Method for improving transformation efficiency of plants by using agrobacterium into which virg mutant gene and nahg gene have been introduced

Abstract

The present invention relates to a method for improving the transformation efficiency of a plant by using Agrobacterium into which a virGN54D gene<sp / >, which is a virG mutant gene, and a NahG(salicylate hydroxylase) gene have been introduced. The Agrobacterium according to the present invention is used to improve transformation efficiency by effectively and stably delivering a gene of interest into a plant, and thus can be effectively used in the fields of transgenic plant production and plant gene editing.

Description

Method to improve plant transformation efficiency using Agrobacterium introduced with virG mutant genes and NahG genes

[0001] The present invention relates to the virG mutant gene, virG N54D This invention relates to a method for improving the transformation efficiency of plants using Agrobacterium into which the NahG (salicylate hydroxylase) gene has been introduced.

[0002]

[0003] This research was conducted with support from the Startup Growth Technology Development Project of the Korea Institute of Technology Information Promotion (KIIT) under the Ministry of SMEs and Startups (Project No.: RS-2024-00468098).

[0004] Agrobacterium is a natural genetic tool useful in the field of plant biotechnology for introducing specific foreign genes into plants. Initially, it was reported that Agrobacterium, containing the Ti (Tumor-inducing) plasmid, caused root nodules by delivering and expressing oncogenes in plants. However, with the advancement of plant biotechnology, it was discovered that the Ti plasmid is separated into a helper plasmid and a binary vector that do not contain oncogenes, leading to the development of techniques to safely introduce specific genes into plants. Nevertheless, transformation using Agrobacterium has not yet been effectively achieved in many plant species.

[0005] In addition, Agrobacterium is positively affected by various substances derived from plants, such as having its unique specific abilities promoted, or negatively affected, such as having its growth inhibited. Plant-derived substances known to have a negative effect on Agrobacterium include GABA (γ-aminobutyric acid), ethylene, IAA (indole acetic acid), or salicylic acid.

[0006] In particular, salicylic acid is a plant hormone that activates plant defense responses against various pathogens and is known to inhibit the replication of Ti plasmids in Agrobacterium by suppressing quorum sensing signals. It is also known to inhibit the expression of vir genes in Agrobacterium by directly inhibiting the two-component signaling system of VirA / VirG. This salicylic acid is oxidized by salicylate hydroxylase or salicylate 1-monooxygenase and degraded into catechol. Accordingly, the inventors intended to investigate whether the salicylic acid activity of the plant could actually change or whether the transfer efficiency of foreign genes could change when plant transformation is induced using Agrobacterium into which salicylic acid-degrading enzymes have been introduced.

[0007] Meanwhile, Korean Registered Patent No. 0948980 discloses 'a method for increasing the transformation efficiency of garlic using Agrobacterium and transformed garlic produced by said method,' and Korean Registered Patent No. 0927135 discloses 'a method for improving plant transformation efficiency through vacuum treatment with a highly concentrated Agrobacterium solution,' but the virG of the present invention N54DThere is no description of a method to improve the transformation efficiency of plants using Agrobacterium into which the gene and NahG gene have been introduced.

[0008] The present invention was derived from the above-mentioned requirements, and in the present invention, virG N54D After constructing a vector expressing the gene and the NahG (salicylate hydroxylase) gene and introducing it into Agrobacterium, and then further introducing a vector expressing the target gene (GFP or RUBY), plants (tobacco or cannabis) were transformed using the Agrobacterium, and the transformation efficiency for the target gene was analyzed, the results showed that virG N54D The present invention was completed by confirming that the transformation efficiency increased when using Agrobacterium into which the gene and the NahG gene were introduced, compared to the control group using Agrobacterium without the NahG gene.

[0009] To solve the above problem, the present invention relates to virG N54D A method for improving the transformation efficiency of a plant is provided, comprising the step of transforming a plant cell using a bacterium of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced.

[0010] In addition, the present invention relates to virG N54D The present invention provides a composition for improving the transformation efficiency of a plant, comprising as an active ingredient bacteria of the genus Agrobacterium into which the gene and the NahG gene have been introduced.

[0011] In addition, the present invention relates to virG N54D We provide bacteria of the genus Agrobacterium into which the gene and NahG gene have been introduced to improve plant transformation efficiency.

[0012] In addition, the present invention relates to virG N54DA method for producing a plant with improved transformation efficiency is provided, comprising the steps of: transforming a plant cell using a bacterium of the genus Agrobacterium into which the gene and the NahG gene have been introduced; and regenerating a transformed plant from the transformed plant cell.

[0013] The virG of the present invention N54D By using Agrobacterium bacteria into which the gene and NahG (salicylate hydroxylase) gene have been introduced, the gene of interest can be effectively and stably delivered to a plant, thereby improving transformation efficiency. Therefore, it can be usefully applied in the fields of manufacturing transgenic plants and plant gene editing.

[0014] FIG. 1 is a schematic diagram of the vectors used in the present invention, including the GabT and AcdS gene expression vector (Tv-GE), GabT, AcdS, and virG N54D Gene expression vector (Tv-GEV), NahG gene expression vector (Tv-S), GabT, AcdS, and NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) and GabT,AcdS, virG N54D This is a schematic diagram of the NahG gene expression vector (Tv-GEVS).

[0015] FIG. 2 shows the results of measuring the salicylic acid (SA) degradation activity (A), GABA amino group transfer activity (B), and ACC deamination activity (C) of Agrobacterium EHA105 strains into which the vectors of the present invention (Tv-GE, Tv-GEV, Tv-S, Tv-GES, Tv-VS, or Tv-GEVS), respectively, were introduced. GV3101(pMP90): Agrobacterium GV3101 strain into which the pMP90 vector was introduced, EHA105(EV): Agrobacterium EHA105 strain into which the covector was introduced.

[0016] FIG. 3 shows the results of evaluating the transformation efficiency for the GFP (green fluorescent protein) gene in tobacco plants using Agrobacterium EHA105 strains into which the vectors of the present invention (Tv-GE, Tv-GEV, Tv-S, Tv-GES, Tv-VS, or Tv-GEVS) were respectively introduced, where A is the Western blot result confirming the GFP protein expression level in tobacco leaves, and B is the quantified Western blot result. LBA4404: Wild-type Agrobacterium LBA4404 strain, EHA105(pRiA4-VIR): Agrobacterium EHA105 strain into which the VIR gene expression vector was introduced, LBA4404(pRiA4-VIR): Agrobacterium LBA4404 strain into which the VIR gene expression vector was introduced.

[0017] Figure 4 shows the results of evaluating the transformation efficiency of the RUBY gene in cannabis plants using Agrobacterium EHA105 strains into which the vectors of the present invention (Tv-GE, Tv-GEV, Tv-S, Tv-GES, Tv-VS, or Tv-GEVS) were respectively introduced. A is a photograph confirming betalain accumulated in immature cannabis embryos, and B and C are the results of measuring the betalain content using a colorimetric method. Mock: Negative control.

[0018] To achieve the objective of the present invention, the present invention relates to virG N54D A method for improving the transformation efficiency of a plant is provided, comprising the step of transforming a plant cell using a bacterium of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced.

[0019] In this invention, the term "transformation efficiency" refers to the ability to stably and effectively deliver a desired target gene to a plant.

[0020] In a method for improving the transformation efficiency of a plant according to the present invention, the NahG gene may be a NahG gene derived from Pseudomonas putida and may consist of the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto.

[0021] In addition, the above virG N54D The gene is a mutant gene in which asparagine (N), the 54th amino acid of the virG gene of Agrobacterium tumefaciens, is substituted with aspartic acid (D), and may consist of the nucleotide sequence of SEQ ID NO. 2, but is not limited thereto.

[0022] In addition, the above Agrobacterium genus may preferably be Agrobacterium tumefaciens, and more preferably may be the Agrobacterium tumefaciens EHA105 strain, but is not limited thereto.

[0023] In addition, in the method according to the present invention, the virG N54D "Plant cells" transformed using bacteria of the genus Agrobacterium into which the gene and the NahG gene have been introduced may be any plant cells. Plant cells are cultured cells, cultured tissues, cultured organs, or whole plants. "Plant tissues" include differentiated or undifferentiated plant tissues, such as, but not limited to, cotyledons, embryos, hypocotyls, roots, stems, leaves, pollen, seeds, female tissues, and various forms of cells used for culture, namely single cells, protoplasts, immature embryos, buds, and callus tissues. Plant tissues may be in planta or in organ culture, tissue culture, or cell culture.

[0024] In a method according to one embodiment of the present invention, the plant body may be a dicotyledonous plant such as tobacco, hemp, tomato, Arabidopsis thaliana, potato, eggplant, chili pepper, burdock, crown daisy, lettuce, balloon flower, spinach, Swiss chard, sweet potato, celery, carrot, water celery, parsley, Chinese cabbage, cabbage, mustard greens, watermelon, Korean melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea, or a monocotyledonous plant such as corn, barley, wheat, rice, oats, rye, or sugarcane, preferably a dicotyledonous plant, more preferably tobacco (Nicotiana benthamiana) or hemp (Cannabis sativa), but is not limited thereto.

[0025] In addition, the method of the present invention includes the step of redifferentiating a transgenic plant from the transgenic plant cell. Any method known in the art may be used for the method of redifferentiating a transgenic plant from the transgenic plant cell.

[0026] A method for improving the transformation efficiency of a plant body according to one embodiment of the present invention is specifically,

[0027] (a) A step of germinating tobacco seeds and culturing them for 25 to 35 days;

[0028] (b) a step of obtaining a fully unfolded leaf from the cultured tobacco plant;

[0029] (c) virG N54D A step of preparing a culture medium by introducing a target gene expression vector into an Agrobacterium tumefaciens strain into which the gene and the NahG gene have been introduced and culturing it; and

[0030] (d) a step of transforming by infiltrating the back surface of the leaf of step (b) with the culture solution of step (c) and then co-culturing for 94 to 98 hours; may be included,

[0031] or,

[0032] (a) A step of germinating cannabis seeds and culturing them for 25 to 35 days;

[0033] (b) a step of obtaining an immature embryo from the cultured cannabis plant;

[0034] (c) virG N54D A step of preparing a culture medium by introducing a target gene expression vector into an Agrobacterium tumefaciens strain into which the gene and the NahG gene have been introduced and culturing it; and

[0035] (d) a step of immersing the immature embryo of step (b) in the culture medium of step (c) for 30 to 40 minutes, and then co-culturing for 70 to 74 hours to transform it; may be included, but is not limited thereto.

[0036] The above-mentioned target gene refers to a portion of DNA intended to be delivered through the present invention, and is not limited to the type of gene and may include both coding and non-coding regions. A person skilled in the art may select the above-mentioned target gene for a transgenic plant to be manufactured according to the purpose.

[0037] The present invention also, virG N54D A composition for improving the transformation efficiency of a plant is provided, comprising as an active ingredient a bacterium of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced.

[0038] In the composition for improving the transformation efficiency of a plant according to the present invention, the virG N54D The gene, NahG gene, and bacteria of the genus Agrobacterium are as described above.

[0039] In the composition for improving the transformation efficiency of a plant according to the present invention, the genus Agrobacterium bacteria may have additionally introduced a GABA aminotransferase gene (GabT) and an ACC deamidase gene (AcdS), but are not limited thereto.

[0040] The present invention also, virG N54DA bacterium of the genus Agrobacterium is provided to improve plant transformation efficiency by introducing the gene and the NahG (salicylate hydroxylase) gene.

[0041] In the bacteria of the genus Agrobacterium according to the present invention, the virG N54D The gene, NahG gene, and bacteria of the genus Agrobacterium are as described above.

[0042] In a bacterium of the genus Agrobacterium according to one embodiment of the present invention, the bacterium of the genus Agrobacterium may additionally have a GABA aminotransferase gene (GabT) and an ACC deamidase gene (AcdS) introduced, but is not limited thereto.

[0043] The present invention also, virG N54D A step of transforming plant cells using bacteria of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced; and

[0044] The present invention provides a method for producing a plant body with improved transformation efficiency, comprising the step of regenerating a transformed plant from the transformed plant cell.

[0045] In the method for producing a plant with improved transformation efficiency according to the present invention, the virG N54D The gene, NahG gene, and bacteria of the genus Agrobacterium are as described above.

[0046]

[0047] The present invention will be explained in detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0048]

[0049] Example 1. virG N54D and NahG gene expression vector manufacturing

[0050] In the present invention, the nucleotide sequence (Sequence No. 1) of the salicylate hydroxylase (NahG) gene was confirmed in the GenBank database (Accession No. M60055.1), and virG N54D The nucleotide sequence of the gene (Sequence No. 2) was amplified from Addgene's vector (#123187) and used in the experiment. In addition, the nucleotide sequence of the GABA aminotransferase gene (γ-aminobutyric acid transaminase, GabT) (Sequence No. 3) was verified in the GenBank database (Accession No. 948067), and the nucleotide sequence of the ACC deamidase gene (1-Aminocyclopropane-1-carboxylic acid deaminase, AcdS) (Sequence No. 4) was verified in the GenBank database (Accession No. AY823987) and used in the experiment.

[0051] A NahG gene expression vector (Tv-S) was prepared by inserting a NahG gene expression cassette, manufactured via the gene synthesis service (Genewiz), into the cloning site of the broad-spectrum host vector pBBR1-MCS5B using GoldenGate cloning.

[0052] In addition, a GabT and AcdS gene expression vector (Tv-GE) was prepared by inserting a GabT and AcdS gene co-expression cassette, manufactured via the gene synthesis service (GenScript), into the cloning site of pBBR1-MCS5B using GoldenGate cloning.

[0053] In addition, virG prepared using the virG-F2 primer (5'-cagtcGGTCTCatagctgtaacctcgaagcgt-3': SEQ ID NO. 5) and the virG-R1 primer (5'-cagtcGGTCTCaAAGCccgtcttggtggtcagtgtg-3': SEQ ID NO. 6) N54DThe gene expression cassette and the GabT and AcdS gene co-expression cassette were inserted into the cloning site of pBBR1-MCS5B via Golden Gate cloning to extract GabT, AcdS, and virG N54D A gene expression vector (Tv-GEV) was prepared.

[0054] In addition, the GabT and AcdS gene co-expression cassette and the NahG gene expression cassette were inserted into the cloning site of pBBR1-MCS5B via Golden Gate cloning to GabT, AcdS, and NahG A gene expression vector (Tv-GES) was prepared.

[0055] Also, virG N54D The gene expression cassette and the NahG gene expression cassette were inserted into the cloning site of pBBR1-MCS5B via Golden Gate cloning to virG N54D A NahG gene expression vector (Tv-VS) was prepared.

[0056] In addition, GabT and AcdS gene co-expression cassette, virG N54D The gene expression cassette and NahG gene expression cassette were inserted into the cloning site of pBBR1-MCS5B via Golden Gate cloning to GabT, AcdS, virG N54D A NahG gene expression vector (Tv-GEVS) was prepared (Fig. 1, Table 1).

[0057] Vector used in the present invention Vector description Tv-GEGabT,AcdS gene insertion Tv-GEVGabT,AcdS, virG N54D Gene insertion Tv-SNahG Gene insertion Tv-GESGabT,AcdS,NahG Gene insertion Tv-VSvirG N54D ,NahG gene insertion Tv-GEVSGabT,AcdS, virG N54D ,NahG gene insertion

[0058] Each vector in Table 1 above was introduced into the Agrobacterium tumefaciens EHA105 strain via electroporation, and then cultured in LB (Luria-Bertani) medium containing 50 mg / L gentamycin and 50 mg / L rifampicin.

[0059]

[0060] Example 2. Measurement of salicylic acid (SA) decomposition activity

[0061] The salicylate hydroxylase gene (NahG) is an enzyme that oxidizes salicylic acid and breaks it down into catechol. Accordingly, the GabT and AcdS gene expression vector (Tv-GE), GabT, AcdS, and virG N54D Gene expression vector (Tv-GEV), NahG gene expression vector (Tv-S), GabT, AcdS, NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) or GabT,AcdS, virG N54D Salicylic acid degradation activity was measured by measuring the catechol content of Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was introduced.

[0062] Specifically, the above Agrobacterium strains are each cultured (OD 600=0.8), bacterial protein extraction reagents BugBuster Master Mix (Novagen) and EDTA-free Protease Inhibitor Cocktail (Roche) were added and dissolved at room temperature for 20 minutes. Then, the mixture was centrifuged at 10,000 rpm for 20 minutes, the supernatant was transferred to a new tube, and the protein concentration of the supernatant was measured using the BCA protein assay (Thermo scientific). Subsequently, the supernatant of each culture with the measured concentration was added to the reaction mixture (200 μM Salicylic acid, 200 μM β-Nicotinamide adenine dinucleotide hydrate (NADH), 20 μM Flavin adenine dinucleotide disodium salt hydrate (FAD), 33 mM potassium phosphate buffer, pH 7.0) in an amount of 50 μg based on protein concentration, and the salicylic acid degradation reaction was induced at 25°C. After the reaction, the remaining salicylic acid forms a purple salicylic acid-FeCl3 complex with iron chloride (FeCl3), which appears purple. The amount of salicylic acid remaining in the reaction solution was measured by measuring this purple solution at a wavelength of 560 nm, and the salicylic acid degradation activity was calculated. Agrobacterium tumefaciens GV3101 strain with the pMP90 vector introduced and Agrobacterium tumefaciens EHA105 strain with the pBBR1 vector introduced were used as controls.

[0063] As a result, NahG gene expression vector (Tv-S), GabT, AcdS, NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) or GabT,AcdS, virG N54DIt was confirmed that the salicylic acid degradation activity of Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was introduced was significantly increased compared to the control group, indicating that the introduced NahG gene was functioning normally within Agrobacterium (Fig. 2A).

[0064]

[0065] Example 3. Measurement of GABA amino group transfer activity

[0066] The GABAeminophilase (GabT) gene is an enzyme that breaks down GABA into glutamate. Accordingly, the GabT and AcdS gene expression vector (Tv-GE), GabT, AcdS, virG N54D Gene expression vector (Tv-GEV), NahG gene expression vector (Tv-S), GabT, AcdS, NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) or GabT,AcdS, virG N54D GABAemin transfer activity was measured by measuring the glutamate content of Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was introduced.

[0067] Specifically, the above Agrobacterium strains are each cultured (OD 600=0.8), bacterial protein extraction reagents BugBuster Master Mix (Novagen) and EDTA-free Protease Inhibitor Cocktail (Roche) were added and dissolved at room temperature for 20 minutes. Then, the mixture was centrifuged at 10,000 rpm for 20 minutes, the supernatant was transferred to a new tube, and the protein concentration of the supernatant was measured using the BCA protein assay (Thermo Scientific). Subsequently, the supernatant of each culture with the measured concentrations was added to the reaction solution (0.1 M NaOH, 1 mM pyridoxalphosphate, 10 mM 2-ketoglutarate, 10 mM GABA) at an amount of 100 μg based on protein concentration, and the GABA degradation reaction was induced at 37°C for 10 minutes. Afterward, the glutamate content was measured using the glutamate assay kit (Sigma). Agrobacterium tumefaciens GV3101 strain with pMP90 vector introduced and Agrobacterium tumefaciens EHA105 strain with pBBR1 vector introduced were used as controls.

[0068] As a result, GabT and AcdS gene expression vectors (Tv-GE), GabT, AcdS, virG N54D Gene expression vector (Tv-GEV), GabT, AcdS, NahG Gene expression vectors (Tv-GES) or GabT,AcdS, virG N54D It was confirmed that the GABA amino group transfer activity of Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was introduced was significantly increased compared to the control group, indicating that the introduced GabT gene was functioning normally within Agrobacterium (Fig. 2B).

[0069]

[0070] Example 4. Measurement of ACC deamination activity

[0071] The ACC deamidase gene (AcdS) is an enzyme that breaks down ACC into α-ketobutyrate and ammonia. Accordingly, the GabT and AcdS gene expression vector (Tv-GE), GabT, AcdS, and virG N54D Gene expression vector (Tv-GEV), NahG gene expression vector (Tv-S), GabT, AcdS, NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) or GabT,AcdS, virG N54D ACC deamination activity was measured by measuring the α-ketobutylate content of Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was introduced.

[0072] Specifically, the above Agrobacterium strains are each cultured (OD 600 =0.8), after additionally culturing for 1 day in DF medium containing 3 mM ACC, the cells were lysed by treatment with toluene. Then, 20 µl of 0.5 M ACC was added to 200 µl of the toluened cell suspension and reacted at 30°C for 30 minutes. After centrifugation at 13,000 rpm for 5 minutes, 300 µl of 0.2% (v / v) 2,4-dinitrophenylhydrazine was added to the supernatant obtained and reacted for 30 minutes, after which the absorbance was measured at 540 nm to determine the content of α-ketobutylate. Agrobacterium tumefaciens GV3101 strain with pMP90 vector introduced and Agrobacterium tumefaciens EHA105 strain with pBBR1 vector introduced were used as controls.

[0073] As a result, GabT and acdS gene expression vectors (Tv-GE), GabT, AcdS, virG N54D Gene expression vector (Tv-GEV), GabT, AcdS, NahG Gene expression vectors (Tv-GES) or GabT,AcdS, virG N54D By confirming that the ACC deamination activity of Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was introduced was significantly increased compared to the control group, it was found that the introduced AcdS gene was functioning normally within Agrobacterium (Fig. 2C).

[0074]

[0075] Example 5. Evaluation of gene transformation efficiency in tobacco plants

[0076] To evaluate the gene transformation efficiency in tobacco (Nicotiana benthamiana) plants, the GFP (green fluorescent protein) gene was delivered to tobacco plants, and the transformation efficiency of the GFP gene was evaluated by measuring GFP expression.

[0077] Specifically, after sowing tobacco seeds in soil, fully unfolded tobacco leaves were prepared by culturing them at 23°C for one month under a photoperiod of 16 hours light / 8 hours dark. GabT and AcdS gene expression vector (Tv-GE), GabT, AcdS, virG N54D Gene expression vector (Tv-GEV), NahG gene expression vector (Tv-S), GabT, AcdS, NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) or GabT,AcdS, virG N54D After introducing binary vectors containing the GFP gene (Addgene #40259, #48015) via electroporation into Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) was respectively introduced, the strains were cultured in LB medium and the OD 600After infiltrating the back of the above tobacco leaf, which had been prepared in advance with a fluorescence level of 0.8, to an area of ​​about 2 cm in diameter using a 1 ml syringe, the leaf was cultured at 23°C for 96 hours under a photoperiod of 16 hours light / 8 hours dark, and the GFP fluorescence signal was confirmed using the Azure 600 Imaging system (Azure Biosystems). In addition, the protein was extracted from 100 mg of the infiltrated leaf that had been cut and quenched in liquid nitrogen, and Western blot was performed using an anti-GFP polyclonal rabbit antibody (1:10,000, abcam-ab6556) to confirm the expression of the GFP protein.

[0078] As a result, virG N54D and It was confirmed that GFP expression in tobacco plants transformed with an Agrobacterium EHA105 strain into which the NahG gene expression vector (Tv-VS) was introduced was significantly increased compared to the control or comparison group (Fig. 3).

[0079] Based on the above results, virG N54D and It was found that the transformation efficiency of the GFP gene in tobacco plants increased due to the expression of the NahG gene.

[0080]

[0081] Example 6. Evaluation of gene transformation efficiency in cannabis plants

[0082] To evaluate the gene transformation efficiency in Cannabis sativa plants, the RUBY gene, which converts tyrosine to betalain, was delivered to immature embryos of Cannabis plants, and the transformation efficiency of the RUBY gene was evaluated by measuring the betalain content.

[0083] Specifically, after sowing cannabis seeds in soil, they were cultured at 23°C for one month under a 16-hour light / 8-hour dark photoperiod, and then transferred to a 12-hour light / 12-hour dark photoperiod to induce flowering. During this process, male and female plants were grown in isolation, and when each plant bloomed, pollen from the male flowers of the male plants was used to pollinate the pistils of the female plants. After 21 days, immature embryos were obtained. GabT and AcdS gene expression vector (Tv-GE), GabT, AcdS, virG N54D Gene expression vector (Tv-GEV), NahG gene expression vector (Tv-S), GabT, AcdS, NahG Gene expression vector (Tv-GES), virG N54D and NahG gene expression vector (Tv-VS) or GabT,AcdS, virG N54D After introducing a binary vector containing the RUBY gene (Addgene #160908) via electroporation into Agrobacterium EHA105 strains into which the NahG gene expression vector (Tv-GEVS) and NahG were respectively introduced, the strains were cultured in LB medium to obtain OD 600 The immature embryos of the above-mentioned cannabis, prepared in advance with a pH of 0.8, were immersed in a medium (1 / 2 MS basal medium, 2% sucrose, 1% glucose, 100 μM acetosyringone, pH 5.2) for 40 minutes, and then co-cultured under dark conditions for 72 hours. Afterward, the transformed Agrobacterium was transferred to 1 / 2 MS basal medium containing tymentin and cultured for 7 days, and then the betalain content was measured using a powder sample prepared by homogenizing with liquid nitrogen.

[0084] As a result, virG N54D and It was confirmed that the beta-rain content in immature cannabis embryos transformed with an Agrobacterium EHA105 strain into which the NahG gene expression vector (Tv-VS) was introduced increased significantly compared to the control group or comparison group (Fig. 4).

[0085] Based on the above results, virG N54D and It was found that the transformation efficiency of the RUBY gene in cannabis plants increased due to the expression of the NahG gene.

Claims

1. virG N54D A method for improving the transformation efficiency of a plant, comprising the step of transforming a plant cell using a bacterium of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced.

2. A method for improving the transformation efficiency of a plant, wherein, in claim 1, the NahG gene is composed of the nucleotide sequence of SEQ ID NO.

1.

3. In paragraph 1, the above virG N54D A method for improving the transformation efficiency of a plant, characterized in that the gene consists of the nucleotide sequence of SEQ ID NO.

2.

4. A method for improving the transformation efficiency of a plant, characterized in that, in claim 1, the bacterium of the genus Agrobacterium is Agrobacterium tumefaciens.

5. In Paragraph 1, (a) A step of germinating tobacco seeds and culturing them for 25 to 35 days; (b) a step of obtaining a fully unfolded leaf from the cultured tobacco plant; (c) virG N54D A step of preparing a culture medium by introducing a target gene expression vector into an Agrobacterium tumefaciens strain into which the gene and the NahG gene have been introduced and culturing it; and (d) a step of infiltrating the back surface of the leaf of step (b) with the culture solution of step (c) and then co-culturing to transform; a method for improving the transformation efficiency of a plant.

6. In Paragraph 1, (a) A step of germinating cannabis seeds and culturing them for 25 to 35 days; (b) a step of obtaining an immature embryo from the cultured cannabis plant; (c) virG N54D A step of preparing a culture medium by introducing a target gene expression vector into an Agrobacterium tumefaciens strain into which the gene and the NahG gene have been introduced and culturing it; and (d) a step of immersing the immature embryo of step (b) in the culture medium of step (c) and then co-culturing to transform it; a method for improving the transformation efficiency of a plant. 7.virG N54D A composition for improving the transformation efficiency of a plant, comprising as an active ingredient bacteria of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced.

8. A composition for improving the transformation efficiency of a plant, wherein, in claim 7, the genus Agrobacterium bacteria further comprises a GABA aminotransferase gene (GabT) and an ACC deamidase gene (AcdS). 9.virG N54D Agrobacterium bacteria into which the gene and the NahG (salicylate hydroxylase) gene are introduced to improve the transformation efficiency of plants.

10. The genus Agrobacterium of claim 9, characterized in that the genus Agrobacterium further has a GABA aminotransferase gene (GabT) and an ACC deamidase gene (AcdS) introduced. 11.virG N54D A step of transforming plant cells using bacteria of the genus Agrobacterium into which the gene and the NahG (salicylate hydroxylase) gene have been introduced; and A method for producing a plant with improved transformation efficiency, comprising the step of regenerating a transformed plant from the transformed plant cell.

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