KR101790010B1 - Gene enhancing pre―harvest sprouting tolerance derived from Oryza sativa and uses thereof - Google Patents

Abstract

The present invention relates to OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene derived from rice ( Oryza sativa ) promoting the resistance to athletic fever and more particularly to a recombinant vector comprising OsPHS1 gene derived from rice, , A method of enhancing the resistance of the plant to avalanche compared to the wild type comprising the step of overexpressing the OsPHS1 gene, and a step of overexpressing the OsPHS1 gene by transforming the plant cell with a recombinant vector containing OsPHS1 gene derived from rice A method for producing a transgenic plant having increased resistance to athrax compared to a wild-type plant, a transgenic plant produced by the above method, a seed thereof, and a composition for enhancing the resistance to athletic resistance of a plant containing a recombinant vector containing OsPHS1 gene derived from rice .
According to the present invention, the plants transformed with OsPHS1 gene derived from rice are more resistant to the aphrodisiac resistance than those of wild-type plants. Therefore, the use of the OsPHS1 gene can be used to develop a plant that can grow strongly even in a poor environment, And it can be useful for constructing environmental stress tolerance crop development platform.

Description

[0002] Rice-derived genes and their uses for promoting resistance to athlete's disease [0002] Gene enhancing pre-harvest sprouting tolerance derived from Oryza sativa and uses thereof [

The present invention relates to OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene derived from rice ( Oryza sativa ) promoting the resistance to athletic fever and more particularly to a recombinant vector comprising OsPHS1 gene derived from rice, , A method of enhancing the resistance of the plant to avalanche compared to the wild type comprising the step of overexpressing the OsPHS1 gene, and a step of overexpressing the OsPHS1 gene by transforming the plant cell with a recombinant vector containing OsPHS1 gene derived from rice A method for producing a transgenic plant having increased resistance to athrax compared to a wild-type plant, a transgenic plant produced by the above method, a seed thereof, and a composition for enhancing the resistance to athletic resistance of a plant containing a recombinant vector containing OsPHS1 gene derived from rice .

Rice, which is the stock of half of all humanity, is the most important food crop in the world. Numerous rice cultivars have been developed as a method of breeding to enhance agricultural characteristics such as increased seed production, good seed production and post-harvest stability after the 20th century (Khush, Plant Mol Biol . , ≪ / RTI > 35: 25-34,1997; Kovach et al ., Trends Genet. , ≪ / RTI > 23: 578-587, 2007). Seed dormancy is a physiological trait that inhibits seed germination under appropriate environmental conditions such as moisture, light, and temperature (Bewley, Plant Cell , 9: 1055-1066, 1997). In general, the dormancy that normally occurs during the development of the seed is maintained for a certain period in the ripened seed, and the dormancy is overcome over time. Seed dormancy is a basic trait necessary for the survival of plants, for example, dormant seeds have the opportunity to spread to a more distant region without germination and are not germinated under adverse environmental conditions, resulting in extreme environmental stress conditions You can also wait until you are in a good environment while maintaining your life.

If the seeds are not dormant or very weak, if the rain is high and the water is constantly high, germination will occur with immature seeds on the ears before harvest. This phenomenon is referred to as pre-harvest sprouting (PHS) (Bewley and Black, Seeds , 1994; Finkelstein et al ., Ann Rev Plant Biol . , 59: 387-415, 2008). Therefore, seed dormancy at harvest time is an important agricultural trait in major crops such as rice, wheat, and corn, and economic losses are serious due to a decrease in grain production and grains quality worldwide. On the other hand, if seed dormancy is too high, germination can not be maintained uniformly on the cultivated land. Therefore, appropriate level of dormancy is an important agricultural characteristic for the development of new varieties.

Generally, the seed dormancy of wild rice is strong, whereas the Japonica type rice cultivars developed by breeding for a long time have weakened dormancy easily for farming, and the degree of dormancy of seeds is very different among developed rice cultivars. In the process of seed development, embryo differentiation is completed within about one week after heading, and about 25 days after embryo maturation is completed, dormancy is obtained. This is called primary dormancy. The primary dormancy of seeds is a genetically complex feature that is influenced by numerous internal and environmental factors (Li and Foley, Trends Plant Sci . , 2: 384-389, 1997). When the seeds mature, the dormancy gradually disappears or is destroyed by cold or other environmental stimuli. It has been reported that rice varieties developed in Korea have a short duration of dormancy (Suh and Kim, J Crop Sci . , 39, 187-192, 1994. Park and Kim, J Crop Sci. , 54: 241-248, 2009).

ABC (abscisic acid), a plant hormone, is known to be an important regulator involved in the process of acquiring and maintaining seed dormancy (Gubler et al ., Curr Op in Plant Biol. , 8: 183-187, 2005; Kermode, J Plant Growth Regulation. , 24: 319-344, 2005). ABA biosynthesis and ABA reactivity are factors affecting the seed dormancy of major crops such as rice and maize. The dormancy of rice seeds is influenced not only by ABA but also by various conditions such as the environmental conditions of seed maturity, postharvest seed storage temperature, moisture content, oxygen, nitrogen and carbon dioxide (Roberts, J Exp Botany , 13: 75-94, 1962 ; Ikehashi, Jap J Breeding , 22: 209-216, 1972). In addition, the dormancy of rice seeds is known to be influenced by the chemical properties of the seed coat and the physical properties of the seed coat (Bewley and Black, Seeds , 1994). Although the dormancy of seeds is known to be regulated by a variety of genes, little is known about the genetic origin of seed dormancy.

Under these circumstances, the inventors of the present invention have made intensive efforts to develop seeds or crops that can be adapted even in a poor environment in which the risk of damage due to abnormal weather has been increased due to an abnormal climate. As a result, they have found that seeds germination characteristics can be controlled, The OsPHS1 gene derived from the transgenic plant or the seed obtained from the transformed plant was obtained by confirming that the resistance to avalanche of the transformed plant or the seed was enhanced in comparison with the wild type plant or seed, Thereby completing the present invention.

Khush, Plant Mol Biol., 35: 25-34, 1997 Kovach et al., Trends Genet., 23: 578-587, 2007 Bewley, Plant Cell, 9: 1055-1066, 1997 Finkelstein et al., Ann Rev Plant Biol., 59: 387-415, 2008 Li and Foley, Trends Plant Sci., 2: 384-389, 1997 Suh and Kim, Korean J Crop Sci., 39, 187-192, 1994 Park and Kim, Korean J Crop Sci., 54: 241-248, 2009 Gubler et al., Curr Op in Plant Biol., 8: 183-187, 2005 Kermode, J Plant Growth Regulation., 24: 319-344, 2005 Roberts, J Exp Botany, 13: 75-94, 1962 Ikehashi, Jap J Breeding, 22: 209-216, 1972

It is an object of the present invention to provide a recombinant vector which promotes the resistance of a plant to the avalanche compared to wild type, including OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene derived from rice ( Oryza sativa ) And to provide a transformant transformed with said vector.

It is another object of the present invention to provide a method for enhancing the resistance of a plant to avalanche compared to a wild type comprising overexpressing the OsPHS1 gene by transforming the recombinant vector into a plant cell, A method for producing the same, a transgenic plant improved in resistance to athlete's foot produced by the method, and seeds thereof.

It is still another object of the present invention to provide a composition for enhancing aberration resistance of a plant containing a recombinant vector comprising OsPHS1 gene derived from rice.

As one embodiment for achieving the above object, the present invention provides a recombinant vector for transfection comprising a gene represented by SEQ. ID.

The term "pre-harvest sprouting (PHS)" in the present invention refers to a phenomenon in which the seeds reaching the physiological maturity germinate with the seeds before harvest in wet conditions caused by rainfall, It is known to be associated with seed dormancy.

The term "seed dormancy" in the present invention refers to a phenomenon in which germination does not occur for a certain period of time even if germination conditions suitable for mature seeds are given. If the seed is not dormant or very weak, Early immature seeds will cause germination with germination on the ears.

In the present invention, "OsPHS1 ( Oryza sativa pre-harvest sprouting 1)" is a gene derived from rice ( Oryza sativa ). OsO3g20770 gene whose function is unknown at chromosome 3 to be.

In one embodiment of the present invention, the rice-derived OsPHS1 gene selected as the causative agent gene of the present invention has been confirmed to be seed-specific and the OsPHS1 gene function unknown to date is closely related to the seed (Figs. 4 and 5). In addition, it was confirmed that the overexpression of the OsPHS1 gene promoted the avalanche resistance of rice seeds (Fig. 9). It was confirmed that the OsPHS1 overexpressed Arabidopsis transformants had increased seed dormancy compared to the control wild type Arabidopsis thaliana (Fig. 11).

The term "wild type" in the present invention means a control group for transgenic plants containing the rice-derived OsPHS1 gene of the present invention. Specifically, the plant is rice or Arabidopsis, and rice includes all varieties of rice known in the art. More specifically, the rice plant may be Dongjin rice, but is not limited thereto.

The term "plant" of the present invention is intended to include a plant type, a whole plant, part of a plant, seed or plant cell.

The term "pre-harvest sprouting tolerance" in the present invention means that seedling before harvesting does not germinate on the ears. Even if the ears reaching the physiological maturity are exposed to wet conditions caused by rainfall, And the resistance of the plant to inhibit seed germination for a certain period of time.

The term "recombinant" as used herein refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a polypeptide encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the wild-type form of the cells, either in sense or antisense form. In addition, recombinant cells can express genes found in wild-type cells, but these genes have been modified and reintroduced intracellularly by artificial means.

In the present invention, the OsPHS1 gene sequence can be inserted into a recombinant vector. The term "vector" of the present invention is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The recombinant vector means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell viral vector, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors containing the respective sequences of the OsPHS1 gene and appropriate transcription / translation control signals can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host, such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

In the recombinant expression vector of the present invention, the promoter may be, but is not limited to, the promoters suitable for transformation, preferably the CaMV 35S promoter, the actin promoter, the ubiquitin promoter, the pEMU promoter, the MAS promoter, the histone promoter or the Clp promoter . The term "promoter" of the present invention means a region above the DNA from the structural gene, and refers to a DNA molecule to which 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. In the present invention, the use of a constant promoter may be desirable. Therefore, the constant promoter does not limit the selectivity.

In the recombinant vector of the present invention, a conventional terminator can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, Agrobacterium tumefaciens Octopine gene A phaseoline terminator of Escherichia coli, and an rrnB1 / B2 terminator of Escherichia coli, but the present invention is not limited thereto. Regarding the need for a terminator, it is generally known that the terminator region increases the certainty and efficiency of gene transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The recombinant vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, hygromycin, chloramphenicol, G418, Bleomycin, Resistant gene, aadA gene, and the like, but are not limited thereto.

In another embodiment, the present invention provides a transgenic plant having enhanced avalanche resistance transformed with a recombinant vector containing OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene of the present invention.

The term "transformation" in the present invention means genetically changing the traits of an individual or a cell by DNA, which is a genetic material given from the outside. In the present invention, the rice ( Oryza sativa ( Oryza sativa pre-harvest sprouting 1) gene derived from OsPHS1.

Transformation with the recombinant expression vector of the present invention in the present invention can be carried out by a transformation technique known to a person skilled in the art. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, Agrobacterium sp. mediated method, and the like, but the present invention is not limited thereto.

In the present invention, the term "transformant" means a host cell transformed with a recombinant expression vector containing OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene derived from rice, which promotes the resistance to sprains of the present invention.

The transformants, and microorganisms are preferred, but are not limited to, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens , and more preferably Agrobacterium tumefaciens . The term " Agrobacterium tumefaciens "

In another embodiment, the present invention relates to a method for producing a plant cell, which comprises transforming a plant cell with a recombinant vector comprising OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene derived from rice ( Oryza sativa ) Wherein the method comprises the steps of:

The method of transforming the plant cell is as described above.

In the method according to an embodiment of the present invention, the OsPHS1 gene may be a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In another embodiment, the present invention relates to a method for producing a recombinant vector comprising the step of transfecting a plant cell with a recombinant vector comprising the OsPHS1 gene derived from rice, which is represented by SEQ ID NO: 1, to overexpress the OsPHS1 gene, A method for producing a transgenic plant is provided.

The method of transforming the plant cell is as described above. Further, the method of the present invention includes a step of regenerating the transformed plant from the transformed plant cell. Regeneration of transgenic plants can be carried out by any method known in the art. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

In the method according to an embodiment of the present invention, the OsPHS1 gene may be a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In another aspect, the present invention provides transgenic plants and seeds thereof produced by the above method with enhanced avalanche resistance.

The plant may be a monocot plant or plant ssangjayeop, preferably rice (Oryza sativa) plants or Arabidopsis (Arabidopsis thaliana , but are not limited thereto.

In yet another embodiment, the present invention provides a composition for promoting aberration resistance of a plant containing a recombinant vector comprising a rice-derived OsPHS1 gene comprising the nucleotide sequence of SEQ ID NO: 1. The above composition is capable of enhancing the aphrodisiac resistance of a plant by transforming a plant with a recombinant vector comprising the OsPHS1 gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient.

According to the present invention, the plants transformed with OsPHS1 gene derived from rice are more resistant to the aphrodisiac resistance than those of wild-type plants. Therefore, the use of the OsPHS1 gene can be used to develop a plant that can grow strongly even in a poor environment, And it can be useful for constructing environmental stress tolerance crop development platform.

Brief Description of the Drawings [ Fig. 1] Fig. 1 is a diagram showing the results of the water- saline examination (A) and the seed germination rate measurement (B) of the male and female mutants phs1 .
FIG. 2 is a schematic diagram showing the position of the Ds transfer agent inserted into the male and female mutant phs1 . FIG.
FIG. 3 is a graph showing the expression level of OsPHS1, a gene related to the resistance to athlete 's foot, in the seed of the male and female mutant phs1 .
FIG. 4 is a diagram showing the results of analysis of the expression pattern of OsPHS1 gene, which is a gene related to the sporadic resistance, through the search of a public database (Public DB).
FIG. 5 is a photograph showing the expression pattern of GUS according to seed development in the transgenic rice seeds into which the OsPHS1 promoter-GUS vector of the present invention was introduced. Here, An is an anther, St is a stigma, Ov is an ovule, Ovt is an ovule track, Em is an embryo, En is an endosperm, (aleurone layer).
FIG. 6 is a micrograph showing fluorescence distribution of the OsPHS1-GFP protein transiently expressed in protoplasts of rice plants into which the OsPHS1-GFP fusion protein of the present invention was introduced.
FIG. 7 is a diagram showing a diagram of the OsPHS1 gene transfection vector for the production of transgenic plants in which the resistance to aster leaf resistance is improved according to the present invention. FIG.
FIG. 8 is a graph showing the results of analysis of the OsPHS1 gene expression level of the overexpressed rice transgenic overexpressed OsPHS1 gene of the present invention using the RT-PCR method. At this time, WT is a wild-type Dongjinbara of the original variety, and 35S: PHS1 / phs1 Transformant (3, 8-2, 10-1, 24-4, 30) is rice in which OsPHS1 gene is introduced and the gene is overexpressed .
FIG. 9 is a graph showing the results of measuring the immature seed germination rate of the overexpressed rice transgenic plants transfected with the OsPHS1 gene of the present invention.
FIG. 10 is a graph showing the results of analysis of the OsPHS1 gene expression level of the overexpressed Arabidopsis thaliana transformed with the OsPHS1 gene of the present invention using the RT-PCR method. In this case, Col-0 is a wild type Arabidopsis thaliana, and 35SP: OsPHS1 (1, 3, 6, 9, 11, 13) is a Arabidopsis transformed with OsPHS1 gene to overexpress the gene.
FIG. 11 is a graph showing the seed germination rate (A) and the seed germination inhibition rate (B) of the Arabidopsis thaliana transgenic overexpressed OsPHS1 gene of the present invention. In this case, Col-0 (Lane 1) is a wild type Arabidopsis thaliana, and 35SP: OsPHS1 (Lane 2, 3, 4, 5) is a Arabidopsis transformed with OsPHS1 gene to overexpress the gene.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: Ds Metastatic factor  Insert mutant population analysis and selection of OsPHS1 resistance gene

Pre-harvest sprouting (PHS) refers to a phenomenon in which Isaac reaching the physiological maturity is germinated due to rainfall and germinated in the ear before harvest. The phenomenon of weeds that occur in harvest crops not only leads to a decrease in the yield of grain but also degrades the quality and causes serious economic losses.

In order to identify genes that regulate the resistance of rice seeds to rice plants, mutant strains with weak susceptibility were selected through analysis of mutant strains in which Ds transferase was inserted into Dongjin rice. Based on the mutant strains, Respectively.

Example  1-1: Ds Metastatic factor  Insert mutant population analysis and Shovel  Mutant ( phs1 ) Selection

In order to select genes for enhancing the resistance of the rice seeds to the aquatic resistance, 52 mutant strains, ie phs1 hydrops mutants, were observed in the packaging of about 15,000 mutant strains in which a Ds transfer factor was inserted in Dongjin - (A in FIG. 1). After 5 days of each of the 52 mutants selected, 10, 25, and 40 days old spikes were collected and cultured at 100% relative humidity and 30 ° C for 7 days And the seed germination ratio was measured (Fig. 1B). At this time, wild type Dongjin bean was used as the control group.

As a result, as shown in Fig. 1, The phs1 mutant exhibited a mite-like phenomenon, showing that the resistance to aphid resistance was weaker than that of the control group (Fig. 1 A). The seed germination rate of the control group, Dong Jinbyeon , was higher at 25 DAH (day after heading) , Whereas the phs1 mutant showed more than 40% seed germination at 25 DAH (day after heading).

Example  1-2: Avalanche resistance gene OsPHS1  Selection

Genomic DNA of the mutant phs1 mutant was isolated and the restriction enzyme Rsa I was used to isolate the genomic information of the insertion site of the Ds transferase in the mutant somatotype phs1 selected in Example 1-1. . Then, FST (flanking sequence tag) gene analysis was performed through the adapter-PCR technique.

As a result of FST analysis of the mutant phs1 mutant, that is, the chromosomal base sequence around the site where the Ds transfer factor was inserted, Similarly, Os03g20770, whose function is unknown It was confirmed that a Ds transfer element was inserted into the second exon of the gene. The gene was finally selected as a candidate gene for resistance to a spore resistance for development of an avalanche-resistant crop and named OsPHS1 ( Oryza sativa pre-harvest sprouting 1) Respectively. The corresponding sequence is shown in SEQ ID NO: 1.

Example  2: OsPHS1  Gene expression analysis

It was confirmed that the Ds transferase was inserted into the second exon of the OsO3g20770 gene, OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene in the mutant somatotype phs1 selected in Example 1-2, RT-PCR analysis confirmed that the OsPHS1 gene was not expressed in the seed of the mature phs1 mutant.

Specifically, the RNA was isolated from each of the seeds of the control group, Dong Jinbyeong and the male phs1 mutant, and cDNA was synthesized. The OsPHS1 gene specific primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '(SEQ ID NO: 2) 618R: 5'-CTAGGCAGAGGAGCTCGATGTTG-3 '(SEQ ID NO: 3)).

As a result, as shown in Fig. 3, unlike the control group, Dongjinbio, the seeds of the subgenus phs1 mutant showed OsO3g20770 It was confirmed that the full-length transcriptome of the gene was not formed. In other words, it was confirmed that the OsPHS1 gene was not expressed in the seeds of the subgenus phs1 mutant.

Based on these results, it was confirmed that OsPHS1 gene derived from rice selected in the Ds rice insertion mutant strain was a causative gene, and thus it was found that the gene can be usefully used for the development of a susceptible crop.

Example  3: OsPHS1  Expression patterns of genes in rice

Through the above Example 2, OsPHS1 gene ( Os03g20770 Genes) were found to be the causative genes, and the expression patterns of the OsPHS1 gene in rice tissues were analyzed by using a public database (Rice Oligonucleotide Array Database) for the development of rice crops and their seeds with resistance to athlete I analyzed it.

As a result of analyzing the expression pattern of the OsPHS1 gene in the rice tissue through public database search, it was found that the OsPHS1 gene was specifically expressed in the embryo of the rice seed, as shown in Fig.

Example  4: OsPHS1  Analysis of GUS expression pattern using gene promoter

As a result of analysis of the expression pattern of OsPHS1 gene ( Os03g20770 gene) in rice tissue using the Rice Oligonucleotide Array Database in Example 3, it was confirmed that it was specifically expressed in the embryo of rice seeds. Thus, expression of OsPHS1 gene To investigate the morphology of GUS, we analyzed the expression pattern of GUS using OsPHS1 gene promoter.

Specifically, a vector (OsPHS1 promoter-GUS vector) in which the 2 kb region upstream of the initiation codon of the OsPHS1 gene was isolated and ligated to a reporter gene GUS was prepared, and the seeds of the transgenic rice into which the transgenic rice was introduced were analyzed by GUS protein staining Respectively. At this time, the site where the promoter of the OsPHS1 gene was activated by GUS staining was stained blue by GUS staining.

As a result of analysis of the expression pattern of GUS using the promoter of OsPHS1 gene, as shown in Fig. 5, as the development of transgenic rice seeds into which OsPHS1 promoter-GUS vector was introduced progressed, The expression of the OsPHS1 gene in the embryo of the seed was increased as well as the results obtained through the analysis.

These results suggest that the OsPHS1 gene selected as a causative gene is actually seed - specific and that the function of the OsPHS1 gene, which is unknown until now, is closely related to the seed.

Example  5: OsPHS1  Encoded by a gene OsPHS1  Analysis of Protein Expression Patterns

It is determined that the protein encoded by the OsPHS1 gene selected as the causative agent gene has a very low homology with the known protein so far and that the expression pattern of the OsPHS1 protein encoded by the OsPHS1 gene .

Specifically, in order to confirm the expression pattern of the OsPHS1 protein, a OsPHS1-GFP fusion protein in which a GFP protein was linked to the C terminus of the OsPHS1 protein was prepared. When transient expression was performed in a protoplast of rice, As shown, it was found to be located in the membrane of the ER (endoplasmic reticulum) or golgi body.

Example  6: for transgenic plants OsPHS1  Over-expression vector and transformant production

In order to prepare a transgenic plant having enhanced avalanche resistance, OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene overexpression vector finally selected as the aberration resistance-related gene in Example 1 was prepared, and the vector was inserted into rice Transgenic rice plants were constructed.

Example  6-1: For plant transformation OsPHS1  Overexpression vector production

To construct an overexpression vector of OsPHS1 gene derived from rice, cDNA was synthesized using RNA isolated from a seed of Dongjinbara as a template and OsPHS1 gene specific primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 ' OsPHS1-618R: 5'-CTAGGCAGAGGAGCTCGATGTTG-3 '(SEQ ID NO: 3)) was used to isolate the start and end of the OsPHS1 gene, or the ORF (open reading frame). As shown in FIG. 7, the OsPHS1 gene was inserted into the pCAMBIA1300 sequence vector, which is a rice transformation vector, to construct a pCAMBIA1300-OsPHS1 overexpression vector.

Specifically, the OsPHS1 gene was linked to the Xba I and BamH I restriction sites downstream of the CaMV35S promoter (35SP), and OsPHS1 overexpression vectors were constructed so that the OsPHS1 gene was constantly expressed in plants. Hygromycin ) Resistance genes were used.

Example  6-2: Construction and analysis of rice plant transformants using Agrobacterium

Among the mutant strains in which the Ds transfer factor was inserted into Dong Jin-jin in Example 1-1, a mutant phs1 with weak resistance to athlete was selected. Based on this, caregiver Shing mutants by inserting a pCAMBIA1300-OsPHS1 vector produced in Example 6-1 to prepare a phs1 the rice transformants so that OsPHS1 gene is overexpressed, and wherein the vector is properly inserted is the gene OsPHS1 RT-PCR was performed to determine whether the transgenic rice plants were overexpressed.

Specifically, phs1 The callus derived from the seed of the mutant was inoculated with Agrobacterium containing the pCAMBIA1300-OsPHS1 vector and cultured in a medium containing hygromycin at a concentration of 30 μg / Ruth was selected and stem and root were induced to produce transgenic rice.

Thereafter, RNAs were isolated from wild-type (WT) Dongjinbye and wild-type transgenic rice leaves prepared to overexpress the OsPHS1 gene, and cDNA was prepared using the RNA as a template. The OsPHS1 gene-specific RT-PCR was performed using a primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '(SEQ ID NO: 2); OsPHS1-618R: 5'-CTAGGCAGAGGAGCTCGATGTTG-3' (SEQ ID NO: 3)).

As shown in FIG. 8, when OsPHS1 gene transgenic rice transgenic plants were wild-type (wild-type, wild-type, wild-type, WT), the OsPHS1 gene, which is a gene related to the spore resistance, was strongly expressed.

Example  7: OsPHS1  Transformation Rice plant  Seedling resistance analysis

The OsPHS1 gene, which is a gene related to the sprains resistance, prepared in Example 6-2, Overgrown rice transgenic plants were cultivated in pavement, and ears of 25 and 40 days after emergence were collected. The seeds were separated from the ears, placed in a petri dish, cultured for 5 days at 100% relative humidity, temperature of 30 ° C and dark, and then seed germination was measured. At this time, the control group used a mutant phs1 having weak resistance to aphthae .

As a result, as shown in Fig. 9, seeds hardly germinated at the time of 25 h DAH (morning after heading) in the case of OsPHS1 overexpressed transgenic rice (35 SP-PHS / phs1) In the case of mutant (phs1), seed germination rate was about 40% even at 25 DAH (day after heading). In addition, the seedling germination rate was more than 2 times higher than that of OsPHS1 overexpressing transgenic rice (35SP-PHS / phs1) at 40 DAH (day after heading)

These results suggest that overexpression of the OsPHS1 gene can enhance the resistance of the rice seeds to weedy.

Example  8: OsPHS1  Genetically overexpressed Arabidopsis production and seeds Dormancy  analysis

In Example 6 and Example 7, OsPHS1 gene overexpression The seeds obtained from the above-prepared rice plants were measured for germination ratio, and OsPHS1 gene overexpression Rice transgenic plants were found to be more resistant to seedling dormancy.

Based on these results, we investigated whether the OsPHS1 gene overexpressed Arabidopsis thaliana was overexpressed and whether it could enhance seed dormancy. The germination rate and ABA responsiveness of the seeds were analyzed.

Example  8-1: OsPHS1  Production and Analysis of Overexpressed Arabidopsis Pole

In order to confirm whether or not the dormancy of the OsPHS1 gene overexpressing strain could be enhanced, the OsPHS1 gene overexpressing Arabidopsis transformants was firstly prepared, and whether the OsPHS1 gene was overexpressed in the Arabidopsis transformants was analyzed by RT-PCR And analyzed.

Specifically, the Agrobacterium (Agrobacterium) containing the gene inserts OsPHS1 pCAMBIA1300-OsPHS1 overexpression vector prepared in the above Examples 6-1 Arabidopsis (Arabidopsis thaliana were transformed by floral dipping method to produce an Arabidopsis transformant overexpressing the OsPHS1 gene. After the preparation, RT-PCR was performed to analyze whether the vector was properly inserted and the OsPHS1 gene was overexpressed in the Arabidopsis transformants. As a control, wild type Arabidopsis thaliana (Col-0) without OsPHS1 gene was used. RT-PCR was performed using OsPHS1 gene specific primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '; OsPHS1-618R: 5'-CTAGGCAGAGGAGCTCGATGTTG-3 '(SEQ ID NO: 3)).

As a result of analysis of the expression level of the OsPHS1 gene in the Arabidopsis thaliana transformant prepared by RT-PCR to overexpress the OsPHS1 gene, it was found that the OsPHS1 gene inserted into the Arabidopsis thaliana transformant -0), it was confirmed that the OsPHS1 gene, which is a gene related to the spore resistance, is strongly expressed.

Example  8-2: OsPHS1  Analysis of seed germination rate and ABA reactivity in overexpressed Arabidopsis thaliana

The seed germination rate and seed dormancy were obtained using the seeds obtained from the overexpressed Arabidopsis thaliana OsPHS1 gene prepared in Example 8-1 in order to confirm whether or not the dormancy of the dysplasia plant overexpressing the OsPHS1 gene could be enhanced ABA (abscisic acid), which is known to be an important regulator involved in the maintenance process, was analyzed by seed germination test. As a control, seeds of wild-type Arabidopsis (Col-0) without OsPHS1 gene were used.

As a result, as shown in Fig. 11, the seed germination rate was significantly inhibited in OsPHS1 transgenic Arabidopsis thaliana as compared with the control group (Fig. 11A), and the germination inhibitory effect by ABA treatment was also inhibited by OsPHS1 Overexpressing transgenic Arabidopsis thaliana (Fig. 11B).

In other words, the results showed that the seed dormancy was increased in the OsPHS1 overexpressing Arabidopsis thaliana transformant (35SP: OsPHS1) compared to the control wild type Arabidopsis thaliana (Col-0), and furthermore, the OsPHS1 gene Overexpression could be used to enhance seed dormancy of dicotyledonous plants.

TL: T-DNA left border
35ST: cauliflower mosaic virus (CaMV) 35S terminator
35SP: cauliflower mosaic virus (CaMV) 35S promotor
NOS: nopaline synthase
PHS1 CDS: Oryza sativa pre-harvest sprouting 1 gene coding sequence
TR: T-DNA right border

<110> Republic of Korea <120> Gene enhancing pre-harvest sprouting tolerance derived from Oryza          sativa and uses thereof <130> P16R12D1540 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1 gene <400> 1 atggcggcaa gccaatacat ggacagtgtg gctgccggcc tccgctcctc cttaatccct 60 ctccaccctg gtgtcggcgt caagctccct ttccgccttc ctctccggtg ggacgtgtgg 120 gtcgaggaaa ttgagaaggc ggcggttgat ggtgctagag cggcaggtgg ggggaggttg 180 ggggagcggt gttctggtgg agggcaaggc attgggggct tgagctccag tgtcagccag 240 ctccagcgga ggcccacgcg catggagatg ggtggatttg gcggcatagt cgctggagga 300 gcgagcatgg ttggggagag caaatctggc agtggggcga ggcgctgggg gctcgagctc 360 cggcccgttg tcgatgggga tgcaatggcg ctcaccaggg caaaacctcc ttgggtagcc 420 atgttccctt tctccatcgc taccgacagg tacatgcgac cgctgaggtt gcggtcgacg 480 ttgtacatgg catccttcca cttgatgcaa caacgcgggg tggatgatca ggacggagct 540 cttgttgtcg tagctcctgc ggcaggcctt gagagagggc gagcgacgcg gcggccaaca 600 tcgagctcct ctgcctag 618 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1-1F <400> 2 atggcggcaa gccaatacat gg 22 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsPHS1-618R <400> 3 ctaggcagag gagctcgatg ttg 23

Claims (9)

1. A recombinant vector for transformation comprising a gene represented by SEQ. ID.
A transgenic plant having enhanced avalanche resistance transformed with the recombinant vector for transformation of claim 1.
1. A method for enhancing the resistance to aberration of a plant, comprising the step of transfecting a plant cell with a recombinant vector comprising the gene represented by SEQ ID NO: 1 to overexpress the gene represented by SEQ ID NO: 1.
1. A method for producing a transgenic plant having enhanced avalanche resistance, comprising the step of transfecting a plant cell with a recombinant vector comprising the gene of SEQ ID NO: 1 to overexpress the gene of SEQ ID NO: 1.
A transgenic plant having enhanced susceptibility to aphid resistance produced by the method of claim 4.
6. The transgenic plant according to claim 5, wherein the plant is a terminal leaf or a twin leaf plant.
7. The transgenic plant according to claim 6, wherein the monocotyledonous plant is rice ( Oryza sativa ) and the dicotyledonous plant is Arabidopsis thaliana .
A transformed seed of a plant according to claim 5.
1. A composition for promoting aberration resistance of a plant containing a recombinant vector comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 1.

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