CN117887740A - OsBCAT2 gene for regulating drought stress resistance of plants and application thereof - Google Patents
OsBCAT2 gene for regulating drought stress resistance of plants and application thereof Download PDFInfo
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Abstract
The invention relates to a rice branched-chain amino acid transaminase 2 (Oryza sativa branched-chain amino acid aminotransferase 2, osBCAT 2) gene for regulating drought stress resistance of plants and application thereof.
Description
Technical Field
The invention relates to a rice branched-chain amino acid transaminase 2 (Oryza sativa branched-chain amino acid aminotransferase 2, osBCAT 2) gene for regulating drought stress resistance of plants and application thereof.
This work was supported by the start-up growth technology development project (TIPS project (No. s 3307687)) sponsored by the small and initial enterprises department (MSS, korea) in 2022.
Background
Unpredictable weather events occur worldwide due to climate change. Abiotic stress refers to a natural event with disastrous consequences for nearby organisms. Moreover, extreme weather disasters bring about crop yield reduction. Drought stress has a considerable impact on international crop production. According to recent reports, drought can bring irreversible damage to rice (Oryza sativa. L) plants during their entire growth period, drastically reducing the agricultural characteristics of the harvested rice. Specifically, in the nutrition stage of rice (Yangliangyou 6, hanyou 113), drought stress is reduced by 23% -24%.
Plants have a variety of strategies for combating abiotic stress. At the molecular level, plants regulate genes associated with stress response through stress-specific signaling pathways. Genetic lesions are caused by the modulation of morphology, biochemistry, and implicit biochemistry. For example, plant amino acids play an essential role in minimizing adverse effects of abiotic stress. This mechanism is associated with the regulation of amino acid content in plant cells. In particular, proline is an amino acid component that accumulates in response to a wide range of abiotic stresses. Proline is known to accumulate in a variety of plants during drought, salt, high temperature, low temperature, and nutrient loss. Gamma-aminobutyric acid (GABA) relieves drought and high temperature stress in sunflower by antioxidant genes. Furthermore, glycine, taurine, arginine and serine are reported to be stress-inducing amino acids, which act as osmotic agents. Interestingly, it is reported that among plant amino acids, branched Chain Amino Acids (BCAA), leucine (Leu), isoleucine (Ile) and valine (Val) are accumulated in plants under stress conditions. Interestingly, in the case of calculating the fold change value of amino acids induced by stress, the accumulation amount of branched-chain amino acids was higher than proline, which positively affects the state of water deficiency.
Branched chain amino acids play an important role in protein synthesis and plant development. And, it is also involved in the formation of various secondary metabolites such as cyanogenic glycosides, thioglucosides, and acylsugars. Branched-chain amino acid synthesis is formed by two different substrates. The preliminary pathway for valine and leucine starts with pyruvic acid produced by the relevant process. In contrast, the synthesis of isoleucine starts with 2-ketobutyric acid after deamination of threonine by threonine deaminase (threonine deaminase, TD, ec 4.2.16). The leucine-producing enzyme of the first valine-related enzyme is known as an acetocarboxylic acid synthetase (acetohydroxyacid synthase, AHAS, EC 2.2.1.6), which catalyzes the formation of acetohydroxybutyrate from valine-related acetolactate and one pyruvate. Ketol-acid reductase (ketol-acid reductoisomerase, KARI, EC 1.1.1.86) and dicarboxylic dehydratase (DHAD, EC 4.2.1.9) continuously catalyze the basic two-stage reaction for synthesis of branched-chain amino acids. Branched-chain amino acid aminotransferases (BCATs, EC 2.6.1.42) act as the final stages of branched-chain amino acid biosynthesis in the plastids.
Catabolism of branched-chain amino acids occurs mainly in mitochondria. BCAT is an enzyme associated with the first stage of decomposition and the last stage of synthesis of branched-chain amino acids. BCAT belongs to pyridoxal 5' phosphate-dependent aminotransferase class IV, converting valine, isoleucine or leucine to alpha-keto acid KIV, KIC or KMV. Branched-chain keto acid dehydrogenases (Branched-chain keto acid dehydrogenase, BCKDH, or BCKDH complex (complex) EC 1.2.4.4) catalyze the formation of linear alpha-keto acids into isobutyryl-CoA (CoA), isovaleryl-CoA or methylbutyl-CoA. Then, isovaleryl-CoA dehydrogenase (Isovalyl-CoA dehydrogenase, IVD, EC 1.3.99.10) identified in various plant species catalyzes the conversion of acyl-CoA to enoyl-CoA. Although not fully described, the substrate specificity of the enzyme is shown differently in different species of these proteins.
Interestingly, such resolvers have multiple BCAT isomers in different plant species. 7 and 6 different BCAT isomer forms were confirmed in arabidopsis thaliana and tomato (Solanum lycopersicum), respectively. BCAT2 (At 1g 10070), slBCAT1 (SGN-U569828), slBCAT2 (SGN-U569830) are restricted to perform important functions in the catabolism of branched chain amino acids in mitochondria. Nevertheless, the pathways associated with the same gene and with the breakdown of protein networks are not completely defined in plants.
On the other hand, korean laid-open patent No. 2021-0063574 discloses "drought stress resistance transformed plant inhibiting expression of AtPR5K2 protein", korean laid-open patent No. 2022-0083464 discloses "novel branched chain amino acid transaminase variant and isoleucine production method using the same", but there is no description about the "BCAT 2 gene regulating plant drought stress resistance" of the present invention.
Disclosure of Invention
The present invention was made in view of the above-described requirements, and the present inventors have studied the functions of BCAT2 derived from rice in plants based on studies of the effects of branched-chain amino acids related to plant resistance under abiotic stress conditions, which have been confirmed in the past.
The present inventors prepared a mutant (osbat 2) that lost the function of rice BCAT2 using CRISPR-Cas9 system, confirmed the drought stress tolerance of the above osbat 2, and confirmed that the osbat 2 mutant exhibited improved drought tolerance and agricultural harvest under various drought stress conditions as compared to the wild type, thereby completing the present invention.
In order to solve the above problems, the present invention provides a method for regulating drought stress resistance of plants, comprising the step of regulating expression of a rice-derived BCAT2 protein-encoding gene in plant cells.
Furthermore, the present invention provides a method for preparing a transformed plant with regulated drought stress resistance, comprising: a step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived BCAT2 protein; and a step of redifferentiating the transformed plant from the above-mentioned transformed plant cells.
Also, the present invention provides a transformed plant having a regulated drought stress resistance prepared by the above-described method for preparing a transformed plant and transformed seeds thereof.
Furthermore, the present invention provides a method for preparing a genome-editing rice plant having increased resistance to drought stress, comprising: step (a), introducing guide RNA (guide RNA) and endonuclease (endonucleoase) proteins specific to a target base sequence of a gene encoding a rice-derived BCAT2 protein into rice plant cells to edit a genome; and (b) subdividing the plant from the rice plant cells editing the genome.
The present invention also provides a genome editing rice plant having increased resistance to drought stress prepared by the above method for preparing a genome editing rice plant, and a seed for editing its genome.
Also, the present invention provides a composition for modulating drought stress resistance of plants comprising a rice-derived BCAT2 protein-encoding gene as an active ingredient.
Ensuring plant resistance to environmental stress is a very important property in the industrial field, and it is expected that the rice-derived BCAT2 gene of the present invention will be usefully used in applications for improving drought stress resistance of plants.
Drawings
FIG. 1 shows the expression pattern of OsBCAT2 for various abiotic stresses and plant hormones. (a) Part is the relative expression level of OsBCAT2 in drought (air-drying), salt (400 mM NaCl) and low temperature (4 ℃) stress conditions, and part (b) is the relative expression level of OsBCAT2 after 100. Mu.M abscisic acid (ABA), methyl jasmonate (MeJA) and Salicylic Acid (SA) treatment, respectively. Using the internal standard for normalizing rice ubiquitin 1 (Os 06g 068100), error bars represent standard deviations of 3 repeated measurements.
FIG. 2 shows the phenotype of rice plants in osmotic stress induced by polyethylene glycol (PEG) after pretreatment with free amino acid.
Figure 3 shows the phenotype of NT plants pretreated with free amino acids under air conditions. Part (a) shows the abiotic stress phenotype induced by air drying (n=10), (b) shows the relative moisture content over time of NT plants of the pre-treated amino acids during the air drying treatment, and (c) shows the total moisture loss during air drying.
Fig. 4 shows the propensity of OsBCAT2 expression in various developmental stages and tissues. Total ribonucleic acid (RNA) was extracted from various tissues and developmental stages (D, dark; L, light; D, day; w, week; M, month; M, meiosis; BH, pre-emergence (before head; AH, post-emergence (after head)), and the expression level of OsBCAT2 was analyzed by real-time quantitative polymerase chain reaction (RT-PCR). Using the internal standard for normalizing rice ubiquitin 1 (Os 06g 068100), error bars represent standard deviations of 3 repeated measurements.
FIG. 5 shows the intracellular location of OsBCAT2 in rice protoplasts, transformed with 35S: osBCAT2-GFP and CD3-991-mCherry as mitochondrial markers, inducing temporary expression. Size column: 10 μm.
Fig. 6 shows the loss of function structure and genotype of OsBCAT2 through CRISPR-Cas9 system. (a) Part (b) shows the design of early stop codons derived from the first exon region of two single guide ribonucleic acids, and part (b) shows the mutation patterns of osbcat2 mutants #3, #4, #5, #10 and # 15. Underlined indicates the pre-spacer adjacent motif (PAM) sequence and hyphens indicate base deletions.
Fig. 7 shows that loss of function of OsBCAT2 confers drought tolerance. (a) In part, 5 other osbcat2 systems were used, including patterns of plants in which NT plants were sequentially subjected to drought stress and re-irrigation in the greenhouse under normal conditions for one month. Days shown in the pictures represent the time of drought treatment and re-irrigation. (b) In part, the optical efficiency measurements during drought treatment, fv/Fm values represent the quantum yield of photosystem II. (c) The performance index of the fractions shows the efficiency of two photosystems (I and ii) that perform potential photosynthesis in the leaves of the plants (n=10). (d) Partly the calculated survival rate recovered by re-irrigation after drought treatment. (e) Part shows soil moisture (n=20) as a function of time during drought treatment. Asterisks indicate significance relative to NT analyzed by unpaired student's t-tests, p <0.05.
Fig. 8 shows that loss of OsBCAT2 in stems and roots promotes branched-chain amino acid content under a variety of drought stress conditions. (a) Part shows the branched-chain amino acid content in the NT plants and the osbcat2 mutants before and after air drying treatment, (b) part shows the accumulation of branched-chain amino acid content in the NT plants and the osbcat2 mutants over time with drought stress. Error bars represent standard deviations of 3 replicates, asterisks indicate significance relative to NT analyzed by unpaired student's t-test, p <0.05, p <0.01, p <0.0001.
Fig. 9 shows that the harvest of the osbcat2 mutant is improved under drought stress conditions, (a) part shows normal conditions, (b) part shows the agricultural properties of NT plants and the osbcat2 mutant under arid field conditions. The average value of NT plants in table 3 was assigned as a reference value of 100%. CL: stem length (cup length); PL: spike length (panicle length); NP: number of ears (number of panicles); NS: number of spikelets (number of spikelets); NTS: total spike number (number of total spikelets); FR: filling rate (filling rate), TGW: total grain weight (total grain weight); 1000GW: thousand grain weight (1000-grain weight).
FIG. 10 shows the results of transcript analysis of the jasmonic acid related genes of NT plants and osbcat2 mutants over drought stress time in a greenhouse environment. Part (a) shows the relative expression level of the core enzyme in JA-Ile formation, (b) shows the relative expression level of the jasmonic-responsive gene, (c) shows the relative expression level of the major gene of the jasmonic acid signal. Using the internal standard for normalizing rice ubiquitin 1 (Os 06g 068100), error bars represent standard deviations of 3 repeated measurements. Asterisks indicate the significance of p <0.05, p <0.01, p <0.0001 analyzed by unpaired student's t-tests relative to NT.
Fig. 11 shows that loss of function of OsBCAT2 in a water deficient state increases the Reactive Oxygen Species (ROS) digestion ability over time. (a) Partly for the detection of H in NT plants and osbcat2 mutants under drought stress conditions 2 O 2 O and O 2 - The results of the histochemical staining analysis of DAB and NBT in (a) were quantified in the stained area. (d) Showing the relative expression levels of L-ascorbate peroxidase2 (L-ascorbate peroxidase2, APX 2), catalase (CAT) and superoxide dismutase1, 2 (superoxide dismutase1, 2 (SOD 1, 2) as antioxidant genes using internal standards for normalizing rice ubiquitin 1 (Os 06g 068100), error bars represent standard deviations of 3 repeated measurements asterisks represent phases analyzed by unpaired student's t-testSignificance for NT, p<0.05，**p<0.01，****p<0.0001。
Detailed Description
To achieve the object of the present invention, the present invention provides a method for regulating drought stress resistance of plants, comprising the step of regulating expression of a rice-derived BCAT2 protein-encoding gene in plant cells.
In the method of modulating drought stress resistance according to an embodiment of the present invention, preferably, the rice-derived BCAT2 protein may consist of the amino acid sequence of sequence 2, but is not limited thereto.
The rice-derived BCAT2 protein of the invention includes, in its scope, a protein having an amino acid sequence represented by sequence 2 and functional equivalents of the above protein. In the present invention, the term "functional equivalent" means a protein which has 70% or more sequence homology, preferably 80% or more sequence homology, more preferably 90% or more sequence homology, and even more preferably 95% or more sequence homology to the amino acid sequence represented by the above sequence 2, as a result of addition, substitution or deletion of amino acids, and which shows substantially the same physiological activity as the protein represented by the sequence 2. "substantially homogenous physiological activity" refers to an activity that modulates drought stress resistance of a plant.
Further, the present invention includes genes encoding rice-derived BCAT2 proteins, and the scope of the genes includes genomic deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), and synthetic deoxyribonucleic acid encoding rice-derived BCAT2 proteins. Preferably, the gene encoding rice-derived BCAT2 protein of the present invention may comprise a rice-derived BCAT2 base sequence represented by sequence 1. Also, a homologue of the above base sequence is included in the scope of the present invention. Specifically, the above gene may contain a base sequence having a sequence homology of 70% or more, preferably a base sequence having a sequence homology of 80% or more, more preferably a base sequence having a sequence homology of 90% or more, most preferably a base sequence having a sequence homology of 95% or more, with the base sequence of sequence 1, respectively. The "% of sequence homology" for a polynucleotide is confirmed by comparing two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., deletions) as compared to a reference sequence (excluding additions or deletions) for optimal alignment of the two sequences.
The method for regulating drought stress resistance of a plant of the present invention suppresses the expression of the above rice-derived BCAT2 protein-encoding gene in plant cells, thereby increasing the drought stress resistance of the plant as compared to non-transformed plants, but is not limited thereto.
In one embodiment of the present invention, the above-mentioned inhibition of the expression of the gene encoding the rice-derived BCAT2 protein may be by inserting T-DNA into the rice-derived BCAT2 protein-encoding gene, translocating (translocating), irradiating with X-rays or gamma-rays to induce mutation, blocking (inhibiting) the expression of the rice-derived BCAT2 protein-encoding gene by ribonucleic acid interference (RNAi) or antisense ribonucleic acid using CRISPR/Cas9 gene editing system, but the present invention is not limited thereto and any method may be used as long as it is a method for inhibiting the expression of a gene, which is usual in the art to which the present invention pertains.
In the recombinant vector of the present invention, the above-mentioned promoter is 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 not limited thereto.
Preferably, the recombinant expression vector may comprise more than one selectable marker. In general, the above-mentioned markers are nucleic acid sequences having chemically selectable properties, and all genes that are distinguishable from non-transformed cells except for the transformed cells correspond thereto. The marker gene may be a dominant drug resistance gene (dominant drug resistance gene), but is not limited thereto.
In the case of transforming the vector of the present invention into eukaryotic cells, yeast (e.g., saccharomyces cerevisiae (Saccharomyce cerevisiae)), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary cell (Chinese hamster ovary)), W138, BHK, COS-7, 293, hepG2, 3T3, RIN, and MDCK cell lines), plant cells, etc., may be used as host cells, and plant cells are preferably used.
Also, the present invention provides a method for preparing a transformed plant with modulated drought stress resistance, comprising: a step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived BCAT2 protein; and a step of redifferentiating the transformed plant from the above-mentioned transformed plant cells.
In the method for producing a transformed plant of the present invention, preferably, the rice-derived BCAT2 protein may include a protein consisting of the amino acid sequence of sequence 2 and functional equivalents of the protein, as described above.
In the method for producing a transformed plant of the present invention, the method for transforming the plant cell is as described above, and any method known in the art of the present invention can be used for differentiating the transformed plant from the transformed plant cell. Transformed plant cells should be subdivided into whole plants. Techniques for redifferentiating mature plants by culture of calli or protoplasts are well known in the art to which the invention pertains.
In the method for preparing a transformed plant according to an embodiment of the present invention, when the expression of the above-described gene encoding rice-derived BCAT2 protein is inhibited in a plant cell, drought stress resistance of the plant can be improved as compared to a non-transformed plant, i.e., as compared to a wild type, but is not limited thereto.
The above-mentioned suppression of the expression of the rice-derived BCAT2 protein-encoding gene may be, but is not limited to, suppression of the expression of the rice-derived BCAT2 protein-encoding gene by transforming a plant cell with a recombinant vector containing sense (sense) or antisense (antisense) deoxyribonucleic acid or microRNA (microRNA) of the rice-derived BCAT2 gene, and a gene expression suppression technique known in the art to which the present invention pertains may be used.
Also, the present invention provides a transformed plant having a regulated drought stress resistance prepared by the above-described method for preparing a transformed plant and transformed seeds thereof.
The transformed plant of the present invention is characterized by increasing resistance to drought stress while inhibiting expression of a gene encoding a rice-derived BCAT2 protein. The plant may be monocotyledonous plants such as rice, barley, wheat, rye, corn, sugarcane, oat, onion, etc., or dicotyledonous plants such as arabidopsis thaliana, potato, eggplant, tobacco, red pepper, tomato, burdock, mustard, lettuce, balloonflower root, spinach, beet, sweet potato, carrot, caraway, chinese cabbage, radish, watermelon, melon, cucumber, pumpkin, cucurbit, strawberry, soybean, mung bean, kidney bean, pea, etc., preferably monocotyledonous plants, more preferably rice plants, but not limited thereto.
Furthermore, the present invention provides a method for preparing a genome-editing rice plant having increased resistance to drought stress, comprising: step (a), introducing a guide ribonucleic acid and an endonuclease protein which have specificity to a target base sequence of a gene encoding a rice-derived BCAT2 protein into a rice plant cell to edit a genome; and (b) subdividing the plant from the rice plant cells editing the genome.
In the present invention, the term "genome/gene editing" refers to a technique capable of introducing a target-directed mutation into a genomic base sequence of an animal or plant cell including a human cell, and refers to a technique capable of knocking out (knock-out) or knocking in (knock-in) a specific gene by deletion (deletion), insertion (insertion), substitution (substitution) or the like of one or more nucleic acid molecules cleaved by deoxyribonucleic acid, or introducing a mutation into a non-coding deoxyribonucleic acid sequence that does not produce a protein. In particular, the genome editing of the present invention may be performed by introducing a mutation into a plant using an endonuclease, for example, cas9 (CRISPR associated protein) protein or ribonucleic acid. Also, "gene editing" may be mixed with "gene modification".
The term "target gene" refers to a part of deoxyribonucleic acid present in the genome of a plant, which is desired to be edited by the present invention, and the kind of the gene is not limited and may include coding regions and non-coding regions. The relevant practitioner can select the target gene according to the desired variation for the genome editing plant to be prepared according to its purpose.
In the present invention, the term "guide ribonucleic acid" refers to a ribonucleic acid having specificity for a deoxyribonucleic acid of a base sequence encoding a target gene, and refers to a ribonucleic acid that functions to guide an endonuclease to a relevant target deoxyribonucleic acid by complementarily binding to all or a part of the base sequence of the target deoxyribonucleic acid. The guide ribonucleic acid means a double-stranded ribonucleic acid (dual RNA) comprising two ribonucleic acids, i.e., a clustered short palindromic repeat ribonucleic acid (crRNA, CRISPR RNA) and a transcriptionally activated clustered short palindromic repeat ribonucleic acid (dual RNA) as structural elements, or means a single-stranded guide ribonucleic acid (sgRNA) form comprising a first site comprising a sequence having complementarity to all or a part of a base sequence within a target gene and a second site comprising a sequence interacting with a ribonucleic acid-guide nuclease, but may be appropriately selected within the scope of the present invention without limitation according to the type of endonuclease to be used together, the microorganism from which the endonuclease is derived, and the like, by a technique known in the art.
The guide ribonucleic acid may be a guide ribonucleic acid transcribed from a plasmid template, transcribed (transcribed) in vitro (in vitro), or synthesized (for example, an oligonucleotide double strand), but is not limited thereto.
In the method for producing a genome editing rice plant of the present invention, the endonuclease protein may be at least one selected from the group consisting of Cas9, clustered regularly interspaced short palindromic repeats 1 (Cpf 1, CRISPR from Prevotella and Francisella 1) derived from prasugrel and francisco, transcription activator-like effector nucleases (TALENs, transcription activator-like effector nuclease), zinc finger nucleases (ZFNs, zinc Finger Nuclease) and functional analogues thereof, and preferably, but not limited thereto, cas9 protein.
The Cas9 protein may be one or more selected from the group consisting of Cas9 protein derived from streptococcus pyogenes (Streptococcus pyogenes), cas9 protein derived from campylobacter jejuni (Campylobacter jejuni), cas9 protein derived from streptococcus thermophilus (s.thermophilus) or staphylococcus aureus (s.aureus), cas9 protein derived from neisseria meningitidis (Neisseria meningitidis), cas9 protein derived from pasteurella multocida (Pasteurella multocida), cas9 protein derived from francissamara novaculum (Francisella novicida), and the like, but is not limited thereto. Information on Cas9 protein or its gene can be obtained from a well-known database such as GenBank of the national center for biotechnology information (NCBI, national Center for Biotechnology Information).
Cas9 protein is a double-stranded deoxyribonucleic acid (dsdna) break induced by ribonucleic acid-guided DNA endonuclease (double stranded DNA break). In order for the Cas9 protein to bind precisely to the target base sequence to cleave the deoxyribonucleic acid fragment, there should be a short base sequence of three bases called a pre-spacer adjacent motif (PAM, protospacer Adjacent Motif) beside the target base sequence, and the Cas9 protein is cleaved by locating the third and fourth base pairs by the pre-spacer adjacent motif sequence (NGG).
In the present invention, the formation of ribonucleoprotein (ribonucleoprotein) complex between the guide ribonucleic acid and the endonuclease protein can be started as ribonucleic acid gene scissors (RNA-Guided Engineered Nuclease, RGEN).
The CRISPR/Cas9 system used in the present invention is a gene editing method by a non-homologous end joining (NHEJ, non-homologous end joining) mechanism of incomplete repair insertion-deletion (InDel) mutation induced during deoxyribonucleic acid repair by introducing double helix cleavage to a specific position of a specific gene to be edited.
In the method for producing a genome editing rice plant of the present invention, in the step (a), when a guide ribonucleic acid and an endonuclease protein are introduced into a rice plant cell, a complex of a guide ribonucleic acid and an endonuclease protein, which are specific for a target base sequence of a rice-derived BCAT2 gene, or a recombinant vector comprising nucleic acid sequences encoding a deoxyribonucleic acid and an endonuclease protein, which encode a guide ribonucleic acid specific for a target base sequence of a rice-derived BCAT2 gene, may be used.
In the method for producing a genome editing rice plant according to an embodiment of the present invention, the target base sequence of the rice-derived BCAT2 gene may be, but is not limited to, the base sequence of sequence 3 or sequence 4.
The present invention also provides a genome editing rice plant having increased resistance to drought stress prepared by the above method for preparing a genome editing rice plant, and a seed for editing its genome.
The genome editing rice plant of the present invention is characterized in that resistance to drought stress is increased by knocking out a rice-derived BCAT2 protein encoding gene.
Also, the present invention provides a composition for modulating drought stress resistance of plants comprising a rice-derived BCAT2 protein-encoding gene as an active ingredient.
The above-described composition of the present invention comprises a gene encoding rice-derived BCAT2 protein or a mutation of a gene encoding rice-derived BCAT2 protein as an active ingredient, and plant drought stress resistance can be modulated by transforming plant cells with a recombinant vector comprising the above-described gene encoding rice-derived BCAT2 protein or a mutation of a gene encoding rice-derived BCAT2 protein.
The present invention will be described in detail with reference to examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited to the following examples.
Materials and methods
1. Plant material
To prepare the loss-of-function mutant (OsBCAT 2) of OsBCAT2 (Os 03g0231600, loc_os03g 12890), rice was edited using the CRISPR-Cas9 system using two guide ribonucleic acids (table 1) (Oryza sativa l. Ssp Japonica cv Dongjin). The guide ribonucleic acid was designed by a web-based tool CRISPR RGEN Tools (http:// www.rgenome.net). In the same manner as described in the prior report (Chung et al 2020, int.J.mol. Sci.21 (24); 9606), an osbcat2 mutant having a total of 18 genotypes was obtained. Mutants of osbcat2 having an early stop codon before amino acid 89 (# 3, #4, #5, #10, # 15) were used in the present invention.
An Dongjin japonica rice variety cultivated under the same conditions as the mutant (osbcat 2) was used as a non-transformed control group.
TABLE 1
Small guide ribonucleic acid (sgRNA) for OsBACT2 gene editing
| Sequence (5 '-3') | |
| sgRNA1 | CGGGAGGAGCGCGCGCTTCG (sequence 3) |
| sgRNA2 | CGCGCTGGCCAGGGCCCTGC (sequence 4) |
2. Abiotic stress and phytohormone treatment
Rice seeds (Oryza sativa cv. Dongjin) were germinated in MS solid medium at 28℃under dark conditions for 4 days. The seedlings were then moved into a growth chamber with 16 hours light/8 hours dark photoperiod, light intensity of 200. Mu. Mol m-2s-1 and relative humidity of 70%. Seedlings were transferred to Yoshida solution for 1 week and continued cultivation for 2 weeks for gene expression analysis. To study the phytohormone-dependent response of OsBCAT2, non-transformed (NT) seedlings were used and transplanted into 50ml tubes containing 100 μm abscisic acid, jasmonic acid, and salicylic acid solution (Sigma, USA). For ribonucleic acid extraction, after each hormone treatment, harvesting is performed every 2 hours up to 6 hours.
To confirm transcript levels of the OsBCAT2 gene under various abiotic stresses, NT plants were grown in soil for 2 weeks under standard greenhouse conditions (16 hours light/8 hours dark cycle at temperatures of 28 ℃ -30 ℃). After the stress treatment is to completely remove the soil of the seedling root, drought stress is induced under air-dried conditions, salt damage stress is applied by culturing in water containing 400mM sodium chloride at a temperature of 28℃and low temperature stress is treated by culturing at a temperature of 4 ℃ (+ -1.5 ℃).
3. Amino acid supply and drought stress management
After culturing NT plants in a dark growth chamber at a temperature of 28 ℃ for 4 days after seeding in MS medium, they were grown at a temperature of 30 ℃ for 2 weeks after moving to a growth chamber with 16 hours light/8 hours dark photoperiod. Seedlings of 2 weeks of age were acclimatized to one day in water before moving to Yoshida solution. Then, the plants were transferred to Yoshida solution for cultivation to 3 weeks of age.
Roots of plants grown to 3 weeks of age in Yoshida solution were immersed in a 10mM FAA solution containing a random selection of L-valine, leucine, isoleucine for 24 hours. For amino acid analysis, plants pretreated with branched-chain amino acids were harvested. The plants were then transferred to 50ml tubes containing 30ml of 21% polyethylene glycol 8000 (Sigma Co.) solution. Macroscopic symptoms induced by polyethylene glycol, such as drying and wilting of leaves, were monitored by imaging using an alpha 5000 camera (Sony corporation). The drought phenotype of FAA-pretreated NT plants due to air drying was recorded by relative moisture content (RWC) and symptom analysis.
RWC(％)＝(FW－DW)/(TW－DW)×100.
[ FW: living body weight, DW: dry weight, TW: expansion weight (turgid weight.)
4. Ribonucleic acid extraction and real-time quantitative polymerase chain reaction (qRT-PCR)
To investigate the spatial Gene transcript pattern of OsBCAT2, total ribonucleic acid samples in roots, stems, flowers of rice at different developmental stages were extracted using Hybrid-R RNA purification kit (Gene All, korea) according to the instructions of the manufacturing company. To detect the expression level of OsBCAT2, total ribonucleic acid of the shoots and roots was isolated using a Hybrid-R RNA purification kit. Use of Reverted aid with oligo (dT) primer (Thermo Scientific Co., USA) TM First Strand cDNA A synthesis kit to synthesize total ribonucleic acid and/or immunoprecipitated ribonucleic acid complementary deoxyribonucleic acid, 20ng cDNA was used as a template for real-time quantitative polymerase chain reaction analysis. Real-time quantitative polymerase chain reaction was performed using 2x real-time quantitative polymerase chain reaction Premix (Premix), 20x EvaGreen (solGent corporation, first-hand (Seoul), korea) and ROX stain (Promega corporation, madison, wisconsin (WI), usa). After reacting a 20. Mu.l volume of the mixture containing 1. Mu.l of Eva Green Mix at a temperature of 95℃for 10 minutes, the reaction was carried out at a temperature of 95℃for 30 seconds, at a temperature of 60℃for 30 seconds, and the reaction was repeated 40 times in total for 30 seconds. Rice ubiquitin 1 (Os 06g 0681400) was used as an internal standard.
TABLE 2
Primer information used in the present invention
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5. Drought stress treatment and stress detection in the nutritional phase
Seedlings of osbcat2 and NT germinated in MS medium in dark growth chambers at 28 ℃ for 4 days were acclimatized to one day in an environment of bright condition prior to transplantation. After 5-day-old seedlings were transferred to pots (4 cm. Times.4 cm. Times.6 cm, 3 plants per pot) in a container (59 cm. Times.38.5 cm. Times.15 cm), they were cultivated under automatically controlled glass greenhouse conditions (temperature of 30.+ -. 3 ℃ C., 16 hours light/8 hours dark) for 5 weeks. Drought treatment for observation or detection is uniformly performed in such a manner that the supply of water is interrupted and then irrigated during cultivation of rice. Soil moisture was monitored during the test, measured by a soil moisture sensor (SM 159, delta-T Devices, cambridge (Cambridge), UK).
To detect drought stress of osbcat2 and NT, the efficiency of light systems I and II was assessed by JIP testing (Straser, R.J., tsimilli-Michael, M., srivastava, A. (2004) Analysis of the Chlorophyll a Fluorescence Transient. In: papageorgiou, G.C., govindjee (eds) chlorphenyl a fluorescence. Advances in Photosynthesis and Respiration, vol 19.Springer, dordrecht. Https:// doi.org/10.1007/978-1-4020-3218-9_12). For optimal dark (dark) adaptation, these data were recorded under dark conditions (minimum 20 minutes) using a handle-PEA fluorometer (Plant Efficiency Analyzer Hansatech Instruments company, uk). After 10 leaves were collected, data were calculated using the handle PEA software (version 1.31) and analyzed according to the calculation formula of the JIP test.
6. Analysis of agricultural Properties of Rice in farmlands
To evaluate the agronomic characteristics and properties of osbcat2 and NT rice, 5 independent T1 isotype mutants and NTs were cultivated in North Qing university farms located in Korea, lewy county (128:34E/36:15N). To minimize microclimate effects, the farmland was divided into three areas, each area being planted with 10 plants in a row. An experimental environment was created for agricultural evaluation of osbcat2 and NT plants under drought conditions. Plants are grown in reservoirs artificially created in such a way that they can block rain. The water level is adjusted before and after the emergence period (head phase) to apply the water shortage condition to the plant. In NT, re-irrigation for recovery was performed once a phenotype due to drought stress was observed. Harvesting elements of plants in three areas were measured under drought conditions in the farmland.
DAB and NBT staining
DAB was stained by dissolving 50mg of 3,3' -diaminobiphenyl (Sigma) in 45ml of hydrogen peroxide and adjusting the pH to 3.8 using 0.1N HCl. To this solution were added 25. Mu. Tween20 (Tween 20) (0.05% v/v) and 2.5ml of Na 2 HPO 4 (200 mM) hydrogen peroxide was added to make the total volume 50ml. Leaf sections of 2.5cm length were cut accurately and immersed in 1% DAB solution. After 30 minutes of vacuum infusion (vacuum infiltrating), the infused leaves were incubated in the dark at ambient temperature for 20 hours. Then, after washing with water until brown spots appear significantly, boiling ethanol is heated to bleach the leaves. The area of the brown spots indicates DAB reactivity with respect to hydrogen peroxide.
To detect accumulation of superoxide (superoxide) using a 0.2% NBT solution in 10mM potassium phosphate buffer (pH 7.7), leaves of about 2.5cm long were cut. After 15 minutes of vacuum infusion, the submerged leaf was incubated overnight at room temperature. After incubation, the mixture was allowed to settle for 30 minutes in alcoholic lactic acid phenol (2:1:1,%) ethanol (ethanol) to lactic acid (phenol) at 65℃and then washed with 50% ethanol and rinsed with water. When NBT in the leaves reacts with superoxide, blue precipitate is formed.
Example 1 OsBCAT2 is an drought-reactive mitochondrial branched-chain amino acid transaminase (aminotransferase)
Multiple amino acid sequence alignment was performed with 12 genes including BCATs (AtBCATs) from previously identified arabidopsis thaliana and BCATs (SlBCATs) from tomato. The phylogenetic analysis results revealed that the BCAT genes of arabidopsis, tomato and rice, including the attat 2 and SlBCAT2 known to be located in mitochondria, have very high homology. In particular, the portions having enzymatic activity (branched amino acid aminotransferase function) have homology of 80% or more, and it is found that the difference between the portions is only present in the N-terminal signal peptide sequence portions which determine the protein site.
In order to find the branched-chain amino acid transaminase gene induced by drought, 5 drought responses (2 each up-regulated, 3 down-regulated, log) were confirmed using the results of transcriptome sequencing (RNA-seq) from the previous study (Chung et al 2016BMC genomics 17:563) 2 A ratio of 2.0 or more and-2.0 or less), the gene related to branched-chain amino acid transferase, was confirmed to be up-regulated with increased drought stress in which OsBCAT2 was found.
To confirm transcript levels of OsBCAT2 in various abiotic conditions, 2 week old seedlings were exposed to drought, high salt, abscisic acid and low temperature, respectively. The analysis results showed that compared to other abiotic stresses, the transcript level of OsBCAT2 was strongly induced in leaves exposed to drought stress within 6 hours (part (a) of fig. 1), which was similar to the transcriptome sequencing results. And, especially after treatment of roots with phytohormones (abscisic acid, jasmonic acid, salicylic acid), transcript levels increased significantly (part (b) of fig. 1). It is therefore thought that OsBCAT2 can act as a variety of enzymes in response to abiotic stress.
Example 2 OsBCAT2 is associated with catabolism of branched chain amino acids
Amino acids are known to be associated with a large portion of metabolic pathways that regulate various developmental processes in plants. The present inventors performed a transcription analysis of OsBCAT2 by quantitative polymerase chain reaction in real time. Expression levels are confirmed in various developmental stages and tissues from vegetative growth to developmental stages. The analysis showed that the OsBCAT2 transcript was expressed upwards in all tissues sleeping. Compared to vegetative growth phase, osBCAT shows higher expression at the developmental stage. High expression was shown especially in dark condition leaves (fig. 4). The transcript pattern described above is similar to genes associated with catabolism of branched-chain amino acids in Arabidopsis thaliana.
The major enzymes involved in branched-chain amino acid biosynthesis and breakdown are known to be located in chloroplasts and mitochondria, respectively. Thus, the present inventors examined whether OsBCAT2 is involved in branched-chain amino acid synthesis or decomposition using an OsBCAT2-GFP fusion protein expressed in rice protoplasts. As a result of analysis by a laser scanning confocal microscope, only a fluorescent signal generated by OsBCAT2-GFP protein was observed in mitochondria. Also, the results of intracellular local analysis showed that the expression of OsBCAT2 protein was similar to the expression of mitochondrial markers (fig. 5). This result suggests that OsBCAT2 can undergo catabolism of branched-chain amino acids in mitochondria.
Example 3 loss of function of OsBCAT2 promotes drought stress resistance
Past studies on osdia over-expression transformants have shown that increasing branched amino acid content in leaf tissue increases tolerance to drought stress. To investigate the biological function of the OsBCAT2 mutant in drought stress, two types of single guide ribonucleic acid (small guide ribonucleic acid (sgRNA)) related to CRISPR-Cas9 system technology were designed to achieve knockout mutation in the OsBCAT2 gene, resulting in 5 homozygously mutants (# 3, #4, #5, #10, # 15) (part (a) of fig. 6). All small guide RNAs were designed in such a way that the first exon (exon) was targeted, with the gRNA1 mutants (# 4, # 10) having a 1-, 2-bp deletion and the gRNA2 mutants (# 3, #5, # 15) having a 1-bp deletion in the other DNA sequences, respectively. Sequencing analysis results revealed a mutation pattern of genetically modified plants including frameshift and early stop codons (FIG. 6 (b)).
Drought tolerance tests were performed on osbcat2 mutants to investigate the response and biological effects to drought stress. Drought stress was obtained in the greenhouse by leaving 4 week old plants out of water supply for several days (part (a) of fig. 7). Soil moisture showed a continuous decrease over three days, indicating that after one day of drought stress was continuously applied to plants, damage due to drought was exhibited in NT plants (fig. 7 (d) part). After 2-3 days of exposure to drought conditions, most NT plants develop unrecoverable symptoms such as wilting, drying out, and withering. In contrast, all osbcat2 variants exhibited tolerance to drought stress after 3 days of exposure to drought, with significantly fewer drought-induced symptoms and higher recovery after re-irrigation than NT plants. To additionally verify drought tolerance of osbcat2 mutants, the maximum quantum efficiency of the photosystem II (Fv/Fm) values and the photosynthetic Performance Index (PI) of photosystem I and photosystem II were determined, which show photochemical efficiency in abiotic stress. Fv/Fm values of NT plants were significantly reduced after 3 days of exposure to stress. In contrast, the osbcat2 mutant showed a slight decrease in Fv/Fm values (FIG. 7 part (b)). The performance index value in NT plants was then also drastically reduced, while the photosynthetic performance index value of osbcat2 was well controlled under drought exposure conditions (fig. 7 part (c)). The above results show that the osbcat2 mutant has higher drought tolerance than NT.
Example 4 significant accumulation of branched-chain amino acid levels in osbcat2 plants
Arabidopsis plants are reported to increase branched-chain amino acids corresponding to drought and osmotic stress.
The branched-chain amino acid levels of NT and osbcat2 mutant plants were evaluated for changes in drought conditions. For High Performance Liquid Chromatography (HPLC) analysis, 6 week old plants were exposed to drought stress conditions due to air drying for 6 hours. The branched-chain amino acid content was then measured in normal and drought conditions. In these plants, branched-chain amino acid levels under drought stress conditions were greatly increased compared to samples under normal conditions. Specifically, the osbcat2 mutant accumulated a much higher branched-chain amino acid content than the NT plant (part (a) of fig. 8). To further verify the amino acid changes over drought treatment time, exposure to drought stress conditions was performed for 3 days in soil. Leucine content of the osbcat2 mutant increased within two days. And, at the branched-chain amino acid level, rapidly accumulated on the fourth day. In contrast, while NT plants induced branched-chain amino acids to the next day, on the third day decreased, especially compared to NT plants, the total branched-chain amino acid content of osbcat2 increased significantly in drought conditions (part (b) of fig. 8).
To additionally confirm whether the osbcat2 gene is associated with catabolism of branched-chain amino acids, dried rice seeds were analyzed for branched-chain amino acid content. The results show that branched-chain amino methionine acid content in the osbcat2 mutant is geometrically increased compared to NT plants. Overall, the above results show that drought stress causes accumulation of branched-chain amino acids in rice species, and that drought stress induces expression of OsBCAT2 associated with catabolism of branched-chain amino acids.
Example 5 external supply of branched-chain amino acids to enhance osmotic shock (osmotic shock) resistance in rice species
To understand the effect of amino acid accumulation in osmotic stress-stressed rice, NT rice was subjected to various amino acids (proline, alanine, aspartic acid, methionine, isoleucine, leucine, valine, and branched-chain amino acid compounds) for 24 hours, respectively. The pretreated plants were confirmed by high performance liquid chromatography analysis. Analysis showed that such external sources increased amino acid levels inside plants (FIG. 2 (a)). 21% polyethylene glycol 8000 was then applied to the pretreated plants to simulate appropriate osmotic stress for observing osmotic pressure induced symptoms. Symptoms associated with osmotic shock, such as She Ganku and wilt, were found in NT plants treated with most of the amino acids except branched chain amino acids and prolines 13 hours after treatment with polyethylene glycol. The onset of the symptoms described above is most severe in the blank group (mock) and is followed by the group of pretreatment alanine (Ala), aspartic acid (Asp), methionine (Met), phenylalanine (Phe), branched chain amino acids, proline (Pro) in this order (part (b) of fig. 2). And, exogenous branched-chain amino acids increase regeneration of plants damaged by treatment of polyethylene glycol after transfer to Yoshida solution, alleviating symptoms of osmotic shock.
In addition, in order to observe the influence of amino acid accumulation under severe osmotic stress in rice, an air drying experiment was performed. First, the amino acid has been pretreated for 24 hours in the same manner as the aforementioned treatment method. After 10 hours of air drying treatment, severe damage due to drought stress was evident in all plants as a whole, but plants treated with branched chain amino acids and proline were less damaged than those pretreated with other amino acid groups (part (a) of fig. 3). The blank plants then showed unrecoverable symptoms after re-irrigation, but the group of pre-treated amino acids recovered. The above results indicate that pretreatment of branched-chain amino acids alone or in combination with treatment of proline shows resistance to drought stress caused by air drying. Similar to the polyethylene glycol experiments described above, the branched amino acid and proline treated groups confirm their recovery potential by a recovery process.
To further understand the effect of external branched-chain amino acid supply, the level of moisture loss per hour due to air drying was monitored. As a result, it was observed that the water loss was reduced in all groups of treated amino acids except for the blank treated plants (part (b) of fig. 3). Wherein the group of 3 branched-chain amino acids and combinations thereof treated alone and the pre-treated proline have a much higher water content retention than alanine, aspartic acid, methionine and phenylalanine (FIG. 3 part (c)). Briefly, branched-chain amino acid accumulation in rice can confer osmotic stress tolerance.
Example 6 genome editing of OsBCAT2 to improve agricultural Properties under drought conditions in farmlands
The above studies indicate that the chart questions of losing the function of OsBCAT2 provide drought tolerance. Thus, to assess whether the osbcat2 mutant can have an effect on total grain harvest under drought stress conditions, osbcat2 and NT plants were cultivated under korean military (128:34 e/36:15 n) packaging (field) conditions with rain shed (rain shed) for a total of 100 DAYS (DAYS) from the transplanting stage to the maturity stage. Then, drought stress is treated twice in succession during flowering and milk maturation. Under normal conditions, the inflorescence (NS) and Filling Rate (FR) of 5 independent osbcat2 were significantly lower than NT. In contrast, under drought conditions, the Total Grain Weight (TGW) of osbcat2 was significantly improved over NT (fig. 9 and table 3). These results show that genome editing of OsBCAT2 affects grain harvest under both normal and drought conditions.
TABLE 3 Table 3
Example 7. An antioxidant gene that induces jasmonic acid mediates drought resistance in the osbcat2 mutant.
Studies on amino acids induced by drought stress, particularly on proline related to drought resistance, have been largely carried out, and the mechanism pathways for abiotic stress are well known. However, there have been few studies on abiotic stress resistance mechanisms using branched-chain amino acids.
The results of confirming the transcript level of rice jasmonic acid resistance 1 (Jasmonate resistance 1) (OsJar 1) in the total ribonucleic acid sample of osbcat2 that treated drought stress showed higher gene expression than NT plants (part (a) of fig. 10). In addition, in osbcat2, an increase in the expression level of the jasmonic acid signal gene and the expression of the jasmonic acid biosynthesis gene was confirmed (parts (b) and (c) of fig. 10).
In osbcat2, it is known that expression of an antioxidant gene such as APX, SOD, POD induced by jasmonic acid signaling is regulated in a highly upward manner (part (d) of fig. 11). Hydrogen peroxide (H) as central redox signalling agent for ROS was then implemented by staining in osbcat2 mutants 2 O 2 ) With superoxide anions (O) 2 - ) Is used for dyeing. By respectively to H 2 O 2 And O 2 Reaction to induce DAB and NBT staining of unrecoverable brown and blue products, corresponding to drought stress, showing H of osbcat2 mutant 2 O 2 The accumulation amount with time was smaller than NT (fig. 11). Taken together with the above results, ROS induced by drought stress are well regulated in osbcat2 by the antioxidant gene induced by jasmonic acid, thus, this means that osbcat2 can confer drought tolerance to plants.
Claims (14)
1. A method of modulating drought stress resistance in a plant comprising the step of modulating expression of a rice-derived BCAT2 protein encoding gene in a plant cell.
2. The method for regulating drought stress resistance of plants according to claim 1, wherein said rice-derived BCAT2 protein consists of the amino acid sequence of sequence 2.
3. The method for regulating drought stress resistance of plants according to claim 1, wherein the expression of the rice-derived BCAT2 protein-encoding gene is suppressed in plant cells by transforming plant cells with a recombinant vector comprising the rice-derived BCAT2 protein-encoding gene, thereby increasing the resistance to drought stress as compared to non-transformed plants.
4. The method for regulating drought stress resistance of plants according to claim 1, wherein said rice-derived BCAT2 protein coding gene is knocked out using a gene editing system to increase drought stress resistance.
5. A method for producing a transformed plant having modulated drought stress resistance, comprising:
a step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived BCAT2 protein; and
a step of redifferentiating the transformed plant from the above-mentioned transformed plant cells.
6. The method for producing a transformed plant according to claim 5, wherein the resistance to drought stress is increased as compared with a non-transformed plant by inhibiting the expression of the gene encoding the rice-derived BCAT2 protein.
7. A transformed plant having modulated drought stress resistance, characterized by being produced by the method for producing a transformed plant according to claim 5 or 6.
8. A transformed seed of the transformed plant having modulated drought stress resistance according to claim 7.
9. A method for preparing a genome-editing rice plant having increased resistance to drought stress, comprising:
step (a), introducing a guide ribonucleic acid and an endonuclease protein which have specificity to a target base sequence of a gene encoding a rice-derived BCAT2 protein into a rice plant cell to edit a genome; and
step (b) of subdividing the plant from the rice plant cells editing the genome.
10. The method of producing a genome-editing rice plant according to claim 9, wherein in the step (a), when introducing a guide ribonucleic acid and an endonuclease protein into a rice plant cell, a recombinant vector comprising a nucleic acid sequence encoding a deoxyribonucleotide and an endonuclease protein or a complex of a guide ribonucleic acid and an endonuclease protein having a specificity for a target base sequence of a rice-derived BCAT2 gene is used, and the guide ribonucleic acid encoding a target base sequence of a rice-derived BCAT2 gene is used.
11. The method for preparing a genome-editing rice plant according to claim 9, wherein the target base sequence of the rice-derived BCAT2 gene is a base sequence of sequence 3 or sequence 4.
12. A genome-editing rice plant having increased resistance to drought stress prepared by the method for preparing a genome-editing rice plant according to any one of claims 9 to 11.
13. A genome-edited seed of a genome-edited rice plant of claim 12.
14. A composition for regulating drought stress resistance of plants, comprising a rice-derived BCAT2 protein-encoding gene as an active ingredient.
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