 28°C, 180–200 rpm in the dark for 12 h). Positive bacterial cultures were verified by PCR and then sequenced. Finally, the bacterial culture was stored at -80°C in a 1:1 glycerol solution.

[0108] 2.8. Agrobacterium tumefaciens GV3101-mediated transformation of Arabidopsis thaliana

[0109] (1) The constructed pCAMBIA1321-MaRBL1 vector plasmid was transformed into GV3101 Agrobacterium competent cells. After electroporation, 1 mL of LB liquid medium was added and mixed well, then transferred to a 1.5 mL centrifuge tube and cultured with shaking at 30°C. 50 μL of the bacterial culture was spread on LB solid medium and cultured at 30°C in the dark for 48 h. The target fragment was amplified by PCR and verified by 1% agarose gel electrophoresis. Positive clones showed specific bands consistent with the control plasmid (negative controls showed no amplification products), thus obtaining the recombinant Agrobacterium positive strain.

[0110] (2) Agrobacterium clones containing the pCAMBIA1321-MaRBL1 recombinant plasmid were inoculated into LB liquid medium (containing 50 μg / mL Kan and 100 μg / mL Rif) and cultured at 28°C with shaking until OD600 = 0.8–1.2. The bacterial cells were collected by centrifugation, resuspended in 5% sucrose solution, and supplemented with 0.02% Silwet-77 surfactant. Arabidopsis inflorescences were then immersed for 2–3 seconds and cultured in a sealed, moist, dark environment for 24 hours. The immersion process was repeated three times at 7-day intervals to complete the genetic transformation cycle.

[0111] (3) After infection, the plants were cultured in a photoperiodic incubator until the seeds matured. Harvested pods were dried at 37℃ for 24 hours, then purified seeds were obtained by sieving through a 60-mesh sieve and stored at 4℃ for later use. T0 generation seeds were surface-sterilized with 95% ethanol (10 min) and 75% ethanol (10 min), rinsed three times with sterile water, and evenly inoculated onto 1 / 2 MS selection medium containing 50 mg / L hygromycin. After vernalization at 4℃ for 48 hours, the seeds were transferred to an incubator for 7–9 days of resistance selection. Surviving seedlings were transplanted to nutrient soil and cultured at 23℃ until seedlings matured.

[0112] (4) Genomic DNA was extracted from the T1 generation Arabidopsis seedlings that had grown for nearly 5 weeks. T1 positive Arabidopsis seedlings were obtained by PCR detection. After the T1 positive Arabidopsis seedlings were harvested, they were dried, dehulled, disinfected, and screened. After 3 to 4 weeks of cultivation, T2 positive Arabidopsis seedlings were obtained. Homozygous lines were obtained by planting and used for disease resistance inoculation verification.

[0113] 3. Experimental Results

[0114] 3.1. Construction of the plant pCAMBIA1321-MaRBL1 vector

[0115] The pCAMBIA1321 plant binary expression vector was double-digested with restriction endonucleases XbaI and SacI (TaKaRa), and then recombined with the target gene MaRBL1 to successfully construct the pCAMBIA132-MaRBL1 overexpression vector. Figure 1 As shown in the image. The constructed pCAMBIA1321-MaRBL1 was then transformed into E. coli competent cells DH5α, and positive identification was performed by colony PCR. Based on the detected and identified target band, the results indicate that the pCAMBIA1321-MaRBL1 plant expression vector has been successfully constructed. Figure 2 As shown.

[0116] 3.2. Expression analysis of the banana MarBL1 gene

[0117] The pCAMBIA1321-MaRBL1 plasmid was successfully transformed into Arabidopsis thaliana using the inflorescence infection method. Figure 3As shown, after selection for resistance to 35 mg / L hygromycin (Hyg), non-transgenic plants withered and died on the culture medium, while T0 generation plants grew normally and were transplanted for seed harvest. After the same resistance selection, PCR testing of T1 generation seeds showed that all six transgenic seedlings amplified a 481 bp target band consistent with the positive control, while wild-type and negative controls showed no band. Figure 4 As shown in the image. Currently, the T2 generation plants have entered the planting stage, and the subsequent development and expression analysis of the T3 generation homozygous lines will be carried out.

[0118] The expression level of MaRBL1 in the obtained T2 generation Arabidopsis transgenic lines was determined by RT-qPCR.

[0119] The results showed that the expression level of the MaRBL1 gene was highest in transgenic Arabidopsis lines #2 and #3, upregulated by 589.1-fold and 414.11-fold, respectively. Figure 5 As shown, this indicates that the MaRBL1 transgenic Arabidopsis thaliana line has been successfully obtained. Further planting and screening of homozygous lines will be conducted for disease resistance inoculation experiments.

[0120] 3.3. Functional verification of the banana MarBL1 gene

[0121] To investigate the function of the banana MaRBL1 gene in plant resistance to Fusarium wilt, this invention used MaRBL1 transgenic Arabidopsis thaliana lines #2 and #3 and wild-type (WT) plants as materials to conduct compatibility Fusarium oxysporum inoculation experiments and disease resistance phenotype analysis.

[0122] The experimental results showed that on the 5th day after inoculation, the leaves of the WT wild-type plants showed significant wilting and yellowing phenotypes; while the inoculated leaves of the MaRBL1 transgenic lines #2 and #3 showed no obvious lesions or wilting symptoms, and the phenotypes were not significantly different from those of the uninoculated healthy plants.

[0123] Further observation was extended to day 11 post-inoculation. The area of ​​leaf wilting in WT plants continued to expand, showing typical Fusarium infection and decline characteristics. In contrast, MaRBL1 transgenic lines #2 and #3 maintained good vegetative growth, with leaf morphology and color remaining basically normal.

[0124] The phenotypic differences mentioned above indicate that overexpression of the MaRBL1 gene significantly enhances the resistance of Arabidopsis thaliana to compatible Fusarium oxysporum. Transgenic plants exhibit superior resistance to Fusarium wilt under pathogen infection conditions, verifying the positive regulatory role of MaRBL1 in plant disease resistance and defense responses.

[0125] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. Banana genes MaRBL1 Application of the banana gene in enhancing Arabidopsis thaliana's resistance to banana wilt disease MaRBL1 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The banana gene according to claim 1 MaRBL1 Its application in enhancing Arabidopsis thaliana's resistance to banana wilt disease is characterized by... In this application, the banana gene MaRBL1 encodes cytidine diphosphate diglyceride synthase.

3. A method for improving plant disease resistance, characterized in that, include: The banana gene encoding CDP-DAG synthase MaRBL1 Transgenic plants with enhanced resistance to banana wilt disease were obtained by introducing the banana gene into target Arabidopsis cells. MaRBL1 The nucleotide sequence is shown in SEQ ID NO.

1.

4. The method for improving plant disease resistance according to claim 3, characterized in that, The method for obtaining the transgenic plant includes: Will contain the banana gene MaRBL1 The expression vector was used to infect Arabidopsis thaliana via Agrobacterium-mediated transformation, and transgenic plants with improved disease resistance were obtained through screening and culture.

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