Plant in-situ transformation vector and application thereof

Abstract

The invention relates to the technical field of plant genetic transformation, in particular to a set of plant in-situ transformation vector and application thereof, the set of plant in-situ transformation vector comprises expression vectors of pMIN-VS1-35S: AmCyan-SlUbi: DRs: NOSter: pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2) and pFZ066 (WIND1); and an expression vector pFZ031 of pMIN-VS1-35S: NptII-35S: AmCyan-Empty-2 * 35S: Lox71-IPT-Lox66-RUBY: 35Ster, and an expression vector pFZ031 of pMIN-VS1-35S: NptII-35S: AmCyan-Empty-2 * 35S: Lox71-IPT-Lox66-RUBY: 35Ster The DRs overexpression vector disclosed by the invention not only can obviously improve the transformation efficiency, but also can utilize a Cre / LoxP system to remove DRs, so as to obtain a DRs-free transgenic material.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic transformation, and specifically relates to a set of in-situ transformation vectors for plants and their applications. Background Art

[0002] Nicotiana benthamiana is an important model plant in plant biological research and biotechnological applications, and is widely used in fields such as plant-pathogen interaction, functional genomics, synthetic biology, and gene editing. Therefore, it is very important to establish an efficient tobacco genetic transformation system. At present, the Agrobacterium-mediated leaf disc transformation method is a commonly used method for creating tobacco transgenic materials. This method is widely used because of its advantages such as high transformation efficiency and good transformation stability. However, the leaf disc transformation method highly depends on plant tissue culture technology. Not only is the transformation period relatively long (it takes 8 - 12 weeks to obtain T0 generation transgenic plants), but also contamination easily occurs during the culture process, and it is not applicable to laboratories lacking tissue culture conditions.

[0003] With the in-depth research on the genetic mechanism of plant cell regeneration, it is found that when somatic cells are transformed into embryonic cells, some genes will be specifically activated or differentially expressed, thereby reprogramming the cells and obtaining embryos or meristems. These genes usually encode transcription factors or signaling proteins, and they are collectively referred to as developmental regulators (DRs). Some studies have shown that the co-expression of developmental regulators (DRs) and sgRNA can induce the generation of gene-edited buds through the induction of de novo meristems. Compared with standard tissue culture, this method has lower costs, less technical difficulty, and shorter required time, providing a new idea for plant genetic transformation. However, the application of this method still faces many problems, such as low transformation efficiency, causing plant developmental malformations and sterility, which affect the application of subsequent transgenic materials. Summary of the Invention

[0004] To solve the above problems, the present invention provides a set of in-situ transformation vectors for plants, and provides an in-situ genetic transformation method for direct injection regeneration and precise removal of transgenic sequences, avoiding the sterile culture process required by most traditional plant genetic transformation methods. The transformation period is short, and T0 generation transgenic plants can be obtained in only 3 - 4 weeks. At the same time, it can efficiently remove unnecessary transgenic elements such as DRs and selectable marker genes to obtain non-transgenic offspring.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A set of in-situ transformation vectors for plants, comprising:

[0007] Expression vectors of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter: pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2) and pFZ066 (WIND1); and

[0008] Expression vector pFZ031 of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster.

[0009] In a set of plant in-situ transformation vectors of the present invention, the expression vector of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter is constructed by the following steps:

[0010] S1.1. Extract the zero-level vector pICH41373 of the 35S promoter, the zero-level vector pJC375 of AmCyan and the zero-level vector pGY269 of the 35S terminator from the Golden Gate cloning vector library, assemble them into the first-level backbone vector pJC268 according to the Modular cloning reaction system, and after correct sequencing verification, save the obtained plasmid to obtain the first-level vector of 35S:AmCyan:35Ster, named pGY336;

[0011] S1.2. Select the zero-level vector pJC434 of the SlUbi promoter, the zero-level vector of DRs and the zero-level vector pICH41421 of the NOS terminator from the vector library, assemble them into the first-level backbone vector pJC269 according to the Modular cloning reaction system, and after correct sequencing verification, save the obtained plasmid to obtain the first-level vector of SlUbi:DRs:NOSter; among them, the zero-level vector of DRs is the zero-level vector pJC297 (IPT) of DRs, the zero-level vector pJPC670 (PLT5) of DRs, the zero-level vector pJC370 (GRF-GIF) of DRs, the zero-level vector pJC253 (WUS2) of DRs, the zero-level vector pJC416 (WIND1) of DRs

[0012] S1.3. The obtained primary vector SlUbi:DRs:NOSter was respectively assembled with pGY336 into the secondary backbone vector pJC337 through the Modular cloning reaction system. After correct sequencing verification, the obtained plasmids were preserved to obtain the expression vectors of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter, named pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2), and pFZ066 (WIND1) respectively.

[0013] In a set of plant in-situ transformation vectors of the present invention, the expression vector pFZ031 of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster was constructed through the following steps:

[0014] Extract the primary vectors pGY336 (35S-AmCyan-35Ster), pGY337 (Empty-2×35S-Lox71), pGY338 (SlUbi-IPT-SlUbiter), and pGY340 (Lox66-RUBY-35Ster), and then connect them to the secondary backbone vector pGY332 according to the Modular cloning reaction system. After correct sequencing verification, the obtained plasmids were preserved to obtain the expression vector of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster, named pFZ031.

[0015] The application of a set of plant in-situ transformation vectors of the present invention can be used to improve the in-situ genetic transformation efficiency of tobacco, including the following steps:

[0016] S1. Inoculate the expression vector of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter into an Agrobacterium strain, and prepare an Agrobacterium infection solution with OD600 = 0.3.

[0017] S2. Infect Nicotiana benthamiana with the above Agrobacterium infection solution to achieve in-situ genetic transformation of tobacco. Specifically: Select Nicotiana benthamiana plants that are 30 to 45 days old under natural growth conditions, remove the existing meristems of the plants (including shoot tips and axillary meristems), and only retain three supporting leaves. Subsequently, inject the Agrobacterium infection solution into the tobacco plants from which the meristems have been removed, and inject it into the phloem of the stems, axils, and apical incisions of the plants from bottom to top in sequence. After the infection is completed, place the plants in a seedling tray, add an appropriate amount of clear water, cover it with a moisturizing cover, and culture it in the dark for 1 to 2 days. After 2 days, transfer it to normal culture conditions for recovery growth (26°C, 16 hours of light, 8 hours of darkness).

[0018] The above method uses DRs to overcome transgenic barriers while significantly shortening the transformation time, expanding the genotypes and explant types available for transgenic operations, and significantly improving the transformation efficiency. However, the continuous expression of DRs may lead to abnormal development of transgenic plants, manifested as problems such as leaf distortion, folding, severe clustering, and reproductive barriers. In order to completely eliminate the influence of DRs on the subsequent application of transgenic materials, the present invention provides a precise recombination method using a site-specific recombinase system (Cre / LoxP) delivered by TRV virus to eliminate DRs in transgenic materials, including the following steps:

[0019] S1. Inoculate the expression vector pFZ031 of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster into Agrobacterium strains to prepare an Agrobacterium infection solution;

[0020] S2. Infect Nicotiana benthamiana with the above Agrobacterium infection solution to achieve in-situ genetic transformation of tobacco and obtain the T1 generation transgenic materials of pFZ031;

[0021] S3. Amplify the CDS sequence of the Cre gene by PCR using the Cre gene sequence;

[0022] S4. Connect the CDS sequence of the Cre gene to the multiple cloning site of pTRV2 to obtain the pTRV2-Cre vector;

[0023] S5. Inoculate pTRV1 and pTRV2-Cre into Agrobacterium GV3101, and after propagation, prepare an infection solution with an OD 600 = 0.6, and then mix them in a ratio of 1:1 for standby;

[0024] S6. Use the leaf injection method to inoculate pTRV2-Cre into the T1 generation transgenic materials of pFZ031.

[0025] S7. Obtain transgenic offspring without DRs. The present invention has the following beneficial effects:

[0026] 1) The in-situ genetic transformation method of tobacco established in the present invention modifies the genome of the whole plant under non-sterile conditions, avoiding the sterile culture process required by most traditional plant genetic transformation methods. The transformation cycle is short, and T0 transgenic plants can be obtained in only 3-4 weeks, greatly reducing the cost of genetic transformation experiments.

[0027] 2) By creating the transgenic material pFZ031

[0028] (pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster), recognition sites Lox71 and Lox66 of Cre recombinase were introduced on both sides of IPT. Subsequently, the Cre recombinase was delivered into the T1 transgenic plants of pFZ031 using the TRV virus vector, successfully achieving the efficient removal of IPT and activating the expression of RUBY. Finally, while cutting the T0 transgenic plants, a transgenic removal experiment was carried out to achieve the removal of contemporary IPT, shortening the creation cycle of transgenic materials without DRs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, purposes, and advantages of the present invention will become more obvious:

[0030] Figure 1 It is a flow chart for constructing an overexpression DRs vector;

[0031] In the figure: BsaI and PaqcI are represented by B and P respectively; pJCLevel0, pFZLevel1, and pFZLevel2 represent the zero-level, first-level, and second-level vectors of IPT, PLT5, GRF-GIF, WUS2, and WIND1 respectively; SpecR is spectinomycin hydrochloride resistance; CarbR is carbenicillin resistance; KanR is kanamycin resistance.

[0032] Figure 2 It is a schematic diagram of using Agrobacterium to infect Nicotiana benthamiana.

[0033] Figure 3 It is the observation of the in-situ transformation phenotype of Nicotiana benthamiana.

[0034] Figure 4 It is the effect of different DRs on the in-situ transformation efficiency of Nicotiana benthamiana;

[0035] In the figure: (A) Phenotype of T0 generation positive plants; (B) Statistical chart of the proportion of T0 generation positive plants (n = 30, different letters indicate significant differences analyzed by One-way ANOVA, P < 0.05).

[0036] Figure 5 It is for PCR identification of transgenic materials.

[0037] Figure 6 It is for the effect of different Agrobacterium strains on the in-situ transformation efficiency of Nicotiana benthamiana.

[0038] In the figure: (A) In-situ transformation efficiency of different Agrobacterium strains (n = 30, different letters indicate significant differences analyzed by One-way ANOVA, P < 0.05); (B) Phenotype of Nicotiana benthamiana 30 days after being infected with Agrobacterium rhizogenes K599.

[0039] Figure 7 It is for the in-situ transformation efficiency of Agrobacterium infection solutions with different concentrations (n = 30, different letters indicate significant differences analyzed by One-way ANOVA, P < 0.05).

[0040] Figure 8 It is the flow chart for constructing the pMIN-VS1-35S:NptII-35S:AmCyan-Lox71-IPT-Lox66-RUBY:35Ster vector.

[0041] Figure 9 It is the schematic diagram of the deletion pattern of the IPT gene in T1 generation transgenic materials mediated by TRV delivering Cre recombinase.

[0042] Figure 10 It is for the phenotype of transgenic plants after removing IPT.

[0043] In the figure: (A) Phenotypes of the above-ground and underground parts of transgenic plants after removing IPT, scale bar: 1 cm; (B) Phenotypes of the flowers and pods of transgenic plants after removing IPT, scale bar: 5 mm; (C) Phenotype of pFZ031ex-T2 generation plants.

[0044] Figure 11 It is for the detection of transgenic elimination efficiency.

[0045] Figure 12 It is for sequencing to verify the IPT elimination result.

[0046] Figure 13 It is the flow chart for the elimination of the IPT gene in T0 generation transgenic materials mediated by TRV delivering Cre recombinase.

[0047] In the figure: (a) Phenotype of the transgenic plant for branch cutting, scale bar: 2 cm; (b) Transgenic lateral branch for branch cutting, scale bar: 1 cm; (c) Use a scalpel to cut an inclined plane at the stem base of the lateral branch, and then smear Agrobacterium rhizogenes on the inclined plane, scale bar: 5 mm; (d) Subsequently, transplant it into a seedling pot and seal it with a transparent bag filled with an appropriate amount of clear water to maintain high humidity, scale bar: 2 cm; (e) Phenotype of the transgenic plant 45 days after IPT removal, scale bar: 4 cm; (f) and (g) Phenotype of the transgenic plant 60 days after IPT removal, scale bar: 4 cm.

[0048] Figure 14 For RT-PCR verification, RUBY was activated and expressed after IPT removal. Detailed implementation manners

[0049] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0050] Example 1

[0051] Method for promoting in-situ genetic transformation of tobacco based on overexpression of developmental regulators (DRs)

[0052] 1. Experimental materials

[0053] 1.1 Plant materials

[0054] Wild-type Nicotiana benthamiana.

[0055] 1.2 Strains

[0056] Escherichia coli strains DH10B, DB3.1, and Agrobacterium tumefaciens strains EHA105, GV3101, AGL1 were purchased from Shanghai Maokang Biotechnology Co., Ltd. The Agrobacterium tumefaciens strain 18R12 was self-made in this laboratory. The Agrobacterium rhizogenes strains Ar1193 and K599 were purchased from Sangon Biotech (Shanghai) Co., Ltd. and stored in the -80 °C refrigerator of this laboratory.

[0057] 1.3 Solution preparation

[0058] Preparation of culture media:

[0059] Table 1 Components of LB liquid medium

[0060]

[0061] Finally, adjust the pH of the LB liquid medium to 7.0, and autoclave it at 121 °C for 20 min. After it cools down to room temperature, add the corresponding antibiotic and shake well, then store it in a 4 °C refrigerator.

[0062] To prepare 1 L of LB solid medium, add 15.0 g of agar powder to 1 L of LB liquid medium, adjust the pH to 7.0, and then autoclave it at 121 °C for 20 min. After sterilization, wait for it to cool naturally to room temperature and store it in a 4 °C refrigerator. When it cools down to about 50 °C, add the required antibiotic and shake well, then pour it into a sterile Petri dish, wait for the medium to solidify, seal the Petri dish with parafilm, and store it in an inverted 4 °C refrigerator.

[0063] Table 2 Components of 1 L of SOC solution

[0064]

[0065] Finally, adjust the pH of the solution to 7.0, and autoclave it at 121 °C for 20 min. After sterilization, wait for the solution to cool to room temperature and store it in a 4 °C refrigerator.

[0066] Preparation of antibiotics:

[0067] Table 3 Antibiotic formula

[0068]

[0069] Filter and sterilize using a 0.22 μm sterile microporous filter in a laminar flow hood. After filtration, aliquot it into 1.5 mL sterile centrifuge tubes and store it in a -20 °C refrigerator for later use.

[0070] Preparation of Agrobacterium resuspension:

[0071] Table 4 Formula of 100 μmol / L acetosyringone

[0072]

[0073] Table 5 Formula of 1 L of Agrobacterium buffer

[0074]

[0075] Prepare 1 L of Agrobacterium buffer, adjust the pH to 5.6, and store it at room temperature for later use.

[0076] 1.4 Primers and sequencing

[0077] The primer synthesis and sequencing were completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0078] 2 Experimental methods

[0079] 2.1 Growth conditions of plant materials

[0080] The wild-type Nicotiana benthamiana used in this example was grown in a light incubator at a temperature of 26 °C, a relative air humidity of 65-75%, and a light intensity of 100-150 μmol m -2 s -1 under a 16-h light / 8-h dark cycle.

[0081] 2.2 Modular cloning

[0082] Modular cloning is an efficient DNA assembly system based on Golden gate cloning technology, which relies on the property that type IIS restriction endonucleases can cleave precisely outside their recognition sites. When these recognition sites are placed in opposite directions at the 5′ and 3′ ends of DNA fragments, they are removed during the digestion process, enabling seamless ligation of DNA fragments with compatible fusion sites. Since type IIS restriction sites can be designed as different fusion sites, directional assembly of multiple DNA fragments can be achieved. During the assembly process, each DNA fragment can be directly used in the reaction as an undigested plasmid and act together with the target vector, T4 DNA ligase, and type IIS restriction endonuclease to complete digestion and ligation in a single reaction system, thus efficiently constructing the target DNA vector.

[0083] The modular cloning strategy defines five basic module types (level-zero modules), including promoters, 5′ untranslated regions (5′UTRs), signal peptides, coding sequences (CDSs), and terminators. Assembling these standardized modules in genetic order yields a complete transcription unit. For assembly using Golden gate technology, specific fusion sites flank each level-zero module. Selecting fusion sites overlapping with the coding sequence can minimize changes to the encoded protein: the fusion site for the start codon is chosen as AATG, and the fusion site between the signal peptide and the coding sequence is AGGT, where GGT encodes glycine, a common amino acid at the -1 position in signal peptides. Since the remaining 4 fusion site sequences (GGAG, TACT, GCTT, and CGCT) are all in untranslated sequences, they are required to be unique and non-palindromic to ensure efficient cloning. For cytoplasmic proteins without a signal peptide, the coding sequence can be cloned as a module with AATG and GCTT fusion sites; this module type replaces the SP and CDS modules for secreted proteins in the assembly reaction. Since all level-zero modules of the same type have the same fusion sites flanking them, they can be freely interchanged, allowing any desired transcription unit to be created by simply selecting the required modules. The assembled transcription unit again represents a module (a higher-order module, a level-one module), which can be directionally assembled into a multi-gene structure (level-two).

[0084] (1) The fusion sites used in the Modular cloning system are shown in the following table (Table 7):

[0085] Table 7 Fusion sites of the Modular cloning system

[0086]

[0087] (2) The Golden gate reaction system is shown in the following table (Table 8):

[0088] Table 8 Golden gate ligation reaction system

[0089]

[0090]

[0091] Note: When using PaqcI, 0.4 μL of PaqcI Activator must be added to the ligation reaction system

[0092] (3) The Golden gate reaction procedure is shown in the following table (Table 9):

[0093] Table 9 Golden gate reaction procedure

[0094]

[0095] 2.3 Preparation of competent cells

[0096] (1) Preparation of competent cells of Escherichia coli DH10B and DB3.1 (CaCl2 method)

[0097] 1) Thaw the Escherichia coli parent strains (DH10B and DB3.1) on ice, then inoculate them on an antibiotic-free LB solid medium, and incubate them overnight (12 - 16 h) in an inverted position in a 37 °C constant temperature incubator;

[0098] 2) Pick freshly activated single colonies of DH10B and DB3.1 respectively, and inoculate them into antibiotic-free LB liquid medium (about 5 mL of LB liquid medium is added to each test tube), and culture them overnight (14 - 16 h) in a shaker at 37 °C at a rotation speed of 200 rpm;

[0099] 3) Inoculate the overnight culture broth into 100 mL of antibiotic-free LB liquid medium at a ratio of 1:50, and then culture it by shaking at 37 °C at a rotation speed of 200 rpm for 2 - 3 h until the OD600 of the broth is about 0.5 (0.4 - 0.6);

[0100] 4) Under sterile conditions, transfer the broth to a pre-cooled 50 mL centrifuge tube, let it stand on ice for 10 min, and centrifuge at 4 °C and 4500 rpm for 10 min;

[0101] 5) Discard the supernatant, invert it on a sterile filter paper for 1 min, gently resuspend the precipitate in each centrifuge tube with 30 mL of pre-cooled 0.1 mol / L CaCl2 solution (gently pipette and mix with a pipette tip), place it on ice for 20 min, and centrifuge at 4 °C and 4500 rpm for 10 min;

[0102] 6) Try to discard the supernatant as much as possible, add 4 mL of pre-cooled 0.1 mol / L CaCl2 solution and 0.8 mL of glycerol to resuspend, place it on ice for 5 min, aliquot it with 1.5 mL centrifuge tubes, 100 μL in each tube, quickly freeze it with liquid nitrogen, and then store it in a -80 °C refrigerator.

[0103] (2) Preparation of competent cells of Agrobacterium (18R12)

[0104] 1) Thaw the Agrobacterium parent strain 18R12 on ice, then inoculate it on an antibiotic-free LB solid medium, and incubate it in an inverted position in a 28 °C constant temperature incubator for 24 h;

[0105] 2) Pick a single colony of Agrobacterium and inoculate it into LB liquid medium without antibiotics. Place it on a shaker at 28 °C and culture it overnight (14 - 16 h) at a rotation speed of 200 rpm.

[0106] 3) Inoculate the overnight cultured bacterial solution into 100 mL of LB liquid medium without antibiotics at a ratio of 1:50. Then, culture it with shaking at 28 °C and a rotation speed of 200 rpm until the OD600 of the bacterial solution reaches 0.6.

[0107] 4) Under sterile conditions, transfer the bacterial solution to a pre-cooled 50 mL centrifuge tube and centrifuge it at 4 °C and 4500 rpm for 10 min.

[0108] 5) Discard the supernatant completely, add 4 mL of pre-cooled 0.1 mol / L CaCl2 solution and 0.8 mL of glycerol, and gently resuspend. After placing it on ice for 5 min, aliquot the resuspended solution into pre-cooled 1.5 mL centrifuge tubes, 100 μL per tube. Quickly freeze it with liquid nitrogen and then store it in a -80 °C refrigerator.

[0109] 2.4 Transformation and screening of competent cells

[0110] (1) Transformation of Escherichia coli DH10B and DB3.1 competent cells

[0111] 1) Place the Escherichia coli DH10B and DB3.1 competent cells taken out from the -80 °C refrigerator on ice. Wait for them to partially melt, add 1 - 2 μL of the target plasmid or 10 - 20 μL of the vector recombinant product, gently mix with a pipette tip, and let it stand on ice for 30 min.

[0112] 2) Heat shock in a 42 °C water bath for 45 - 60 s, and then ice bath for 2 min.

[0113] 3) In a laminar flow hood, pipette 400 μL of SOC solution into the ice-bathed competent cells, and culture them with shaking at 37 °C and 200 rpm for 1 h.

[0114] 4) In a laminar flow hood, pipette 150 - 200 μL of the bacterial solution and spread it evenly on the LB solid medium containing the corresponding antibiotics. Invert the medium and culture it overnight (12 - 16 h) in a 37 °C incubator.

[0115] (2) Transformation of Agrobacterium competent cells

[0116] 1) Take out the Agrobacterium competent cells from the -80 °C refrigerator and let them melt on ice.

[0117] 2) Add 1 μL of plasmid to 100 μL of competent cells, and gently pipette to mix well. Subsequently, place the competent cells on ice for 5 min, freeze in liquid nitrogen for 5 min, incubate in a 37 °C water bath for 5 min, and finally let it stand on ice for another 5 min;

[0118] 3) Add 400 μL of SOC solution, and incubate at 28 °C with shaking at 200 rpm for 2 - 3 h;

[0119] 4) Pipette 150 - 200 μL of the bacterial solution in a laminar flow hood, and evenly spread it on an LB solid medium containing the corresponding antibiotic. Finally, place it in an incubator at 28 °C and incubate it upside down for 2 - 3 days.

[0120] 2.5 Plasmid Extraction and Sequencing Verification

[0121] (1) Plasmid Extraction:

[0122] 1) Pick a single colony and inoculate it into an LB liquid medium containing the corresponding antibiotic, and incubate it overnight at 37 °C with shaking at 200 rpm for 14 - 16 h;

[0123] 2) Add 1 - 5 mL of the overnight culture to a sterile centrifuge tube, centrifuge at 12000 rpm for 1 min to collect the bacteria, and discard the supernatant;

[0124] 3) Add 250 μL of Buffer P1 (RNase A has been added) to the centrifuge tube, and vortex or pipette repeatedly to fully suspend the bacterial pellet;

[0125] 4) Add 250 μL of Buffer P2, gently invert it up and down 7 - 10 times, and let it stand at room temperature for 2 - 5 min until the solution becomes clear and viscous, indicating that the bacteria have been fully lysed;

[0126] 5) Add 350 μL of Buffer N3, immediately gently invert it 7 - 10 times, and a white flocculent precipitate will form at this time. Centrifuge at 12000 rpm for 5 min to separate the precipitate;

[0127] 6) Place the adsorption column in the collection tube, then transfer the supernatant from step 5 to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and then place the adsorption column back into the collection tube;

[0128] 7) Add 500 μL of Buffer PB to the adsorption column, centrifuge at 12000 rpm for 30 s, and pour out the waste liquid in the collection tube;

[0129] 8) Add 600 μL of Buffer PW (anhydrous ethanol has been added), centrifuge at 12000 rpm for 30 s, and pour out the waste liquid. Repeat step 8 once to ensure complete washing;

[0130] 9) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min to remove the residual Buffer PW.

[0131] 10) Place the adsorption column into a sterile 1.5 mL centrifuge tube, and pipette 50 - 100 μL of preheated ddH2O dropwise (avoid piercing the filter membrane with the pipette tip), then let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 1 min. The plasmid extraction solution collected can be used immediately or stored in a -20 °C refrigerator.

[0132] (2) Sequence verification

[0133] Sequencing and primer synthesis were completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0134] 2.6 Preparation and infection of Agrobacterium infection solution

[0135] 1) Pick a single colony of Agrobacterium and inoculate it into an LB liquid medium containing the corresponding antibiotic (5 mL of LB liquid medium in a test tube), then place it in a shaker at 28 °C and 200 rpm for 14 - 16 h until OD600 = 1.2 - 1.6.

[0136] 2) Place the propagated Agrobacterium solution in a centrifuge and centrifuge at 5,000 rpm at room temperature for 8 min to collect the bacterial cells.

[0137] 3) Discard the supernatant, add 5 mL of Agrobacterium buffer, vortex to mix evenly, centrifuge at 5,000 rpm at room temperature for 8 min to collect the bacterial cells, and repeat this step twice.

[0138] 4) Add 1.5 mL of Agrobacterium buffer, vortex to mix evenly. Pipette an appropriate amount of the resuspended solution to prepare a 5 mL resuspended solution with OD600 = 0.3, and finally add 5 μL of 100 μmol / L acetosyringone.

[0139] 5) Keep the resuspended solution in the dark in an incubator at 28 °C for 2 h. After standing, it can be used for subsequent infection experiments.

[0140] 2.7 Screening of transgenic Nicotiana benthamiana plants

[0141] To facilitate the screening of transgenic plants, all expression vectors used for transformation employed the AmCyan fluorescent protein as a screening marker. A Fluorescence Flashlight System was used to screen transgenic plants. The light source wavelength of the fluorescence flashlight was consistent with the excitation light wavelength range (430 - 450 nm) of the AmCyan fluorescent protein. When the ultraviolet light emitted by the fluorescence flashlight irradiated the transgenic plants, the plants emitted green fluorescence. Researchers could wear filter glasses that could filter the excitation light and transmit the emission light (475 nm) of the AmCyan fluorescent protein, thereby clearly observing the green fluorescence signal of the plants.

[0142] 2.8 Extraction of Nicotiana benthamiana genomic DNA

[0143] The CTAB method was used to extract the genomic DNA of transgenic Nicotiana benthamiana. The specific steps were as follows:

[0144] 1) Take an appropriate amount of tobacco leaves (0.1 - 0.5 g) and put them into a 2 mL centrifuge tube. Then add two steel beads for grinding (both the centrifuge tube and the steel beads have been sterilized). After making marks on the lid and the tube wall of the centrifuge tube, immediately put it into liquid nitrogen for freezing;

[0145] 2) Use a sample grinder to grind the sample thoroughly into a powder. Add 0.8 mL of 2×CTAB extraction buffer and incubate in a water bath at 60 °C for 1 h. During this period, gently invert and mix every 20 min;

[0146] 3) Add 0.8 mL of a mixture of phenol:chloroform:isopropanol (25:24:1), gently invert and mix, and then centrifuge at 12000 rpm for 10 min;

[0147] 4) Transfer the upper aqueous phase (about 600 - 700 μL) to a new centrifuge tube;

[0148] 5) Add 2 / 3 volume of isopropanol solution from the previous step, gently invert and mix up and down, and store in a -20 °C refrigerator for 1 h;

[0149] 6) At room temperature, centrifuge at 12000 rpm for 15 min;

[0150] 7) Discard the supernatant completely, add 0.5 mL of 70% alcohol, centrifuge at 12000 rpm at room temperature for 15 min, and repeat this step twice;

[0151] 8) Discard the supernatant completely and dry in a vacuum;

[0152] 9) Add 100 μL of ddH2O or TE to dissolve the DNA and store in a -20 °C refrigerator.

[0153] 2.9 Statistical analysis of in - situ transformation efficiency of Nicotiana benthamiana

[0154] In-situ transformation efficiency (%) = (Total number of explants producing transgenic buds / Total number of infected explants) × 100

[0155] 3 Results and analysis

[0156] 3.1 Construction of overexpression vectors for DRs (IPT, PLT5, GRF-GIF, WUS2, and WIND1)

[0157] The application of developmental regulators (DRs) plays a core role in plant genetic transformation and is of great significance for promoting transformation and regeneration and obtaining transgenic plants. In this example, five DRs (IPT, PLT5, GRF-GIF, WUS2, and WIND1) were selected, and overexpression vectors were constructed based on the modular cloning system. To facilitate the screening of transgenic plants, each expression vector contained an AmCyan fluorescent protein expression cassette driven by the 35S promoter. At the same time, the DRs in each vector were driven by the SlUbi promoter. The vector construction process is as Figure 1Shown as follows: First, extract the zero-level vectors of the 35S promoter (pICH41373), AmCyan zero-level vector (pJC375), and 35S terminator zero-level vector (pGY269) from the Golden Gate cloning vector library, and assemble them into the first-level backbone vector pJC268 according to the Modular cloning reaction system. After correct sequencing verification, save the obtained plasmid, that is, the 35S:AmCyan:35Ster first-level vector is successfully constructed and named pGY336. Subsequently, select the SlUbi promoter zero-level vector (pJC434), zero-level vectors of five DRs pJC297 (IPT), pJPC670 (PLT5), pJC370 (GRF-GIF), pJC253 (WUS2), pJC416 (WIND1), and NOS terminator zero-level vector (pICH41421) from the vector library, and assemble them into the first-level backbone vector pJC269 according to the Modular cloning reaction system. After correct sequencing verification, save the obtained plasmid, and the SlUbi:DRs:NOSter first-level vector is successfully constructed and named pFZ044 (IPT), pFZ051 (PLT5), pFZ052 (GRF-GIF), pFZ054 (WUS2), and pFZ055 (WIND1). Finally, assemble pFZ044, pFZ051, pFZ052, pFZ054, and pFZ055 with pGY336 into the second-level backbone vector pJC337 through the Modular cloning reaction system respectively. After correct sequencing verification, save the obtained plasmid, and the expression vector of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter is successfully constructed, and these vectors are named pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2), and pFZ066 (WIND1) respectively. In addition, a control vector pMIN-VS1-35S:AmCyan-SlUbi:Empty:NOSter without DRs was constructed according to the above vector construction method and named pFZ036 (No DRs). Finally, inoculate these vectors into the Agrobacterium strain 18R12 for subsequent infection experiments.

[0158] 3.2 Establishment of Agrobacterium-mediated in situ transformation method for tobacco

[0159] As Figure 2As shown, select Nicotiana benthamiana plants at 30 to 45 days of natural growth. Use a scalpel to excise the existing meristems of the plants (including shoot tips and axillary meristems), and only retain three supporting leaves. Subsequently, use a syringe equipped with a 31G needle to inject the Agrobacterium infection solution into the tobacco plants from which the meristems have been removed, injecting successively from bottom to top into the phloem of the stems, axils, and apical incisions of the plants. After the infection is completed, place the plants in a seedling tray, add an appropriate amount of clear water, cover with a humidity-keeping cover, and culture in the dark for 1 to 2 days. After 2 days, transfer to conventional culture conditions for recovery growth (26 °C, 16 hours of light, 8 hours of darkness).

[0160] The infection results are as Figure 3 shown. AmCyan fluorescence signals can be detected 3 days after infection. Within 7 days after infection, new buds mainly germinate from the original meristems, and these newly germinated buds should be continuously removed within the first 20 days. After 20 days, the buds germinating from the infected sites include spontaneously formed wild-type buds and buds induced by developmental regulators. Transgenic buds emit green fluorescence under ultraviolet light, which helps to distinguish them from wild-type buds. To ensure the growth advantage of transgenic buds, use a scalpel to thoroughly remove wild-type buds.

[0161] 3.3 Effects of different developmental regulators on the in situ transformation efficiency of Nicotiana benthamiana

[0162] To explore the effects of different developmental regulators on the in situ transformation efficiency of Nicotiana benthamiana, in this example, overexpression vectors of five DRs were constructed: pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2), and pFZ066 (WIND1), and the vector pFZ036 (No DRs) without DRs was used as a control. Subsequently, using Nicotiana benthamiana as the experimental material, in situ genetic transformation experiments were carried out using these vectors respectively. Each experimental treatment was 30 plants, with 3 replicates. The in situ genetic transformation results are as Figure 4 shown. The experiments showed that PLT5, WIND1, IPT, and GRF-GIF could all effectively promote the transformation and regeneration process. Among them, the transformation efficiency of IPT was the highest, reaching 46.7%; PLT5 was the second, with a transformation efficiency of 40%. In contrast, the control group without using DRs had the lowest transformation efficiency, only 4.0%, and all the obtained transgenic plants were chimeras. In addition, no transgenic plants overexpressing WUS2 were obtained.

[0163] To further verify the transgenic events, DNA was extracted from wild-type and tobacco leaves with green fluorescence, and specific primers were designed based on the gene sequences of AmCyan, IPT, PLT5, WIND1, and GRF-GIF. Among them, the amplified fragment of oFZ047-048 (AmCyan) was 690 bp, oFZ024-025 (IPT) was 545 bp, oFZ049-050 (PLT5) was 1656 bp, oFZ051-052 (WIND1) was 1906 bp, and oFZ053-054 (GRF-GIF) was 921 bp. The PCR results were as Figure 5 shown, all of which met the expectations, further verifying the successful creation of transgenic materials pFZ036 (No DRs), pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), and pFZ066 (WIND1).

[0164] 3.4 Effects of Different Agrobacterium Strains on the in Situ Transformation Efficiency of Nicotiana benthamiana

[0165] To improve the transformation efficiency, the Agrobacterium strains were screened and optimized in this example. The overexpression vector pFZ080 of IPT was inoculated into Agrobacterium strains 18R12, GV3101, EHA105, and AGL1 respectively, and then the in situ transformation experiment of tobacco was carried out. The results of the in situ transformation experiment were as Figure 6 shown in Figure A. All of these four strains could effectively infect tobacco and induce the generation of transgenic buds. Among them, the transformation efficiency of 18R12 was the highest, reaching 46.7%, which was significantly higher than the other three strains. There was no significant difference in the transformation efficiency of GV3101, EHA105, and AGL1, all of which were between 32% and 38.6%. Since 18R12 was modified from the Agrobacterium rhizogenes strain K599, the in situ transformation effect of K599 was further explored in this experiment. As Figure 6 shown in Figure B, the Nicotiana benthamiana plants infected with K599 only formed callus and could not induce the generation of transgenic buds. In summary, the 18R12 strain screened in this example had the highest transformation efficiency in the in situ transformation experiment of tobacco, while its parental strain K599 could not induce the formation of transgenic buds, indicating that 18R12 had better genetic transformation ability.

[0166] 3.5 Effects of Agrobacterium Infection Solution Concentration on the in Situ Transformation Efficiency of Nicotiana benthamiana

[0167] The concentration of Agrobacterium infection solution is a key factor affecting the in - planta transformation efficiency of tobacco. When the concentration of the infection solution is too low, the efficiency of delivering exogenous DNA to recipient cells will be reduced; while when the concentration of the infection solution is too high, it may inhibit the normal growth and development of plants and even cause plant death. In order to optimize the concentration of Agrobacterium infection solution and improve the in - planta transformation efficiency of tobacco, five different concentrations of Agrobacterium infection solutions were set in this example, namely OD600 = 0.1, 0.15, 0.3, 0.6, 0.9. The vector used in the experiment was pFZ080(IPT), and the Agrobacterium strain was 18R12.

[0168] As Figure 7 shown, the concentration of Agrobacterium infection solution has a significant impact on the in - planta transformation efficiency of Nicotiana benthamiana. With the increase of the concentration of the infection solution, the transformation efficiency gradually increases. When OD600 = 0.3, the transformation efficiency is the highest, reaching 46.7%; when the OD600 of the infection solution is greater than 0.3, the transformation efficiency shows a downward trend. The above results indicate that the optimal concentration of Agrobacterium infection solution is OD600 = 0.3.

[0169] Example 2

[0170] Removal of transgenic DRs mediated by Tobacco rattle virus (TRV)

[0171] 4 Experimental materials

[0172] 4.1 Plant materials

[0173] Wild - type Nicotiana benthamiana.

[0174] 4.2 Strains and plasmids

[0175] Agrobacterium tumefaciens 18R12 and GV3101, Agrobacterium rhizogenes K599. The Tobacco rattle virus (TRV) vectors pTRV1(pGY038) and pTRV2(pGY088) used in this example were donated by Professor Huajinping of China Agricultural University and stored in the - 80 °C refrigerator of this laboratory.

[0176] 4.3 Solution preparation

[0177] LB medium, antibiotics, Agrobacterium buffer, etc. refer to Example 1. The formula of 1 L of 1 / 2MS solid medium is shown in Table 10:

[0178] Table 10 Formula of 1 L of 1 / 2MS solid medium

[0179]

[0180] Adjust the pH to 5.6 - 5.8, and place it in an autoclave for autoclaving at 121 °C for 20 min.

[0181] 5 Experimental methods

[0182] 5.1 Cloning of Cre gene

[0183] (1) Cloning of Cre gene

[0184] The PCR amplification system is as follows (Table 11):

[0185] Table 11 PCR reaction system

[0186]

[0187] PCR reaction procedure:

[0188] Table 12 PCR reaction procedure

[0189]

[0190] The PCR amplification products were identified by agarose gel electrophoresis, and the correct bands were cut and recovered for subsequent use.

[0191] (2) Gel recovery of target fragment

[0192] Use the Gel Extraction Kit of Yeasen Biotech Co., Ltd. (Shanghai) for DNA recovery and purification. The steps are as follows:

[0193] 1) Weigh the agarose gel containing the target fragment.

[0194] 2) Add 100 μL of sol solution BD to every 100 mg of 1% agarose. For high-concentration agarose gels, the addition amount of sol solution BD needs to be increased proportionally.

[0195] 3) Incubate in a water bath at 56 °C for 10 min. Invert gently every 3 min during this period until the gel block is completely melted.

[0196] 4) Add 100 μL of buffer AC to the DNA adsorption column G1, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0197] 5) Add the sample mixture to the DNA adsorption column G1, let it stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0198] 6) Put the DNA adsorption column G1 back into the collection tube, add 600 μL of wash buffer W* (anhydrous ethanol has been added), centrifuge at 12000 rpm for 1 min at room temperature, and discard the waste liquid.

[0199] 7) Add 600 μL of Wash Buffer W*, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the waste liquid.

[0200] 8) Place the DNA adsorption column G1 back into the collection tube, centrifuge at 12,000 rpm for 2 min at room temperature to remove the residual Wash Buffer W*.

[0201] 9) Place the DNA adsorption column G1 into a new centrifuge tube, add 30 - 50 μL of elution buffer preheated to 70 °C to the center of the DNA adsorption column G1, and let it stand at room temperature for 2 min. Then centrifuge at 12,000 rpm for 1 min. Collect the filtrate, which is the DNA solution.

[0202] 10) Store the DNA solution in a -20 °C refrigerator.

[0203] 5.2 Plasmid Extraction and Sequencing Verification

[0204] Refer to Example 1.

[0205] 5.3 Preparation and Infection of Agrobacterium Infection Solution

[0206] Refer to Example 1 for the specific steps.

[0207] 5.4 Screening of Transgenic Nicotiana benthamiana Plants

[0208] Refer to Example 1.

[0209] 5.5 Extraction of Genomic DNA from Nicotiana benthamiana

[0210] Refer to Example 1 for the specific steps.

[0211] 5.6 Extraction of RNA from Tobacco Tissue and Synthesis of cDNA

[0212] (1) Use the Trizol method to extract RNA from tobacco tissue. The detailed steps are as follows:

[0213] 1) Weigh approximately 0.1 g of tobacco leaf tissue into a 2 mL centrifuge tube, then add two steel beads for grinding (both the centrifuge tube and the steel beads have been sterilized), make a mark on the tube wall, and immediately freeze it in liquid nitrogen;

[0214] 2) Use a grinder to thoroughly grind the sample into a powder, add 1 mL of Trizol extraction solution, and mix well by shaking to ensure that the powder does not freeze - thaw before contacting the Trizol solution;

[0215] 3) Let it stand on ice for 5 min (if there is a large amount of sample, it can be extended to 1 h);

[0216] 4) Add 0.25 mL of pre - cooled chloroform, mix well using a vortex mixer, and let it stand on ice for 5 min;

[0217] 5) Centrifuge at 4°C, 13,000 rpm for 15 min;

[0218] 6) Repeat steps 4) and 5) once each;

[0219] 7) Aspirate the supernatant and transfer it to a newly prepared 2 mL centrifuge tube (pre-cooled), then add 2 / 3 volume of pre-cooled isopropanol, gently mix, and incubate on ice for 10 min;

[0220] 8) Centrifuge at 4°C, 13,000 rpm for 10 min, discard the supernatant completely, and a small amount of white precipitate can be observed at the bottom of the centrifuge tube;

[0221] 9) Add 1 mL of 75% ethanol to wash the precipitate 2 - 3 times (use a pipette to aspirate ethanol and gently blow up the precipitate, cover the centrifuge tube cap, gently shake to wash the tube wall, and then centrifuge at 13,000 rpm for 2 min);

[0222] 10) Discard the supernatant, after drying, add 50 - 100 μL of RNase-free ddH2O to dissolve the RNA;

[0223] 11) Use a NaNoDrop to detect the RNA concentration and quality, and then store it in a -80°C refrigerator.

[0224] (2) Synthesis of cDNA:

[0225] Use the kit of YEASEN Biotech Co., Ltd. ( AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR) to synthesize the first-strand cDNA. The specific experimental steps are detailed in the instruction manual.

[0226] 6 Results and Analysis

[0227] 6.1 The application of the Cre / LoxP system has no effect on the in-situ transformation efficiency

[0228] The Cre / LoxP system consists of the Cre recombinase (Cyclization recombination enzyme) and the LoxP (Locus of x-over P1) site, and it is one of the most widely used site-specific recombinase systems. The Cre recombinase is a 38 kDa DNA recombinase produced by the cyclization recombinase gene of bacteriophage P1. It can recognize the specific DNA fragment sequence of the LoxP site and mediate the site-specific deletion of the DNA sequence between two LoxP sites. The LoxP site is 34 bp in length and consists of two 13 bp inverted palindromic repeat sequences and an 8 bp core sequence. The core sequence determines the directionality of recombination after recognition by the Cre recombinase, while the two repeat sequences provide binding sites for the Cre recombinase. The Cre / LoxP system is mainly used for gene knockout, but it can also induce DNA inversion and translocation between two LoxP sites according to the orientation and position of the LoxP sites. When two LoxP recognition sites are on the same chromatin and in the same direction, the DNA fragment between the recognition sites will be deleted under the catalysis of the Cre recombinase; when the two recognition sites are on the same chromatin and in opposite directions, recombination results in the inversion of the target DNA. Although the DNA recombination reaction catalyzed by the Cre recombinase is reversible, intramolecular DNA deletion is kinetically more advantageous than intermolecular DNA reintegration, so the gene deletion reaction is basically irreversible.

[0229] First, download the Cre gene sequence on NCBI (https: / / www.ncbi.nlm.nih.gov / ), and design specific primers according to the sequence information. Then, obtain the CDS sequence of the Cre gene by PCR amplification. Finally, ligate the CDS sequence of the Cre gene to the multiple cloning site of pTRV2, and the pTRV2-Cre vector is obtained.

[0230] As Figure 8 shown, extract the primary vectors pGY336 (35S-AmCyan-35Ster), pGY337 (Empty-2×35S-Lox71), pGY338 (SlUbi-IPT-SlUbiter), and pGY340 (Lox66-RUBY-35Ster) from the vector library, and then ligate them to the secondary backbone vector pGY332 according to the Modular cloning reaction system. After correct sequencing verification, save the obtained plasmid, and the expression vector of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster is successfully constructed and named pFZ031. Finally, inoculate pFZ031 into the 18R12 strain for subsequent infection experiments.

[0231] Using the in situ transformation method of tobacco established in 3.2, transgenic material pFZ031 (pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster) was successfully created. The statistical results of in situ transformation efficiency showed that the in situ transformation efficiency of pFZ031 was 44.5%, which was not significantly different from the transformation efficiency (46.7%) of pFZ080 (SlUbi:IPT). The above results proved that the application of the Cre / LoxP system had no significant effect on the in situ transformation efficiency.

[0232] 6.2 IPT clearance mediated by Cre recombinase delivered remotely by TRV virus

[0233] Positive plants with green fluorescence were screened out using a fluorescence flashlight system, and then the transgenic plants overcame the reproductive barrier through branch cutting, and T0 generation transgenic seeds were harvested. The T0 generation seeds were propagated, and then the fluorescence flashlight system was used again to screen T1 generation positive plants for transgenic clearance experiments. Although cutting alleviated the abnormal development problem of transgenic plants to a certain extent, the continuous expression of DRs still caused abnormal development of T1 generation transgenic plants, thus limiting their subsequent application. In addition, as a non-essential transgenic element, the complete clearance of DRs is of great significance.

[0234] pTRV1 and pTRV2-Cre were inoculated into Agrobacterium tumefaciens GV3101, and after propagation, an infection solution with an OD 600 = 0.6 was prepared and then mixed and reserved in a 1:1 ratio. As Figure 9 shown, pTRV2-Cre was inoculated into the T1 generation transgenic material of pFZ031 by leaf injection. In the pFZ031 vector, the directions of Lox71 and Lox66 were the same. Under the catalysis of Cre recombinase, Lox71 and Lox66 recombined to generate LoxP, thereby deleting the IPT gene between Lox71 and Lox66. After deleting IPT, the RUBY gene was activated and expressed. The results of transgenic clearance are as Figure 10As shown, 7 days after inoculation with pTRV2-Cre, RUBY signals appeared in the stems and roots of transgenic plants and then spread to the leaves through the vascular system. Over time, the RUBY signals gradually increased. 60 days after inoculation, RUBY signals were also observed in reproductive organs (such as red streaks on petals, sepals, and pods). The transgenic material pFZ031 that completed the transgenic removal experiment was named pFZ031ex. The seeds of pFZ031ex-T1 generation were disinfected with 5% sodium hypochlorite solution and then inoculated onto 1 / 2MS solid medium without antibiotics and cultured in an artificial climate incubator. 7 days after culture, the proportion of red plants in pFZ031ex-T2 generation plants was counted. The statistical results showed that the red plants accounted for about 50%. The above results indicate that by delivering Cre recombinase through the TRV system, the IPT gene was successfully removed and the expression of the RUBY gene was activated.

[0235] In this experiment, genomic DNA of transgenic materials pFZ031-T1, pFZ031ex-T1, and pFZ031ex-T2 was extracted, and then the removal effect of IPT was evaluated by RT-PCR. As Figure 9 shown, primers oFZ045-oFZ046 and oFZ010-oFZ011 were designed on the CDS sequences of AmCyan and IPT respectively, and the amplified fragment lengths were both 530 bp. In addition, in order to detect the transgenic removal effect, primers oFZ043 and oFZ044 were designed on both sides of the Lox site. Before IPT was removed, the sequence length between oFZ043 and oFZ044 was 2891 bp, and the length of the recombinant fragment obtained after IPT was removed was 530 bp. RT-PCR experiments were carried out with the AmCyan gene as an internal reference. The results were as Figure 11 shown. Using the genomic DNA of transgenic material pFZ031-T1 as a template, a target fragment of 2891 bp was obtained by PCR with oFZ043 and oFZ044. Using the genomic DNA of transgenic materials pFZ031-T1 and pFZ031ex-T1 as templates, PCR was carried out with primers oFZ010 and oFZ011, and IPT fragments of 530 bp were amplified, but the gel electrophoresis band brightness of pFZ031ex-T1 (very weak) was significantly lower than that of pFZ031-T1. Therefore, the removal efficiency of IPT should be above 90%. Using the genomic DNA of pFZ031ex-T2 as a template, PCR was carried out with primers oFZ043-oFZ044 and oFZ010-oFZ011 respectively, and only a recombinant fragment of 530 bp was obtained, and the target fragment of IPT was not detected. Subsequently, the PCR products were subjected to sanger sequencing. The results were as Figure 12 shown. Only the recombinantly formed LoxP site was detected in pFZ031ex-T2, and the ITP gene was not detected.

[0236] In summary, the TRV virus was able to successfully deliver the Cre recombinase into pFZ031-T1 tobacco plants, achieving efficient clearance of IPT, and transgenic tobacco plants with complete clearance of IPT were identified in the pFZ031ex-T2 generation. We refer to this process of direct in-plant transformation regeneration and precise non-selective clearance as DipTRANS (Direct in-plant transformation regeneration and accurate needlessness scavenging).

[0237] 6.3 Transgene clearance mediated by the TRV virus

[0238] This example further explored the effect of conducting transgene clearance experiments in the T0 generation transgenic materials of pFZ031. Based on the characteristic of severe clustering of T0 generation transgenic plants, transgene clearance experiments were carried out simultaneously during branch cutting. First, pTRV1 and pTRV2-Cre were inoculated into Agrobacterium rhizogenes K599 for standby. As shown in Figure 13 a, b, c, and d, a scalpel was used to separate the lateral branches from the transgenic plants, an oblique cut surface was made at the base of the stem of the lateral branches, and then monoclonal colonies of pTRV1 and pTRV2-Cre were picked, mixed evenly in equal proportions, and applied to the cut surface. The treated lateral branches were transplanted into a seedling pot, and then sealed with a transparent plastic bag filled with an appropriate amount of clear water and cultured under weak light conditions. As the cut lateral branches regenerated, the RUBY signal gradually appeared on the newly formed tissues, and finally the RUBY signal was observed in both flowers and pods ( Figure 13 e, f, and g).

[0239] To detect the transgene clearance effect, RNA was extracted from the roots, stems, and leaves of the transgenic material pFZ031ex-T0, reverse transcribed into cDNA, and stored in a -20°C refrigerator for standby. Then, according to the sequence information of the RUBY gene in the figure, specific primers oHL019 and oHL020 were designed, and the amplified fragment length was 1457 bp. The RT-PCR results are as shown in Figure 14 RUBY was expressed in the roots, stems, and leaves of the transgenic material pFZ031ex-T0, among which the expression level in the stem was the highest and the expression level in the root was the lowest. The above results indicate that the TRV virus successfully delivered the Cre recombinase into pFZ031ex-T0 plants, achieved efficient clearance of IPT, and activated the expression of the RUBY gene.

[0240] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A set of in - situ plant transformation vectors, characterized in that: Including: Expression vectors of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter: pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2), and pFZ066 (WIND1); and Expression vector pFZ031 of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster.

2. The set of in-situ plant transformation vectors according to claim 1, characterized in that: The expression vector of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter is constructed by the following steps: S1.

1. Extract the zero-level vector pICH41373 of the 35S promoter, the zero-level vector pJC375 of AmCyan, and the zero-level vector pGY269 of the 35S terminator from the Golden Gate cloning vector library, assemble them into the first-level backbone vector pJC268 according to the Modular cloning reaction system. After sequencing verification is correct, save the obtained plasmid to obtain the 35S:AmCyan:35Ster first-level vector, named pGY336; S1.

2. Select the zero-level vector pJC434 of the SlUbi promoter, the zero-level vector of DRs, and the zero-level vector pICH41421 of the NOS terminator from the vector library, assemble them into the first-level backbone vector pJC269 according to the Modular cloning reaction system. After sequencing verification is correct, save the obtained plasmid to obtain the SlUbi:DRs:NOSter first-level vector; S1.

3. Assemble the obtained SlUbi:DRs:NOSter first-level vector and pGY336 into the second-level backbone vector pJC337 through the Modular cloning reaction system respectively. After sequencing verification is correct, save the obtained plasmid to obtain the expression vector of pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter, named pFZ080 (IPT), pFZ062 (PLT5), pFZ063 (GRF-GIF), pFZ065 (WUS2), and pFZ066 (WIND1) respectively.

3. The set of in-situ plant transformation vectors according to claim 1, characterized in that: The DRs zero-level vector in the step S1.2 is the DRs zero-level vector pJC297 (IPT), the DRs zero-level vector pJPC670 (PLT5), the DRs zero-level vector pJC370 (GRF-GIF), the DRs zero-level vector pJC253 (WUS2), the DRs zero-level vector pJC416 (WIND1).

4. The set of in - situ plant transformation vectors according to claim 1, characterized in that: The expression vector pFZ031 of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster is constructed by the following steps: Extract the first-level vectors pGY336(35S-AmCyan-35Ster), pGY337(Empty-2×35S-Lox71), pGY338(SlUbi-IPT-SlUbiter) and pGY340(Lox66-RUBY-35Ster), and then ligate them to the second-level backbone vector pGY332 according to the Modular cloning reaction system. After verifying the correctness by sequencing, save the obtained plasmid to obtain the expression vector pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster, named pFZ031.

5. Use of a set of in-situ plant transformation vectors according to claim 1, characterized in that: For improving the in-situ genetic transformation efficiency of tobacco.

6. Use of a set of in-situ plant transformation vectors according to claim 5, characterized in that: Including the following steps: S1. Inoculate the expression vector pMIN-VS1-35S:AmCyan-SlUbi:DRs:NOSter into an Agrobacterium strain, and prepare an Agrobacterium infection solution with OD600 = 0.

3. S2. Infect Nicotiana benthamiana based on the Agrobacterium infection solution to achieve in-situ genetic transformation of tobacco. Specifically: Select Nicotiana benthamiana plants that are 30 to 45 days old and growing naturally, excise the existing meristems of the plants, and only retain three supporting leaves. Subsequently, inject the Agrobacterium infection solution into the tobacco plants from which the meristems have been removed, and inject it into the phloem of the stems, leaf axils, and apical incisions of the plants from bottom to top. After the infection is completed, place the plants in a seedling tray, add an appropriate amount of water, cover with a humidity-keeping cover, and culture in the dark for 1 to 2 days. After 2 days, transfer them to conventional culture conditions to resume growth.

7. Use of a set of in-situ plant transformation vectors as described in claim 1, characterized in that: For removing DRs from transgenic materials.

8. The application of a set of in-situ plant transformation vectors as claimed in claim 7, characterized in that: Use the precise recombination method mediated by the TRV virus delivery site-specific recombinase system (Cre / LoxP) to remove DRs from transgenic materials.

9. Use of a set of in-situ plant transformation vectors according to claim 8, characterized in that: Including the following steps: S1. Inoculate the expression vector pFZ031 of pMIN-VS1-35S:NptII-35S:AmCyan-Empty-2×35S:Lox71-IPT-Lox66-RUBY:35Ster into an Agrobacterium strain, and prepare an Agrobacterium infection solution. S2. Infect Nicotiana benthamiana based on the Agrobacterium infection solution to achieve in-situ genetic transformation of tobacco and obtain the T1 generation transgenic materials of pFZ031. S3. Amplify the CDS sequence of the Cre gene by PCR to obtain the Cre gene sequence. S4. Ligate the CDS sequence of the Cre gene to the multiple cloning site of pTRV2 to obtain the pTRV2-Cre vector. S5. Inoculate pTRV1 and pTRV2-Cre into Agrobacterium tumefaciens GV3101, and after propagation, prepare an infection solution with an OD 600 of 0.6, and then mix them in a ratio of 1:1 for standby; S6. Use the leaf injection method to inoculate pTRV2-Cre into the T1 generation transgenic materials of pFZ031. S7. Obtain transgenic offspring without DRs.

Images