KR20030084677A - Method for improving herbicide resistance of crops by expressing protoporphyrinogen oxidase both in chloroplasts and mitochondria and method for selecting transgenic cell lines using the said gene as herbicide-resistant selection marker - Google Patents

Abstract

PURPOSE: A method for improving herbicide resistance of crops by expressing protoporphyrinogen oxidase both in chloroplasts and mitochondria and a method for selecting transgenic cell lines using the same gene as a herbicide-resistant selection marker are provided, thereby improving herbicide resistance of crops. CONSTITUTION: A method for improving herbicide resistance of crops by expressing protoporphyrinogen oxidase both in chloroplasts and mitochondria is characterized by transforming crops with a host microorganism transformed by a vector containing protoporphyrinogen oxidase gene, wherein the protoporphyrinogen oxidase changes the transit sequence of protoporphyrinogen oxidase; the protoporphyrinogen oxidase is isolated from Myxococcus xanthus; the vector contains the protoporphyrinogen oxidase gene, an ubiquitin promoter and a hygromycin phosphotranferase selection marker; the vector is pGA1611:MP; the host microorganism is Agrobacterium tumefaciens; and the herbicide is protoporphyrinogen oxidase inhibiting herbicide.

Description

Method for enhancing herbicide resistance of crops using chloroplast and mitochondrial co-expression of protoporpyrronogen oxidase and method for selecting herbicide resistance of crops by expressing protoporphyrinogen oxidase both in chloroplasts and mitochondria and method for selecting transgenic cell lines using the said gene as herbicide-resistant selection marker}

The present invention relates to a method for increasing herbicide resistance in crops using chloroplast and mitochondrial co-expression of protoporpyrronogen oxidase, and a method for selecting a transformed cell line using the gene as a herbicide resistance selection marker. A vector system containing a herbicide-sensitive protoporpyrronogen oxidase gene from the genus (Myxococcus sp.) Is introduced into a plant host and resistance to herbicides by allowing the protoporpynogen oxidase protein to co-express in the chloroplast and mitochondria of the plant host. And a method for selecting a transformed cell line using the gene as a herbicide resistance selection marker.

Protoporphyrinogen oxidase (also referred to as Protox), which catalyzes the oxidation process from Protogen IX to Proto IX, is called chlorophyll and heme. It is the last step of the branch point in the biosynthetic porphyrin pathway. A substance that inhibits the protox enzyme inhibits the protox enzyme activity, inhibits the biosynthesis of chlorophyll and heme, and protophorpyrinogen IX, a substrate of the protox enzyme, is released from the normal porphyrin synthesis pathway, thereby protoxifying the plasma membrane. Allow enzymes to accumulate in Protoporphyrin IX. The accumulated protoporphyrin IX kills plants by generating singlet oxygen ( ¹ O ₂ ) from oxygen molecules in the light state, causing plant membrane destruction (Jacobs et al., Plant Physiol. 97, 197-). 302, 1991; Lee et al., Plant Physiol. 102, 881-889, 1993).

Protox inhibitors known to date include diphenyl ether, pyrimidinyl, piperidine, LS analogue, N-phenyl imide, M & B analogues, oxadiazon, F-6285, pyrazole phenyl ether and pyrrolidine are known (Lee, Kor. J. Weed Sci. 17, 243-255, 1997). These protox inhibitors are commonly used as herbicides and are widely used to control annual flowers, weeds and broadleaf weeds, which are common in the world's major crops such as soybean, rice, cotton and peanuts. (Lee, Kor. J. Weed Sci. 17, 243-255, 1997).

To date, dozens of Protox genes have been cloned and characterized from prokaryotic and eukaryotic microorganisms. Among each of these protoxes there was a low amino acid identity between the protoxes isolated from different organisms, but it was found to have high homology between the protoxes isolated from closely related organism populations (Dailey et al. , J. Biol. Chem. 271, 8714-8718, 1996; Lermontova et al., Proc. Natl. Acad. Sci. USA 94, 8895-8900, 1997; Corrigall et al., Arch. Biochem. Biophys. 358, 251-256, 1998).

In terms of herbicide resistance, Protox genes can be classified into two types. That is, it can be classified into a group of genes that are resistant to protox inhibitory herbicides and a group of genes that are sensitive. The former family of resistant Protox genes include the Protox enzyme gene cloned in Escherichia coli and Bacillus subtilis and point-mutated plant-derived Protox genes (Sasarman et al, Can. J.). Microbiol. 39, 1155-1161, 1993; Dailey et al., J. Biol. Chem. 269, 813-815, 1994; United States Patent No. 6,308,458 B1; International Publication No. WO9833927A1), belonging to the latter The sensitive Protox gene family includes all plant-derived Protox genes, including Myxococcus xanthus, Arabidopsis and Tobacco, as well as human and rat Protox genes (Dailey and Dailey, J. Biol. Chem). 271, 8714-8718, 1996; Nishimura et al., J. Biol. Chem. 270, 8076-8080, 1995; Dailey et al., Arch. Biochem. Biophys. 324, 379-384, 1995; Narita et al. ., Gene, 182, 169-175, 1996; and Lermontova et al., Proc. Natl. Acad. Sci. US A, 94, 8895-8900, 1997).

Conventionally, as an example of manufacturing a herbicide-tolerant plant transformed with a Protox gene using genetic engineering techniques, diphenyl is produced by transforming Protox gene derived from Bacillus subtilis resistant to Protox inhibitory herbicide to tobacco or rice. Reports of resistance to oxyfluorfen belonging to ether herbicides (Choi et al., Biosci. Biotechnol. Biochem. 62, 558-560, 1998; Lee et al., Plant Cell Physiol. 41, 743 -749, 2000) and a report on susceptibility to acifluorfen by transforming the protox gene from Arabidopsis, a sensitive protox gene, into tobacco (Lemontova and Grimm, Plant Physiol. 2000, 75-83, 2000).

To date, approaches that impart herbicide resistance to crops using the Protox genes have either the Protox gene cloned in the plant or the plant-derived Protox gene modified to be resistant to herbicides, or Bacillus subtilis resistant to herbicides. This study focuses on the possibility of giving crop selectivity to weed control or herbicides by limiting only overexpression of chloroplasts in host plants using protox genes derived from E. coli and Escherichia coli as main target genes (Choi et. al., Biosci. Biotechnol.Biochem. 62, 558-560, 1998; Lee et al., Plant Cell Physiol. 41, 743-749, 2000; US Pat. No. 5,767,373; US Pat. No. 6,308,458 B1; and International Publication WO9833927A1).

However, to date, no report has been made on the production of highly herbicide tolerant crops by using the Protox gene to allow the Protox protein to be expressed simultaneously in the chloroplasts and mitochondria of host plants.

Therefore, the present inventors have over-expressed this gene by transforming a plant with a Mytococcus-derived Protox gene which is sensitive to a Protox inhibitory herbicide, and as a result, investigates whether the transgenic crop is provided with Protox inhibitory herbicide resistance. It was. To this end, transgenic rice that overexpresses Protox through Agrobacterium-mediated transformation techniques were grown and their herbicide resistance properties were investigated in T ₁ and T ₂ generations. The herbicide resistance of the converted rice was found to be markedly increased due to the vector-host system of the present invention, and the fact that such herbicide resistance was conferred was confirmed by confirming that the Protox protein was co-expressed in the chloroplast and mitochondria of host plants. Was completed.

It is an object of the present invention to provide a method of increasing the crop's resistance to herbicides.

Another object of the present invention is to provide a method for selecting a transformed cell line using herbicide resistance selection marker.

1 is a schematic diagram of a T-DNA region (A) of a binary vector, pGA1611: C, pGA1611: P, and pGA1611: MP, and an overall vector schematic diagram (B) of pGA1611: MP.

Ubi-P: corn ubiquitin promoter; Bs Protox: Bacillus subtilis protoporpyrinogen oxidase gene; TS: transit sequence; Mx Protox: Mysococcus protoporpyrinogen oxidase gene; Tnos: noparin synthetic terminator; P _35S : Cauliflower 35S promoter; HPT: hygromycin phosphotransferase; TiA6-7: Octopine type TiA6-7 terminating gene.

2 is a photograph showing the herbicide resistance results of transgenic and non-transgenic null segregants in T ₁ generation. A is a photograph after foliage treatment of oxyfluorfen herbicide of 1 mM concentration in rice, B is a photograph after foliage treatment of 1 mM carfentrazone herbicide in rice.

NT: untransformed rice; T: transformed rice

3 is a photograph showing a result of Northern blot analysis of T ₁ transformed rice.

W: control group (wild type rice), M2 to M8: myosococcus Santos protox expression transformed rice.

Figure 4 shows the results of the oxyfluorophene resistance germination assay in the medium of transgenic rice expressing various Protox genes, the oxyfluorene concentration is 5 ~ M, it is taken on the 7th day after the tooth.

A: control group (wild-type rice), B: myococcus protox expression transgenic rice, C: cytoplasmic expression Arabidopsis protox expression transgenic rice, D: plastid expression Arabidopsis protox transgenic rice.

Figure 5 shows the results of the foliage treatment of the transgenic rice expressing Protox, a photograph showing the results of treatment of 100 ~ M or 10 mM concentration of oxyfluorophene in the rice seedlings of each of the 3 to 4 leaves.

W: control (wild-type Dongjin rice), BP: Bacillus subtilis protox expression transformed rice, MP: myosococcus protox expression transformed rice, ACP: cytoplasmic expression Arabidopsis protox expression transformed rice, APP: chromatin expression Arabidopsis Protox transformed rice.

6 is a diagram showing the intracellular expression site (A) and protox activity (B) of the transgenic rice expressing Protox.

M: protein standard marker, Ep: chloroplast protein, Mit: mitochondrial protein, Pc: purified protox protein, W: control (wild-type rice), M7: myxococcus Santos protox expressing transformed rice, Ox: oxyfluorfen .

In order to achieve the above object, the present invention provides a protoporphyrinogen oxidase (protoporphyrinogen oxidase) by a vector containing a gene encoding a protoporphyrinogen oxidase (protoporphyrinogen oxidase) so that co-expression in the chloroplast and mitochondria of plants Provided is a method of transforming a crop with a transformed host microorganism to increase the crop's resistance to herbicides.

In addition, in order to achieve another object of the present invention, the present invention is a transgenic cell line characterized by using a gene encoding a protoporphyrinogen oxidase coexpressed in the chloroplast and mitochondria as herbicide resistance selection marker Provide a selection method.

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

The method for increasing herbicide resistance of a crop according to the present invention can be achieved by introducing a gene encoding protoporpyrogenogen oxidase into a crop, thereby co-expressing it in the chloroplasts and mitochondria of the host plant. At this time, the gene encoding the rotoporpyrinogen oxidase includes herbicide sensitive gene or herbicide resistance gene. In the present invention, herbicide-sensitive genes are preferred, particularly herbicide-sensitive genes derived from Myococcus. More preferably, it is a herbicide sensitive protox coding gene derived from Myxococcus xanthus.

The Protox gene derived from Mycoccus Santos is registered as an unidentified gene on top of the csgA gene (GenBank Gen. No. M73709) involved in sporulation (Li et al., Genes Cev. 6, 401-410, 1992). ). In addition, Daily (Dailey and Dailey, J. Biol. Chem. 271, 8714-8718, 1996), the top gene of the Myosococcus Santos M73709 gene was prototyping by methods such as E. coli expression, protein purification and protox enzyme activity analysis. It was first identified that the phosphinogenogen oxidase was encoded, and the gene sequence was registered under GenBank gene accession number AF098938.

Protox from Myxococcus santos is a membrane protein consisting of 471 amino acids, containing three membrane helices, and having a molecular weight of about 50,000 Daltons. Purified protox enzymes have the property of strongly inhibiting their enzymatic activity by acifluorfen herbicides of about 1 M concentration (Dailey and Dailey, J. Biol. Chem. 271, 8714-8718). , 1996; Takatsugu et al., Bioinformatics, 14: 378-379, 1998).

As mentioned above, protox derived from Mycoccus santos has been known to exhibit a very high herbicide sensitivity due to the strong inhibition of its enzymatic activity by protox inhibitory herbicides. The present invention is characterized in that, when such a protox inhibitory herbicide-sensitive gene is introduced into other plants, it shows a rather significant resistance to the protox inhibitory herbicide. In order to elucidate the related mechanism, Western blotting was performed by separating chloroplasts and mitochondria of crops transformed with Protox gene in the present invention. Protox protein was co-expressed in the chloroplasts and mitochondria of host plants and confirmed that the resistance to herbicides was greatly increased. In other words, Protox from Mycoccus Santos has an ambiguous transition sequence at the amino terminus, and when Protox is expressed in plants, it is simultaneously transferred to the mitochondria and chloroplasts of the host plant and stably overexpressed in two different organelles. It exhibits strong resistance during herbicide treatment. As is known in the art, the expression of herbicide-resistant protoxes on chloroplasts can prevent the inactivation of chloroplast protoxes to herbicides, while at the same time herbicides inactivate mitochondrial protoxes and thus prevent the inhibition of mitochondrial heme synthesis Eventually, such plants will not have high resistance to herbicides. Therefore, no matter how much resistant protoxes are developed, sufficient herbicide resistance may not be achieved when these protoxes are expressed only in either chloroplast or mitochondria. Therefore, the present invention is characterized by the first time that protox is transformed into crops and expressed simultaneously in chloroplasts and mitochondria of host crops to increase herbicide resistance of crops. The protox gene includes both herbicide sensitive or herbicide resistance genes. When the herbicide-resistant protox gene or herbicide-sensitive protox gene is transformed and simultaneously expressed in the chloroplast and mitochondria of the host crop, the herbicide resistance of the crop is included in the present invention.

There are many examples of the co-expression of proteins in two organelles in a cell. For example, a protein may be expressed in the cytoplasm and mitochondria by having an inefficient targeting sequence, and because it has ambiguous targeting signals that are not clear to either side. Cases of simultaneous expression in chloroplasts have been reported. Or when two proteins are transferred from one gene and the two proteins are targeted to different organelles (Small et al., Plant Mol. Biol. 38, 265-277, 1998). Therefore, coexpression of protox proteins with mitochondria and chloroplasts can be easily achieved by combining transgenic sequences that are simultaneously targeted to these mitochondria and chloroplasts or sequences that allow two proteins to transfer from one gene to the Protox gene. These plants will be able to exert high resistance to protox inhibitory herbicides.

In the present invention, the mycoccus Santos protox gene was inserted into the vector using the binary vector pGA1611: C (Accession No. KCTC 0692BP). The binary vector pGA1611: C (Accession No. KCTC 0692BP) was prepared by inserting the Bacillus subtilis protox gene into a known vector pGA1611 (Kang et al., Plant Mol. Biol. 38, pp 1021-1029, 1998). . Molecular biological techniques well known in the art were used to remove the Bacillus subtilis protox gene from the binary vector pGA1611: C, and to construct the vector pGA1611: MP containing the myxococcus Santos protox full-length gene. After that, it was used for further transformation.

Specifically, the hygromycin phosphotransferase used as a constitutive ubiquitin promoter and selection marker known to be capable of inducing high expression of foreign genes when transformed into crops. Subcloning the Myococcus Santos protox full-length gene into a binary vector pGA1611: C (Accession No. KCTC 0692BP) containing the gene to prepare a vector pGA1611: MP (see FIG. 1) and use the vector to host microorganism Agrobacterium Tome Pacific Enschede (Agrobacteriumtumefaciens) was transformed with LBA4404. The transformed Agrobacterium tumefaciens LBA4404 was introduced into the host crop to prepare a crop having increased resistance to herbicides. There is no restriction on the crop transformed by the method of the present invention, but it is preferably a monocotyledonous or dicotyledonous plant. The monocotyledonous crops include barley, rape, corn, wheat, rye, oats, grass, forage, millet and rice, and the dicotyledonous crops include sugar cane, soybeans, tobacco and cotton.

The transformation can be carried out using conventional methods known in the art. Plants can be transformed using Agrobacterium-mediated transformation, and the methods illustrated in Horsch et al., Science 227: 1229-1231, 1985 can be used. For example, Agrobacterium-mediated transformation methods for rice are disclosed in An et al., Plant Molecular Biology Mannual A3: 1-19, 1988.

The monocotyledonous plants can be transformed by using a method of directly transplanting genes into protoplasts using PEG or electroporation, or using particle bombardment into callus tissue. It may also be transformed using transformations using single DNA and cotransformation using multiple DNA. Transformed cells can be re-differentiated into whole plants using standard techniques well known in the art.

In one embodiment of the present invention, Agrobacterium tumefaci transformed with the vector pGA1611: MP prepared according to the method of the present invention to obtain a crop having increased herbicide resistance of the present invention through regeneration of the transformed crop. Scutellum calli derived from Ens and rice (breed: Dongjin) were co-cultured. As a result, on average, about 10 to 15% of callus survived in the selection medium containing 50 g / mL hygromycin. In order to induce leaves from such hygromycin resistant callus, 1-5% of the selected callus was re-differentiated into shoots (young shoots) by transferring the callus to a regeneration medium and incubating for a period of time. Subsequently, the replanted shoots were transferred to root induction medium and cultured to obtain complete herbicide resistant plants (T ₁ transgenic) (Lee et al., Plant Cell Physiol. 41, 743-749, 2000).

In another embodiment of the present invention, the herbicide resistance of transgenic rice expressing rice transformed by the method of the present invention and conventional herbicide resistant Bacillus subtilis protox and herbicide sensitive Arabidopsis protox were compared. As a result, the herbicide resistance of the transgenic rice expressing the herbicide-sensitive Myococcus Santos protox of the present invention was found to be superior to those of the transgenic rice listed above.

On the other hand, the herbicide is preferably a protox inhibitory herbicide. At this time, the protox inhibitory herbicides include axifluorfen, aclonifen, acfenifen, bifenox, pomesafen, lactofen and oxyfluorfen. Diphenylethers; Oxadiazoles, including oxadiazon; N-phenylphthalimides including flumioxazin and flumiclorac-pentyl; Triazolinones including carfentrazone, sulfentrazone, etc .; And thiadiazoles including fluthiacet-methyl, thidiazimin, and the like.

In addition, the chloroplast and mitochondrial coexpression genes according to the present invention can be used for selection of transformed cell lines as herbicide resistance selection markers. At this time, the chloroplast and mitochondrial co-expression gene is preferably a Protox gene derived from Myococcus Santos. In addition, the herbicide is preferably a protox inhibitory herbicide.

Specifically, Agrobacterium into which the vector pGA1611: MP containing the Mycoccus Santos Protox gene prepared according to the present invention is introduced into Agrobacterium tumefaciens, and the vector is introduced in a selection medium containing a certain amount of antibiotic. Tumefaciens can be cultured to select transformed Agrobacterium tumefaciens. At this time, the antibiotic may be used in the tetracycline and hygromycin respectively or simultaneously. In addition, after co-cultivation of transgenic Agrobacterium tumefaciens and scutellum calli derived from rice (breed: Dongjin) selected by the above method, in a selection medium containing a certain amount of a protox inhibitory herbicide. When cultured, only the callus of the transformed herbicide resistant cell line can survive, thereby selecting only the transformed cell line.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Preparation of Rice Transformation Vector

Bacillus subtilis Protox gene prepared in Korean Patent Application No. 99-52492, the applicant of the present applicant, was prepared in order to prepare a vector containing a Mytococcus santos-protox gene used for rice transformation. Binary vector pGA1611: C (Accession No. KCTC 0692BP) was used. The Bacillus subtilis protox gene included in the binary vector pGA1611: C (Accession No. KCTC 0692BP) was cleaved using Sac I and Kpn I, and the mycosoctus Santos protox gene was inserted into the cleaved site. A binary vector pGA1611: MP capable of expressing the full-length gene of Sococus Santos protox was prepared (see FIG. 1).

Specifically, the Mycoccus Santos Protox gene ( Mx Protox) was PCR amplified using primers having the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2. At this time, the primers were added to the Sac I and Kpn I primer restriction enzyme sites for easy subcloning of the amplification gene, respectively, the underlined portion of the sequence list means the restriction enzyme site.

PMx-PPO plasmid DNA (Dailey and Dailey, J. Biol. Chem) containing the aforementioned two anterior (SEQ ID NO: 1) and posterior (SEQ ID NO: 2) primers and the mycoccus Santos protose gene as a template 271, 8714-8718, 1996) were used to PCR amplify the Myococcus Santos Protox gene. The PCR product was digested with Sac I and Kpn I, gel purified, and inserted into the same restriction enzyme site in the vector pBluescript (Strategene, USA) to confirm the abnormality of the inserted nucleotide sequence. Sac I and Kpn I fragments of the protox sequence were linked to pGA1611: C (Accession No. KCTC 0692BP) vector previously treated with the same restriction enzyme using a ligase to regulate corn ubiquitin promoter (Ubi-P). By placing it underneath, a binary vector pGA1611: MP was produced (see Fig. 1).

<Example 2>

Rice Transformation and Regeneration

Plasmid pGA1611: MP was transformed into the Agrobacterium tumefaciens LBA4404 strain. Transformed Agrobacterium tumefaciens LBA4404 strains were prepared in YEP medium (1% Bacto-peptone, 1% Bacto-yeast extract and 0.5) containing 5 ° g / mL tetracycline and 40 ° G / mL hygromycin. % NaCl) was incubated overnight at 28 ?? conditions. The culture was centrifuged and the pellets suspended in AA medium (Hiei et al., Plant Mol. Biol. 35, 205-218, 1997) containing 100 μM of acetosyringone in the same volume. A converted Agrobacterium tumefaciens LBA4404 suspension was prepared. Callus of rice was collected from N6 medium (Rashid et al., Plant Cell Rep. 15: 727-730, 1996; Hiei et al., Plant Mol. Biol. 35, 205-218, 1997). Derived from betrayal (breed: Nakdong). Callus of dense rice, 3-4 weeks old, was deposited in the bacterial suspension for 3 minutes, and then excess bacteria were removed from the suspension by contacting the suspension with sterile filter paper to absorb and dry the suspension. The callus in suspension was then transferred to the coculture medium and co-cultured for 2 to 3 days in the dark at 25 °. The co-cultured callus was washed with sterile water containing 250 mg / L of Cefotaxime to remove residual bacteria, and the removed callus was 250 mg / L of Cefotaxime and 50 mg / hygromycin. It was transferred to N6 medium containing L and cultured. Selected callus was incubated for 3 to 4 weeks in the regeneration medium for germination and root growth. Once the roots of the callus had grown sufficiently, these transgenic crops were transferred to the greenhouse and kept growing.

<Example 3>

T _One Isolation of Hygromycin Resistant Plants from Generation

The T ₁ generation assay was performed by selecting transgenic rice having a large amount of seed from the self-pollinating rice seed. The transmission of the trans gene was tested using eight transgenic rice (M1, M2, M3, M4, M5, M7, M8 and M10). In addition, for all transgenic cell lines tested, the hygromycin resistant and susceptible individuals of each transgenic cell line exhibited when germinated brown rice at half the concentration of MS solidification medium containing 50 g / mL of hygromycin. The separation rate was investigated. Table 1 below describes the hygromacin resistance separation patterns of the transgenic cell lines tested.

Isolation of Hygromycin Resistant Plants from T1 Generation Transgenic Rice Transformed rice Resistance (seed count) Susceptibility (Number of Seeds) Separation ratio (resistance: sensitivity) X ² M1 8 12 - M2 14 3 3: 1 0.49 M3 18 3 3: 1 1.29 M4 5 2 3: 1 0.05 M5 18 3 3: 1 1.29 M7 14 7 3; 1 0.78 M8 12 One 15: 1 0.05 M10 2 3 -

X ² : The value used as the test method for the deviation in the X ² test.It is a statistical method for testing the goodness of fit to see if the actual phenotype can be regarded as separated by the theory. do.

X ² = ?? (observation frequency-expected frequency) ² / expected frequency

As a result, the M2, M3, M4, M5, and M7 cell lines, except for the M8 cell line, had a resistance to sensitivity ratio of about 3: 1, indicating that transgenes inserted in the rice genome were subject to Mendel's law. Thus isolated and expressed. In the M1 and M10 cell lines, there was a high percentage of susceptible plants whose isolation rates did not match Mendel's law.

<Example 4>

T _One Herbicide Resistant Packaging Assay in Separate Generations

To carry out herbicide resistant packaging assays in T ₁ isolated generations, T ₁ seeds were harvested from the transformed rice into which the Myococcus protox gene was introduced, and 20 seeds were seeded from each T ₁ transformant line. Then, it was transplanted to the packaging and grown.

FIG. 2 shows 1 mM of Oxyfluorfen (FIG. 2A) and Carfentrazone (FIG. 2B), a protox inhibitory herbicide, in rice grown again at the bottom after harvesting the T ₁ transformant. It is a photograph taken 5 days after the foliage treatment. Considering the results of the photograph, it was confirmed that the growth of the transformants (Transgenic, T) was not affected by the herbicide treatment, but the non-transgenic null segregants (NT) were completely killed by the herbicide treatment. From these results, it was confirmed that the transformants introduced with Mycoccus Santos Protox were resistant to Protox inhibitory herbicides, and that the non-transformant isolates were sensitive to herbicides.

Example 5

Transgene Insertion Assay by Genomic DNA Gel Blot Analysis

Genomic DNA was extracted according to standard methods to determine whether the Mycoccus Santos Protox gene was stably introduced into the rice genome of a reprogrammed transgenic cell line from hygromycin selection media and the number of insertions thereof (Ausubel). et al., Current Protocol in Molecular Biology, 1st ed., 1987). 5 μg of genomic DNA extracted from 8 transgenic T ₁ rice transformed with pGA1611: MP and untransformed wild-type rice, respectively, were digested with Hin dIII restriction enzyme and 0.8% (w / v) agarose gel. Electrophoresis was carried out in phases and fractionated by size. These fractions were blotted onto nylon membranes (Nylon 66 plus, Pharmacia Biotech.) And then hybridized with ³² P-labeled Myococcus Santos protox.

If there is no Hin dIII restriction enzyme recognition site inside the probe transgene, the number of hybridized bands will match the number of trans genes inserted into the genome of the transgenic plant. Accordingly, in the case of genomic DNA extracted from wild type rice, which is a control, the hybridized band was not detected, which means that the mycoccus Santos protox gene does not exist, and that the transgenic rice transformants (M2 to M8) In each case, hybridization bands of 5 kb or more were detected, and it was confirmed that myococcus Santos protox gene was inserted into the rice genome (not shown).

<Example 6>

Investigation of mRNA Expression of Trans Gene by RNA Gel Blot Analysis

An individual (myococcus protox expression strains M2 to M8) whose insertion of the myococcus protox gene was confirmed in hygromycin medium was selected using an expression strain having a high seed amount from the transformed rice, and selected. Expression stocks were grown in greenhouses. Total RNA of the selected expression strains was extracted from the leaves of the cultivated rice using TRI reagent (obtained from Sigma). Total RNA (10 μg) was fractionated using 20 mM Mops [3- (N-morpholino) -propanesulfonic acid] solution in development buffer on 1% agarose gel with formaldehyde. The developed gel was blotted onto the nylon membrane and hybridized with the full-length Myococcus Santos protox, and the results are shown in the photograph of FIG. 3. Prior to blotting of the gels, the identity of RNA samples developed on a 1% agarose gel was checked using ethidium bromide (not shown).

Based on the concentration of the detected hybridization bands, the expression of Mycoccus Santos Protox mRNA was examined by RNA gel block analysis. As a result, the Mycoccus Santos Protox mRNA was not expressed in the untransformed control group (W). (See Figure 3). On the other hand, the transgenic cell line was overexpressed in Mycoccus Santos Protox mRNA. In particular, M3 and M4 transformed cell lines showed the highest expression rate, and M7, M5, M8, M2 transformed cell lines, etc. showed the difference in the degree of expression.

<Example 7>

T ₂ Herbicide Resistant Germination Assay in Separate Generations

To carry out herbicide resistant germination assays in T ₂ isolated generations, T ₂ seeds were obtained from autologous pollination from T ₁ myococcus expressing transformants. The obtained T ₂ seeds were selected from an independent seed line that is 100% resistant, i.e. presumed Homozygous, via a hygromycin germination assay. Seeds are removed from these Mysokocus protox expressing Homozygous T ₂ seeds, followed by disinfection of the seeded homozygos T ₂ seed with 70% ethanol and NaOCl. This was implanted in 1/2 MS medium containing oxyfluorfen 5 ?? M herbicide, 28 ?? After culturing for 2 days in a state in which the light is blocked under the culture temperature, and then for 7 days in the state exposed to light, the growth state thereof was investigated. At the same time, wild type rice, Arabidopsis protox cytoplasmic expression transformed rice T ₁ seed and Arabidopsis protox chromatin expressing rice T ₁ seed were simultaneously wounded (refer FIG. 4).

Figure 4 A is a photograph showing the results of the growth of wild type rice control. As can be seen in the photo, the wild type control was found to be completely lethal. 4B is a photograph showing the results of the growth of mycoccus protox expressing T ₂ , and its growth was not inhibited at all by herbicides. Figure 4C is a photograph showing the results of the growth of transgenic rice expressing Arabidopsis protox cytoplasm, Figure 4D is a photograph showing the results of the growth of transgenic rice expressing Arabidopsis protox cytoplasm. Arabidopsis Protox plastid expressing rice (FIG. 4D) showed higher herbicide resistance than Arabidopsis Protox cytoplasmic expressing transgenic rice (FIG. 4C), but Myococcus protox transformant (FIG. 4) Compared with B), herbicide inhibition was significantly higher.

<Example 8>

Herbicide Resistance Tests of Transgenic Rice

For herbicide resistance experiments of transformed rice, transformed rice MP was prepared according to the method of Example 2 using a vector (PGA1611: MP) comprising the myococcus Santos protox gene of the present invention prepared in Example 1. Prepared. For comparative experiments, the chromatin expressing Arabidopsis Protox gene APP (Genebank Gene Accession No. D83139) and the cytoplasmic expressing Arabidopsis Protox Gene ACP (Genebank Gene Accession No. D83139) from which the transient sequence was removed were prepared according to the method of Example 1. A vector (pGA1611: ACP and pGA1611: APP) containing the herbicide sensitive Arabidopsis protox gene was prepared by substitution with the Bacillus subtilis Protox gene of binary vector pGA1611: C (Accession No. KCTC 0692BP). Then, according to the method of Example 2, the transformed rice (ACP) to which the pGA1611: ACP vector was introduced and the transformed rice (APP) to which the pGA1611: APP vector was introduced were prepared. In addition, transgenic rice BP was prepared according to the method of Example 2 using the binary vector pGA1611: P (Accession No. KCTC 0693BP) prepared in Korean Patent Application No. 99-52492, the applicant of the present applicant. The herbicide resistance of the thus prepared transgenic rice MP, BP, ACP and APP was compared. As a control, rice (W) that was not treated with the Protox gene was used.

As a result, when the leaves were treated with the herbicide oxyfluorfen 5 ?? M in the control nursery of Dongjinbyeong (W) in the 3-4 leaf phase, it was observed that rice (W) died (not shown). In addition, when the oxyfluorophene 100 ?? M was foliage-treated on the nacres of Dongjin rice (W), it was also observed that Dongjin rice (W) died (see FIG. 5).

In addition, transgenic rice (BP) expressing Bacillus subtilis protox, which has been reported to be resistant to oxyfluorfen through in-flight experiments with mature leaf disk assays (Lee et al. al., Plant Cell Physiol. 41, 743-749, 2000) were examined for resistance by foliage treatment of the oxyfluorfen. As a result, rice was killed similarly to the control group by foliage treatment of 5 to M oxyfluorophene herbicide to the BP seedlings of the 3 to 4 leaf stages (not shown). In addition, seedlings (BP) were also killed when oxyfluorophene 100 ?? M was applied to transgenic rice seedling (BP) seedlings (see FIG. 5). In other words, these results indicate that although Bacillus subtilis Protox transformed rice (BP) showed herbicide resistance in in-flight experiments using a leaf disk assay (Dailey et al., J. Biol. Chem). 269, 813-815, 1994), which indicated that the herbicide resistance of 5 or more M in the direct seedlings showed little herbicide resistance.

Protox inhibitory herbicide-sensitive herbaceous transgenic rice (ACP and APP) expressing protox genes in the cytoplasm and plastids showed significant herbicide resistance compared to controls (not shown). When the leaves were treated with M oxyfluorfen, both ACP and APP rice were killed. In contrast, in case of transgenic rice (MP) expressing Mycoccus protox, the growth of MP (MP) itself was not inhibited at all when the leaves were treated with 100 mM M as well as 10 mM oxyfluorfen herbicide. It showed strong herbicide resistance without receiving (see FIG. 5).

From these results, the present inventors have confirmed that mycoccus Santos protox transgenic rice (MP) according to the present invention has a herbicide resistance of about 2000 times higher than that of the control (W) rice, and herbicide resistant Bacillus It was confirmed that the herbicide resistance was higher than the transgenic rice (BP) introduced with the subtilis protox gene and the transgenic rice (ACP and APP) with the herbicide-sensitive Arabidopsis gene.

Example 9

Protox protein expression site assay and enzyme activity of transgenic rice

In order to elucidate the mechanism of herbicide resistance of transgenic rice, intracellular expression sites of protox were examined. Chloroplasts and mitochondria were isolated from the leaves of transgenic rice (Lee and Duke, Agric. Food Chem. 42, 2610-2618; Neuburger et al., Arch. Biochem. Biophys. 217, 312-323), Western blotting Was performed (see FIG. 6). 6A, it was confirmed that protox proteins were simultaneously observed in chloroplasts and mitochondria, and the enzymatic activity of these purified chloroplasts and mitochondria was 35 and 25 times higher than that of control, respectively. The addition of 100 ?? M oxyfluorfen to the chloroplasts and mitochondria isolated from the leaves showed higher enzyme activity than the wild-type control (FIG. 6B). Therefore, the herbicide resistance of the transformed plants was confirmed that the mycoccus protox protein was obtained by co-expression in chloroplast and mitochondria.

As described above, the present invention by introducing a vector system containing the protox gene into the crop, by co-expressing protox in the chloroplast and mitochondria of the host crop, thereby significantly increasing the resistance of the crop to protox inhibitory herbicide Weed control and seed production were possible. In addition, the chloroplast and mitochondrial coexpression Protox genes of the present invention can be usefully used as a herbicide resistance selection marker for selection of transformed cell lines.

Claims (19)

Crop is transformed into a host microorganism transformed with a vector containing a gene encoding protoporphyrinogen oxidase such that protoporphyrinogen oxidase is co-expressed in the chloroplasts and mitochondria of plants. To increase the resistance to herbicides. 2. The herbicide of crops according to claim 1, wherein said protoporphyrinogen oxidase alters the transient sequence of said protoporphyrinogen oxidase such that it is co-expressed in the chloroplasts and mitochondria of plants. How to increase resistance to. The method according to claim 1 or 2, wherein the protoporpyrinogen oxidase is a protoporphyrinogen oxidase derived from the herbicide sensitive mysococcus genus. 4. The method of claim 3, wherein the genus Myococcus is Myxococcus xanthus. 4. The vector according to claim 3, wherein the vector comprises a protoporpynogen oxidase gene, a ubiquitin promoter and a hygromycin phosphotransferase selection marker, and has a restriction map of B of FIG. How to. The method of claim 5, wherein said vector is pGA1611: MP. The method of claim 1, wherein the host microorganism is Agrobacterium tumefaciens . A method according to claim 1, wherein the crop is monocotyledonous crop or dicotyledonous crop. 9. The method of claim 8, wherein the monocotyledonous crop is selected from the group consisting of barley, rapeseed, corn, wheat, rye, oats, grass, forage, millet and rice. 9. The method of claim 8, wherein the dicotyledonous crop is selected from the group consisting of sugar cane, soybeans, tobacco and cotton. 2. The method of claim 1, wherein the herbicide is a protoporpyrronogen oxidase inhibitory herbicide. The method of claim 11, wherein the protoporpyrogen oxidase inhibitory herbicides are diphenylethers, oxadiazoles, phenylphthalamides, triazolinones, and thiadiazoles. (thiadiazoles). 13. The diphenyl ether herbicide of claim 12, wherein the diphenyl ether herbicide is acifluorfen, alonifen, bifenox, fomesafen, lactofen and oxyfluorfen. The method comprising a). 13. The method of claim 12, wherein said oxadiazole herbicide comprises oxadidiazon. 13. The method of claim 12, wherein the phenylphthalamide herbicide comprises flumioxazin and flumiclorac-pentyl. 13. The method of claim 12, wherein said triazolinone system comprises carfentrazone and sulfentrazone. 13. The method of claim 12, wherein the thiadiazole system comprises fluthiacet-methyl and thidiazimin. A method for selecting a transformed cell line, characterized by using a gene encoding protoporphyrinogen oxidase co-expressed in chloroplasts and mitochondria as herbicide resistance selection marker. The gene encoding the protoporphyrinogen oxidase co-expressed in the chloroplast and the mitochondria is a gene encoding the protoporphyrinogen oxidase derived from the herbicide-sensitive myococcus. Transgenic cell line selection method, characterized in that.