CN118547001A - Herbicide resistance genes and their applications - Google Patents

Abstract

The present invention belongs to the field of transgenic technology, and discloses a herbicide resistance gene and its application. The present invention utilizes genetic engineering technology, and through an Agrobacterium-mediated genetic transformation method, a glyphosate resistance gene ( EPSPS gene) and a glufosinate resistance gene ( Bar gene, "BlpR" in the plasmid map) are transferred into bermudagrass callus tissue. The callus tissue is differentiated, rooted, and seedlings are grown to obtain bermudagrass plants with dual resistance to glyphosate and glufosinate. The EPSPS gene and the Bar gene are successfully transferred through GUS staining, PCR detection, RT-PCR, leaf smearing and other detection methods. The present invention directionally transforms the genetic traits of bermudagrass, and provides a solution for the prevention and control of various weeds during lawn establishment and management.

Description

Herbicide resistance genes and their applications

Technical Field

The invention belongs to the technical field of transgenic technology and relates to a herbicide resistance gene and application thereof.

Background Art

Cynodon dactylon (L.) Persoon is an important greening plant, playing an active role in urban greening, sports field construction, and ecological environment protection. The application scope of lawns is constantly expanding, but pests and diseases and abiotic stress have seriously affected the function and appearance of lawns.

Using genetic engineering technology to improve grass lawns is a convenient and effective way. The improvement directions of lawn grasses mainly include disease resistance, insect resistance, and herbicide resistance. Among them, herbicide resistance is a useful agronomic trait of transgenic lawn grasses. Herbicide resistance in lawn grasses can not only be used as an effective tool to control the growth of weeds, but also reduce the maintenance and management burden of lawns such as urban greening and sports fields. On the other hand, it can also control the occurrence of certain diseases. For example, in the existing document "Establishment of the Genetic Transformation System of Perennial Ryegrass and the Acquisition of Transformed Plants" (Yi Zili et al., Journal of Grassland Science, 2006-08-20), genetic transformation research was carried out with the Bar gene as the exogenous target gene and a widely used lawn-type perennial ryegrass new variety as the receptor material, and herbicide-resistant transgenic ryegrass plants were obtained.

At present, most of the research on lawn grass with herbicide resistance is based on one resistance gene. The transgenic lawn grass obtained has a single resistance function and is only resistant to one herbicide, which is greatly limited in practical application. How to obtain transgenic lawn grass with resistance to two or even multiple herbicides and expand the practical application of existing transgenic lawn grass is a problem that needs to be solved urgently.

Summary of the invention

In view of the technical problems that the existing technology has a single resistance function for lawn grass and poor resistance effect to the combined application of multiple herbicides, the present invention obtains a lawn grass variety with dual resistance to glyphosate and glufosinate by genetic engineering technology. In order to achieve this technical purpose, the present invention specifically provides the following technical solutions.

The present invention provides an application of a herbicide resistance gene in cultivating a herbicide-resistant lawn grass variety, specifically, a glyphosate resistance gene and a glufosinate resistance gene are simultaneously transferred into lawn grass to obtain a transgenic lawn grass, wherein the transgenic lawn grass exhibits resistance to both glyphosate and glufosinate. The glyphosate resistance gene is an EPSPS gene having a nucleotide sequence as shown in SEQ ID NO: 1; and the glufosinate resistance gene is a Bar gene having a nucleotide sequence as shown in SEQ ID NO: 2.

Furthermore, in the above application, the lawn grass is Bermuda grass.

On the other hand, the present invention also provides a method for breeding lawn grass varieties with dual resistance to glyphosate and glufosinate, which specifically includes: constructing a recombinant plasmid containing a glyphosate resistance gene and a glufosinate resistance gene, transferring the recombinant plasmid into lawn grass callus by using an Agrobacterium-mediated genetic transformation method, and the callus is differentiated, rooted, and grows seedlings to obtain a lawn grass variety with dual resistance to glyphosate and glufosinate. The glyphosate resistance gene is an EPSPS gene, and the EPSPS gene has a nucleotide sequence as shown in SEQ ID NO: 1; the glufosinate resistance gene is a Bar gene, and the Bar gene has a nucleotide sequence as shown in SEQ ID NO: 2.

Furthermore, in the above-mentioned lawn grass variety breeding method, the upstream primer used to amplify the EPSPS gene is SEQ ID NO: 3, and the downstream primer is SEQ ID NO: 4. The upstream primer used to amplify the Bar gene is SEQ ID NO: 5, and the downstream primer is SEQ ID NO: 6.

Furthermore, in the above-mentioned lawn grass variety breeding method, the lawn grass is Bermuda grass.

Compared with the prior art, the "herbicide resistance gene and its application" of the present invention has the following beneficial effects:

The present invention utilizes genetic engineering technology to transfer a glyphosate-resistant gene ( EPSPS gene) and a glufosinate-resistant gene ( Bar gene) into bermudagrass plants through an Agrobacterium-mediated genetic transformation method, thereby obtaining transgenic bermudagrass plants with dual resistance to glyphosate and glufosinate, and through GUS staining, PCR detection, RT-PCR, leaf smearing and other detection methods, it is proved that both the EPSPS gene and the Bar gene are successfully integrated.

The present invention has directed the modification of the genetic traits of Bermuda grass, providing an environmentally friendly and efficient way to control weeds in grass lawns and even pastures. In actual weed control work, more than one weed is usually faced with control, and transgenic lawn grasses resistant to a single herbicide are significantly limited in actual application. The technical solution provided by the present invention successfully obtains transgenic Bermuda grass varieties with dual resistance to glyphosate and glufosinate, providing a solution for the control of multiple weeds during lawn establishment and management.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the electrophoresis result of EPSPS gene after PCR amplification. In Figure 1, lane M is the DNA standard molecule; lanes 1 to 4 are the EPSPS gene.

Figure 2 is a schematic diagram of the structure of the pCAMBIA3301-35S plasmid containing the Bar gene. In Figure 2, kanR is the kanamycin resistance gene, LB T-DNA repeat is the two border sequences of T-DNA, CaMV poly (A) signal is the terminator, BlpR is the Bar gene, CaMV 35S promoter (enhanced) and CaMV 35S promoter are 35S promoters, and EcorR I is a restriction endonuclease cleavage site.

Figure 3 is a schematic diagram of the structure of the p3301-35S-EPSPS recombinant plasmid containing the EPSPS gene. In Figure 3, kanR is a kanamycin resistance gene, LB T-DNA repeat is two border sequences of T-DNA, CaMV poly (A) signal is a terminator, BlpR is a Bar gene, CaMV 35S promoter (enhanced) and CaMV 35S promoter are 35S promoters, EcorR I and Hind III are restriction endonuclease cleavage sites, and EPSPS is an EPSPS gene.

Figure 4 is the electrophoresis result of E. coli colony PCR. In Figure 4, lane M is the DNA standard molecule; lanes 1 to 4 are the tested bands.

Figure 5 is a graph showing the electrophoresis results of Agrobacterium colony PCR. In Figure 5, lane M is the DNA standard molecule; lane 1 is the tested band.

Figure 6 shows the process of callus tissue growing into transgenic bermudagrass plants. Figure 6 a shows callus tissue subculture screening; Figure 6 b shows callus tissue differentiation; Figure 6 c shows callus tissue rooting and seedling growth; Figure 6 d shows the survival of transgenic bermudagrass plants after transplantation.

Figure 7 shows the results of GUS histochemical staining. Figure 7 a is the CK control group; Figure 7 b and c are instantaneous detection after 10 minutes of infection; Figure 7 d is the detection of leaves of transgenic Bermuda grass plants.

Figure 8 is the electrophoresis result of PCR amplification of EPSPS gene. Lane M is the DNA standard molecule; Lanes 1 to 6 are transgenic Bermuda grass plants; Lane 7 is the wild-type plant; Lane 8 is the EHA105-p3301-35S-EPSPS strain.

Figure 9 is the electrophoresis result of PCR amplification of the Bar gene. Lane M is a DNA standard molecule; Lanes 1 to 6 are transgenic Bermudagrass plants; Lane 7 is a wild-type plant; Lane 8 is the EHA105-p3301-35S-EPSPS strain.

Figure 10 is a graph showing the results of RT-PCR electrophoresis of the EPSPS gene. Lane M is a DNA standard molecule; Lanes 1 to 6 are transgenic Bermudagrass plants; Lane 7 is the EHA105-p3301-35S-EPSPS strain; and Lane 8 is a wild-type plant.

Figure 11 is a diagram of the RT-PCR electrophoresis results of the Bar gene. Lane M is a DNA standard molecule; Lanes 1 to 6 are transgenic Bermudagrass plants; Lane 7 is the EHA105-p3301-35S-EPSPS strain; Lane 8 is a wild-type plant.

Figure 12 shows the appearance of leaves of Bermuda grass plants 7 days after being smeared with two concentrations of glyphosate. Figure 12 a shows the result of 0.4% glyphosate smearing, and Figure 12 b shows the result of 0.8% glyphosate smearing; 1 and 2 represent transgenic Bermuda grass plants, and 3 and 4 represent wild-type plants.

Figure 13 shows the appearance of leaves of Bermuda grass plants 7 days after being smeared with two concentrations of glufosinate ammonium. Figure 13 a shows the result of 0.2% glufosinate ammonium smearing, and Figure 13 b shows the result of 0.4% glufosinate ammonium smearing; 1 and 2 represent transgenic Bermuda grass plants, and 3 and 4 represent wild-type plants.

DETAILED DESCRIPTION

The technical solution of the present invention is described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Culture medium and solution used in the examples:

1) LB solid medium: weigh 5 g yeast extract, 10 g NaCl, 10 g tryptone, and 15 g agar, add ddH ₂ O to make up to 1 L, and sterilize at 121°C for 20 min.

2) YEB solid medium: weigh 1g yeast extract, 5g tryptone, 5g beef extract, 5g sucrose, 0.5g MgSO ₄ ·7H ₂ O, add 900mL ddH ₂ O, adjust pH to 7.2 with NaOH, make up to 1L, and sterilize at 121℃ for 20min.

3) MS solid medium: weigh 4.43 g of MS basic medium, 30 g of sucrose, 9 g of agar, add 900 mL of ddH ₂ O, adjust the pH to 6.0 with NaOH, make up to 1 L, and sterilize at 121°C for 20 min.

4) Kanamycin solution: Prepare to a concentration of 50 mg/mL, filter through a 0.22 μm water filter for sterilization, store at -20°C, and use at a concentration of 50 mg/L.

5) Thioproterenol solution: prepare to a concentration of 500 mg/mL, filter through a 0.22 μm water filter to sterilize, store at -20°C, and use at a concentration of 500 mg/L.

6) Acetosyringone solution: Weigh 392 mg of acetosyringone and dissolve it in 20 mL of DMS. After sufficient dissolution and mixing, filter and sterilize through a 0.22 μm organic filter membrane to obtain a 100 mM stock solution, which is stored at -20°C for future use.

The plant tissue culture medium used in the embodiment is:

1) Callus induction medium: MS basic medium, 3 mg/L 2,4-D, 0.01 mg/L 6-BA, 1 mg/L chitosan, sucrose (30 g/L).

2) Callus subculture medium: MS basic medium, 1 mg/L 2,4-D, 0.01 mg/L 6-BA, 2 mg/L ABA, 0.2 mg/L GA3.

3) Differentiation medium: MS basic medium, 2 mg/L 6-BA, 0.5 mg/L NAA.

4) Rooting medium for callus tissue: MS basic medium, 0.3 mg/L NAA.

5) Co-culture medium: MS basic medium, 1 mg/L 2,4-D, 100 µM acetosyringone.

6) Screening medium: subculture medium, 20 mg/L kanamycin, 500 mg/L thiophanate-methyl.

The pH value of the above culture medium was adjusted to 6.0 and sterilized by high pressure at 121°C for 20 min.

The experimental materials and reagents used in the examples are shown in Table 1, and the strains and plasmids used are shown in Table 2.

Table 1. Experimental materials and reagents used in the examples

Table 2. Strains and plasmids used in the examples

Example 1

This example describes the nucleotide sequence information of the EPSPS gene and the Bar gene.

The nucleotide sequence of the EPSPS gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the Bar gene is shown in SEQ ID NO: 2.

Example 2

This embodiment provides the use of EPSPS gene and Bar gene in breeding herbicide-resistant lawn grass varieties.

1. Amplification and purification of target gene

1) PCR amplification of EPSPS gene

According to the EPSPS gene (i.e., glyphosate-resistant gene) sequence described in Example 1, the upstream primer EPSPS- F and the downstream primer EPSPS- R of the EPSPS gene were designed (see Table 3).

Table 3. EPSPS gene primer information

Note: Sequences with lowercase letters are homologous sequences.

Using the EPSPS gene sequence as a template, the above primers were used for PCR amplification. The amplification system was: template DNA, 2µL, EPSPS- F, 2µL, EPSPS- R, 2µL, 2×Phanta Max Master Mix, 25µL, ddH ₂ O, 19µL. Reaction conditions: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 55℃ annealing for 15s, 72℃ extension for 1.5min, 35 cycles; 72℃ post-extension for 5min, and storage at 4℃.

2) Column recovery

Take 5 µL of PCR product for 1% agarose gel electrophoresis and observe it in a UV gel imaging system after 25 min of electrophoresis. Recover the PCR product according to the column recovery method (Cycle-Pure Kit) as follows.

① Place the PCR product in a 1.5 mL centrifuge tube and add 4-5 times the volume of CP buffer. Vortex and centrifuge briefly to mix.

② Add 700µL DNA wash buffer to the adsorption column, centrifuge at 12000rpm for 1min, and discard the filtrate.

③Repeat step ②.

④ Place the adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 minutes at room temperature.

⑤ Transfer the adsorption column to a sterilized clean centrifuge tube, open the cover of the adsorption column and let it stand for 2 minutes, then add 30~50µL of preheated ddH ₂ O in the center of the adsorption membrane, centrifuge at 12000rpm for 1 minute at room temperature to obtain the purified DNA product, and store it at -20℃.

The agarose gel electrophoresis results of the EPSPS gene after PCR amplification are shown in Figure 1. In Figure 1, lane M is the DNA standard molecule; lanes 1 to 4 are the EPSPS gene. As shown in Figure 1, there is a target band between 1000 bp and 2000 bp, which is consistent with the expected size of the EPSPS gene, indicating that the EPSPS gene was successfully amplified.

2. Construction of recombinant plasmid

1) Plasmid propagation

① Take the pCAMBIA3301-35S plasmid containing the Bar gene, purchased from Miaoling Biotechnology Co., Ltd. (see Figure 2 for a schematic diagram of the structure), centrifuge at 5000 rpm for 1 min at room temperature, add 20 µL of ddH ₂ O to dissolve, and let stand for 1 min.

②Take out the E. coli DH5α competent cells from the -80℃ refrigerator and thaw them on ice.

③ Add 2µL of pCAMBIA3301-35S plasmid to 100µL of DH5α competent cells, incubate on ice for 30min, heat shock at 42℃ for 90s, and immediately place on ice for 2min.

④ Add 900 µL of LB liquid culture medium without antibiotics and culture in a shaking incubator at 37°C for 45 min.

⑤ Centrifuge at 5000rpm for 5min, discard 800µL of supernatant, mix the remaining liquid with the bacterial pellet, spread the mixed bacterial liquid on a plate containing 50mg/L kanamycin (Kan), and culture inverted at 37℃ for 12h.

2) Plasmid extraction

Select the strain with the correct sequencing result and use the Plasmid mini Kit to extract the pCAMBIA3301-35S plasmid. The steps are as follows:

① Centrifuge the bacterial solution at 12000rpm for 2min at room temperature, discard the supernatant and retain the bacterial pellet.

②Add 250 μL of Solution I and vortex until there is no white bacterial precipitate.

③Add 250μL Solution II, mix and invert 5 times (there will be sticky threads at the mouth of the bottle). Invert slowly and for no more than 5 minutes.

④ Add 350 μL Solution III and immediately rotate and invert for 10 to 20 times until white flocs are precipitated. Be careful to avoid violent shaking.

⑤ Centrifuge at 12000rpm for 12min at room temperature.

⑥ Pipette 750 µL of supernatant into the filter column (the filter column is placed in a 2 mL collection tube), centrifuge at 12000 rpm for 1.5 min at room temperature, and discard the filtrate.

⑦ Add 500 μL of HBC Buffer to the filter column, centrifuge at 12000 rpm for 1.5 min, and discard the filtrate.

⑧Add 700 μL DNA Wash Buffer to the filter column, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

⑨Repeat step ⑧.

⑩ Centrifuge at 12000rpm for 2.5min, place the filter column in a new sterilized centrifuge tube, add sterile water preheated at 65℃, let it stand for 1min, then centrifuge at 12000rpm for 1.5min, and store at -20℃.

3. Ligation of EPSPS gene with pCAMBIA3301-35S plasmid

The pCAMBIA3301-35S plasmid was double-digested with EcoR I and Hind III to obtain a linearized pCAMBIA3301-35S plasmid. After digestion, electrophoresis was performed to verify the target band. The gel block was cut out and recovered with a gel extraction kit (Gel Extraction Kit). The purified linearized pCAMBIA3301-35S plasmid was connected to the EPSPS gene by homologous recombinase C112, and the recombinant plasmid obtained by connection was named p3301-35S-EPSPS, and its structural schematic diagram is shown in Figure 3.

The ligation reaction system is shown in Table 4. Ligation reaction conditions: 37°C water bath for 30 min, then cool to 4°C or immediately place on ice.

Table 4. Ligation reaction system of EPSPS gene and linearized pCAMBIA3301-35S plasmid

The ligation product was transformed into E. coli DH5α competent cells. The transformation and culture methods were described in the above plasmid expansion steps. Pick the cultured single colony and extract the p3301-35S-EPSPS recombinant plasmid. Use EPSPS gene primers to amplify it by PCR. The size of EPSPS gene is about 1500bp. The electrophoresis results of PCR amplification products are shown in Figure 4. Lane M in Figure 4 is a DNA standard molecule; Lanes 1 to 4 are tested colonies. In Figure 4, it can be seen that there are target bands between 1000bp and 2000bp, which are in line with the expected size. The colony with the correct band was expanded and cultured to obtain a recombinant E. coli strain. Use 30% glycerol to preserve the bacteria and store at -80℃.

4. Transformation of recombinant plasmid into Agrobacterium

Take the recombinant E. coli strain stored at -80℃, extract the p3301-35S-EPSPS recombinant plasmid and transform it into Agrobacterium EHA105. Transformation method:

① Take out EHA105 Agrobacterium competent cells from -80℃ freezer and thaw on ice.

② Take 5µL of p3301-35S-EPSPS recombinant plasmid and add it to 100µL Agrobacterium competent cells and mix well.

③ Place on ice for 5 min, place in liquid nitrogen for 5 min, place in 37°C water bath for 5 min, and place in ice bath for 5 min.

④ Add 700 µL of YEB medium into the centrifuge tube without adding antibiotics to the medium, and culture in a shaker at 28°C for 3 h.

⑤ Centrifuge at 5000rpm for 5min, discard 700µL of supernatant, mix the remaining liquid with the bacterial pellet, spread the bacterial solution on the plate containing Kan, and invert it in a 28℃ incubator for 2d.

A single colony was picked and verified by colony PCR. The bacterial solution with the correct band was sent for testing. After the sequencing result was correct, the culture was expanded and the Agrobacterium was stored at -80°C with 30% glycerol. The recombinant Agrobacterium strain was named EHA105-p3301-35S-EPSPS.

The electrophoresis result of Agrobacterium colony PCR is shown in Figure 5. In Figure 5, lane M is the DNA standard molecule; lane 1 is the tested colony. In Figure 5, it can be seen that there is a target band between 1000bp and 2000bp, indicating that EHA105-p3301-35S-EPSPS Agrobacterium was successfully obtained.

5. Cultivation of transgenic Bermuda grass plants

1) Agrobacterium activation culture

Cultivate EHA105-p3301-35S-EPSPS Agrobacterium on solid YEB plates, select single colonies, add 1mL YEB liquid medium (containing 50mg/L Kan), and place in a 28℃ shaker for 1d. After the culture is completed, transfer the liquid medium to 30mL YEB liquid medium (containing 50mg/L Kan), and continue to shake and culture in a 28℃ shaker until the bacterial concentration OD600=0.5. Add 100µM acetosyringone and let it stand for 3h in the dark at 28℃ before infection.

2) Infection

① Soak the Bermudagrass callus in good condition in Agrobacterium solution and shake it for 10 minutes.

② Use sterile filter paper to absorb excess bacterial liquid from the callus tissue, transfer it to the co-cultivation medium padded with filter paper, and culture it in the dark at 25℃ for 2 days.

③ Rinse the co-cultivated callus tissue with sterile water for several times, add 500 mg/L cefotaxime (Cef) to the sterile water, mix well and soak the callus tissue in it for 30 minutes, then replace it with sterile water and rinse 1 to 2 times, each time for 2 minutes, and use sterile filter paper to absorb the droplets on the callus tissue.

④ The callus tissue was inoculated into the screening medium and cultured at 25°C, L:D=16h:8h under light. The medium was changed every two weeks. After changing the screening medium twice, it was transferred to the differentiation medium at 30 days. After differentiation and rooting, green bermudagrass seedlings grew. The callus tissue that was not successfully infected gradually died on the screening medium and was discarded.

Figure 6 shows the process of callus tissue growing into transgenic bermudagrass plants. Figure 6 a shows callus tissue subculture screening; Figure 6 b shows callus tissue differentiation; Figure 6 c shows callus tissue rooting and seedling growth; Figure 6 d shows the survival of transgenic bermudagrass plants after transplantation.

Example 3

This example provides identification of transgenic Bermuda grass plants.

1. GUS Histochemical Staining

The expression product of the reporter gene GUS , GUS, can hydrolyze X-Gluc. The hydrolysis product is a colorless indole derivative, which undergoes an oxidation reaction with K ₄ Fe(CN) ₆ , making the tissue part appear blue. The preparation of GUS dye is shown in Table 5.

Table 5. Preparation of GUS staining solution

After the infected callus tissue was co-cultured for 2 days, it was taken out and placed in a centrifuge tube, and the prepared dye solution was added to immerse the callus tissue, and placed at 37°C in the dark for 24 hours. The dye solution was discarded, and the callus tissue was rinsed with 50% and 70% anhydrous ethanol respectively, and finally decolorized by soaking in anhydrous ethanol, and observed and photographed.

Figure 7 shows the results of GUS histochemical staining. Figure 7 a is the CK control group; Figure 7 b and c are transient detections after 10 minutes of infection, indicating that the GUS gene was successfully expressed in the callus tissue.

The transgenic bermudagrass plants were transplanted to an incubator at 25°C, with a light intensity of 1000-2000 lx and L:D=16h:8h. Leaves of wild-type bermudagrass and transgenic bermudagrass were selected for staining. After decolorization with anhydrous ethanol, the leaves of wild-type bermudagrass were yellow, while the leaves of transgenic bermudagrass had blue spots, indicating that the GUS gene was stably expressed in bermudagrass plants (Fig. 7d).

2. PCR detection of transgenic Bermuda grass plants

1) CTAB method to extract plant DNA

① Add 500µL of DNA lysis buffer and grind thoroughly.

② Add 350µL of balanced phenol (pH>7, lower solution), 340µL of chloroform, and 20µL of isopropanol, shake vigorously, let stand for 5 minutes, and centrifuge at 12000rpm for 15 minutes at room temperature.

③ Take 200µL of the supernatant, add 400µL of anhydrous ethanol and 20µL of ammonium acetate, mix well and place at -80℃ for 5min, centrifuge at 12000rpm for 5min, discard the filtrate and keep the precipitate at the bottom of the centrifuge tube.

④ Wash the precipitate twice with 700 µL of 75% ethanol, centrifuge at 12,000 rpm for 2 min, and dry it with a pipette tip.

⑤ Add 20~30µL of Eluen water preheated at 65℃ to dissolve the precipitate and store at -20℃.

2) Gene PCR and electrophoresis detection

According to the Bar gene (i.e., glufosinate-resistant gene) sequence, specific primers for the Bar gene were designed and synthesized using primer 5 software, as shown in Table 6.

Table 6. Bar gene primer information

The transgenic bermudagrass DNA was extracted by the method of step 2, and the extracted DNA was amplified by PCR with reference to the method of step 1 of Example 2, and the Bar gene and EPSPS gene were detected by using the specific primers of the Bar gene and EPSPS gene, respectively. The wild-type bermudagrass and the EHA105-p3301-35S-EPSPS strain were used as negative and positive controls. 5 µL of the amplified product was taken for gel electrophoresis, and observed and photographed after 25 minutes.

Figure 8 is the electrophoresis result of PCR amplification of EPSPS gene. Lane M is DNA standard molecule; Lanes 1 to 6 are transgenic Cynodon dactylon plants; Lane 7 is wild-type plants; Lane 8 is EHA105-p3301-35S-EPSPS strain. As shown in Figure 8, the bands in lanes 1 to 6 are consistent with those in lane 8, and both have target bands at 1500 bp, while no band at 1500 bp appears in lane 7, indicating that the transgenic Cynodon dactylon plants contain EPSPS gene fragments.

Figure 9 is the electrophoresis result of PCR amplification of the Bar gene. Lane M is a DNA standard molecule; Lanes 1 to 6 are transgenic Cynodon dactylon plants; Lane 7 is a wild-type plant; Lane 8 is an EHA105-p3301-35S-EPSPS strain. As shown in Figure 9, the bands in lanes 1 to 6 are consistent with those in lane 8, and both have target bands at 500 bp, while no target band appears in lane 7, indicating that the transgenic Cynodon dactylon plants contain Bar gene fragments. Figures 8 and 9 show that both the EPSPS gene and the Bar gene were successfully transferred into Cynodon dactylon plants.

3. RT-PCR detection of transgenic Bermuda grass plants

1) RNA extraction

① Pre-cool the centrifuge tube containing the Bermudagrass leaves in liquid nitrogen, grind them and add 1000 mL of RNAex to mix.

② Add 200µL of chloroform, mix well, and let stand for 5 minutes.

③ Centrifuge at 12000rpm in a 4℃ centrifuge for 15min, aspirate the top layer of liquid, and transfer it to a new centrifuge tube.

④ Add 450 µL of isopropanol to the supernatant, mix well and let stand at room temperature for 10 min.

⑤ Centrifuge at 12000rpm in a 4℃ centrifuge for 10min and discard the supernatant.

⑥ Add 1000 µL of 75% ethanol (pre-cooled) to the centrifuge tube, centrifuge at 7500 rpm for 5 min in a 4°C centrifuge, discard the filtrate, and repeat twice.

⑦ Dry at room temperature for 5 minutes and blow off the residual ethanol.

⑧Add 20µL of RNA-free water to dissolve the precipitate.

2) Reverse transcription

The extracted RNA from Cynodon dactylon was reverse transcribed into cDNA, and the reverse transcription system was as follows: RNA, 1000µL, gDNA remover, 1µL, 4×AllinOne RT Mixture IV, 5µL, Free H ₂ O, up to 20µL. Reverse transcription procedure: 37℃, 5min, 55℃, 35min, 90℃, 1min, and stored at 4℃.

3) RT-PCR detection

RT-PCR was performed using cDNA as a template to amplify EPSPS gene and Bar gene fragments, with wild-type Bermuda grass as a negative control and EHA105-p3301-35S-EPSPS strain as a positive control. The amplification system was referred to step 1 in Example 2.

Figure 10 is a graph showing the RT-PCR electrophoresis results of the EPSPS gene, and Figure 11 is a graph showing the RT-PCR electrophoresis results of the Bar gene. Lane M is a DNA standard molecule; lanes 1 to 6 are transgenic Cynodon dactylon plants; lane 7 is an EHA105-p3301-35S-EPSPS strain; and lane 8 is a wild-type plant. As shown in Figure 10, target bands that match the size of the EPSPS gene fragment appear in lanes 1 to 6 and 7, while no band appears in lane 8. As shown in Figure 11, target bands that match the size of the Bar gene fragment appear in lanes 1 to 6 and 7, while no band appears in lane 8. Figures 10 and 11 indicate that both the EPSPS gene and the Bar gene were successfully transferred into Cynodon dactylon plants.

4. Leaf smear resistance test of transgenic plants

Transgenic bermudagrass plants were smeared with glufosinate and glyphosate, respectively, and wild-type bermudagrass plants were used as controls. Glyphosate was set at two concentrations of 0.4% and 0.8%, and glufosinate was set at two concentrations of 0.2% and 0.4%, and observations were made 1, 3, 5, and 7 days after smearing. The results are shown in Table 7.

Table 7. Changes in leaves of Bermuda grass plants after application of glufosinate and glyphosate

Figure 12 shows the appearance of leaves of Bermuda grass plants 7 days after being smeared with two concentrations of glyphosate. Figure 12 a shows the result of 0.4% glyphosate smearing, and Figure 12 b shows the result of 0.8% glyphosate smearing; 1 and 2 represent transgenic Bermuda grass plants, and 3 and 4 represent wild-type plants.

Figure 13 shows the appearance of leaves of Bermuda grass plants 7 days after being smeared with two concentrations of glufosinate. Figure 13 a shows the result of 0.2% glufosinate smear, and Figure 13 b shows the result of 0.4% glufosinate smear; 1 and 2 represent transgenic Bermuda grass plants, and 3 and 4 represent wild-type plants. As shown in Figures 12 and 13, transgenic plants have dual resistance to glyphosate and glufosinate.

The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims (5)

1. Application of herbicide resistance genes in breeding herbicide-resistant lawn grass varieties, characterized in that glyphosate resistance genes and glufosinate resistance genes are simultaneously transferred into lawn grass to obtain transgenic lawn grass, wherein the transgenic lawn grass exhibits resistance to both glyphosate and glufosinate; The glyphosate-resistant gene is an EPSPS gene having a nucleotide sequence as shown in SEQ ID NO: 1; The glufosinate-resistant gene is a Bar gene having a nucleotide sequence as shown in SEQ ID NO:2. 2. The use according to claim 1, characterized in that the lawn grass is Bermuda grass. 3. A method for breeding a lawn grass variety with dual resistance to glyphosate and glufosinate, characterized in that it comprises: constructing a recombinant plasmid containing a glyphosate resistance gene and a glufosinate resistance gene, transferring the recombinant plasmid into lawn grass callus by using an Agrobacterium-mediated genetic transformation method, and causing the callus to differentiate, root, and grow seedlings to obtain a lawn grass variety with dual resistance to glyphosate and glufosinate; The glyphosate-resistant gene is an EPSPS gene, and the EPSPS gene has a nucleotide sequence as shown in SEQ ID NO: 1; The glufosinate-resistant gene is a Bar gene, and the Bar gene has a nucleotide sequence as shown in SEQ ID NO:2. 4. The method for breeding lawn grass varieties according to claim 3, characterized in that the upstream primer used to amplify the EPSPS gene is SEQ ID NO: 3, and the downstream primer is SEQ ID NO: 4; The upstream primer used to amplify the Bar gene is SEQ ID NO:5, and the downstream primer is SEQ ID NO:6. 5 . The method for breeding lawn grass varieties according to claim 4 , characterized in that the lawn grass is Bermuda grass.