KR100742192B1 - Chromosome transformation method of plant - Google Patents

Abstract

The present invention relates to a method for transforming a pigment in a plant. More specifically, the present invention relates to a method for transforming a pigment of a plant, characterized in that the callus derived from the plant is transformed with a vector for transforming a pigment.

The method according to the present invention does not disappear even when the transformant is developed into an adult, and there is an effect that the gene transplanted into the pigment is transferred to the next generation.

Callus, Transgenic, Plant, Callus

Description

Method for plastid transformation in plants             

Figure 1a is a schematic diagram showing the manufacturing process of the vector pLD-RCtV for transforming the pigment according to the present invention does not contain the sgfp gene.

Figure 1b is a schematic diagram of the vector pLD-RCtV-sGFP for chromosomal transformation according to the present invention.

Figure 1c is a schematic diagram showing the homologous recombination process between the chromatin transformation vector according to the present invention and the natural chromatin genome.

Figure 2a is a callus of rice and the streptomycin was added to each MS (Murachige and Skoog) medium with different concentrations and cultured under light conditions.

a: Add 0 mg / L streptomycin

b: Add 100 mg / L streptomycin

c: 200 mg / L of streptomycin added

Figure 2b is a callus of rice was incubated in dark and MS conditions in which streptomycin was added at different concentrations, respectively.

a: Add 0 mg / L streptomycin

b: Add 500 mg / L streptomycin

c: 1000 mg / L addition of streptomycin

Figure 3a is a photograph of the shoots induced in the callus showing resistance to streptomycin when the callus of rice transformed with a vector for transforming the chromosomes according to the method of the present invention and cultured in a selective medium added with streptomycin .

Figure 3b is a photograph showing a transformant obtained by transforming the callus of rice with a vector for transforming the pigment in accordance with the method of the present invention, selected and re-differentiated in a selection medium added with streptomycin.

Figure 4 shows the effect of the treatment concentration of streptomycin when the transformants selected according to the method of the present invention to be purified in the soil.

Figure 5a is a schematic diagram showing the size of the trnI and trnA gene used in homologous recombination of the present invention, the aadA gene as a selection gene, the sgfp gene used as the target gene.

Figure 5b is a result of PCR amplification of genomic DNA of the transformed rice in order to confirm the presence of the sgfp gene in the transformed rice in accordance with the method of the agarose gel.

6 is a result of analyzing the genomic DNA of the transformed rice by Southern blot to confirm the presence of aadA gene in the transformed rice according to the method of the present invention.

Figure 7a is a result of PCR amplification of genomic DNA of rice in order to confirm the presence of the left interface fragment of the homologous recombination site in the transformed rice in accordance with the method of the present invention.

Figure 7b is a result of PCR amplification of genomic DNA of rice in agarose gel to determine whether the right interface fragment of the homologous recombination site in the transformed rice according to the method of the present invention.

Figure 8 is the result confirmed by Western blot analysis whether sGFP is expressed in the transformed rice according to the method of the present invention.

9 is a photograph confirming the expression of sGFP in T1 generation rice obtained by culturing seeds harvested by self-modifying the transformed rice according to the method of the present invention (light green: sGFP, red: Chloroplast, yellow: sGFP expressed in chloroplast).

The present invention relates to a method for transforming a pigment in a plant. More specifically, the present invention relates to a method for transforming a pigment of a plant, characterized in that the callus derived from the plant is transformed with a vector for transforming a pigment.

In general, nuclear transformation technology used for plant transformation involves problems such as low expression level of transplanted gene, position effect of gene insertion position, and gene silencing. There is this. In addition, recombinant plants produced by nuclear transformation techniques can cause serious ecosystem problems because the exogenous genes used for transformation can be transferred through pollen to other similar wild plant species. Recently, in order to solve the above problems, the chromatin transformation technology has attracted attention as an alternative to the nuclear transformation technology.

The chromosomes are chloroplasts responsible for photosynthesis, chromoplasts involved in the color of flowers and fruits, amyloplasts that perform starch storage, leucoplasts and pigments that do not contain pigments. The above-mentioned pigments are divided into prochromosomes (proplastids) which are the mothers. A chromosome has its own genome, and its genetic information is transmitted later by its maternal genetics. Therefore, the use of chromatin transformation can prevent the transfer of foreign genes introduced into plants to other plant species. In addition, there is no need to select and maintain a transformed strain by genetic separation of Mendel, there is an advantage that can produce a large number of uniform seeds. Furthermore, there are approximately 100 plastids in plant cells, and since each plastid has 10-100 genomes, there are 1,000-10,000 copy genes per cell. Therefore, compared to the technology for transforming a nucleus having 1-2 genomes per cell, when transforming a chromosome, the expression level of the transplanted gene is expected to be much higher and stable expression of the gene can be expected. In addition, the plastid has a prokaryotic gene expression system has the advantage that can be expressed in multiple genes.

Plant chromosomal transformation has been reported for the first time in tobacco, cephalopods (Sikdar, S. et al., Plant Cell Rep . 18, 20-25, 1998), potato (Sidorov. V. et al., Plant J 19, 209-216, 1999), tomatoes (Ruf, S. et al., Nat. Biotechnol . 19, 870-875, 2001) and Lesquerella (Skarjinskaia M. et al., Transgenic Res. , 12, 115-122, 2003) have been reported for the transformation of pigments in plant species.

On the other hand, all of the plants that have been reported to be successful in the transformation of pigments belong to the dicotyledonous plants. So far, only the case of monochromatic transformation of monocotyledonous plants using cell suspension of rice has been reported (Khan M. et al. , Nat. Biotechnol , 17, 910-915, 1999). However, rice cells transformed with a chromosome by setting the cell suspension of rice to be transformed have a possibility that only a part of the chromosome is transformed and the transformed chromosome may gradually disappear in the process of development of the transformant as an adult. There is a disadvantage that the gene transplanted into the chromosome cannot be transferred to the next generation.

Therefore, the present inventors, while studying an effective method for transforming a plant in the plant, selects a plant-derived callus as a target for transformation and transforms the plant with a vector for transforming the plant, even if the transformant develops as an adult. The present invention was completed by confirming that genes transplanted into the chromosomes are not destroyed and are transferred to the next generation.

Accordingly, it is an object of the present invention to provide a method for transforming a pigment of a plant, characterized in that the callus derived from the plant is transformed with a vector for transforming a pigment.

In order to achieve the above object, the present invention provides a method for transforming a pigment of a plant, characterized in that the callus derived from the plant is transformed with a vector for transforming the pigment.

Hereinafter, the present invention will be described in detail.

Chromosome transformation method of the plant of the present invention is characterized in that the callus derived from the plant is transformed with a vector for transforming the pigment. Until now, the successful transformation of the pigment of a plant has been based on the plant leaf tissue or cell suspension, but the present invention is characterized in that the callus derived from the plant is selected as the target of transformation.

When transforming a plant's plastid, only a small number of plastid genomic copies of approximately 10,000 plastid DNA copies present in the plastid cells of the plastid are to be transformed. Thus, primarily transformed cells are present in a mixture of wild-type and transformed pigments. Such cells, tissues and plants are called heteroplasmy states. The heterocytoplasmic state is generally unstable and requires homoplasmy that results in the genome being transformed into either a wild type or a transformed state. Therefore, there is a need to increase the genetic safety of transformed plants and to induce an isoplasmic state in which all cells of the chromosomes are completely transformed. Such homocytic cytoplasmic state can be obtained by sufficient number of cell divisions under high selection pressure.

In this regard, the present inventors selected plant-derived callus as a target for transformation, transformed with a vector for transforming into a pigment, and then implanted in a selection medium to re-differentiate under high selection pressure to induce homogenous cytoplasm. As a result, even when the callus was differentiated into adults, the transgenic plastids were not destroyed.

Specifically, the method for transforming a pigment in a plant according to the present invention comprises: (a) inducing callus by culturing a fish selected from the group consisting of seed, tissue and cell suspension of a plant in a callus induction medium;

(b) transforming the callus of the plant of step (a) with a vector for transforming the pigment;

(c) selecting the transformed callus by culturing the callus transformed in step (b) in a selection medium; And

(d) culturing the callus selected in step (c) in a rooting medium to induce the roots and transfer to the soil, characterized in that it comprises a step of purifying.

The plant in step (a) is not particularly limited and may be any kind of plant. Preferably, it may be a monocotyledonous plant. Examples of the monocotyledonous plant include, but are not limited to, rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onions, garlic, leeks, leeks, soothing, hemp and ginger. Preferably, rice is used as the monocotyledonous plant.

The method of inducing callus in the step (a) may be carried out using conventional methods known in the art. Preferably, any one selected from the group consisting of seed, tissue and cell suspension of plants is cultured in a callus induction medium. The tissue of the plant may be part of the leaves, stems and roots. Preferably, the callus can be derived from the seed or cell suspension of the plant if the plant is a monocotyledonous plant and from the part of the tissue if the plant is a dicotyledon. The callus induction medium may be N6 medium, MS (Murashige & Skoog) medium, WHITE medium, B5 medium, R2 medium and the like. In this case, it is preferable to select a medium suitable for the plant to be cultured in consideration of the species of the plant, the age of the plant, the maturity of the cultured seed and the like. In addition, growth regulators may be further added to the medium. The growth regulators include, but are not limited to, IAA (3-indoleacetic acid), IAl (3-indoleacet aldehyde), IPA (3-indolepyruvic acid), IAN (3-indoleacetonitrile), IAE (ethyl ester of indoleacetic acid) Auxin and 2,4-D (2,4-dichlorphenoxy acidic acid), derivatives of the auxin such as NAA (naphthaleneacetic acid); And cytokinins such as kinetin, benzoic acid (BA), CPPU ((N-2-chloro-4-pyridyl) -N'-phenylurea), and derivatives thereof. Preferred callus induction method is as follows. Plant seeds are obtained and cultured in solid medium containing organic constituents such as carbohydrates and amino acids, inorganic nutrients such as nitrogen, phosphoric acid and potassium, and plant hormones such as auxin, and then cell division from plant seeds results in cell lumps. Once generated, callus is separated from the medium and sown on fresh solid medium. Subsequently, it is subcultured at regular intervals.

In a preferred embodiment of the present invention, the seedlings of hwacheongpum seed in N6 medium and incubated for 2-3 weeks under dark conditions at 28 ℃ to obtain a callus of rice (see Example 2).

Meanwhile, in the present invention, a total callus or an embryogenic callus may be used, but a total callus including all callus which is not re-differentiated with the embryogenic callus is used. This is because, in the preliminary experiment of the present invention, the regeneration efficiency was higher than that of the total callus compared to the case of using the embryogenic callus (result not shown).

In the step (b), the vector for transforming the chromosomes may be purchased and used or prepared according to methods known in the art. Preferably, the chromosomal transformation vector comprises a left interface region, a selection marker, a target gene and a right interface region of the homologous recombination site in the plant chromosome.

In general, transformation of a pigment is performed by inserting a gene of interest into a target region of the pigment genome by homologous recombination. Therefore, in the chromatin transformation vector, the same chromatin genomic sequence as the site to be introduced into the chromatin genome is adjacent to both sides of the target gene. The left interface region and the right interface region of the homologous recombination site may vary depending on the plant of interest, and may be prepared from the chromatin genomic sequence of a known plant. For example, the base sequences of the chromatin genome of plants such as rice, tobacco and corn are all known. On the other hand, since the gene array structure of the plant is known to be very similar between plants, the homologous recombination site can be used to cross between different plant species. Preferably, as the homologous recombination site, a trnI-trnA region sequence of rice which is a sequence highly conserved among higher plants can be used.

The selection marker refers to a gene serving as an indicator for confirming that a foreign gene has been introduced into a chromosome of a target plant. The selection markers include, but are not limited to, antibiotic resistance genes, herbicide resistance genes, metabolic genes, luminescent genes, GFP (green fluorescence protein), GUS (β-glucuronidase) gene, GAL (β-galactosidase) gene, etc. Can be used. Preferably, antibiotic resistance genes are used. The antibiotic resistance genes include, but are not limited to, aadA (Svab Z. et al., Proc. Natl. Acad. Sci . USA 90, 913-917, 1993), kanamycin, which is a streptomycin and spectinomycin resistance gene . Resistance genes neo (Kuroda H. et al., Plant Physiol . 125, 430-436, 2001; Kuroda H. et al., Nucleic Acids Res . 29, 970-975, 2001)) and aphA-6 (Huang FC et al., Mol. Genet. Genom . 268, 19-27, 2002).

The target gene is not particularly limited and may be used as long as it can be introduced into a plant's plastid so that the plant can obtain useful traits. The gene of interest may be obtained by recombination from a known sequence by a method known in the art or by commercially purchasing a plasmid containing the gene.

In addition to the above-described genes, the plant transformed vector for the chromosomal transformation of the plant may further include a control sequence for controlling gene expression. For example, it may further comprise a promoter that initiates transcription of the gene and a terminator sequence that terminates transcription.

In one embodiment of the present invention, from the genomic DNA of homologous recombinant rice to the left interface region of the homologous recombination site, the trnI gene of rice, Prrn of the chromatin rRNA promoter, aadA gene of antibiotic resistance gene, sgfp gene used as the target gene, psbA terminator and A rice chromatin transformation vector was prepared such that the trnA gene of rice was sequentially connected to the right interface region of the homologous recombination site (see Example 1 and FIG. 1). The use of sgfp (green fluorescent protein) as the target gene is for visually confirming whether the rice is transformed into a pigment.

In the step (b), the transformation of callus using the vector for transforming the chromosomes may be performed using a method known in the art. For example, but not limited to, particle bombardment, polyethylene glycerol (PEG) treatment, electroporation, and microinjection may be used. Preferably, a particle bombardment method can be used.

On the other hand, the present invention may further comprise the step of culturing the cancer condition in a medium containing no antibiotics before transforming the callus in the step (b) and before the screening step of the transformant. This step is to allow damaged cells to recover during the transformation process. Preferably, the present invention further comprises the step of incubating the transformed callus in step (b) under conditions of 25 to 30 ° C. under dark conditions in MS or N6 medium to which growth regulator is not added without antibiotics. It can be included.

The selection of the callus transformed in step (c) may be performed by selectively selecting only the callus growing by culturing the callus in a selection medium. The selection medium may vary depending on the selection marker of the callus. For example, when an antibiotic resistance gene is used as the selection marker, a medium containing antibiotics can be used as the selection medium. Meanwhile, the callus selection step may be performed in multiple steps to induce homozygous forms of transformants. Preferably, the screening of the transformed callus is to first select the callus showing antibiotic resistance by culturing the transformed callus in a medium containing 100 ~ 500mg / L antibiotics in the light condition in step (b) and then again It can be carried out by transferring to a medium containing 300 to 500 mg / L of antibiotics and culturing to secondary selection of callus showing antibiotic resistance.

In the above antibiotics, all kinds known in the art can be used, and preferably, streptomycin is used.

In one embodiment of the present invention, in order to determine the optimal concentration of streptomycin used for the selection of rice transformed plastid, 2-3 weeks after germination, the callus is dentured in a medium containing various concentrations of streptomycin and subjected to light conditions or cancer. Re-differentiated under conditions. As a result, the growth of callus was inhibited as the concentration of streptomycin was increased under light condition, but the concentration of streptomycin did not significantly affect the growth of callus under dark condition. Therefore, in the transformant of the present invention, the present inventors selected the transformants under light conditions, and the optimal concentration of streptomycin was 100-500 mg / L at the initial stage of selection, and 300-500 mg / L at the subsequent stage of selection. It was.

In another embodiment of the present invention, the transformed callus was selected using a selection medium containing antibiotics at the concentration determined above. In other words, the transformed callus was cultured in a medium containing 200 mg / L of streptomycin to induce re-differentiation and select callus showing antibiotic resistance. The selected callus was then transferred to a fresh selection medium containing 300 mg / L streptomycin, cultured for one month, and secondary shoots showing streptomycin resistance were selected and rooted in a medium containing 500 mg / L streptomycin (Example 3). As a result, two independent streptomycin resistant strains were obtained and named sGFP-1 and sGFP-2, respectively (see FIG. 3B).

In step (d), the chromosomal transformed callus selected in step (c) was cultured in a rooting medium to induce roots and transfer to the soil to purify. In the above rooting medium, it is preferable to use MS medium containing 300-1000 mg / L of antibiotics and no hormone added thereto. The antibiotics included in the medium may be selected and used according to the type of selection marker. Illustrated in the present invention is streptomycin.

When the root-derived plant is purified and transferred to soil, 500-1500 mg of antibiotic per liter of soil is treated every week until the plant harvests seeds so that all the cells in the chromosome are transformed into a transformed state. It is desirable to induce. More preferably, the antibiotic can be treated at a concentration of 500 to 1000 mg per liter of soil.

In an embodiment of the present invention, when the transformed callus is cultured in the rooting medium to induce the roots, and then the seedlings are grown to a sufficient number, the seedlings are grown in separate soils and streptomycin is obtained. After treatment by concentration, the growth of the seedlings was examined to determine the appropriate treatment concentration (see Example 4). As a result, it was confirmed that streptomycin can be treated in the range of 500 to 1000 mg per 1 L of soil (see FIG. 4).

Therefore, the present inventors investigated whether the chromosomes of the transformants prepared and selected according to the method of the present invention were stably transformed by the vector for transforming the chromosomes. In one embodiment of the present invention, the genomic DNA of the transformed rice selected according to the method of the present invention was analyzed to determine whether the sgfp gene, which is a target gene, exists in the transformed rice (see Example 6). As a result, it was confirmed that the presence of the sgfp gene in all selected transgenic rice (see Figs. 5a and 5b).

In another embodiment of the present invention, Southern blot analysis was performed using a probe specific to the aadA gene to determine whether the aadA gene, which is an antibiotic resistance gene, was present in the transformed rice (see Example 7). As a result, it was confirmed that the aadA gene exists in the transformed rice (see Fig. 6).

In another embodiment of the present invention, it was examined whether the site-specific sgfp gene and the antibiotic resistance gene aadA gene were integrated in the chromosomal genome of transformed rice (see Example 8). The experimental results, the aadA gene and sgfp gene and confirmed that exists between trnI and trnA genes in the plastid genome of a rice plant (FIG. 7).

In another embodiment of the present invention, whether or not sGFP is expressed in transgenic rice was examined by Western blot analysis (see Example 9). As a result, it was confirmed that sGFP is expressed in the transformed rice (see Fig. 8).

The present inventors can confirm that the target gene was introduced into the chromosomal genome of rice transformed according to the method of the present invention from the above experimental results, and confirmed that the transformed chromosome does not disappear even when the transformant is grown to adult. Could.

Furthermore, the present inventors investigated whether the genes transplanted into the chromosomes of the transformed rice are transferred to the next generation (T1 progeny) by self-modifying the transformed rice, obtaining seeds, and seeding and culturing the seed (Example 10). Reference). As a result, according to the method of the present invention it was confirmed that the gene transplanted into the rice chromosome is transferred to the next generation (see Figure 9)

Hereinafter, the present invention will be described in more detail by the following examples.

The following examples illustrate the invention and are not intended to limit the scope of the invention.

<Example 1>

Preparation of Rice Chromosome Transformation Vector

Total genomic DNA was extracted from the leaf tissue of Hwacheong rice (provided by Professor Ko Heejong, Seoul National University) to prepare a vector for rice chromosomal transformation (Doyle J. et al. Phytochem. Bull . 19 : 11-15, 1987). Using the genomic DNA isolated above as a template, the following primer pairs (SEQ ID NO: 3 and SEQ ID NO: 4) are used for the trnI gene (SEQ ID NO: 1) and trnA gene (SEQ ID NO: 2) to be used as a homologous recombination site. PCR amplification using. PCR amplification conditions were repeated 30 times, consisting of denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, stretching for 2 minutes at 72 ℃.

Forward primer (SEQ ID NO: 3)

5'- CGAGGCCTCCCAAAGGGAGGCCCGCGCGA-3 '

Reverse primer (SEQ ID NO: 4)

5'-GCTGATATCCAACTTTCATCGTACTGTGCTCT-3 '

The PCR amplification products were treated with Stu I and Eco RV restriction enzymes and then cloned into the Pvu II restriction enzyme site of the pUC19 vector (strataene). Next, the 16S rRNA promoter Prrn , the antibiotic resistance gene aadA gene, psbA terminator in the pZS197 (Svab, Z et al., Proc. Natl. Acad. Sci . USA, 90, 913-917, 1993), a chromosomal transformation vector by treatment with the restriction enzymes Not I was isolated as a Not I- Not I fragment. The pLD - RCtV vector was prepared by treating this with clinow enzyme and then introducing it into the Pvu II restriction site present between trnI and trnA of the pUC19 vector prepared above (FIG. 1A).

In order to visually distinguish the transformation, a green fluorescent protein (GFP) gene was introduced into the pLD-RCtV vector as a selection marker gene. To this end, the sgfp gene, a variant of the gfp gene known to be nontoxic in rice, was amplified by PCR. At this time, the sgfp gene was to have a ribosomal binding site (ribosome binding site). PCR amplification of the sgfp gene (SEQ ID NO: 5) is based on the pSK-RG plasmid (Jang E. et al., Plant Physiol , 129, 1-9, 2002) and the following primer pairs (SEQ ID NO: 6 and SEQ ID NO: 7) was used. Underscores in the following primer sequences refer to Xba I sites and portions indicated in italics denote ribosomal binding regions.

Forward primer (SEQ ID NO: 6)

5'- GC TCTAGA GTTGTAG GGAGG GACGTATGGCCAAGGGCGAGGAGCTGTT-3 '

Reverse primer (SEQ ID NO: 7)

5'-GC TCTAGA TTACTTGTACAGCTCGTCCATG-3 '

The PCR amplification product was introduced into the Xba I site existing between the aadA gene of the pLD-RCtV vector and the 3'UTR of psbA to prepare a pLD-RCtV-sGFP vector (FIGS. 1B and 1C).

<Example 2>

Optimization of Streptomycin Content and Selection Conditions in Selected Medium for Selecting Transformed Rice

To determine the optimal concentration of streptomycin used for the selection of transformed rice, callus 2-3 weeks after germination were dentured and re-differentiated in medium containing various concentrations of streptomycin. The callus is Hwachung rice seed 2,4-dichlorophenoxy acetic acid (2,4-D) 2mg / L, casein hydrolysate 1g / L, sucrose (30g / L) sucrose (gucrite) It was seeded in N6 medium consisting of 2g / L and obtained by incubating for 2 to 3 weeks under 28 ℃, dark conditions.

The callus was wound on MS (Murachige and Skoog) medium to which streptomycin was added at concentrations of 0, 100, 200, 500 and 1000 mg / L, respectively, and cultured under light conditions for 2 months. In addition, callus was dentured in the same medium as above and cultured for 1 month under dark conditions.

As a result, the growth of callus was partially inhibited when cultured in the medium added with 100 mg / L of streptomycin under light conditions, and roots were not induced. In addition, growth was further inhibited when streptomycin was added at 200 mg / L (FIG. 2A). Callus growth was also inhibited when streptomycin was added at a concentration of 500 mg / L. When streptomycin was at 1000 mg / L, all callus turned brown within 3 weeks.

In contrast, callus growth was not significantly inhibited even when 1000 mg / L of streptomycin was added under dark conditions (FIG. 2B).

From the experimental results, it was found that callus is not sensitive to antibiotics under cancer conditions, and therefore, it was found that culturing callus under cancer conditions was not suitable for selection of transformants.

Therefore, the transformant of the present invention is preferably selected under light conditions, and the optimal concentration of streptomycin added to the medium is 100 mg / L, which is shown to partially inhibit growth, and 1000 mg / L when callus is hardly grown. Except for the concentration range of 200mg / L to 500mg / L was found to be suitable.

<Example 3>

Transformation and Regeneration of Rice Pigment by Particle Impact Method

0.6 μm diameter gold particles coated with pLD-RCtV-sGFP DNA, which is a rice pigment transformant vector prepared in Example 1, from callus derived from rice seeds in the same manner as in Example 2, and The PDH-1000 / He gene transfer system (Bio-Rad) was used to transform under conditions of acceleration power (1,100 psi) and target distance (6 cm). The callus was then incubated for two days at 28 ° C. under dark conditions, and in 50 ml of MS (Murachige and Skoog) medium containing 2 mg / L NAA, 2 mg / L kinetin, 14 g / L phytagar and 200 mg / L streptomycin. Re-differentiation was induced by incubating under light conditions of μE. As a result, streptomycin-resistant callus appeared as green spots after one month in the selection medium and shoots developed in the callus the following month (FIG. 3A).

When young leaves were formed in the callus showing resistance to streptomycin, the cells were transferred to fresh medium having increased streptomycin concentration to 300 mg / L, and then cultured for one month under the same light conditions. Thereafter, root formation was induced by incubating shoots showing resistance to streptomycin in MS medium containing 500 mg / L of streptomycin, which is a rooting medium, and without hormones.

Ten independent experiments were performed in this manner using about 4000 callus, resulting in two independent strains selected and redifferentiated (FIG. 3B). The two streptomycin-resistant strains were named sGFP-1 and sGFP-2, respectively.

<Example 4>

Determination of Optimal Throughput of Streptomycin in Soil Purification of Chromosome Transformed Rice

The sGFP-1 and sGFP-2 strains selected in Example 3 were transferred to a pot containing soil and purified, and 500 mg of streptomycin per 1 liter of soil was treated at weekly intervals. When the number of seedlings of sGFP-1 and sGFP-2 is sufficient, the seedlings are separated into three pots each containing separate soil and planted with 500, 1000 and 1500 mg of streptomycin per 1 liter of soil at weekly intervals. The seeds were seeded and treated separately until harvested. At this time, a seedling of untransformed rice was used as a control.

As a result, the treatment of 500 mg of streptomycin per liter of soil inhibited growth, whereas the growth of seedlings of sGFP-1 and sGFP-2 was not affected and seeds were produced. When 1000 mg of streptomycin was treated per 1 L of soil, the growth of untransformed rice was significantly inhibited. The trophic growth of sGFP-1 and sGFP-2 seedlings was not significantly affected. Produced. Treatment of 1,500 mg of streptomycin per 1 liter of soil was shown to slightly inhibit the growth of sGFP-1 and sGFP-2 seedlings, and no seeds were produced (FIG. 4).

Seedlings of sGFP-1 and sGFP-2 which showed antibiotic resistance in each of the above treated with different concentrations of streptomycin were selected to obtain 6 individuals.

<Example 6>

Genomic DNA Analysis of Chromosome Transformed Rice

In order to confirm whether the sgfp gene is present in the transformed rice selected in Example 5, genomic DNA of rice was analyzed by PCR amplification.

Total genomic DNA was extracted from the mouth tissue of the transformed rice according to a known method (Doyle J. et al., Phytochem. Bull . 19, 11-15, 1987). In addition, the total genomic DNA was extracted in the same manner from the untransformed wild rice mouth tissue for use as a control. The following primer pairs (SEQ ID NO: 8 and SEQ ID NO: 9) capable of amplifying the sgfp gene using 100 ng of genomic DNA extracted above as a template and DNA polymerase (Excel Taq DNA polymerase, Corebio, Seoul, South Korea) PCR amplification using. PCR amplification conditions were repeated 30 times, consisting of denaturation for 30 seconds at 94 ℃, annealing for 30 seconds at 63 ℃, elongation for 40 seconds at 72 ℃. The PCR amplification product was identified on agarose gel (1%).

Forward primer of sgfp (SEQ ID NO: 8)

5'-GACCCTGAAGTTCATCTGAC-3 '

Reverse primer of sgfp (SEQ ID NO: 9)

5'-ACTTGTACAGCT CGTCCATG-3 '

As a result, it was confirmed that the six transformants selected in Example 5 had a 717 bp sgfp gene (FIGS. 5A and 5B).

<Example 7>

Southern blot analysis of chromosomal transgenic rice

Southern blot analysis was performed to confirm the presence of the aadA gene in the chromosomal genome of the transformed rice selected in Example 5. At this time, two individuals selected by treating streptomycin at a concentration of 500 mg / L in the soil among the six transformed individuals selected in Example 5 were used as samples.

Total genomic DNA extracted in the same manner as in Example 6 was digested with BamH I and 18 μg of DNA was used as a sample. The samples were separated on a 0.7% agarose gel and transferred to a high-bond-N + nylon membrane (Amersham), hybridized with probes specific for aadA labeled with [α-32P] dCTP and photosensitive with X-ray film. specific probe in the aadA was produced by PCR amplification with the primers of Example 1 below in the pLD-RCtV vector as the template (SEQ ID NO: 10 and SEQ ID NO: 11) containing the aadA gene. PCR amplification conditions were repeated 30 times, consisting of denaturation for 30 seconds at 94 ℃, annealing for 30 seconds at 60 ℃, elongation for 40 seconds at 72 ℃.

Forward primer of aadA probe (SEQ ID NO: 10)

5'-GATCGCCGAAGTATCGACTC-3 '

Reverse primer of aadA probe (SEQ ID NO: 11)

5'-ATTTGCCGACTACCTTGGTG-3 '

As a result, 3.3 kb fragments were detected in sGFP-1 and sGFP-2 (FIG. 6). From this it was confirmed that the aadA gene is present and expressed in the sGFP-1 and sGFP-2.

<Example 8>

Investigation of site-specific introduction of the target gene into the chromosomal genome of transgenic rice

In order to confirm whether site-specific sgfp and aadA genes were integrated in the chromosomal genome of the transformed rice selected in Example 5, genomic DNA of rice was analyzed by PCR amplification.

The total genomic DNA of the transformed rice was extracted in the same manner as in Example 6, and the following primer pairs (SEQ ID NOs 12 to 12) capable of amplifying the left and right interfaces of rice plastids, respectively, using 100-500 ng of genomic DNA as a template. PCR amplification using SEQ ID NO: 15).

Left interface forward primer (SEQ ID NO: 12)

5'-TCAGCCATACGGCGGTGAATCCG-3 '

Left interface reverse primer (SEQ ID NO: 13)

5'-CCGCGTTGTTTCATCAAGCCTTACG-3 '

Right interface forward primer (SEQ ID NO: 14)

5'-CGGGATCACTCACGGCATGGACGAG-3 '

Right interface reverse primer (SEQ ID NO: 15)

5'-TACCATAGAGGCCAACGATAGACAATAA-3 '

PCR amplification of the left interface was carried out in 35 cycles consisting of denaturation at 94 ° C. for 30 seconds, annealing at 63 ° C. for 30 seconds, and extension at 72 ° C. for 2 minutes.

PCR amplification of the right interface was repeated 10 times with denaturation at 94 ° C for 30 seconds, annealing at 63 ° C for 30 seconds, and extension at 72 ° C for 1 minute and 30 seconds, followed by denaturation at 94 ° C for 30 seconds, and 30 ° at 60 ° C. The process consisting of annealing for 1 second and stretching for 1 minute and 30 seconds at 72 ° C. was repeated 25 times.

After confirming the PCR amplification product on an agarose gel (1%), only DNA on the gel was purified and cloned into pGEM-T Easy vector (Promega, Madison, WI) and commissioned by Coabio Co., Ltd., a sequencing company. The analysis confirmed that the insertion position of the aadA and gfp genes is the part between trnI and trnA in the rice chromosomal genome.

As a result of the experiment, the left interface fragment (Fig. 7a) and the right interface fragment of the rice pigment body of 1.9kb and 1.3kb size were detected in the transformed rice (Fig. 7b). As a result of analyzing the sequence of the PCR amplification product, it was confirmed that aadA gene and sgfp gene exist between trnI and trnA genes in rice chromosomal genome (result not shown).

Example 9

Western Blot Analysis of Chromosome Transformed Rice

Whether or not GFP is expressed in the transformed rice selected in Example 5 was examined by Western blot analysis.

Soluble protein is extracted from the leaf tissues of the transformed rice using protein extraction buffer (0.1M Tris-HCl, pH 8.3, 0.5M NaCl, 5mM dithiothreitol, 5mM EDTA, 2mM phenylmethylsulfonylfluoride) It was. Protein was extracted in the same manner as above from the leaves of untransformed rice for use as a control. 2 μg of the soluble protein extracted above was electrophoresed on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Amersham) for immunoblotting. As a primary antibody, monoclonal anti-GFP (Sigma) was diluted 1: 500, and as a secondary antibody, alkaline phosphatase-binding goat-antimouse IgG (Sigma) was diluted 1: 10,000. Detection of GFP was performed using BCIP / NBP combo (Gibco BRL).

As a result, it was confirmed that a band of 27 kDa was generated in the sGFP-1 and sGFP-2 lines, and sGFP was generated therefrom (FIG. 8).

<Example 10>

Acquisition and Analysis of Progeny of Chromosome Transformed Rice

Seeds were obtained by self-correcting the chromosomal transformed rice selected in Example 5 and seeding the seed in MS medium having a streptomycin concentration of 100 mg / L and no hormone, and incubated under light conditions at 28 ° C. for 2 weeks. . The cultured rice was observed by confocal fluorescence microscopy to investigate the expression of GFP. In addition, it was confirmed whether the GFP is located in the chloroplast of transformed rice. In other words, using a 488 nm excitation wavelength and a 510 nm emission wavelength filter on the progeny of the chromosomal transgenic rice, the laser was irradiated and the green GFP fluorescence was expressed using a confocal fluorescence microscope (Carl Zeiss 510 CLSM, Jena, Germany). Confirmed by.

Experimental results showed sGFP fluorescence in chloroplasts, young chloroplasts and leaf epidermis (FIG. 9). Therefore, it was confirmed that the gene inserted into the rice plastid is transferred to the next generation.

In the method for transforming a plant in accordance with the present invention, the plant's callus is transformed with a vector for transforming the plant, and even when the transformant is developed into an adult, the transformed pigment is not destroyed and the gene transplanted into the pigment is transferred to the next generation. It works.

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Claims (11)

(a) inducing callus by culturing any one selected from the group consisting of rice seed, tissue and cell suspension in a callus induction medium; (b) transforming the callus of the rice of step (a) with a vector for transforming the pigment; (c) selecting the transformed callus by culturing the callus transformed in step (b) in a selection medium; (d) culturing the callus selected in step (c) induce the roots by incubating the medium for rooting and transfer to the soil, characterized in that it comprises the step of transforming the pigments of rice. delete delete delete The method of claim 1, wherein the vector for transforming the chromatin in step (b) comprises a left boundary region of the homologous recombination site, a selection marker, a target gene, and a right interface region of the homologous recombination site. The method of claim 5, wherein the selection marker is a streptomycin resistance gene and the left and right interface regions of the homologous recombination site are trnI and trnA genes of rice, respectively. The method of claim 1, wherein the transformation of step (b) is performed by particle bombardment. The method of claim 1, further comprising the step of transforming the callus in the step (b) and then incubating for 1 to 3 days at 25 to 30 ℃ under cancer conditions in N6 or MS medium containing no antibiotics. How to. The method of claim 1, wherein the selection of the callus transformed in step (c) is resistant to antibiotics by culturing the callus transformed in step (b) under light conditions in MS medium containing 100-500 mg / L of antibiotics. The method is characterized in that it is carried out by first selecting the callus showing the first and then incubated in the MS medium containing an antibiotic of 300 to 500mg / L and then cultured to secondary screening antibiotic-resistant callus. The method of claim 1, wherein the rooting medium of step (d) is characterized in that the MS medium containing 300 to 1000mg / L of antibiotics and no hormone is added. The method according to claim 1, wherein in the step (d), the plant is treated with 500 to 1500 mg of antibiotic per liter of soil at a weekly interval until the plant is seeded and harvested.

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