Application of Arabidopsis thaliana proPep1 mutant gene and its encoded protein in resistance to potyvirus

Abstract

The invention relates to the application of a mutant Arabidopsis proPep1 gene and its encoded protein in resistance to potyviruses, belonging to the technical field of plant genetic engineering. The present invention aims to solve the technical problems in the prior art of the limited resources for resistance to potyviruses, the difficulty in prevention and control, and the environmental pollution caused by chemical control insect vectors. The invention provides an application of a mutant Arabidopsis proPep1 gene and its encoded protein in resistance to potyviruses. The nucleotide sequence of the mutant Arabidopsis proPep1 gene is shown in SEQ ID NO.2, and the encoded protein sequence is shown in SEQ ID NO.4. An expression vector for a plant elicitor peptide Pep1 matured by hydrolysis of NIa-Pro protease and a transgenic disease-resistant plant are constructed. The disease resistance system provided by the present invention achieves the purpose of enhancing the ability of plants to resist potyvirus diseases and can be used to prevent and control diseases caused by potyviruses.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and relates to the application of an Arabidopsis proPep1 mutant gene and its encoded protein in resisting potyvirus. Background Art

[0002] The genus Potyvirus is the largest known genus of plant RNA viruses, containing at least 200 members, accounting for approximately 30% of the total number of plant RNA viruses (Riechmann JL, Lain S and García JA. Highlights and prospects of Potyvirus molecular biology. Journal of General Virology, 1992, 73(1), 1-16). Potyviruses have a wide host range and diverse transmission pathways, infecting host plants through seeds, vectors, or mechanical transmission. Typical viruses in the genus Potyvirus include turnip mosaic virus (TuMV), soybean mosaic virus (SMV), and potato virus Y (PVY). TuMV is mainly distributed in temperate and subtropical regions and has a very wide range of transmission, infecting approximately 320 plant species in 43 families and 156 genera, such as rapeseed, cabbage, and Arabidopsis. When plants are infected with TuMV, their leaves develop mosaic and wrinkling, seriously affecting the quality and commercial value of the crop (Walsh JA, Jenner CE. Turnip mosaic virus and the quest for durable resistance. Molecular Plant Pathology, 2002, 3(5), 289-300). Because this genus of viruses has a wide host range and strong transmission ability, it causes significant economic losses to agriculture, animal husbandry, horticulture, and ornamental crops. Therefore, enhancing plant disease resistance is crucial for controlling potyviruses.

[0003] The genome of the potato virus genus Potyvirus has two open reading frames (ORFs) and two untranslated regions. The polyprotein encoded by the ORFs is processed by the protease encoded by the ORFs to produce multiple mature proteins. [9]From left to right: first protein (P1), helper component-proteinase (HC-Pro), third protein (P3), first 6K protein (6K1), cytoplasmic inclusion protein (CI), second 6K protein (6K2), viral protein linked to the genome (VPg), nuclear inclusion body a protein (NIa-Pro), nuclear inclusion body b protein (NIb), and coat protein (CP) (Basso J, Dallaire P, Charest PJ, et al. Evidence for an internal ribosome entry site within the 5′ non-translated region of turnipmosaic Potyvirus RNA. Journal of General Virology, 1994, 75(11), 3157-3165). In addition, there is a polymerase slippage motif inside P3, which produces the 11th protein: P3N-PIPO (Chung BY, Miller WA, Atkins JF, et al (2008). An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences, 105(15), 5897-5902).

[0004] NIa-Pro, a trypsin-like cysteine ​​protease, is primarily responsible for the maturation of seven proteins between P3 and CP. Currently, research on the hydrolysis sites of NIa-Pro has been extensive. Goh and Admas's study confirmed that the NIa-Pro recognition sequence is highly conserved, with a consensus sequence of V-(EYR)-(HF)-(QE) / (SGA) (Adams MJ, Antoniw JF and Beaudoin F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Molecular Plant Pathology, 2005, 6(4), 471-487; Goh CJ, Hahn Y. Analysis of proteolytic processing sites in Potyvirus polyproteins revealed differential amino acid preferences of NIa-Pro protease in each of seven cleavage sites. PLoS One, 2021, 16(1), e0245853). Given that the NIa-Pro protease recognition sequence is highly conserved, it can be used to design broad-spectrum resistance against potyviruses.

[0005] Plants have evolved their own immune mechanisms to resist the invasion of pathogens. Plant innate immunity is divided into two layers: pattern-triggered immunity (PTI) triggered by pathogen-associated molecular patterns and effector-triggered immunity (ETI) triggered by plant effectors (Qi Y, Tsuda K, Glazebrook J, et al. Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis. Molecular plant pathology, 2011, 12(7), 702-708). PTI is the first line of defense in plants. Pattern-recognition receptors (PRRs) can sense pathogen / microbe-associated molecular patterns (PAMPs / MAMPs) or damage-associated molecular patterns (DAMPs) to trigger plant PTI (Boller T, Felix GA Renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology, 2009, 60, 379-406). Plant elicitor peptides (Pep) are a class of plant-encoded damage-associated DAMPs that play an important role in monocotyledonous and dicotyledonous plants, including tomato, maize, and Arabidopsis. Pep protein is translated in the form of precursor protein (proPep), with a size of 10.4kDa and composed of 92 amino acids. It is located on the vacuole membrane, with Pep located at its C-terminus. When the plant is damaged, glutamate is released from the damaged area, which binds to glutamate receptors through long-distance transport, triggering Ca 2+The PepR receptor in the plant is recognized by the PepR receptor, triggering the Ca 2+ Signal transduction, reactive oxygen species burst, accumulation of plant hormones such as salicylic acid and ethylene, etc., trigger plant immunity (Huffaker A, Dafoe N J. and Schmelz E A. ZmPep1, an ortholog of Arabidopsis elicitor peptide 1, regulates maize innate immunity and enhances disease resistance. Plant Physiology, 2011, 155(3), 1325-1338).

[0006] The present invention aims to provide a disease resistance system for enhancing plant resistance to potyviruses, thereby addressing the current problems of limited potyvirus-resistant resources, difficulty in prevention and control, and environmental pollution caused by chemical control of insect vectors. Summary of the Invention

[0007] The present invention aims to solve the technical problems in the prior art of the limited resources for resistance to potato virus Y, the difficulty in prevention and control, and the environmental pollution easily caused by chemical control of insect vectors. The present invention provides the use of the Arabidopsis proPep1 mutant gene and the encoded protein thereof in resistance to potato virus Y.

[0008] One of the objectives of the present invention is to provide a proPep1 mutant gene, wherein the proPep1 mutant gene sequence has a 9-base mutation compared with the proPep1 gene whose nucleotide sequence is shown in SEQ ID NO.1, and the mutation is that the bases 199-207 of the sequence shown in SEQ ID NO.1 are mutated from ACGAGTAGA to TATCACCAG.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the proPep1 mutant gene is shown as SEQ ID NO.2.

[0010] In a preferred embodiment of the present invention, the proPep1 mutant gene is expressed through the 35S constitutive promoter of the cauliflower mosaic virus (CaMV).

[0011] A second object of the present invention is to provide a protein encoded by a proPep1 mutant gene, wherein the protein sequence encoded by the proPep1 mutant gene has three amino acid mutations compared with the protein sequence encoded by the proPep1 gene shown in SEQ ID NO.3, and the mutation is that the amino acids 67-69 of the sequence shown in SEQ ID NO.3 are mutated from TSR to YHQ.

[0012] In a preferred embodiment of the present invention, the protein sequence encoded by the proPep1 mutant gene is shown as SEQ ID NO.4.

[0013] A third object of the present invention is to provide a vector comprising the aforementioned proPep1 mutant gene or expressing the protein encoded by the aforementioned proPep1 mutant gene.

[0014] A fourth object of the present invention is to provide a method for preparing a transgenic plant resistant to potyvirus, the method comprising the following steps: constructing a recombinant vector expressing a mutant proPep1 gene, transforming the recombinant vector into Agrobacterium GV3101 to obtain recombinant Agrobacterium, and then transferring the recombinant Agrobacterium into a plant to obtain a transgenic plant.

[0015] In a preferred embodiment of the present invention, the plant is wild-type Arabidopsis thaliana Col-0.

[0016] A fifth object of the present invention is to provide use of the proPep1 mutant gene, the protein encoded by the proPep1 mutant gene, or the vector in resisting plants infected by potyvirus.

[0017] In a preferred embodiment of the present invention, the potyvirus is Turnip mosaic virus TuMV.

[0018] Beneficial effects of the present invention:

[0019] Studies have shown that when plants are not stressed by the turnip mosaic virus TuMV, the plant elicitor peptide Pep1 generally exists in the form of the plant elicitor peptide precursor proPep1, and the plant elicitor peptide precursor proPep1 cannot bind to its receptor PepR to trigger plant immunity; when plants are stressed by the turnip mosaic virus TuMV, the TuMV NIa Pro protease recognizes and hydrolyzes the recognition site on the plant elicitor peptide precursor proPep1, thereby releasing the plant elicitor peptide Pep1 and exerting its disease resistance.

[0020] The present invention provides the use of a mutant Arabidopsis proPep1 gene and its encoded protein in resisting potyviruses. The mutant proPep1 gene comprises a 9-base mutation compared to the proPep1 gene whose nucleotide sequence is shown in SEQ ID NO.1, wherein the ACGAGTAGA at bases 199-207 of SEQ ID NO.1 is mutated to TATCACCAG; and a 3-amino acid mutation compared to the protein sequence encoded by the proPep1 gene shown in SEQ ID NO.3, wherein the TSR at amino acids 67-69 of SEQ ID NO.3 is mutated to YHQ. The present invention constructs an expression vector for a plant elicitor peptide Pep1 matured by hydrolysis of NIa-Pro protease and names it pEarley101-proPep1. TuMV -YFP; based on pEarley101-proPep1 TuMV -YFP, transgenic Arabidopsis thaliana 35S::proPep1 was obtained TuMV -YFP.

[0021] The present invention used fluorescence detection of the 6K2mCherry infection area in transgenic Arabidopsis thaliana 35S::proPep1-YFP plants after injection with TuMV-6K2mCherry. The results showed that the virus-infected area of ​​transgenic Arabidopsis thaliana 35S::proPep1-YFP was approximately one-third of that of wild-type Arabidopsis. qPCR detection results showed that the accumulation of TuMV genomes in transgenic Arabidopsis thaliana 35S::proPep1-YFP was reduced compared to wild-type Arabidopsis. This indicates that the transgenic Arabidopsis provided by the present invention has stronger disease resistance than wild-type Arabidopsis.

[0022] The disease resistance system provided by the present invention achieves the purpose of enhancing the ability of plants to resist potyvirus diseases and can be used to prevent and control diseases caused by potyviruses, wherein the potyvirus is turnip mosaic virus (TuMV). This system solves the environmental pollution caused by the use of chemical agents during the prevention and control process, achieves green and environmentally friendly prevention and control, and plays an important role in the research field of transgenic plant disease resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the construction of related vectors in Example 1; A is pEarley101-proPep1 TuMV -YFP vector construction diagram, B is the pBA-TuPro vector construction diagram, C is the pBA-TuPro vector construction diagram C151AVector construction diagram; LB and RB are the left and right border sequences of T-DNA, N is the NOS terminator sequence, 35S is the CaMV 35S promoter, FLAG-4×Myc and YFP are the tag sequences, respectively;

[0024] Figure 2 is proPep1 in Example 2 TuMV -YFP, NIa-Pro, and NIa-Pro C151A Western blotting results of proPep1 TuMV -YFP indicates pEarley101-proPep1 TuMV -YFP; FLAG-4×MYC-Pro indicates pBA-TuPro; FLAG-4×Myc-Pro C151A pBA-TuPro C151A ;

[0025] Figure 3 The untreated transgenic Arabidopsis thaliana 35S::proPep in Example 3 TuMV -Comparison of the phenotypes of YFP and wild-type Arabidopsis Col-0; scale bar = 1 cm;

[0026] Figure 4 The transgenic Arabidopsis thaliana 35S::proPep1m in Example 3 TuMV -YFP and wild-type Arabidopsis Col-0 resistance phenotypes to TuMV and resistance analysis results; White light refers to white light, UV light refers to laser light of a specific wavelength, and Pseudo color is generated after ImageJ processing; red indicates virus-free areas, and yellow indicates virus-infected areas;

[0027] Figure 5 The transgenic Arabidopsis thaliana 35S::proPep1m in Example 3 TuMV -The percentage of diseased area in the whole plant after YFP and wild-type Arabidopsis Col-0 were inoculated with TuMV-mcherry;

[0028] Figure 6 For transgenic Arabidopsis thaliana 35S::proPep1m in Example 3 TuMV -YFP and TuMV-CP gene qPCR detection diagram of wild-type Arabidopsis Col-0; At-EF1α is the internal reference gene, the viral genome accumulation in wild-type Arabidopsis Col-0 is 1, and the bar graph represents the mean ± standard deviation (n = 3);

[0029] Figure 7 The transgenic Arabidopsis thaliana 35S::proPep1m in Example 3 TuMV-YFP and chlorophyll fluorescence detection results of wild-type Arabidopsis Col-0. DETAILED DESCRIPTION

[0030] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0032] The English name of the present invention:

[0033] Potyvirus: Potyvirus;

[0034] Potato virus Y: potato virus Y, PVY;

[0035] Turnip mosaic virus: turnip mosaic virus, TuMV;

[0036] Nuclear inclusion protein a protease: nuclear inclusion protein a protease, NIa-Pro;

[0037] Immune response stimulated by pathogen-associated molecular patterns: pattern-triggered immunity, PTI;

[0038] Plant elicitor peptide precursor proPep1: proPep1;

[0039] Plant elicitor peptides Pep1: plant elicitor peptides, Pep1;

[0040] Capsid protein gene: coat protein, CP;

[0041] Acetosyringone: acetosyringone, AS;

[0042] 2-Morpholinoethanesulfonic acid: 2-(4-Morpholino)ethanesulfonic acid, MES.

[0043] Example 1: Construction of vectors in disease resistance system

[0044] (1)pEarley101-proPep1 TuMV -YFP vector construction:

[0045] S1: The mRNA sequence of the Arabidopsis proPep1 gene was obtained from NCBI GenBank (GenBank accession number: AT5G64900). Using wild-type Arabidopsis cDNA as a template, the first half of the proPep1 gene region (1-211 bp) was amplified using primers proPep1-F (SEQ ID NO. 5) and Pep-Pro-R (SEQ ID NO. 6) to obtain a first-half PCR amplification product. The second half of the proPep1 gene region (192-276 bp) was amplified using primers Pep-Pro-F (SEQ ID NO. 7) and proPep1-R (SEQ ID NO. 8) to obtain a second-half PCR amplification product. The two PCR amplification products were ligated by overlapping PCR to obtain a complete proPep1 gene (nucleotide sequence shown in SEQ ID NO. 1) PCR amplification product.

[0046] S2: The bases ACGAGTAGA at positions 199-207 of the PCR amplification product of the proPep1 gene obtained in S1 were mutated to TATCACCAG, and the amino acids TSR at positions 67-69 of the protein sequence encoded by the proPep1 gene (amino acid sequence shown in SEQ ID NO. 3) were mutated to YHQ (amino acid sequence shown in SEQ ID NO. 3), thereby obtaining the modified mutant proPep1 gene, hereinafter referred to as proPep1. TuMV (The nucleotide sequence is shown in SEQ ID NO.2);

[0047] S3: Using the pDONR207-TuPro plasmid as a template, primers 207-F (SEQ ID NO.9) and 207-R (SEQ ID NO.10) were used for PCR amplification to obtain the pDONR207 vector; the proPep1 obtained in S2 was used to TuMV The PCR product was used as a template and primers proPep1-F and Pep-proR were used for PCR amplification to obtain proPep1 TuMV The PCR amplification product of the fragment was then connected by homologous recombination to the above pDONR207 vector and proPep1 TuMVThe fragments were ligated to obtain pDONR207-proPep1 TuMV Then, the pEarley101-proPep1 was obtained by LR recombination. TuMV -YFP, such as Figure 1 As shown in A.

[0048] (2) Construction of TuMV-encoded NIa-Pro expression vector:

[0049] Using the pDONR207m vector (SEQ ID NO.11) as a template, primers 207-F and 207-R were used for PCR amplification to obtain a linearized pDONR207m vector; using the TuMV-6K2mCherry infectious clone as a template, primers 207Pro-F (SEQ ID NO.12) and 207Pro-R (SEQ ID NO.13) were used to amplify the NIa-Pro gene fragment of TuMV (GenBank accession number: AB194802.1) to obtain a PCR amplification product of the NIa-Pro gene fragment of TuMV; the linearized pDONR207m vector and the NIa-Pro gene fragment of TuMV were then ligated by homologous recombination to obtain a recombinant vector pDONR207-TuPro, and the TuMV NIa-Pro gene fragment (SEQ ID NO.11) was ligated by LR recombination. NO.20) The recombinant vector pDONR207-TuPro was connected to the pBA-FlAG-4×Myc-DC expression vector to obtain the recombinant vector pBA-TuPro. Figure 1 As shown in B, (Cotton S, Grangeon R, Thi ivierge K, et al. Turnip mosaic virus RNA replication complex vesicles are mobile, aligned with microfilaments, and are each derived from a single viral genome. Journal of Virology, 2009, 83(20), 10460-10471).

[0050] (3) Enzyme-inactive TuMV NIa-Pro mutant NIa-Pro C151A Construction of expression vector:

[0051] Studies have shown that mutation of the 151st amino acid of TuMV NIa-Pro from C to A will cause NIa-Pro to lose its protease activity (Xiao H, Lord E, H (2022). Proteolytic processing of plant proteins by Potyvirus NIa proteases (Proteolytic processing of plant proteins). Journal of Virology, 2022, 96 (2), e01444-21), the nucleotide sequence of the TuMV mutant NIa-Pro gene is shown in SEQ ID NO. 21. In this example, primers NIa-Prom-F (SEQ ID NO. 14) and 207Pro-R, NIa-Prom-R (SEQ ID NO. 15) and 207Pro-F were used to perform PCR amplification using pDONR207-TuPro as a template, and then primers 207Pro-F and 207Pro-R were used for overlapping PCR to obtain NIa-Pro C151A Gene fragment; using homologous recombination to C151A The gene fragment was ligated with the linearized pDONR207m vector obtained in (2) to obtain pDO NR207-TuPro C151A vector; pDONR207-TuPro was then recombined by LR recombination C151A The vector was connected to the pBA-FlAG-4×Myc-DC expression vector to obtain the recombinant expression vector pBA-TuPro C151A ,like Figure 1 As shown in C.

[0052] Example 2: Verification of the disease resistance system induced by TuMV NIa-Pro

[0053] S1: The vector pEarley101-proPep1 obtained in Example 1 was electroporated. TuMV -YFP, pBA-TuPro, and pBA-TuPro C151AThe target vectors were respectively transformed into Agrobacterium GV3101, and the target vectors were spread on solid LB medium (peptone Tryptone 2g, yeast extract 1g, sodium chloride NaCl 2g, agar Agar 3g, diluted to 200mL with water) containing 50mg / mL corresponding antibiotics and 10mg / mL rifampicin, and cultured at 28°C until single colonies appeared. The positive single colonies were respectively picked and placed in liquid LB medium containing 50mg / L corresponding resistance and 10mg / L rifampicin (peptone Tryptone 10g, yeast extract 5g, sodium chloride NaCl 10g, diluted to 1000mL with water), cultured at 28°C overnight, centrifuged to enrich the bacteria, and resuspended in a washing buffer composed of 0.01mol / L MES and 0.01mol / l MgCl2, and adjusted to OD 600 When the value was between 0.4 and 0.8, AS was added at a ratio of 1000:1 and allowed to stand at room temperature for 1 h to obtain an Agrobacterium suspension;

[0054] S2: To verify whether TuMV NIa-Pro can recognize proPep1 TuMV The hydrolysis site on the lipopolysaccharide was detected and Pep1 was released. Four-week-old Nicotiana benthamiana was taken and the Agrobacterium suspension obtained in S1 was injected into the lipopolysaccharide suspension using a 1 mL sterile syringe without a needle as follows: Figure 2 The indicated combination was injected into the back of leaves, and the inoculated Nicotiana benthamiana was cultured in a light incubator. Samples were taken 48 hours after inoculation, and the samples were subjected to Western blotting.

[0055] Test results such as Figure 2 As shown, GFP-N antibody and Myc antibody were used to detect proPep1-YFP, TuMV NIa-Pro or TuMV NIa-Pro, respectively. C151A The expression of pEarley101-proPep1 alone TuMV -YFP or pEarley101-proPep1 TuMV -YFP and pBA-TuPro C151A Only a single band was observed in the co-infiltrated Nicotiana benthamiana sample, and the size of the band was similar to that of proPep1. TuMV -YFP fusion protein size corresponds to; and pEarley101-proPep1 TuMV Two specific bands were detected in the leaf samples of Nicotiana benthamiana co-infiltrated with pBA-TuPro or TuMV, and the positions of the bands were respectively TuMV-YFP and Pep1-YFP protein sizes are similar. The above results prove that TuMV NIa-Pro can recognize and cleave proPep1 in this system. TuMV and activates Pep1 expression.

[0056] Example 3: Transgenic Arabidopsis thaliana 35S::proPep1 TuMV Preparation of -YFP

[0057] S1: Prepare wild-type Arabidopsis Col-0 with good growth and lush shoots, remove the Arabidopsis seed pods, and place in a constant temperature incubator for later use; take 100 μl of the pEarley101-proPep1 TuMV -YFP vector Agrobacterium suspension was placed in 3 mL of liquid LB medium containing 50 mg / mL Kana and 25 mg / mL Rif resistance and cultured overnight; the bacterial solution was then placed in 200 mL of LB liquid medium with the same resistance and shaken overnight at 28°C constant temperature shaker until the bacterial solution became turbid; the bacterial solution was enriched four times in a 50 mL centrifuge tube, centrifuged at 6000 rpm / min for 10 min at room temperature, and the supernatant was discarded; 10 mL of 5% sucrose solution was added to the centrifuge tube to completely dissolve the precipitate, and the mixture was gently pipetted with a Pasteur pipette, and 5% sucrose solution was added according to the OD value, and the sucrose solution was diluted to OD 600 The concentration of Silwet L-77 was 1.0. 15 μl of Silwet L-77 was added to every 50 mL of liquid, and the mixture was mixed by pipetting. The inflorescence was fully immersed for 20 seconds. The inflorescence was fixed. The Arabidopsis thaliana was wrapped with plastic wrap on the outside of the bamboo stick and placed in a dark environment for 18 hours. The Arabidopsis thaliana was cultured in a constant temperature incubator and waited for harvest.

[0058] S2: The Arabidopsis seed pods cultured in S1 were harvested after maturity, and Arabidopsis seeds and sandy soil were evenly spread in the soil at a ratio of 1:3. After vernalization at 4°C, they were cultured in a constant temperature incubator. After the Arabidopsis seeds germinated, they were sprayed with 0.08% Basta antibiotics three times, once every other day. Arabidopsis with good growth were selected for transplanting. After the Arabidopsis grew to an appropriate size, transgenic Arabidopsis 35S::proPep1 was obtained. TuMV -YFP.

[0059] Effect experiment:

[0060] In this example, transgenic Arabidopsis thaliana 35S::proPep1 grown to four weeks old was selected. TuMV -YFP and wild-type Arabidopsis Col-0 plants were compared in size, and the results were as follows Figure 3 As shown, the results showed that overexpression of the proPep1 gene did not affect the normal growth of Arabidopsis thaliana.

[0061] This example further verifies the transgenic Arabidopsis thaliana 35S::proPep1 prepared in the present invention. TuMV To investigate the disease resistance of four-week-old wild-type Arabidopsis Col-0 and transgenic Arabidopsis 35S::proPep1 TuMV -YFP, the above two Arabidopsis thaliana were injected with TuMV-6K2mCherry and the fluorescence signal was observed after 11 days of culture. Figure 4 As shown, both wild-type Arabidopsis Col-0 and 35S::proPep1-YFP can detect 6K2mCherry fluorescence signals. ImageJ was used to process the fluorescence area, and the results are shown in Figure 2. Figure 5 As shown, the virus-infected area of ​​transgenic Arabidopsis 35S::proPep1-YFP accounts for about 1 / 20 of the total plant area, and the virus-infected area of ​​wild-type Arabidopsis Col-0 accounts for about 1 / 3 of the total plant area; compared with wild-type Arabidopsis, transgenic Arabidopsis has stronger disease resistance.

[0062] In this study, qPCR was performed to detect the expression of the TuMV-CP gene using AtEF1α-F (SEQ ID NO.16) and AtEF1α-R (SEQ ID NO.17) as primers, AtEF1α of Arabidopsis thaliana as an internal reference gene, and TuMV CP-RTF (SEQ ID NO.18) and TuMV CP-RTR (SEQ ID NO.19) as primers. Figure 6 As shown in Figure 2, compared with wild-type Arabidopsis Col-0, the accumulation of TuMV genome in transgenic Arabidopsis 35S::proPep1-YFP plants was significantly reduced. The chlorophyll content of the plants was then tested, and the test results were as follows: Figure 7 As shown in Figure 2, the chlorophyll fluorescence parameters of wild-type Arabidopsis Col-0 were found to be lower than those of transgenic Arabidopsis 35S::proPep1. TuMV -YFP, indicating that the stress level of wild-type Arabidopsis Col-0 is higher than that of transgenic Arabidopsis 35S::proPep1 TuMV -YFP is more serious.

[0063] In summary, the present invention provides transgenic Arabidopsis thaliana 35S::proPep1 expressing a proPep1 mutant gene. TuMV- YFP has good disease resistance without affecting its own growth and development, avoiding problems such as dwarf plants and delayed development caused by the plant's own growth-defense trade-off strategy.

[0064] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A proPep1 A mutant gene, characterized in that described proPep1 The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

2. according to claim 1 proPep1 A mutant gene, characterized in that described proPep1 The mutant gene was expressed through the cauliflower mosaic virus CaMV 35S Expression is driven by a constitutive promoter.

3. A proPep1 The protein encoded by the mutant gene is characterized in that described proPep1 The protein sequence encoded by the mutant gene is shown in SEQ ID NO.

4.

4. A carrier, characterized in that The carrier contains the proPep1 Mutant gene or expression as claimed in claim 3 proPep1 The protein encoded by the mutant gene.

5. A method for preparing a transgenic plant resistant to potato virus Y, characterized in that: The preparation method comprises the following steps: constructing an expression proPep1 The recombinant vector of the mutant gene is transformed into GV3101 Obtaining recombinant Agrobacterium from Agrobacterium, and then transferring the recombinant Agrobacterium into plants to obtain transgenic plants; The potyvirus is Turnip mosaic virus TuMV; The plant is Arabidopsis thaliana; described proPep1 The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

6. The preparation method according to claim 5, characterized in that The plant is wild-type Arabidopsis thaliana Col-0.

7. The method according to any one of claims 1 to 2 proPep1 Mutant gene, as claimed in claim 3 proPep1 Use of the protein encoded by the mutant gene or the vector according to claim 4 in resisting plants infected by potato virus; The plant is Arabidopsis thaliana; The potato Y virus is turnip mosaic virus TuMV.

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