KR100781061B1 - Toxoflavin-degrading enzyme as selectable marker for plant transformation and selection method of transformed plant - Google Patents

Abstract

A toxoflavin-degrading enzyme tf1A is provided to select a transgenic plant simply and efficiently. And a method for selecting a transgenic plant and a method for preparing a transgenic plant using the tf1A are provided. A recombinant Arabidopsis thaliana expression vector includes a tf1A gene consisting of a sequence of SEQ ID : NO. 1 and has a cleavage map depicted in Fig. 2. A method for selecting a transgenic plant comprises the steps of: (a) transforming a plant using the recombinant vector including the tf1A gene; and (b) proliferating the transgenic plant in a culture medium containing toxoflavin. A method for preparing a transgenic plant comprises the steps of: (a) transforming a plant cell using the recombinant vector including the tf1A gene; (b) proliferating the transformed plant cell in the culture medium containing toxoflavin; and (c) redifferentiating the transformed plant cell into a transgenic plant.

Description

Toxoflavin-degrading enzyme as selectable marker for plant transformation and selection method of transformed plant}

1 is a schematic of T-DNA in pCamLA.

2 is a schematic of T-DNA in pTflA.

Figure 3 shows the hygromycin (30ug / ml) secondary selection of rice callus transformed with pCamLA (left) and pTflA (right) vectors, respectively.

FIG. 4 shows toxoflavin (5ug / ml) selection prior to redifferentiation of rice callus transformed with pCamLA (left) and pTflA (right) vectors, respectively.

FIG. 5 shows toxoflavin (7.5 ug / ml) selection at 4 weeks after regeneration of rice callus transformed with pCamLA vector.

FIG. 6 shows selection of toxoflavin (7.5 ug / ml) at 4 weeks after regeneration medium of rice callus transformed with pTflA vector.

Figure 7 shows the result of agarose electrophoresis of the PCR product by performing PCR from the isolated genomic DNA of the selected transformant.

8 shows a transgenic plant in which pCamLA: tflA, pCamLA (Δhpt): tflA, pMBP1: tflA and pMBP1 vectors are inserted, respectively.

Figure 9 shows the results of agarose electrophoresis of the PCR product by performing PCR from the isolated genomic DNA of the selected transformant.

10 shows the decolorizing effect of the transformants.

The present invention provides a plant transformation selection marker expression cassette comprising the toxoflavin degrading enzyme tflA, tflA gene, a recombinant vector comprising the expression cassette, a plant transformed with the vector, a tflA gene, which is used as a plant transformation selection marker. It relates to a method for selecting a transformed plant using and a method for producing a transformed plant using the tflA gene.

Expression vectors include one or more genetic markers that select for transformed cells by inhibiting the growth of cells that do not contain a selectable marker gene. Most of the selection marker genes commonly used for plant transformation have been isolated from bacteria and encode enzymes that metabolically detoxify select chemicals, which may be antibiotics or herbicides.

The most widely used plant transformation selectable marker gene is the neomycin phosphotransferase II (nptII) gene isolated from Tn5, and another marker gene is hygromycin phosphotransferra, which confers resistance to the antibiotic hygromycin. Aze gene.

Although many selectable markers have been used for selecting transformed plant tissues, such selection systems using toxic chemicals can have disadvantages or limitations. First, it may be difficult to directly recover normal viable transgenic plants from chemical selection. Secondly, not all selectable marker systems work for all tissues and for all plant species. Third, some of the compounds that must be added to allow for selection are antibiotics. The propagation of genes that confer resistance to antibiotics or herbicides should be minimized as much as possible to avoid the risk of tolerating the pathogen. Fourth, some of the compounds that must be added to enable selection are relatively expensive and therefore require cheaper selection markers.

Based on the prior art as described above, while studying plant transformation selection markers, the present inventors can conveniently select plant transformants by using the toxoflavin degrading enzyme tflA, which degrades toxoflavin. The present invention was completed.

It is an object of the present invention to provide toxoflavin degrading enzyme tflA which can select plant transformants simply and efficiently.

It is also an object of the present invention to provide a plant transformation selection marker expression cassette comprising the tflA gene.

It is also an object of the present invention to provide a recombinant vector comprising the expression cassette.

It is also an object of the present invention to provide a plant transformed with the vector.

It is also an object of the present invention to provide a method for selecting a transgenic plant using the tflA gene.

It is also an object of the present invention to provide a method for producing a transgenic plant using the tflA gene.

In order to achieve the object of the present invention, the present invention provides toxoflavin degrading enzyme tflA which is used as a plant transformation selection marker.

The toxoflavin degrading enzyme tflA of the present invention confers resistance to the compound toxoflavin. Toxoflavin is the most important pathogenic component of rice bran disease. The transformant of the present invention may be a plant, preferably the plant may be rice or Arabidopsis thaliana.

In the selection marker according to one embodiment of the invention, the toxoflavin degrading enzyme tflA may comprise the amino acid sequence of SEQ ID NO: 2. In addition, variants of such sequences are included within the scope of the present invention. Variants are amino acid sequences that vary in amino acid sequence but have functional and immunological properties similar to those of SEQ ID NO: 2. Specifically, the toxoflavin degrading enzyme has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the amino acid sequence of SEQ ID NO: 2. Sequences may be included.

In the selection marker according to the embodiment of the present invention, the toxoflavin degrading enzyme tflA may be encoded by the nucleotide sequence of SEQ ID NO: 1. In addition, variants of such sequences are included within the scope of the present invention. Variants are base sequences that vary in base sequence but have functional and immunological properties similar to those of SEQ ID NO: 1. Specifically, the toxoflavin degrading enzyme is a base having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence of SEQ ID NO: 1 Sequences may be included.

The "% sequence homology" for polynucleotides and polypeptides is determined by comparing two optimally arranged sequences with a comparison region, wherein a portion of the polynucleotide and polypeptide sequences in the comparison region are the reference sequences for the optimal alignment of the two sequences. It may include additions or deletions (ie gaps) compared to (not including additions or deletions). The% identifies the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences, yielding the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison region, and subtracting 100 for the result. Calculated by multiplying to yield% sequence homology. Optimal alignment of sequences for comparison is by computerized implementation of known algorithms (e.g. GAP, BESTFIT, FASTA and TFAST in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science). Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information), or by examination.

The term "substantial identity" or "substantial similarity" means that the polypeptide includes sequences that can hybridize with the target polypeptide under stringent conditions. Stringent conditions mean a solution of 2 × SSC and a temperature of 65 ° C.

“Substantially similar” polypeptides share the sequence except that non-identical residue positions may differ by conservative amino acid changes. Conservative amino acid substitutions mean interchangeability of residues having similar side chains. For example, the amino acid groups having aliphatic side chains are glycine, alanine, valine, leucine and isoleucine, the amino acid groups having aliphatic hydroxyl side chains are serine and threonine, and the amino acid groups having amide containing side chains are asparagine and glutamine, aromatic The amino acid groups with side chains are phenylalanine, tyrosine and tryptophan, the amino acid groups with basic side chains are lysine, arginine and histidine, and the amino acid groups with sulfur containing side chains are cysteine and methionine.

Substantial identity of the polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, most preferably at least 95%. Another meaning is that the nucleotide sequences are substantially identical when the two molecules specifically hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in other situations. In general, stringent conditions are selected to be about 10 ° C. below the thermal melting point (Tm) for a particular sequence at a given ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a fully matched probe. Hybrid Tm, a function of both the length and base composition of the probe, is described in Sambrook, T. et al., (1989) Molecular Cloning-A Laboratory Manual (second edition), Volume 1-3, Cold Spring Harbor Laboratory, Cold Spring) can be calculated using information. Typically, stringent conditions for the Southern blot procedure include washing at 65 ° C. with 0.2 × SSC. For preferred oligonucleotide probes, wash conditions are typically about 42 ° C. at 6 × SSC.

To achieve another object of the present invention, the present invention provides a plant transformation selection marker expression cassette comprising the following sequences operably linked in the 5 'to 3' direction:

(i) promoter sequence;

(ii) toxoflavin degrading enzyme coding sequence; And

(iii) 3 ′ untranslated terminator sequence.

In order for the expressed protein to be expressed in the host cell in such a way that it can tolerate the toxic selection agent, the toxoflavin degrading enzyme coding sequence according to the present invention is generally recognized by the biochemical machinery of the host. Will be provided as an expression cassette with regulatory factors that allow for this and allow the open reading frame to be transcribed and translated into the host cell. It will generally include a transcription initiation region for ribosomal recognition and attachment as well as a transcription initiation region that can be appropriately derived from any gene that can be expressed in a selected host cell. In eukaryotic plant cells, the expression cassettes generally further comprise a transcription termination region located downstream of the open reading frame, where transcription is terminated and polyadenylation of the primary transcript occurs. In addition, the codon usage may be appropriate to the allowed codon usage of the selected host. The principles governing the expression of hybrid DNA constructs in selected host cells are generally understood by those skilled in the art and the construction of hybrid DNA constructs that can be expressed is common for any type of host cell that is a prokaryotic or eukaryotic organism.

In the expression cassette according to an embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the configuration promoter does not limit the selection possibilities.

In the expression cassette according to an embodiment of the present invention, the terminator may be nopaline synthase (NOS) or rice α-amylase RAmy1 A terminator, but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

In the expression cassette according to the embodiment of the present invention, the toxoflavin degrading enzyme coding sequence may include the nucleotide sequence of SEQ ID NO: 1. In addition, it may include a base sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence of SEQ ID NO: 1.

In the present invention, "operably linked" refers to a component of an expression cassette that functions as a unit for expressing a heterologous protein. For example, a promoter operably linked to heterologous DNA encoding a protein promotes the production of functional mRNA corresponding to the heterologous DNA.

In an expression cassette according to one embodiment of the invention, (i) a promoter sequence; (ii) the protein encoding sequence of interest; And (iii) a 3 ′ untranslated terminator sequence. Proteins of interest include erythropoietin (EPO), tissue plasminogen activator (t-PA), urokinase and prourokinase, growth hormone, cytokines, factor VIII, epoetin-α, granulocyte colony stimulating factor and vaccines Important therapeutic proteins and polypeptides that are commercially available.

In the expression cassette according to an embodiment of the present invention, the target protein expression cassette may be constructed in tandem as one expression cassette together with the selection marker expression cassette. That is, the target protein expression cassette and the selection marker expression cassette are linked side by side together in one expression cassette. It is also possible that the target protein expression cassette and the selection marker expression cassette are each present in separate expression cassettes.

In order to achieve another object of the present invention, the present invention provides a recombinant vector comprising the expression cassette according to the present invention. In order for the open reading frame to be maintained in the host cell, it will generally be provided in the form of a replicon comprising the open reading frame according to the invention linked to the DNA to be recognized and replicated by the selected host cell. Thus, the choice of replicon is highly dependent on the host cell of choice. Principles that govern the selection of suitable replicons for the particular host selected are within the scope of those skilled in the art.

A particular type of replicon is one that can transfer itself or part of it to another host cell, such as a plant cell, thereby allowing the simultaneous transmission of the open reading frame according to the invention to said plant cell. Replicons with such a possibility are referred to herein as vectors. An example of such a vector is a Ti-plasmid vector capable of transferring part of itself, the so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host. Such may be the case with woody species, especially trees and vines.

In order to achieve another object of the present invention, the present invention provides a host cell transformed with the recombinant vector of the present invention. Preferably, the host cell may be of the genus Agrobacterium, even more preferably Agrobacterium tumerfaciens.

In order to achieve another object of the present invention, the present invention provides a plant transformed with the recombinant vector of the present invention. The plant may be rice or Arabidopsis.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

In order to achieve another object of the present invention, the present invention provides a transgenic seed of a plant transformed with the recombinant vector of the present invention.

In order to achieve another object of the present invention, the present invention comprises the steps of transforming a plant or plant part or plant cells with a recombinant vector according to the present invention; And

Provided is a method for selecting a transformed plant comprising propagating the transformant in a toxoflavin-containing medium. The method comprises transforming a plant or plant part or plant cell with the recombinant vector according to the invention, wherein the transformation can be mediated by Agrobacterium tumefiaciens. The method also includes propagating the transformant in a toxoflavin-containing medium. The transformants will survive in toxoflavin containing medium and the non-transformants will not survive and die in the medium. Therefore, the transgenic plant can be selected simply.

In order to achieve another object of the present invention, the present invention comprises the steps of transforming plant cells with a recombinant vector according to the present invention;

Propagating the transgenic plant cells in toxoflavin-containing medium; And

It provides a method for producing a transformed plant comprising the step of regenerating the transformed plant from the transformed plant cells. The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, wherein the transformation can be mediated by Agrobacterium tumefiaciens. The method also includes propagating the transgenic plant cells in a toxoflavin-containing medium. The transformants will survive in toxoflavin containing medium and the non-transformants will not survive and die in the medium. The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example

Example  One: Toxoflavin ( Toxoflavin A) breakdown of genes Cloning

About 500 kinds of bacteria were isolated from wild field soil, paddy and field soil, and rice seeds, and after incubation in nutrient minimal medium containing toxoflavin, bacteria were grown after toxin degradation. Bacteria degrading toxoflavin among the bacteria isolated from rice seeds were isolated purely and identified using 16s DNA sequencing, fatty acid analysis, Biolog analysis system. Identification result Penny were identified as Bacillus miksa poly (Paenibacillus polymyxa) was named JH2. A chromosomal DNA library was constructed in E. coli HB101 from P. polymyxa JH2 and colonies were grown on toxin medium in library clones. Minimal DNA fragments required for toxin digestion were identified by clones made by restriction enzyme digestion from isolated DNA clones. The minimum clone 1.5kb Eco RI fragment required for toxin degradation was identified by sequencing the ORF of 666bp (SEQ ID NO: 1). NCBI BLAST analysis showed 67.3% similarity with the ring-cleavage extradiol dioxygenase of Exiguobacterium sp. The gene tflA involved in toxoflavin degradation has been found to be a novel useful gene that has not been reported yet.

Example  2: Construction of the transformation vector

Vectors used for transformation are pCamLA and pTflA. pCamLA (FIG. 1) contains Hygromicin phophotransferase (Hyg ^R ) inside T-DNA and has kanamycin resistance gene outside T-DNA. pTflA contains toxoflavin degrading enzyme (tflA) inside the T-DNA and has a 35S promoter and a NOS terminator (FIG. 2).

The vector used for the hygromycin selection is pCamLA derived from the binary vector pCAMBIA1300, which contains the hygromycin phosphotransferase (HigR) gene in the T-DNA. It has a kanamycin resistance gene outside the DNA. The growth of pCamLA was grown using Escherichia coli DH5α ( E. coli DH5α) as a host, followed by extraction of plasmid DNA. The extracted pCamLA was agrobacterium tumerfaciens LBA4404 by electroporation. ( Agrobacterium tumerfaciens LBA4404) was transformed.

Toxoplasma flavin (Toxoflavin) screening the vectors pTflA the Xho I adapter on to the pCamLA Xho I digest cloth (digestion) to remove the HygR gene and the start codon (start condon) and termination codon (stop codon) there (adaptor used for TflA with).

Construction of tflA with Xho I adapter consists of pJ90 (1.2kb Hind III fragment containing tflA in pBluscript II SK (+)) as template DNA (J90XhoI-F (5`-CTCGAGATGACTTCGATTAAACAGCTTAC-3`) and J90XhoI After PCR amplification of tflA using -R (CTCGAGTTAGATCACCAGTTCACC-3`) using a primer, the amplified PCR product was cloned into the Xho I site of pBluscript II SK (+) and sequenced. This was named pJX90-6. pTflA was prepared by replacing pJX90-6 with Xho I digested fragments and pCamLA from which the HygR gene was removed.

Example  3: rice transformation

One) Callus  Judo

Seeds were used in the previous year. The process of inducing callus is as follows.

1. Pick only good rice. (Be careful not to drop the snow)

2. Put in Falcon tube and shake for 30 seconds with 100% ethanol.

3. Add 1/2 Lax and incubate for 20 minutes in a shake incubator.

4. Wash 4-5 times with sterile water.

5. Raise the seed face up when transferred to callus induction media (2N6 free media).

Callus induction was appropriate for 3-4 weeks at 28 ℃, best induction for 4 weeks, not more than 5 weeks. The Petri dish used a larger one (100 / 20mm) than the normal Petri dish.

TABLE 1

Sucrose 30 g Showa 1900-3260 Cassamino 300mg  Difco 0230-01-1 Proline 2.878mg  Sigma CHU 3.981 g  Sigma C1416 N6-vitamin 1ml 2,4-D (stock 2 mg / ml) 1ml  Sigma D2999 (100 g) pH 5.8 adjustment Gellan sword 2 g Kanto 17611-13

TABLE 2

Stock solution (× 1000)

ingredient Medium (mg / 100ml) MS N6 R2 B5 Inositol 10000 - - 10000 Nicotinic acid 50 50 - 100 Pyridoxine-HCl 50 50 - 100 Thiamine-HCl 100 100 100 1000

2) Callus  Transformation

(One) Co-inoculation (cancer condition)

Callus derived 3 days prior to co-inoculation were placed in fresh 2N6 medium and cultured agrobacterium the next day.

1. Incubate Agrobacterium for 48 hours.

2. Centrifuge for 5 minutes at 3000 rpm to precipitate the cells.

3. Dilute in AAM medium to OD ₆₆₀ = 0.1.

4. Place an appropriate amount of callus in the tube and soak in AAM medium for 30 seconds.

5. After 30 seconds, the supernatant is well drained and sprinkled on filter paper to dry.

6. While drying for 20-30 minutes, place the filter paper in a Petri dish, moisten 1-2 ml of AAM and let it flow.

7. Place dry callus on filter paper, wrap with foil, and incubate at 24 ° C for 3 days.

(2) First choice (dark conditions)

 1. Make 2N6 + hygromycin (plant selection marker) 10mg / L (200ul-50mg / ml) + Sephatoxin 250mg / L (1ml-250mg / ml) medium.

 2. Place the callus incubated for 3 days in a sterile tube and wash once with sterile water.

 3. Wash 3 times with 50ml of sterile water + 50ul of Sephatoxin each for 20 minutes.

 4. Sprinkle over filter paper to dry.

 5. Get on the first selection medium.

 6. Wrap in foil and incubate at 28 ° C. and observe for 7 days.

(3) Second choice (dark conditions)

1.Create 2N6 + Hygromycin 30mg / L (600ul-50mg / ml) + Sephatoxin 250mg / L (1ml-250mg / ml) medium.

2. The darkened areas are not resisted and the white areas are removed on the second selection medium.

3. Observe for three weeks.

(4) first differentiation medium (light conditions) for three weeks

(5) second differentiation medium (light conditions) for 3 weeks

(6) Bottle 1/2 MS

TABLE 3

AAM Badge

1L 500 ml MSAA 100 ml 50 ml MS vitamin 0.5ml 0.5ml Cassamino 0.5g 0.25g Sucrose 65.8 g 32.9 g Glucose 36 g 36 g Autoclave Acetosyringone (Stock 20ml / ml) 1ml

TABLE 4

10X MSAA Badge

1L 200 ml CaCl ₂ · 2H ₂ O 4.4 g 0.88 g MgSO _{4 7} H ₂ O 3.7 g 0.74 g KH ₂ PO ₄ 1.7 g 0.34 g L-Glutamine 8.769 g 1.7538 g L-aspartic acid 2.662 g 0.5324 g L-arginine 1.762 g 0.3524 g Glycine 0.075 g 0.0150g Autoclave

Callus  First choice badge

2N6 Medium 1L-Autoclave

Hygromycin (50mg / ml) 200ul (final concentration 10mg / L)

Sephatoxin (250mg / ml) 1ml (final concentration 250mg / L)

Callus  Second choice badge

2N6 Medium 1L-Autoclave

Hygromycin (50mg / ml) 600ul

Sephatoxin (250mg / ml) 1ml

TABLE 5

Callus first regeneration medium

1L 250 ml MS 4.3 g 1.075 g Sigma M5524 (10L) MS vitamin 1ml 250ul Sucrose 30 g 7.5g Sorbitol 30 g 7.5g Cassamino 2 g 0.5g MES 11 g 2.75 g Acros 172595000 pH 5.8 Gellan sword 4 g 1 g After autoclave NAA (2mg / ml) 20ul 5ul kinetin (1mg / ml) 10ul 2.5ul Sigma Sephatoxin 1ml 25.ul Bioworld  Hygromycin 600ul 150ul

TABLE 6

Callus second regeneration medium

1L 500 ml MS 4.3 g 2.15g MS vitamin 1ml 5ul Sucrose 30 g 15 g pH 5.8 Gellan sword 4 g 2 g After autoclave

TABLE 7

Final medium (callus production-1/2 MS medium using bottle)

1L 500 ml 250 ml MS 2.15g 1.07 g 0.54 g Sucrose 15 g 7.5g 3.75 g Gela gum 4 (3) g 2 (1.5) g 1 (0.75) g

FIG. 3 shows a second selection of hygromycin (30 ug / ml) of rice callus transformed with pCamLA and pTflA vectors, respectively. It was found that the killing of rice callus transformed with the pTflA vector in the right medium.

4 shows toxoflavin (5ug / ml) selection prior to redifferentiation of rice callus transformed with pCamLA and pTflA vectors, respectively. The rice callus transformed with pCamLA on the left side of the medium did not have toxoflavin degrading enzymes, and thus, most of callus was killed after treatment with toxoflavin. However, it can be seen that only part of the rice callus transformed with pTflA mounted on the right medium dies.

FIG. 5 shows toxoflavin (7.5 ug / ml) selection at 4 weeks after regeneration of rice callus transformed with pCamLA vector. As can be seen in Figure 5, since the rice callus transformed with the pCamLA vector does not have a toxoflavin degrading enzyme, it was found that the majority of callus was killed as a result of treating toxoflavin.

FIG. 6 shows selection of toxoflavin (7.5 ug / ml) at 4 weeks after regeneration medium of rice callus transformed with pTflA vector. As can be seen in Figure 6, since the rice callus transformed with the pTflA vector has a toxoflavin degrading enzyme, it can be seen that the callus is normally re-differentiated even after treatment with toxoflavin.

Regeneration rate of toxoflavin (7.5 ug / ml) was examined at 4 weeks after regeneration of rice callus transformed with pCamLA and pTflA vectors, respectively. The results are shown in Table 8 below.

TABLE 8

Regeneration rate

pCamLA (hpt) pTflA Secondary transformation 2/11 (18.18%) 28/84 (33.3%) 3rd transformation 11/109 (10.09%) 26/192 (13.54%)

In Table 8 above, the regeneration rate represents the ratio of the number of regeneration plants to the total callus number. As can be seen in Table 8, it can be seen that the re-differentiation rate of pTflA is higher than that of pCamLA.

After the selected plants were transferred to pots, the tflA gene was transformed and confirmed by tissue PCR. PCR primers were designed based on the tflA sequence and run at annealing temperature 55 ° C. PCR primers were used with tfla140-U (SEQ ID NO: 5'-TGCAGCTGCTGATGGAACAAA-3 ') and TFLA370-L (SEQ ID NO: 4'-TTATCCAGTACAGGTGCAGCT-3'). Figure 7 shows the result of agarose electrophoresis of the PCR product by performing PCR from the isolated genomic DNA of the selected transformant. In Figure 7, lanes 12, 16, 17, 18, 21 and 36 did not produce PCR products. As can be seen in Figure 7, the transformant generates a PCR product at a desired position, it was found that the toxoflavin degradation gene was stably inserted into the rice genome.

Example  4: Arabidopsis transformation

1) Comparative Analysis of Existing Transformation Vectors and New Transformation Vectors Using tflA

Comparative analysis of the existing transformation vector and the novel transformation vector using tflA was performed. PMBP1 with pCamLA (Δ hpt): tflA and tflA inserted with the binary vector pMBP1 deleted from pCamLA: tflA and pCamLA: tflA carrying toxoflavin and hygromycin resistance genes: Comparative analysis was performed with tflA and pMBP1, which is widely used as a conventional transformation vector.

2) Experiment Method

Arabibacterium containing each construct is transformed into Arabidopsis Col-0, respectively. Transformation was carried out as follows.

1. Grow Arabidopsis Col-0 in the pot until it blossoms. When the flower beds come out and the flowers start to bloom, cut them with scissors.

2. Agrobacterium containing the gene to be transformed is incubated overnight in LB broth.

3. Centrifuge the cultured Agrobacterium and add O.D. in 5% sucrose. = 0.8% suspend and add 0.03% silwet.

4. Cut the stalk and after 1 week immerse the new stalk in 5% sucrose suspension for 2-3 seconds.

5. Wrap the plants in plastic bags. After two days, remove the vinyl completely.

After harvesting all the seeds, constructs with tflA resistance genes were spread on MS plates containing 20 uM (3.84 μg / ml) toxoflavin, respectively, and pMBP1 containing kanamycin resistance genes was 50 μg / ml of kanamycin. Smear the seed onto this embedded MS plate. After 10 days, transformants are selected from the plates. About 1,000 seeds were plated on each plate, and about 10 transformants were obtained on each plate.

8 shows a transgenic plant in which pCamLA: tflA, pCamLA (Δhpt): tflA, pMBP1: tflA and pMBP1 vectors are inserted, respectively. Plants in which the toxoflavin resistance gene had been inserted had normal germination and root development in 20 uM toxoflavin medium. However, most of the non-transformants did not germinate, and some germinated plants did not grow roots normally. Compared with the pMBP1 vector that was used a lot, the transformation rate was similar.

2) PCR and photo-bleaching test of selected transformants

(1) PCR of selected transformants

After the selected plants were transferred to pots, the tflA gene was transformed and confirmed by tissue PCR. PCR primers were designed based on the tflA sequence and run at annealing temperature 55 ° C. PCR primers were used with tfla140-U (SEQ ID NO: 5'-TGCAGCTGCTGATGGAACAAA-3 ') and TFLA370-L (SEQ ID NO: 4'-TTATCCAGTACAGGTGCAGCT-3'). Figure 9 shows the results of agarose electrophoresis of the PCR product by performing PCR from the isolated genomic DNA of the selected transformant. Transformations containing constructs of pCamLA: tflA (1-1 to 1-10), pCamLA (Δhpt): tflA (2-1 to 2-12) and pMBP1: tflA (3-1 to 3-11) When the sieves were confirmed by PCR, the plants that germinated normally and developed roots on the plate containing toxoflavin were able to identify the same band as the control (tflA gene), and the plants that did not normally develop roots could not identify the bands. . Therefore, it was confirmed that toxoflavin could be used as a selection marker.

(2) Transformant Identification Using Photobleaching Method

Toxoflavin, a factor that determines the pathogenicity of rice almond pathogens, has a light-dependent depigmenting effect that is commonly seen when treated in plants. Arabidopsis Col-0 was used to treat various concentrations of toxoflavin, which resulted in decolorization at low concentrations. This phenomenon was used to test transformants. 10 shows the bleaching effect of transformants and non-transformants. The selected transformants showed no photobleaching effect and the non-transformants showed photobleaching effect, which was confirmed to be consistent with the above PCR results. Therefore, the use of toxoflavin as a selection marker is expected to be able to construct a system capable of transforming a desired gene into plants without using antibiotics, compared to markers previously used with antibiotics.

According to the present invention, transgenic plants can be selected using inexpensive toxoflavin instead of expensive antibiotics.

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Claims (24)

delete delete delete delete delete delete delete delete A recombinant Arabidopsis plant expression vector comprising a tflA gene consisting of the nucleotide sequence of SEQ ID NO: 1. delete delete delete delete The vector of claim 9, wherein the vector has a cleavage map as described in FIG. 2. delete delete A host cell transformed with the vector according to claim 9. 18. The host cell of claim 17, wherein said host cell is of the genus Agrobacterium. Arabidopsis plant transformed with the vector according to claim 9. delete Transgenic seed of the plant according to claim 19. transforming a plant or plant part or plant cell with a recombinant vector comprising a tflA gene; And Propagating the transformant in a toxoflavin-containing medium. The method of claim 22, wherein the tflA gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. transforming the plant cell with a recombinant vector comprising a tflA gene; Propagating the transgenic plant cells in toxoflavin-containing medium; And Regenerating a transformed plant from the transformed plant cells.

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