CN116286870B - Arabidopsis NPR1 mutant gene and application of protein thereof in inhibiting turnip mosaic virus - Google Patents
Arabidopsis NPR1 mutant gene and application of protein thereof in inhibiting turnip mosaic virus Download PDFInfo
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Abstract
The invention discloses an arabidopsis NPR1 mutant gene and application of protein thereof in inhibiting turnip mosaic virus, and belongs to the technical field of plant genetic engineering. The invention provides an NPR1 mutant gene and a coding protein thereof, wherein the NPR1 mutant gene is obtained by taking an NPR1 gene sequence shown as SEQ ID NO.21 as a starting sequence and mutating 31 st-33 rd, 43 rd-45 th and 1033 th-1043 th positions. The invention also discloses a protein encoded by the NPR1 mutant gene, wherein the amino acid sequence of the NPR1 mutant protein has the following mutations: which corresponds to the mutation of amino acids 11, 15 and 345-348 of the amino acid sequence of the wild-type Arabidopsis NPR1. The NPR1 mutant gene is expressed in Arabidopsis thaliana of cruciferae by a transgenic method, so that the resistance to turnip mosaic virus can be obtained.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to an arabidopsis NPR1 mutant gene and application of protein thereof in inhibiting turnip mosaic virus.
Background
Plant viruses are one of the main pathogens causing plant diseases and can cause huge economic losses in agriculture. Turnip mosaic virus (turnip mosaic virus, tuMV) is a potato virus belonging to the family Y virusPotyviridae) Genus potyvirusPotyvirus) Is also the only important member of the genus capable of infecting brassicaBrassicaL.) virus seed of the plant. TuMV host RangeWidely, it can infect at least 43 plants of 156 genus 310 including brassica crucifers such as Chinese cabbage, cabbage and rape (described in WALSH JA, JENNER CE. Turnip mosaic virus and the quest for durable resistance, molecular Plant Pathology, 2022, 3:289-300.). In asia, north america and parts of europe, vegetables are most severely damaged by TuMV, the second most common vegetable virus disease. The virus is distributed in a plurality of areas such as Hebei, sichuan, shandong, shanxi and Fujian of China, and is harmful to a plurality of vegetables such as Chinese cabbage, radish, cabbage, rape, chilli, pseudostellaria root and the like, and the serious areas of epidemic year disease are almost produced, so that huge economic losses are caused (recorded in Liu Genxin, worn by a person, xu Yuanyuan and the like. The current research situation of turnip anti-turnip mosaic virus is that of Yangtze vegetables, 2016, 406 (08): 31-34.). Therefore, the excavation and creation of broad-spectrum disease-resistant germplasm resources is critical to the development of the agricultural industry.
The protein encoded by the NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1) gene is a receptor for salicylic acid in plants, plays an important role in maintaining basal resistance and establishing systemic acquired resistance (Systemic acquired resistance, SAR) in plants, is responsible for defense genes (pathway-related genes,PRgene) is expressed (described in CAO H, GLAZEBROOK J, CLARKE JD, et al The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats, cell, 1997, 88:57-63.). Previous studies have found that NPR1 requires phosphorylation and hematoxylin modifications in the induction of PR gene expression, wherein the phosphorylation may occur at serine at positions 11 and 15, hematoxylin modifications are associated with amino acids 345-348 (described in SPOEL SH, MOU Z, TADA Y, et al, proteasmem-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity Cell, 2009, 137:860-872; SALEH A, WITHERS J, MOHAN R, et al, posttranslational modifications of the master transcriptional regulator NPR1 enable dynamic but tight control of plant immune responses Cell Host)&Microbe, 2015, 18:169-182). Transgenic overexpression of Arabidopsis wildThe raw NPR1 gene can improve the resistance of various nematodes, bacteria, oomycetes and fungi such as arabidopsis thaliana, rice, wheat, carrot, rape, citrus, cotton, strawberry and the like to rice bacterial blight, rice blast, fusarium, sclerotinia sclerotiorum, phloem of Citrus, canker, verticillium, pseudomonas syringae, root knot nematode and the like (described in SILVA KJP, BRUNINNGS A, PERES NA, et al The Arabidopsis NPR1 gene confers Broad-spectrum disease resistance in strawberry; BUTT M, BARTHE G, IREY M, et al Transgenic Citrus expressing an Arabidopsis NPR.1 gene exihibit enhance resistance against Huanglongbing (HLB; citrus Greening). PLoS ONE, 2015, 10:e 0137134; KUMAR V, JOSHI SG, BELL A, et al Enhance resistance against Thielaviopsis basicola in transgenic cotton plants expressing Arabidopsis NPR1 gene. Transgenic Research, 2013, 22:359-368; PRIYA DB, SOMASEKECHAR N, PRASAD JS, et al Transgenic tobacco plants constitutively expressing Arabidopsis NPR1 show enhanced resistance to root-knot new code, meloidogyne incognita. BMC Research Notes, 2011, 4:231; MEUR G, BUDATHA M, SRINIVASAN T, et al Constitutive expression of Arabidopsis NPR1 confers enhanced resistance to the early instars of Spodoptera litura in transgenic tobacco Physiologia Plantarum, 2008, 133:765-775; WALLY O, JAJ J, PUNJA ZK. Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Dauccarota L.) expressing an Arabidopsis NPR 1.11:11, plant, 2009, 231-131; KKHAR V, UMAR V, CAMPBELL LM, et al Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis NPR1. Transgenic Research, 2010, 19:959-975). However, the role of the NPR1 gene in combating plant viruses (especially turnip mosaic virus) is not yet known.
Mutation of different sites of NPR1 may alter the function of NPR1. For example, transgenic plants that overexpress mutant NPR1 with serine to aspartic acid mutations at positions 11 and 15 are resistant to P.syringae comparable to wild type (described in SPOEL SH, MOU Z, TADA Y, et al Proteasiome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity Cell, 2009, 137:860-872); transgenic plants that overexpress mutant NPR1, which mutates amino acids 345-348, have significantly reduced resistance to P.syringae compared to wild-type plants (described in SALEH A, WITHERS J, MOHAN R, et al Posttranslational modifications of the master transcriptional regulator NPR1 enable dynamic but tight control of plant immune responses, cell Host & Microbe, 2015, 18:169-182). However, the resistance of transgenic plants overexpressing mutant NPR1 in which serine 11 and 15 and amino acids 345 to 348 are mutated simultaneously to different pathogens, in particular plant viruses, is not known. How to modify NPR1 to make plants have stronger resistance, and the technical difficulty of modifying NPR1 is further improved.
Disclosure of Invention
The invention aims to provide a method for resisting turnip mosaic virus.
The invention provides a method for improving the turnip mosaic virus resistance of arabidopsis thaliana by mutating an NPR1 gene, wherein the sequence of the NPR1 gene is shown as SEQ ID NO. 21.
Further defined, the NPR1 mutant gene is obtained by mutating positions 31-33, 43-45 and 1033-1043 with the NPR1 gene sequence shown in SEQ ID NO.21 as a starting sequence.
Further defined, the mutation of the NPR1 mutant gene is as follows:
(a) Mutating tct at positions 31-33 in the gene sequence shown in SEQ ID NO.21 to gac;
(b) The agc at the 43 th to 45 th positions in the gene sequence shown in SEQ ID NO.21 is mutated into gat;
(c) Atactatctct at positions 1033-1043 in the gene sequence shown in SEQ ID NO.21 was mutated to gcagcatctgc.
The invention provides application of NPR1 mutant gene coding protein in inhibiting turnip mosaic virus, wherein the sequence of the NPR1 protein is shown as SEQ ID NO. 22.
Further limited, the NPR1 mutant gene encoded protein takes the amino acid sequence shown in SEQ ID NO.22 as a starting sequence, and mutates amino acids at 11 th, 15 th, 345 th, 346 th and 348 th positions.
Further defined, the NPR1 mutant gene-encoded protein comprises the following amino acid changes:
(1) Mutating serine (S) at position 11 in the amino acid sequence shown in SEQ ID No.22 to aspartic acid (D);
(2) Mutating serine (S) at position 15 in the amino acid sequence shown in SEQ ID No.22 to aspartic acid (D);
(3) Isoleucine (I) at position 345 in the amino acid sequence shown in SEQ ID NO.22 was mutated to glycine (A), and leucine at positions 346 and 348 were mutated to glycine (A).
The invention provides a method for preparing a plant resistant to turnip mosaic virus, which comprises the steps of connecting a gene sequence shown in SEQ ID NO.27 to a pEarley gate103 vector to obtain a recombinant vector, transforming the recombinant vector into agrobacterium to obtain recombinant agrobacterium, and transferring the recombinant agrobacterium into the plant.
Further defined, the plant is arabidopsis thaliana.
The beneficial effects are that: the growth of transgenic arabidopsis plants expressing NPR1 mutant genes was completely consistent with non-transgenic control plants, but resistance to turnip mosaic virus was significantly enhanced.
Drawings
FIG. 1 shows that NPR1 inhibits TuMV infection. a, tuMV-GFP infects wild type Arabidopsis Col-0 ecology (WT) and 3npr1Phenotype at 14 dpi of the mutant. Mock represents injection of inoculation buffer; red for virus-free areas and green for virus-infected areas; b, WT and 3 NPR1 mutants 14 dpi infection leaf area to rosette total area ratio. Columns represent mean ± standard deviation (n=5); c, RT-qPCR detects relative accumulation of viral genome at 14 dpi of WT and 3 NPR1 mutants. Columns represent mean ± standard deviation (n=5); d, relative virion numbers at 14 dpi for WT and 3 NPR1 mutants were detected by ELISA. Columns represent mean ± standard deviation (n=5);
FIG. 2 shows RT-qPCR detection of WT and NPR1 mutants PR1 expression levels. a-c, RT-qPCR detection of WT and 3npr1When the mutant is inoculated with TuMV 48 hpiPR1Gene expression level. Reference genes are respectively selected from Arabidopsis thalianaActin Ⅱ（a），UBQ（b），GAPDH(c) A. The invention relates to a method for producing a fibre-reinforced plastic composite Columns represent mean ± standard deviation (n=3);
FIG. 3 is a phenotypic and expression analysis of NPR1 and mutant NPR1 transgenic plants. a, WT, NPR1-1, NPR1-0, transgenic plants overexpressing NPR1 (35S:: NPR 1-GFP-2), and transgenic plants expressing NPR1 mutant genes mutated to glycine at positions 345, 346 and 348 (sim 3) (35S::: sim3-GFP-1 3) were phenotyped at three weeks of age; b-c, WB detection 35S:: NPR1-GFP (b) and 35S:: sim3-GFP (c) transgenic anaplerotic line protein expression.
FIG. 4 is a graph showing phenotypes when TuMV-GFP infects WT, 35S:: NPR1-GFP-3, 35S:: NPR1-GFP-4, 35S:: sim3-GFP-1 and 35S:npr1sim3 18 dpi. Red for virus-free areas and green for virus-infected areas; b, relative accumulation of viral genomes when TuMV-6K2-mCherry infects WT, 35S:: NPR1-GFP-3, 35S:: NPR1-GFP-4, 35S:: sim3-GFP-1 and 35S:npr1sim3 18 dpi were detected by RT-qPCR. Columns represent mean ± standard deviation (n=3); c, RT-qPCR detection of PR1 expression level of TuMV-6K2-mCherry friction inoculation of WT and transgenic plants 48 hpi. Columns represent mean ± standard deviation (n=3).
FIG. 5 is a phenotypic and expression analysis of transgenic plants of different NPR1 mutant genes. a, WT, transgenic plants expressing NPR1 mutant genes mutated to glycine (A) at positions 11 and 15 (sim 3|S11/15A) at positions 345, 346 and 348 (sim 3|S11/15A-GFP-4), transgenic plants expressing NPR1 mutant genes mutated to aspartic acid (D) at positions 11 and 15 (sim 3|S11/15D) at positions 345, 346 and 348 (sim 3|S11/15D) at positions 11 and 15 (sim 3|S11/15D-GFP-2), transgenic plants expressing NPR1 mutant genes mutated to aspartic acid (D) at positions 11 and 15 (S11/15D) (35S: S11/15D-GFP-3, 35S: S11/15D-GFP-6 and 35S: S11/15D-GFP-8) at three week-old phenotypes; b-D WB detection 35S:: sim3|S11/15D-GFP (b), 35S:: sim3|S11/15A-GFP (c)And 35S: S11/15D-GFP (c) expression. e, RT-qPCR detection of 35S: S11/15D-GFP transgenic line endogenousPR1Gene expression level. Columns represent mean ± standard deviation (n=3);
FIG. 6 shows that phosphorylation of NPR1 Ser11/Ser15 inhibits TuMV infection. a, tuMV-GFP infects WT, 35S:: sim3|S11/15A-GFP, 35S:: sim3|S11/15D-GFP and 35S:: S11/15D-GFP 18 dpi. Red for virus-free areas and green for virus-infected areas; b, graph of leaf area infected to total rosette area ratio of WT and transgenic lines. Columns represent mean ± standard deviation (n=5); c, RT-qPCR detecting the relative accumulation of viral genome at 18 dpi of WT and transgenic plants. Columns represent mean ± standard deviation (n=5); and d, detecting the expression level of the virus particles when the WB detects the WT and the transgenic plant 18 dpi.
Detailed Description
npr1-1 is a product obtained by screening after Ethyl Methanesulfonate (EMS) induced mutation, and sequencing results show that His 334 is mutated to Tyr, which is described in CAO H, bowling SA, gordon AS, et al Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance [ J ]. The Plant Cell, 1994, 6:1583-1592;
NPR1-0 (SALK_ 204100) is a null mutant, a T-DNA insert is present in the first exon of the NPR1 gene, and the inability of the gene to translate into a protein is described in SKELLY MJ, FURNISS JJ, GREY H, et al Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR [ J ]. Elife, 2019, 8, e47005;
NPR1-6 (SAIL_708_F09) contains a T-DNA insert in the third exon of the NPR1 gene, allowing the expression of NPR1 1-432 aa (NPR1ΔC) as described in DING Y, DOMMEL M R, WANG C, et al Differential quantitative requirements for NPR1 between basal immunity and systemic acquired resistance inArabidopsis thaliana[J]. Frontiers in Plant Science, 2020, 11: 570422。
Example 1 method for preparing recombinant vector containing NPR1 Gene
Amplification of NPR1 gene: extracting Arabidopsis RNA, carrying out reverse transcription to obtain cDNA as a template, and amplifying an NPR1 gene by using an upstream primer 207-NPR1F1-F (SEQ ID NO. 1) and a downstream primer 207-NPR1F3-R (SEQ ID NO. 2); and (3) connecting the NPR1 gene homologous recombination method obtained in the step (1) into the pDONR207 vector to obtain the pDONR207-NPR1 vector. EXAMPLE 2 method for preparing recombinant vector of NPR1 mutant Gene
1. Construction of NPR1 mutant (S11/15D) recombinant vector 1 in which serine at positions 11 and 15 was mutated to aspartic acid:
1. amplification of NPR1 mutant fragment 1: the mutant fragment was amplified using the primers NPR1-pd-11.15D-F (SEQ ID NO. 5) upstream and NPR1-pd-R (SEQ ID NO. 4) downstream using pDONR207-NPR1 as template.
2. Amplification of AtNPR1 mutant gene vector 1: the vector was amplified using pDONR207-NPR1 as template, the upstream primer NPR1-zt-F (SEQ ID NO. 3) and the downstream primer NPR1-zt-11.15D-R (SEQ ID NO. 6).
3. The fragment obtained in the step 1 is connected with the vector obtained in the step 2 by a homologous recombination method to obtain a plasmid pDONR207-S11/15D; S11/15D gene sequence (SEQ ID NO. 23).
2. Construction of NPR1 mutant (sim 3) recombinant vector 2 in which isoleucine (I) at position 345 was mutated to glycine (a), leucine at positions 346 and 348 was mutated to glycine (a):
amplification of AtNPR1 mutant fragment 2: the mutant fragment was amplified using pDONR207-NPR1 as template, the upstream primer NPR1-pd-sim3-F (SEQ ID NO. 9) and the downstream primer NPR1-pd-R (SEQ ID NO. 4). Amplification of NPR1 mutant gene vector 2: the vector was amplified using pDONR207-NPR1 as template, the upstream primer NPR1-zt-F (SEQ ID NO. 3) and the downstream primer NPR1-zt-sim3-R (SEQ ID NO. 10). 3. Connecting the fragment obtained in the step 1 with the vector obtained in the step 2 by a homologous recombination method to obtain a plasmid pDONR207-sim3; sim3 gene sequence (SEQ ID NO. 25).
3. Construction of NPR1 mutant (sim 3|S11/15A) recombinant vector 3 in which serine at positions 11 and 15 was mutated to glycine, isoleucine (I) at position 345 was mutated to glycine (A), leucine at positions 346 and 348 was mutated to glycine (A):
1. amplification of NPR1 mutant fragment 3: the mutant fragment was amplified using the primers NPR1-pd-11.15A-F (SEQ ID NO. 7) and the downstream primer NPR1-pd-R (SEQ ID NO. 4) using pDONR207-sim3 as template.
2. Amplification of NPR1 mutant gene vector 3: the vector was amplified using pDONR207-sim3 as template, the upstream primer NPR1-zt-F (SEQ ID NO. 3) and the downstream primer NPR1-zt-11.15A-R (SEQ ID NO. 8).
3. The fragment obtained in the step 1 is connected with the vector obtained in the step 2 by a homologous recombination method to obtain a plasmid pDONR207-sim3|S11/15A; the sim3|s11/15A gene sequence (SEQ ID No. 29).
4. Construction of NPR1 mutant (sim 3|S11/15D) recombinant vector 4 in which serine at positions 11 and 15 was mutated to aspartic acid, isoleucine (I) at position 345 was mutated to glycine (A), leucine at positions 346 and 348 was mutated to aspartic acid:
amplification of NPR1 mutant fragment 4: the mutant fragment was amplified using the primers NPR1-pd-sim3-F (SEQ ID NO. 9) and the downstream primer NPR1-pd-R (SEQ ID NO. 4) using pDONR207-11.15D as template. 2. Amplification of NPR1 mutant gene vector 4: the vector was amplified using pDONR207-11.15D as template, the upstream primer NPR1-zt-F (SEQ ID NO. 3) and the downstream primer NPR1-zt-sim3-R (SEQ ID NO. 10).
3. The fragment obtained in the step 1 is connected with the vector obtained in the step 2 by a homologous recombination method to obtain a plasmid pDONR207-sim3|S11/15D; the sim3|s11/15D gene sequence (SEQ ID No. 27).
5. Constructing a recombinant final vector:
1. plasmids pDONR207-NPR1, pDONR207-11.15D, pDONR207-sim3, pDONR207-sim3|S11/15A and pDONR207-sim3|S11/15D were ligated into vector pEarley gate103, respectively, using LR recombination, to give 35S promoter-driven expression vectors, respectively: 103-NPR1, 103-11.15D, 103-sim3, 103-sim3|S11/15A and 103-sim3|S11/15D.
TABLE 1 primer sequences
Example 3 obtaining transgenic plants
The 5 recombinant vectors obtained in example 2 were transformed into Agrobacterium GV3101, respectively, to obtain 5 recombinant Agrobacterium, and the recombinant Agrobacterium was transformed into npr1-1 mutants, respectively, by an Arabidopsis inflorescence infection method (described in CLOUGH SJ, BENT AF. Flora dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thiana. Plant Journal, 1998, 16: 735-743.), to finally obtain homozygous anaplerotic transgenic plants.
Example 4.
1. The wild type Arabidopsis Col-0 ecology (WT), npr1-1, npr1-0 and npr1-6 were grown and inoculated with the green fluorescent-tagged infectious clone TuMV-GFP (described in GARCIA-RUIZ H, TAKEDA A, CHAPANE EJ, et al Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during turnip mosaic virus in section Plant Cell 2015, 27 (3): 944-5) using Agrobacterium infiltration at 3 weeks of age.
TuMV infection was observed 14 days after inoculation (dpi), and Image J software was used for plant fluorescence area statistics and generation of a plant fluorescence pattern graph. The results show (a and b in FIG. 1) that the TuMV infected leaf areas to Arabidopsis total rosette leaf area ratios of npr1-1, npr1-0 and npr1-6 were 49.43%,41.91% and 35.69%, respectively, significantly higher than 21.61% of WT. Total plant RNA was extracted from whole plant samples and inverted to cDNA, and real-time quantitative PCR (RT-qPCR) experiments were performed using specific primers (Table 2). The RT-qPCR assay was performed using a relative quantification method (FIG. 1 c), and the amounts of accumulated viral genomic RNA were set to 1, npr1-0 and npr1-6, respectively 2.06,1.89 and 1.78, and were 1.5 times or more than that of WT inoculated with TuMV-GFP. Sample extraction Total plant protein specific antibody TuMV-CP recognizes antigen and ELISA assay is used to detect the level of viral particle accumulation. ELISA results showed (d in FIG. 1) that the accumulation of virions of NPR1-1, NPR1-0 and NPR1-6 increased 1.8-2.1 fold over WT, demonstrating that TuMV invasion was restricted only when NPR1 had complete function. When NPR1 was deleted, both truncated or nonfunctional NPR1 mutants were expressed, plant susceptibility increased, indicating that NPR1 inhibited TuMV infestation (FIGS. 1 and 2).
TABLE 2 primer sequences
RT-qPCR assay detects the expression level of the defense gene PR1 after the inoculation of WT, npr1-1, npr1-0 and npr1-6 with virus. Since agrobacterium activates plant immunity, mechanical inoculation was selected to tribo-inoculate TuMV-GFP with 4 week old WT and 3 npr1 mutants, and 48 hpi was extracted from the inoculated leaves and total RNA was inverted to cDNA for RT-qPCR detection. It was found experimentally (a-c in FIG. 2) that both WT and 3 NPR1 mutants induced PR1 expression after friction inoculation with TuMV-GFP. The WT increased the expression level of PR1 more than 5-fold compared to the uninoculated control, whereas the expression level of PR1 in NPR1-1, NPR1-0 and NPR1-6 plants increased from below to a similar level as the WT uninoculated control, indicating that NPR1 plays a dominant role in inducing PR1 expression when TuMV infects plants. Taken together, NPR 1-dependent immune responses suppress infection by TuMV.
2. Transgenic plants 35S:: NPR1-GFP and 35S:: sim3-GFP of NPR1 or sim3 expressed by the CaMV 35S promoter and fused with GFP tag at the C-terminal were prepared in the NPR1-1 background, and analyzed for resistance to TuMV.
All homozygous lines of 35S:: NPR1-GFP and 35S:: sim3-GFP transgenic plants exhibited normal phenotypes similar to WT (a in FIG. 3). The WB test detects 35S:: NPR1-GFP and 35S:: sim3-GFP homozygous lines are expressed, and the results show (b and c in FIG. 3) 35S: only line 1 anaplerotic protein in NPR1-GFP is not expressed, and lines 2, 3 and 4 are expressed normally; 35S, only strain 1 of sim3-GFP expresses anaplerotic protein. 35S:: NPR1-GFP-3, 35S:: NPR1-GFP-4 and 35S:: sim3-GFP-2 were finally selected for subsequent experiments, and the transgenic line 35S: npR1sim3 (NPR 1-2) which has been reported was also used for this experiment (described in SALEH A, WITHERS J, MOHAN R, et al Posttranslational modifications of the master transcriptional regulator NPR1 enable dynamic but tight control of plant immune responses Cell Host Microbe, 2015, 18:169-82). Planting WT, 35S, NPR1-GFP-3, 35S, NPR1-GFP-4, 35S, sim3-GFP-1 and 35S, NPR1sim3, inoculating TuMV-GFP by Agrobacterium infiltration method at 3 weeks of age. 18 Photographs were taken at dpi and analyzed using Image J software, and the results showed that 35S:: NPR1-GFP-3, 35S:: sim3-GFP-1, and 35S:: NPR1sim3 was substantially consistent with the ratio of the area of the infected leaves to the total rosette leaf area of the WT, while the 35S:: NPR1-GFP-4 strain exhibited a slight decrease in the ratio (a in FIG. 4). Transgenic complementation plants were inoculated by selecting a TuMV invasive clone with the mCherry tagged 6K2 protein inserted between P1 and HcPro, and the effect of RNA silencing on the assay was excluded (described in COTTON S, GRANGEON R, THIVIERGE K, et al Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments, and are each derived from a single viral genome. J Virol, 2009, 83:10460-71). Whole plant samples were taken at 18 dpi and tested for RT-qPCR to detect viral genome accumulation. As a result, all transgenic lines accumulated similar levels of viral genome to WT (b in FIG. 4), indicating that plants overexpressing wild-type NPR1 or sim3 were similar to WT in susceptibility. NPR1 mediated restoration of immune pathways in the anaplerotic transgenic lines was further analyzed by mechanical inoculation of TuMV-6K2-mCherry with 4 week old WT, 35S:: NPR1-GFP-3, 35S::: NPR1-GFP-4, 35S:: sim3-GFP and 35S::: NPR1sim3 to detect the expression of the defense gene PR1. 48 The RNA extracted from the inoculated leaf sample at the time of hpi is reversed to cDNA for RT-qPCR test. The results showed that PR1 expression levels were up-regulated after friction inoculation of TuMV on all transgenic plants and similar to the expression levels after WT inoculation with virus (c in FIG. 4). The above experiments indicate that transgenic overexpression of NPR1 or sim3 does not increase resistance of plants to TuMV when the NPR1 or sim3 muteins are back supplemented.
3. Transgenic plants 35S:: S11/15D-GFP, 35S:: sim3|S11/15D-GFP and 35S:: sim3|S11/15D-GFP expressed by the CaMV 35S promoter fused C-terminal with GFP tags were constructed in the npr1-1 background, and their disease resistance to TuMV was analyzed.
Homozygous 35S:: sim3|S11/15D-GFP and 35S:: sim3|S11/15A-GFP plants were similar to the Arabidopsis wild type phenotype (a in FIG. 5), and the WB test examined the protein expression levels of 35S:: sim3|S11/15D-GFP and 35S:: sim3|S11/15A-GFP homozygous lines, and the results showed (FIGS. 5b and c) that only line 1 was not expressed and that 2, 3 and 20 were expressed normally in 35S:: sim3|S11/15D-GFP; 35S, the strain 1 and the strain 4 in the sim3|S11/15A-GFP are expressed normally. Depending on the number of seeds and the germination level, 35S:: sim3|S11/15D-GFP-2/-20 and 35S:: sim3|S11/15A-GFP-1/-4 were finally selected for the subsequent experiments. While 35S 11/15D-GFP showed two completely different phenotypes, most of which showed self-immune mutant phenotypes, such as smaller plant size, rosette leaf curl, and increased PR1 expression (a and e in FIG. 5). The WB assay detects protein expression levels (fig. 5 d), where the expression levels of strain 3 and 6 proteins with autoimmune mutant phenotypes are high, while the normal phenotype strain 8 protein is essentially not expressed. In addition, the severity of the phenotype and PR1 expression levels were found to be positively correlated with the S11/15D-GFP protein expression levels (a, D and e in FIG. 5), which also confirmed that phosphorylation of serine at positions 11 and 15 was necessary for NPR1 to induce PR gene expression. WT and the two independent lines of each transgene described above (35S:: sim3|S11/15D-GFP-2/-20, 35S:: sim3|S11/15A-GFP-1/-4 and 35S:: S11/15D-GFP-3/-6) were observed and plant fluorescence area statistics were performed at 3 weeks of age by Agrobacterium infiltration and a plant fluorescence pattern map was generated (a and b in FIG. 6) with both lines WT and 35S:: sim3|S11/15A-GFP almost all had GFP fluorescence signals on all rosette leaves, whereas both lines 35S:: sim3|S11/15D-GFP had GFP fluorescence on only the center rosette leaf, accounting for 9.29% and 7.12% of the total rosette leaf area, respectively, whereas both lines 35S: SIm3|S11/15A-GFP were not observed to date. The whole plant samples were subjected to RT-qPCR experiments (c in FIG. 6), 35S that the viral genome accumulation amounts of the two strains of sim3|S11/15A-GFP were not significantly different from that of WT, 35S that the S11/15D-GFP-1/-4 transgenic plants accumulated the lowest viral genome, and next 35S that the sim3|S11/15D-GFP-3/6 plants. WB detected the virion expression and analyzed the band gray scale (D in FIG. 6), 35S:: S11/15D-GFP the presence of TuMV CP protein was undetectable in the transgenic line, whereas 35S:: sim3|S11/15D-GFP had a relative expression of about half of WT. In addition, 35S: sim3|S11/15A-GFP showed very weak elevation of virions compared to WT. These results indicate that transgenic plants expressing S11/15D are highly resistant to TuMV, but the plants grow abnormally; transgenic plants expressing sim3|s11/15D have very good resistance to TuMV while not affecting plant growth; transgenic plants expressing sim3|s11/15A were comparable to WT for TuMV resistance.
NPR1 gene sequence: (SEQ ID NO. 21)
ATGGACACCACCATTGATGGATTCGCCGATTCTTATGAAATCAGCAGCACTAGTTTCGTCGCTACCGATAACACCGACTCCTCTATTGTTTATCTGGCCGCCGAACAAGTACTCACCGGACCTGATGTATCTGCTCTGCAATTGCTCTCCAACAGCTTCGAATCCGTCTTTGACTCGCCGGATGATTTCTACAGCGACGCTAAGCTTGTTCTCTCCGACGGCCGGGAAGTTTCTTTCCACCGGTGCGTTTTGTCAGCGAGAAGCTCTTTCTTCAAGAGCGCTTTAGCCGCCGCTAAGAAGGAGAAAGACTCCAACAACACCGCCGCCGTGAAGCTCGAGCTTAAGGAGATTGCCAAGGATTACGAAGTCGGTTTCGATTCGGTTGTGACTGTTTTGGCTTATGTTTACAGCAGCAGAGTGAGACCGCCGCCTAAAGGAGTTTCTGAATGCGCAGACGAGAATTGCTGCCACGTGGCTTGCCGGCCGGCGGTGGATTTCATGTTGGAGGTTCTCTATTTGGCTTTCATCTTCAAGATCCCTGAATTAATTACTCTCTATCAGAGGCACTTATTGGACGTTGTAGACAAAGTTGTTATAGAGGACACATTGGTTATACTCAAGCTTGCTAATATATGTGGTAAAGCTTGTATGAAGCTATTGGATAGATGTAAAGAGATTATTGTCAAGTCTAATGTAGATATGGTTAGTCTTGAAAAGTCATTGCCGGAAGAGCTTGTTAAAGAGATAATTGATAGACGTAAAGAGCTTGGTTTGGAGGTACCTAAAGTAAAGAAACATGTCTCGAATGTACATAAGGCACTTGACTCGGATGATATTGAGTTAGTCAAGTTGCTTTTGAAAGAGGATCACACCAATCTAGATGATGCGTGTGCTCTTCATTTCGCTGTTGCATATTGCAATGTGAAGACCGCAACAGATCTTTTAAAACTTGATCTTGCCGATGTCAACCATAGGAATCCGAGGGGATATACGGTGCTTCATGTTGCTGCGATGCGGAAGGAGCCACAATTGATACTATCTCTATTGGAAAAAGGTGCAAGTGCATCAGAAGCAACTTTGGAAGGTAGAACCGCACTCATGATCGCAAAACAAGCCACTATGGCGGTTGAATGTAATAATATCCCGGAGCAATGCAAGCATTCTCTCAAAGGCCGACTATGTGTAGAAATACTAGAGCAAGAAGACAAACGAGAACAAATTCCTAGAGATGTTCCTCCCTCTTTTGCAGTGGCGGCCGATGAATTGAAGATGACGCTGCTCGATCTTGAAAATAGAGTTGCACTTGCTCAACGTCTTTTTCCAACGGAAGCACAAGCTGCAATGGAGATCGCCGAAATGAAGGGAACATGTGAGTTCATAGTGACTAGCCTCGAGCCTGACCGTCTCACTGGTACGAAGAGAACATCACCGGGTGTAAAGATAGCACCTTTCAGAATCCTAGAAGAGCATCAAAGTAGACTAAAAGCGCTTTCTAAAACCGTGGAACTCGGGAAACGATTCTTCCCGCGCTGTTCGGCAGTGCTCGACCAGATTATGAACTGTGAGGACTTGACTCAACTGGCTTGCGGAGAAGACGACACTGCTGAGAAACGACTACAAAAGAAGCAAAGGTACATGGAAATACAAGAGACACTAAAGAAGGCCTTTAGTGAGGACAATTTGGAATTAGGAAATTCGTCCCTGACAGATTCGACTTCTTCCACATCGAAATCAACCGGTGGAAAGAGGTCTAACCGTAAACTCTCTCATCGTCGTCGGTGA；
NPR1 amino acid sequence: (SEQ ID NO. 22)
MDTTIDGFADSYEISSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSPDDFYSDAKLVLSDGREVSFHRCVLSARSSFFKSALAAAKKEKDSNNTAAVKLELKEIAKDYEVGFDSVVTVLAYVYSSRVRPPPKGVSECADENCCHVACRPAVDFMLEVLYLAFIFKIPELITLYQRHLLDVVDKVVIEDTLVILKLANICGKACMKLLDRCKEIIVKSNVDMVSLEKSLPEELVKEIIDRRKELGLEVPKVKKHVSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCNVKTATDLLKLDLADVNHRNPRGYTVLHVAAMRKEPQLILSLLEKGASASEATLEGRTALMIAKQATMAVECNNIPEQCKHSLKGRLCVEILEQEDKREQIPRDVPPSFAVAADELKMTLLDLENRVALAQRLFPTEAQAAMEIAEMKGTCEFIVTSLEPDRLTGTKRTSPGVKIAPFRILEEHQSRLKALSKTVELGKRFFPRCSAVLDQIMNCEDLTQLACGEDDTAEKRLQKKQRYMEIQETLKKAFSEDNLELGNSSLTDSTSSTSKSTGGKRSNRKLSHRRR；
S11/15D Gene sequence (SEQ ID NO. 23)
ATGGACACCACCATTGATGGATTCGCCGATGACTATGAAATCGATAGCACTAGTTTCGTCGCTACCGATAACACCGACTCCTCTATTGTTTATCTGGCCGCCGAACAAGTACTCACCGGACCTGATGTATCTGCTCTGCAATTGCTCTCCAACAGCTTCGAATCCGTCTTTGACTCGCCGGATGATTTCTACAGCGACGCTAAGCTTGTTCTCTCCGACGGCCGGGAAGTTTCTTTCCACCGGTGCGTTTTGTCAGCGAGAAGCTCTTTCTTCAAGAGCGCTTTAGCCGCCGCTAAGAAGGAGAAAGACTCCAACAACACCGCCGCCGTGAAGCTCGAGCTTAAGGAGATTGCCAAGGATTACGAAGTCGGTTTCGATTCGGTTGTGACTGTTTTGGCTTATGTTTACAGCAGCAGAGTGAGACCGCCGCCTAAAGGAGTTTCTGAATGCGCAGACGAGAATTGCTGCCACGTGGCTTGCCGGCCGGCGGTGGATTTCATGTTGGAGGTTCTCTATTTGGCTTTCATCTTCAAGATCCCTGAATTAATTACTCTCTATCAGAGGCACTTATTGGACGTTGTAGACAAAGTTGTTATAGAGGACACATTGGTTATACTCAAGCTTGCTAATATATGTGGTAAAGCTTGTATGAAGCTATTGGATAGATGTAAAGAGATTATTGTCAAGTCTAATGTAGATATGGTTAGTCTTGAAAAGTCATTGCCGGAAGAGCTTGTTAAAGAGATAATTGATAGACGTAAAGAGCTTGGTTTGGAGGTACCTAAAGTAAAGAAACATGTCTCGAATGTACATAAGGCACTTGACTCGGATGATATTGAGTTAGTCAAGTTGCTTTTGAAAGAGGATCACACCAATCTAGATGATGCGTGTGCTCTTCATTTCGCTGTTGCATATTGCAATGTGAAGACCGCAACAGATCTTTTAAAACTTGATCTTGCCGATGTCAACCATAGGAATCCGAGGGGATATACGGTGCTTCATGTTGCTGCGATGCGGAAGGAGCCACAATTGATACTATCTCTATTGGAAAAAGGTGCAAGTGCATCAGAAGCAACTTTGGAAGGTAGAACCGCACTCATGATCGCAAAACAAGCCACTATGGCGGTTGAATGTAATAATATCCCGGAGCAATGCAAGCATTCTCTCAAAGGCCGACTATGTGTAGAAATACTAGAGCAAGAAGACAAACGAGAACAAATTCCTAGAGATGTTCCTCCCTCTTTTGCAGTGGCGGCCGATGAATTGAAGATGACGCTGCTCGATCTTGAAAATAGAGTTGCACTTGCTCAACGTCTTTTTCCAACGGAAGCACAAGCTGCAATGGAGATCGCCGAAATGAAGGGAACATGTGAGTTCATAGTGACTAGCCTCGAGCCTGACCGTCTCACTGGTACGAAGAGAACATCACCGGGTGTAAAGATAGCACCTTTCAGAATCCTAGAAGAGCATCAAAGTAGACTAAAAGCGCTTTCTAAAACCGTGGAACTCGGGAAACGATTCTTCCCGCGCTGTTCGGCAGTGCTCGACCAGATTATGAACTGTGAGGACTTGACTCAACTGGCTTGCGGAGAAGACGACACTGCTGAGAAACGACTACAAAAGAAGCAAAGGTACATGGAAATACAAGAGACACTAAAGAAGGCCTTTAGTGAGGACAATTTGGAATTAGGAAATTCGTCCCTGACAGATTCGACTTCTTCCACATCGAAATCAACCGGTGGAAAGAGGTCTAACCGTAAACTCTCTCATCGTCGTCGG；
S11/15D amino acid sequence (SEQ ID NO. 24)
MDTTIDGFADDYEIDSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSPDDFYSDAKLVLSDGREVSFHRCVLSARSSFFKSALAAAKKEKDSNNTAAVKLELKEIAKDYEVGFDSVVTVLAYVYSSRVRPPPKGVSECADENCCHVACRPAVDFMLEVLYLAFIFKIPELITLYQRHLLDVVDKVVIEDTLVILKLANICGKACMKLLDRCKEIIVKSNVDMVSLEKSLPEELVKEIIDRRKELGLEVPKVKKHVSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCNVKTATDLLKLDLADVNHRNPRGYTVLHVAAMRKEPQLILSLLEKGASASEATLEGRTALMIAKQATMAVECNNIPEQCKHSLKGRLCVEILEQEDKREQIPRDVPPSFAVAADELKMTLLDLENRVALAQRLFPTEAQAAMEIAEMKGTCEFIVTSLEPDRLTGTKRTSPGVKIAPFRILEEHQSRLKALSKTVELGKRFFPRCSAVLDQIMNCEDLTQLACGEDDTAEKRLQKKQRYMEIQETLKKAFSEDNLELGNSSLTDSTSSTSKSTGGKRSNRKLSHRRR；
sim3 gene sequence (SEQ ID NO. 25)
ATGGACACCACCATTGATGGATTCGCCGATGACTATGAAATCGATAGCACTAGTTTCGTCGCTACCGATAACACCGACTCCTCTATTGTTTATCTGGCCGCCGAACAAGTACTCACCGGACCTGATGTATCTGCTCTGCAATTGCTCTCCAACAGCTTCGAATCCGTCTTTGACTCGCCGGATGATTTCTACAGCGACGCTAAGCTTGTTCTCTCCGACGGCCGGGAAGTTTCTTTCCACCGGTGCGTTTTGTCAGCGAGAAGCTCTTTCTTCAAGAGCGCTTTAGCCGCCGCTAAGAAGGAGAAAGACTCCAACAACACCGCCGCCGTGAAGCTCGAGCTTAAGGAGATTGCCAAGGATTACGAAGTCGGTTTCGATTCGGTTGTGACTGTTTTGGCTTATGTTTACAGCAGCAGAGTGAGACCGCCGCCTAAAGGAGTTTCTGAATGCGCAGACGAGAATTGCTGCCACGTGGCTTGCCGGCCGGCGGTGGATTTCATGTTGGAGGTTCTCTATTTGGCTTTCATCTTCAAGATCCCTGAATTAATTACTCTCTATCAGAGGCACTTATTGGACGTTGTAGACAAAGTTGTTATAGAGGACACATTGGTTATACTCAAGCTTGCTAATATATGTGGTAAAGCTTGTATGAAGCTATTGGATAGATGTAAAGAGATTATTGTCAAGTCTAATGTAGATATGGTTAGTCTTGAAAAGTCATTGCCGGAAGAGCTTGTTAAAGAGATAATTGATAGACGTAAAGAGCTTGGTTTGGAGGTACCTAAAGTAAAGAAACATGTCTCGAATGTACATAAGGCACTTGACTCGGATGATATTGAGTTAGTCAAGTTGCTTTTGAAAGAGGATCACACCAATCTAGATGATGCGTGTGCTCTTCATTTCGCTGTTGCATATTGCAATGTGAAGACCGCAACAGATCTTTTAAAACTTGATCTTGCCGATGTCAACCATAGGAATCCGAGGGGATATACGGTGCTTCATGTTGCTGCGATGCGGAAGGAGCCACAATTGGCAGCATCTGCATTGGAAAAAGGTGCAAGTGCATCAGAAGCAACTTTGGAAGGTAGAACCGCACTCATGATCGCAAAACAAGCCACTATGGCGGTTGAATGTAATAATATCCCGGAGCAATGCAAGCATTCTCTCAAAGGCCGACTATGTGTAGAAATACTAGAGCAAGAAGACAAACGAGAACAAATTCCTAGAGATGTTCCTCCCTCTTTTGCAGTGGCGGCCGATGAATTGAAGATGACGCTGCTCGATCTTGAAAATAGAGTTGCACTTGCTCAACGTCTTTTTCCAACGGAAGCACAAGCTGCAATGGAGATCGCCGAAATGAAGGGAACATGTGAGTTCATAGTGACTAGCCTCGAGCCTGACCGTCTCACTGGTACGAAGAGAACATCACCGGGTGTAAAGATAGCACCTTTCAGAATCCTAGAAGAGCATCAAAGTAGACTAAAAGCGCTTTCTAAAACCGTGGAACTCGGGAAACGATTCTTCCCGCGCTGTTCGGCAGTGCTCGACCAGATTATGAACTGTGAGGACTTGACTCAACTGGCTTGCGGAGAAGACGACACTGCTGAGAAACGACTACAAAAGAAGCAAAGGTACATGGAAATACAAGAGACACTAAAGAAGGCCTTTAGTGAGGACAATTTGGAATTAGGAAATTCGTCCCTGACAGATTCGACTTCTTCCACATCGAAATCAACCGGTGGAAAGAGGTCTAACCGTAAACTCTCTCATCGTCGTCGGTGA；
sim3 amino acid sequence (SEQ ID NO. 26)
MDTTIDGFADSYEISSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSPDDFYSDAKLVLSDGREVSFHRCVLSARSSFFKSALAAAKKEKDSNNTAAVKLELKEIAKDYEVGFDSVVTVLAYVYSSRVRPPPKGVSECADENCCHVACRPAVDFMLEVLYLAFIFKIPELITLYQRHLLDVVDKVVIEDTLVILKLANICGKACMKLLDRCKEIIVKSNVDMVSLEKSLPEELVKEIIDRRKELGLEVPKVKKHVSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCNVKTATDLLKLDLADVNHRNPRGYTVLHVAAMRKEPQLAASALEKGASASEATLEGRTALMIAKQATMAVECNNIPEQCKHSLKGRLCVEILEQEDKREQIPRDVPPSFAVAADELKMTLLDLENRVALAQRLFPTEAQAAMEIAEMKGTCEFIVTSLEPDRLTGTKRTSPGVKIAPFRILEEHQSRLKALSKTVELGKRFFPRCSAVLDQIMNCEDLTQLACGEDDTAEKRLQKKQRYMEIQETLKKAFSEDNLELGNSSLTDSTSSTSKSTGGKRSNRKLSHRRR；
sim3|11.15D gene sequence: (SEQ ID NO. 27)
ATGGACACCACCATTGATGGATTCGCCGATGACTATGAAATCGATAGCACTAGTTTCGTCGCTACCGATAACACCGACTCCTCTATTGTTTATCTGGCCGCCGAACAAGTACTCACCGGACCTGATGTATCTGCTCTGCAATTGCTCTCCAACAGCTTCGAATCCGTCTTTGACTCGCCGGATGATTTCTACAGCGACGCTAAGCTTGTTCTCTCCGACGGCCGGGAAGTTTCTTTCCACCGGTGCGTTTTGTCAGCGAGAAGCTCTTTCTTCAAGAGCGCTTTAGCCGCCGCTAAGAAGGAGAAAGACTCCAACAACACCGCCGCCGTGAAGCTCGAGCTTAAGGAGATTGCCAAGGATTACGAAGTCGGTTTCGATTCGGTTGTGACTGTTTTGGCTTATGTTTACAGCAGCAGAGTGAGACCGCCGCCTAAAGGAGTTTCTGAATGCGCAGACGAGAATTGCTGCCACGTGGCTTGCCGGCCGGCGGTGGATTTCATGTTGGAGGTTCTCTATTTGGCTTTCATCTTCAAGATCCCTGAATTAATTACTCTCTATCAGAGGCACTTATTGGACGTTGTAGACAAAGTTGTTATAGAGGACACATTGGTTATACTCAAGCTTGCTAATATATGTGGTAAAGCTTGTATGAAGCTATTGGATAGATGTAAAGAGATTATTGTCAAGTCTAATGTAGATATGGTTAGTCTTGAAAAGTCATTGCCGGAAGAGCTTGTTAAAGAGATAATTGATAGACGTAAAGAGCTTGGTTTGGAGGTACCTAAAGTAAAGAAACATGTCTCGAATGTACATAAGGCACTTGACTCGGATGATATTGAGTTAGTCAAGTTGCTTTTGAAAGAGGATCACACCAATCTAGATGATGCGTGTGCTCTTCATTTCGCTGTTGCATATTGCAATGTGAAGACCGCAACAGATCTTTTAAAACTTGATCTTGCCGATGTCAACCATAGGAATCCGAGGGGATATACGGTGCTTCATGTTGCTGCGATGCGGAAGGAGCCACAATTGGCAGCATCTGCATTGGAAAAAGGTGCAAGTGCATCAGAAGCAACTTTGGAAGGTAGAACCGCACTCATGATCGCAAAACAAGCCACTATGGCGGTTGAATGTAATAATATCCCGGAGCAATGCAAGCATTCTCTCAAAGGCCGACTATGTGTAGAAATACTAGAGCAAGAAGACAAACGAGAACAAATTCCTAGAGATGTTCCTCCCTCTTTTGCAGTGGCGGCCGATGAATTGAAGATGACGCTGCTCGATCTTGAAAATAGAGTTGCACTTGCTCAACGTCTTTTTCCAACGGAAGCACAAGCTGCAATGGAGATCGCCGAAATGAAGGGAACATGTGAGTTCATAGTGACTAGCCTCGAGCCTGACCGTCTCACTGGTACGAAGAGAACATCACCGGGTGTAAAGATAGCACCTTTCAGAATCCTAGAAGAGCATCAAAGTAGACTAAAAGCGCTTTCTAAAACCGTGGAACTCGGGAAACGATTCTTCCCGCGCTGTTCGGCAGTGCTCGACCAGATTATGAACTGTGAGGACTTGACTCAACTGGCTTGCGGAGAAGACGACACTGCTGAGAAACGACTACAAAAGAAGCAAAGGTACATGGAAATACAAGAGACACTAAAGAAGGCCTTTAGTGAGGACAATTTGGAATTAGGAAATTCGTCCCTGACAGATTCGACTTCTTCCACATCGAAATCAACCGGTGGAAAGAGGTCTAACCGTAAACTCTCTCATCGTCGTCGGTGA；
sim3|11.15D amino acid sequence: (SEQ ID NO. 28)
MDTTIDGFADDYEIDSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSPDDFYSDAKLVLSDGREVSFHRCVLSARSSFFKSALAAAKKEKDSNNTAAVKLELKEIAKDYEVGFDSVVTVLAYVYSSRVRPPPKGVSECADENCCHVACRPAVDFMLEVLYLAFIFKIPELITLYQRHLLDVVDKVVIEDTLVILKLANICGKACMKLLDRCKEIIVKSNVDMVSLEKSLPEELVKEIIDRRKELGLEVPKVKKHVSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCNVKTATDLLKLDLADVNHRNPRGYTVLHVAAMRKEPQLAASALEKGASASEATLEGRTALMIAKQATMAVECNNIPEQCKHSLKGRLCVEILEQEDKREQIPRDVPPSFAVAADELKMTLLDLENRVALAQRLFPTEAQAAMEIAEMKGTCEFIVTSLEPDRLTGTKRTSPGVKIAPFRILEEHQSRLKALSKTVELGKRFFPRCSAVLDQIMNCEDLTQLACGEDDTAEKRLQKKQRYMEIQETLKKAFSEDNLELGNSSLTDSTSSTSKSTGGKRSNRKLSHRRR；
Sim3|S11/15A Gene sequence (SEQ ID NO. 29)
ATGGACACCACCATTGATGGATTCGCCGATGCTTATGAAATCGCTAGCACTAGTTTCGTCGCTACCGATAACACCGACTCCTCTATTGTTTATCTGGCCGCCGAACAAGTACTCACCGGACCTGATGTATCTGCTCTGCAATTGCTCTCCAACAGCTTCGAATCCGTCTTTGACTCGCCGGATGATTTCTACAGCGACGCTAAGCTTGTTCTCTCCGACGGCCGGGAAGTTTCTTTCCACCGGTGCGTTTTGTCAGCGAGAAGCTCTTTCTTCAAGAGCGCTTTAGCCGCCGCTAAGAAGGAGAAAGACTCCAACAACACCGCCGCCGTGAAGCTCGAGCTTAAGGAGATTGCCAAGGATTACGAAGTCGGTTTCGATTCGGTTGTGACTGTTTTGGCTTATGTTTACAGCAGCAGAGTGAGACCGCCGCCTAAAGGAGTTTCTGAATGCGCAGACGAGAATTGCTGCCACGTGGCTTGCCGGCCGGCGGTGGATTTCATGTTGGAGGTTCTCTATTTGGCTTTCATCTTCAAGATCCCTGAATTAATTACTCTCTATCAGAGGCACTTATTGGACGTTGTAGACAAAGTTGTTATAGAGGACACATTGGTTATACTCAAGCTTGCTAATATATGTGGTAAAGCTTGTATGAAGCTATTGGATAGATGTAAAGAGATTATTGTCAAGTCTAATGTAGATATGGTTAGTCTTGAAAAGTCATTGCCGGAAGAGCTTGTTAAAGAGATAATTGATAGACGTAAAGAGCTTGGTTTGGAGGTACCTAAAGTAAAGAAACATGTCTCGAATGTACATAAGGCACTTGACTCGGATGATATTGAGTTAGTCAAGTTGCTTTTGAAAGAGGATCACACCAATCTAGATGATGCGTGTGCTCTTCATTTCGCTGTTGCATATTGCAATGTGAAGACCGCAACAGATCTTTTAAAACTTGATCTTGCCGATGTCAACCATAGGAATCCGAGGGGATATACGGTGCTTCATGTTGCTGCGATGCGGAAGGAGCCACAATTGGCAGCATCTGCATTGGAAAAAGGTGCAAGTGCATCAGAAGCAACTTTGGAAGGTAGAACCGCACTCATGATCGCAAAACAAGCCACTATGGCGGTTGAATGTAATAATATCCCGGAGCAATGCAAGCATTCTCTCAAAGGCCGACTATGTGTAGAAATACTAGAGCAAGAAGACAAACGAGAACAAATTCCTAGAGATGTTCCTCCCTCTTTTGCAGTGGCGGCCGATGAATTGAAGATGACGCTGCTCGATCTTGAAAATAGAGTTGCACTTGCTCAACGTCTTTTTCCAACGGAAGCACAAGCTGCAATGGAGATCGCCGAAATGAAGGGAACATGTGAGTTCATAGTGACTAGCCTCGAGCCTGACCGTCTCACTGGTACGAAGAGAACATCACCGGGTGTAAAGATAGCACCTTTCAGAATCCTAGAAGAGCATCAAAGTAGACTAAAAGCGCTTTCTAAAACCGTGGAACTCGGGAAACGATTCTTCCCGCGCTGTTCGGCAGTGCTCGACCAGATTATGAACTGTGAGGACTTGACTCAACTGGCTTGCGGAGAAGACGACACTGCTGAGAAACGACTACAAAAGAAGCAAAGGTACATGGAAATACAAGAGACACTAAAGAAGGCCTTTAGTGAGGACAATTTGGAATTAGGAAATTCGTCCCTGACAGATTCGACTTCTTCCACATCGAAATCAACCGGTGGAAAGAGGTCTAACCGTAAACTCTCTCATCGTCGTCGGTGA；
The amino acid sequence of sim3|S11/15A (SEQ ID NO. 30)
MDTTIDGFADDYEIDSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSPDDFYSDAKLVLSDGREVSFHRCVLSARSSFFKSALAAAKKEKDSNNTAAVKLELKEIAKDYEVGFDSVVTVLAYVYSSRVRPPPKGVSECADENCCHVACRPAVDFMLEVLYLAFIFKIPELITLYQRHLLDVVDKVVIEDTLVILKLANICGKACMKLLDRCKEIIVKSNVDMVSLEKSLPEELVKEIIDRRKELGLEVPKVKKHVSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCNVKTATDLLKLDLADVNHRNPRGYTVLHVAAMRKEPQLAASALEKGASASEATLEGRTALMIAKQATMAVECNNIPEQCKHSLKGRLCVEILEQEDKREQIPRDVPPSFAVAADELKMTLLDLENRVALAQRLFPTEAQAAMEIAEMKGTCEFIVTSLEPDRLTGTKRTSPGVKIAPFRILEEHQSRLKALSKTVELGKRFFPRCSAVLDQIMNCEDLTQLACGEDDTAEKRLQKKQRYMEIQETLKKAFSEDNLELGNSSLTDSTSSTSKSTGGKRSNRKLSHRRR。
Claims (3)
- The application of NPR1 mutant genes in inhibiting turnip mosaic virus in arabidopsis thaliana is characterized in that the NPR1 mutant genes take a gene sequence shown in SEQ ID NO.21 as a starting sequence, and tct at 31 st-33 rd positions in the gene sequence shown in SEQ ID NO.21 is mutated into gac; the agc at the 43 th to 45 th positions in the gene sequence shown in SEQ ID NO.21 is mutated into gat; atactatctct at 1033-1043 in the gene sequence shown in SEQ ID NO.21 is mutated into gcagcatctgc;the NPR1 mutant gene is expressed by the CaMV 35S promoter.
- 2. A method for preparing a turnip mosaic virus resistant plant is characterized in that NPR1 mutant genes are connected into a plant expression vector to obtain a recombinant vector, the recombinant vector is transformed into agrobacterium to obtain recombinant agrobacterium, and then the recombinant agrobacterium is transferred into the plant; the sequence of the NPR1 mutant gene is shown in SEQ ID NO. 27; the plant is arabidopsis thaliana; the NPR1 mutant gene is expressed by the CaMV 35S promoter.
- 3. The application of the plant expressing the NPR1 mutant gene in improving the turnip mosaic virus resistance of the plant or in breeding the turnip mosaic virus resistance of the plant; the sequence of the NPR1 mutant gene is shown as SEQ ID NO.27, and the plant is Arabidopsis thaliana; the NPR1 mutant gene is expressed by the CaMV 35S promoter.
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| CN102127550A (en) * | 2010-11-24 | 2011-07-20 | 江苏省农业科学院 | Plant NPR1 (Non-Expressor of PR (Pathogenesis-Related 1)) gene, encoded protein and applications thereof |
| CN111635455A (en) * | 2020-05-20 | 2020-09-08 | 中国农业大学 | Plant disease-resistant related protein and application thereof |
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| US6706952B1 (en) * | 1999-12-15 | 2004-03-16 | Syngenta Participations Ag | Arabidopsis gene encoding a protein involved in the regulation of SAR gene expression in plants |
| US20080172765A1 (en) * | 2007-01-16 | 2008-07-17 | Fumiaki Katagiri | Plant genes involved in defense against pathogens |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102127550A (en) * | 2010-11-24 | 2011-07-20 | 江苏省农业科学院 | Plant NPR1 (Non-Expressor of PR (Pathogenesis-Related 1)) gene, encoded protein and applications thereof |
| CN111635455A (en) * | 2020-05-20 | 2020-09-08 | 中国农业大学 | Plant disease-resistant related protein and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity;Liu 等;nature communications;第14卷;1-16 * |
| Sumoylation of Turnip mosaic virus RNA Polymerase Promotes Viral Infection by Counteracting the Host NPR1-Mediated Immune Response;Cheng 等;The Plant Cell;第29卷(第3期);508-525 * |
| TYLCV编码的V3功能鉴定及TuMV编码蛋白与AtRIN13互作的机制研究;龚攀;中国博士学位论文全文数据库基础科学辑(第04（2022）期);A006-73 * |
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