Application of arabidopsis thaliana proPep1 mutant gene and encoded protein thereof in resisting potyvirus virus

Abstract

The invention discloses application of an arabidopsis proPep1 mutant gene and an encoded protein thereof in resisting potyvirus viruses, and belongs to the technical field of plant genetic engineering. In order to solve the technical problems that in the prior art, potyvirus resistance resources are few, prevention and control are difficult, and environmental pollution is easily caused by chemical prevention and control of insect propagation mediators, the invention provides an arabidopsis proPep1 mutant gene and an application of an encoding protein thereof in resisting potyvirus viruses, the nucleotide sequence of the arabidopsis proPep1 mutant gene is as shown in SEQ ID NO.2, and the encoding protein of the arabidopsis proPep1 mutant gene has a nucleotide sequence as shown in SEQ ID NO.1. The arabidopsis proPep1 mutant gene has a nucleotide sequence as shown in SEQ ID NO.2. And a coding protein sequence is shown as SEQ ID NO.4, and a plant elicitor small peptide Pep1 expression vector mature by NIa-Pro protease hydrolysis and a transgenic disease-resistant plant are constructed. The disease-resistant system provided by the invention achieves the purpose of enhancing the potyvirus virus resistance of plants, and can be applied to prevention and treatment of diseases caused by potyvirus viruses.

Description

Technical Field

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

[0002] Potyvirus is the largest known genus of plant RNA viruses, with at least 200 members, accounting for about 30% of the total 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). Potyvirus has a wide host range and diverse transmission pathways, and can infect host plants through seeds, vectors or mechanical transmission. Typical viruses in the Potyvirus genus include turnip mosaic virus (TuMV), soybean mosaic virus (SMV) and potato virus Y (PVY). TuMV is mainly distributed in temperate and subtropical zones, with a very wide range of transmission, and can infect about 320 plant species in 43 families and 156 genera, such as rapeseed, cabbage, Arabidopsis, etc. When plants are infected with TuMV, the leaves will show mosaic and wrinkle, which seriously affects the quality and commercial value of the crops (Walsh JA, Jenner CE. Turnip mosaic virus and the quest for durable resistance. Molecular Plant Pathology, 2002, 3(5), 289-300). Since this genus of viruses has a wide host range and strong transmission ability, it has caused huge economic losses to agriculture, animal husbandry, horticulture and ornamental crops. Therefore, enhancing plant disease resistance is crucial for the prevention and control of potato Y virus.

[0003] Potyvirus genome 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 is a trypsin-like cysteine ​​protease that is mainly responsible for the maturation of seven proteins between P3-CP. At present, the research on the hydrolysis site of NIa-Pro has been quite in-depth. Goh and Admas' research 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). Since 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, namely, immune responses triggered by pathogen-associated molecular patterns (pattern-triggered immunity, PTI) and plant immunity triggered by effector factors (effector-triggered immunity, ETI) (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). Among them, PTI is the first layer of defense response of 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 member of the plant's own self-encoded damage-related DAMPs, which play an important role in monocotyledonous and dicotyledonous plants such as tomato, corn, 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, and Pep is located at its C-terminus. When plants are damaged, glutamate is released from the damaged site, which binds to glutamate receptors through long-distance transport, triggering Ca 2+The PepR receptor in the plant is recognized by the PepR receptor, which triggers 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 the ability of plants to resist potyvirus, so as to solve the problems that there are few potyvirus resistance resources, prevention and control are difficult, and chemical control of insect vectors easily causes environmental pollution. Summary of the invention

[0007] The present invention aims to solve the technical problems in the prior art that there are few potato virus resistance resources, prevention and control are difficult, and chemical control of insect transmission vectors easily causes environmental pollution. The present invention provides the use of Arabidopsis proPep1 mutant gene and its encoded protein in resistance to potato virus.

[0008] One of the purposes of the present invention is to provide a proPep1 mutant gene, wherein the proPep1 mutant gene sequence has a mutation of 9 bases compared with the proPep1 gene with the nucleotide sequence shown in SEQ ID NO.1, and the mutation is that the 199-207 bases of the sequence shown in SEQ ID NO.1 mutate 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] The 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 at positions 67-69 of the sequence shown in SEQ ID NO.3 mutate 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, wherein the vector contains the above-mentioned proPep1 mutant gene or expresses the protein encoded by the above-mentioned proPep1 mutant gene.

[0014] A fourth object of the present invention is to provide a method for preparing a transgenic plant resistant to potato virus Y, the preparation method comprising the following steps: constructing a recombinant vector expressing a proPep1 mutant gene, transforming the recombinant vector into GV3101 Agrobacterium to obtain a 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 Turnip mosaic virus TuMV, the plant elicitor peptide Pep1 generally exists in the form of 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 Turnip mosaic virus TuMV, 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 an application of Arabidopsis proPep1 mutant gene and its encoded protein in resisting potato Y virus. The proPep1 mutant gene is used. The proPep1 mutant gene sequence has 9 base mutations compared with the proPep1 gene whose nucleotide sequence is shown in SEQ ID NO.1, and the mutation is that the bases ACGAGTAGA at positions 199-207 of SEQ ID NO.1 are mutated to TATCACCAG; the protein sequence encoded by the proPep1 mutant gene has 3 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 TSR at positions 67-69 of SEQ ID NO.3 are mutated to YHQ. The present invention constructs an expression vector of plant elicitor peptide Pep1 matured by NIa-Pro protease hydrolysis, and it is named pEarley101-proPep1. TuMV -YFP; based on pEarley101-proPep1 TuMV -YFP, obtained transgenic Arabidopsis 35S::proPep1 TuMV -YFP.

[0021] The present invention performs fluorescence detection of 6K2mCherry infection area on transgenic Arabidopsis 35S::proPep1-YFP after injection and inoculation of TuMV-6K2mCherry. The results show that the virus infection area of ​​transgenic Arabidopsis 35S::proPep1-YFP accounts for about 1 / 3 of the infection area of ​​wild-type Arabidopsis. The qPCR detection results show that compared with wild-type Arabidopsis, the accumulation of TuMV genome in transgenic Arabidopsis 35S::proPep1-YFP is reduced. It can be seen that compared with wild-type Arabidopsis, the transgenic Arabidopsis provided by the present invention has stronger disease resistance.

[0022] The disease resistance system provided by the present invention achieves the purpose of enhancing the ability of plants to resist potato virus diseases, and can be applied to the prevention and treatment of diseases caused by potato virus, wherein the potato virus is turnip mosaic virus TuMV, and solves the problem of environmental pollution caused by the use of chemical agents in the prevention and treatment process, thereby achieving green and environmentally friendly prevention and treatment, and plays an important role in the research in the field of transgenic disease-resistant breeding of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the construction of the relevant vector 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 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 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 is white light, UV light is a laser with 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 of ​​the whole plant after YFP and wild-type Arabidopsis Col-0 were inoculated with TuMV-mcherry;

[0028] Figure 6 For the 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 article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously 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 scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. 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 by those skilled in the art through commercial channels.

[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 triggered 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 vector in disease resistance system

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

[0045] S1: The mRNA sequence of Arabidopsis proPep1 gene was obtained from NCBI GenBank (GenBank accession number: AT5G64900). The wild-type Arabidopsis cDNA was used as a template, and the primers proPep1-F (SEQ ID NO.5) and Pep-Pro-R (SEQ ID NO.6) were used to amplify the first half region (1-211 bp) of the proPep1 gene to obtain the first half PCR amplification product; the primers Pep-Pro-F (SEQ ID NO.7) and proPep1-R (SEQ ID NO.8) were used to amplify the second half region (192-276 bp) of the proPep1 gene to obtain the second half PCR amplification product; the above two PCR amplification products were connected by overlapping PCR to obtain the complete proPep1 gene (nucleotide sequence is 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 are mutated to TATCACCAG, and the amino acids TSR at positions 67-69 of the protein sequence encoded by the proPep1 gene (the amino acid sequence is shown in SEQ ID NO.3) are mutated to YHQ (the amino acid sequence is shown in SEQ ID NO.3), thereby obtaining a modified mutant proPep1 gene, hereinafter referred to as proPep1 TuMV (The nucleotide sequence is shown in SEQ ID NO.2);

[0047] S3: Using pDONR207-TuPro plasmid as template, PCR amplification was performed using primers 207-F (SEQ ID NO.9) and 207-R (SEQ ID NO.10) to obtain pDONR207 vector; using proPep1 obtained in S2 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 method to the above pDONR207 vector and proPep1 TuMVThe fragments were connected 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] The pDONR207m vector (SEQ ID NO.11) was used as a template and primers 207-F and 207-R were used for PCR amplification to obtain a linearized pDONR207m vector; the NIa-Pro gene fragment of TuMV (GenBank accession number: AB194802.1) was amplified using the TuMV-6K2mCherry infectious clone as a template and primers 207Pro-F (SEQ ID NO.12) and 207Pro-R (SEQ ID NO.13) were used to amplify the 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 connected by homologous recombination to obtain a recombinant vector pDONR207-TuPro, and the TuMV NIa-Pro gene fragment (SEQ ID NO.12) was connected by LR recombination to obtain a recombinant vector pDONR207-TuPro. 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 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 C151ATransformed into Agrobacterium GV3101 respectively, the target vector was spread on solid LB medium (Tryptone 2g, yeast extract 1g, sodium chloride Nacl 2g, agar 3g, diluted to 200mL with water) containing 50mg / mL corresponding antibiotics and 10mg / mL rifampicin, cultured at 28°C until single colonies appeared, and the positive single colonies were picked in liquid LB medium containing 50mg / L corresponding resistance and 10mg / L rifampicin (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 bacteria washing buffer composed of 0.01mol / L MES and 0.01mol / l MgCl2, and adjusted to OD 600 The value was between 0.4 and 0.8, and AS was added at a ratio of 1000:1, and the mixture was 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 surface of the tuberculin was used to release Pep1. Four-week-old Nicotiana benthamiana was taken and the Agrobacterium suspension obtained in S1 was injected into the tube using a 1 mL sterile syringe without a needle as follows: Figure 2 The combination was injected into the back of the leaf, and the inoculated Nicotiana benthamiana was placed in a light incubator for culture. Samples were taken 48 hours after inoculation, and the samples were detected by 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 N. benthamiana sample, which was similar in size to proPep1 TuMV -YFP fusion protein size corresponds to; while 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 correspond. The above results prove that TuMV NIa-Pro can recognize and cut proPep1 in this system TuMV and activates Pep1 expression.

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

[0057] S1: Prepare wild-type Arabidopsis Col-0 with good growth and luxuriant shoots, remove the Arabidopsis seed pods, and place in a constant temperature incubator for use; take 100 μl of the pEarley101-proPep1 obtained in Example 2 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 for overnight culture; 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 gently pipetted with a Pasteur pipette to mix, and 5% sucrose solution was added according to the OD value, and then the sucrose solution was diluted to OD 600 The concentration of silwet L-77 was 1.0, and 15 μl of silwet L-77 was added to every 50 mL of liquid. The mixture was mixed by blowing and the inflorescence was fully immersed for 20 seconds. The inflorescence was fixed, and the Arabidopsis thaliana was wrapped with plastic wrap on 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 harvesting.

[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 for 3 times, once every other day. Arabidopsis with good growth were selected for transplanting, and transgenic Arabidopsis 35S::proPep1 was obtained after the Arabidopsis grew to a suitable size. 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 -YFP disease resistance efficiency, wild-type Arabidopsis Col-0 and transgenic Arabidopsis 35S::proPep1 were cultured to four weeks old. TuMV -YFP, the above two Arabidopsis were injected with TuMV-6K2mCherry, and the fluorescence signal was observed after 11 days of culture. Figure 4 As shown in Figure 2, wild-type Arabidopsis Col-0 and 35S::proPep1-YFP can both 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 is about 1 / 20 of the total plant area, and the virus-infected area of ​​wild-type Arabidopsis Col-0 is about 1 / 3 of the total plant area; compared with wild-type Arabidopsis, transgenic Arabidopsis has stronger disease resistance.

[0062] In this embodiment, AtEF1α-F (SEQ ID NO.16) and AtEF1α-R (SEQ ID NO.17) were used as primers, AtEF1α of Arabidopsis thaliana was used as the internal reference gene, and TuMV CP-RTF (SEQ ID NO.18) and TuMV CP-RTR (SEQ ID NO.19) were used as primers to detect the expression level of TuMV-CP gene by qPCR. 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 transgenic Arabidopsis thaliana 35S::proPep1 expressing the proPep1 mutant gene provided by the present invention TuMV- YFP has good disease resistance without affecting its own growth and development, avoiding problems such as short plants and slow development caused by the plant's own growth-defense trade-off strategy.

[0064] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A proPep1 mutant gene, characterized in that: The proPep1 mutant gene sequence has a mutation of 9 bases compared to the proPep1 gene with the nucleotide sequence shown in SEQ ID NO.1, and the mutation is that the bases at positions 199-207 of the sequence shown in SEQ ID NO.1 mutate from ACGAGTAGA to TATCACCAG.

2. The proPep1 mutant gene according to claim 1, characterized in that The nucleotide sequence of the proPep1 mutant gene is shown in SEQ ID NO.

2.

3. The proPep1 mutant gene according to claim 1, characterized in that The proPep1 mutant gene is expressed through the 35S constitutive promoter of the cauliflower mosaic virus CaMV.

4. A protein encoded by a proPep1 mutant gene, characterized in that: 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 at positions 67-69 of the sequence shown in SEQ ID NO.3 mutate from TSR to YHQ.

5. The protein encoded by the proPep1 mutant gene according to claim 4, characterized in that: The protein sequence encoded by the proPep1 mutant gene is shown in SEQ ID NO.

4.

6. A carrier, characterized in that The vector contains the proPep1 mutant gene according to any one of claims 1 to 3 or expresses the protein encoded by the proPep1 mutant gene according to any one of claims 4 to 5.

7. A method for preparing a transgenic plant resistant to potato virus Y, characterized in that: The preparation method comprises the following steps: constructing a recombinant vector expressing a proPep1 mutant gene, transforming the recombinant vector into GV3101 Agrobacterium to obtain a recombinant Agrobacterium, and then transferring the recombinant Agrobacterium into a plant to obtain a transgenic plant.

8. The preparation method according to claim 7, characterized in that: The plant is wild-type Arabidopsis thaliana Col-0.

9. Use of the proPep1 mutant gene according to any one of claims 1 to 3, the protein encoded by the proPep1 mutant gene according to any one of claims 4 to 5, or the vector according to claim 6 in resisting plants infected by potyvirus.

10. The use according to claim 9, characterized in that: The potato Y virus is turnip mosaic virus TuMV.

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