Rootstock and scion dual-purpose transgenic anti-TMV tomato and cultivation method thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a rootstock and scion dual-purpose transgenic anti-TMV tomato and a cultivation method thereof, a TMV MP RNAi overexpression vector, the structure of which is: a CmYLCV promoter, a TMV MP positive sequence, a PDK intron sequence, a TMV MP inverse sequence and an octopus alkali terminator; the TMV MP RNAi overexpression vector is introduced into a tomato through an agrobacterium transformation method, and the transgenic anti-TMV tomato with both the rootstock and the scion is obtained through screening. TMV MP RNAi tomatoes are used as rootstocks or scions, sRNA or mRNA from transgenic plants can be inevitably received at non-transgenic rootstocks or scions due to overexpression of RNAi, and corresponding RNAi is generated through the received sRNA or mRNA, so that breeding of viruses at non-transgenic plant parts is inhibited.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a transgenic TMV-resistant tomato used as both a rootstock and a scion and a cultivation method thereof. Background Art

[0002] A 2019 survey revealed that 25 tomato-infecting viruses were detected (Liu et al. 2019). Tomato virus diseases often present a multi-virus complex infection pattern, with CMV having the highest detection rate, followed by TMV, followed by ToMV, TYLCV, and TSWV, all with average detection rates exceeding 5% (Liu et al. 2019). In tomato production, the distribution and infection rates of viruses vary across regions, with the highest detection rate of CMV in Shanxi (Jing Chenchen et al. 2024), and the highest detection rate of TYLCV in cherry tomatoes in Hainan (Che Haiyan et al. 2024). At the same time, the tomato industry faces severe challenges due to the recent detection and outbreak of new tomato viruses. Since TYLCV was introduced into my country in 2006, it has rapidly spread throughout the country within a decade and has dealt a devastating blow to the domestic tomato industry (Su Mingming et al. 2013). It was not until the development of TYLCV-resistant tomato varieties represented by Xi’an Agricultural University 2011 (Li Jigang et al. 2012) and the introduction of the resistance gene “TY-1” from Solanum habrochaites into tomatoes through hybrid breeding (Ma et al 2024, Prabhandakavi et al 2020) that the outbreak of TYLCV in China was gradually successfully controlled. However, in 2019, the first case of ToBRFV (Tomato Brown Wrinkled Fruit Virus) was reported in Yucheng, Shandong (Guo et al 2023). In 2021, the General Administration of Customs of China (Announcement No. 91 of the General Administration of Customs of 2021) issued inspection and quarantine requirements for this virus, showing that the new virus poses a serious threat to the tomato industry. Although ToBRFV belongs to the genus Tobacco mosaic virus in taxonomy, it overcomes TM-2 by the C68H mutation at position 68 of the movement protein MP. 2 Resistance to it (Cheng et al 2018, Hak and Spiegelman 2021, 2023). The continuous emergence of new viruses and the continuous breakthroughs in existing resistance genes have put the tomato industry under tremendous pressure.

[0003] Early discoveries revealed that when the chalcone synthase gene, responsible for anthocyanin synthesis, was introduced into petunias, the purple color of the petunias was largely lost in the perianth (Napoli et al. 1990). This phenomenon was subsequently replicated in species such as Drosophila and Alternaria (Matzke and Matzke 2004). It wasn't until 1998, when Fire et al. (Fire et al. 1998) demonstrated that injecting double-stranded RNA (dsRNA) contaminated with sense or antisense RNA could suppress the expression of the par-1 gene in Caenorhabditis elegans, a phenomenon known as RNA interference (RNAi).

[0004] One essential prerequisite for RNAi is dsRNA, or double-stranded RNA. Double-stranded RNA is an RNA with an antiparallel structure. After cleavage by DICER, dsRNA generates 21-24 nt sRNAs. These sRNAs then form RISC with AGO, leading to RNA cleavage or translational inhibition. RNAi can also be produced in organisms by artificially synthesizing hpRNA or sRNA with parallel and antiparallel structures. Within plants, sRNAs can also travel long distances through the vascular system to transmit their effects (Voinnet and Baulcombe 1997).

[0005] Post-transcriptional silencing of viral RNA is mediated by small RNAs (sRNAs) containing 21 or 22 nucleotides (nt) and Argonaute proteins (AGOs), resulting in cleavage of the target viral RNA or inhibition of translation (Carbonelland and Carrington 2015). AGOs are loaded with a 21nt single-stranded sRNA to form the silencing complex RISC, which then complementarily pairs with the viral RNA to specifically cleave the viral RNA. After the AGO protein is loaded with the 22nt sRNA, the additional base length allows its 3' end to extend from RISC. After binding to the 22nt RISC and recruiting SGS3, translating ribosomes bound to the viral mRNA are stopped by RISC, leading to the termination of translation (Baulcombe 2022). At the same time, SGS3 also recruits RNA-dependent RNA polymerase (RDR) to complete the viral single-stranded RNA into a double-stranded RNA, allowing DICER to cut it into secondary 22nt sRNA, amplifying the number of sRNA and effectively enhancing the cutting or translation inhibition of viral RNA (Lopez-Gomollon and Baulcombe 2022).

[0006] There are two general strategies for making transgenic plants resistant to viruses. One involves cloning "resistance genes" (not strictly limited to R genes) from other plants or the same plant species and then transferring them into the recipient plant, conferring resistance to certain viral diseases. The other involves introducing viral proteins or nucleic acid sequences to achieve antiviral effects (Yakufujiang Asimu et al.).

[0007] One of the earliest examples of plant resistance mediated by the introduction of viral components was the overexpression of the TMV capsid protein (CP) in tobacco, conferring TMV resistance to the transgenic tobacco plant (Yakufujiang Asimu et al.). However, this approach subsequently proved to be limited in effectiveness. For example, papaya transgenic for resistance to Cyclosporin virus (Cyclosporin) failed to confer immunity to another virus (Mo Cuiping et al. 2022, Zhang Yuliang et al. 2013). Ultimately, overexpression of the viral protein coding sequence led to co-suppression, inhibiting the reproduction of the virus itself. Transgenic introduction of antisense RNA sequences, or RNAi, into plants can also effectively inhibit viral reproduction and outbreaks. Examples include the US transgenic papaya variety "Rainbow" and my country's first transgenic papaya variety, "Huanong No. 1" (Jia Ruizong et al. 2024). Overexpression of RNAi against the CMV C2 gene in tobacco has also been reported to confer CMV resistance (Shen Baoming 2011, Zhang Qian et al. 2024). Interference targeting the overlapping 3'-UTR region of the CMV genome, 2a and 2b, can also alleviate symptoms and restore yield in tomatoes infected with CMV (Zhang Xiaohui 2010). RNA interference targeting the TMV replicase can also effectively protect tomatoes (Yang Gong et al. 2001, Yin Lei et al. 2016). Furthermore, synthetic sRNAs can be used as pesticides. Previous studies have shown that spraying dsRNA on the surface of vegetables or flowers can confer resistance to gray mold (Wang et al. 2016). Currently, pesticides using synthetic dsRNA as tobacco mosaic virus inhibitors (interferons) have been registered and approved (Gao Ying et al. 2024).

[0008] In view of the above analysis, the technical problems that urgently need to be solved in the existing technology are: there are currently transgenic RNAi disease-resistant tomatoes targeting CMV, and transgenic RNAi tomatoes targeting TMV, but without exception, RNAi is targeted at a certain gene or the entire genome of the virus. There are currently no transgenic RNAi tomatoes that only target a certain gene core sequence of TMV and the core gene sequence of members of the tobacco mosaic virus genus, nor are there tomatoes that enhance TMV virus disease resistance by grafting small RNA. Summary of the Invention

[0009] In response to the problems existing in the prior art, the present invention provides a transgenic TMV-resistant tomato that can be used as both a rootstock and a scion, and a cultivation method thereof.

[0010] The present invention is achieved by providing a TMV MP RNAi overexpression vector having the structure of: CmYLCV promoter-TMVMP positive sequence-PDK intron sequence-TMV MP reverse sequence-octopine terminator; the nucleotide sequence of the TMV MP RNAi overexpression vector is shown in SEQ ID NO.1.

[0011] SEQ ID NO.1.

[0012] The method for constructing the TMVMPRNAi overexpression vector comprises:

[0013] (1) Double-digest 1 μg of the vector PICSL4723_eGFP~3xFlag with 1 U of Xho1 and Xba1 at 37°C for 8 hours (PICSL4723 vector backbone from: Weber E, Engler C, Gruetzner R, et al, 2011)

[0014] (2) Separate the digestion products using 1% agarose gel electrophoresis: vector backbone fragment (~7400 bp) and discarded fragment (~500 bp);

[0015] (3) Using high-fidelity enzyme amplification, the reverse-transcribed TMV-infected tomato material was amplified to obtain the TMV viral MP sequence (with homologous recombination linker);

[0016] (4) Amplify the PDK (pyruvate dehydrogenase kinase) intron sequence of Flaveria trinervia using a high-fidelity enzyme to obtain the PDK intron;

[0017] (5) amplifying the vector backbone using a high-fidelity enzyme to impart a linker to the vector backbone;

[0018] (6) Separate the PCR products using 1% agarose gel electrophoresis: TMV_MP_RNAi sequence (~330 bp); PDKintr_HR sequence (~830 bp); PICSL4723_HR sequence (~7500 bp);

[0019] (7) Using a gel recovery kit (CW2302M), the above-mentioned nucleic acid fragments with adapters were recovered in sequence;

[0020] (8) assembling the above three fragments by homologous recombination;

[0021] (9) The homologous recombination product was transformed into Escherichia coli DH5α by heat shock method:

[0022] (10) The plasmid was extracted from Escherichia coli by alkaline lysis.

[0023] Furthermore, in step (4), primers are used:

[0024] TMV MP RNAi_HR_F:

[0025] 5-ggtataccgtcagtgATGGCTCTAGTTGTTAAAGGAAAAG-3

[0026] TMV MP RNAi_HR_R:

[0027] 5-tccgattgtaagaagGGCTCTTTCCATCCTTTTGTC-3 (lowercase bases are linker sequences)

[0028] Furthermore, in step (5), primers are used:

[0029] PDKintr_HR_F:

[0030] 5-cttcttacaatcggaGTAAGGAAAATAATTATTTTCTTTTTTCCTTTTAG-3

[0031] PDKintr_HR_R:

[0032] 5-cttcttacaatcggaCTGTAATCAATCCAAATGTAAGATCAATG-3 (lowercase bases are linker sequences)

[0033] Furthermore, in step (6), primers are used:

[0034] Cmps_HR_R:

[0035] 5-cggtataccCTCGAGCTACTTCTAGGCTAC-3

[0036] OCSter_HR_F:

[0037] 5-cactgacggtataccTCTAGAGGATCCCCGGGCGAGC-3 (lowercase bases are linker sequences).

[0038] Another object of the present invention is to provide a transgenic TMV-resistant tomato for both rootstock and scion. The TMV MP RNAi overexpression vector is introduced into tomatoes through Agrobacterium transformation, and the transgenic TMV-resistant tomato for both rootstock and scion is obtained through screening.

[0039] Another object of the present invention is to provide a method for cultivating transgenic TMV-resistant tomatoes for dual-purpose rootstock and scion, comprising the following steps:

[0040] S1, the extracted plasmid was transformed into Agrobacterium GV3101 by heat shock method, and resuspended Agrobacterium was obtained;

[0041] S2, wild-type tomatoes were sown and cultured on MS medium;

[0042] S3, infect the target plants with resuspended Agrobacterium and screen the positive plants.

[0043] Furthermore, solid MS medium: 10 g / L glucose was added as a seeding medium; 30 g / L sucrose and 0.2 μg / mL 2,4-chlorophenoxyacetic acid were added as a dedifferentiation medium; 30 g / L sucrose, 100 μg / L kanamycin, 0.1 μg / L indoleacetic acid, 25 μg / L cefotaxime, 320 μg / L timentin and 2 μg / L trans-zein nucleoside were added as redifferentiation medium 1; 30 g / L sucrose, 80 μg / L kanamycin, 25 μg / L cefotaxime, 265.6 μg / L timentin and 0.2 μg / L trans-zein nucleoside were added as redifferentiation medium 2; 30 g sucrose, 50 μg / L kanamycin, 2 μg / L indolebutyric acid and 320 μg / L timentin were added as a rooting medium.

[0044] Another object of the present invention is to provide a transgenic TMV-resistant tomato for use as a rootstock or scion.

[0045] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0046] First, the present invention designs TMV MP RNAi targeting the core sequence of TMV movement protein, which has a clear role, good effect, small vector size and high expression efficiency.

[0047] The present invention constructs a transgenic overexpression vector of TMV MP RNAi and transfers it into tomatoes; thus, a transgenic TMV-resistant tomato for both rootstock and scion is obtained. The TMV MP RNAi tomato is used as the rootstock or scion. Due to the overexpression of RNAi, the non-transgenic rootstock or scion part can inevitably receive sRNA or mRNA from the transgenic plant, and then generate corresponding RNAi through the received sRNA or mRNA, thereby inhibiting the reproduction of the virus in the non-transgenic plant part.

[0048] Second, as auxiliary evidence for the inventiveness of the present invention's claims, it is also reflected in the following important aspects:

[0049] (1) The technical solution of the present invention fills the technical gap in the industry at home and abroad: there is currently no tomato that enhances TMV virus disease resistance by grafting small RNA. The current industry mainly focuses on cultivating tomatoes with TMV resistance genes TM-1 / TM-2 / TM-2A, but with the breakthrough of ToBRFV on TM-2A, there are basically no new ToBRFV resistance genes that can be confirmed (except those still under development and breeding). Due to the homology of the tobacco mosaic virus MP, the RMAi tomatoes developed in the same way can also obtain ToBRFV resistance (Cheng et al 2018, Hak and Spiegelman 2021, 2023), filling the industry gap in the inability to obtain new virus resistance before the development of resistance genes.

[0050] (2) The technical solution of the present invention overcomes technical prejudice: Currently, most of the existing transgenic virus-resistant tomatoes are directly produced and harvested as the production target of transgenic virus-resistant tomatoes. Previously, there were no more reports on transgenic rootstocks transferring virus disease resistance to non-transgenic scions for production (or transgenic scions / non-transgenic rootstocks). The technical solution of the present invention expands the scope of application of transgenic plants in production, complies with the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms", and can effectively address consumers' concerns about transgenic tomatoes through grafting. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the construction of the TMVMP RNAi vector provided in an embodiment of the present invention;

[0052] Figure 2 This is a schematic diagram of the real-time fluorescence quantitative PCR detection of TMV virus gene expression in tomato plants provided by an embodiment of the present invention;

[0053] Figure 3 This is a schematic diagram of TMVMPRNAi provided in an embodiment of the present invention as a scion's resistance to viruses;

[0054] Figure 4 This is a schematic diagram of TMVMPRNAi provided in an embodiment of the present invention as a rootstock's resistance to viruses. DETAILED DESCRIPTION

[0055] 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 the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0056] In the embodiment of the present invention, the common wild-type tomato variety "M82" is used as the genetic transformation recipient material, and the obtained plant is used as the rootstock or scion, named TMV MP RNAi tomato. The rootstock or scion varieties used to verify the function are the wild-type tomato M82 and wild-type Nicotiana benthamiana Nb retained in the laboratory.

[0057] The tomato genetic transformation selection laboratory maintained vector PICSL4723_eGFP~3xFlag.

[0058] Primers were designed directly based on the vector's existing sequences and sequences obtained from the NCBI website, and their TM values were estimated using a formula. Primer synthesis services were provided by Quintiles (Wuhan) Biotechnology Co., Ltd. Primers were synthesized at a concentration of 10 pmol / μL and stored at -20°C.

[0059] The present invention provides a method for constructing a TMV MP RNAi vector, comprising:

[0060] 1. Use 1 unit (1U) of Xho1 and Xba1 purchased from NEB (New England Biolabs) to double-digest 1 μg of vector PICSL4723_eGFP~3xFlag at 37°C for 8 hours.

[0061] 2. Use 1% agarose gel electrophoresis to separate the digestion products: vector backbone fragment (~7400 bp) and discarded fragment (~500 bp).

[0062] 3. Use high-fidelity enzyme amplification to obtain the TMV viral MP sequence (with homologous recombination linker) from the reverse transcribed TMV-infected tomato material using the following primers:

[0063] TMV MP RNAi_HR_F:

[0064] 5-ggtataccgtcagtgATGGCTCTAGTTGTTAAAGGAAAAG-3 (SEQ ID NO.2)

[0065] TMV MP RNAi_HR_R:

[0066] 5-tccgattgtaagaagGGCTCTTTCCATCCTTTTGTC-3 (SEQ ID NO. 3) (lowercase bases are linker sequences)

[0067] 4. Use high-fidelity enzyme to amplify the PDK (pyruvate dehydrogenase kinase) intron sequence of Flaveria trinervia to obtain PDK intron using primers:

[0068] PDKintr_HR_F:

[0069] 5-cttcttacaatcggaGTAAGGAAAATAATTATTTTCTTTTTTCCTTTTAG-3(SEQ ID NO.4)

[0070] PDKintr_HR_R:

[0071] 5-cttcttacaatcggaCTGTAATCAATCCAAATGTAAGATCAATG-3 (SEQ ID NO. 5) (lowercase bases are linker sequences)

[0072] 5. Use high-fidelity enzyme to amplify the vector backbone and give the vector backbone adapter: Use primers:

[0073] Cmps_HR_R:

[0074] 5-cggtataccCTCGAGCTACTTCTAGGCTAC-3(SEQ ID NO.6)

[0075] OCSter_HR_F:

[0076] 5-cactgacggtataccTCTAGAGGATCCCCGGGCGAGC-3 (SEQ ID NO.7) (lowercase bases are linker sequences)

[0077] 6. Separate PCR products using 1% agarose gel electrophoresis:

[0078] TMV_MP_RNAi sequence (~330 bp):

[0079] ggtataccgtcagtgATGGCTCTAGTTGTTAAAGGAAAAGTGAATATCAATGAGTTTATCGACCTGACAAAAATGGAGAAGATCTTACCGTCGATGTTTACCCCTGTAAAGAGTGTTATGTGTTCCAAAGTTGATAAAATAATGGTTCATGAGAATGAGTCATTGTCAGAGGTGAACCTTCTTAAAGGAGTTAAGCTTATTGATAGTGGATACGTCTGTTTAGCCGGTTTGGTCGTCACGGGCGAGTGGAACTTGCCTGACAATTGCAGAGGAGGTGTGAGCGTGTGTCTGGTGGACAAAAGGATGGAAAGAGCCcttcttacaatcgga(SEQ ID NO.8);

[0080] PDK intr_HR sequence (~830bp):

[0081] cttcttacaatcggaCTGTAATCAATCCAAATGTAAGATCAATGATAACACAATGACATGATCTATCATGTTACCTTGTTTATTCATGTTCGACTAATTCATTTAATTAATAGTCAATCCATTTAGAAGTTAATAAAACTACAAGTATTATTTAGAAATTAATAAGAATGTTGATTGAAAATAATACTATATAAAATGATAGATCTTGCGCTTTGTTATATTAGCATTAGATTATGTTTTGTTACATTAGATTACTGTTTCTATTAGTTTGATATTATTTGTTACTTTAGCTTGTTATTTAATATTTTGTTTATTGATAAATTACAAGCAGATTGGAATTTCTAACAAAATATTTATTAACTTTTAAACTAAAATATTTAGTAATGGTATAGATATTTAATTATATAATAAACTATTAATCATAAAAAAATATTATTTTAATTTATTTATTCTTATTTTTACTATAGTATTTTATCATTGATATTTAATTCATCAAACCAGCTAGAATTACTATTATGATTAAAACAAATATTAATGCTAGTATATCATCTTACATGTTCGATCAAATTCATTAAAAATAATATACTTACTCTCAACTTTTATCTTCTTCGTCTTACACATCACTTGTCATATTTTTTTACATTACTATGTTGTTTATGTAAACAATATATTTATAAATTATTTTTTCACAATTATAACAACTATATTATTATAATCATACTAATTAACATCACTTAACTATTTTATACTAAAAGGAAAAAAGAAAATAATTATTTCCTTACtccgattgtaagaagG(SEQ ID NO.9);

[0082] PICSL4723_HR sequence: (~7500bp)

[0083]

[0084] 7. Use the gel recovery kit (CW2302M) purchased from Kangwei Century Company to recover the above-mentioned nucleic acid fragments with linkers in sequence.

[0085] 8. Assemble the above three fragments by homologous recombination: Measure the concentration of the recovered nucleic acid fragments on a micro-spectrophotometer and calculate the number of nucleic acid fragments required by the following formula:

[0086]

[0087] The calculated nucleic acid fragments were placed into a 0.2 mL microcentrifuge tube and mixed thoroughly by pipetting.

[0088] 5 μL of the mixed fragments was pipetted into a 0.2 mL microcentrifuge tube and mixed with 5 μL of ABclonal 2X MultiFSeamless Assembly Mix (RK21020) by pipetting. Homologous recombination was performed at 50°C for 60 minutes.

[0089] 9. The homologous recombination product was transformed into Escherichia coli DH5α (CC96102) purchased from Tolo Biotechnology Co., Ltd. by heat shock method:

[0090] (1) Place 100 μL of competent E. coli DH5α in an ice bath until thawed;

[0091] (2) Pipette 5 μL of homologous recombination transformation product and slowly inject it into the competent medium;

[0092] (3) Gently shake the competent E. coli centrifuge tube to evenly mix the product and competent cells;

[0093] (4) ice bath for 30 minutes;

[0094] (5) Competent state: 42°C water bath for 60 to 90 seconds;

[0095] (6) ice bath for 5 minutes;

[0096] (7) Add 1000 μL of liquid LB medium without antibiotics to the competent cells and resuscitate at 37°C in a shaker at 200 RPM for 2 h;

[0097] (8) The transformed E. coli was spread onto LB plates containing 1:1000 kanamycin (50 mg / mL) and incubated at 37°C for 18-36 hours.

[0098] 10. Select E. coli monoclones and perform genotyping. After identification, send them to Quintiles for Sanger sequencing.

[0099] 11. After obtaining the correct plasmid with the correct structure and sequence of: CmYLCV promoter-TMV MP positive sequence-PDK intron sequence-TMV MP reverse sequence-octopine terminator (sequence alignment was performed on the NCBI BLAST website), the plasmid was extracted from Escherichia coli by alkaline lysis method.

[0100] 12. Alkaline lysis method

[0101] (1) Preparation of reagents (final concentrations): solution 1: 50 mM glucose + 25 mM Tris-HCl (pH = 8.0) + 10 mM EDTA (pH = 8.0); solution 2: 0.1 M NaOH + 0.5% SDS; solution 3: 3 M Kac + 2 M Hac;

[0102] (2) Use a 1.5 mL conical-bottom centrifuge tube to collect 1 mL of E. coli pellet at 5000 RPM for 1 min, discard the supernatant, and repeat twice;

[0103] (3) Add 200 μL of solution 1 to each pellet and mix thoroughly.

[0104] (4) Add 300 μL of solution 2 to the mixture and mix until slightly clear;

[0105] (5) Add 600 μL of the mixture and invert the tube until a filamentous mass appears;

[0106] (6) Centrifuge the mixture at 13000 RPM for 8 min, and transfer 750 μL of the supernatant to a new 1.5 mL conical centrifuge tube;

[0107] (7) Add an equal volume of isopropanol (750 μL) to the supernatant, mix well, let stand at 4°C for 10 min, and centrifuge at 13,000 RPM for 10 min.

[0108] (8) Discard the supernatant and retain the precipitate, wash the precipitate twice with 70% ethanol, and air dry;

[0109] (9) Add ultrapure water to the dried precipitate for 20 min.

[0110] The present invention provides a method for obtaining transgenic TMV-resistant tomatoes for both rootstock and scion purposes, comprising:

[0111] 1. The correctly extracted plasmid was transformed into Agrobacterium GV3101 (CC96304) purchased from Tolo Biotechnology Co., Ltd. by heat shock method:

[0112] (1) Place 100 μL of competent Agrobacterium GV3101 on ice until thawed;

[0113] (2) Pipette 10 μL of plasmid and slowly inject it into the competent medium;

[0114] (3) Gently shake the competent Agrobacterium centrifuge tube to evenly mix the product and competent cells;

[0115] (4) ice bath for 30 minutes;

[0116] (5) Liquid nitrogen bath for 5 minutes;

[0117] (6) 37℃ water bath for 5 minutes;

[0118] (7) ice bath for 5 minutes;

[0119] (8) Add 1000 μL of liquid LB medium without antibiotics to the competent cells and resuscitate at 28°C in a shaker at 200 RPM for 2 h;

[0120] (9) The transformed Agrobacterium was spread onto LB plates containing 1:1000 kanamycin (50 mg / mL) and 1:1000 rifampicin (50 mg / mL, DMSO) and incubated at 28°C for 48-72 hours.

[0121] 2. Select Agrobacterium single clones, identify their genotypes, and preserve them.

[0122] 3. Transform the overexpression vector TMV MP RNAi into tomato M82.

[0123] 4. Prepare MS medium (hereinafter referred to as MS): ammonium nitrate 1650 mg / L; potassium iodide 0.83 mg / L; boric acid 6.2 mg / L; potassium nitrate 1900 mg / L; anhydrous calcium chloride 332.2 mg / L; potassium dihydrogen phosphate 170 mg / L; cobalt chloride hexahydrate 0.025 mg / L; zinc sulfate heptahydrate 8.6 mg / L; copper sulfate pentahydrate 0.025 mg / L; glycine 2 mg / L; disodium edetate dihydrate 37.26 mg / L; inositol 100 mg / L; ferrous sulfate heptahydrate 27.8 mg / L; nicotinic acid 0.5 mg / L; anhydrous magnesium sulfate 180.7 mg / L; pyridoxine hydrochloride 0.5 mg / L; manganese sulfate monohydrate 16.9 mg / L; thiamine hydrochloride 0.1 mg / L; sodium molybdate dihydrate 0.25 mg / L; use 1M KOH was used to adjust the pH to 5.80-5.82; 7 g / L agar was added to the solid.

[0124] 5. Prepare solid MS medium and add 10 g / L glucose as the sowing medium; add 30 g / L sucrose and 0.2 μg / mL 2,4-chlorophenoxyacetic acid as the dedifferentiation medium; add 30 g / L sucrose, 100 μg / L kanamycin, 0.1 μg / L indoleacetic acid, 25 μg / L cefotaxime, 320 μg / L timentin and 2 μg / L trans-zein nucleoside as the redifferentiation medium 1; add 30 g / L sucrose, 80 μg / L kanamycin, 25 μg / L cefotaxime, 265.6 μg / L timentin and 0.2 μg / L trans-zein nucleoside as the redifferentiation medium 2; add 30 g sucrose, 50 μg / L kanamycin, 2 μg / L indolebutyric acid and 320 μg / L timentin as the rooting medium.

[0125] 6. After disinfecting wild-type tomatoes with 0.5% (available chlorine content) sodium hypochlorite, sow them on the sowing medium and infect them 7 to 8 days after the seedlings germinate.

[0126] 7. Take out the developed sterile tomato seedlings from the tissue culture bottle in a sterile clean bench, cut off the cotyledons with scissors in a sterilized glass culture dish, cut each cotyledon into two parts, and soak them in clean water.

[0127] 8. Remove the cotyledons from the culture dish in clean water and transfer them to a new culture dish.

[0128] 9. Precipitate the Agrobacterium that has been expanded in advance at 3600RPM*5min, discard the supernatant, and resuspend it in liquid MS to OD600=0.5

[0129] 10. Add resuspended Agrobacterium to the culture dish and infect for 5 minutes.

[0130] 11. Remove the cotyledons from the Agrobacterium infection solution, dry them with sterile filter paper, spread them flat on the dedifferentiation medium, and culture them in the dark for 3 days.

[0131] 12. Transfer the cotyledons on the dedifferentiation medium to the redifferentiation medium 1 and culture for 14 days.

[0132] 13. Transfer the explants that have differentiated and sprouted on the redifferentiation medium to redifferentiation medium 2 and culture for 14 to 21 days.

[0133] 14. Transfer the explants with buds from Redifferentiation Medium 2 to Redifferentiation Medium 2 again and culture for 14 days.

[0134] 15. Cut off the complete buds differentiated from the explants, transfer them to rooting medium, and culture for 14 to 21 days.

[0135] 16. Remove the rooted plants from the culture medium, wash off the attached agar, and transplant them in vermiculite.

[0136] 17. Identify the genotype of the transplanted transgenic plants, retain the transgenic positive plants and transplant them.

[0137] 18. Take leaves from the transgenic T0 generation plants, extract RNA for reverse transcription, and identify the expression of the vector.

[0138] 19. Transgenic plants of generation T0 are transplanted into the field and propagated to obtain transgenic plants of generation T1, which are then retained.

[0139] 20. Self-pollinate the T1 generation of transgenic plants, select the lines whose genotypes are all transgene-positive among the offspring, repeat the selection for one generation, and obtain a stable TMV MP RNAi inbred line.

[0140] 1. Specific application fields or related products of the present invention.

[0141] The embodiment of the present invention provides a transgenic TMV-resistant tomato for dual-purpose rootstock and scion, which can be used as a rootstock or a scion.

[0142] 2. Relevant evidence of the technical effects obtained by the embodiments of the present invention.

[0143] Using RNAi for antiviral purposes is a proven method, but in practice the full-length viral coding sequence is always considered as one of the necessary conditions for RNAi construction.

[0144] RNAi plants must be genetically modified plants, which are restricted by the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms" and are also strictly managed by the Ministry of Agriculture.

[0145] It has been proven that sRNA and mRNA in plants can be transferred to the rootstock or scion through grafting, that is, the mRNA or sRNA of the rootstock can move to the scion through the graft healing site, and vice versa.

[0146] According to the above conditions, the present invention proposes:

[0147] The construction of RNAi does not require the full length of the virus's coding sequence, but only requires the most conserved and critical parts of the virus to be included in the RNAi interference range.

[0148] RNAi plants are necessarily transgenic plants, but non-transgenic plants grafted onto RNAi are necessarily non-transgenic plants.

[0149] A non-transgenic plant grafted onto an RNAi transgenic plant will receive the mRNA or sRNA from the transgenic RNAi plant through the graft callus.

[0150] When non-transgenic plants receive mRNA or sRNA from transgenic plants, the mRNA or sRNA can produce corresponding RNAi effects at the target site.

[0151] According to the above technical solution, a transgenic overexpression vector of TMV MP RNAi was constructed and transferred into tomatoes. Among them, the RNAi part of TMV MP RNAi selected a 300 base pair length sequence of TMV MP (mobile protein). There is evidence that due to the mutation of amino acid No. 68 of the ToBRFV mobile protein MP, which also belongs to the tobacco mosaic virus genus, ToBRFV has acquired the ability to break through the TMV-related resistance gene TM-22, thereby escaping the plant's immunity. Therefore, it is speculated that the TMV conserved sequence must be located near the 68th amino acid, that is, near the 201st to 204th bases of MP. Therefore, the designed RNAi vector must contain the base sequence of positions 201 to 204, and it must be the TGT (triplet codon) of cysteine.

[0152] Based on the conclusions obtained, it can be inferred that the sequence of the tobacco mosaic virus near the 68th amino acid must be conserved. Therefore, TMV RNAi covering this region must be effective and long-lasting.

[0153] When TMV MP RNAi tomatoes are used as rootstocks or scions, due to the overexpression of RNAi, the non-transgenic rootstock or scion part will inevitably receive sRNA or mRNA from the transgenic plant, and then produce corresponding RNAi through the received sRNA or mRNA, thereby inhibiting the reproduction of the virus in the non-transgenic plant part.

[0154] Example 1

[0155] TMV MP RNAi demonstrates resistance to TMV virus

[0156] 1. Sow the TMV MP RNAi strain and inoculate the seedlings with TMV virus by friction 7 days after germination.

[0157] 2. Seven days after virus inoculation, leaves of tomato plants inoculated with TMV were removed and total RMA was extracted by the trizol method.

[0158] 3. After obtaining the tomato total RNA, reverse transcribe the obtained RNA to obtain cDNA.

[0159] 4. Use real-time fluorescence quantitative PCR to detect the expression of TMV virus genes in tomato plants.

[0160] Example 2

[0161] TMV MP RNAi as a demonstration of scion resistance to viruses (Nicotiana benthamiana demonstration)

[0162] 1. Sow Nicotiana benthamiana (Nb) and transplant the seedlings 14 days later.

[0163] 2. After Nicotiana benthamiana has grown normally for 4 weeks, it is ready for use.

[0164] 3. Sow the TMV MP RNAi strain and cut a small amount of cotyledon tissue 7 days after the seedlings germinate for genotyping.

[0165] 4. Retain plants with positive genotype identification.

[0166] 5. After the plants have grown for 4 weeks, TMV MP RNAI tomatoes are used as scions of Nicotiana benthamiana using the modified cleft grafting method.

[0167] 6. Induce graft healing for 2 weeks.

[0168] 7. After healing is complete, the grafted combination grows normally for 1 week.

[0169] 8. Inoculate TMV into Nicotiana benthamiana leaves by rubbing and keep them moist for 3 days.

[0170] 9. Observe the phenotype after one week.

[0171] When wild-type tomato plants were grafted onto Nicotiana benthamiana as scions and inoculated with TMV, none of the Nicotiana benthamiana plants developed normally. However, when RNAi-treated Nicotiana benthamiana plants were inoculated with TMV, some combinations maintained normal development, while others experienced leaf curl and death, demonstrating that the RNAi-treated tomato scion provided a significant protective effect on the rootstock.

[0172] Example 3

[0173] TMV MP RNAi as a rootstock to demonstrate virus resistance (Nicotiana benthamiana demonstration)

[0174] The method is the same as above, but tomato is used as the rootstock and Nicotiana benthamiana is used as the scion.

[0175] 1. Sow Nicotiana benthamiana (Nb) and transplant the seedlings 14 days later.

[0176] 2. After Nicotiana benthamiana has grown normally for 4 weeks, it is ready for use.

[0177] 3. Sow the TMV MP RNAi strain and cut a small amount of cotyledon tissue 7 days after the seedlings germinate for genotyping.

[0178] 4. Retain plants with positive genotype identification.

[0179] 5. After the plants have grown for 4 weeks, Nicotiana benthamiana is used as the scion for TMV MP RNAi tomatoes using the modified cleft grafting method.

[0180] 6. Induce graft healing for 2 weeks.

[0181] 7. After healing is complete, the grafted combination grows normally for 1 week.

[0182] 8. Inoculate TMV into Nicotiana benthamiana leaves by rubbing and keep them moist for 3 days.

[0183] 9. Observe the phenotype after one week.

[0184] When Nicotiana benthamiana scions were grafted onto wild-type tomatoes and inoculated with TMV, all plants failed to develop normally (e.g., yellowing, wilting, and death). In the Nicotiana benthamiana / RNAi graft, leaves withered and curled after TMV inoculation, but the plants continued to develop, demonstrating that the RNAi tomato rootstock also provided considerable protection for the scion.

[0185] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A TMV MPRNAi overexpression vector, characterized in that: The structure is: CmYLCV promoter-TMV MP positive sequence-PDK intron sequence-TMVMP reverse sequence-octopine terminator; the nucleotide sequence of the TMV MP RNAi overexpression vector is shown in SEQ ID NO.

1.

2. The method for constructing the TMVMP RNAi overexpression vector according to claim 1, wherein include: (1) 1 μg of the vector PICSL4723_eGFP-3xFlag was double-digested with 1 U of Xho1 and Xba1 at 37°C for 8 hours; (2) Separate the digestion products using 1% agarose gel electrophoresis: vector backbone fragment (~7400 bp) and discarded fragment (~500 bp); (3) Using high-fidelity enzyme amplification, the reverse-transcribed TMV-infected tomato material was amplified to obtain the TMV viral MP sequence (with homologous recombination linker); (4) Amplify the PDK (pyruvate dehydrogenase kinase) intron sequence of Flaveria trinervia using a high-fidelity enzyme to obtain the PDK intron; (5) amplifying the vector backbone using a high-fidelity enzyme to impart a linker to the vector backbone; (6) Separate the PCR products using 1% agarose gel electrophoresis: TMV_MP_RNAi sequence (~330 bp); PDKintr_HR sequence (~830 bp); PICSL4723_HR sequence (~7500 bp); (7) Using a gel recovery kit (CW2302M), the above-mentioned nucleic acid fragments with adapters were recovered in sequence; (8) assembling the above three fragments by homologous recombination; (9) The homologous recombination product was transformed into Escherichia coli DH5α by heat shock method: (10) The plasmid was extracted from Escherichia coli by alkaline lysis.

3. The method for constructing the TMV MP RNAi overexpression vector according to claim 2, wherein: Primers used in step (4): TMV MP RNAi_HR_F: 5-ggtataccgtcagtgATGGCTCTAGTTGTTAAAGGAAAAG-3 TMV MP RNAi_HR_R: 5-tccgattgtaagaagGGCTCTTTCCATCCTTTTGTC-3 (lowercase bases are linker sequences) Primers used in step (5): PDKintr_HR_F: 5-cttcttacaatcggaGTAAGGAAAATAATTATTTTCTTTTTTCCTTTTAG-3 PDKintr_HR_R: 5-cttcttacaatcggaCTGTAATCAATCCAAATGTAAGATCAATG-3 (lowercase bases are linker sequences) Primers used in step (6): Cmps_HR_R: 5-cggtataccCTCGAGCTACTTCTAGGCTAC-3 OCSter_HR_F: 5-cactgacggtataccTCTAGAGGATCCCCGGGCGAGC-3 (lowercase bases are linker sequences).

4. A transgenic TMV-resistant tomato for dual-purpose rootstock and scion, characterized in that: The TMV MPRNAi overexpression vector according to claim 1 is introduced into tomatoes by Agrobacterium transformation, and transgenic TMV-resistant tomatoes for both rootstock and scion are obtained by screening.

5. A method for cultivating transgenic TMV-resistant tomatoes for both rootstock and scion purposes according to claim 4, characterized in that: The following steps are involved: S1, the extracted plasmid was transformed into Agrobacterium GV3101 by heat shock method, and resuspended Agrobacterium was obtained; S2, wild-type tomatoes were sown and cultured on MS medium; S3, infect the target plants with resuspended Agrobacterium and screen the positive plants.

6. The method for cultivating transgenic TMV-resistant tomatoes for both rootstock and scion purposes according to claim 5, characterized in that: Solid MS medium: add 10 g / L glucose as the seeding medium; add 30 g / L sucrose and 0.2 μg / mL 2,4-chlorophenoxyacetic acid as the dedifferentiation medium; add 30 g / L sucrose, 100 μg / L kanamycin, 0.1 μg / L indoleacetic acid, 25 μg / L cefotaxime, 320 μg / L timentin and 2 μg / L trans-zein nucleoside as the redifferentiation medium 1; add 30 g / L sucrose, 80 μg / L kanamycin, 25 μg / L cefotaxime, 265.6 μg / L timentin and 0.2 μg / L trans-zein nucleoside as the redifferentiation medium 2; add 30 g sucrose, 50 μg / L kanamycin, 2 μg / L indolebutyric acid, and 320 μg / L timentin as the rooting medium.

7. Use of the transgenic TMV-resistant tomato for dual-purpose rootstock and scion according to claim 4 as a rootstock or scion.

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