KR101145689B1 - Transgenic bentgrass plants with herbicide resistance and disease resistance - Google Patents

Abstract

The present invention relates to a transformed grass having herbicide resistance and disease resistance, and more particularly, to a herbicide resistant and disease resistant transformed grass into which a producer resistance gene bar and a disease resistance gene PepEST1 are introduced. Transition turf maintains growth stability by having herbicide resistance and disease resistance at the same time, while not only fostering a system of grass with increased resistance to biological and abiotic stresses, but also to leisure and sports industries. It will contribute greatly to the development of high value-added genetically engineered grass varieties that are rapidly increasing in the first half.

Description

Transgenic bentgrass plants with herbicide resistance and disease resistance

The present invention relates to a transgenic grass having herbicide resistance and disease resistance, and more particularly, to a herbicide resistant and disease resistant transgenic grass into which a producer resistance gene bar and a disease resistance gene PepEST1 are introduced.

Ventgrass is an economically important cold-land grass that is mainly used for golf courses on putting greens or golf courses due to its soft surface texture, dense growth and high resistance to low mowing. In addition, it can be used for landscaping grass squares, grass around roads and livestock feed, and the grass color of Bentgrass is light green from olive green.

Proper drainage, irrigation, and disease management are especially important for the ventgrass cultivation and management. In particular, in case of having a high temperature and humidity climate in summer, as in Korea, the disease caused by the fungus of the soil is a big problem, moreover, in the case of poorly breathable and permeable grass due to dense growth, such as bentgrass has a high incidence rate It is showing.

In addition, grass is not only planted, but management is very important. Especially, the grass is weakened by weeds such as crocus or wormwood with strong fertility and roots. As a result, there is a problem of replanting the grass, which is a costly maintenance crop, and the need for and development of high value-added genetic engineering grass varieties using biotechnology is increasing.

The present inventors, while maintaining stability on the growth of lawns in particular bentgrass, while introducing a gene that gives useful agricultural characteristics or functionality, while conducting research on efficient breeding methods of grass at the same time herbicide resistance and disease resistance Eggplants have developed transgenic grass and completed the present invention.

An object of the present invention is to reduce the damage caused by the excessive use of various types of herbicides used for weed removal, and to provide a functional transgenic grass simultaneously having resistance to fungal diseases caused by high temperature and high humidity in summer.

Transformed grass according to the invention is characterized in that the transformed using a vector containing the PepEST1 gene and Bar gene, the grass is characterized in that the Bentgrass (Bentgrass).

Transformed grass according to the present invention is characterized by having a resistance to the manufacture of glufosinate (glufosinate) as an active ingredient and to brown leaf blight or coin blight.

Hereinafter, the present invention will be described in detail.

The present invention provides a transgenic grass having both herbicide resistance and disease resistance transformed with a vector containing the PepEST1 gene and the Bar gene.

The PepEST1 protein has been reported to prevent disease from occurring because it inhibits mold formation and infected mycelium, preventing mold from entering the plant (Kim et al., 2001). The Bar (bialaphos resistance gene) protein encodes a gene of phosphinothricin acetyltransferase, an enzyme that acetylates and detoxifies phosphinothricin produced by Streptomyces hygroscopyus itself. Resistance to phosphorus phosphinothricin and bialaphos has been reported.

More specifically, the transgenic grass of the present invention is transformed with a vector including the PepEST1 gene and Bar gene, and the PepEST1 gene and Bar gene are ubiquitin promoters (P _ubi ) or 35S promoters (P _35S ) for expression. As a transcription terminator, ARBCS (T _Arbcs ) Terminator or 35S Terminator (T _35S ) is used. As a specific example, the PepEST1 gene is between the ubiquitin promoter and the ARBCS terminator, and the Bar gene is between the 35S promoter and the 35S terminator. In the present invention, a transgenic grass having both herbicide resistance and disease resistance is produced by a vector containing the PepEST1 gene and the Bar gene.

And characterized by having the vector is the restriction enzyme map of Fig. 1 according to the present invention, the vector is Agrobacterium to be transformed into a grass embryogenic capacity callus by tumefaciens (agrobacterium), wherein the callus is from 0.2 to 0.8 ㎎ / It is characterized in that cultured in callus induction medium containing l of kinetin (kinetin).

More specifically, after disinfecting the grass seed, seed is placed in callus induction medium to obtain embryogenic callus through cancer culture at 25 ° C. for 4 weeks, and the callus suitable for transformation is selected and 0.5 mg / L kinetin After culturing for one week in the callus-inducing medium to which (kinetin) was added, a transformed grass having both herbicide resistance and disease resistance was prepared using the transformed Agrobacterium EHA105 which is a host microorganism. The transformed Agrobacterium EHA105 is transformed into a host microorganism Agrobacterium EHA105 by a conventional method of the grass transformation vector having the restriction enzyme map shown in FIG. 1B according to the present invention.

In the present invention, genomic DNA, total RNA and total protein were isolated from the leaves of the transgenic grass and molecular biological analysis was performed.

More specifically, the genomic polymerase chain reaction was performed to confirm the introduction of the transgenic grass and confirmed the introduction of bar and PepEST1 genes, and the genomic DNA of the grass of the individual identified by the genomic polymerase chain reaction was confirmed. Southern blot analysis was performed using a bar gene fragment amplified by restriction enzyme Hind III or EcoR I and amplified by polymerase chain reaction to confirm that the bar gene was efficiently introduced. See FIGS. 1 and 2.

In addition, Northern blot (Northern) using a bar gene or PepEST1 gene fragment amplified by polymerase chain reaction using total RNA of the individuals identified by Southern blot analysis to confirm the expression of the introduced gene. blot), and confirmed the mRNA expression of the introduced gene, using the total protein of the individuals identified by the Northern blot analysis using the PepEST1-specific polyclonal antibody and anti-rabbit monoclonal antibody (Sigma, USA) Western blot using was confirmed that the efficient expression of the protein of the introduced gene. See FIG. 3.

Transformed grass according to the invention is resistant to herbicides containing glufosinate (glufosinate) as an active ingredient, the preparation is D, L-glufosinate, D, L-glufosinate-ammonium, L -Glufosinate, L-glufosinate-ammonium, bialaphos and bialaphosium-sodium, characterized in that any one or more selected from the group consisting of.

As a specific example, 0.8% (v / v) BASTA (including 18% glufosinate ammonium) was applied to transgenic grass and non-transgenic grass grown for two weeks in a greenhouse, and 10 days later, we checked herbicide resistance. Normal grass dries yellow after 10 days, whereas individuals in transgenic grass still remain green before or after herbicide treatment. See FIG. 6.

Transgenic grass according to the present invention is characterized by having resistance to brown leaf blight or coin blight, and the grass is characterized in that the Bentgrass (Bentgrass).

Rizoctonia is a representative fungus that causes disease in the bentgrass. solani AG1-1A, Sclerotinia homoeocarpa , Phythium aphanidermatum , and each of these fungi causes brown leaf blight, Rizoctonia. solani AG1-1A), Dollar spot ( Sclerotinia homoeocarpa ), Phythium blight ( Phythium) aphanidermatum ), etc. In particular, brown leaf blight, which is a major problem in golf courses, is mainly affected by the high temperature (22-28 ° C) of early autumn (July-September) in summer, and has a feature of showing round, dark brown patches of several tens of centimeters in diameter on the lawn. have. The occurrence of this disease may be caused by continuous rainfall or excessive fertilization of nitrogenous fertilizers, and the grass of the diseased area is wilted in the form of clouds, and germs are invaded in the lower part of the upright diameter so that the stem is easily pulled out. Coin blight is irregularly yellowish green or yellowish brown spots on grass leaves or stems, and sometimes mycelium may be visible when early morning dew remains. As the disease progresses, the area around the bed will partially show a barley straw-colored discoloration, and the leaves will dry out and the grass will die. The size of the lesions is characterized by an enlargement of US dollar coins (3-5 cm in diameter). Unlike other diseases, the disease is caused by nitrogen deficiency and dry soil conditions, and is mainly caused by high, humid weather in early summer and early autumn. It is characterized by the fact that the lesions form barley straws and dark brown around them (Kim, 2001).

Bentgrass (Bentgrass) as described above exhibits a very high sensitivity to brown leaf blight or coin blight, transgenic grass according to the invention is characterized in that it has resistance to the disease.

As a specific example, three types of fungi, Rizoctonia , which cause major fungal diseases in Ventras to investigate the antifungal activity of the transgene. solani AG1-1A), coin blight (Sclerotinia homoeocarpa), Fish Umm Blight disease (the mycelium inoculation and vaccination with the purified protein concentration by checking the degree of inhibition of mycelium extending the results of Phythium aphanidermatum), a concentration-dependent manner Brown Rizoctonia solani AG1-1A) or Sclerotinia homoeocarpa was found to inhibit the incidence of. See FIG. 4.

In addition, in the resistance of crops of the transformed grass and disease of the whole plant of the transformed grass, healthy leaves without lesions caused by the fungal disease in the transgenic grass, unlike the general grass that leaves with a large number of lesions caused by fungal disease infection Could be observed. See FIGS. 5 and 7.

Transformed turf according to the present invention has the herbicide resistance and disease resistance, while maintaining stability to growth, can not only cultivate a system of grass with increased resistance to biological and abiotic stress, but also leisure and sports It will contribute greatly to the development of high value-added genetically engineered grass varieties, which are rapidly increasing in industry and society as a whole.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

[ Example  1] Development of vector for grass transformation

Gene vector for turf transformation was used by inserting the gene cassette required for turf transformation in the pCAMBIA 3301 vector (http://www.cambia.org). The ubiquitin gene promoter (P _ubi ) of corn was used for the transformation of grass, a monocotyledonous plant, and the terminator (T _Arbcs ) of RBCS gene (ARBCS) of Arabidopsis was used as a transcriptional terminator. It was. Pepper's PepEST1 gene was inserted between the ubiquitin promoter and the ARBCS terminator using BamH I and Sac I restriction enzymes. At this time, the PepEST1 gene was amplified using a polymerase chain reaction, and the polymerase chain reaction was performed using the following primers. Restriction enzyme sequences used for gene insertion ( BamH I, Sac I) are underlined.

PepEST1 forward primer: 5'- CC GGATCC ATGGCTACGCAAAGTTTTG-3 '

PepEST1 reverse primer: 5'- GG GAGCTC TTAATTTGTAGTAGCAC-3 '

In addition, as shown in FIG. 1, the gene vector for turf transformation includes not only the PepEST1 gene and the bar gene, which is a herbicide resistance gene, but also a beta glucosidase including a catalase intron as a reporter gene. intron-GUS) gene, which was used for transformation.

[ Example  2] Manufacture of Transgenic Grass with Herbicide Resistance and Disease Resistance

(1) Agrobacterium EHA105 Transformation using

In order to transform the grass using the transformation vector prepared in Example 1, first, after treating the surface of the grass seed (Bentgrass "Crenshaw" varieties) with 70% ethanol (v / v) for 10 minutes, After 20 minutes of disinfection with 2% (w / v) sodium hypochlorite, the seeds were seeded with MS basal salts, vitamins, 30 g / L sucrose, 2 mg / L 2,4-dichlorophenoxyacetic acid ( 2,4-D), 3 g / L gelrite; pH 5.8).

Embryonic callus was obtained through the culture of grass seeded in the medium for 4 weeks at 25 ℃, the callus was cultured once every two weeks and cultured in callus induction medium for one month. During passage culture, callus suitable for transformation was selected and cultured in callus induction medium to which 0.5 mg / L kinetin was added for 1 week, and then prepared in Example 1 on Agrobacterium EHA105 by a conventional method. The vector for transformation was transformed. The transformed Agrobacterium EHA105 is OD ₆₀₀ After incubation in LB medium (Luria-Bertani medium; Difco, USA), and centrifuged at 3,000 rpm for 15 minutes in the same volume of LB medium (infection media; 1/2 MS media, 2% sucrose, 1 % glucose, 100 mM acetosyringone, pH 5.2) and incubated for 4 hours. Callus cultured in a callus induction medium containing 0.5 mg / L kinetin for 1 week was mixed with a medium containing transformed Agrobacterium EHA105, incubated with gentle shaking for 15 minutes, and then placed on the filter paper and dried on callus while drying. Removed more Agrobacterium than necessary. Inoculated callus was then co-cultivated media (1/2 MS medium, 2 mg / L 2,4-D, 2% sucrose, 1% glucose, 100 μM acetosyringone, 3 g / L gelrite, pH 5.8). ), And cultured in a cancerous state at 25 ℃ for about 3.5 days.

(2) GUS staining  Confirmation of transformation through analysis

After co-cultured transformed callus of (1) was confirmed by the GUS staining analysis for transformation, 5 mg / L phosphinothricin (PPT; Duchefa) and 250 mg / L cell taxim ( cefotaxime) was cultured in a callus induction medium containing cancer at 25 ° C. for 2 weeks. Callus cultured for 2 weeks in a cancer state was incubated in a 100 μmolm ^-2 s ^-1 cool-white fluorescent lamp condition for 8 to 10 weeks in the same callus induction medium to induce re-differentiation, and to phosphinothricin Roots were induced by transferring resistant green shoots to a root induction medium containing 10 mg / L phosphinothricin and 250 mg / L cytotaxy. Subsequently, the candidates for re-differentiated transgenic grass were transferred to soil and grown in greenhouse conditions for further study.

[ Example  3] Analysis of genomic polymerase chain reaction

In order to confirm the gene introduction of the grass transformed by the method of Example 2 was analyzed using genomic polymerase chain reaction. Genomic DNA was extracted from normal turf and transgenic turf, and then polymerase chain reaction was performed using primer sets of bar, PepEST1, and actin genes.

bar forward primer: 5'-CTACCATGAGCCCAGAACGACG-3 '

bar reverse primer: 5'-CTGCCAGAAACCCACGTCATGCCAGTTC-3 '

PepEST1 forward primer: 5'-CCTGCAGCTTACGATGCC-3 '

PepEST1 reverse primer: 5'-GCATGATAACCATCTCCAAAGAGC-3 '

In addition, the actin (actin) gene was used to confirm the quantification of the reacted DNA, genomic polymerase chain reaction was performed using the following primer set.

actin forward primer: 5'-AACTGGGATGATATGGAGAA-3 '

actin reverse primer: 3′-CCTCCAATCCAGACACTGTA-3 ’

Polymerase chain reaction conditions were used for genomic DNA 200 ng or 5 ng turf transformation vector, 400 μM dNTPs, 200 μM primer set and 2 units of EX Taq DNA polymerase (Takara, Japan). The process of denaturation for 45 seconds, binding for 45 seconds at 62 ° C., and expansion for 1 minute at 72 ° C. was performed 30 times.

As a result, as can be seen in Figure 2A, it can be confirmed that the herbicide resistance gene bar and PepEST1 genes were introduced in the transgenic individuals (# 6, 7, 15, 18, 19, 21, 24).

[ Example  4] Molecular Biological Analysis of Transgenic Grass

(1) Southern Blot ( Southern blot ) analysis

Southern blot analysis was performed to confirm the number of transgenes. The procedure was as follows. First, 20 μg of genomic DNA was cut with Hind III or EcoR I, followed by electrophoresis on 0.8% agarose gel, and then transferred to Hybond N ⁺ membrane (Amersham Biosciences, UK) using capillary action. The bar gene used as a probe was amplified by polymerase chain reaction and labeled using the Radiprime ™ II random primer Labeling System (Amersham Biosciences, UK) and [α32-P] dCTP. Unlabeled nucleotides were removed using a Microspin ™ G-50 column (Amersham Biosciences, UK). Hybridization was used by mixing Quick Hyb (Stratagene, USA) with 5 × SSC in a 1: 1 ratio. Pre-hybridization, hybridization, and washing steps were performed at 65 ° C. After hybridization, wash the membrane twice with washing buffer I (2 × SSC, 0.1% SDS) for 5 minutes, then wash buffer III (1 × SSC, 0.1% SDS) for 5 minutes, and wash buffer III (0.5 × SSC). , 0.1% SDS) for 5 minutes, and finally wash with Washing buffer IV (0.1 × SSC, 0.1% SDS) for 15 minutes. Membrane was inserted into an X-ray film (Agfa, Belgium) and exposed after exposure at -70 ° C. for 3 to 4 days.

As a result, as shown in B of FIG. 2, lines # 6 and # 21 have one transgene, lines # 24 and # 19 have two transgenes, and lines # 18 and # 7 have three transgenes. It was confirmed that the above introduced gene.

(2) Northern Blot ( Northern blot ) analysis

Northern blot analysis was performed to confirm the mRNA expression of the transgene. Total RNA was extracted according to the experimental method using Trizol reagent (Invitrogen, USA). 10 μg of the extracted total RNA was electrophoresed on a 1.2% agarose gel containing formaldehyde and then transferred to a Hybond N ⁺ membrane (Amersham Biosciences, UK) using capillary action. Probes were amplified bar or PepEST1 gene using polymerase chain reaction and labeled using Radiprime ™ II random primer Labeling System (Amersham Biosciences, UK) and [α32P] dCTP, and the unlabeled nucleotides were microspin ™ G- Removed using 50 columns (Amersham Biosciences, UK). Hybridization proceeded in the same process as the Southern blot analysis. Membrane was inserted into an X-ray film (Agfa, Belgium) and exposed at -70 ° C. for 3 to 4 days.

As a result, as shown in FIG. 3A, gene expression was confirmed in all lines (# 6, 21, 15, 2, 24, 19) having one or two introduced genes as a result of the Southern blot. On the other hand, the gene expression was not found in # 18 in which three or more genes were introduced.

(3) Weston Blot ( Western blot ) analysis

Western blot analysis was performed to confirm the protein expression of the transgene. The procedure was as follows. Total protein was extracted using extraction buffer (70 mM Tris-HCl, pH 8.3), 35% ethylene glycol, 98 mM (NH ₄ ) ₂ SO ₄ , 7 mM sodium metabissulfate, 0.07% polyethyleneimine). 30 μg of protein was separated by electrophoresis on 8% SDS-PAGE and then transferred to Hybond-P membrane (Amersham Biosciences, UK). PepEST1-specific polyclonal antibody and anti-rabbit monoclonal antibody (Sigma, USA) were used to confirm the expression level of the transgene, and polyclonal βtubulin antibody (Santacruz, USA) and anti-goat were used to confirm the expression level of βtubulin, a loading control. Monoclonal antibody (Sigma, USA) was used. And it was analyzed using ECL ^TM advance western blotting analysis system (Amersham Bioscience, UK). The purified protein GST-PepEST1 was used as a positive control, and the pure purified GST-fused PepEST1 protein PC was used as a positive control.

As can be seen in B of FIG. 3, the result of protein expression analysis shows that the protein is expressed in both lines (# 6, 21, 15, 2, 24, and 19) having one or two introduced genes as in the northern blot. This was confirmed, but the expression of the protein was confirmed that the three or more genes were introduced in # 18.

[ Example  5] Antifungal Activity Analysis of Vector Protein for Grass Transformation

The antifungal activity of the transgene was examined and found to confirm the degree of inhibition of fungal mycelial growth. Three types of fungi, Rizoctonia , which cause major fungal diseases in Ventras to identify antifungal activity solani AG1-1A), coin blight (Sclerotinia homoeocarpa ) and Physium aphanidermatum , and the three fungal myceliums were cut into 20.5 cm after incubation in each medium (potato dextrose agar) and used for antifungal activity assays. . Mycelia were inoculated at both the upper and lower ends of the PDA medium, and sterile paper disks were placed in the middle of the medium, and the purified proteins were inoculated at each concentration (0, 38, 75, 150 μg). PDA medium inoculated with the fungus mycelium was placed at 25 ° C. for 72 hours to confirm the degree of inhibition of mycelial growth around the disc.

As a result you can see the results in incidence of 4, had a brown blight (Rizoctonia solani AG1-1A) inhibit fungal growth is the most effective born up in a concentration-dependent manner Coin blight (Sclerotinia homoeocarpa) occurring in inhibiting the growth of fungi Could be observed.

[ Example  6] Herbicide Resistance Survey

BASTA herbicide was used to confirm herbicide resistance to the transformed grass. 0.8% (v / v) BASTA (including 18% glufosinate ammonium) was applied to the transformed lawn candidates grown in the greenhouse for 2 weeks, and 10 days later, the herbicide resistance was confirmed.

As can be seen from the results of FIG. 6, the general grass (NT, Non-transgenic) is dried yellow after 10 days, while the transformed grass (# 2, 6, 15, 18, 19, 21, 24) still remains It was confirmed that the greenness remained unchanged before or after treatment with the herbicide.

[ Example  7] bottle resistance probe

Investigation into fungal disease resistance of the transformed grass was carried out using grass leaves and whole plants.

(1) Disease Resistance Survey Using Transgenic Grass Leaves

To investigate the disease resistance using transgenic grass leaves, cultured pathogens of brown blight ( Rizoctonia solani AG1-1A) in PDA (potato dextrose agar) medium, and put mycelium of brown blight pathogens in PDA broth in a 25 ℃ thermostat. The OD ₆₀₀ was prepared to be 0.9. 5 ml each of the brown blight pathogens was inoculated on the leaves cut from the grass grown for 4 weeks. Observation confirmed the degree of infection of the grass. In addition, the staining method was used to more accurately determine the degree of fungal infection, lactophenol-blue staining proceeded as follows. Grass leaves infected with the brown blight pathogen were fixed in fixed solution (50% EtOH 100 ml, formalin 6.5 ml, acetic acid 2.5 ml) for 3 days, and then 500 µl of lactophenol-blue solution was dropped onto the fixed grass leaves. Leave for a minute and wait for the staining of brown blight. Thereafter, the dyed grass leaf was placed on the slide glass, the curved glass was covered, and the pathogen of the brown blight was dyed at 200 (2010) magnification under a microscope.

As a result, as can be seen from the results of Figure 5, the lesions were found in the transgenic grass lines (# 6, 21, 24) in which the protein expression of the introduced gene was verified compared to the general grass (NT), which is infected with the fungus and observed the lesion. Not observed.

(2) Disease resistance investigation using whole plants of transgenic grass

Investigation of disease resistance using the whole plant of transformed grass was carried out in a similar manner to the investigation of disease resistance using grass leaves of the above (1). First, the pathogens of brown blight were prepared to have an OD ₆₀₀ of 0.9, and 10 ml of each of the pathogen cultures of brown blight was sprayed on the grass plants grown for 4 weeks. After the grass was grown for 4 weeks in 25 ℃ long conditions (16 hours light conditions / 8 hours dark conditions), the pathogenesis of the pathogen of brown blight on the plant was observed to confirm the degree of infection of the grass.

As a result of observing the lesion formation by the pathogen of brown blight disease, as shown in FIG. 7, it was observed that the leaves withered together with many lesions caused by the fungal disease infection in the general turf, but the lesion caused by the fungal disease in the transgenic grass. You could observe healthy leaves without these.

Figure 1 shows a schematic diagram of the vector used in the transgenic grass according to the present invention,

(A: Schematic diagram of the T-DNA region of the vector, B: Schematic diagram of the whole vector)

Figure 2 is a result confirming the gene introduction of the transgenic grass according to the present invention,

(A: genomic polymerase chain reaction analysis, B: Southern blot analysis)

3 is a result confirming the expression of the transgene introduced transgenic grass according to the present invention,

(A: Northern blot analysis, B: Western blot analysis)

Figure 4 shows the results confirming the antifungal activity of the vector protein for turf transformation according to the present invention,

5 is a disease using the leaves of the transgenic grass according to the present invention ( Rizotonia solani ) is the result of investigating the resistance,

(A: lesion after 4 days of inoculation, B: fungal infection after 4 days of inoculation)

6 is a result of examining the herbicide resistance using the outpost of the transformed grass according to the present invention,

Figure 7 is a result of examining the resistance of the disease ( Rizoctonia solani ) using the outpost of the transformed grass according to the present invention.

Claims (7)

Glufosinate- resistant, Rizoctonia solani and Sclerotinia homoeocarpa- resistant Bentgrass varieties Crenshaw, which are transformed with a vector having a restriction map of FIG. 1. grass. delete The method of claim 1, The vector is a grass characterized in that the grass embryogenic calli cultivated in a callus induction medium containing 0.2 to 0.8 mg / l of kinetin (kinetin) by agrobacterium ( Agrobacterium ) strains EHA105. delete delete delete delete

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