Application of arabidopsis thaliana AtUPL5 gene in aspect of improving plant resistance

Abstract

Application of an arabidopsis thaliana AtUPL5 gene in the aspect of improving plant resistance belongs to the technical field of genetic engineering, the invention provides application of the arabidopsis thaliana AtUPL5 gene, and the gene can significantly enhance the resistance of arabidopsis thaliana to sclerotiniose; compared with wild arabidopsis thaliana, the T-DNA insertion deletion mutant strain of the AtUPL5 gene shows weaker disease resistance when suffering from sclerotiniose infection, and the expression level of the main defense related gene in the mutant strain is more significant in change before and after pathogen infection. The AtUPL5 gene of arabidopsis thaliana is closely related to the pathogenesis of sclerotiniose, so that the gene and a homologous gene thereof in other cruciferous plants can be applied to breeding of the cruciferous plants, and a wide application prospect is shown.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of Arabidopsis thaliana AtUPL5 gene in improving plant resistance. Background Art

[0002] Sclerotinia is a fungal disease caused by the genus Sclerotinia, which widely harms a variety of economic crops, including rapeseed, beans, sunflowers and various vegetables. Its pathogen Sclerotinia sclerotiorum (Lib.) de Bary achieves long-term survival by forming stress-resistant sclerotia. The sclerotia germinate under suitable environmental conditions and release ascospores to infect the host plant. The disease is characterized by water-soaked rot spots on plant leaves, stems and fruits, and in severe cases, the whole plant can die. Sclerotinia not only directly leads to crop yield reduction or total loss, but also weakens the overall growth of the plant, reduces stress resistance, and increases sensitivity to other diseases and pests. In addition, sclerotia can persist in the soil for many years. Once Sclerotinia breaks out in a certain area, the management and control of the disease will be extremely complicated.

[0003] Sclerotinia caused by Sclerotinia poses a major threat to a variety of crops, and its resistance to pesticides makes chemical control strategies increasingly complex. Sclerotinia easily adapts to pesticide pressure and evolves new resistance mechanisms, resulting in a weakening of the effectiveness of traditional chemical control methods, an increase in pesticide use, and increased risks to the environment and human health. Therefore, the development and application of plant-derived disease resistance genes has become a sustainable and environmentally friendly control strategy.

[0004] Arabidopsis thaliana is a model plant widely used in plant biology research. With its small genome, short life cycle and easy genetic manipulation, it has become an ideal model for studying plant physiological functions and disease resistance mechanisms. In the field of plant pathology, Arabidopsis thaliana is used to discover and study disease resistance genes, and has broad application prospects, especially in the discovery and application of homologous genes in other economic crops. Disease resistance genes found in Arabidopsis thaliana usually have corresponding homologous genes in crops, laying an important foundation for cross-species disease resistance research and improvement.

[0005] Ubiquitination plays a key role in the interaction between plants and microorganisms. As a core protein post-translational modification mechanism in plant cells, it regulates the response of plants to microorganisms. Through ubiquitination, plants can mark specific proteins for degradation or functional regulation, and quickly adjust their own physiological and defense states to cope with the invasion of foreign microorganisms. This process requires the participation of three types of enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). E1 activates ubiquitin and transfers it to E2, and then E3 recognizes specific target proteins and assists E2 in transferring ubiquitin to the target protein. Ubiquitination regulates protein degradation, localization, activity and signal transduction pathways, and is crucial to cellular functions such as protein degradation, signal transduction and cell cycle control, thereby affecting the ability of cells to cope with various biological and abiotic stresses.

[0006] E3 ubiquitin ligase has the ability to specifically recognize and mark substrate proteins, and therefore plays a central role in the ubiquitination process. In the interaction between plants and pathogens, E3 ubiquitin ligase participates in multiple signal transduction pathways, regulating the stability and function of key proteins involved in plant disease resistance. For example, E3 ubiquitin ligase can enhance the plant's defense capabilities by marking proteins that inhibit plant immunity for degradation. Therefore, in-depth analysis of the specific functions and regulatory mechanisms of E3 ubiquitin ligases in the interaction between plants and pathogens is of great significance for the development of new disease management strategies and the promotion of high-quality agricultural development. Summary of the invention

[0007] The present invention aims to provide a gene for regulating plant immunity and a protein encoded by the gene.

[0008] The invention provides application of an Arabidopsis thaliana AtUPL5 gene in improving the resistance of Arabidopsis thaliana to sclerotinia sclerotiorum. The ID number of the Arabidopsis thaliana AtUPL5 gene in NCBI is At4g12570.

[0009] According to the above scheme, the nucleotide sequence of the Arabidopsis thaliana AtUPL5 gene is shown in SEQ ID NO:1.

[0010] The present invention provides an application of Arabidopsis thaliana E3 ubiquitin ligase AtUPL5 in enhancing the resistance of Arabidopsis thaliana to sclerotinia sclerotinia. The Arabidopsis thaliana AtUPL5 protein has an amino acid sequence shown in Ⅰ) or Ⅱ):

[0011] 1) the amino acid sequence shown in SEQ ID NO: 2; or

[0012] II) A protein having the same function obtained by replacing, deleting and / or adding one or more amino acids based on the above amino acid sequence.

[0013] The present invention provides the application of the Arabidopsis thaliana AtUPL5 gene or its corresponding biological material in plant disease resistance. Specific applications include:

[0014] I) Silencing or knocking out the AtUPL5 gene in plants increases the susceptibility of plants to Sclerotinia sclerotiorum;

[0015] II) The T-DNA insertion mutation of the AtUPL5 gene can promote the formation of lesions by Sclerotinia sclerotiorum on the leaves of mutant Arabidopsis thaliana; before and after pathogen infection, the expression of defense-related genes in T-DNA insertion mutant plants is more significant than that in the wild type, which can be used in the field of plant disease resistance.

[0016] The present invention proves that the deletion of AtUPL5 gene can significantly enhance the immunity of plants, indicating that AtUPL5 gene is an important gene for Arabidopsis to enhance immunity to pathogens. Therefore, screening for compounds that enhance the expression or modification of this gene and its synthetic protein can effectively inhibit the spread of sclerotinia disease in cruciferous plants. That is, an important use provided by the present invention is that this gene and its homologous genes in other cruciferous plants can be used as important target sites in plants to enhance the disease resistance of plants, providing new resources for green integrated prevention and control of plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the AtUPL5 gene overexpression vector;

[0018] Figure 2 This is a photo of the subcellular localization of the AtUPL5 gene;

[0019] Figure 3 It is the agarose gel electrophoresis of the T-DNA insertion mutant SALK_116446C;

[0020] Figure 4 The photos and the statistical bar graph of leaf lesions of AtUPL5 gene T-DNA insertion mutant SALK_116446C and wild-type Arabidopsis thaliana infected by Sclerotinia sclerotiorum are shown;

[0021] Figure 5 This is a bar graph showing the quantitative analysis of the main defense genes and photosynthetic genes in Columbia wild Arabidopsis and AtUPL5 gene T-DNA insertion mutants at 0h (0hpi) and 24h (24hpi) after infection with Sclerotinia sclerotiorum. DETAILED DESCRIPTION

[0022] The present invention is described below with reference to the accompanying drawings.

[0023] The gene for regulating plant immunity provided by the present invention is derived from Arabidopsis thaliana, named AtUPL5, and its gene number in the NCBI database is At4g12570. The amino acid sequence of the gene is shown in SEQ ID NO: 2, and its encoding nucleotide sequence (CDS sequence) is shown in SEQ ID NO: 1. The gene name and gene number are both derived from the Arabidopsis gene sequencing database (www.arabidopsis.org), specifically from the wild-type Arabidopsis Col-0 strain.

[0024] The Arabidopsis thaliana T-DNA insertion mutant SALK_206973C used in the present invention was purchased from Fuzhou Baiersent Technology Co., Ltd., address:

[0025] No. 522, S15, China Resources MixC (Phase III), No. 52, Hongshanyuan Road, Hongshan Town, Gulou District, Fuzhou

[0026] Website: https: / / www.arashare.cn / index / Product / index

[0027] Mutant ID: SALK_116446C

[0028] Example 1 Construction of AtUPL5 gene overexpression vector

[0029] 1. Double restriction digestion of pCAMBIA1301-RFP vector

[0030] The pCAMBIA1301-RFP vector was double-digested with restriction endonucleases KpnI and SacI, and the digestion products were separated by agarose gel electrophoresis, and the corresponding high molecular weight bands were cut out and gel recovered;

[0031] 2. Preparation of Arabidopsis thaliana total cDNA

[0032] Extract RNA using the full-form gold RNA extraction kit: To ensure that there is no RNase contamination, the mortar, pestle and small iron spoon must be wrapped with tin foil in advance, baked in a 180℃ constant temperature box for 3 hours to cool to room temperature, and finally transferred to a -20℃ freezer for precooling and standby use; in addition to the reagents included in the kit, prepare RNase Free 1.5mL centrifuge tubes, 1mL, 200μL, 10μL tips and pipettes, liquid nitrogen, and centrifuge tube racks; take 2-3 fresh leaves of 30-day-old Arabidopsis, place them in a precooled mortar, add liquid nitrogen and grind them 2-3 times until they are powdery, transfer them to labeled centrifuge tubes, and number them; add 1mL TransZol UP and 200μL RNA Extraction Reagent to the centrifuge tube, vortex to mix for 5 minutes, and centrifuge at 12,000g at 4℃ for 15 minutes. Transfer 400 μL of supernatant to a new centrifuge tube, add an equal volume of anhydrous ethanol and mix thoroughly, then transfer to the adsorption column provided with the kit and centrifuge at 12,000 g for 30 seconds at room temperature. After discarding the waste liquid in the collection tube, add 500 μL of CB solution to the adsorption column, centrifuge at 12,000 g for 30 seconds again and discard the waste liquid (repeat the operation twice). Subsequently, add 500 μL of WB solution containing anhydrous ethanol to the adsorption column, centrifuge at 12,000 g for 30 seconds and discard the waste liquid (repeat the operation twice), and centrifuge again at 12,000 g for 1 minute to remove residual ethanol. Finally, elute RNA with 50 μL ddH2O and detect the RNA concentration for later use.

[0033] 3. Preparation of cDNA

[0034] cDNA was synthesized using the TOLOBIO reverse transcription kit. The reaction system was: 5×All-in-one RT Buffer 4μL, All-in-one Enzyme Mix 1μL, template (RNA) 1μL, RNase-free ddH2O 14μL, a total of 20μL system. Reaction conditions: 50℃ for 15 minutes, 85℃ for 5 seconds. After the product concentration was tested, it was stored at -20℃ for future use.

[0035] 4. Design specific primers (primer sequences are shown in Table 1)

[0036] The CDS sequence of the AtUPL5 gene was amplified using the high-fidelity enzyme KOD produced by TOYOBO, and the resulting PCR product was separated by 0.8% agarose gel electrophoresis, and the target band of the corresponding molecular weight was cut and recovered.

[0037] 5. Homologous recombination ligation

[0038] The linearized vector and the AtUPL5 gene fragment purified by PCR were connected by homologous recombination. Specific operation: Use the Novozyme single fragment homologous recombination kit C112 to prepare the reaction system: 5×CEⅡBuffer 4μL, ExaseⅡ2μL, 200ng double enzyme digestion recovered linearized vector, 20ng target CDS fragment, and ddH2O to make up the total reaction volume to 20μL. React at 37℃ for 30 minutes.

[0039] 6. Transformation of E. coli

[0040] The above recombinant reaction solution was transferred into DH5α competent cells and cultured overnight at 37°C in a medium containing kanamycin. After picking a single colony and placing it in LB liquid medium containing kanamycin and shaking it for 3 hours, the bacterial solution was taken for PCR detection. The electrophoresis results showed that the amplified band was correct. The plasmid was extracted and sequenced for verification. The map of the successfully constructed recombinant vector is shown in the figure below. Figure 1 The verified bacterial solution was stored at -80°C for future use. Figure 1 shown.

[0041] Table 1 Primers used for construction of heterologous expression vectors

[0042]

[0043] Example 2 Transient expression of AtUPL5 gene in tobacco leaves using recombinant vector

[0044] 1. Transformation of recombinant vector into Agrobacterium GV3101

[0045] Take Agrobacterium GV3101 competent cells and thaw them on ice. After they are fully thawed, add about 100 ng (about 0.5 μL) of recombinant plasmid to 33 μL Agrobacterium competent cells, mix gently and let stand on ice for 5 minutes; then place the mixture in liquid nitrogen for 5 minutes, take it out and incubate it in a 37°C water bath for 5 minutes, and then let it stand on ice for 5 minutes; add 500 μL LB or YEP medium to the above system and culture it at 28°C and 200 r / min for 3 hours. ; Centrifuge the culture product at 5,000r / min for 1 minute, discard the supernatant and resuspend the remaining liquid, transfer it to a YEP plate containing rifamycin (Rif) and kanamycin (Kan), and culture it upside down at 28°C for about 40 hours; pick a single colony in 700μL YEP liquid culture medium containing the corresponding antibiotics, culture it at 28°C with shaking for 18 hours and perform bacterial liquid PCR verification, add an equal volume of 50% glycerol to the positive bacteria with the correct band, mix well, and freeze it at -80°C for use.

[0046] 2. Tobacco transient expression

[0047] Take the 20μL glycerol bacteria stored at -80℃, inoculate it in YEP containing the corresponding antibiotics, and culture it at 28℃ and 200r / min for 18 hours; then take 50μL of the activated bacterial solution and add it to 12.5mL YEP containing the corresponding antibiotics, and culture it at 28℃ and 200r / min until OD600 is about 0.8; centrifuge it at 5,000r / min for 10 minutes at room temperature to recover the bacteria. Prepare the infiltration buffer: add 98mL ddH2O, 1mL MES, 1mL MgCl2, and 100μL AS to every 100mL buffer. Resuspend the bacteria in the buffer to a final OD600 of 0.6-0.8, and let it stand at 28℃ in the dark for 3 hours. Select Nicotiana benthamiana that has grown for 34 weeks, and slowly inject the resuspended bacterial solution from the back of the leaf with a syringe to fully penetrate the entire leaf. Move the treated tobacco into the greenhouse for routine maintenance, and subsequent experiments can be carried out after 48 hours.

[0048] Example 3 Subcellular localization analysis of AtUPL5 protein based on recombinant vector and transient expression in tobacco

[0049] According to the methods described in Examples 1 and 2, the target gene was fused with the RFP reporter gene coding region; the vector containing the exogenous target gene was introduced into tobacco leaves using Agrobacterium and transient expression was achieved. The fluorescence signal in the leaf epidermal cells was observed using a laser confocal microscope. The subcellular localization results are shown in Figure 2. Figure 2 As shown, the fluorescence signal of the AtUPL5 protein fusion protein expressed with RFP was detected in the nuclei of epidermal cells of tobacco leaves and partially overlapped with the nucleus marker dye DAPI.

[0050] Example 4 Screening of T-DNA insertion mutant materials

[0051] 1. CTAB method to extract Arabidopsis DNA

[0052] Take 2-3 wild-type or mutant Arabidopsis leaves and 2 0.5 mm steel balls, place them in a 1.5 mL centrifuge tube, and use a high-throughput grinder to fully grind them into tissue powder. Add CTAB solution preheated to 65°C, vortex mix, and place in a water bath at 65°C for 30 minutes. During this period, prepare a chloroform: isoamyl alcohol (24:1) mixed solution. After the water bath ends, add an equal volume of chloroform: isoamyl alcohol solution to the sample in a fume hood, mix well, and centrifuge at 12,000 r / min for 15 minutes; aspirate the supernatant, add an equal volume of chloroform: isoamyl alcohol solution, mix well, and repeat the centrifugation step. Transfer the supernatant to a new centrifuge tube, add 2.5 times the volume of pre-cooled isopropanol (-20°C), gently invert to mix, place at -20°C for 30 minutes, and then centrifuge at 12,000 r / min for 15 minutes. After discarding the supernatant, aspirate the residual liquid and add 1mL 75% ethanol, invert and mix, and centrifuge at 12,000r / min for 5 minutes. Discard the supernatant and aspirate the residual liquid, finally add 30μL ddH2O to the precipitate, heat in a 65℃ metal bath for 30 minutes to dissolve the DNA, and measure the concentration, then store at -20℃.

[0053] 2. Primer design for PCR amplification using the three-primer method

[0054] According to the T-DNA insert sequence number SALK_116446C provided by https: / / signal.salk.edu / , the left primer (LP) and right primer (RP) sequences provided by the website were consulted, and the nucleotide sequences are shown in Table 2. BP represents a specific fragment in the T-DNA insert sequence, and three primers were designed. According to the sequence number category, LBb1.3 was selected as the BP primer of the target gene, and its corresponding nucleotide sequence is shown in Table 2.

[0055] Table 2 Primers used for screening of T-DNA insertion mutant materials

[0056]

[0057] 2. PCR reaction system and procedure

[0058] PCR reaction system: Primer LP or BP: 0.5 μL; Primer RP: 0.5 μL; Template DNA: 1 μL; Deionized water; ddH2O): 3 μL; 2×TransFast Taq PCR SuperMix (containing dye): 5 μL.

[0059] PCR reaction program: initial denaturation: 94°C, for 3 minutes; denaturation stage: 94°C, for 5 seconds; annealing stage: 56°C, for 15 seconds; extension stage: 72°C, extension rate is 6 kb per minute; number of cycles: 35 times; final extension: 72°C, for 5 minutes.

[0060] 3. Agarose gel electrophoresis

[0061] The PCR amplification products were separated by electrophoresis in 1% agarose gel. For the primer combination LP+RP, no band appeared; while the Arabidopsis strains that showed only a single band for the BP+RP combination were confirmed to be homozygous SALK_116446C mutants. The screening results are as follows Figure 3 Homozygous mutant seeds were collected for subsequent experiments.

[0062] Example 5 Sclerotinia infection and phenotypic observation

[0063] 1. Activation of Sclerotinia sclerotiorum

[0064] Take a small piece of Sclerotinia sclerotiorum from the preserved slant culture aseptically and inoculate it in the center of PDA (potato dextrose agar) medium without antibiotics for activation. When the mycelium growth is close to covering the surface of the medium, collect a small piece from the edge area containing active mycelium and transfer it to a new PDA medium without antibiotics. When the new round of mycelium growth is close to covering the plate, use a 0.5 cm diameter puncher to punch holes at the edge of the mycelium and take a cylindrical cake (medium).

[0065] 2. Sclerotinia sclerotiorum infection of AtUPL5 gene-silenced mutants and wild-type Arabidopsis

[0066] Homozygous T-DNA insertion mutant plants SALK_116446C were selected and sown at the same time as wild-type Arabidopsis. They were transplanted when they grew two true leaves and cultured in a greenhouse for 3 to 4 weeks. After preparing the bacterial cake, three healthy SALK_116446C mutants and three wild-type Arabidopsis were selected, and three leaves from each plant were selected for inoculation. The cylindrical bacterial cake containing sufficient hyphae was covered with the hyphae side facing down on the back of the Arabidopsis leaf, and the culture container was closed to maintain humidity. After 24 hours of infection, the lesions formed on the leaves were photographed and recorded, and the lesion area was measured using ImageJ software. According to the measurement results, statistical analysis methods were used to evaluate the significance of the differences in the incidence between different groups.

[0067] Results (see Figure 4 ) showed that compared with the wild-type Arabidopsis control group, the SALK_116446C mutant Arabidopsis plants showed a phenotype of reduced resistance to Sclerotinia infection. Statistical analysis by ImageJ and SPSS software showed that the lesion area on the leaves of the SALK_116446C mutant plants was significantly larger than that of the wild-type Arabidopsis, indicating that its disease resistance was significantly reduced.

[0068] Example 6 Real-time fluorescence quantitative polymerase chain reaction (qPCR)

[0069] According to the method described in Example 5, Sclerotinia sclerotiorum and Botrytis cinerea were inoculated on leaves of mutant and wild-type Arabidopsis thaliana. Leaf samples were collected at 0 hours (0h) and 24 hours (24h) after inoculation, respectively, and the bacterial cakes were removed and wrapped with aluminum foil. The samples were quickly frozen with liquid nitrogen and stored in an environment of -80°C. After all samples were collected, total RNA was extracted, complementary DNA (cDNA) was synthesized by reverse transcription reaction, and then quantitative polymerase chain reaction (qPCR) analysis was performed according to the above method. The primer sequences used are listed in Table 3.

[0070] Experimental results (see Figure 5 ) showed that at 0h after Sclerotinia inoculation, the expression levels of defense-related genes PR1, PR2, PR4, PAD3, EDS5, ERD1 and FRK1 in the mutant Arabidopsis were significantly lower than those in the wild-type Arabidopsis; at 24h after Sclerotinia inoculation, although the expression levels of the above defense genes in the mutant were still lower than those in the wild-type 24h after inoculation, they were higher than those at 0h after inoculation. Compared with the wild-type, the upregulation of the above defense genes in the mutant was more significant, indicating that the mutant had a higher sensitivity to Sclerotinia infection and an enhanced defense response.

[0071] Table 3 Primers used for qRT-PCR analysis of Arabidopsis plants

[0072]

Claims

1. An application of the disease-resistant AtUPL5 gene of Arabidopsis thaliana in improving plant resistance, specifically: the deletion of the AtUPL5 gene can significantly increase the area of ​​sclerotinia lesions and reduce the expression of plant defense-related genes, and the nucleotide sequence of the AtUPL5 gene is shown in SEQ ID NO: 1.

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