KR101120406B1 - Novel glutelin RNAi sequence and Method for increasing the accumulation of target protein by using the same - Google Patents

Abstract

The present invention relates to a method for increasing the accumulation of a target protein by inhibiting the expression of a storage protein of a plant, and more particularly, a polynucleotide capable of showing RNA interference (RNAi) in a plant, a recombinant expression vector comprising the same, and the recombination. The present invention relates to a method of inhibiting expression of gluterin using an expression vector and a method of increasing accumulation of a target protein. In a plant transformed with the recombinant vector of the present invention, the expression of gluterin is suppressed, and the accumulation thereof is increased when the gene of the target protein is transformed together. In particular, the polynucleotide of the present invention, a recombinant vector using the same, a method of inhibiting the expression of gluterin using the same or a method of increasing the expression of the target protein is effective to increase the accumulation of the target protein by effectively inhibiting all the group of gluterin genes. Therefore, the method of inhibiting the expression of the polynucleotide of the present invention, a recombinant vector using the same, a storage protein using the same or a method of increasing the accumulation of the target protein may be usefully used for the purpose of increasing the expression of the target protein in a plant during production of the target protein. Can be.

Description

Novel glutelin RNAi sequence and method for increasing the accumulation of target protein by using the same

The present invention relates to a method for increasing the accumulation of a target protein by inhibiting the expression of a storage protein of a plant, and more particularly, a polynucleotide capable of showing RNA interference (RNAi) in a plant, a recombinant expression vector comprising the same, and the recombination. The present invention relates to a method of inhibiting expression of a storage protein using an expression vector and to a method of increasing accumulation of a target protein.

Crop molecular breeding is the key to lead the next generation of agriculture in that it is possible to use all kinds of genes as a material and to fine-tune the effects of infinite breeding by pulling down the breeding techniques previously available only in the genome. Technology. In order to maximize the effect of this crop molecular breeding technology, a number of genes that can represent genes of various plants must be secured, a transformation system of various crops must be established, and an expression system that can control the expression of the gene inserted from outside. Should be optimized. Protein expression control technology that can directly control the amount or accumulation of the expression protein of the introduced gene, or directly control the intracellular microorganisms of the expression protein material is also essential.

Glutelin is a generic term for proteins that belong to simple proteins and are insoluble in water and neutral salt solutions and are soluble in dilute acids and dilute alkalis. Cereals are abundant in grains, rice is oreginine, wheat is glutenin, and is a very important storage protein that accounts for 80% of the total rice protein. Wheat glutenin, together with gliadin, is involved in gluten formation and is characterized by high glutamic acid content. The results of SDS-PAGE electrophoresis show that rice gluterine has a molecular weight of about 54 kDa, and is composed of an alpha subunit having a basic chain of 34-37 kDa and an beta subunit having an acidic chain of 21-22 kDa. Gluterin genes form more than 10 gene groups and show a diversity of 10% to 40% of each other.

The demand for functional grains is increasing steadily despite the high price compared to general grains, and its manufacturing methods are also diversified. In general, a method of coating a specific ingredient on grains or cultivating in an environment containing a specific ingredient from the germination stage is widely used, and in recent years, a method of allowing a large amount of a specific ingredient to be contained through a transformation method has been put into practical use. to be. However, in this transformation method, grains have to express all genes introduced by transformation while making all the substances that are supposed to be produced in-house, so it is limited to increase the content of specific components of interest. There was.

The inventors of the present invention, while studying a method for increasing the accumulation efficiency of the target protein in the plant, when the inhibition of the expression of gluterin gene in rice, confirmed that the accumulation of transformed foreign protein is increased and the present invention Completed.

Accordingly, an object of the present invention is to provide a polynucleotide having a nucleotide sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide.

Still another object of the present invention is to provide a bacterium transformed with the recombinant expression vector.

Still another object of the present invention is to provide a plant transformed with the recombinant expression vector.

Still another object of the present invention is to provide a method for inhibiting expression of gluterin, comprising introducing into a plant a recombinant expression vector comprising a promoter and a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked thereto. will be.

Still another object of the present invention is to introduce a recombinant expression vector comprising (a) a promoter and a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked thereto, and (b) a promoter and operably linked thereto. It is to provide a method for increasing the accumulation of the target protein comprising the step of introducing the target gene into the plant of (a).

In order to solve the above problems, the present invention provides a polynucleotide having a nucleotide sequence of SEQ ID NO: 1.

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

In order to achieve another object of the present invention, the present invention provides a bacterium transformed with the recombinant expression vector.

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

In order to achieve another object of the present invention, the present invention is to inhibit the expression of gluterin comprising the step of introducing into the plant a recombinant expression vector comprising a promoter and a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked thereto It provides a method to make it.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) a promoter and a recombinant expression vector comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked to the plant and (b) promoter and It provides a method for increasing the accumulation of the target protein comprising the step of introducing a target gene operably linked to the plant of (a).

Hereinafter, the present invention will be described in detail.

The present invention provides a polynucleotide having a nucleotide sequence of SEQ ID NO: 1.

The polynucleotide of the present invention encodes RNA having RNA interference (RNAi) function for the gluterin gene.

RNA interference (RNAi) refers to selective intercellular degradation of RNA (also refers to gene silencing). RNAi also includes translational repression by fragmentation by microRNAs, siRNAs acting like microRNAs or diserase of dsRNA. RNAi can be triggered by the introduction of small interfering RNAs (siRNAs) into cells or the generation of siRNAs to silence the expression of one or more target genes. RNAi occurs naturally in cells and removes foreign RNAs (eg viral RNAs). Natural RNAi proceeds through dicer-directed fragmentation of precursor dsRNAs that direct desensitization mechanisms to other cognate RNA sequences.

The polynucleotide of the present invention includes a nucleotide sequence common to all gluterin genes among the gluterin gene sequences included in plants. Therefore, RNAi using the polynucleotide of the present invention can inhibit the expression of all gluterin genes in plants.

The polynucleotide of the present invention may include all of the nucleotide sequences common to all gluterin genes among the gluterin gene sequences included in plants, or may include a part thereof. Preferably, the polynucleotide of the present invention may include the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a recombinant expression vector comprising the polynucleotide. The recombinant expression vector of the present invention can be prepared to include the polynucleotide and the polynucleotide of the base sequence further complementary thereto. The expression vector is a vector capable of expressing a target RNA in a host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert. Expression vectors include expression control sequences and signal or leader sequences for membrane targeting or secretion and can be prepared in various ways according to the purpose. Expression control sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. In addition, the expression vector may include a selection marker for selecting a host cell including the vector, and in the case of a replicable expression vector, may include a replication origin.

The expression vector according to the present invention is a polynucleotide having a polynucleotide base sequence of the present invention and a complementary base sequence of the present invention inserted into the promoter and downstream of the promoter using a conventional vector used for protein expression as a backbone It can manufacture by inserting. Preferably, the expression vector of the present invention may be a pGlu-RNAi recombinant expression vector described in FIG. 1A.

A promoter is a gene region generally located upstream of a coding region that includes a point of initiation of transcription. The promoter of the present invention affects gene expression in response to TATA boxes, CAAT box regions, and external stimuli that regulate gene expression. Notes include enhancers that promote expression of almost all genes, regardless of site, location and orientation.

Existing vector used for the expression of the protein may be a recombinant expression vector for plant transformation, which can be used in the production of transgenic plants, in particular as a known recombinant expression vector. Examples include E. coli-derived plasmids, Bacillus subtilis-derived plasmids, plasmid vectors such as yeast-derived plasmids and Ti plasmids, cosmid vectors, bacteriophage vectors and viral vectors, and the like. A binary vector or cointegration vector can be used. There are a wide variety of binary vectors used in plant transformation, and almost all binary vectors are international centers and university laboratories such as the Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra ACT2601, Australia (CAMBIA). Available at

A recombinant expression vector comprising a promoter of the present invention and a nucleotide sequence encoding a protein of interest linked to the promoter can be introduced into a host using methods known in the art. Examples include, but are not limited to, calcium chloride and heat shock, particle gun bombardment, silicon carbide whiskers, sonication, electroporaion, PEG- Fusion method, microinjection method, microinjection method, liposome mediated method and vacuum infiltration method can be used.

The present invention therefore also provides a bacterium transformed with the recombinant expression vector according to the invention.

The host cell is preferably a bacterium, more preferably E. coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus, Agrobacterium tumefaciens and Agrobacte It may be Leeum lyzogenes. Most preferably, it may be Agrobacterium tumefaciens, and in one embodiment of the present invention, Agrobacterium LBA4404 (Agrobacterium tumefaciens) was used.

In addition, the recombinant expression vector according to the present invention can be introduced into plants using methods known in the art. Examples include, but are not limited to, Agrobacterium sp. Mediation, particle gun bombardment, silicon carbide whiskers, sonication, and electroporation. (electroporaion), PEG-mediated fusion method, microinjection method, microinjection method, liposome mediated method, vacuum infiltration method, and powder dip method. Preferably, the method by Agrobacterium sp. Mediation can be used and the method exemplified in the literature can be used (Horsch et al., Science 227: 1229-1231, 1985; An et al., EMBO J). 4: 227-288, 1985).

Accordingly, the present invention provides a plant transformed with the recombinant expression vector of the present invention.

The plant includes all plants, parts of plants, callus, plant tissues, plant cells and plant seeds. The plant of the present invention means rice.

The plant cell may be a hairy root cell, and additionally, the plant cell may be cultured according to a conventional method to be a drug culture, callus, protoplast, and may be differentiated to be a plant tissue or a plant. That is, the transformed hairy roots into which the recombinant vector of the present invention is introduced can be re-differentiated into plants through processes such as callus induction, rooting and soil purification using standard techniques known in the art, and asexuality such as folding and grafting It can be produced by well breeding method using breeding method and seed.

The present invention also provides a method for inhibiting expression of gluterin, comprising the step of introducing into a plant a recombinant expression vector comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked to a promoter. Operationally linked means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment.

Gluterin is the major storage protein of rice and consists of about 10 genes per haploid genome and is classified as a subfamily of GluA and GluB. In general, in order to suppress the expression of a specific protein, individuals whose gene expression is reduced by mutating or mutating the selected gene are selected, but in this case, it is not guaranteed that there is no abnormality in other genes. Only some of them are suppressed and the effect is not excellent. According to the method of inhibiting the expression of the storage protein of the present invention, the expression of these genes can be suppressed, and the degree of inhibition is superior to the existing method, and other unexpected modifications other than the inhibition of the storage protein expression are not performed by directly mutating the target plant. None can be guaranteed.

The present invention also provides a method of introducing a recombinant expression vector comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked to a promoter, and introducing a promoter and a target gene operably linked thereto to the plant. It provides a method for increasing the accumulation of the target protein, including.

The protein of interest according to the invention can be any target protein of external origin, ie an exogenous protein or an endogenous protein and a reporter protein. The exogenous protein refers to a protein that does not naturally exist in a specific tissue or cell of the plant, and the endogenous protein refers to a protein expressed by a gene naturally present in a specific tissue or cell. In addition, the reporter protein is a protein expressed by the reporter gene and refers to a labeling protein that indicates its activity in the cell by its presence. Proteins are useful functional and bioactive proteins, such as antibiotic-resistant proteins, herbicide-resistant proteins, virus-resistant proteins, pest-resistant proteins, metabolic-related proteins, antigens, antibodies, luminescent proteins, GFP (green fluorescence protein), and RFP (red fluorescence protein). , GUS (β-glucuronidase), GAL (β-galactosidase) and the like, but are not limited thereto.

The gene of interest refers to a gene encoding the protein of interest, and when it is expressed in plant seeds, a vector containing a gene of interest linked functionally downstream of a promoter guaranteeing expression in plant seeds can be preferably used. .

As a promoter used for the expression of a foreign gene, for example, in the case of expression of rice seed, a promoter of gluterin gene (Takaiwa, F. et al., Plant Mol. Biol. 17: 875-885,1991) is used. It is possible. Seeds of soybean crops such as string beans, horse beans, and peas, and oilseed crops for flow rates such as peanuts, sesame seeds, rapeseeds, cottonseeds, sunflowers, and safflowers. When expressed in the promoter of glycinin gene or promoter of the major storage protein gene of each crop, for example, kidney beans, the promoter of phaseolin gene (Murai, N. et al., Science 222: 476-482,1983), if the rapeseed can be used promoter of the cruciferin gene (Rodin, J. et al., Plant Mol. Biol. 20: 559-563, 1992). Specific examples of these promoters are illustrative only, and it may be considered to use a promoter for constitutive expression such as, for example, a 35S promoter.

In one embodiment of the present invention to prepare a polynucleotide of SEQ ID NO: 1 of the present invention using a primer for the homology region of the gluterin gene group of rice and then cytochrome for constitutive expression in rice The recombinant expression vector (pGlu-RNAi) was produced by operably linking with a promoter. The recombinant expression vector also includes the complementary nucleotide sequence of the polynucleotide of the present invention was produced so that dsRNA can be formed during transcription. In addition, a recombinant expression vector (pGlu-RFP) containing an RFP gene operably linked to a gluterin promoter for rice seed specific expression was prepared to inject red fluorescent protein (RFP) to be used as a target for increased expression. The recombinant expression vector thus prepared was introduced into Agrobacteria LBA4404 to prepare transformed bacteria (see Example 1).

In another embodiment of the present invention, the bacteria transformed with the recombinant expression vector were infected with rice callus to prepare rice transformed with the recombinant expression vector. Individuals into which the recombinant expression vector (pGlu-RNAi) of the present invention was introduced into the plant were selected using hygromycin, and the recombinant expression vector (pGlu-RFP) containing the RFP gene was transferred into the plant using phosphinothricin. Entering subjects were selected (see Example 2).

In another embodiment of the present invention was confirmed the color of the rice transformant, it was confirmed whether the pGlu-RNAi entered into the rice chromosome by Southern blot analysis. As a result, compared with the rice transformed with pGlu-RFP alone, the rice transformed with pGlu-RFP and pGlu-RNAi emitted more fluorescence and confirmed that the accumulation of RFP was increased (see Example 3-1). In addition, genomic DNA was isolated from the leaves of plants transformed with pGlu-RNAi, digested with restriction enzymes, subjected to electrophoresis, and Southern blot with gluterin gene, resulting in pGlu-RNAi structure in the chromosome of the transformed plant. It was confirmed that was inserted (see Example 3-2).

In another embodiment of the present invention whether the expression of the gluterin gene is inhibited by the inserted pGlu-RNAi was confirmed through Northern blot and RT-PCR. Whole RNA was extracted and electrophoresed from rice transformed with pGlu-RNAi, followed by Northern blot using gluterin gene. As a result, it was confirmed that gluten transcripts were detected in non-transformants, whereas glutin transcripts were not found in rice transformed with pGlu-RNAi. In addition, in order to determine whether expression of the entire group of gluterin genes is suppressed, cDNA was synthesized using the SuperScript III First-strand system for RT-PCR kit (Invitrogen) and then specific for each gene of the group of gluterin genes. Red primer was prepared and PCR was performed. As a result, the RT-PCR product of the entire gluterin gene was detected in the non-transformant, whereas the expression of the gluterin gene group was suppressed in the rice transformed with pGlu-RNAi (see Example 4). ).

In another embodiment of the present invention, the protein accumulation patterns in pGlu-RNAi transformed rice were analyzed. The whole protein of rice seed introduced with pGlu-RNAi and pGlu-RFP simultaneously and rice seed with pGlu-RFP alone were separated and subjected to electrophoresis, and the relative amount was calculated by image analysis of the bend. As a result, in the case of rice seed introduced with pGlu-RNAi and pGlu-RFP at the same time, other storage proteins did not change, but gluterin protein was 62 ~ 75% based on the total protein amount of the seed compared to rice introduced with only pGlu-RFP. As a result, it was confirmed that the reduction was 52-57% based on the 80kDa protein (see Example 5). Therefore, the method of inhibiting the expression of the polynucleotide, the recombinant expression vector of the present invention or the storage protein using the same, it was confirmed that the protein expression was significantly suppressed by effectively inhibiting the gluterin gene group.

In another embodiment of the present invention, the red fluorescent protein increase in pGlu-RNAi transformed rice was analyzed. The protein extracted in Example 5 was quantified and electrophoresed, and then subjected to Western blot using anti-RFP monoclonal antibody, and the results were analyzed. As a result, it was confirmed that the amount of RFP increased twice as much as that of the rice in which pGlu-RNAi and pGlu-RFP were introduced at the same time (see Example 6). Therefore, the polynucleotide, the recombinant expression vector of the present invention, or the method of increasing the accumulation of the target protein using the same, it was confirmed that the accumulation of the target protein is remarkably increased by effectively inhibiting the gluterin gene group.

Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual) , 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984) and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

Therefore, the polynucleotide of the present invention and the recombinant vector using the same can effectively suppress the expression of gluterin in a plant. In addition, in plants transformed with the recombinant vector of the present invention, expression of gluterin is suppressed, and when the genes of the target protein are transformed together, their accumulation is increased. In particular, the polynucleotide of the present invention, a recombinant vector using the same, a method of inhibiting the expression of gluterin using the same or a method of increasing the accumulation of the target protein is effective in increasing the accumulation of the target protein by effectively inhibiting all the group of gluterin genes. Therefore, the polynucleotide of the present invention, a recombinant vector using the same, a method of inhibiting the expression of gluterin using the same or a method of increasing the accumulation of the target protein can be useful for the purpose of increasing the target protein accumulation in the plant during the production of the target protein Can be.

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Preparation of plant transformation vector and Agrobacterial transformant

<1-1> Plant transformation vector production

Primer for homologous region (229bp) of Glu-A subfamily gene group (M28156, Y00687, X05661, L36819) in rice hull storage protein gluterin gene group for rice storage protein gene expression inhibition (RNAi) vector production Was prepared by PCR and confirmed by sequencing (SEQ ID NO: 1). The Gluterin RNAi vector of FIG. 1 was prepared using a gateway system as follows.

Primer a designed from the cDNA synthesized from RNA isolated from rice immature seeds to specifically amplify gluterin gene fragments, including a part of the recombinant nucleic acid sequence which is specific to bacteria when infected by bacteriophage And primer b were prepared to isolate 229bp gluterin gene fragment (SEQ ID NO: 1) through a polymerase chain reaction (PCR).

Primer sequence used for vector construction Primer name SEQ ID NO: order Primer a 2 5'-AAAAAGCAGGCTCAGTTTGCTTGTTCCTCT-3 ' Primer b 3 5'-AGAAAGCTGGGTACTCGGCGGACAACAG-3 ' Primer c 4 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 ' Primer d 5 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3 '

The amplified PCR products were re-PCRed using primers c and d, which contain the entire bacterial-specific recombinant nucleic acid sequence site when the bacteria were infected with bacteriophages.

The secondary PCR products were a mixture of pDONR221 vector containing Bacteriophage-specific recombinant nucleic acid sequence site and BP recombination reaction, ie BP reaction buffer, 150ng pDONR221 vector and gateway BP clonease enzyme mixture when bacteria were infected with bacteriophage. 200ng of the PCR product was mixed and reacted overnight at 25 ° C. to produce a cloning vector of pENTR-glu. Subcloned carriers were again subjected to LR recombination reactions, LR reaction buffers and gateway LR clones, with the pMJ202 (GGBio Inc.) vector into which the gluterin gene fragment could be introduced into the RNAi vector to produce the final plant transformation carrier. The mixture of enzyme and the cloning vector (300 ng) was mixed and reacted overnight at 25 ° C. to complete the final pGlu-RNAi carrier (FIG. 1). At this time, pMJ202, which is used as a binary vector, has a spectinomycin resistance gene for microbial selection, and contains a herbicide-resistant hygromycin resistance gene as a plant selection factor.

In addition, by using a glutinin promoter, a vector connecting the red fluorescent protein (RFP) gene, which is a reporter gene, was prepared. First, the RFP gene was digested with plasmid pDsRed2 (BD Bioscience Clonetech) with restriction enzymes BamHI and NotI and recovered by agarose electrophoresis (0.7 kb). RFP gene cleaved with BamHI and NotI after cleaving the vector (pSK-GluB1) inserted into pBluscript SK vector by gluterin B1 promoter (Takaiwa, Plant Mol. Biol. 17, 875-885, 1991) Was inserted into pSK-GluB1 vector through ligation reaction at 16 ° C. for 30 minutes using T4DNA ligase to prepare pSK-PGluB1-RFP vector. The fragments linked to the GluB1 promoter and the RFP gene obtained by cleaving the pSK-PGluB1-RFP vector with XhoI and NotI were synthesized by pSBG700 (Jang et al. , Plant Physiol. 129 (4): 14731481, 2002). Finally, pGlu-RFP was prepared as shown in FIG. 1B by connecting to XhoI and NotI.

<1-2> Agrobacterium transformant preparation

The produced transformant carriers pGlu-RNAi and pGlu-RFP were each triparental mating method (Glevin et al., Plant Molecular Biology Manual.Kluwer academic Publishers B1, 1-19,1994), respectively, using Agrobacterial LBA4404 (pSB1) (Hiei et al., Plant J 6: 271-282.204, 1994). Subsequently, the transformed Agrobacterium was used for 2 days in AB medium [AB buffer (60 g / LK ₂ HPO ₄ , 20 g / L NaH ₂ PO ₄ ), AB salts (20 g / L NH ₄ Cl, 6 g / L MgSO ₄ 7H ₂ O, 3 g / L KCl.0.2 g / L CaCl ₂ , 0.05 g / L FeSO ₄ 7H ₂ O), 5 g / L glucose, 15 mg / ml tetracycline, 50 mg / ml spectinomycin, 50 mg / L cultured in ml kanamycine] and then AAM medium [1X N6 macro elements, 1X N6 micro elements, 1X AA amino acids (877 mg / L glutamine, 228mg / L arginine), 75mg / L glycine ), 266 mg / L aspartic acid), 1X MS vitamins, 500 mg / L casamino acids, 36 g / L glucose, 68.5 g / L sucrose , 100 / L acetosyringone] was resuspended at 1.5 at absorbance 600 nm.

<Example 2>

Preparation of Rice Transformant

Agrobacterium LBA4404 (Glevin et al., Plant Mmolecular Biology Manual.Kluwer academic Publishers B1, 1-19,1994; Hiei et al., Prepared in Example 1 to inhibit storage protein expression and express the RFP gene in rice seeds Plant J 6: 271-282.204, 1994) was transformed into rice callus as follows.

Sterilized rice seeds were sown in 2N6 medium to collect fresh callus cultured aseptically for 4 weeks under dark conditions. Agrobacteria, quantified with absorbance 1.5, were co-cultured in dark conditions for 3 days at room temperature in 2N6 medium after infection with the callus for 20 minutes. After 3 days, the cells were washed with distilled water and then cultured for 3 weeks in 2N6-vitamin-containing medium (Maxim-Bio) containing 250 mg / L cefotaxime. In this case, 50 mg / L hygromycin was used as a selection marker when co-cultured with pGlu-RNAi transformed agrobacterium and 6 mg / L phosphinotricin when co-cultured with pGlu-RFP transformed agrobacterium. , ppt) were added, and in the case of co-culture of pGlu-RNAi and pGlu-RFP agrobacteria, 50 mg / L hygromycin and 6 mg / L ppt were added and cultured. Callus that survived without browning was incubated for 2 weeks in the same medium above. Resistant callus was collected in MSR medium [(4.4 g / L MS salt, 30 g / L cross-link, 2 mg / L kinetin, 1 mg / L NAA, 2.5 g phytagel, 250 mg / L cells Taksim, 50 mg / L hygromycin ± 6 mg / L ppt)] was incubated for one month to induce differentiation, callus was passaged to the same medium every 14 days. Stem-derived plants induced roots in MS medium, and after a week after rooting, they were grown in a greenhouse after being purified in a culture room. In order to determine the transformed rice, surviving transformants were selected by continuing passage in a medium containing hygromycin ± ppt.

<Example 3>

Confirmation of Gene Introduction by Seed Color and Southern Blot Analysis of Rice Transformant

<3-1> Confirmation of Gene Introduction by Seed Color Comparison of Rice Transformants and Increase of RFP Expression by Suppression of Storage Protein Expression

The rice transformant selected in Example 2 was grown to determine whether expression by introduction of a gene was first performed by red fluorescence analysis. The pGlu-RFP vector transgenic rice in which the RFP gene was linked to the gluterin promoter was subjected to fluorescence analysis of RFP in seeds. The RFP expressed by the glutinin promoter, a neutrophil specific expression promoter, strongly expressed the red fluorescent protein in the seed, so that transformed individuals could be selected visually without using a fluorescence microscope. Particularly, the pGlu-RNAi vector and the pGlu-RFP were selected. It was confirmed that the rice seed double transformed with the vector emits stronger fluorescence than the rice seed transformed with pGlu-RFP alone (see FIG. 2). This suggests that by inhibiting the expression of intrinsic storage proteins, the protein (RFP) accumulation of foreign genes can be increased.

<3-2> Confirmation of gene introduction by Southern blot analysis

Meanwhile, 10 ug of each genomic DNA isolated from the leaves of gluten RNAi-transformed rice T1 plants into which the pGlu-RNAi vector was introduced was digested with restriction enzyme XhoI and electrophoresed on a 0.8% agarose gel. membrane), and hybridization was performed at 65 ° C with a ³² P-labeled gluterin gene (225 bp PCR amplified in Example 1). Washed with 2 × SSC, 0.1% SDS solution and 0.1 × SSC, 0.1% SDS solution and exposed to an image analysis screen for analysis with an image analyzer.

Southern blot analysis showed that a non-transformant, a negative control, was not detected, whereas a transformant detected a pGlu-RNAi gene fragment of the same size as a positive control, the pGlu-RNAi vector. It was confirmed that the pGlu-RNAi structure that can inhibit the insertion (see Figure 3).

<Example 4>

Inhibition of Gluterin Protein Expression by Northern Blot and RT-PCR of Transgenic Rice Seeds

To confirm whether the expression of the gluterin gene was inhibited by gluterin RNAi, 10 ug of total RNA extracted from immature seeds of Nakdong rice was subjected to electrophoresis on 1% formaldehyde agarose gel as a control. Hybridization was performed at 65 ° C by transferring RNA to a nylon membrane in 20XSSC solution and labeling the same gluterin gene fragment used in the Southern blot of Example 3 with a radioisotope. Washed with 2 × SSC, 0.1% SDS solution and 0.1 × SSC, 0.1% SDS solution and exposed to an image analysis screen for analysis with an image analyzer.

As a result, it was confirmed that gluterin transcript was detected in the seed of non-transformed rice, whereas expression of gluterin gene was completely suppressed in the seed of gluterin RNAi transformant as shown in FIG. 4.

In addition, in FIG. 3, the expression of the gluterin gene used as a probe for the gluterin RNAi transformant was confirmed, but in order to analyze the expression inhibition level of the entire gluterin gene group, the total RNA 1g was superscript III First-strand system for RT. CDNA was synthesized using a PCR kit (Invitrogen). In addition, specific primers were prepared using sequence information for each gene gluA1, gluA2, gluA3, gluB1, gluB2, gluB3, and gluB4 in the gluterin gene group (see Table 2). Using the synthesized cDNA as a template, using 10 pmol of each primer pair of the gluterin gene group, 20mMTris-HCl, pH8.3, 100mM KCl, 2.5mM dNTP, 5 unit Taq DNA polymerase (Takara), 5mM MgCl2 solution PCR was synthesized 25 cycles at 94 ℃ 30 seconds, 59 ℃ 30 seconds, 72 ℃ 1 minutes.

As a result, as shown in FIG. 5, RT-PCR products for all gluterin A and gluterin B subfamily genes were detected in Nakdong rice, whereas no PCR products were detected in pGlu-RNAi transgenic rice. As a result, it was confirmed that pGlu-RNAi introduced into rice inhibited the expression of the entire group of gluterin genes composed of gluterin A and gluterin B subfamily with very high DNA homology.

Primer Sequences Used to Validate Gluterin Expression Primer Name SEQ ID NO: Primer Sequence Specificity GeneBank No Primer e 6 5'-TTCTTCGATGTCTCTAATGAGCAA-3 ' GluA1 F1 L36819 Primer f 7 5'-AGCTAACAAGAAATCCCTTTGCCT-3 ' GluA1 R1 Primer g 8 5'-TTCTTCGATGTCTCTAATGAGTTG-3 ' GluA2 F1 M28156 Primer h 9 5'-AGCTAACAAGAAATCCCTCTGTCG-3 ' GluA2 R1 Primer i 10 5'-AACGCACTCCTTTCACCT-3 ' GluA3 F1 M28158 Primer j 11 5'-GCCTCCTAGCTTCTTCTCTT-3 ' GluA3 R1 Primer k 12 5'-CCTTTTGGTACCTCGATACACA-3 ' GluB1 F1 X54314 Primer l 13 5'-AAGCCTCACTTAGCATCTCAAC-3 ' GluB1 R1 Primer m 14 5'-CCTTTTGGTACCTCGATATAGC-3 ' GluB2 F1 X54192 Primer n 15 5'-AAGCCTCACTTAGTAGCTCAGT-3 ' GluB2 R1 Primer o 16 5'-TGGTTCTACAATGATGGTGATGC-3 ' GluB3 F1 X54193 Primer p 17 5'-GGCCTCTTTGCTGCTACTGTG-3 ' GluB3 R1 Primer q 18 5'-GTTGCAACGGTTTAGATGAG-3 ' GluB4 F1 AB093593 Primer r 19 5'-ACGTAGTGTTGCGGAATGA-3 ' GluB4 R1

<Example 5>

Glutin RNAi Transgenic Rice Seed Protein Accumulation Analysis

Total protein accumulation patterns in T1 strains in which gluterin RNAi and RFP were introduced simultaneously were analyzed. One transformed seed was transferred to SDS-urea buffer (4% SDS, 8M urea, 0.25M Tris pH 6.8, 20% glycerol, 0.01% bromophenolblue, 5% mercaptoethanol). ) 350ul was added and mixed, and then allowed to stand overnight at room temperature, followed by centrifugation at 12000 rpm for 5 minutes, and 3ul of the supernatant was electrophoresed on a 14% SDS-PAGE gel. After electrophoresis, gluten protein, which accounts for 60-80% of the seed storage protein, was identified by distinguishing alpha (37-39 kDa) and beta (22-23 kDa) gluterin subunits by CBB staining. Beta subunits were identified by two bands.

As a result, as compared to the accumulation patterns of gluterin, globulin, and prolamin, the main storage proteins in transgenic seed introduced only RFP as shown in FIG. 6, the seed (RFP X glu RNAi) in which both gluterine RNAi and RFP were simultaneously introduced In the case of other storage proteins, there was no change, but the expression of both gluterin alpha and beta was significantly reduced.

The intensity of the band on SDS-PAGE was quantitatively analyzed by SCION image analysis to analyze the rate of glutelin protein inhibition in the seed into which glutin RNAi and RFP were simultaneously introduced (hereinafter referred to as 'gluterin RNAi X RFP seed'). .

As a result, as shown in FIG. 7, the relative quantitative value of the storage protein was analyzed based on the 80KDa band. As a result, it was confirmed that the accumulation of gluterin protein was reduced to 52-57% in the gluterin RNAi X RFP seed compared to the RFP transgenic seed. Could. Based on the total protein amount of the seeds, the amount of gluterin protein in gluterin RNAi X RFP seed was reduced to 62-75%.

<Example 6>

Analysis of Red Fluorescent Protein Increase in Gluterin-Inhibited Transgenic Rice Seeds

Western blot analysis was performed using RFP antibodies to determine whether the accumulation of foreign protein RFP protein in gluterin RNAi X RFP seeds was increasing.

The rice seed protein extracted in Example 5 was quantified, electrophoresed into 50 ug by 12% SDS PAGE, transferred to nitrocellulose membrane, and reacted with 5% skim milk solution for 1 hour, followed by anti-RFP monoclonal antibody. (1: 500, Sigma). 3 washes with TBST buffer (100 mM Tris pH7.5, 150 mM sodium chloride, 0.05% Tween-20) followed by anti-rabbit IgG alkaline phosphatase conjugate (promega) ( 1: 1000) was reacted for one hour, washed three times with TBST buffer solution, and then the alkaline phosphatase color developing reagent was poured to check the color band. An antibody of AGPase enzyme protein expressed in rice seeds was used as an RFP antibody and a positive control antibody.

As a result, RFP and AGPase protein bands were detected in both RFP and gluterin RNAi X RFP seeds. The detection intensity of RFP was more strongly detected in gluterin RNAi X RFP seeds than in RFP transgenic seeds (see FIG. 8). As a result of relative quantitative analysis of RFP band intensities based on AGPase band intensities using an image analyzer, it was confirmed that the expression level of RFP was increased by two-fold in glutin RNAi X RFP seeds compared to RFP transgenic seeds (FIG. 9). Reference). As a result, it was confirmed that the protein accumulation of the foreign transgene can be increased by inhibiting the expression of gluterin and the useful protein accumulation can be increased by the storage protein expression suppression technology during the production of the useful protein.

As described above, the polynucleotide of the present invention and a recombinant vector using the same may effectively inhibit expression of gluterin in a plant. In addition, in plants transformed with the recombinant vector of the present invention, expression of gluterin is suppressed, and when the genes of the target protein are transformed together, their accumulation is increased. In particular, the polynucleotide of the present invention, a recombinant vector using the same, a method of inhibiting the expression of gluterin using the same or a method of increasing the accumulation of the target protein is effective in increasing the accumulation of the target protein by effectively inhibiting all the group of gluterin genes. Therefore, the polynucleotide of the present invention, a recombinant vector using the same, a method of inhibiting the expression of gluterin using the same or a method of increasing the accumulation of the target protein can be useful for the purpose of increasing the target protein accumulation in the plant during the production of the target protein Can be.

1 is a schematic diagram of a carrier for inhibiting rice storage protein gene expression and a carrier for expressing a red fluorescent protein gene, which is a foreign protein.

<Code description>

Figure 1A

POsCc1: rice cytochrome promoter;

Glu: rice gluterin gene;

Int: Synthetic GPA1 intron containing splice donors, β-lactamase gene;

Amp ^R : ampicillin resistant β-lactamase gene gene;

Tnos: nofarin synthase terminator;

P35S: Cauliflower virus CaMV 35S promoter;

T35S: cauliflower virus 35S terminator;

Hyg ^R : hygromycin resistance gene;

Sp ^R : spectinomycin resistance gene:

MAR: matrix attachment site;

BR: Right border;

BL: Left border

Figure 1B

PgluB1: rice gluterin promoter;

RFP: red fluorescent protein gene;

T pinII: pinII terminator;

P35S: Cauliflower virus CaMV 35S promoter;

Bar: herbicide resistance gene;

Tnos: nofarin synthase terminator;

MAR: matrix attachment site;

P35S: cauliflower virus 35S promoter;

BR: Right border;

BL: Left border

FIG. 2 is a photograph of a rice seed that was tested to increase the accumulation of foreign protein by a gluterin expression inhibitory vector (WT: non-transformed rice; Pglu :: RFP: rice transformed with pGlu-RFP; RFP × glu RNAi : rice simultaneously transformed with pGlu-RFP and pGlu-RNAi).

Figure 3 confirms the gluterin protein expression inhibitory gene insertion by Southern blot analysis of the transformed rice (P: glu-RNAi plasmid; WT: non-transformant; 1 to 10: transformant)

Figure 4 shows the results of testing the inhibition of gluterin expression by gluterin expression inhibitory vectors (Nakdong rice: control Nakdong rice; GluRNAi: rice transformed with pGlu-RNAi; Glu: gluterine; Ubq: ubiquitin protein) .

Figure 5 shows the results of testing the inhibition of the expression of the entire group of genes gluten by the glutinin expression inhibitory vector by RT-PCR (Nakdong rice: control Nakdong rice; GluRNAi: rice transformed with pGlu-RNAi; GluA1-3, GluB1 4: subfamily of gluterin A and gluterin B; Ubq: ubiquitin protein).

Figure 6 shows the results of testing protein accumulation patterns of strains transformed with gluterin expression inhibitory vectors (WT: non-transformed rice; RFP: rice transformed with pGlu-RFP; RFP X glu RNAi: pGlu-RFP and rice simultaneously transformed with pGlu-RNAi).

7 is a result of comparing the accumulation pattern of gluterin by measuring the amount of storage protein relative to 80kDa protein or total protein (R1, R2: rice transformed with pGlu-RFP; LR1105, LR1105, LR1106, LR1113: Rice transformed simultaneously with pGlu-RFP and pGlu-RNAi; Cont: Average protein content of rice transformed with pGlu-RFP; RNAi: Corresponding protein of rice transformed simultaneously with pGlu-RFP and pGlu-RNAi Content average value).

Figure 8 is a result of comparing the amount of RFP protein accumulation in the seed transformed with gluterin expression inhibitory vector by Western blot image analysis. In the rice seed transformed only with the RFP expression vector and the rice transformed with the RFP expression vector and pGlu-RNAi simultaneously, the expression level of RFP and AGPase was measured by Western blot and the image was corrected to equal the amount of AGPase. (RFP (ng): red fluorescent protein (ng); RFP: rice transformed with pGlu-RFP; WT: nontransformed rice; RFP X glu RNAi: pGlu-RFP and pGlu-RNAi) Simultaneously transformed rice; AGPase: ADP-glucose pyrophosphorylase; RFP (~ 38 kD): red fluorescent protein).

9 is a graph comparing red fluorescent protein accumulation by Western blot image analysis (WT: non-transformed rice; RFP: rice transformed with pGlu-RFP; RFP X glu RNAi: pGlu-RFP and pGlu) Rice simultaneously transformed with RNAi).

<110> Republic of Korea (Management: Rural Development Administration) <120> Novel glutelin RNAi sequence and method for increasing the          accumulation of target protein by using the same <160> 19 <170> Kopatentin 1.71 <210> 1 <211> 229 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide for Glutelin RNA interference <400> 1 cagtttgctt gttcctcttg tgcgatggct ccctagccca gcagctatta ggccagagca 60 ctagtcaatg gcagagttct cgtcgtggaa gtccgagagg atgtagattt gataggttgc 120 aagcatttga gccaattcgg agtgtgaggt ctcaagctgg cacaactgag ttcttcgatg 180 tctctaatga gttgtttcaa tgtaccggag tatctgttgt ccgccgagt 229 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer A <400> 2 aaaaagcagg ctcagtttgc ttgttcctct 30 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer B <400> 3 agaaagctgg gtactcggcg gacaacag 28 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer C <400> 4 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer D <400> 5 ggggaccact ttgtacaaga aagctgggt 29 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer E: forward primer for GluA1 <400> 6 ttcttcgatg tctctaatga gcaa 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer F: reverse primer for GluA1 <400> 7 agctaacaag aaatcccttt gcct 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer G: forward primer for GluA2 <400> 8 ttcttcgatg tctctaatga gttg 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> Primer H: reverse primer for GluA2 <400> 9 agctaacaag aaatccctct gtcg 24 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer I: forward primer for GluA3 <400> 10 aacgcactcc tttcacct 18 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer J: reverse primer for GluA3 <400> 11 gcctcctagc ttcttctctt 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer K: forward primer for GluB1 <400> 12 ccttttggta cctcgataca ca 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer L: reverse primer for GluB1 <400> 13 aagcctcact tagcatctca ac 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer M: forward primer for GluB2 <400> 14 ccttttggta cctcgatata gc 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer N: reverse primer for GluB2 <400> 15 aagcctcact tagtagctca gt 22 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer O: forward primer for GluB3 <400> 16 tggttctaca atgatggtga tgc 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer P: reverse primer for GluB3 <400> 17 ggcctctttg ctgctactgt g 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer Q: forward primer for GluB4 <400> 18 gttgcaacgg tttagatgag 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer R: reverse primer for GluB4 <400> 19 acgtagtgtt gcggaatga 19  

Claims (9)

Polynucleotide having the nucleotide sequence of SEQ ID NO: 1. Recombinant expression vector comprising the polynucleotide of claim 1. The recombinant expression vector of claim 2, wherein the recombinant expression vector is pGluRNAi described in FIG. 1A. A bacterium transformed with the recombinant expression vector of claim 2. The method of claim 4, wherein the transformed bacteria are E. coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus, Agrobacterium tumefaciens and Agrobacterium lyzogenes Transformed bacteria selected from the group. Rice transformed with the recombinant expression vector of claim 2 or 3. delete A method of inhibiting expression of gluterin, comprising introducing into a plant a recombinant expression vector comprising a promoter and a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked thereto. (a) introducing into a plant a recombinant expression vector comprising a promoter and a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 operably linked thereto; and (b) introducing a promoter and a gene of interest operably linked thereto into the plant of (a).

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