Application of arabidopsis thaliana AtUBP6 gene in aspect of improving plant resistance

Abstract

The invention discloses application of an arabidopsis thaliana AtUBP6 gene in the aspect of improving plant resistance, belongs to the technical field of genetic engineering, and provides application of the arabidopsis thaliana AtUBP6 gene which can improve the sclerotiniose resistance of arabidopsis thaliana. Compared with wild arabidopsis thaliana, the T-DNA insertion deletion mutant strain of the AtUBP6 gene shows weaker disease resistance to sclerotiniose, and the expression quantity of main defensive genes in the mutant has more obvious difference before and after pathogen infection. The AtUBP6 of arabidopsis thaliana is closely related to sclerotiniose, and the gene and a homologous gene thereof in other cruciferous plants can be applied to cruciferous plant breeding, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to the application of Arabidopsis thaliana AtUBP6 gene in improving plant resistance. Background Art

[0002] Sclerotinia sclerotiorum (Lib.) de Bary is a fungal plant disease that is serious and difficult to control. It has a wide host range and can infect a variety of vegetable crops such as rapeseed, beans, sunflowers, etc., up to more than 600 species. Sclerotinia sclerotiorum survives for a long time in the soil or diseased residues by forming stress-resistant sclerotia. The sclerotia germinate under suitable environmental conditions, release a large number of ascospores, and infect the host plant. The disease usually manifests as water-soaked rot spots on plant leaves, stems and fruits, and in severe cases can cause the death of the entire plant. The damage of Sclerotinia sclerotiorum can not only directly lead to crop yield reduction or total loss, but also weaken the overall growth of the plant, reduce stress resistance, and increase the sensitivity of the plant to other diseases and pests. In addition, since the sclerotia of Sclerotinia sclerotiorum can survive in the soil for many years, once Sclerotinia sclerotiorum occurs in a certain area, disease prevention and control will become extremely difficult.

[0003] Sclerotinia sclerotiorum, caused by Sclerotinia sclerotiorum, poses a major threat to a variety of crops, and the resistance of this pathogen makes chemical control increasingly difficult. Sclerotinia sclerotiorum can quickly adapt to pesticide pressure and evolve new resistance mechanisms, which leads to a decrease in the effectiveness of traditional chemical control methods, an increase in pesticide use, and increased risks to the environment and human health. Therefore, exploring and utilizing the plant's own 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. Due to its small genome, short life cycle and easy genetic manipulation, it has become an ideal object for studying plant physiological functions and disease resistance mechanisms. Arabidopsis is particularly important in the field of plant pathology. Scientists use it as a model system to discover and study disease resistance genes. These disease resistance gene studies have broad application prospects, especially in the discovery and utilization of homologous genes in other economic crops. Disease resistance genes found in Arabidopsis often have corresponding homologous genes in crops, which provides an important basis for cross-species disease resistance research and improvement.

[0005] Ubiquitination plays a crucial role in plant-microbe interactions. It is a core protein post-translational modification mechanism in plant cells to regulate plant responses to microorganisms. Through the ubiquitination process, plants can mark specific proteins for degradation or change their functions, thereby quickly adjusting their own physiological and defense states to cope with the invasion of foreign microorganisms. Deubiquitination is a process that reverses ubiquitination modification, mainly achieved by deubiquitinating enzymes (DUBs), which can cut and remove ubiquitin molecules attached to proteins. This process plays a key regulatory role in many functions of cells, including protein stability, cell signaling, and gene expression regulation. 64 deubiquitinating enzymes have been found in Arabidopsis, of which ubiquitin-specific proteases (USP / UBP) are the largest subfamily with 27 members. At present, research on deubiquitinating enzymes mainly focuses on the field of plant physiology, while in the field of plant pathology, little is known about the pathways and mechanisms of how they regulate plant immunity. Summary of the invention

[0006] The purpose of the present invention is to provide a gene for regulating plant immunity and a protein encoded by the gene.

[0007] The invention provides application of an Arabidopsis thaliana deubiquitinase AtUBP6 gene in improving the resistance of Arabidopsis thaliana to sclerotinia sclerotiorum. The ID number of the gene in NCBI is At1g51710.

[0008] The nucleotide sequence of the Arabidopsis thaliana deubiquitinase AtUBP6 gene is shown in SEQ ID NO:1.

[0009] The present invention provides an application of an Arabidopsis thaliana deubiquitinase AtUBP6 gene in improving the resistance of Arabidopsis thaliana to sclerotinia sclerotinia. The Arabidopsis thaliana AtUBP6 transcription factor has an amino acid sequence shown in Ⅰ) or Ⅱ):

[0010] 1) the amino acid sequence shown in SEQ ID NO: 2;

[0011] II) A protein with the same function obtained by replacing, deleting and / or adding one or more amino acids based on the amino acid sequence.

[0012] The present invention provides the use of the above-mentioned Arabidopsis thaliana AtUBP6 or its corresponding biological material in plant disease resistance, specifically:

[0013] Knocking out the AtUBP6 gene in plants increased the susceptibility of plants to sclerotinia disease.

[0014] The immune regulatory gene AtUBP6 from Arabidopsis was found to be upregulated when infected by Sclerotinia sclerotiorum through fluorescent real-time quantitative PCR detection; compared with the wild type, the T-DNA insertion mutation of AtUBP6 resulted in a larger lesion area on the leaves of the mutant Arabidopsis thaliana when infected by Sclerotinia sclerotiorum; and the expression of defense-related genes in the AtUBP6 mutant plants was significantly reduced before and after pathogen infection, thereby weakening the plant's immunity.

[0015] The present invention proves that the deletion of the gene can significantly reduce the immunity of plants, indicating that the AtUBP6 gene plays an important role in regulating plant immunity. Therefore, by screening compounds that can enhance the expression of the gene, or cultivating varieties with overexpression of the gene, sclerotinia can be effectively prevented and treated, that is, an important use provided by the present invention is that the gene and its homologous genes in other cruciferous plants can be used as key targets for enhancing plant immunity, providing new resources for green integrated prevention and control of plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of the AtUBP6 overexpression vector;

[0017] Figure 2 This is the subcellular localization map of the AtUBP6 gene;

[0018] Figure 3 The agarose gel electrophoresis diagram of the T-DNA insertion mutant SALK_206973C;

[0019] Figure 4 The photos and lesion statistics of Arabidopsis thaliana plants infected with S. sclerotiorum and the AtUBP6 T-DNA insertion mutant SALK_206973C and wild type were shown;

[0020] Figure 5 The quantitative analysis graph of AtUBP6 at 6h (6hpi), 12h (12hpi), 18h (18hpi) and 24h (24hpi) after Sclerotinia sclerotiorum infection of wild-type Arabidopsis thaliana;

[0021] Figure 6 Figure 2 shows the fluorescence quantitative analysis of the main defense genes in wild-type Arabidopsis thaliana and AtUBP6 T-DNA insertion mutant plants at 0h (0hpi) and 24h (24hpi) infected 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 of the present invention is derived from Arabidopsis thaliana, named AtUBP6, and the gene number in the NCBI database is At1g51710. Its amino acid sequence is shown in SEQ ID NO: 2, and the corresponding coding nucleotide sequence (CDS) is shown in SEQ ID NO: 1. The gene name and gene ID are both taken from the Arabidopsis sequencing database (www.arabidopsis.org), specifically taken 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: S15#522, China Resources MixC (Phase III), No. 52, Hongshanyuan Road, Hongshan Town, Gulou District, Fuzhou;

[0025] Website: https: / / www.arashare.cn / index / Product / index

[0026] Mutant ID: SALK_206973C

[0027] Example 1: Construction of AtUBP6 overexpression vector

[0028] 1. Double restriction digestion of pCAMBIA1301-GFP vector

[0029] The pCAMBIA1301-GFP vector was double-digested with restriction endonucleases KpnI and SacI, and the digested products were separated by agarose gel electrophoresis, and the corresponding high molecular weight bands were cut out and gel recovered;

[0030] 2. Preparation of Arabidopsis thaliana total cDNA

[0031] (1) 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 in tin foil in advance, baked in a 180℃ constant temperature oven for 3 hours to cool to room temperature, and finally transferred to a -20℃ freezer for precooling. 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. Take 400μl of supernatant and transfer it to a new centrifuge tube. Add an equal volume of anhydrous ethanol and mix thoroughly. Then transfer it to the adsorption column provided with the kit and centrifuge it at 12,000g for 30 seconds at room temperature. After discarding the waste liquid in the collection tube, add 500μl of CB solution to the adsorption column, centrifuge it at 12,000g for 30 seconds again and discard the waste liquid (repeat the operation twice). Subsequently, add 500μl of WB solution containing anhydrous ethanol to the adsorption column, centrifuge it at 12,000g for 30 seconds and discard the waste liquid (repeat the operation twice), and centrifuge it again at 12,000g for 1 minute to remove residual ethanol. Finally, elute RNA with 50μl ddH2O and detect the RNA concentration for later use.

[0032] (2) Preparation of cDNA: cDNA was synthesized using the TOLOBIO reverse transcription kit. The reaction system was: 4 μl of 5× All-in-one RT Buffer, 1 μl of All-in-one Enzyme Mix, 1 μl of template (RNA), 14 μl of RNase-free ddH2O, a total of 20 μl system. Reaction conditions: 50°C for 15 minutes, 85°C for 5 seconds. After the product concentration was tested, it was stored at -20°C for future use.

[0033] 3. Design specific primers (primer sequences are shown in Table 1)

[0034] The CDS sequence of AtUBP6 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.

[0035] 4. Homologous recombination ligation

[0036] The linearized vector and the AtUBP6 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.

[0037] 5. Transformation of E. coli

[0038] The above recombinant reaction solution was transferred into DH5α competent cells by heat shock method and cultured overnight at 37℃ in a medium containing kanamycin. After picking a single colony and placing it in LB liquid medium containing kanamycin for shaking culture 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 As shown, the verified bacterial solution was stored at -80°C for future use.

[0039] Table 1 Primers used for construction of heterologous expression vectors

[0040]

[0041] Example 2 Transient expression of AtUBP6 in tobacco leaves using a recombinant vector

[0042] 1. Transformation of recombinant vector into Agrobacterium GV3101

[0043] 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.

[0044] 2. Tobacco transient expression

[0045] Take 20 μl of the glycerol bacteria stored at -80℃, inoculate it in YEP containing the corresponding antibiotics, and culture it at 28℃ and 200 r / min for 18 hours; then take 50 μl of the activated bacterial solution and add it to 12.5 mL of YEP containing the corresponding antibiotics, and culture it at 28℃ and 200 r / min until the OD 600 About 0.8; centrifuge at 5,000r / min for 10 minutes at room temperature to recover the cells. Prepare the infiltration buffer: add 98mL ddH2O, 1mL MES, 1mL MgCl2, and 100μl AS to every 100mL buffer. Resuspend the cells in the buffer to a final OD 600 When the concentration reaches 0.6-0.8, place it 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 to the greenhouse for cultivation, and subsequent experiments can be carried out after 48 hours.

[0046] Example 3 Subcellular localization analysis of AtUBP6 based on recombinant vector and transient expression in tobacco

[0047] According to the methods described in Examples 1 and 2, the target gene was fused with the coding region of the GFP reporter gene; the vector containing the exogenous target gene was introduced into tobacco leaves using Agrobacterium and transient expression was achieved. Subsequently, the fluorescence signal in the epidermal cells of the leaves was observed using a laser confocal microscope. The results are shown in FIG. Figure 2 As shown, the signals of AtUBP6 and GFP fusion protein can be detected in the nucleus and cytoplasm of tobacco leaf epidermal cells.

[0048] Example 4 Screening of T-DNA insertion mutant materials

[0049] 1. CTAB method to extract Arabidopsis DNA

[0050] 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, remove the residual liquid and add 1mL 75% ethanol, invert and mix, centrifuge at 12,000r / min for 5 minutes. Discard the supernatant and remove 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℃.

[0051] 2. Primer design for PCR amplification using the three-primer method

[0052] According to the SALK_206973C insertion site information provided by http: / / signal.salk.edu / , the LP and RP primer sequences obtained by query are shown in Table 2; in addition, a BP primer corresponding to the T-DNA insertion sequence was designed, and LBb1.3 was selected as the BP primer of the gene, and the nucleotide sequence is shown in Table 2.

[0053] Table 2 Primers used for screening of T-DNA insertion mutant materials

[0054]

[0055] 3. PCR reaction system and procedure

[0056] The PCR reaction system was: primer LP or BP 0.5 μl; primer RP 0.5 μl; DNA template 1 μl; ddH2O 3 μl; 2xTransFast Taq PCR SuperMix (+dye) 5 μl. The reaction program was: 94°C pre-denaturation for 3 minutes; followed by 35 cycles, each cycle including 94°C for 5 seconds, 56°C for 15 seconds, 72°C (6 kb / min); and finally 72°C extension for 5 minutes.

[0057] 4. Agarose gel electrophoresis

[0058] The above PCR products were separated by 1% agarose gel electrophoresis. If no band was detected by the LP+RP combination, and a single band was detected by the BP+RP combination, it was determined to be homozygous material for the SALK_206973C mutant. Homozygous seeds were collected for subsequent experiments.

[0059] Example 5 Observation of the incidence and phenotype of Sclerotinia sclerotiorum infection

[0060] 1. Activation of Sclerotinia sclerotiorum

[0061] Take the Sclerotinia sclerotiorum preserved on the slant culture medium, pick a small amount of the strain and place it in the center of the PDA plate without antibiotics for activation. When the mycelium grows close to the edge of the plate, cut a bacterial cake (cylindrical PDA culture medium) with a diameter of about 0.5 cm from the front area of ​​mycelium growth and transfer it to a new PDA plate. Cultivate again until the mycelium is about to fill the plate, use a 0.5 cm aperture puncher to drill the bacterial cake at the edge of mycelium growth for later use.

[0062] 2. Sclerotinia sclerotiorum infection of AtUBP6 mutants and wild-type Arabidopsis

[0063] The seeds of the T-DNA insertion mutant SALK_206973C and wild-type Arabidopsis thaliana with clear phenotype and homozygous genotype detected by three-primer PCR were sown at the same time, and after growing two true leaves, they were transplanted to the greenhouse for further cultivation for 3 to 4 weeks. For the infection test, water was first sprayed on the surface of Arabidopsis thaliana leaves to increase the humidity; then the above-mentioned activated Sclerotinia sclerotiorum was prepared into a 0.5 cm pore cake, and 3 mutants with strong growth and 3 wild-type Arabidopsis thaliana were selected, and 3 leaves of each plant were selected for inoculation. The specific operation was: the cake (with the hyphae side) was turned upside down on the leaf surface facing the back of the leaf, and then the culture dish cover was covered and the humidity was maintained. After 24 hours, the occurrence of leaf lesions was observed and photographed, and the lesion area was determined by ImageJ software, and the difference in lesion area among the treatment groups was statistically analyzed by SPSS software.

[0064] The results are as follows Figure 4 The results showed that compared with the wild-type Arabidopsis control, the mutant SALK_206973C exhibited a significantly reduced phenotype in resistance to Sclerotinia sclerotiorum; the statistical analysis results using Image J and SPSS showed that the lesion area on the leaves of the SALK_206973C Arabidopsis line was significantly larger than that of the wild-type Arabidopsis.

[0065] Example 6 Real-time fluorescence quantitative PCR

[0066] 1. Real-time fluorescence quantitative PCR analysis of changes in AtUBP6 expression before and after pathogen infection

[0067] According to the scheme of Example 5, the activated Sclerotinia sclerotiorum was inoculated into the leaves of wild-type Arabidopsis, and samples were taken at 0h, 6h, 12h, 18h and 24h of infection. When sampling, the bacterial cake was removed, the leaves were wrapped with tin foil, quick-frozen in liquid nitrogen and stored at -80°C. After all the samples were collected, the total RNA was extracted and reverse transcribed to synthesize cDNA. The expression changes of AtUBP6 at different time points were detected by qPCR. The primers used for qPCR were designed by Primer Premier 6 (as shown in Table 3); the reaction system was 20μl, including: 2×Q3 SYBR qPCR Master Mix 10μl, 0.4μl each of forward and reverse primers, 1μl template, 8.6μl ddH2O; the amplification program was 95°C pre-denaturation for 30 seconds, followed by 40 cycles (95°C for 10 seconds, 60°C for 45 seconds). AtActin2 was used as the internal reference gene, and the melting curve analysis confirmed the amplification specificity. The relative expression of the gene was based on 2 -ΔΔCt Algorithm calculation. Three biological replicates were set during sampling to ensure the reliability of statistical results.

[0068] Figure 5 The results showed that the AtUBP6 gene was differentially expressed 6 hours after Sclerotinia infection and reached the maximum upregulation at 18 hours; although the expression level declined at 24 hours, it was still higher than the levels at 0 hours and 6 hours.

[0069] 2. Real-time fluorescence quantitative PCR analysis of the expression changes of major defense genes and photosynthetic genes in wild-type and mutant Arabidopsis before and after pathogen infection

[0070] Referring to the operation of Example 5, Sclerotinia sclerotiorum and Botrytis cinerea were inoculated into mutant and wild-type Arabidopsis leaves, respectively, and samples were taken at 0h and 24h of infection. After removing the bacterial cake, the samples were wrapped in tin foil, quickly frozen in liquid nitrogen, and stored at -80°C. After all the samples were collected, total RNA was extracted and reverse transcribed to synthesize cDNA, and the expression level of each gene was detected by qPCR (primers are shown in Table 3).

[0071] Figure 6 The results showed that the expression levels of defense genes PR1, PR2, PR4, PAD3, EDS5, FRK1, and ICS1 in the mutant were significantly lower than those in the wild-type Arabidopsis thaliana 24 hours after infection with Sclerotinia sclerotiorum. This result indicates that after losing the function of the AtUBP6 gene, the immune response ability of Arabidopsis thaliana to pathogens is significantly weakened, and insufficient expression of defense genes makes it more susceptible to pathogen infection.

[0072] Table 3 Primers used for qRT-PCR analysis of Arabidopsis plants

[0073]

[0074]

Claims

1. An application of the Arabidopsis thaliana AtUBP6 gene in improving plant resistance, specifically: when Sclerotinia sclerotiorum infects Arabidopsis thaliana, the AtUBP6 gene is upregulated; the AtUBP6 gene deletion can increase the area of ​​lesions infected by Sclerotinia sclerotiorum and reduce the expression of plant-related defense genes, and the nucleotide sequence of the AtUBP6 gene is shown in SEQ ID NO: 1.

Images