KR101511190B1 - OsHIR1 gene increasing heavy metal stress tolerance and decreasing heavy metal uptake of plant and uses thereof - Google Patents

Abstract

The present invention relates to an OsHIR1 gene for increasing heavy metal stress resistance and heavy metal absorption of a plant and to use of the same. A plant in which a gene for coding an OsHIR1 protein is overexpressed exhibits a phenotype that is insensitive to arsenic and cadmium, has more excellent root growth than a control group plant grown under the same conditions, and has a remarkably reduced arsenic and cadmium absorption amount. Accordingly, the expression of a gen for coding an OsHIR1 protein in a plant is adjusted, thereby increasing heavy metal resistance and productivity and developing a safe plant having reduced heavy metal absorption.

Description

[0001] The present invention relates to an OsHIR1 gene and a use thereof for decreasing heavy metal stress tolerance and heavy metal uptake of plants,

More particularly, the present invention relates to a gene encoding OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) protein derived from rice, and more particularly, to a OsHIR1 gene and a use thereof for decreasing heavy metal stress tolerance and heavy metal absorption of plants. methods by transforming a recombinant vector into a plant cell as compared to the wild type comprises the step of overexpressing OsHIR1 gene to reduce the heavy metal stress tolerance increase, and heavy metal absorption of the plant, heavy metals than the wild type with the recombinant vector including the OsHIR1 gene A method for producing a transgenic plant having increased stress tolerance and reduced heavy metal absorption rate, a transgenic plant having increased heavy metal stress tolerance, reduced heavy metal absorption rate, seeds thereof, and OsHIR1 gene derived from rice Plants containing the recombinant vector It relates to heavy metal for increased stress tolerance and reduce heavy metal absorption composition.

Arsenic and cadmium exist everywhere in the world and their toxicity causes serious environmental problems. In addition, they pose a serious risk to human health, but they are used agriculturally by means of herbicides, fertilizers and pesticides. Crop cultivation in soil contaminated with arsenic and cadmium is accumulated in humans through the food chain (Verbruggen et al. , 2009, Curr Opin Plant Biol 12: 364-372). In particular, arsenic and cadmium absorbed from the soil through the roots during rice cultivation accumulate at high concentrations in the kernels. Recently, a high amount of arsenic was detected in the urine of pregnant women in the US who consumed rice (Gilbert-Diamond et al. , 2011, Proc Natl Acad Sci USA 108: 20656-20660).

Arsenate (AsO ₄ ^3- ) is mainly present in aerobic soil, and arsenate (AsO ₃ ^3- ), which is a reduced form of arsenate, is more present in rice. These two types of arsenic absorption mechanisms differ in plants. For example, arsenate in rice cells is absorbed by a high affinity inorganic phosphate (Pi) delivery system. On the other hand, arsenite is similar in molecular size and structure to silicic acid and is absorbed by silicate input carriers such as OsNIP2; 1 (Catarecha et al. , 2007, Plant Cell 19: 1123-1133). The major intrinsic proteins (MIPs) of plants, commonly referred to as aquaporins, are nodulin 26-like intrinsic membrane protein (NIP), plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP) and small basic intrinsic protein ). To date, there has been no report of the absorption of arsenate in other groups except NIP, but the possibility that the proteins of the TIP group carry the arsenate in vacuoles (Zhao et al. , 2009, The New phytologist 181: 777-794 ). Cadmium has been reported to be absorbed from the soil by Zn ²⁺ , Mn ²⁺ , Fe ²⁺ and Ca ²⁺ carriers (Clemens, 2006, Biochimie 88: 1707-1719).

Protease hydrolysis by proteasomes in higher plants appears to play an important role in many stress response pathways, including heavy metal stress. As a post-translational modification, ubiquitination is an essential step in the hydrolysis pathway by 26S proteasome. The ubiquitin, consisting of 76 amino acids, is covalently attached to the lysine residue of the target protein. Three enzymes dependent on ATP (E1, E2, and E3) sequentially act to attach the ubiquitin molecule to the target protein and induce post-translational modification. E3 is classified into two groups, the first group consisting of the APC complex and the SCF complex, and these groups are composed of a plurality of subunits. The second group is the HECT and RING / U-box E3 ligase, which consists of one subunit (Vierstra, 2009, Nat. Rev. Mol. Cell Biol. 10: 385-397), E3 ligase Recognizes that the ubiquitin molecule is attached to the surface of the target substrate and specifically interacts with it. The binding of a poly- or mono-ubiquitin chain to a substrate protein determines the function and location of many target proteins (Roos-Mattjus and Sistonen, 2004, Ann Med. 36 (4): 285-295 ).

RING (Really Interesting New Gene) E3 ligase is a common sequence consisting of cysteine and histidine residues (Cys-x ₂ -Cys-x ₉ -Cys-x _1-3 -His-x _2-3 -Cys / His-x ₂ -Cys-x _4-48 -Cys-x ₂ -Cys, where x may be any amino acid, and these structures are associated with two zinc atoms. This structure is generally known to be classified into two basic forms, including RING-H2 and RING-HC, based on the cysteine or histidine position of the RING-finger proteins. Several variants such as RING-D, RING-V, RING-S / T, RING-G and RING-C2 have also been found in higher plants including Arabidopsis or rice genomes. The researchers demonstrated that the E3 ubiquitin ligase with the RING-domain plays an important role in adapting to extreme environments, defense mechanisms against pathogens, and plant growth. For example, the function of E3 ubiquitin ligase (i. E., Rma1H1) in red pepper involves the inhibition of the transport of aquaporin to the plasma membrane, and hydrolysis by proteasomes subsequently provides enhanced resistance to drought stress . Arabidopsis SDIR1 (salt- and drought-induced RING finger1) is thought to regulate stress-responsive ABA signaling. Similarly, transgenic rice plants overexpressing OsSDIR1 have been shown to be resistant to drought (Gao et al. , 2011, Plant Mol Biol 76: 145-156). However, the role of RING E3 ubiquitin ligase in arsenic and cadmium stress remains to be elucidated, and post-sexual control of arsenic and cadmium accumulation by RING E3 ligase has not been reported to date.

The present inventors confirmed the expression levels of 47 OsRFP ( Oryza sativa RING finger protein) genes according to excessive arsenic and cadmium concentrations by semi-quantitative RT-PCR. OsRFPH2-11 located in the cell membrane was markedly increased by treatment with arsenic and cadmium and was renamed OSHIR1 ( O. sativa heavy metal induced RING E3 ligase 1). Arabidopsis overexpressing OsHIR1 showed arsenic and cadmium - insensitive phenotypes and significantly decreased arsenic and cadmium uptake. The present inventors have identified proteins that interact with OsHIR1 ubiquitin E3 ligase using a yeast double hybridization method. In addition, the degradation analysis showed that the OsHIR1 E3 ligase migrates OsTIP4; 1 located in the tonoplast to the cell membrane and causes protein degradation through the 26S ubiquitin proteasome pathway. These results suggest that OsHIR1 E3 ligase plays an important role in the regulation of the absorption of arsenic and cadmium.

Korean Patent No. 0515520 discloses " a method for producing a transformant having improved heavy metal resistance and decreased absorption, " and Korean Patent No. 1039793 discloses a method for producing a heavy metal resistance Pcr gene and a transformant &Quot; However, as in the present invention, the OsHIR1 gene and its use for reducing heavy metal stress tolerance and heavy metal absorption of plants have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have selected OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) gene which is remarkably increased in expression by arsenic and cadmium treatment in rice and OsHIR1 gene The present inventors have completed the present invention by confirming that arsenic and cadmium stress resistance in overexpressed Arabidopsis plants is increased and the absorption rate of arsenic and cadmium is decreased.

In order to solve the above problems, the present invention includes a step of overexpressing OsHIR1 gene by transforming a plant cell with a recombinant vector comprising a gene encoding rice-derived OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) Compared to the wild-type plants, which are resistant to heavy metals, and to reduce heavy metal absorption.

The present invention also provides a method for producing a transgenic plant having increased heavy metal stress tolerance and reduced heavy metal absorption rate compared to the wild type using a recombinant vector comprising the gene encoding the OsHIR1 protein.

In addition, the present invention provides transgenic plants and seeds thereof, wherein the heavy metal stress tolerance produced by the method is increased and the heavy metal absorption rate is decreased.

The present invention also provides a composition for increasing heavy metal stress tolerance and reducing heavy metal absorption of a plant containing a recombinant vector comprising OsHIR1 gene derived from rice.

The plants overexpressing the gene encoding the OsHIR1 protein of the present invention exhibit a phenotype insensitive to arsenic and cadmium and exhibit superior roots growth as compared with the control plants grown under the same conditions and remarkably reduce arsenic and cadmium uptake, By modulating the expression of the gene encoding the OsHIR1 protein, it can contribute to the development of safe crops with increased heavy metal tolerance and productivity and reduced heavy metal uptake.

Figure 1 shows the expression patterns of 47 OsRFP genes in plants treated with arsenic (As) or cadmium (Cd). Germinated seeds were treated with diluted Na ₂ HAsO ₄ · 7H ₂ O and CdSO ₄ , respectively. Samples were sampled 4 weeks after treatment and Os18S-rRNA was used as an internal control. The red arrow indicates the OsHIR1 gene.
Fig. 2 shows the amino acid sequence analysis of rice OsHIR1 and the in vitro E3 ligase activity of the RING-H2 domain. A, the deduced amino acid sequence of rice OsHIR1 and other RING-H2 type proteins, Brachypodium derived Bradi3g25680 (registration number XM_003573851), maize derived GRMZM5G879737 (BT055823), sorghum derived Sb01g022360 (XM_002467225) and Arabidopsis thaliana derived AT1G14180 ) Protein. Sorting was performed using the ClustalW2 software (http://www.ebi.ac.uk/clustalw/). The conserved metal-ligand residues of cysteine (C) or histidine (H) that form the RING-H2 domain in the red box are marked with an asterisk. B, MBP-OsHIR1 ^285-343 or Empty-MBP were incubated with ubiquitination reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , pH 7.4) with or without yeast E1 or Arabidopsis E2 (AtUBC10 and 11) 0.05 mM ZnCl ₂ , 1 mM ATP, 0.2 mM DTT, 10 mM phosphocreatine and 0.1 unit of creatine kinase). The ubiquitinated bands were detected by immunoblot analysis using anti-ubiquitin antibody.
Figure 3 shows the results of measuring increased arsenic and cadmium resistance and decreased arsenic and cadmium uptake of OsHIR1 -overexpressing Arabidopsis thaliana. A, OsHIR1- overexpressing Arabidopsis seeds and control (blank vector) seeds were germinated and seeded in 1/2 MS solid medium containing 1.5% sucrose with or without 150 μM arsenic or 50 μM cadmium for 16 hours And cultured under light / 8 hour arm (22 ° C / 18 ° C). Root length was analyzed using Image J software at day 12 after culture (DAP). Data were expressed as means ± SD (n = 30). The asterisk indicates a significant difference (* P <0.05 and ** P <0.01, t- test) between the mean values of the respective OsHIR1 -overexpressing plants compared to the control. B, OsHIR1- overexpressing Arabidopsis seeds and control (blank vector) seeds were germinated and seeded in 1/2 MS solid medium containing 1.5% sucrose with or without 150 μM arsenic or 50 μM cadmium for 16 hours And cultured for 2 weeks under light / 8 hour arm (22 ° C / 18 ° C). Data were expressed as means ± SD (n = 250). The asterisk indicates a significant difference (* P <0.05 and ** P <0.01, t- test) between the mean values of the respective OsHIR1 -overexpressing plants compared to the control.
Figure 4 shows that OsTIP4; 1 protein levels are reduced by OsHIRl E3 ligase in a 26S proteasome-dependent manner. AB, OsHIR1-cMyc reduced OsTIP4; 1-EYFP protein. The indicated constructs were inoculated on tobacco ( N. benthamiana ) leaves and protein extracts of leaves cultured for varying times (0, 1/2, 1 and 2 hours) were incubated with an anti-cMyc antibody or an immunoglobulin Respectively. C, MG132 inhibits protein degradation of OsTIP4; 1-EYFP by OsHIR1-cMyc. The extracted OsTIP4; 1-EYFP protein was mixed with OsHIR1 E3 ligase containing MG132 and the level of OsTIP4; 1-EYFP protein was analyzed using anti-GFP antibody. Rubisco protein stained with Ponceau S was indicated as the loading control.

In order to accomplish the object of the present invention, the present invention provides a method for producing a recombinant vector comprising the step of overexpressing the OsHIR1 gene by transforming a plant cell with a recombinant vector comprising a gene encoding OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) To increase tolerance of heavy metal stress in plants and to reduce heavy metal absorption.

The scope of the OsHIR1 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 isolated from rice ( Oryza sativa ) and functional equivalents of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means an activity that increases resistance to heavy metal stress and decreases the absorption rate of heavy metals. The invention also includes fragments, derivatives and analogues of the OsHIR1 protein. As used herein, the terms "fragments", "derivatives" and "analogs" refer to polypeptides having substantially the same biological function or activity as the OsHIR1 polypeptides of the invention.

The gene coding for OsHIR1 protein derived from rice of the present invention may be DNA or RNA, and DNA includes cDNA, genomic DNA or artificial synthetic DNA. Preferably, the OsHIR1 protein coding gene of the present invention may comprise the nucleotide sequence of SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the &lt; / RTI &gt; Methods for sequencing and means for determining sequence identity or homology (e.g., BLAST) are well known in the art.

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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The recombinant plant expression vector of the present invention can be used as a transient expression vector capable of transient expression in a plant into which the foreign gene is introduced and a plant expression vector capable of permanently expressing the foreign gene in the introduced plant.

In the present invention, the gene sequence encoding the OsHIR1 protein may be inserted into a recombinant vector. The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element. Enhancers, ribosome binding sites, termination signals, polyadenylation signals, and promoters, which are available in eukaryotic cells, are well known in the art. The expression vector can be constructed by a method known in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

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

In the recombinant vector of the present invention, the promoter may be a promoter suitable for transformation, preferably a CaMV 35S promoter, an actin promoter, a ubiquitin promoter, a pEMU promoter, a MAS promoter or a histone promoter, preferably a CaMV 35S promoter But is not limited thereto. The term "promoter " refers to the region of DNA upstream from the 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. Since the selection of the transformant can be carried out by various tissues at various stages, a constant promoter may be preferable in the present invention. Therefore, the constant promoter does not limit the selectivity.

In the recombinant vector of the present invention, the terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens Agrobacterium tumefaciens , Octopine gene terminator of Agrobacterium tumefaciens , and the like. Regarding the need for a terminator, it is generally known that the terminator region increases the certainty and efficiency of gene transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

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

Introduction of a gene according to the present invention into a plant can be accomplished using a suitable vector comprising the OsHIR1 protein coding gene of the present invention, and methods for carrying the vector of the present invention into a plant host cell are well known in the art. For example, gene-mediated transformation (bombardment), Agrobacterium-mediated transformation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and DEAE-dextran treatment But is not limited thereto.

In one embodiment of the present invention, the heavy metal may be, but is not limited to, arsenic or cadmium.

The present invention also provides a method for producing a recombinant vector comprising the steps of: transforming a plant cell with a recombinant vector comprising a gene encoding OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) And

The present invention provides a method for producing a transgenic plant having increased heavy metal stress tolerance and reduced heavy metal absorption rate as compared to the wild type comprising regenerating the plant from the transformed plant cell.

In one embodiment of the present invention, the OsHIR1 protein may be an amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The method of transforming plant cells is as described above. Regeneration of transgenic plants from the transformed plant cells can be carried out by any method known in the art.

In one embodiment of the present invention, the heavy metal may be arsenic or cadmium, but is not limited thereto.

In addition, the present invention provides transgenic plants and seeds thereof, wherein the heavy metal stress tolerance produced by the method is increased and the heavy metal absorption rate is decreased.

In one embodiment of the present invention, the plants used in the present invention are selected from the group consisting of Arabidopsis, potato, eggplant, cigarette, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, Such as rice, barley, wheat, rye, corn, sugar cane, oats, onions, etc., such as Chinese cabbage, cabbage, gangbu, watermelon, melon, cucumber, amber, pak, strawberry, soybean, mung bean, It may be monocotyledons, preferably Arabidopsis thaliana or rice ( Oryza sativa ), but is not limited thereto.

The present invention also provides a composition for increasing heavy metal stress tolerance and reducing heavy metal absorption of a plant comprising a recombinant vector comprising a gene encoding an OsHIR1 protein derived from rice, the amino acid sequence of SEQ ID NO: 2. The composition includes a recombinant vector containing the gene coding for the amino acid sequence of SEQ ID NO: 2 as an active ingredient. The gene can be transformed into a plant to increase heavy metal stress tolerance of the plant and reduce heavy metal absorption . The heavy metal may be, but is not limited to, arsenic or cadmium.

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.

Materials and methods

1. Plant material and growth condition

The seeds of Oryza sativa cv. Donganbyeo were germinated in water-soaked paper, and seeds were cultivated in soil (Baroker, Korea) with or without added arsenic (Na ₂ HAsO ₄ ) and cadmium (CdSO ₄ ) Hour light / 8 hours arm (22 ° C / 18 ° C). The rice plant height was measured by measuring rules after 4 weeks of sowing in order to confirm plant growth response to various concentrations of arsenic and cadmium treatment. At the same time, plants were sampled to measure levels of 47 RFP gene transcripts by arsenic and cadmium.

OsHIR1 - over-expressing Arabidopsis thaliana (ecotype Columbia, Col-0) to develop the plants, the entire coding sequence of the forward OsHIR1 5'-GAGATCTAGAATGGGGTCATTGTGCTG-3 '(SEQ ID NO: 3) and reverse 5'-GAGAGGTACCCATGCAGGCAGTTCTTGA-3' (SEQ ID NO: 4) RT-PCR using primers. The PCR product was inserted into a 35S: EGFP vector and transformed with the floral-dip method using Agrobacterium strain GV3101 containing 35S: OsHIR1-EGFP or 35S: EGFP (control) Respectively. For selection of foreign homozygous lines, T ₃ seeds were grown in MS solid medium containing 50 μg / ml of kanamycin, and the transcription level of OsHIR1 was confirmed by RT-PCR. Root lengths were determined by culturing the Arabidopsis seeds and the Arabidopsis seeds overexpressing OsHIR1 in 1/2 MS solid medium containing 1.5% sucrose with or without 150 μM arsenic or 50 μM cadmium added under the same conditions as above Respectively. Plant growth was monitored for 12 days and root length was analyzed using ImageJ software (http://rsbweb.nih.gov/ij/).

2. Semi-quantitative RT - PCR  ( Semi - quantitative RT - PCR )

Total RNA was extracted using (Invitrogen, Carlsbad, CA, USA ) TRIzol, cDNA was synthesized using the ^{PrimeScript TM RTase (Takara-Bio,} Ohtsu, Japan) according to the manufacturer's protocol. The design of primers for 47 OsRFP genes for semi-quantitative RT-PCR was performed by the method described in the previous study (Lim et al. al . , 2013b, DNA Res 20: 299-314). Os18S - rRNA was used as an internal control.

3. In vitro Ubiquitination  analysis ( In vitro ubiquitination assay )

The RING-H2 domain region (amino acids 285 to 343) of OsHIR1 was amplified by PCR using the forward 5'-GAGACATATGATGTGCGACCTTTGCGAGAGGC-3 '(SEQ ID NO: 5) and reverse 5'-GAGAGTCGACCTAGCAGGCAGGGCAAGGAG-3' (SEQ ID NO: 6) primers. PCR product and pMAL-c5x expression vector (New England Biolabs, Beverly, Mass., USA) were digested with Nde I and Sal I and then ligated, respectively. Recombinant MBP-OsHIR1 ^285-343 vector and non-recombinant MBP vector (negative control) were expressed in E. coli BL21 (DES) pLysS (Promega, Madison, Wis., USA) Purification by chromatography (New England Biolabs, Beverly, Mass., USA). The purified MBP-OsHIR1 ^285-343 , non-recombinant MBP, Arabidopsis E2 (AtUBC10 and AtUBC11) and yeast E1 (Boston Biochemicals, Boston, Mass., USA) underwent an in vitro self-ubiquitination assay (Lim et al. , 2013b, DNA Res 20: 299-314). MBP-OsHIR1 ^{285 -343} fusion protein (300 ng) was ^incubated with ubiquitination reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 0.05 mM ZnCl ₂ , 1 mM ATP, 0.2 mM DTT, 10 mM phosphocreatine And 0.1 unit of creatine kinase) with 50 ng of E1, 250 ng of E2 and 10 μg of bovine ubiquitin (Sigma-Aldrich, St. Louis, MO, USA). The reaction mixture was reacted at 30 ° C. for 3 hours, and the reaction was terminated by adding 6 × sample buffer. The reaction mixture was then heated at 95 ° C for 5 minutes, and each sample (15 μl) was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. Anti-immune blot analysis with ubiquitin antibodies (Sigma-Aldrich, St. Louis, MO, USA) was performed by the method described in previous studies (Lim et al . , 2013a, J Exp Bot 64: 2899-2914).

4. Proteolytic analysis

The full-length cDNA of OsHIR1 was amplified using the forward 5'-GAGAGGATCCATGGGGTCATTGTGCTG-3 '(SEQ ID NO: 7) and the reverse 5'-GAGAGGTACCCATGCAGGCAGTTCTTGA-3' (SEQ ID NO: 4) primer and then cloned into 35s :: 8xcMyc vector. Agrobacterium strain GV3101 containing 35S: OsHIR1 -8 xcMyc and 35S: OsTiP4: 1-EYFP , respectively, was inoculated on tobacco (Nicotiana benthamiana) leaves for proteolytic analysis. The leaves inoculated with Agrobacterium were sampled and homogenized and then harvested in a circular extraction buffer 1 (NB1; 50 mM TRIS-MES, pH 8.0, 0.5 M sucrose, 1 mM MgCl ₂ , 10 mM EDTA, 5 mM DTT, CompleteMini tablet [Roche, Germany]) at 4 [deg.] C for 5 minutes. Each sample was centrifuged at 16,000 x g for 30 minutes at 4 ° C, and the supernatant was used for analysis. OsHIR1-8xcMyc or empty-8xcMyc extracts were mixed with the OsTIP4; 1-EYFP extract at a volume ratio of 1: 1, followed by the final concentration of ATP adjusted to 10 μM. Each sample was reacted at 4 [deg.] C for 3 hours with or without 50 [mu] M MG132 added. 20 μl of each of the reactants were analyzed by immunoblot using antibodies against c-Myc (sc-40, Santa Cruz, CA, USA) and GFP (G1544, Sigma-Aldrich, MO, USA).

5. Total arsenic and cadmium analysis

OsHIR1 -overexpressing Arabidopsis seeds and control plant seeds were germinated by incubation for 2 days in 1/2 MS solid medium containing 1.5% sucrose. Germinated seeds were transferred to 1/2 MS solid medium with or without 50 μM arsenic (V) or 50 μM cadmium, and grown for 2 weeks in a controlled growth chamber. Plant tissues (root and stem) were washed with cold buffer (1 mM K ₂ HPO ₄ , 0.5 mM Ca (NO ₃ ) ₂ and 5 mM Mes, pH 5.6) to remove apoplastic arsenic and cadmium Arsenic and cadmium contents were measured. The samples were dried at 60 ° C for 3 days and mineralized with nitric acid (HNO ₄ ) -hydrogen peroxide (H ₂ O ₂ ) using a microwave accelerated reaction system (MARS-X, HP-500 plus, CEM, USA) (ICP-OES, Optima 7300 DV, Perkin Elmer, USA) were used to analyze total arsenic and cadmium contents in a central laboratory of Kangwon National University.

Example 1. Expression patterns of 47 OsRFP genes in response to arsenic and cadmium

To identify the phenotype of cultivar Donganbyeo caused by arsenic and cadmium, plants were treated with various concentrations of arsenic and cadmium for 4 weeks each. As a result, it was confirmed that significant difference in plant growth occurred between 600 ~ 1600 μM arsenic and 130 ~ 650 μM cadmium treatment. The samples were then treated with arsenic (30, 300 and 1000 μM) or cadmium (13, 130 and 390 μM), respectively. The amount of expression of 47 OsRFP genes in each sample was confirmed by semi-quantitative RT-PCR analysis (FIG. 1). Among them, 5 OsRFP genes such as OsRFPH2-3 , OsRFPH2-11 , OsRFPH2-18 , OsRFPHC-3 and OsRFPHC-4 showed an increase in the expression level as the throughput of arsenic and cadmium increased. In addition, OsRFPH2-5 , OsRFPH2-12, and OsRFPv-2 genes showed decreased expression levels with increasing amounts of arsenic and cadmium. OsRFPH2-22 and OsRFPHC-14 genes increased in expression level with increasing arsenic content, but gene expression decreased with increasing cadmium throughput. Therefore, the present inventors were OsRFPH2-11 select genes that are highly expressed as the increase in the throughput in both the case of arsenic and cadmium processing two, OsHIR1 was renamed as (O ryza s ativa h eavy metal i nduced R ING E3 ligase 1).

Example 2. Function of OsHIR1 as E3 ligase

The OsHIR1 gene is composed of 433 amino acids, and the molecular weight of the OsHIR1 protein is estimated to be 47 kDa. Subsequent alignment of the homologous genes of the OsHIR1 gene and the brachypodium, maize, sorghum and Arabidopsis lines confirmed the well conserved C3H2C3-type RING-H2 domain (amino acids 285-343) in the C terminal residue ). Proteins with the RING domain are known for their function as E3 ubiquitin ligases. Therefore OsHIR1 the E3 Riga to verify that represents the kinase activity, battlefield OsHIR1 The fusion protein with MBP (maltose-binding protein) was expressed in E. coli (BL21). The full-length OsHIR1-MBP protein exhibited low expression levels or was expressed in an insoluble form due to its hydrophobic character. Next, the present inventors confirmed that the RING-H2 domain of OsHIR1 protein has E3 ubiquitin ligase activity. Recombinant MBP-OsHIR1 ^285-343 or non-recombinant empty-MBP were expressed in E. coli , respectively, and each of the purified proteins was purified using the ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme UBC10 11) was reacted with the enzyme in ubiquitination buffer at 30 ° C for 3 hours. The ubiquitination activity was measured using an anti-ubiquitin antibody. MBP-OsHIR1 ^285-343 showed a large number of ubiquitinated bands whereas Empty-MBP did not show ubiquitinated bands. It was also confirmed that MBP-OsHIR1 ^285-343 does not play a role as ubiquitin ligase in the absence of E1 and E2 (FIG. 2B). These results indicate that the OsHIR1 protein containing the RING-H2 domain functions as an E3 ubiquitin ligase.

Example 3. OsHIR1 - Improvement of arsenic and cadmium resistance in overexposed Arabidopsis thaliana

Three independent transfection lines (# 1, # 11, and # 12) confirmed to express the OsHIR1 gene were selected by RT-PCR and treated with 150 μM arsenic and 50 μM cadmium, Respectively. OsHIR1 In the case of overexpressed lines, there was no phenotypic difference in the normal environment as compared to the control plant in which only the vector was inserted, but it was confirmed that the root length was longer when treated with arsenic and cadmium than in the control plant (Fig. 3A ). In order to investigate whether the absorption of arsenic and cadmium by OsHIR1 ubiquitin E3 ligase in transgenic plants was affected, the accumulation levels of arsenic and cadmium in control plants and OsHIR1 overexpressing transgenic plants were compared. The arsenic concentrations measured in the stem and root of the OsHIR1 overexpressed plants were 14.1% and 46.6%, respectively, which was lower than that of the control plants (Fig. 3B). Cadmium was also measured 12.3% and 3.8% lower in the stem and root of OsHIR1 overexpressing plants than the control plants, respectively. These results suggest that when the OsHIR1 gene is overexpressed in the Arabidopsis thaliana, the absorption of arsenic and cadmium by arsenic and cadmium can be controlled by controlling the substrate protein that controls the absorption of arsenic and cadmium.

Example 4 OsHIR1-promoted OsTIP4; 1 degradation by the ubiquitin 26S proteasome system (OsHIR1-promoted OsTIP4; 1 degradation)

To confirm whether the OsHIR1 gene promotes OsTIP4; 1 protein degradation, the present inventors expressed OsHIR1-cMyc and OsTIP4; 1-EYFP, respectively, and performed proteolytic analysis. OsTIP4; 1-EYFP protein levels were measured by immunoblot analysis using an anti-GFP N-terminal antibody with or without the OsHIR1-cMyc protein. When OsHIR1-cMyc was reacted together, the protein level of OsTIP4: 1-EYFP was dramatically reduced after 3 hours of reaction compared to the control (empty-cMyc vector) (Fig. 4A, B). Subsequently, MG132, an inhibitor of 26S proteasome, was used to determine whether proteolysis occurred via the ubiquitin proteasome pathway. OsTIP4; 1-EYFP and OsHIR1-cMyc were reacted for 3 hours with or without MG132, respectively. As shown in FIG. 4C, when all of the reactions were treated with the same amount of OsHIR1-cMyc protein, the proteolysis of OsTIP4; 1-EYFP did not occur when MG132 was reacted together. These results indicate that OsHIR1 ubiquitin E3 ligase binds to OsTIP4; 1 through the 26S proteasome degradation pathway and mediates proteolysis of OsTIP4; 1.

<110> KNU-Industry Cooperation Foundation <120> OsHIR1 gene increased heavy metal stress tolerance and          decreasing heavy metal uptake of plant and uses thereof <130> PN13270 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 1302 <212> DNA <213> Oryza sativa <400> 1 atggggtcat tgtgctgcgt cgcgtcgagg ccccatgggg ccagcacggc tagccgggag 60 tggtcctcga ttgggcggag cgatccgctc tggcggacta acgctggctt ttcgccgccg 120 ctgtcgagga ggtgggagta ccgcatcaat tctgaggggc tgtcgtatgg ctcccagggt 180 gacagcgggg ctgctgcgca ctacggttcg tcgctatcct cgaatagtaa agagcctagt 240 aggagctggg agaggagtga tgttccaccg gatcatcatc gttactccac ctcggaaggt 300 gccatttcgt attttaacag tccggatgtt accttccaga accaccatat tatgcttcct 360 atgctgcagg attctggcat tgatgaatac atgcgagtct cggtggctga gccaattggt 420 gcattgttat tgtcagaggg gatatctgga cagcaaaaca gtggtggttc cacttcctct 480 cgctctgatg gaagtgaata tgatattgta ccaaaatcat attcctctac cccacgcaac 540 ttcccaagtc gtcgatcatt cctgtcaaag ccaatccatc cgctctcgtt tccagagcat 600 gcactagaag ggcaagaaac tgatagccct gttgccaatg caagcaccag cagtcctatg 660 ccctcggagt tcaaggcaat aggggagatc cgcccgtcag gtctgatgga ctacgcctac 720 gccagtggca gtcatgggga gtcagcaaac tggagtgccg caagtagcat ggatcttacc 780 gatttatcag aacgacatga tgctgaacgc tctggaccac tgcgttccaa taacataatg 840 gatagaacaa ggtgcgacct ttgcgagagg ctcttgtcca agagatcgcc ctggggctcc 900 cgccgaatcg tccgtactgg agatctgcct gttgctggtg tgcttccctg ctgccatgtc 960 taccatgccg agtgcttgga gcgaaccact cccaaggggc agaagcatga ccctccttgc 1020 cctgcctgcg ataggctgtc cggcaaagac accgagcagt ggtcgatctg caggctaaga 1080 aacggattcc cgaggctaag gtcacttggg gaaggccctt ccagggtctg gagctgtgcc 1140 caagctggag actgcgtggc tggcgctgtc cagataccaa gagcaagcag catctctctg 1200 ctgagccgga gtggccacaa gaggcatcat gctgcttcca agggagaatc cggcaaagac 1260 tgggctgaga catcatcatc atcaagaact gcctgcatgt ag 1302 <210> 2 <211> 433 <212> PRT <213> Oryza sativa <400> 2 Met Gly Ser Leu Cys Cys Val Ala Ser Arg Pro His Gly Ala Ser Thr   1 5 10 15 Ala Ser Arg Glu Trp Ser Ser Ile Gly Arg Ser Asp Pro Leu Trp Arg              20 25 30 Thr Asn Ala Gly Phe Ser Pro Pro Leu Ser Arg Arg Trp Glu Tyr Arg          35 40 45 Ile Asn Ser Glu Gly Leu Ser Tyr Gly Ser Gln Gly Asp Ser Gly Ala      50 55 60 Ala Ala His Tyr Gly Ser Ser Leu Ser Ser Asn Ser Lys Glu Pro Ser  65 70 75 80 Arg Ser Trp Glu Arg Ser Asp Val Pro Pro Asp His His Arg Tyr Ser                  85 90 95 Thr Ser Glu Gly Ala Ile Ser Tyr Phe Asn Ser Pro Asp Val Thr Phe             100 105 110 Gln Asn His His Ile Met Leu Pro Met Leu Gln Asp Ser Gly Ile Asp         115 120 125 Glu Tyr Met Val Val Ser Ala Glu Pro Ile Gly Ala Leu Leu Leu     130 135 140 Ser Glu Gly Ile Ser Gly Gln Gln Asn Ser Gly Gly Ser Thr Ser Ser 145 150 155 160 Arg Ser Asp Gly Ser Glu Tyr Asp Ile Val Pro Lys Ser Tyr Ser Ser                 165 170 175 Thr Pro Arg Asn Phe Pro Ser Arg Arg Ser Phe Leu Ser Lys Pro Ile             180 185 190 His Pro Leu Ser Phe Pro Glu His Ala Leu Glu Gly Gin Glu Thr Asp         195 200 205 Ser Pro Val Ala Asn Ala Ser Thr Ser Ser Pro Met Pro Ser Glu Phe     210 215 220 Lys Ala Ile Gly Glu Ile Arg Pro Ser Gly Leu Met Asp Tyr Ala Tyr 225 230 235 240 Ala Ser Gly Ser Gly Gly Ser Ala Asn Trp Ser Ala Ala Ser Ser                 245 250 255 Met Asp Leu Thr Asp Leu Ser Glu Arg His Asp Ala Glu Arg Ser Gly             260 265 270 Pro Leu Arg Ser Asn Asn Ile Met Asp Arg Thr Arg Cys Asp Leu Cys         275 280 285 Glu Arg Leu Leu Ser Lys Arg Ser Pro Trp Gly Ser Arg Arg Ile Val     290 295 300 Arg Thr Gly Asp Leu Pro Val Ala Gly Val Leu Pro Cys Cys His Val 305 310 315 320 Tyr His Ala Glu Cys Leu Glu Arg Thr Thr Pro Lys Gly Gln Lys His                 325 330 335 Asp Pro Pro Cys Pro Ala Cys Asp Arg Leu Ser Gly Lys Asp Thr Glu             340 345 350 Gln Trp Ser Ile Cys Arg Leu Arg Asn Gly Phe Pro Arg Leu Arg Ser         355 360 365 Leu Gly Glu Gly Pro Ser Arg Val Trp Ser Cys Ala Gln Ala Gly Asp     370 375 380 Cys Val Ala Gly Ala Val Gln Ile Pro Arg Ala Ser Ser Ile Ser Leu 385 390 395 400 Leu Ser Arg Ser Gly His Lys Arg His His Ala Ala Ser Lys Gly Glu                 405 410 415 Ser Gly Lys Asp Trp Ala Glu Thr Ser Ser Ser Ser Thr Ala Cys             420 425 430 Met     <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gagatctaga atggggtcat tgtgctg 27 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gagaggtacc catgcaggca gttcttga 28 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gagacatatg atgtgcgacc tttgcgagag gc 32 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gagagtcgac ctagcaggca gggcaaggag 30 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gagaggatcc atggggtcat tgtgctg 27

Claims (9)

Comprising transfecting a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) protein consisting of the amino acid sequence of SEQ ID NO: 2 and overexpressing the OsHIR1 gene. Wherein the arsenic or cadmium resistance of the plant is increased and the arsenic or cadmium absorption is reduced. delete delete Transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsHIR1 ( Oryza sativa heavy metal induced RING E3 ligase 1) protein comprising the amino acid sequence of SEQ ID NO: 2; And
Wherein the arsenic or cadmium resistance is increased and the rate of arsenic or cadmium absorption is reduced as compared to the wild type comprising regenerating the plant from the transformed plant cell. delete delete An improved transgenic plant having increased resistance to arsenic or cadmium produced by the method of claim 4 and reduced arsenic or cadmium absorption. 8. A seed of a transgenic plant according to claim 7. A composition for increasing arsenic or cadmium resistance and reducing arsenic or cadmium absorption of a plant containing a recombinant vector comprising a gene encoding an OsHIR1 protein derived from rice, the amino acid sequence of SEQ ID NO: 2.

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