Use of pub gene and its encoded protein in regulating plant disease resistance and / or breeding disease-resistant plants

Abstract

This invention belongs to the field of agricultural genetic engineering technology, specifically relating to the application of the PUB gene and its encoded protein in regulating plant disease resistance and / or cultivating disease-resistant plants. The PUB25 and / or PUB26 genes provided by this invention are closely related to clubroot disease in cruciferous plants. Therefore, knocking out the PUB25 and / or PUB26 genes in plants not only improves plant resistance to clubroot disease but also allows for the cultivation of clubroot-resistant plants, providing scientific genetic resources for the effective control of clubroot disease.

Description

Technical Field

[0001] This invention belongs to the field of agricultural genetic engineering technology, specifically relating to the application of the PUB gene and its encoded protein in regulating plant disease resistance and / or cultivating disease-resistant plants. Background Technology

[0002] Clubroot is an important plant disease that primarily affects the roots of plants, causing tumors of varying sizes to form. This disease has been reported in over 80 countries worldwide (Javed MA, Schwelm A, Zamani-Noor N, Salih R, Silvestre). M, Wu J, González García M, Heick TM, Luo C, Prakash P, Pérez-López E. The clubroot pathogen Plasmodiophora brassicae: A profile update. Mol Plant Patholog, 2023, 24: 89-106.), The pathogen of clubroot disease is Plasmodiophora brassicae, which is an obligate parasite of cruciferous plants such as rapeseed, cabbage, radish, cauliflower and mustard, seriously affecting the yield and quality of the plants.

[0003] Existing research often focuses on controlling clubroot disease in plants through the application of chemical fertilizers. For example, Chinese patent CN105900749A describes a method for controlling clubroot disease by providing chemical agents in conjunction with crop rotation. However, this method is not only environmentally unfriendly but also time-consuming and labor-intensive. Furthermore, although existing research has reported on E3 ligases PUB25 and PUB26, their relationship with clubroot disease remains unreported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the PUB gene and its encoded protein in regulating plant disease resistance and / or cultivating disease-resistant plants, which can not only improve the plant's resistance to clubroot disease, but also cultivate plants resistant to clubroot disease.

[0005] This invention provides the application of the PUB gene and the PUB protein encoded by the PUB gene in regulating plant disease resistance and / or cultivating disease-resistant plants; the PUB gene includes the PUB25 gene and / or the PUB26 gene; the disease resistance includes resistance to clubroot disease.

[0006] Preferably, the regulation includes: enhancing plant disease resistance by inhibiting the expression of the PUB gene or reducing the content of PUB protein.

[0007] Preferably, the biomaterials used for the regulation include biomaterials that inhibit the PUB gene.

[0008] Preferably, the biomaterial that inhibits the PUB gene includes: biomaterial that inhibits the PUB25 gene and / or biomaterial that inhibits the PUB26 gene.

[0009] Preferably, the plant includes plants of the Brassicaceae family.

[0010] Preferably, the cruciferous plants include one or more of Arabidopsis thaliana, rapeseed, Chinese cabbage, radish, cauliflower, and mustard.

[0011] This invention provides a method for regulating plant disease resistance and / or cultivating disease-resistant plants, comprising the following steps: enhancing the disease resistance of the target plant or cultivating disease-resistant plants by knocking out the PUB gene in the target plant.

[0012] Preferably, the plant includes Arabidopsis thaliana.

[0013] Beneficial effects:

[0014] This invention provides the application of the PUB gene and the PUB protein encoded by the PUB gene in regulating plant disease resistance and / or cultivating disease-resistant plants; the PUB gene includes the PUB25 gene and / or the PUB26 gene; the disease resistance includes resistance to clubroot disease; the PUB25 gene and / or the PUB26 gene are closely related to clubroot disease in plants. Therefore, this invention, by knocking out the PUB25 gene and / or the PUB26 gene in plants, is beneficial to improving the plant's resistance to clubroot disease.

[0015] Based on the aforementioned technical advantages, this invention also provides a method for regulating plant disease resistance and / or cultivating disease-resistant plants. By knocking out the expression of the PUB gene in the target plant, the plant's disease resistance can be improved, and plants with good disease resistance can be cultivated. Experiments have shown that, compared with wild-type Arabidopsis, the disease index of clubroot disease in Arabidopsis decreased by 37.2% to 50.0% after adopting the technical solution provided by this invention. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The phenotype of the Col-0 and pub mutant plants in Example 1 after inoculation with Plasmodium falciparum for 21 days;

[0018] Figure 2 The disease index of the Col-0 and pub mutant plants 21 days after inoculation with clubroot bacteria in Example 1;

[0019] Figure 3 The content of clubroot bacteria in the diseased roots of Arabidopsis thaliana in Example 1. Detailed Implementation

[0020] This invention provides the application of the PUB gene and the PUB protein encoded by the PUB gene in regulating plant disease resistance and / or cultivating disease-resistant plants; the PUB gene includes the PUB25 gene and / or the PUB26 gene, preferably the PUB25 gene and the PUB26 gene; the disease resistance includes resistance to clubroot disease.

[0021]

[0022] The amino acid sequence of the PUB25 protein encoded by the PUB25 gene described in this invention is shown in SEQ ID NO.2, and is: MPRNIEPLDLGIQIPYHFRCPISLELMQDPVTVCTGQTYDRASIESW VSIGNNTTCPVTRAPLSDDFTLIPNHTLRRLIQEWCVANRSNGVERIPTPKQPADPTSVRALLSQASAITGTHVSVRSRAAALRRLRGFARDSDKNRVLIAAHNATEILIKILFSETTSSELVSESLALLVMLPITEPNQFVSISSDPGRVEFLTRLLFDSSIETRVNAAALIEIVSTGTKSADLKGSI SNSESVFEGVLDLLRNPISSRRALKIGIKTLFALCSVKSTRHIAITAGAPEILIDRLAADFDRCDTERALATVELLCRTPEGCAAFGEHALTVPLLVKTILRVSDRATEYAAGALLALCTAEERWREEAAGAGVVVQLLLMVQSECTERAKKKAQKLLKLLRDSWPDYNSFANSDDFGCSSQVVPF*.

[0023]

[0024] The amino acid sequence of the PUB26 protein encoded by the PUB26 gene described in this invention is shown in SEQ ID NO.4, and is: MPGNLEPLDLGIQIPYHFRCPISLDLMSDPVTISTGQTYDRTSIDSWI AMGNTTCPVTRVALSDFTLIPNHTLRRLIQEWCVANRSNGVERIPTPKQPADPISVRSLLSQASAITGTHVSVRSRAAAIRRLRGLARDSEKNRVLIAGHNAREILVRILFADIETTSLSSELVSESLALLVLLHMTETECEAVASDPSRVGFMTRLLFDSSIEIRVNAAALIEMVLTGAKSMDLKLIISGSDSIFEGVLDLLKNPISSRRALKIGIKAIFALCLVKQTRHLAISAGAPGILIDRLAADFDRCDTERGLATVELLCRLPEGCAAFGEHALTVPLMVKTILRVSDRATEYAAGALLALCTAEERCRDEAAAAGLVTQLLLLVQSDCTERAKRKAQMLLKLLRDSWPDDSTVHSDDFNRSEVAPF*; The above-mentioned PUB25 gene, PUB26 gene, PUB25 protein, and PUB26 protein are all associated with clubroot disease in plants.

[0025] The regulation described in this invention preferably includes: enhancing plant disease resistance by inhibiting the expression of the PUB gene or reducing the content of PUB protein; the biological material used for the regulation preferably includes biological material that inhibits the PUB gene; the biological material that inhibits the PUB gene preferably includes: biological material that inhibits the PUB25 gene and / or biological material that inhibits the PUB26 gene, more preferably including biological material that inhibits the PUB26 gene; the preparation method of the biological material that inhibits the PUB gene has no special requirements and can use techniques well known in the art.

[0026] The plants described in this invention preferably include cruciferous plants; the cruciferous plants preferably include one or more of Arabidopsis thaliana, rapeseed, Chinese cabbage, radish, cauliflower and mustard greens, more preferably Arabidopsis thaliana or rapeseed, and more preferably Arabidopsis thaliana.

[0027] This invention provides a method for regulating plant disease resistance and / or cultivating disease-resistant plants, comprising the following steps: enhancing the disease resistance of the target plant or cultivating disease-resistant plants by knocking out the PUB gene in the target plant; the plant preferably includes Arabidopsis thaliana; the knockout method has no special requirements and can be any technique well known in the art.

[0028] Experiments have shown that, compared with wild-type Arabidopsis thaliana, the disease index of the pub25 mutant (with only the PUB25 gene knocked out) decreased by 37.2% after adopting the technical solution provided by this invention; the disease index of the pub26 mutant (with only the PUB26 gene knocked out) decreased by 50.0%; and the disease index of the pub25 / 26 double mutant (with both PUB25 and PUB26 genes knocked out) decreased by 39.5%. Therefore, this invention lays a scientific foundation for improving plant resistance to clubroot disease and cultivating clubroot-resistant plants.

[0029] To further illustrate the present invention, the application of the PUB gene and its encoded protein provided by the present invention in regulating plant disease resistance and / or cultivating disease-resistant plants is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Preparation before the experiment:

[0031] 1. Plant material: The Arabidopsis thaliana ecotype (Col-0) was preserved in our laboratory and is designated as Col-0;

[0032] The Arabidopsis PUB25 T-DNA insertion mutant pub25 (mutant number: SALK_147032), i.e. the plant with the PUB25 gene knocked out, is denoted as pub25;

[0033] The Arabidopsis PUB26 T-DNA insertion mutant pub26 (mutant number: GABI_308D07), i.e. the plant with the PUB26 gene knocked out, is denoted as pub26;

[0034] The Arabidopsis thaliana double mutant pub25 / 26, i.e., the plant in which both the PUB25 and PUB26 genes are knocked out, is denoted as pub25 / 26.

[0035] The methods for constructing pub25, pub26, and pub25 / 26 have been disclosed in the literature (Wang J, Grubb LE, Wang J, et al. A regulatory module controlling homeostasis of a plant immune kinase. Mol Cell, 2018, 69:493-504.), and the specific plants were donated by Researcher Zhou Jianmin from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0036] 2. Extraction of dormant spores of *Plasmodiophora stylosa*: Chop 10g of clubroot tissue, add 50mL of sterile water, and grind finely in a mortar or juicer; then filter through 4 layers of gauze, transfer the filtrate to a clean 50mL centrifuge tube, centrifuge at 3100r / min for 15min, and discard the supernatant; redissolve the precipitate in 50mL of sterile water, centrifuge at 3100r / min for 10min, this step can be repeated 3 times, discard the supernatant; add to the precipitate... Mix 5 mL of 50% sucrose solution thoroughly and centrifuge at 3100 rpm for 10 min. Carefully transfer the supernatant to a clean centrifuge tube using a pipette, add 30 mL of sterile water, centrifuge at 3100 rpm for 10 min, and discard the supernatant. Redissolve the precipitate in 30 mL of sterile water and centrifuge at 3100 rpm for 10 min. This step can be repeated 3 times. Dissolve the precipitate in 5 mL of sterile water and store at 4°C in the dark for later use.

[0037] 3. Inoculation with *Plasmodiophora stylosa*: The above-mentioned *Arabidopsis thaliana* plants (38 Col-0 plants, 37 pub25 plants, 39 pub26 plants, and 39 pub25 / 26 plants) were cultured for approximately 2 weeks (14 days). The culture conditions were: 22℃, 12h light and 12h darkness, and 60% relative humidity. 1 mL of a 1×10⁻⁶ solution was pipetted into the solution. 6 Or 1×10 7 Inoculate the plant roots with dormant spores of *Plasmodiophora* at a concentration of 1 spore / mL, and count the disease index 21 days after inoculation.

[0038] 4. Statistical Standards for Clubroot Disease Severity Index: Reference (Li C, Luo S, Feng L, Wang Q, Cheng J, Xie J, Lin Y, Fu Y, Jiang D, Chen T. Protistubiquitin ligase effector PbE3-2 targets cysteine ​​protease RD21A to impede plant immunity. Plant) Physiol.2024Feb29;194(3):1764-1778.doi:10.1093 / plphys / kiad603.PMID:38035763.) The statistical standard of clubroot disease severity index is based on a five-level grading standard: Level 0, no disease; Level 1, a very small number of tumors on the lateral roots, with negligible damage to the main root; Level 2, the main root and a few lateral roots are covered with small tumors; Level 3, the main root has medium to large spherical tumors; Level 4, the lateral roots and main root have severe tumors, the main root is completely swollen, and the fine roots on the lateral roots are completely damaged; Level 5, the roots are completely swollen and rotten.

[0039] 5. Total DNA extraction of clubroot bacteria and detection of relative biomass of clubroot bacteria in diseased roots were performed according to the method in the reference (Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc. 2006;1(5):2320-5. doi:10.1038 / nprot.2006.384.PMID:17406474.).

[0040] Total DNA extraction from clubroot (CTAB method): Clean the diseased roots of clubroot, cut them into small pieces, place them in a clean mortar, flash-freeze in liquid nitrogen, grind thoroughly, and transfer to a 2 mL centrifuge tube. Add 800 μL of 2% CTAB extraction buffer preheated to 65°C, quickly invert to mix, and incubate at 65°C for 15–30 min, gently inverting to mix every 5 min. Add 400 μL of chloroform and Tris-saturated phenol solution to the mixture, centrifuge at 12000 rpm for 15 min, and transfer 500 μL of the supernatant. Transfer the extract to another 2 mL centrifuge tube, add an equal volume of chloroform, centrifuge at 12000 rpm for 15 min, take 450 μL of supernatant, add an equal volume of isopropanol, mix well, and precipitate at -20℃ for 10-15 min; centrifuge at 12000 rpm for 15 min, discard the supernatant and keep the precipitate, wash the precipitate twice with 1 mL of 75% ethanol; dry in a 37℃ oven, dissolve the DNA in 30 μL of deionized water (containing 25 μg / mL RNase A), measure the DNA concentration, and store at -20℃.

[0041] Example 1: Detection of relative biomass of clubroot bacteria in diseased roots:

[0042] Arabidopsis thaliana was inoculated according to the above-described method for inoculating with *Plasmodiophora stylosa* (38 *Col-0* plants, 37 *pub25* plants, 39 *pub26* plants, and 39 *pub25 / 26* plants), with each plant constituting one treatment and 37–39 biological replicates per treatment. Plant growth was observed 21 days post-inoculation, and the results are shown in Table 1. Figure 1 and Figure 2 ( Figure 1 This indicates the phenotype of Col-0 and pub mutant plants 21 days after inoculation with *Plasmodium*. Figure 2 The disease index represents the disease severity of Col-0 and pub mutant plants 21 days after inoculation with *Cladophora*; the figure shows the percentage of plants at each disease severity level.

[0043] Table 1 Disease index under different treatments

[0044] deal with Disease index Col-0 86 pub25 54 pub26 43 pub25 / 26 52

[0045] From Table 1, Figure 1 and Figure 2 It can be seen that, compared with the control Col-0, the pub25, pub26, and pub25 / 26 mutants showed significantly enhanced resistance to clubroot. The aboveground parts of the Col-0 plants inoculated with clubroot turned yellow and wilted, and the underground parts formed obvious tumors, with the lateral roots almost completely destroyed. In contrast, the aboveground parts of the pub mutants were almost unaffected, and only the main root or lateral roots showed slight swelling in the underground parts. In terms of disease index, compared with the disease index of 86 for the control Col-0, the disease index of the pub25, pub26, and pub25 / 26 mutants was significantly reduced to 54, 43, and 52, respectively. The proportion of plants with disease grades 4 and 5 in Col-0 was 86.8%, while that of the pub25, pub26, and pub25 / 26 mutants was 29.7%, 15.4%, and 28.2%, respectively.

[0046] Roots of diseased Arabidopsis thaliana were used as experimental materials. Three diseased roots were taken from each plant (family) as one sample. Total DNA was extracted from the diseased roots of Arabidopsis thaliana with clubroot disease using the CTAB method. The DNA concentration was determined using Nanodrop, and the total DNA concentration of each treatment was diluted to 100–200 ng / μL. Using the diluted DNA as a template, the content of the clubroot bacteria internal reference gene relative to the Arabidopsis thaliana internal reference gene was detected by qPCR. The relative biomass of clubroot bacteria was expressed as the content of the clubroot bacteria ACTIN gene relative to the Arabidopsis thaliana ACTIN2 gene. The results are shown in Table 2 and [Table data missing]. Figure 3 ( Figure 3 The ordinate represents the content of clubroot bacteria in diseased roots of Arabidopsis thaliana, with the vertical axis representing the multiple of clubroot bacteria content in different strains relative to Col-0. The relative biomass of clubroot bacteria is expressed as the content of the clubroot bacteria ACTIN gene relative to the Arabidopsis thaliana ACTIN2 gene. Data are expressed as mean ± standard deviation (SD). One-way ANOVA was used (significance was set at P ≤ 0.05). Different letters indicate significant differences.

[0047] The ACTIN gene has an accession number of AY452179.1 on NCBI; the upstream primer for amplifying the ACTIN gene is Pbactin_qF: 5'-CACCGACTACCTGATGAA-3' (SEQ ID NO.5), and the downstream primer for amplifying the ACTIN gene is Pbactin_qR: 5'-CAGCTTCTCCTTGATGTC-3' (SEQ ID NO.6);

[0048] The accession number of the Arabidopsis thaliana ACTIN2 gene on NCBI is AT3G18780; the upstream primer for amplifying the ACTIN2 gene is: ACTIN2_qF: 5'-GCACCCTGTTCTTCTTACGGA-3' (SEQ ID NO.7), and the downstream primer for amplifying the ACTIN2 gene is: ACTIN2_qR: 5'-GTGAGACACACCATCACCAGA-3' (SEQ ID NO.8).

[0049] Table 2. Relative content of clubroot bacteria in different treatments

[0050] deal with Relative content of clubroot bacteria Col-0 103.15 pub25 7.12 pub26 7.13 pub25 / 26 1.00

[0051] From Table 2 and Figure 3 It can be seen that the biomass of *Pub* in the diseased roots of the pub mutant was significantly lower than that in the Col-0. Inoculation with *Pub* revealed increased resistance of the pub mutant to *Pub*, indicating that PUB25 / 26 negatively regulates the resistance of *Arabidopsis thaliana* to *Pub*.

[0052] In summary, the single mutants pub25 and pub26, and the double mutant pub25 / 26 showed higher resistance to clubroot fungus than the wild type. The disease index of mutant pub25 decreased by 37.2% compared to the control, the disease index of mutant pub26 decreased by 50.0% compared to the control, and the disease index of the pub25 / 26 double mutant decreased by 39.5% compared to the control. Furthermore, the content of clubroot fungus in the diseased roots was significantly reduced.

[0053] Therefore, E3 ubiquitin ligases PUB25 and PUB26 can serve as gene resources for cultivating clubroot resistance. The ability of plants to resist clubroot can be improved by knocking out the PUB25 gene or the PUB26 gene in Arabidopsis thaliana or cruciferous hosts, or by knocking out both the PUB25 gene and the PUB26 gene simultaneously, through gene editing.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of the PUB gene and the PUB protein encoded by the PUB gene in regulating plant disease resistance and / or cultivating disease-resistant plants, characterized in that, By knocking out the PUB gene in the target plant, the disease resistance of the target plant can be enhanced or disease-resistant plants can be cultivated. The PUB gene is the PUB25 gene and / or the PUB26 gene; The disease resistance mentioned is resistance to clubroot disease; The plant in question is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The regulation includes: enhancing plant disease resistance by inhibiting the expression of the PUB gene or reducing the content of PUB protein.

3. The application according to claim 1 or 2, characterized in that, The biological materials used for this regulation include those that suppress the PUB gene.

4. A method for regulating plant disease resistance and / or cultivating disease-resistant plants, characterized in that, Includes the following steps: By knocking out the PUB gene in the target plant, the disease resistance of the target plant can be enhanced or disease-resistant plants can be cultivated. The PUB gene is the PUB25 gene and / or the PUB26 gene; The disease resistance mentioned is resistance to clubroot disease; The plant in question is Arabidopsis thaliana.

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