WO2016104937A1 - Scp mutant gene, and secretion regulating method for secretory protein using same - Google Patents

Abstract

The present invention relates to: an SCP gene; an SCP mutant gene; a production method for a genetically modified plant using same; and a method for regulating the secretion of a secretory protein from a genetically modified plant cell body. When the SCP gene or SCP mutant gene of the present invention is used, it is possible to regulate the migration of TLP, which is a protein wherein secretion is induced by stress, and thus it is possible to use a vector comprising the gene of the present invention in order to produce a genetically modified plant having enhanced stress resistance.

Description

SCC Mutant Gene and Method of Regulating Secretion Proteins Using the Same

The present invention relates to a SCP gene, a SCP mutant gene, a method for producing a transformed plant using the same, and a method for controlling secretion of secreted proteins in a transformed plant cell body.

Secretory protein is a generic term for proteins secreted out of a cell. Cells recognize signals from certain hormones or stresses to activate transcription factors, thereby producing mRNAs for specific proteins. Based on the information of this mRNA, proteins are made. Secretory proteins are induced in the rough endoplasmic reticulum and translated into proteins before being translated into proteins by the signals that are present in the cell substrate. There, proteins in precursor form are made. The produced protein is transported to the Golgi apparatus by the carrier at the exit of the endoplasmic reticulum, and the transferred protein is transported to the cell plasma membrane or vacuole through the cis- / trans-Golgi apparatus and secreted or stored. Extracellular enzymes, peptide antibiotics, extracellular matrix proteins, and the like are contrasted with structural proteins that form and maintain the structure of a living body (cell).

Thaumatin-Like Protein (TLP) is a type of secreted protein that reports the characteristics and functions of TLP (Plant Cell Reports, vol. 29, no. 5, pp. 419-436 (2010)). . TLP is known to influence the flavor of wine (Deutsche Lebensmittel-Rundschau, vol. 109, no. 6, pp.319-323 (2013)), and studies on the separation of TLP from Barley Physiological and Molecular Plant Pathology, vol. 35, no. 1, pp. 45-52 (1989).

On the other hand, dephosphatase (phosphatase) is an enzyme that removes the phosphate group of the substrate phosphorylated by the dephosphorase. These dephosphoryases play an important role in metabolism, intercellular signaling, protein regulation, cell transport, secretion and many other cellular pathways.

The present inventors have completed the present invention by studying the effect of the gene and mutant genes encoding the dephosphoryase of rice on the secretion of secreted proteins.

(Non-Patent Document 1) Liu, JJ et al. "The Superfamily of Thaumatin-Like Proteins: Its Origin, Evolution, and Expression Towards Biological Function.", Plant Cell Reports, vol. 29, no. 5, pp. 419-436. (2010).

(Non-Patent Document 2) Pfeiffer, P. et al., "Do Thaumatin-Like Proteins in Wine Influence Its Flavor Profile?", Deutsche Lebensmittel-Rundschau, vol. 109, no. 6, pp.319-323 (2013).

(Non-Patent Document 3) Bryngelsson, T. et al., "Characterization of a Pathogenesis-Related, Thaumatin-Like Protein Isolated from Barley Challenged with an Incompatible Race of Mildew.", Physiological and Molecular Plant Pathology, vol. 35, no. 1 , pp. 45-52 (1989).

It is an object of the present invention to provide an SCP gene for secretion of secretory proteins.

Another object of the present invention is to provide an SCP protein.

Another object of the present invention is to provide an SCP mutant gene.

Another object of the present invention is to provide an SCP mutant protein.

Another object of the present invention is to provide a recombinant vector comprising the SCP gene or SCP mutant gene.

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

Still another object of the present invention is to provide a method for producing a transgenic plant having enhanced resistance to stress.

It is another object of the present invention to provide a method for controlling the secretion of secretory proteins in a transgenic plant cell body.

The present invention provides a SCP (Secretion Controlling Phosphatase) gene for secretion regulation of secreted proteins, consisting of the nucleotide sequence of SEQ ID NO: 1.

The gene may be isolated from rice ( Oryza sativa L. ).

As used herein, the term "Secretion Controlling Phosphatase" (SCP) refers to secretory regulatory dephosphatase.

As used herein, the term "dephosphorylase" refers to an enzyme that removes phosphoric acid by breaking down an ester bond of a phosphoric acid to a hydroxyl group of a specific serine, threonine or tyrosine residue.

The present invention also provides a SCP (Secretion Controlling Phosphatase) protein encoded from the SCP gene.

The protein is a protein consisting of the amino acid sequence of SEQ ID NO: 2.

The protein is an SCP protein for secretion of secreted proteins.

In addition, the present invention is SCP (Secretion Controlling Phosphatase) gene consisting of the nucleotide sequence of SEQ ID NO: 1, T (thymine) at position 136 is G (guanine), C (cytosine) at position 137 is A (adenin) Thus, the SCP mutation gene (SCP-SSEE) is represented by SEQ ID NO: 3 in which T in position 139 is G, C in position 140 is A, and 141th T is substituted with A.

The SCP mutant gene means SCP-SSEE (SEQ ID NO: 3) and is a non-processed mutation of SCP. In the wild type of SCP, there is a form of 'full length form' and 'processed form' in which the N-terminal of the protein is cut. SCP-SSEE, which is a non-processed mutant type of SCP, is mostly There is only a 'full length form'.

The gene is rice ( Oryza sativa L. ).

The present invention also provides a SCP (Secretion Controlling Phosphatase) mutant protein encoded from the SCP mutant gene (SCP-SSEE).

The protein is a protein consisting of the amino acid sequence of SEQ ID NO: 4.

The protein is an SCP mutant protein in which the serine, an amino acid residue at positions 46 and 47, is replaced with glutamic acid in the amino acid sequence of the SCP (Secretion Controlling Phosphatase) protein consisting of the amino acid sequence of SEQ ID NO: 2. Since two amino acid residues at positions 46 and 47 were replaced with glutamic acid (E) in serine (S), the SCP mutant gene consisting of the SCP mutant protein and the nucleotide sequence encoding it was named SCP-SSEE.

The present invention also provides a recombinant vector comprising the SCP gene or SCP mutant gene.

The recombinant vector may be for controlling secretion of secretory proteins, preferably for promoting secretion of secretory proteins, and most preferably for promoting secretion of TLP (Thaumatin-like protein).

As used herein, the term "secretory protein" refers to a protein that is secreted out of a cell in a cell. Cells activate transcription factors by recognizing signals from certain hormones or stresses, thereby producing mRNAs for specific proteins. Based on the mRNA information, proteins are made. Secretory proteins are induced by the endoplasmic reticulum and translated into proteins before being translated into proteins by the signal-recognition proteins present in the cell substrate, and then move into the rough cells. There, proteins in precursor form are made. The produced protein is transported to the Golgi apparatus by the carrier at the exit of the endoplasmic reticulum, and the transferred protein is secreted through the carrier through the stages of the Golgi apparatus (cis- / trans-Golgi apparatus) to the plasma plasma membrane. Types of secretory proteins include TLP (Thaumatin-like protein, Os03g46070, apoplast secretion protein), CA6 (Carbonic anhydrases 6, Os08g0423500, apoplast secretion protein), Glu2 (O-Glycosyl hydrolases, Os01g51570, apoplast secretion protein ), But is not limited thereto.

As used herein, the term "TLP (Thaumatin-like protein)" is a type of secretory protein known to be secreted by stress secretion (Sun Tae Kim et al. "Secretome analysis of differentially induced proteins in rice suspension- cultured cells triggered by rice blast fungus and elicitor ", Proteomics, Vol. 9, no.5, pp.1302-1313 (2009);" Plant pathogenesis-related (PR) proteins: A focus on by Sels, J. et al. PR peptides ", Plant physiology and biochemistry: PPB, Vol. 46, no. 11, pp. 941-950 (2008); Liu et al." The family 10 of plant pathogenesis-related proteins: Their structure, regulation, and function in response to biotic and abiotic stresses ", Physiological and molecular plant pathology, vol. 68, no. 1/3, pp.3-13 (2006); and" Proteomic analysis of pathogen-responsive proteins from Sun Tae Kim, et al. rice leaves induced by rice blast fungus, Magnaporthe grisea ", Proteomics, vol. 4, no. 11, pp. 3569-3578 (2004). Therefore, when stress is induced by TLP, the plant's stress resistance is enhanced by the secreted TLP (Datta). , K. et al., "Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease", Theoretical and applied genetics, vol. 98, no .6 / 7, pp. 1138-1145 (1999); Munis et al. "A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticillium dahliae and other stresses in transgenic tobacco", Biochemical and biophysical research communications, vol. 393, no. 1, pp. 38-44 (2010); "Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases" by Soon FF et al., Science, vol. 335, no.6064, pp. 85-88 (2012); and Velezhahan, R. et al., "Transgenic Tobacco Plants Constitutively Overexpressi ng a Rice Thaumatin-like Protein (PR-5) Show Enhanced Resistance to Alternaria alternata ", Biologia plantarum, vol. 47, no. 3, pp.347-354 (2003)).

As used herein, the term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

As used herein, the term "vector" refers to a DNA preparation having a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases integrate into the genome itself. In particular, “plant transformation vector” means a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular plant host organism. Promoters, enhancers, termination signals and polyadenylation signals available in plant cells are known to those skilled in the art. The vector of the present invention is most preferably a vector for plant transformation, but is not limited thereto.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the recombinant vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription may be used. It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

Methods for carrying vectors of the present invention into host cells include microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, Agrobacterium-mediated transfection, DEAE-dextran treatment, and gene balm. The vector can be injected into a host cell by a body or the like.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, kanamycin, G418, bleomycin, hygromycin ), But is not limited to antibiotic resistance genes such as chloramphenicol.

In the vector 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. The constant expression promoter may be preferred in the present invention because the selection of the transformants may be made by various tissues at various stages. Thus, the expression promoter does not limit the possibility of selection.

In one embodiment of the invention, the production of the vector used a Gateway vector system. Specifically, both sequences of the gene sequence to be expressed were amplified by PCR (Polymerase chain reaction) using a primer sequence. To insert into the pENTR / Directional TOPO vector (Invitrogen) containing the att L1 and att L2 sites, the CACC is inserted in front of the forward primer among the bidirectional primers so that the amplified gene can enter the pENTR ™ Directional TOPO vector with direction. (PENTR ™ Directional TOPO Cloning Kits, Invitrogen). And again, the target gene was inserted into the target vector using the LR Clonase II Enzyme Mix in the target gateway vector system having att R1 and att R2. In this way, a vector was produced which overexpresses the gene of interest. It uses site-specific recombination (SSR) through a specific sequence called att .

The present invention also provides a transformed plant transformed with the recombinant vector.

The plant may be one in which the secretion of secreted proteins from the cells of the plant is regulated to improve resistance to stress.

Secretion control of the secretory protein may be to promote the secretion of TLP. Specifically, since TLP is a protein inducing secretion by stress, promoting the release of TLP enhances the stress resistance of plants (Theoretical and applied genetics, vol. 98, no. 6/7, pp. 1138-1145 ( 1999); Biochemical and biophysical research communications, vol. 393, no. 1, pp. 38-44 (2010); Science, vol. 335, no.6064, pp. 85-88 (2012); and Biologia plantarum, vol. 47, no. 3, pp.347-354 (2003)).

The stress may be, but is not limited to, biological stresses such as molds, bacteria, viruses, or abiotic stresses such as salt, dryness, and low temperature.

The plant may be rice ( Oryza sativa L. ) or callus of rice, but is not limited thereto.

The transgenic plant according to the present invention can be obtained through a sexual breeding method or an asexual breeding method which is a conventional method in the art. More specifically, the plant of the present invention may be obtained through oily breeding, which is a process of producing seeds from the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant expression vector according to the present invention it can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of the callus according to a conventional method. That is, the explants of plants transformed with the recombinant expression vector of the present invention are inoculated in a suitable medium known in the art, and then cultured under appropriate conditions to induce the formation of callus, and when the shoots are formed, they are transferred to a hormone-free medium. Incubate. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. The transformed plant according to the present invention can be obtained by inducing roots and then transplanting them into the soil to purify them. Transgenic plants in the present invention may include whole plants as well as tissues, cells or seeds that can be obtained therefrom.

In addition, the present invention

1) preparing a recombinant vector comprising an SCP gene or an SCP mutant gene; And

2) provides a method for producing a transformed plant is improved resistance to stress by the secretion of the secreted protein comprising the step of transforming the vector into a plant.

Secretion control of the secretory protein may be to promote the secretion of TLP. Specifically, since TLP is a protein inducing secretion by stress, promoting the release of TLP enhances the stress resistance of plants (Theoretical and applied genetics, vol. 98, no. 6/7, pp. 1138-1145 ( 1999); Biochemical and biophysical research communications, vol. 393, no. 1, pp. 38-44 (2010); Science, vol. 335, no.6064, pp. 85-88 (2012); and Biologia plantarum, vol. 47, no. 3, pp.347-354 (2003)).

The stress may be, but is not limited to, biological stresses such as molds, bacteria, viruses, or abiotic stresses such as salt, dryness, and low temperature.

In addition, the present invention

1) preparing a recombinant vector comprising the gene of claim 1;

2) transforming the vector into a plant; And

3) It provides a method for controlling the secretion of secretory protein in the transformed plant cell body comprising the step of controlling the secretion of secreted protein in the cells of the transformed plant.

Secretion control of the secretory protein may be to promote the secretion of TLP.

In one embodiment of the present invention, as a result of overexpressing the SCP gene in rice protoplasts, changes in the rate of transfer of the secreted proteins Glu2, AALP, TLP, CA6 occurred (A and B of Fig. 2). In addition, as a result of overexpression of SCP-SSEE gene, it was confirmed that a significant migration inhibition of Glu2 and a significant increase of TLP, and a significant increase in secretion of TLP protein outside the protoplasts (A and C of Fig. 3). In another embodiment of the present invention, it was confirmed that the secretion of TLP in SCP knockdown transformants or knockout transformants was weakened (A and B of FIG. 5). These results indicate that SCP plays a role in promoting TLP secretion, and that SCP non-processed mutations result in stronger TLP secretion promoting function.

In another embodiment of the present invention, as a result of confirming the interaction between SCP and Sar1p serving as a switch to form a carrier at the vesicle exit, it was confirmed that SCP and Sar1p play their role in the same position (FIG. 5) A). These results indicate that SCP interacts with Sar1p and that SCP can be involved in protein transport at the endoplasmic reticulum exit.

By using the SCP gene or SCP mutant gene of the present invention can control the movement of TLP, a protein that is induced by stress, using the vector containing the gene of the present invention industrially useful secretion protein TLP (wine, etc.) A sweet protein that can affect the flavor of fermented food or substitute sugar, and a substance having a function of stress-promoting protein) can be improved in the production of plant cells, and a transgenic plant can be produced with enhanced stress resistance.

1 is a diagram showing the cDNA sequence of the SCP:

Bolt-shaped marking sequence, SCP-SSEE mutation site; And

Underlined sequence, SCP-G121D mutation site.

2 is a diagram showing changes in the secretion protein movement by SCP overexpression:

A, GFP-labeled proteins CA6-GFP, TLP-GFP, AALP-GFP, and Glu2-GFP together with SCP-HA labeled with HA (Hemaglutinin) or with SCP-HA-free controls in rice protoplasts Western blot assay results after expression for 20 hours; And

B, a graph showing the relative proportions of proteins moved.

Figure 3 shows the effect of overexpression of the SCP gene, SCP-SSEE gene (Non-processed mutation) and SCP-G121D gene (dephosphorylation inhibiting mutation) on secreted protein:

A, Western blot assay results;

B, a graph showing the relative proportion of migrated protein; And

C, the result of precipitation of the protein secreted out of the protoplasts using Trichloroacetic acid (TCA).

4 is a graph comparing SCP RNA levels of SCP knock-down transformants and SCP knock-out transformants by qPCR analysis:

DongJin, control group;

SCP-RNAi-1-5, SCP knockdown transformants;

SCP-RNAi-4-2, SCP knockdown transformants; And

scp, SCP knockout transformant.

FIG. 5 shows a control (DJ), SCP knockdown rice (SCP-RNAi-1-5, SCP-RNAi-4-2) and SCP knockout rice (scp) treated with 10 μM of JA (jasmonic acid) for 6 hours. The following figure shows the secretory regulatory effects of TLP, CA6 and Glu2 proteins:

A, Western blot assay results; And

B, graph showing the amount of protein secreted.

6 shows the results of confirming the interaction between the SCP and Sar1p:

A, venus fluorescence assay results;

B, Western blot assay results; And

C, Co-immunoprecipitation results.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples merely illustrate the contents of the present invention and do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention.

Example 1 Amplification of SCP battlefield cDNA

Primers were prepared to amplify the full-length cDNA of SCP (Secretion Controlling Phosphatase) protein in rice. Amplifying cDNA by PCR using 5-topo-SCP (5'-CACCATGCATGGGAGGCCGT-3 ', forward; SEQ ID NO: 6) and 3-SCP-ns (5'-TGAGTTTTGGTCGCTGGCG-3', reverse; SEQ ID NO: 7) primers PENTR ™ Directional TOPO Inserted into vector ( invitrogen ) and cloned ( pENTR - SCP ) . Full length cDNA sequencing of the SCP (SEQ ID NO: 1) is shown in FIG. The amino acid sequence of the SCP protein is shown in SEQ ID NO: 2.

Example 2 Assay of Change of Protein Movement by SCP

Experiments have been conducted to determine whether SCP can influence the transport of proteins from the endoplasmic reticulum through the Golgi.

<2-1> Construction of SCP Overexpression Vector

PENTR-SCP vector and pGEM - gw - x3HA to express hemagglutinin (HA) labeled SCP protein The vector was subjected to LR recombination reaction to generate a pGEM-SCP-HA vector.

Specifically, the pGEM-SCP-HA vector was prepared as follows.

First, three kinds of reverse primers, 3-HA-1 (5'-ACGAGCTCTCAGCACTGAGCAGCGTAATCTGGAACGTCATATGGATAGGATC-3 ', reverse; SEQ ID NO: 18), 3-HA-2 (5'-CGTCATATGGATAGGATCCTGCATAGTCCGGGACGTCATAGGGATAGCCCGCATAGTCAGGAAC-3'), reverse; And using 3-HA-SacI-3 (5'-CCCGCATAGTCAGGAACATCGTATGGGTAAACTTCGATATCAACCACTTTGTACAAGAA-3 ', reverse; SEQ ID NO: 20) and forward primer 5-pGA2897- att R1 (5'-CCGAAGCTTGATATCAACAAGTTTGTACAAAAA-3', forward; SEQ ID NO: 21) pGA2897 vector (Kim, H. et al., "A Rice Orthologue of the Aba Receptor, Ospyl / Rcar5, Is a Positive Regulator of the Aba Signal Transduction Pathway in Seed Germination and Early Seedling Growth." J Exp Bot 63, no. 2 ( 2012): 1013-24) were PCR amplified into a template. At this time, the forward primer used the same 5-pGA2897- att R1 (SEQ ID NO: 21), the reverse primer is 3-HA-1 (SEQ ID NO: 18), 3-HA-2 (SEQ ID NO: 19), 3-HA- Triple HA was expanded by PCR amplification using SacI-3 (SEQ ID NO: 20). Subsequently, pGA2897-HA vector was prepared by reinsertion into pGA2897 vector using HindIII and Sac I restriction enzyme sites.

Next, maize ubiquitin of pGA2897-HA vector Promoter , att R1 , Chloramphenicol resistance gene (CmR), ccdB The part containing the gene , att R2 , triple HA and nos- terminator was primers, 5-Ubi (5'-CTGCAGTGCAGCGTGACC-3 ', forward; SEQ ID NO: 22) and 3-nos-t (5'-GATCTAGTAACATAGATGACACCGC- 3 ', reverse; SEQ ID NO: 23) to make a fragment by PCR amplification, and then inserted into the pGEM T-easy vector (promega). In this way, a pGEM - gw- x3HA vector was produced.

PENTR - SCP Vector and pGEM - gw - x3HA The vector was subjected to LR recombination reaction to create a pGEM-SCP-HA vector that overexpresses HA labeled SCP protein.

<2-2> Change of secretion protein movement by SCP

PGEM - SCP- HA vector prepared in Example <2-1> was expressed on rice protoplasts and SCP was overexpressed, and the change of secretion proteins was confirmed by Western blot analysis. .

G6 (green fluorescent protein) labeled CA6 (Carbonic anhydrases 6, Os08g0423500, Apoplast secretion protein), TLP (Thaumatin like proteins, Os03g46070, Apoplast secretion protein), Glu2 (O- Genomes encoding Glycosyl hydrolases, Os01g51570, Apoplast secretion protein), AALP ( Arabidopsis thaliana Aluenin like protein, labeling proteins that migrate to vacuole), or rice alone or with pGEM - SCP -HA vector After expression on protoplasts and incubation for 20 hours, protein status was confirmed by Western blot analysis (A of FIG. 2).

Proteins secreted out of the cell tend to migrate to vacuoles in the protoplasts, the protein shown at the bottom in the upper figure of A of FIG. 2 is separated after being moved after the Golgi apparatus, and the full size protein shown above is released from the endoplasmic reticulum. It is a protein that is not.

2 shows the ratio of the protein moved to B. As shown in FIG. 2B, when the SCP was overexpressed, each secreted protein had a difference in migration rate compared to the control.

Therefore, it was confirmed that the presence of SCP affects the protein migration rate.

<Example 3> TLP secretion promoting effect test of SCP-SSEE (Non-processed mutant)

<3-1> Non-processed Mutation of SCP, Creation of SCP-SSEE

Created SCP - SSEE , a non-processed mutation of SCP . Non-processed mutations in SCP are those in which the N-terminus of the SCP protein is not cut and appears only in full length.

Specifically, primers for SCP cDNA of <Example 1> 5 - SCP-SSEE (5'-CGGGTCGGGAGGAATGCTGGTG-3 ', forward; SEQ ID NO: 8) and 3-SCP-ns (SEQ ID NO: 7), and 3-SCP-SSEE (5'-CACCAGCATTCCTCCCGACCCG-3', reverse; sequence No. 9) and 5-topo-SCP (SEQ ID NO: 6) were used to amplify the C-terminal and N-terminal fragments by PCR, respectively. PCR was performed using AccuPrime Pfx DNA Polymerase (Invitrogen) under conditions of repeating 35 cycles at 95 ° C denaturing temperature, 57 ° C annealing temperature and 68 ° C extension temperature. Two amplified fragments were used as templates and re-amplified using 5-topo-SCP (SEQ ID NO: 6) and 3-SCP-ns (SEQ ID NO: 7) primers. The full length DNA (SEQ ID NO: 3) of the SCP - SSEE mutant was changed from TCGTCT to GAGGAA. In addition, the SCP-SSEE protein encoded by the nucleotide sequence of SEQ ID NO: 3 is a protein in which serine (Serine, S), the 46th and 47th amino acid residues of SCP protein, is mutated to glutamic acid (E) (SEQ ID NO: 2). Number 4).

<3-2> Construction of SCP-G121D, a Mutant That Inhibits SCP Dephosphoryase Function

To construct a mutant that inhibited SCP dephosphorylation, SCP-G121D was constructed in which the 121st amino acid of SCP protein was mutated from glycine (G) to aspartic acid (D) (SEQ ID NO: 2). Number 5).

Specifically, 5-SCP-G121D (5'-CGGCCATGACGGAGCT-3 '; SEQ ID NO: 10) instead of 5-SCP-SSEE, and 3-SCP instead of 3-SCP-SSEE by the method described in Example <3-1>. -G121D (5'-AGCTCCGTCATGGCCG-3 '; SEQ ID NO: 11) using the 362nd sequence of SCP gene sequence of changes in G (guanine) to a (adenine), SCP - G121D Full length DNA was obtained (SEQ ID NO: 5).

<3-3> Change in secretion protein movement by SCP-SSEE

DNA of each mutant obtained in Examples <3-1> and <3-2> was placed in pENTR ™ Directional TOPO vector (Invitrogen), and then pGEM - SCP - SSEE through LR recombination reaction with pGEM - gw - 3xHA vector. made G121D -HA vectors, respectively - -HA vector pGEM - SCP. The pGEM-SCP-SSEE-HA vector overexpresses the SCP-SSEE gene labeled HA (Hemaglutinin), and the p-GEM-SCP-G121D-HA vector overexpresses the HA-labeled SCP-G121D gene.

And, GFP (green fluorescent protein) overexpress the labeled Glu2 gene or TLP gene pGEM - Glu2 - GFP vector or pGEM - TLP - GFP vector with pGEM - SCP -HA vector, pGEM-SCP-SSEE-HA vector or pGEM - SCP - the G121D -HA vector was expressed for 20 hours in rice protoplasts. The protein was separated by separating the medium from the cells, and the expression was confirmed by Western blot analysis (FIG. 3A).

Secretory proteins expressed in protoplasts tend to migrate to vacuoles in most cases. Comparing the secreted protein Glu2 and TLP, Glu2 showed a significant inhibition of movement in the place where SCP-SSEE was expressed together, and it was confirmed that the movement was significantly increased in the case of TLP. In contrast, where SCP-G121D, which inhibited dephosphorylation, was co-expressed, there was no appreciable difference from the control group expressing only the labeled protein (FIG. 3B).

In addition, the protein secreted out of the protoplasts was confirmed by precipitating with Trichlororoacetic acid (TCA). As a result, it was confirmed that when expressed with SCP-SSEE, a marked increase in secretion of TLP protein and a weak increase in secretion of Glu2 appeared (FIG. 3C).

This suggests that SCP-SSEE has the ability to regulate the transport of stress-induced TLP or Glu2 proteins. The effects on Glu2 and TLP appear different, which can be expected due to the nature of each protein. In the case of TLP, when a motif was searched using an amino acid sequence, only a simple tyrosine-based sorting signal (Yxx [LMVIF]) was found. Glu2, on the other hand, is a tyrosine-based sorting signal (Yxx [LMVIF]) as well as an FxDxF motif and N-glycosylation that can bind to AP-2 involved in endocytosis. It even had a site. This suggests that SCP-SSEE may have a stronger effect on the secretion of secreted proteins that contain only Tyrosine-based sorting signals (Yxx [LMVIF]), such as TLP. In addition, SCP could be expected to have the same effect as SCP-SSEE.

< Example  4> SCP  gene Knockdown  Transformants and Knockout  Induced by JA (Jasmonic Acid) in transformants Secreted protein  Shift change test

To determine how SCP affects changes in secretion protein movement, SCP knock-down transformants with significantly reduced expression of SCP genes and SCP knock-out without SCP gene expression The shift of secretion protein in the transformants was assayed.

<4-1> Construction of SCP Gene Knockdown Transformant

SCP gene knock-down transformants were constructed as follows.

First, using primers, 5-UTR Fr (5'-CTCCTCCTCCTCTCTCTCTCT-3 ', forward; SEQ ID NO: 24) and 5-UTR Re (5'-TGCCTTGACATATTCAGCCGC-3', reverse; SEQ ID NO: 25) The N-terminal region containing the '-UTR was cloned into the pENTR ™ Directional TOPO vector (invitrogen). Subsequently, LR recombination of the vector and the pPANDA vector ("Simple RNAi vectors for stable and transient suppression of gene function in rice", Plant Cell Physiol 45: 490-495 (2004)) by SCP-RNAi Vectors were produced. The completed vector was transformed into Agrobacteria LBA4404.

The rice seed of Dongjin rice was used for rice transformation. The peeled brown rice was sterilized in 70% ethanol for 1 minute and then used for 50 minutes in 50% lacx. Sterilized rice seed is 2N6 medium (Chu's salt 4g / L, Sucrose 30g / L, Casamino acids 1g / L, Proline 0.5g / L, Glutamine 0.5g / L, N6 1x, 2,4-D 2mg / L of vitamins and 4g / L of pytagel, pH 5.8) were injured and incubated at 20 ° C under dark conditions to induce callus.

Transformed Agrobacterium into AB solid medium (K ₂ HPO ₄ 3g / L, NaH ₂ PO ₄ 2H ₂ O 1.3g / L, NH ₄ Cl 3g / L, MgSO ₄ 7H ₂ O 1.15g / L, KCl 0.15g / L, CaCl ₂ 2H ₂ O 0.01g / L, FeSO ₄ 7H ₂ O 27.8 mg / L, Na ₂ EDTA2H ₂ O 37.3 mg / L, Glucose 5 g / L and Agar 20 g / L for 3 days, followed by AAM liquid medium (CaCl ₂ 0.33 g / L, KH ₂ PO ₄ 0.17g / L, MgSO ₄ 7H ₂ O 0.37g / L, KCl 2.94g / L, L-Glutamine 0.885g / L, L-Aspartic Acid 0.266g / L, L-Arginine (Arginine ) 0.228 g / L, glycine 0.075 g / L, MnSO ₄ 7H ₂ O 16.9 mg / L, ZnSO ₄ 7H ₂ O 8.6 mg / L, H ₃ BO ₃ 6.2 mg / L, KI 0.83 mg / L, Na ₂ MoO ₄ 2H ₂ O 0.25mg / L, FeSO ₄ 7H ₂ O 27.8mg / L, Na ₂ EDTA2H ₂ O 37.3mg / L, Nicotinic Acid 0.5mg / L, Pyridoxine-HCl 0.5mg / L, Thiamine-HCl 0.2mg / L, myo-inositol 100mg / L, CuSO ₄ 5H ₂ O 0.01875mg / L, CoCl ₂ 6H ₂ O 0.01875mg / L, Casamino acids ), 0.3125 g / L, Sucrose 68.5 g / L, and D-glucose 26 g / L, pH 5.8) to make a suspension. Optical Density After making 0.1 at 600 nm and diluting 10 times with AAM medium, the callus induced above was immersed for 2 minutes and gently shaken for 2 minutes to induce contact with Agrobacterium. Callus with Agrobacterium was incubated for 7 days at 25 ° C. in 2N6-AS medium (2N6 medium, 10 g / L glucose, and acetosyringone 100 uM, pH 5.2). After cancer cultivation, washing with distilled water containing carbenicillin 500 mg / L, followed by 2N6-CH (2N6 medium, 200 mg / L cefotaxime, and 40 mg / L Hygromycin) Transfer was incubated for 14 days at 28 ℃ transformed callus was induced. Transformed callus was MSR medium (Sorbitol 30g / L, Maltose 20g / L, MS salt 4.4g / L, Mes 0.5g / L NAA 20mg / L, Kinetin to induce regeneration) 2 mg / L, and 200 mg / L of Cefaxime) were used to obtain transformed SCP-RNAi individuals. SCP-RNAi instances thus randomly numbered, SCP-RNAi-1-5 and SCP-RNAi-4-2 transformants were used in the experiment.

<4-2> Preparation of SCP Gene Knockout Transformant

SCP knock-out transformants received mutated rice seeds (PEG_2D-21407) from Kyung Hee University, College of Life Sciences, which are expected to have T-DNA inserted into the SCP gene (The Plant Journal, vol. 22, no. .6, pp. 561-570 (2000)). Preformed transformants were named scp and used in the following experiments.

<4-3> Identification of SCP gene knockdown transformants and knockout transformants

Knock-down Knock-down transformants with significantly reduced expression of SCP, SCP-RNAi-1-5 and SCP-RNAi-4-2, and knock-out without T-DNA insertion into the SCP gene Out) In order to identify mutated rice ( scp , PFG_2D-21407), the expression level of SCP was confirmed at the RNA level.

RNA was isolated from each transformant using the RNA Purification Kit (RNeasy Plant Mini Kit, Qiagen) according to the manufacturer's instructions, followed by the manufacturer's instructions using the cDNA Synthesis Kit (RNA to cDNA EcoDry Premix, Clontech). cDNA was synthesized. And qSCP-F (5'-GAACAACGACTGGTGGTGGA-3 ', forward; SEQ ID NO: 12) and qSCP-R (5'-AGGAGCATGAAAGCGAGGAC-3', reverse; SEQ ID NO: 13) capable of specifically amplifying some sequences of SCP QPCR was performed using the primers to confirm the expression level of the SCP gene. qPCR mixes each cDNA and primer in a Master Mix (Prime Q-Master Mix, Genet Bio), and denaturing temperature 94 ℃, annealing on the qPCR machine 40 cycles were repeated at a temperature of 60 ° C. and an extension temperature of 72 ° C.

As a result, the expression levels of SCP genes were significantly decreased in the knockdown transformants SCP-RNAi-1-5 and SCP-RNAi-4-2, and scp (knockout mutant rice) showed almost no expression of SCP. (FIG. 4). This confirmed that these transformants were knock-down and knock-out mutants of SCP.

<4-4> SCP  gene Knockdown  Transformants and Knockout  In a transformant Secreted protein  Shift change test

In general, the treatment of JA (Jasmonic acid, Jasmonic acid) to plants induces the secretion of secreted proteins. After treatment with JA (10 uM) to Dongjin rice (control), RNAi-1-5, RNAi-4-2 and scp plants, 6 hours later, apoplasts were isolated by infiltration. It was. Secretory proteins TLP, Glu2 and CA6 were then confirmed by Western blot analysis.

As a result, it was confirmed that the secretion of CA6 and Glu2 is slightly reduced in the SCP-RNAi-1-5, SCP-RNAi-4-2 and scp than Dongjinbyeo. On the contrary, in the case of TLP showing strong responsiveness to JA, it was confirmed that the secretion is significantly weakened in SCP-RNAi-1-5, SCP-RNAi-4-2 and scp (FIG. 5A and B).

This confirms that SCP is an important factor in the secretion of TLP. In addition, when the results are combined with the results of Example <3-3>, the secretion of TLP is weakened by knockdown or knockout mutants of SCP, and the secretion of TLP is increased by non-processed mutants of SCP. It is confirmed that SCP plays a role in promoting the secretion of TLP, and that SCP non-processed mutations make TLP secretion promoting function stronger.

Example 5 Test of Interaction between SCP and Sar1p

<5-1> BiFC test to confirm interaction between SCP and Sar1p

BiFC (Bimolecular fluorescence complementation) was performed to confirm the intracellular interaction of Sar1p, which serves as a switch in the formation of the COPII vesicle at SCP and endoplasmic reticulum exit.

When the N-terminus (VN) and Venus C-terminus (VC) of the fluorescent protein, respectively, are expressed by labeling different proteins, when the different proteins are present near each other, VN and VC are combined to fluoresce between proteins. You can see the interaction. In order to test the interaction between SCP and Sar1p, VN and VC were labeled on SCP and Sar1p respectively. SCP inserted into pENTR ™ Directional TOPO vector (invitrogen) is subjected to LR recombination reaction with pGEM - gw - VN vector and then p GEM - SCP - VN Created a vector. pGEM - gw - VN In the vector, gw means gate way cassette ( att R1- att R2). P GEM - SCP - VN The vector expresses the SCP labeled VN.

Sar1p was harvested using primers 5-topo-Sar1p (5'-CACCATGTTCCTGGTTGACTGGTT-3 ', forward; SEQ ID NO: 14) and 3-topo-Sar1p-ns (5'-TTTGATGTACTGGGACATCCATT-3', reverse; SEQ ID NO: 15) It was amplified from the cDNA, the cloning of the amplified cDNA to the pENTR Directional TOPO ™ vector and then, pGEM - made -Sar1p pGEM-VC vector using the vector and the gw -VC LR recombination reaction. The thus produced pGEM - Sar1p -VC vectors expressing Sar1p the VC is labeled.

PGEM - gw - VN used for the experiment Vector and pGEM - gw -VC vector, and pGEM - VN - gw The vector is pDEST - gw - VYNE , pDEST - gw - VYCE And pDEST - VYNE (R) - gw The 35S promoter and nos-terminator, including the gateway system (invitrogen) of each vector, were amplified and cloned into pGEN- T vectors (Promega) (Gehl C et al., "New GATEWAY vectors for high throughput analyses. of protein-protein interactions by bimolecular fluorescence complementation ", Mol Plant 2: 1051-1058 (2009)). Primers 5-35S (5'-AGCTTGCATGCCTG-3 ', forward; SEQ ID NO: 16) and 3-Nos-T (5'-CCGATCTAGTAACATAGATG-3', reverse; sequence SEQ ID NO: 17 to amplify the gateway system including the promoter and terminator PCR amplification using

VN - - gw pGEM vector and to express the Sar1p-VC - only expressed pGEM to VN one pairs of the Sar1p -VC vector, and SCP-VN pGEM expressing - SCP - VN PGEM vector and expressing Sar1p-VC - with the Sar1p -VC vector of a pair were transformed to protoplasts of each rice plant. Transfection result of observation through a microscope after 15 hours, pGEM - VN - fluorescence signal that can be seen by the non-specific binding between the VC caused by the over-expression of the VN is hardly visible from the protoplasts into a Sar1p -VC - gw and pGEM. On the other hand, pGEM - SCP - confirmed that the recovery point type venus In the fluorescent looks into the protoplast Sar1p -VC - VN and pGEM.

From this, SCP and Sar1p confirmed that the labeled VN and VC were close to or attached to a position close to the fluorescence of venus by combining (Fig. 6A). This shows that Sar1p and SCP can play their role in the same location.

<5-2> Western blot analysis to confirm interaction between SCP and Sar1p

Samples subjected to BiFC were identified by Western blot analysis using an anti-GFP antibody to identify expression forms of three genes. As a result, the amount of SCP-VN is smaller than that of the VN put as a control which does not emit fluorescence signal, and the strong fluorescence signal of dot shape shows that SCP-VN does not interact with Sar1p-VC due to overexpression. It can be confirmed that it exists in the position to interact (B of FIG. 6).

<5-3> Mutual Immunoprecipitation Test to Verify Interaction Between SCP and Sar1p

Co-immunoprecipitation was performed to confirm the actual interaction between SCP and Sar1p.

Sar1p and pGEM - gw - GFP inserted in the pENTR ™ Directional TOPO for this purpose. The vector was made pGEM - Sar1p - GFP using LR recombination reaction. PGEM - gw - gfp above Vectors include promoters and terminators (2x35S), including the gateway system of the pMDC83 binary vector ("A gateway cloning vector set for high-throughput functional analysis of genes in planta", Curtis MD, et al., Plant Physiol 133: 462-469 (2003)). Promoters, att R1- att R2, GFP and nos-terminators were prepared using primers 5-pMDC (5'-GACGGCCAGTGCCA-3 ', forward, SEQ ID NO: 26) and 3-pMDC (5'-GTAACATAGATGACACCGCGC-3', Reverse amplification using SEQ ID NO: 27) was completed by cloning into a pGEM- T vector (Promega). In this way, a pGEM-Sar1p-GFP vector was produced that overexpressed Sar1p protein labeled with GFP (green fluorescent protein).

With SCP -HA vector pGEM - - A pGEM manufactured in the GFP - gw The vector and pGEM - Sar1p - GFP vector were introduced into rice protoplasts and incubated for 20 hours, and GFP trap, a bead coated with a change portion of an antibody capable of binding to GFP protein. Sar1p-GFP was precipitated using (ChromoTek). As a result, SCP-HA was found to settle together. When seeing that SCP-HA did not precipitate with GFP used in the control (control), it can be seen that SCP-HA binds to Sar1p and precipitates (FIG. 6C). This confirms the interaction between Sar1p and SCP-HA.

These results show that SCP can be involved in protein transport at the endoplasmic reticulum by revealing that it interacts with Sar1p, which plays an important role in the formation of protein-carrying carriers at the endoplasmic reticulum exit (ERES).

SCP: Secretion Controlling Phosphatase;

SCP-SSEE: SCP [S46E, S47E];

TLP: Thaumatin-like protein, Os03g46070, apoplast secreted protein;

CA6: Carbonic anhydrases 6, Os08g0423500, apoplast secreted protein;

Glu2: O-Glycosyl hydrolases, Os01g51570, apoplast secreted protein;

AALP: Arabidopsis thaliana Aluenin like protein, a marker protein that moves into vacuoles;

SCP-RNAi: knockdown mutant of SCP; And

scp : knockout mutant of SCP.

Claims (17)

1. SCP (Secretion Controlling Phosphatase) gene for secretion regulation of secreted protein, consisting of the nucleotide sequence of SEQ ID NO: 1.
2. SCP (Secretion Controlling Phosphatase) protein consisting of the amino acid sequence of SEQ ID NO: 2 encoded from the gene of claim 1.
3. In the SCP (Secretion Controlling Phosphatase) gene consisting of the nucleotide sequence of SEQ ID NO: 1, T (thymine) at position 136 is G (guanine), C (cytosine) at position 137 is A (adenin), and position 139 SCP mutant gene (SCP-SSEE) represented by SEQ ID NO: 3 with T of G, C of 140th position of A, and 141th of T replacing of A.
4. 4. The SCP mutant gene of claim 3, wherein the gene is isolated from rice ( Oryza sativa L. ).
5. SCP (Secretion Controlling Phosphatase) mutant protein encoded from the gene of claim 3.
6. 6. The SCP mutant protein of claim 5, wherein said protein consists of the amino acid sequence of SEQ ID NO.
7. A recombinant vector comprising the gene of claim 1 or 3.
8. According to claim 7, wherein the recombinant vector is a recombinant vector, characterized in that for controlling the secretion of secreted proteins.
9. The recombinant vector according to claim 8, wherein the regulation of secretion protein promotes secretion of Thaumatin-like protein (TLP).
10. A transformed plant transformed with the recombinant vector of claim 7.
11. The method of claim 10, wherein the plant is transformed plants characterized in that the secretion of the secreted protein secreted from the cells of the plant is enhanced resistance to stress.
12. The transgenic plant of claim 11, wherein the regulation of secretion protein is promoted by the secretion of TLP.
13. 12. The transgenic plant of claim 11, wherein the plant is rice.
14. 1) preparing a recombinant vector comprising the gene of claim 1 or 3; And

    2) A method for producing a transgenic plant comprising the step of transforming the vector into a plant, the secretion of the secreted protein is controlled to improve the resistance to stress.
15. 15. The method of claim 14, wherein the regulation of secretion protein secretion is facilitated by the secretion of TLP.
16. 1) preparing a recombinant vector comprising the gene of claim 1 or 3;

    2) transforming the vector into a plant; And

    3) controlling the secretion of the secreted protein in the transformed plant cell body, comprising the step of controlling the secretion of the secreted protein in the cells of the transformed plant.
17. 17. The method of claim 16, wherein the regulation of secretion protein secretion of the TLP is promoted, the method of controlling the secretion of secretory protein in the transformed plant cell body.

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