A method for rapid and accurate screening of transgenic potato plants

Abstract

The application discloses a method for quickly and accurately screening potato transgenic plants, and the method comprises the following steps: constructing a recombinant expression vector by using Rpi-vnt1.1 fused with a GFP-Myc label, performing an agrobacterium-mediated genetic transformation experiment on potato plants, extracting leaf proteins after the transgenic seedlings grow under normal culture, and verifying positive potato transgenic seedlings by using Western blotting and combining with a late blight fungus inoculation experiment. By constructing the overexpression vector fused with the label, the potato transgenic seedlings can be quickly and accurately detected by Western blotting, a large number of false positives generated by PCR detection can be effectively avoided, and a large amount of work in the screening process of the potato transgenic seedlings is reduced. The application is accurate and efficient, and can be widely applied to the technical field of biology.

Description

Technical Field

[0001] This invention belongs to the field of transgenic plant detection technology, specifically relating to a method for rapidly and accurately screening transgenic potato plants. Background Technology

[0002] Potatoes, as the world's third largest food crop, play a vital role in ensuring global food security and promoting economic development in some regions. Potato late blight, caused by Phytophthora blight, seriously threatens potato production. Therefore, identifying late blight resistance genes and creating resistant materials is a sustainable and effective means of controlling late blight.

[0003] Due to the long breeding cycle and the highly heterozygous genome of potatoes, the transfer of late blight resistance genes (R) into susceptible varieties through traditional breeding methods is extremely limited. With the continuous development of biotechnology, several methods have emerged to overcome hybridization barriers and shorten the time required to introduce resistance genes into susceptible varieties, such as somatic cell hybridization and genetic engineering. Genetic engineering can significantly shorten the cycle of introducing resistance genes into tetraploid potatoes. Researchers have introduced the resistance genes Rpi-vnt1.1 and Rpi-sto1 into Atlantic, Bintje, and Potae 9, respectively, and tests have shown that these transgenic materials are resistant to most strains. Other researchers have transferred Rpi-vnt1.1 into the susceptible variety Desiree, and the transgenic material exhibits complete resistance to late blight. Still other researchers have used genetic engineering to simultaneously introduce multiple resistance genes into a single resistant variety, enabling the transgenic material to develop broader and more durable resistance to late blight pathogens. Although transgenic materials exhibit excellent resistance to late blight, the screening process for transgenic materials is extremely labor-intensive, particularly due to the high probability of false positives. Currently, potato transgenic engineering is conducted using Agrobacterium-mediated transformation systems. Through years of research and optimization, the induction conditions for Agrobacterium-mediated transformation have been determined, and transformation efficiency has been improved by altering the species of Agrobacterium tumefaciens, changing the T-DNA insertion sequence, and enhancing the expression of virulence genes. However, research on the detection of transgenic plants after transformation is limited, and most studies rely on PCR to detect the target gene or the vector backbone to determine whether the target gene has been successfully introduced. When Agrobacterium is introduced into the plant but fails to insert the T-DNA into the plant genome, false positives occur, and these false positives are very high. This increases the workload for researchers in detecting transgenic lines, thus requiring significant investment of human and material resources.

[0004] Current research on the detection of transgenic plants is limited, and the existing methods are often simplistic, leading to numerous false positives and increased workload. To address this issue, this paper presents a vector incorporating the GFP-Myc tag, enabling rapid and accurate screening of transgenic materials. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapidly and accurately screening transgenic potato plants.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for a method for rapidly and accurately screening transgenic plants, wherein, during the genetic transformation of plants, an expression vector containing a GFP-Myc fusion gene driven by a 35S promoter inserted into the T-DNA region is used; the nucleotide sequence of the GFP-Myc fusion gene is shown in SEQ ID NO:1.

[0008] Furthermore, the method for constructing the expression vector includes the following steps:

[0009] (1) Using amplification primers, DNA fragments of the 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were obtained by PCR amplification;

[0010] The upstream and downstream primers used to amplify the 35S promoter are shown in SEQ ID NO:3 and SEQ ID NO:4; the upstream and downstream primers used to amplify the GFP coding sequence are shown in SEQ ID NO:5 and SEQ ID NO:6; the upstream and downstream primers used to amplify the Myc coding sequence are shown in SEQ ID NO:7 and SEQ ID NO:8; and the upstream and downstream primers used to amplify the NOS terminator are shown in SEQ ID NO:9 and SEQ ID NO:10. The specific sequences of each primer are shown below:

[0011] 35S promoter amplification primers:

[0012] Upstream primer: 5'-GTCGCGAGCGATCGCGGTACCGCAAGAATTCAAGCTTGGAGGG-3' (SEQ ID NO:3)

[0013] Downstream primer: 5'-GGGGTCGACTGCAGAATTCG-3' (SEQ ID NO:4)

[0014] GFP amplification primers:

[0015] Upstream primer: 5'-CGAATTCTGCAGTCGACcccATGGTGAGCAAGGGCGAGGA-3' (SEQ ID NO:5)

[0016] Downstream primer: 5'-TACAAGAAAGCTGGGTCcccCTTGTACAGCTCGTCCATGCC-3' (SEQ ID NO:6)

[0017] Myc amplification primers:

[0018] Upstream primer: 5'-GGGGACCCAGCTTTCTTGTACAAAGTG-3' (SEQ ID NO:7)

[0019] Downstream primer: 5'-GCGGACTCTAGATCAGGTGGATC-3' (SEQ ID NO:8)

[0020] NOS terminator amplification primers:

[0021] Upstream primer: 5'-CCACCTGATCTAGAGTCCGCGATCTAGTAACATAGATGACACCGC-3' (SEQ ID NO: 9)

[0022] Downstream primer: 5'-ATGTGCATCCTCTAGTAGCGGATCGTTCAAACATTTGGCAAT-3' (SEQ ID NO:10);

[0023] (2) The 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were sequentially linked using homologous recombination technology and inserted into the T-DNA region of the plant transformation plasmid to construct the intermediate vector pPZP-RCS::GFP-Myc;

[0024] (3) Insert the DNA sequence of the target gene into the T-DNA region of the intermediate vector to obtain the expression vector.

[0025] Secondly, the present invention claims protection for an expression vector for rapid screening of transgenic plants, the expression vector being a vector in which a GFP-Myc fusion gene driven by a 35S promoter is inserted into the T-DNA region; the nucleotide sequence of the GFP-Myc fusion gene is shown in SEQ ID NO:1.

[0026] Furthermore, the expression vector is constructed using the following method:

[0027] (1) Using amplification primers, DNA fragments of the 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were obtained by PCR amplification;

[0028] The upstream and downstream primers used to amplify the 35S promoter are shown in SEQ ID NO:3 and SEQ ID NO:4; the upstream and downstream primers used to amplify the GFP coding sequence are shown in SEQ ID NO:5 and SEQ ID NO:6; the upstream and downstream primers used to amplify the Myc coding sequence are shown in SEQ ID NO:7 and SEQ ID NO:8; and the upstream and downstream primers used to amplify the NOS terminator are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0029] (2) The 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were sequentially linked using homologous recombination technology and inserted into the T-DNA region of the plant transformation plasmid to construct the intermediate vector pPZP-RCS::GFP-Myc;

[0030] (3) Insert the DNA sequence of the target gene into the T-DNA region of the intermediate vector to obtain the expression vector.

[0031] The tag GFP mentioned in this invention is a green fluorescent protein sequence, and Myc is composed of 10 consecutive Myc gene sequences.

[0032] In the technical solution of this invention, the plant transformation plasmid is pPZP-RCS.

[0033] In this invention, the plant is a member of the Solanaceae family, preferably tobacco, tomato, or potato. The target gene is a plant disease resistance gene. Taking potato as an example, the target gene is a potato disease resistance gene, such as the potato late blight resistance gene Rpi-vnt1.1.

[0034] In a specific embodiment of the present invention, the GFP-Myc fusion gene driven by the 35S promoter is inserted into the T-DNA region of the pPZP-RCS vector, with the restriction enzyme site being Kpn I, to construct the pPZP-RCS::GFP-Myc vector. The Rpi-vnt1.1 sequence is inserted into pPZP-RCS::GFP-Myc via the Asc I restriction site, and the recombinant vector is pPZP-RCS::Rpi-vnt1.1-GFP-Myc.

[0035] Thirdly, the present invention claims protection for a recombinant microorganism comprising the aforementioned expression vector. Preferably, the recombinant microorganism is obtained by introducing the aforementioned recombinant vector into a host cell, wherein the host cell is preferably an *Escherichia coli* cell or an *Agrobacterium* cell.

[0036] Fourthly, the present invention claims protection for the use of the aforementioned expression vector in any of the following:

[0037] (1) Screening for disease-resistant transgenic plants;

[0038] (2) Prepare a kit for screening disease-resistant transgenic plants;

[0039] (3) Create new plant germplasm that is resistant to plant diseases.

[0040] Fifthly, the present invention seeks protection for the use of biological materials comprising the aforementioned expression vector in any of the following:

[0041] (1) Screening for disease-resistant transgenic plants;

[0042] (2) Prepare a kit for screening disease-resistant transgenic plants;

[0043] (3) Create new plant germplasm that is resistant to plant diseases;

[0044] The biological material containing the expression vector is at least one of the following (a1) to (a7):

[0045] (a1) Recombinant microorganisms containing the expression vector;

[0046] (a2) A transgenic plant cell line containing the expression vector described above;

[0047] (a3) Transgenic plant tissue containing the expression vector;

[0048] (a4) Transgenic plant organs containing the expression vector described above;

[0049] (a5) Transgenic plant plants containing the expression vector described above;

[0050] (a6) Tissue cultures produced from regenerative cells of the transgenic plant described in (a5);

[0051] (a7) Protoplasts produced from the tissue culture described in (a6).

[0052] Furthermore, the above application involves using the expression vector in the genetic transformation process of plants to detect the expression of GFP or Myc proteins by Western blotting, thereby screening out transgenic plants that have successfully introduced and expressed the target gene.

[0053] Furthermore, the above applications include screening transgenic plant materials for whether they contain functional disease-resistant genes and whether these genes are expressed, thereby improving plant resistance to late blight or creating new plant germplasm resistant to late blight for production application.

[0054] In the technical solution of the present invention, the plant disease is a disease caused by oomycete pathogens (such as late blight); the disease resistance is preferably resistance to oomycete pathogens (such as Phytophthora infestans) or diseases caused by oomycete pathogens (such as late blight); more preferably, resistance to late blight.

[0055] The beneficial effects of this invention are:

[0056] This invention enables rapid and accurate detection of whether disease-resistance genes have been successfully introduced into transgenic materials, and simultaneously determines whether these genes are successfully expressed. Detection can be performed on transgenic materials at the seedling stage. Proteins are extracted from leaves, and Western blot analysis reveals bands containing GFP or Myc. Observing the band size indicates successful introduction and expression of the disease-resistance gene. This invention avoids false positives and rapidly screens for target transgenic plants, significantly improving the later-stage resistance identification work for transgenic plants. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of the recombinant vector; a GFP-Myc tag driven by a 35S promoter is inserted into the T-DNA region of pPZP-RCS, and the recombinant vector is pPZP-RCS::GFP-Myc. Then, the disease resistance gene Rpi-vnt1.1 is inserted, and the vector is finally recombined into pPZP-RCS::Rpi-vnt1.1-GFP-Myc.

[0058] Figure 2 To detect the agarose gel electrophoresis images of transgenic potato seedlings and to assess the introduction of disease resistance genes into 18 transgenic potato seedlings, forward primers were designed on the vector, and reverse primers were designed for the Rpi-vnt1.1 promoter region. PCR was performed using genomic DNA from the transgenic material as a template. The negative control (-) was the non-transgenic potato plant Desiree; the positive control (+) was the pPZP-RCS::Rpi-vnt1.1-GFP-Myc plasmid.

[0059] Figure 3 Western blot images of transgenic potato seedlings were obtained to detect the introduction of disease resistance genes in 18 transgenic potato seedlings. The antibody used for detection was Myc antibody. The negative control (-) was the non-transgenic potato plant Desiree; the positive control (+) was the tobacco protein transiently expressed by pPZP-RCS::Rpi-vnt1.1-GFP-Myc.

[0060] Figure 4 This study tested the resistance of transgenic potato seedlings; the negative control (-) was the non-transgenic potato plant Desiree; the positive control (+) was the disease-resistant variety. Detailed Implementation

[0061] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The primers involved in the embodiments of the present invention were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0062] Example 1. Construction of pPZP-RCS::Rpi-vnt1.1-GFP-Myc expression vector

[0063] Primers were designed, and PCR amplification was performed using plasmids containing GFP and Myc tags (pBinGFP2, NovoPro; pBin308, CN120484081B) as templates, respectively. The PCR products were recovered by electrophoresis and gel excision to obtain the GFP coding sequence and the Myc coding sequence, respectively.

[0064] PCR amplification was performed using pBin308 (CN120484081B) as a template, and the PCR product was recovered by electrophoresis and gel extraction to obtain the 35S promoter.

[0065] PCR amplification was performed using pBin308 (CN120484081B) as a template, and the PCR product was recovered by electrophoresis and gel excision to obtain the NOS terminator.

[0066] Using homologous recombination technology and the Novizan ClonExpress II One Step Cloning Kit, the 35S promoter, GFP coding sequence, Myc coding sequence, and NOS terminator obtained by PCR amplification were ligated together into the Kpn I digested pPZP-RCS vector (pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants.) to obtain the pPZP-RCS::GFP-Myc recombinant vector, which served as an intermediate vector.

[0067] Then, using potato genomic DNA as a template, PCR amplification was performed on the Rpi-vnt1.1 sequence containing its own promoter and terminator. The PCR product was recovered by electrophoresis gel excision and ligated into the Asc I digested pPZP-RCS::GFP-Myc recombinant vector to obtain the pPZP-RCS::Rpi-vnt1.1-GFP-Myc recombinant expression vector. Figure 1 The constructed recombinant expression vector was introduced into Agrobacterium competent cells using Agrobacterium transformation technology to obtain Agrobacterium containing the pPZP-RCS::Rpi-vnt1.1-GFP-Myc recombinant expression vector.

[0068] The primers designed for PCR amplification are shown below:

[0069] (1) Primers for 35S promoter amplification:

[0070] Upstream primer: 5'-GTCGCGAGCGATCGCGGTACCGCAAGAATTCAAGCTTGGAGGG-3' (SEQ ID NO:3)

[0071] Downstream primer: 5'-GGGGTCGACTGCAGAATTCG-3' (SEQ ID NO:4)

[0072] (2) GFP amplification primers:

[0073] Upstream primer: 5'-CGAATTCTGCAGTCGACcccATGGTGAGCAAGGGCGAGGA-3' (SEQ ID NO:5)

[0074] Downstream primer: 5'-TACAAGAAAGCTGGGTCcccCTTGTACAGCTCGTCCATGCC-3' (SEQ ID NO:6)

[0075] (3) MYC amplification primers:

[0076] Upstream primer: 5'-GGGGACCCAGCTTTCTTGTACAAAGTG-3' (SEQ ID NO:7)

[0077] Downstream primer: 5'-GCGGACTCTAGATCAGGTGGATC-3' (SEQ ID NO:8)

[0078] (4) NOS terminator amplification primers:

[0079] Upstream primer: 5'-CCACCTGATCTAGAGTCCGCGATCTAGTAACATAGATGACACCGC-3' (SEQ ID NO: 9)

[0080] Downstream primer: 5'-ATGTGCATCCTCTAGTAGCGGATCGTTCAAACATTTGGCAAT-3' (SEQ ID NO:10)

[0081] (5) Rpi-vnt1.1 amplification primers:

[0082] Upstream primer: 5'-CATTTAAATTCTAGAGGCACTAGTGATTAGTTATACACCCTAC-3' (SEQ ID NO:11)

[0083] Downstream primer: 5'-TTAAGAGAGGATATCGGCTTTGAAAAGAGGCTTCATACTCC-3' (SEQ ID NO:12).

[0084] Example 2: Transformation of potatoes with the pPZP-RCS::Rpi-vnt1.1-GFP-Myc recombinant expression vector to construct transgenic potato seedlings

[0085] (1) Agrobacterium preparation: Agrobacterium containing the pPZP-RCS::Rpi-vnt1.1-GFP-Myc recombinant expression vector was taken from -80℃ and streaked on LB plates containing spectinomycin (50 mg / L) and rifampin (50 mg / L) to grow single colonies. The cells were cultured at 28-30℃ for 2 days and then cultured by shaking. The buffer used in this process was MS20 containing a final concentration of 0.1 mM AS. Therefore, the cells were resuspended in MS20 liquid medium and the concentration of Agrobacterium was adjusted to OD600=0.5. The volume was adjusted to 20 mL to infect the potato stem segments.

[0086] (2) Pre-culture of explants: In Z1N2 medium, lay two sterile filter papers, add 2 ml of PACM, cut a certain number of potato stem segments, about 20 explants per plate, arrange them neatly, and culture under light for 60-72 h.

[0087] (3) Agrobacterium infection of potato explants: Place the pre-cultured stem segments in a bacterial solution with an OD600 of 0.5 and infect for 10-15 min, shaking continuously during the process. Spread a filter paper on a Z1N2AS culture plate and place the infected stem segments in it. Incubate in the dark at 24℃ for 60-72 h.

[0088] (4) Callus differentiation: Explants were cultured in recovery medium for one week, and then placed on differentiation medium Z2N0.01 to continue culture to form callus. The Z2N0.01 medium was changed every two weeks until the callus differentiated into shoots.

[0089] (5) Screening of transformed lines on rooting medium: When the callus differentiates into buds, the buds are cut off and transferred to a rooting medium containing a high concentration of antibiotics for screening. Successfully transformed plants will grow roots from the cut. A bud tip of about 1-1.5 cm is cut off from the normally rooted plant and then inserted into the screening medium for secondary screening. Finally, transgenic potato seedlings are obtained.

[0090] The reagents, antibiotics, and culture medium formulations used in the experiment:

[0091] A: 1M (i.e., 1mol / L) NaOH: Weigh 2g of NaOH and add water to 50mL.

[0092] B: Naphthaleneacetic acid (NAA) stock solution: (1) 10 mg / mL: Weigh 100 mg NAA, add a small amount of 1M NaOH to aid dissolution, and add water to 10 mL. Filter sterilize and dispense into sterile 1.5 mL centrifuge tubes. Store at -20℃ for later use. (2) 0.1 mg / mL: Take 100 μL of 10 mg / mL NAA stock solution, dilute to 9.9 mL of water, filter sterilize and dispense into sterile 1.5 mL centrifuge tubes. Store at -20℃ for later use.

[0093] C. Acetyleugenone AS (40 mg / mL): Weigh 4g AS (Solepro Cat# IA2900), dissolve in 100 mL methanol or DMSO (dimethyl sulfoxide), filter sterilize after dissolution, dispense into 15 mL centrifuge tubes, wrap in aluminum foil to protect from light, and store at -20℃ for later use.

[0094] D: Zeazone ZT (1 mg / ml): Weigh 0.2g ZT (Solepro Cat#T8110), dissolve in a small amount of 1M NaOH, add 200 mL of sterile ddH2O, filter sterilize and dispense into 15mL centrifuge tubes, and store at -20℃ for later use.

[0095] E: TMT (100 mg / ml): Weigh 10 g TMT (Solepro, Cat# T8660), dissolve in 100 mL sterile ddH2O, filter sterilize, dispense into 15 mL centrifuge tubes, and store at -20℃ for later use.

[0096] F: 2,4-Dichlorophenoxyacetic acid 2,4-D (1 mg / mL): Weigh 0.01 g of 2,4-D, add a small amount of 1M NaOH to aid dissolution, dissolve in 10 mL of sterile ddH2O, filter sterilize, dispense into 1.5 mL centrifuge tubes, and store at -20℃ for later use.

[0097] G: Rifampicin (25 mg / mL): 0.25 g of rifampicin (Solepro, cat# R8011) was dissolved in 10 mL of methanol or ethanol, filtered and sterilized, and dispensed into sterile 1.5 mL centrifuge tubes.

[0098] H: Spectinomycin (50 mg / mL): 1 g spectinomycin (Solepro, cat# S8040) was dissolved in 20 mL of sterile ddH2O, filtered and sterilized, and dispensed into 1.5 mL centrifuge tubes. Store at -20℃ for later use.

[0099] I: Kanamycin (Kana) (50 mg / mL): Weigh 5 g of Kana (Solepro, cat# K8020), dissolve in 100 mL of sterile ddH2O, filter sterilize, and dispense into 1.5 mL centrifuge tubes. Store at -20℃ for later use.

[0100] J: MS20 liquid medium (1L): Weigh 4.43g MS powder (PhytoTech LABS, cat#M516), 20g sucrose, and bring the volume to 1L.

[0101] K: PACM: 0.44g MS powder, 0.2g hydrolyzed casein (caseine hydrolysate, a standard reagent), 3g sucrose, 100 μL 2,4-D (1 mg / mL), 50 μL kinetine (1 mg / mL), pH 6.5, bring to a final volume of 100 mL.

[0102] L: MS20 solid medium: Add 9 g of agar to MS20 liquid and bring the volume to 1 L.

[0103] M: Z1N2: 2.215 g MS powder, 10 g sucrose, 1.55 g plant gel, 500 μL ZT, 100 μL NAA (10 mg / ml), bring to a final volume of 500 mL.

[0104] N: Z1N2AS (1L): Add 1 mL ZT, 200 μL NAA (10 mg / ml), and 1 mL AS to 1 L MS20.

[0105] O: Recovery medium (1L): Add 2 mL ZT, 100 μL NAA (0.1 mg / ml), and 2 mL TMT to 1L MS20.

[0106] P: Z2N0.01 (1L): Add 2 mL ZT, 100 μL NAA (0.1 mg / ml), 2 mL TMT, and 2 mL Kana to 1 L MS20.

[0107] Q: Rooting medium (1L): 1 L MS20, 2 mL TMT, 2 mL Kana.

[0108] Example 3. The resistance of the transgenic potato line Rpi-vnt1.1 was detected by PCR and Western blot, respectively.

[0109] Genomic DNA and protein were extracted from leaves of 4-5 week old regenerated seedlings obtained from the transformation. Transgenic potato seedlings were cultivated in pots containing vermiculite and nutrient soil (mass ratio 1:1) and cultured under alternating light and dark conditions at 22℃ for 16 h of light and 18℃ for 8 h of darkness.

[0110] Genomic DNA extraction: Potato leaves were taken, flash-frozen in liquid nitrogen, and then ground into powder using a sampler. The DNA concentration was then measured using the Novizan Plant Genomic DNA Extraction Kit and stored at -20°C.

[0111] Protein extraction: Take potato leaves, freeze them in liquid nitrogen, grind them into powder using a sample grinder, add protein extraction solution (25 μl of 4×Laemmli sample buffer, 10 μl of 1 M DTT, 65 μl of ddH2O), mix immediately, boil at 95℃ for 10 min, then centrifuge and store at -20℃.

[0112] (1) Detection of transgenic potato seedlings by PCR amplification. Upstream primers (5'-TGGCTGGTGGCAGGATAT-3') and downstream primers (5'-GGATGAGGAAGGGAAAGTAAGAG-3') were used on the vector and the target gene, respectively, with a total length of 918 bp. Amplification was performed using the genome as a template, with plasmids as positive controls and non-transgenic potatoes as negative controls. Results showed that, except for the negative control, all other transgenic seedlings amplified bands of the same size as the positive control. Figure 2 ).

[0113] (2) Detection of transgenic potato seedlings by Western blot. 10 μl of sample was subjected to denaturing electrophoresis on an SDS-PAGE gel. The gel was run at 80 V for 30 minutes and then at 120 V for 1.5 hours. After the reaction, the sample was transferred to PVDF and incubated in 5% (g / 100ml) TBST skim milk powder for 1 hour. MYC primary antibody (anti-c-Myc antibody, Abmart, M20002L) diluted 1:5000 was added and incubated for 2 hours. The membrane was washed three times for 5 minutes with TBST. Then, mouse antibody (IRDye 800CW Goat anti-Mouse IgG Secondary Antibody, LICORbio) diluted 1:20000 was added and incubated for 1 hour. The membrane was washed three times with TBST, and the membrane was scanned and photographed. The presence or absence of bands was used to determine whether the seedling was transgenic. Figure 3 ).

[0114] (3) Detection of resistance of transgenic potato seedlings to late blight strain. After the transgenic potato cultivar Desiree carrying Rpi-vnt1.1 was grown under the above conditions for 5-6 weeks, three-leaf sections were inoculated with zoospores. Zoospores of late blight strain JH19 were inoculated onto the inoculation tray with the dorsal side facing up, and the resistance phenotype was observed 4-5 days later. Compared with the negative control, the vast majority of transgenic seedlings did not exhibit resistance, and the resistant transgenic seedlings were consistent with those showing the target band detected by Western blot (…). Figure 4 ).

[0115] This invention uses an expression vector containing a GFP-Myc fusion gene driven by a 35S promoter inserted into the T-DNA region to transfer the disease resistance gene into potatoes. The success of the introduction and expression of the disease resistance gene can be determined by observing the size of the GFP or Myc bands detected by Western blot. Compared to the PCR amplification method used in step (1) of Example 3, the detection of transgenic potato seedlings by Western blot in step (2) of Example 3 demonstrates that protein extraction is faster and simpler than DNA extraction, protein detection avoids false positives, and ensures that the T-DNA region is inserted into the genome.

[0116] sequence list

[0117]

[0118] GFP-Myc fusion (SEQ ID NO:2):MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFKSAMPEG YVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGDPAFLYKVVDNSGLINGEQLISEEDLNGEQLISEEDLNGGEQKLI SEEDLNGEQLISEEDLNGEQLISEEDLNGLDGEQLISEEDLNGEQLISEEDLNGEQLISEEDLNGLDGEQLISEEDLNGEQLISEEDLNGA。

Claims

1. A method for screening transgenic potato plants, characterized in that, In the genetic transformation of potatoes, an expression vector containing a GFP-Myc fusion gene driven by a 35S promoter inserted into the T-DNA region was used; the nucleotide sequence of the GFP-Myc fusion gene is shown in SEQ ID NO:1; the construction method of the expression vector includes the following steps: (1) Using amplification primers, DNA fragments of the 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were obtained by PCR amplification; The upstream and downstream primers used to amplify the 35S promoter are shown in SEQ ID NO:3 and SEQ ID NO:4; the upstream and downstream primers used to amplify the GFP coding sequence are shown in SEQ ID NO:5 and SEQ ID NO:6; the upstream and downstream primers used to amplify the Myc coding sequence are shown in SEQ ID NO:7 and SEQ ID NO:8; and the upstream and downstream primers used to amplify the NOS terminator are shown in SEQ ID NO:9 and SEQ ID NO:

10. (2) The 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were sequentially linked using homologous recombination technology and inserted into the T-DNA region of the plant transformation plasmid to construct an intermediate vector; (3) Insert the DNA sequence of the target gene into the T-DNA region of the intermediate vector to obtain the expression vector; (4) The recombinant vector is introduced into the host cell to obtain recombinant microorganisms, the recombinant microorganisms are used to transform plant tissues, and the expression of GFP or Myc protein is detected by Western blotting, thereby screening out transgenic plants that have successfully introduced and expressed the target gene.

2. The method according to claim 1, characterized in that, The plant transformation plasmid is pPZP-RCS; the target gene is a plant disease resistance gene.

3. An expression vector for screening transgenic potato plants, characterized in that, This expression vector is a vector in which a GFP-Myc fusion gene driven by a 35S promoter is inserted into the T-DNA region; the nucleotide sequence of the GFP-Myc fusion gene is shown in SEQ ID NO:1; the expression vector is constructed by the following method: (1) Using amplification primers, DNA fragments of the 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were obtained by PCR amplification; The upstream and downstream primers used to amplify the 35S promoter are shown in SEQ ID NO:3 and SEQ ID NO:4; the upstream and downstream primers used to amplify the GFP coding sequence are shown in SEQ ID NO:5 and SEQ ID NO:6; the upstream and downstream primers used to amplify the Myc coding sequence are shown in SEQ ID NO:7 and SEQ ID NO:8; and the upstream and downstream primers used to amplify the NOS terminator are shown in SEQ ID NO:9 and SEQ ID NO:

10. (2) The 35S promoter, GFP coding sequence, Myc coding sequence and NOS terminator were sequentially linked using homologous recombination technology and inserted into the T-DNA region of the plant transformation plasmid to construct an intermediate vector; (3) Insert the DNA sequence of the target gene into the T-DNA region of the intermediate vector to obtain the expression vector.

4. The expression vector according to claim 3, characterized in that, The plant transformation plasmid is pPZP-RCS; the target gene is a plant disease resistance gene.

5. A recombinant microorganism, characterized in that, The recombinant microorganism comprises the expression vector as described in claim 3 or 4.

6. The use of the expression vector according to claim 3 in any of the following: (1) Screening for disease-resistant transgenic potato plants; (2) Prepare a kit for screening transgenic potato plants resistant to disease; (3) Create new potato germplasm that is resistant to plant diseases.

7. Use of the biomaterial comprising the expression vector of claim 3 in any of the following: (1) Screening for disease-resistant transgenic potato plants; (2) Prepare a kit for screening transgenic potato plants resistant to disease; (3) Create new potato germplasm that is resistant to plant diseases; The biological material containing the expression vector is at least one of the following (a1) to (a7): (a1) Recombinant microorganisms containing the expression vector; (a2) A transgenic potato cell line containing the expression vector described above; (a3) Transgenic potato tissue containing the expression vector described above; (a4) Transgenic potato organs containing the expression vector described above; (a5) Transgenic potato plants containing the expression vector described above; (a6) Tissue cultures produced from regenerative cells of the transgenic plant described in (a5); (a7) Protoplasts produced from the tissue culture described in (a6).

8. The application according to claim 6, characterized in that, In the genetic transformation of potatoes, the expression vector was used to detect the expression of GFP or Myc protein by Western blotting, thereby screening out transgenic plants that successfully introduced and expressed the target gene.

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