KR100780485B1 - Rice osagps1 gene or osagps2a gene and plant transformed with the same - Google Patents

Abstract

A gene coding two small subunits of ADP-glucose pyrophosphorylase(OsAGP)S1 and OsAGPS2a controlling the starch synthesis of Oryza sativa L. is provided which is specifically located at a plastid and specifically expressed in an endosperm and a leaf tissue. A gene coding a small subunit of OsAGP derived from Oryza sativa L. is characterized in that a coding protein of each gene is specifically located at a plastid in an Oryza sativa L. cell and includes a sequence of SEQ ID : NO. 1 specifically expressed in each an endosperm and a leaf tissue. A method for preparing a transgenic Oryza sativa L. comprises the steps of: (a) transforming a Oryza sativa L. cell using a recombinant plant expression vector including the gene; and (b) redifferentiating the transgenic Oryza sativa L. plant cell into the transgenic Oryza sativa L..

Description

Rice Osgps1 gene or OssgPS2a gene and plant transformed with the same gene {Rice OsAGPS1 gene or OsAGPS2a gene and plant transformed with the same}

1 shows a vector map of the OsAGP :: GFP fusion protein for determining the intracellular location of AGP isoforms in rice and corn protoplasts.

A: Schematic diagram of OsAGP :: GFP fusion constructs used in the present invention;

B, C: expression signal of OsAGPS1 :: GFP fusion construct in rice calli;

D, E: position of expression of OsAGP2a :: GFP fusion construct in rice leaves;

F, G: expression signal of OsAGP2b :: GFP fusion construct in rice roots;

H, I: expression signal of OsAGPL1 :: GFP fusion construct in rice leaves;

J, K: expression signal of OsAGPL2 :: GFP fusion construct in rice leaves;

L, M: expression patterns of OsAGPL3 :: GFP fusion constructs in maize protoplasts;

N, O: Expression patterns of OsAGPL3 :: GFP fusion constructs in maize protoplasts.

Figure 2 shows a model of spatiotemporal complex formation between OsAGP small subunit and large subunit isoforms present in the endosperm of rice and leaves.

A: OsAGPS2a (S2a) / OsAGPL3 (L3) heterotetramer complex formed from chloroplasts in the leaves of rice;

B: OsAGPS1 (S1) / OsAGPL1 (L1) and early stages of endosperm production of rice and

OsAGPS2b (2b) / OsAGPL2 (L2) complex;

C: OsAGPS1 / OsAGPL1 and OsAGPS2b / OsAGPL2 complexes in the middle stage of endosperm production of rice.

In the present invention, the coding protein of each gene is specifically located in the chromosomes in rice cells, and each has an OsAGPS1 gene or a nucleotide sequence of SEQ ID NO: 2 having a nucleotide sequence of SEQ ID NO: 1 specifically expressed in endosperm and leaf tissue. A gene encoding a small subunit of rice-derived ADP-glucose pyrophosphorylase (OsAGP) consisting of an OsAGPS2a gene, a recombinant plant expression vector containing the gene, and a transformed vector with the expression vector. A method for determining the intracellular location of a plant, the seed of the plant, the gene product, the method of producing the transgenic plant, and the starch isolated from the transgenic plant produced by the method.

Starch, a polysaccharide, is a polymer made from chemically homogeneous basic components, ie, glucose molecules, and constitutes various types of molecular complexes having different degrees of polymerization and branching of glucose chains. Starch is the end product of photosynthesis in the plant's production organs, and in the storage organs it is the main storage form. Therefore, it is particularly important to fully understand the procedures involved in starch synthesis, to provide plants that can synthesize starch that can be modified for a variety of uses, increasing productivity in grains such as rice, corn and wheat. The possibility of providing these plants with different breeding methods lies in the specific genetic modification of starch metabolism of starch-producing plants by recombinant DNA technology. However, the necessary condition is not only to isolate each DNA molecule encoded by the enzyme, but also to identify the gene.

The synthesis of starch in plants is accomplished by the major enzyme, adenosine 5 'diphosphate glucose (ADP-Glc) pyrophosphorylase (AGP). AGP catalyzes the production of ADP-Glc and pyrophosphate (PPi) using glucose-1-phosphate (Glc-1-P) and adenosine 5 'triphosphate (ATP). The resulting ADP-Glc acts as a glucosyl donor activated during α-1,4-glucan synthesis.

The bacterial AGP consists of a homotetramer structure consisting of four identical subunits, while higher plants have a heterotetramer consisting of two large subunits (LSUs) and two small subunits (SSUs). ) To form a structure. Small subunits (SSUs) are catalytic subunits and the function of large subunits (LSUs) is not well understood but is known to be involved in both catalysis and regulation.

AGP activity is mainly present in the cytoplasm, not in the chromosome of grain endosperm, and in the chromosome in other tissues of the grain and other plant tissues. Several studies have shown that most of the AGP activity is mainly in the cytoplasm of grain endosperm and rarely in the white body of endosperm. In the developing embryo of maize, AGP activity was measured after separation by organelles, indicating that only 5% of AGP activity was present in the chromosomes and most of the cytoplasm was present (Denyer et al., (1996) Plant Physiol 112: 779-). 785). In barley endosperm, only 15% of the activity was present in the plastids (Thorbjrnsen et al. (1996a) Plant J 10: 243-2).

The rice AGP gene population consists of two small subunit (SSU) genes, OsAGPS1 and OsAGPS2, four large subunit (LSU) genes, OsAGPL1, OsAGPL2, OsAGPL3, OsAGPL4 (Akihiro et al., (2005) Plant Cell Physiol 46: 937- 94; Ohdan et al., (2005) J Exp Bot 56: 3229-3244; Hirose et al., (2006) Physiol Plant 128: 425-435. OsAGPS2 produces two transcripts, which are named OsAGPS2a and OsAGPS2b, respectively (Ohdan et al. (2005) J Exp Bot 56: 3229-3244). As in rice, barley and wheat are known to produce two transcripts for AGPS2 (Luo et al. (1997) Z Naturforsch [C] 52: 807-811).

The present inventors have isolated the genes for the two small subunit proteins OsAGPS1 and OsAGPS2a, which are AGP proteins, enzymes that regulate starch synthesis in rice, respectively, and the protein is a rice cell through the GFP fusion construct. It has been found to be specifically located in the plastids within and to express specifically in endosperm and leaf tissue.

Accordingly, an object of the present invention is a small subunit of rice-derived ADP-glucose pyrophosphorylase (OsAGP), which is specifically located in the plastids in rice cells and is expressed specifically in embryonic and leaf tissues. Gene is provided.

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

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

It is also an object of the present invention to provide a method for determining the intracellular location of the gene product using the recombinant plant expression vector.

It is also an object of the present invention to provide a method for producing a transformed plant using the recombinant plant expression vector.

It is also an object of the present invention to provide starch isolated from the transgenic plant produced by the method.

 In order to achieve the above object, the present invention provides an OsAGPS1 gene or SEQ ID NO, which has a nucleotide sequence of SEQ ID NO: 1, wherein the coding protein of each gene is specifically located on a plastid in a rice cell, and is specifically expressed in endosperm and leaf tissue. Provided is a gene encoding a small subunit of rice-derived ADP-glucose pyrophosphorylase (OsAGP) consisting of an OsAGPS2a gene having a nucleotide sequence of 2.

The protein encoded by the gene of the present invention is specifically located in the plastids in rice cells, OsAGPS1 is specifically expressed in the embryonic tissue, OsAGPS2a is characterized in that it is specifically expressed in the leaf tissue. In the genetic notation of the present invention, Os represents rice (Oryza sativa L.), AGP represents ADP-glucose pyrophosphorylase, and S represents small subunits.

Through the BLAST search of the rice genome (IRGSP, 2005), two small subunits (SSU) (OsAGPS1 and OsAGPS2) and four large subunits (LSU) (OsAGPL1, OsAGPL2, OsAGPL3, and OsAGPL4), a total of six AGPs Genes were isolated (see Table 1). In this study, AGP genes were named according to the nomenclature of [Ohdan et al. (2005) J Exp Bot 56: 3229-324] and [Hirose et al. (2006) Physiol Plant 128: 425-43].

In rice endosperm, AGP activity is known to be mainly in the cytoplasm. It is also known that starch synthesis in leaves is regulated by the AGP activity of the plastids. To determine which OsAGP subunits are important for starch synthesis in leaves and seeds, we first track the intracellular locations of all OsAGP subunits.

OsAGPS2a and OsAGPS2b differ only in the protein sequence corresponding to the first exon and all the exons up to the tenth are identical. Analysis using the Target P program showed that OsAGPS2a had a transit peptide site at the N-terminus while OsAGPS2b did not (Emanuelsson et al., (2000) J Mol Biol 300: 1005-101, http: // www. cbs.dtu.dk/services/TargetP). OsAGPS2a is similar to the transgenic peptide region of OsAGPS1 that has already been identified (Sikka et al., (2001) Plant Sci 161: 461-46; data not shown). This finding suggests that by using different exons 1 the OsAGPS2 gene expresses two proteins, which makes it possible to have different cytoplasmic positions. It is believed that the N-terminal region of OsAGPL1, OsAGPL3, OsAGPL4 among the large subunit (LSU) isoforms has a transgenic peptide. OsAGPL2 does not appear to have a transit peptide and is therefore thought to be present in the cytoplasm.

In order to achieve another object of the present invention, the present invention provides a recombinant plant expression vector comprising the gene according to the present invention.

In the present invention, to determine the intracellular location of the AGP active gene isotype, an OsAGP :: GFP fusion vector was constructed (see FIG. 1). Genomic DNA or cDNA containing the predicted signal sequence was used in the experiment. These vectors were transformed into rice or to protoplasts of maize. About 10 different transformants for each vector were obtained and tested.

As a result of the present invention, OsAGPS1 :: GFP fusion protein was found to be present in the white body of the transgenic calli and the chloroplast of the leaf (see FIGS. OsAGPS2a :: GFP fusion protein was found to be present in the chloroplasts of leaves (see FIGS. 1D, E). In contrast, the OsAGPS2b :: GFP fusion protein was found to be present in the cytoplasm in rice roots (see Figure 1F, G). Of the OsAGPL :: GFP fusion proteins, only OsAGPL2 :: GFP fusion proteins were found to be present in the cytoplasm of leaves (see FIG. 1J, K). The remaining OsAGPL1, OsAGPL3, OsAGPL4 and GFP fusion proteins were found to be present in the leaf chloroplasts through rice leaves (see FIGS. 1H, I) and maize protoplasts (see FIGS. 1L-O).

In the present invention, the pJJ461 vector, which is one of plant expression vectors, was used. Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. 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.

The expression vector will preferably comprise one or more selectable markers. The marker includes dihydrofolate reductase or neomycin resistance to eukaryotic cell culture. The most widely used plant transformation marker is the neomycin phosphotransferase II (nptII) gene isolated from Tn5 and another marker gene is the hygromycin phosphotransferase gene that confers resistance to the antibiotic hygromycin. to be. In the present invention, the hygromycin phosphotransferase gene was used.

In the plant expression vector 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 present invention, the CaMV 35S promoter was used.

Terminators may be, but are not limited to, nopaline synthase (NOS) or rice α-amylase RAmy1 A terminator. 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 present invention, a NOS terminator was used.

In order to achieve another object of the present invention, the present invention provides a plant transformed with the recombinant vector according to the present invention. The plant can be a dicotyledonous plant as well as a monocotyledonous plant. These are preferably useful plants, in particular starch-storage plants or starch-synthetic plants such as cereals (wheat, barley, oats, rye, etc.), rice, corn, soybeans or potatoes. Most preferably the plant is rice.

The present invention may also include the propagation material of the plant of the present invention, such as seeds, seedlings, and grasps.

Recombinant constructs can be introduced into cultured host cells using well known techniques such as infection, transduction, transfection, transfection, electroporation, and transformation. Representative examples of hosts include bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium cells; Fungal cells such as yeast cells; Insect cells, such as Drosophila S2 and Spodhodera Sf9 cells; Animal cells such as CHO, COS and Bowes melanoma cells; And plant cells. Suitable culture media and conditions for host cells are known in the art. In the present invention, rice was transformed using Agrobacterium tumerfaciens mediated transformation using a recombinant vector containing an OsAGP gene.

In order to achieve another object of the present invention, the present invention provides a seed of the transgenic plant according to the present invention. Preferably, the plant is rice, but is not limited thereto.

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; And

It provides a method for producing a transformed plant comprising the step of regenerating the transformed plant from the transformed plant cells.

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.

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 "plant cells" used for plant transformation may be any plant cells. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

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.

In a method according to one embodiment of the invention, the plant cells are derived from rice, but not limited thereto.

In order to achieve another object of the present invention, the present invention provides a method for determining the intracellular location of OsAGPS1 or OsAGPS2a protein comprising the step of transforming plant cells with the vector according to the present invention. When the OsAGPS1 or OsAGPS2a gene of the present invention is operably linked to a detectable marker such as a green fluorescent protein, and transformed into a plant cell, preferably a rice cell, and its expression pattern is observed, the OsAGPS1 or OsAGPS2a protein The location of the cell can be known. As a result, it was found that the protein was specifically distributed in the pigment body in the rice cells. In a similar manner, the intracellular location of mRNA, the product of expression of the OsAGPS1 or OsAGPS2a gene, can also be determined.

In order to achieve another object of the present invention, the present invention provides a starch isolated from the transformed plant prepared by the method for producing a transformed plant according to the present invention.

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.

As a plant material, all the rice plants were grown in nature during summer in the rice fields owned by Kyung Hee University.

Example 1 Preparation of OsAGP :: GFP Fusion Protein for Intracellular Location Tracking

To identify the intracellular location of the OsAGP isotype, partial genomic DNA or cDNA containing the N-terminus of the protein was fused with the green fluorescent protein (GFP) to fit the frame.

OsAGPS1 and OsAGPL2 genomic DNA sites are forward primers comprising XbaI restriction enzyme sites present in the 5 'untranslated region (UTR) and 5'-GCTCTAGATGAAGCCTCTGACACTCCACTGTA-3' for OsAGPS1 (SEQ ID NO: 3) and 5 'for OsAGPL2. -GCTCTAGACAGGTTTGTCTGGGACTTTGAGTA-3 '(SEQ ID NO: 4)) Reverse primer containing the SmaI or BamHI restriction enzyme site present at the appropriate exon site believed to contain the inferred signal sequence (5'-TCCCCCGGGTTGCAAATTCAATGATCCTCCCTTC-3' (SEQ ID NO: 6) 5) and 5'-CGGGATCCTATAGCTGAGGAA GCTGGTATCAAC-3 '(SEQ ID NO: 6) for OsAGPL2 were used for PCR.

The 5'-side portion of the cDNA of OsAGPS2a, OsAGPS2b, OsAGPL1, corresponding to the N-terminal side of the protein, is composed of a forward primer containing a KpnI or XbaI restriction enzyme site present in the 5 'untranslated region (UTR) and (for OsAGPS2a). 5'-GCTCTAGAAATGGCGATGGCGGCAGCCATGGG-3 '(SEQ ID NO: 7), 5'-GCTCTAGATGCA ACCATGAATGTATTGGCATC-3' (SEQ ID NO: 8), and 5'-GCTCTAGAAAC GATGCAGTTCAGCAGTG-3 '(SEQ ID NO: 9) for OsAGPS2b) Reverse-action primer (5'-CGGGATCCTGACTTCAACGAACCCTTCATTCTT-3 'for SEQ ID NO: 10) and 5'- for OsAGPL1, including a BamHI or XhoI restriction site present at the appropriate exon site that is thought to contain a signal sequence CGGGATCCTGACTTCAACGAACCCTTCATTCTT-3 '(SEQ ID NO: 11)) was used for PCR.

The amplified fragment was cut with a suitable restriction enzyme and inserted between the promoter and sGFP of the pJJ461 vector made from the double vector pC1300intC (Ouwerkerk et al., (2001) Planta 213: 370-3; GenBank Accession Number AF234296). The resulting vector was transformed into rice by Agrobacterial mediation (Jeon et al., (2000) Plant J 22: 561-5). Transformed rice was selected in medium containing 40 mg / L hygromycin B.

In order to confirm the intracellular positions of OsAGPL3 and OsAGPL4, the N-terminal region was fused with the green fluorescent protein according to the frame. Forward primer (5'-GCTCTAGATAGACGCGTAGT GATGGCGGCGAT-3 '(SEQ ID NO: 12) for OsAGPL3 and 5'-GCTCTAGAATGGCGACTGCTCGTC containing a 5' site thought to contain the inferred signal sequence of OsAGPL3 and OsAGPL4 cDNAs) (SEQ ID NO: 13)) and reverse primers (5'-CGGGA TCCAGATGACTAATCCATCTGCAATTA-3 'for SEQ ID NO: 3) and 5'-CGGGATCCAGATAACTGTACCATCTGGA-3' for OsAGPL4 (SEQ ID NO: 15)) were used for PCR. The amplified DNA was cut with an appropriate restriction enzyme and inserted between the 35S promoter of pJJ1450 made from pBluescript and the green fluorescent protein. The completed OsAGPL3 :: GFP and OsAGPL4 :: GFP vectors were identified by transient expression in maize protoplasts according to the method of Jang and Sheen (1994).

As a result, it was shown that OsAGPS1 :: GFP fusion protein is present in amyloplasm of transgenic calli (FIGS. 1B and C) and chloroplasts of leaves (data not shown). In OsAGPS2a :: GFP transgenic plants, GFP signals were also found in the chloroplasts of leaves (FIGS. 1D and E), whereas in OsAGPS2b :: GFP transgenic plants, GFP fusion proteins were present in the cytoplasm of roots (FIGS. 1F and G). Of the OsAGPL :: GFP fusion proteins tested, only OsAGPL2 :: GFP was found to be exclusively located in the cytoplasm of the leaves (FIGS. 1J and K), indicating that OsAGPL2 is the only large subunit (LSU) isoform present in the cytoplasm. Indicates that The remaining GFP fusion proteins of OsAGPL1, OsAGPL3 and OsAGPL4 were detected in the chloroplasts of leaves (FIGS. 1H and I) and corn protoplasts (FIG. 1L-O), indicating that the OsAGPL isotype is a chromatin targeted protein.

In addition, tissue-specific expression analysis revealed that there are many transcripts of OsAGPS2b and OsAGPL2 in rice seeds. These results suggest that cytoplasmic OsAGP small subunit (SSU) and large subunit (LSU) may play an important role in starch synthesis in seed seeding of rice.

The seven isolated genes are summarized in Table 1 below.

TABLE 1 Seven Gene Types and Features Isolated.

gene Locus  cDNA length Intracellular location Large distribution of mRNA and protein in leaf and growth embryos mRNA protein OsAGPS1 LOC_Os09g12660 AK073146 Plastid Initial stage of drainage Oil OsAGPS2a LOC_Os08g25734 AK071826 Plastid leaf leaf OsAGPS2b LOC_Os08g25734 AK103906 cytoplasm  Late to mid-drain Oil OsAGPL1 LOC_Os05g50380 AK100910 Plastid Initial stage of drainage ND OsAGPL2 LOC_Os01g44220 AK071497 cytoplasm Late to mid-drain Oil OsAGPL3 LOC_Os03g52460 AK069296 Plastid leaf ND OsAGPL4 LOC_Os07g13980 AK121036 Plastid Leaf and endosperm ND

ND: Not confirmed.

According to the present invention, first, the expression of the gene is different depending on the tissue of the rice, and second, the role of the AGP active protein expression position is also different.

Attach sequence list electronic file  

Claims (11)

The coding protein of each gene is specifically located in the chromosomes in the rice cells, and the OsAGPS1 gene having the nucleotide sequence of SEQ ID NO. 1 or the nucleotide sequence of SEQ ID NO. A gene encoding a small subunit of rice-derived ADP-glucose pyrophosphorylase (OsAGP). A recombinant plant expression vector comprising the gene according to claim 1. An Oryza sativa L. plant transformed with the recombinant plant expression vector according to claim 2. delete delete Seed of the rice plant (Oryza sativa L.) according to claim 3. Transforming rice (Oryza sativa L.) plant cells with the vector according to claim 2; And regenerating the transformed rice (Oryza sativa L.) plant from the transformed rice (Oryza sativa L.) plant cell. A method of targeting an OsAGPS1 or OsAGPS2a protein to a specific location in an Oryza sativa L. cell, comprising transforming an Oryza sativa L. plant cell with the vector according to claim 2. delete The method of claim 8, wherein the intracellular location of the protein is a chromosome. Starch isolated from a transformed rice (Oryza sativa L.) plant prepared by the method of claim 7.

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