Methods for increasing plant stress tolerance

Abstract

This invention relates to a method for enhancing plant stress tolerance, and more specifically to a method for enhancing plant stress tolerance and / or preventing reduced plant growth by introducing a polynucleotide encoding a repeat proline enrichment protein (RePRP) into plants.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 472,669, filed March 17, 2017, pursuant to 35 U.SC §119, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the use of repeat proline enriched protein (RePRP) for modifying the characteristics of transgenic plants that overexpress the protein. Background Technology

[0004] With the worsening of global climate change in recent years, abiotic stresses such as drought, extreme temperatures, and floods have led to significant reductions in agricultural yields. It is estimated that two-thirds of major crop yields are frequently lost due to adverse / stressful conditions (see Boyer, 1982). In-depth research is underway to understand how plants survive and how to minimize the impact of abiotic stresses on crop yields.

[0005] Most tolerance mechanisms associated with water stress are related to osmotic regulation, maintenance of ion homeostasis, and removal of destructive reactive oxygen species (see Kar, 2011). Basic architectural features of plants are also known to be adjusted under stress, such as increased surface wax, sunken stomata, and promotion of leaf curling.

[0006] Abscisic acid (ABA) is the most common stress hormone during periods of water stress. ABA can (i) cause stomata in leaves to close to reduce transpiration, and (ii) enhance the expression of other genes required to establish stress tolerance.

[0007] The growth and development of rice plants are readjusted to avoid unnecessary branch growth, thereby reducing water loss caused by severe water shortage or high concentrations of ABA treatment. Rice roots become shorter, thicker, and heavier due to the accumulation of biomass synthesized from residual nutrients transported from the buds.

[0008] A group of highly proline-rich proteins (“OsRePRPs”) were highly induced in rice roots by ABA, salinity, and drought (see Tseng et al., 2013). OsRePRPs are located in the cell membrane, where they interact with cell wall polysaccharides, which are both necessary and sufficient for shortening and thickening roots. Their effects on plant stress tolerance and growth have not yet been reported.

[0009] Several methods have been developed in this field to improve plant stress tolerance. However, plant survival under stress often manifests as stunted growth, which is detrimental to productivity and fails to meet agricultural needs. Therefore, there remains a need to provide a method for breeding plants that can tolerate a variety of stresses while still maintaining growth and productivity. Summary of the Invention

[0010] This invention is based, at least in part, on the unexpected discovery that the expression of a repetitive proline-rich protein (RePRP) leads to enhanced stress tolerance in transgenic plants that overexpress this protein, particularly without substantial growth retardation. Compared to control (wild-type) plants without RePRP gene conversion, the transgenic plants of this invention exhibit a significantly increased survival rate under stress without substantial growth retardation. Compared to control (wild-type) plants, the transgenic plants of this invention also show less yield reduction under stress.

[0011] Therefore, the present invention provides a method for enhancing plant stress tolerance and / or preventing plant growth loss, comprising:

[0012] (a) Transform plant cells with a vector containing a nucleic acid operatively linked to a promoter to obtain recombinant plant cells expressing a repeat proline enrichment protein (RePRP), wherein the nucleic acid encodes the RePRP protein;

[0013] (b) Culturing the recombinant plant cells obtained in (a) to produce a majority of transgenic plants; and

[0014] (c) Select transgenic plants from the majority of transgenic plants produced in (b) that exhibit enhanced tolerance to adversity or substantially no reduction in growth or a combination thereof, when compared with their non-transgenic counterparts grown under the same conditions.

[0015] In some specific instances, the transgenic plants exhibited less growth impairment compared to their non-transgenic counterparts.

[0016] In some specific instances, the RePRP protein contains the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

[0017] In some specific instances, the promoter is heterologous to the naturally occurring gene encoding the RePRP protein.

[0018] In some specific instances, the promoter is a constitutive promoter or an inducible promoter.

[0019] In some specific instances, the promoter is a constitutive promoter selected from the group consisting of the maize ubiquitin (Ubi) promoter, the rice actin (Act1) promoter, and the cauliflower mosaic virus 35S (CaMV35S) promoter.

[0020] In some specific instances, the promoter is an inducible promoter selected from the group consisting of the Arabidopsis thaliana corl SA promoter, the Arabidopsis thaliana kin1 promoter, the Arabidopsis thaliana heat shock factor (HSF) promoter, the Arabidopsis thaliana kin1 promoter, the Arabidopsis thaliana rd29A promoter, the α-amylase promoter, and the synthetic ABRC321 promoter.

[0021] In some specific instances, the inducible promoter is 3XABRC321.

[0022] In some specific instances, the carrier contains SEQ ID NO: 12, 13 or 14.

[0023] In some specific instances, the genetically modified plant is a monocotyledonous plant.

[0024] In some specific instances, the monocotyledonous plant is rice, barley, wheat, rye, oats, corn, bamboo, sugarcane, onion, leek, or ginger.

[0025] In some specific instances, the genetically modified plant is a dicotyledonous plant.

[0026] In some specific instances, the genetically modified plant is Arabidopsis thaliana, soybean, peanut, sunflower, safflower, cotton, tobacco, tomato, pea, chickpea, pigeon pea, or potato.

[0027] In some specific instances, the adversity is selected from a group of abiotic adversities consisting of osmotic adversity, drought adversity, salinity adversity, or combinations thereof.

[0028] Details of one or more embodiments of the invention are set forth in the specification, accompanying drawings, and the examples described below. Other features, objects, and advantages of the invention will become apparent from the detailed description of several embodiments and the claims. All publications and patent documents cited herein are incorporated herein by reference in their entirety. Attached Figure Description

[0029] The foregoing overview and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. Preferred embodiments are shown in the drawings to illustrate the objectives of the invention. However, it should be understood that the invention is not limited to the precise arrangements and means shown.

[0030] Figure 1OsRePRP2.1 overexpression enhanced salt tolerance in rice. Three-leaf seedlings were treated with 250 mM NaCl for 5 days and then allowed to recover to normal conditions for 12 days. The survival rate of OsRePRP2.1OX transgenic plants was significantly higher than that of TNG67. Data are mean ± SD from three experimental replicates.

[0031] Figure 2 Overexpression of OsRePRP2.1 in rice enhanced drought tolerance. Three-leaf seedlings were treated with 30% PEG for 20 hours and then restored to normal conditions for 10 days. The survival rate of OsRePRP2.1OX transgenic plants was significantly higher than that of TNG67. Data are mean ± SD from three experimental replicates.

[0032] Figure 3 The study showed that OsRePRP2.1 overexpression enhanced plant recovery after drought treatment in rice. Watering was stopped for 13 days after the two-week-old plants were watered again for 12 days. Half of the OsRePRP2.1OX plants survived and grew new leaves, but only a small number of TNG67 plants survived.

[0033] Figure 4 The OsRePRP RNAi plants were shown to be sensitive to abiotic stress treatments. Three-leaf seedlings were treated with 250 mM NaCl for 5 days or with 30% PEG for 24 hours. Survival rates were recorded after 12 days of recovery. In both stress treatments, the mean survival rate of the RNAi lines was significantly lower than that of WT (TNG67), while the OsRePRP2.1 overexpression lines showed a survival rate as high as 80%.

[0034] Figure 5A and Figure 5B The OsRePRP2.1 transgenic rice plants were shown to tolerate PEG treatment after induced expression. Two-week-old plants were treated with 30% PEG for 18 hours and then returned to normal conditions for 10 days. Figure 5A The survival rate of ABRC321:OsRePRP2.1 transgenic plants (T2 generation) was significantly higher than that of TNG67. Data are the mean ± SD from two experimental replicates. Figure 5B The root images of 14-day-old rice seedlings showing wild-type and ABRC321:OsRePRP2.1 plants are similar.

[0035] Figures 6A to 6C The results showed that OsRePRP2.1 overexpression in Arabidopsis thaliana plants resulted in normal growth, similar to that of non-transgenic Arabidopsis thaliana plants under normal conditions. Figure 6A This shows 50-day-old mature Arabidopsis thaliana plants grown under normal conditions. Figure 6B Images of the roots of 10-day-old Arabidopsis thaliana seedlings grown on normal agar plates are shown. Figure 6CThe root length data for each plant line is expressed as mean ± SD. Detailed Implementation

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] As used in this article, the definite articles “a” and “an” refer to one or more (i.e., at least one) of the grammatical objects of an item. For example, “an element” refers to one or more elements.

[0038] The words “comprise” or “comprising” are generally used in the sense of including, indicating that one or more features, ingredients, or components are allowed to exist. The words “comprise” or “comprising” include “consists” or “consisting of”.

[0039] This study unexpectedly found that repetitive proline-enriched protein (RePRP) enhanced the characteristics of transgenic plants that overexpressed this protein, such as growth characteristics and / or stress tolerance. Therefore, this paper provides transgenic plants that overexpress RePRP as described herein, vectors for expressing RePRP, methods for preparing such transgenic plants, and methods for improving plant growth characteristics or stress tolerance by overexpressing the RePRP protein.

[0040] I. Repetitive Proline-Enriched Proteins (RePRPs)

[0041] Repetitive proline-rich proteins (RePRPs) are ABA-induced glycoproteins in plants. These proteins include a signal peptide at the N-terminus, followed by a proline-rich domain, which constitutes approximately 70% of the protein. The proline-rich domain contains numerous repeating PX1PX2 motifs (SEQ ID NO: 25) and forms a hydrophilic region, where P is a proline residue, X1 and X2 are any amino acid residues other than proline, and more specifically, multiple proline-derived hydrogen-proline residues are highly glycosylated via arabinose and glucose. The motif can be repeated "n" times, denoted by (PX1PX2)n. It is understood that X1 or X2 can be the same or different in each repeat, and the identification of X1 or X2 residues is not necessarily completely consistent throughout the "n" repeats of the residues. It is further understood that each repeat can be linked end-to-end (directly) or linked to one or more indirect amino acids (indirectly). In some embodiments, n is an integer of 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, or 80 or more. In some embodiments, based on the total number of amino acid residues in the protein, the RePRP has a proline content, for example, 20% or more, 30% or more, 40% or more, or 50% or more. In some embodiments, X1 or X2 is selected from the group consisting of: lysine (Lys, K), glutamic acid (Glu, E), asparagine (Asn, N), aspartic acid (Asp, D), tyrosine (Tyr, Y), valine (Val, V), histidine (His, H), isoleucine (Ile, I), glycine (Gly, G), threonine (Thr, T), glutamine (Gln, Q), and serine (Ser, S). In some embodiments, the RePRP has a length of 200 to 500 amino acids, for example, about 200, 250, 300, or 350 amino acids.

[0042] According to this disclosure, as used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymers formed from amino acid residues, one or more of which are naturally occurring amino acids or imitations of artificial chemicals.

[0043] The RePRPs described herein can be naturally occurring proteins of any suitable species. Exemplary RePRPs can be derived from plants, preferably monocotyledons, including but not limited to rice, barley, wheat, maize, and sorghum. In rice, there are two subclasses of RePRPs: (1) RePRP1, including RePRP1.1 (SEQ ID NO: 1) and RePRP1.2 (SEQ ID NO: 2); and (2) RePRP2, including RePRP2.1 (SEQ ID NO: 3) and RePRP2.2 (SEQ ID NO: 4).

[0044] In some embodiments, the RePRP may comprise the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. Alternatively, the RePRP may be a naturally occurring protein highly homologous to SEQ ID NO: 1, 2, 3, or 4, for example, sharing at least 85% sequence identity across its full length (e.g., at least 90%, at least 93%, at least 95%, or at least 97%). Such a RePRP can be readily identified from publicly available gene databases (e.g., GenBank) using SEQ ID NO: 1, 2, 3, or 4 as a query.

[0045] Understandably, polypeptides can have a limited number of alterations or modifications that can be made within specific parts of the polypeptide, wherein such alterations or modifications are unrelated to their activity or function and still produce molecules with acceptable levels of equivalent biological activity or function. Modifications and alterations can be made within the structure of such polypeptides and molecules with similar or desired characteristics can still be obtained. For example, certain amino acids in a peptide / polypeptide structure (except for conserved regions) can be substituted for other amino acids without a significant loss of activity. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. For example, arginine (Arg), lysine (Lys), and histidine (His) are all positively charged residues; alanine (Ala), glycine (Gly), and serine (Ser) are all of similar size. Therefore, based on these considerations, arginine (Arg), lysine (Lys), and histidine (His); and alanine (Ala), glycine (Gly), and serine (Ser) can be defined as biologically equivalent. Recombinant genes can be easily designed and prepared for microbial expression of peptides with equivalent amino acid residues.

[0046] Therefore, in some embodiments, RePRP can be a functional variant of naturally occurring RePRP. Such a functional variant can share a high degree of sequence identity with the wild-type counterpart, for example, having at least 85% (e.g., 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of the wild-type counterpart, and having substantially similar biological activities to the wild-type counterpart.

[0047] To determine the percentage of identity between two amino acid sequences, sequence alignment can be performed for optimal comparison (e.g., a gap can be introduced into the first amino acid sequence to achieve optimal alignment with the second). When calculating percentage identity, an exact match is typically calculated. The determination of percentage homology or identity between two sequences can be accomplished using mathematical algorithms known in the art, such as the BLAST and Gapped BLAST procedures, NBLAST and XBLAST procedures, or the ALIGN procedure.

[0048] II. Carriers encoding RePRPs

[0049] In some aspects, the present invention provides a vector comprising a nucleic acid encoding any RePRP described herein. The term "nucleic acid" or "polynucleotide" refers to a polymer composed of nucleotide units, including naturally occurring deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), and any analogues thereof. For example, polynucleotides can be synthesized using an automated DNA synthesizer. The term "nucleic acid" generally refers to a large polynucleotide. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences where "U" is replaced by "T" (i.e., A, U, G, C). The term "cDNA" refers to DNA that is complementary to or identical to mRNA in single-stranded or double-stranded form.

[0050] The term "encoding" refers to the inherent property of a specific nucleotide sequence (e.g., a gene, cDNA, or mRNA) in a polynucleotide that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological characteristics. Thus, if the mRNA produced by a gene is transcribed and translated to produce a protein in a cell or other biological system, then that gene encodes a protein. Those skilled in the art will understand that many different polynucleotides and nucleic acids can encode the same polypeptide due to the degeneracy of the genetic code. It should also be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotide, thereby reflecting the use of codons in any particular host organism expressing the polypeptide. Therefore, unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0051] In some implementations, the nucleic acid encoding the RePRP protein as described herein is SEQ ID NO: 5 (encoding OsRePRP1.1), SEQ ID NO: 6 (encoding OsRePRP1.2), SEQ ID NO: 7 (encoding OsRePRP2.1), or SEQ ID NO: 8 (encoding OsRePRP2.2).

[0052] As used herein, "vector" can be a basic recombinant nucleic acid-based vehicle for artificially carrying foreign genetic material into a host cell, in which the foreign genetic material can be replicated and / or expressed. Vectors described herein can be cloning and / or expression vectors. Vectors can be linear or circular. When introduced into a host cell, it may remain episomal or be integrated into the host cell genome. In some embodiments, the vector can be a viral vector or a non-viral vector (e.g., a plasmid). In particular examples, the vector can be a plant vector or an Agrobacterium vector.

[0053] In some implementations, the nucleic acid encoding RePRP can be operatively linked to a promoter in a vector to drive the expression of RePRP in vitro or in vivo. As used herein, the term "operatively linked" can mean that a polynucleotide is linked to an expression control sequence, such that the polynucleotide can be expressed when an appropriate molecule (e.g., a transcription factor) binds to the expression control sequence. As used herein, the term "expression control sequence" or "regulatory sequence" refers to a DNA sequence that regulates the expression of an operatively linked nucleic acid sequence in a host cell.

[0054] Examples of vectors include, but are not limited to, plasmids, granules, phages, YACs, or PACs. Typically, in a vector, a given nucleotide sequence is operatively linked to a regulatory sequence such that when the vector is introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. The regulatory sequence may include, for example, but not limited to, promoter sequences (e.g., cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter), start codons, origin of replication, enhancers, operon sequences, secretion signal sequences (e.g., alpha-mating factor signal), and other control sequences (e.g., Shine-Dalgano sequences and termination sequences). Preferably, the vector may further contain a marker sequence (e.g., an antibiotic resistance marker sequence) for subsequent screening / selection processes.

[0055] In some instances, the promoters described herein may be heterologous to the nucleic acid encoding RePRP in the vector. As used herein, a promoter heterologous to the coding sequence (gene) refers to a promoter that is not a natural promoter that controls (drives) gene expression in its natural state. For example, the vectors disclosed herein may contain promoters derived from non-RePRP genes.

[0056] In some cases, the promoters described herein can be constitutive, meaning their initiation is independent of the transcriptional influences of regulation. Exemplary constitutive promoters include, but are not limited to, the maize ubiquitin (Ubi) promoter, the rice actin (Act1) promoter, and the cauliflower mosaic virus 35S (CaMV35S) promoter.

[0057] In other cases, the promoters described herein may be inducible, initiating transcription in a regulated manner, for example, in the presence or absence of a specific factor. Exemplary inducible promoters include ethanol-inducible promoters (e.g., the AlcR / AlcA promoter) or β-estradiol-inducible promoters (e.g., the XVE promoter, see the Examples section below). Exemplary promoters that can be induced by biotic or abiotic stresses (e.g., osmotic stress, drought stress, salinity stress, high or low temperature, hypoxia, anoxic, hydration, pH, chemicals, hormones, or combinations thereof) include the Arabidopsis rd29A promoter, the Arabidopsis SA promoter, the Arabidopsis kin1 promoter, the Arabidopsis heat shock factor (HSF) promoter, the alfa-amylase promoter, and the synthetic ABRC321 promoter.

[0058] In some embodiments, the promoter sequence used in this invention is a synthesized ABRC321 promoter having SEQ ID NO: 9 (1xABRC321), SEQ ID NO: 10 (2xABRC321), or SEQ ID NO: 11 (3xABRC321), preferably SEQ ID NO: 11 (3xABRC321).

[0059] In some embodiments, the vector comprising a nucleic acid encoding the RePRP protein and operatively linked to a promoter includes a fusion promoter / coding region fragment of SEQ ID NO: 12 (Ubi: OsRePRP2.1).

[0060] In some implementations, the vector containing a nucleic acid encoding the RePRP protein and operatively linked to a promoter contains a fusion promoter / coding region fragment of SEQ ID NO: 13 (3xABRC321i: OsRePRP2.1).

[0061] In some implementations, the vector containing a nucleic acid encoding the RePRP protein and operatively linked to a promoter contains a fusion promoter / coding region fragment of SEQ ID NO: 14 (35S: OsRePRP2.1).

[0062] Any of the vectors described in this article can be prepared using conventional recombination techniques.

[0063] III. Host cells, transgenic plants and their preparation methods

[0064] Some aspects of this invention provide host cells (e.g., Agrobacterium cells or plant cells) comprising any vector as described herein. These host cells (or recombinant cells) carry exogenous / exogenous genetic material (e.g., vectors as described herein) which can be introduced into the host cell through conventional practice. As used herein, “exogenous genetic material” can mean genetic material that is not originally present in the cell but is artificially introduced into the cell of its parent. In some cases, the exogenous genetic material can be from a species different from the host cell. In some other cases, the exogenous genetic material can be from the same species as the host cell and introduced into the host cell, resulting in recombinant cells containing an additional number of copies of the genetic material compared to the wild-type counterpart. As used herein, the term “transformation” or “transformation” refers to the introduction of exogenous genetic material into a host cell, such as a plant cell.

[0065] In some embodiments, the host cell may be an Agrobacterium host cell. In some embodiments, the host cell may be a plant cell, such as a cell from a monocot or dicotyledonous plant.

[0066] Suitable conventional methods can be used to prepare the recombinant cells described herein. Examples of such methods include electroporation, PEGylation, particle bombardment, microinjection of plant cell protoplasts or embryogenic callus or other plant tissues, or Agrobacterium-mediated transformation.

[0067] RePRP expression can be detected using methods known in the art (e.g., before and after transformation of the vector presented herein into host cells). For example, reverse transcription polymerase chain reaction (RT-PCR) can be used to determine RePRP mRNA expression. Other detection methods include western blot analysis and enzyme-linked immunosorbent assay (ELISA) using anti-RePRP antibodies for protein detection.

[0068] The present invention also provides a transgenic plant comprising a foreign nucleic acid operatively linked to a promoter, wherein the foreign nucleic acid (transgenic) encodes any RePRP described herein.

[0069] As used herein, a plant can be a whole plant or a part thereof, including fruit, branches, stems, roots, leaves or seeds, or various types of cells in culture (e.g., single cells, protoplasts, embryos, callus, protocorms and other cell types). As mentioned above, the plants disclosed herein can be monocotyledonous or dicotyledonous plants.

[0070] In some implementations, the plants described herein are monocotyledonous plants. Examples of monocotyledonous plants include, but are not limited to, rice, barley, wheat, rye, oats, corn, bamboo, sugarcane, onion, leek, and ginger.

[0071] In other embodiments, the plants described herein are dicotyledonous plants. Exemplary dicotyledonous plants include Arabidopsis thaliana, soybean, peanut, sunflower, safflower, cotton, tobacco, tomato, pea, chickpea, pigeon pea, and potato.

[0072] Various procedures that can be used in the art to construct stable transgenic plants are available. In one embodiment of the invention, transgenic plants are produced by transforming plant tissues (such as protoplasts or leaf discs) with recombinant Agrobacterium cells containing nucleic acids (e.g., RePRP) encoding a desired protein, and whole plants are generated from the transformed plant tissues. In another embodiment, the nucleic acid encoding the desired protein can be introduced into the plant using gene gun technology, particularly if transformation with recombinant Agrobacterium cells is ineffective.

[0073] Specifically, the term "transgenic plant" as used herein can refer to a plant containing a transgenic gene that allows the expression of the RePRP gene in the transgenic plant (e.g., a foreign nucleic acid containing a RePRP gene operatively linked to a suitable promoter).

[0074] In some implementations, the transgenic plants described herein overexpress RePRPs. As used herein, the term "overexpression" can refer to the production of gene products (e.g., RePRPs) in transgenic plants at levels exceeding those in their non-transgenic (wild-type) counterparts, including but not limited to constitutive or inducible expression. For example, the level of RePRPs in transgenic plants may be at least 10% higher (e.g., 20%, 30%, 50%, 1-fold, 2-fold, 5-fold, 10-fold, or higher) compared to their non-transgenic (wild-type) counterparts. In some cases, the wild-type parent does not express RePRPs.

[0075] According to the present invention, transgenic plants as disclosed herein may exhibit enhanced tolerance to stress (e.g., biotic or abiotic stress). Biotic stress can be stress caused by damage to the plant by other organisms, such as pathogens, for example, bacteria, viruses, fungi, parasites, beneficial and harmful insects. Abiotic stress may be the negative impact of abiotic factors on living organisms in a particular environment. In some embodiments, abiotic stress is osmotic stress, drought stress, salinity stress, or a combination thereof.

[0076] In some implementations, enhancing plant stress tolerance refers to increasing the plant's ability to survive under stress. For example, the survival rate of the transgenic plants disclosed herein can be at least 20% higher (e.g., 30%, 50%, 1x, 2x, 5x, 10x, 20x, 50x, 100x, or more) compared to the survival rate of the wild-type counterpart during stress and / or recovery from stress.

[0077] In some embodiments, the transgenic plants disclosed herein exhibit substantially no growth or yield reduction under stress and / or during recovery from stress, compared to non-stress conditions. For example, the grain yield reduction of transgenic plants disclosed herein may be less than 20% (e.g., less than 15%, less than 10%, less than 5% or less) under stress or unaffected (remaining the same) and / or during recovery from stress, compared to normal non-stress conditions.

[0078] In some implementations, the transgenic plants disclosed herein show substantially no reduction in growth compared to their wild-type counterparts under the same conditions. In other words, the presence of the transgene has no materially adverse effect on plant growth. For example, the plant height, spike length, spike number, or root number / length of the transgenic plants disclosed herein may be reduced by less than 15% (e.g., less than 10%, less than 5%, or less) or remain unaffected (remain the same) compared to their wild-type counterparts under the same growth conditions.

[0079] In some embodiments, the transgenic plants disclosed herein exhibit substantially no yield reduction under stress and / or during recovery from stress compared to normal, non-stressful conditions. For example, the grain yield reduction of the transgenic plants disclosed herein during stress and / or recovery from stress is less than 15% (e.g., less than 10%, less than 5% or less) compared to normal, non-stressful conditions. In particular, the yield reduction of the transgenic plants of the present invention relative to normal, non-stressful conditions during stress and / or recovery from stress is less than that of their wild-type counterparts.

[0080] In some embodiments, such as those disclosed herein, the transgenic plants can exhibit enhanced survival under salt stress. Salt stress can be simulated by exposure to 100 mM or higher sodium chloride (NaCl) (e.g., 150 mM or higher, 200 mM or higher, 250 mM or higher). In some embodiments, the plants can be allowed to recover from salt stress.

[0081] In some embodiments, the transgenic plants disclosed herein can exhibit enhanced survival under osmotic stress. Osmotic stress can be simulated by exposure to 20% or higher (e.g., 30% or higher) of polyethylene glycol (PEG). In some embodiments, osmotic pressure is simulated at 30% PEG6000. In some embodiments, the plants can be allowed to recover from osmotic stress.

[0082] In some implementations, the transgenic plants disclosed herein can exhibit enhanced survival under drought stress. Drought stress may be simulated through dehydration. In some implementations, recovery from drought stress can be achieved through rehydration.

[0083] Therefore, the present invention also provides a method for producing the transgenic plants described herein. The method may include: (a) transforming plant cells with a nucleic acid operatively linked to a promoter to obtain recombinant plant cells expressing a RePRP protein, wherein the nucleic acid encodes the RePRP protein; and (b) culturing the recombinant plant cells obtained in (a) to produce the transgenic plants.

[0084] The present invention further provides a method for enhancing plant growth (e.g., under stress and / or during recovery from stress) or stress tolerance. The method may include: (a) transforming plant cells with a nucleic acid operably linked to a promoter to obtain recombinant plant cells expressing a RePRP protein, wherein the nucleic acid encodes the RePRP protein; (b) culturing the recombinant plant cells obtained in (a) to produce a variety of transgenic plants; and (c) selecting transgenic plants from the majority of transgenic plants produced in (b) that exhibit enhanced characteristics in terms of stress tolerance or growth, including higher survival rates to abiotic stresses, substantially no growth reduction, or combinations thereof, compared to non-transgenic plant counterparts grown under the same conditions. In some embodiments, the selected transgenic plant exhibits substantially no growth / yield reduction under stress and / or during recovery from stress compared to normal non-stress conditions. In some embodiments, the selected transgenic plant exhibits similar growth characteristics and / or a lower degree of yield reduction under stress conditions compared to its non-transgenic plant counterpart.

[0085] Without further detailed description, it is believed that those skilled in the art can fully utilize the invention based on the disclosure herein. Therefore, the specific examples below are to be interpreted only as descriptive and not in any way limiting the remainder of this disclosure.

[0086] Example

[0087] In this study, we generated transgenic plants overexpressing OsRePRP2.1 and found that OsRePRP2.1 overexpression made the plants more tolerant to drought and salinity conditions. In field trials under semi-arid conditions, the OsRePRP2.1 overexpressing plant lines also showed no significant growth retardation and less grain yield reduction compared to wild-type plants. To further reduce yield loss in OsRePRP2.1 overexpressing plants, stress-induced expression of OsRePRP2.1 controlled by a synthetic 3XABRC321 promoter was introduced into transgenic rice. These transgenic plants with ABA / stress-induced OsRePRP2.1 overexpression showed significantly higher drought tolerance and maintained normal plant growth under non-stress conditions.

[0088] 1. Materials and Methods

[0089] 1.1 Plant materials and growth conditions

[0090] Wild-type rice (Oryza sativa L., cv. Taiinung 67; "TNG67") seeds were sterilized with 2% sodium hypochlorite for 30 minutes and then thoroughly washed with distilled water. To obtain uniform germination, the rice seeds were soaked in distilled water at 37°C for 1 day in the dark, and then germinated in 20cm petri dishes containing distilled water at 37°C.

[0091] Transgenic rice seeds were germinated for 3 days at 28°C in water containing hygromycin-B (30 μg / ml; Invitrogen) to select transgenic seedlings. Uniformly germinated seeds were then selected and cultured in beakers containing a half-strength Kimura B solution (Hsu et al., 2003). Hydroponic seedlings were grown under 14-hour light / 10-hour dark conditions at 28°C and 90% relative humidity, with a light intensity of 100–105 μmol photons m⁻² sec⁻¹.

[0092] Arabidopsis seeds were sterilized with 0.6% sodium hydrochloride for 15 minutes and thoroughly washed with distilled water. To select transgenic plants, seeds were sown in the dark at 4°C for 2 days in half-strength Murashige and Skoog (MS) medium containing 1% sucrose and 0.8% agar (pH 5.7) with kanamycin (50 μg / ml; Invitrogena), followed by 5 days of growth on plates at 22°C with a 16-hour light / 8-hour dark cycle. Homozygous transgenic plants and wild-type seeds were grown for 10 days in normal half-strength MS medium without antibiotics containing 1% sucrose and 0.8% agar (pH 5.7) and then transferred to soil conditions for phenotypic observation.

[0093] 1.2 The Production of Transgenic Plants

[0094] Transgenic rice overexpressing OsRePRP2.1 was produced as follows. The coding region of OsRePRP2.1, amplified by genomic PCR, was cloned following the maize Ubi1 promoter, with its first intron located in the pPZP binary vector. This vector contained a promoter / coding region fragment of SEQ ID NO: 12 (Ubi: OsRePRP2.1). Essentially as described by Hong et al., 2004, in their rice transformation laboratory, transgenic rice lines were generated using this construct via Agrobacterium-mediated transformation.

[0095] Transgenic Arabidopsis thaliana with OsRePRP2.1 overexpression was produced as follows. The OsRePRP2.1 coding region was cloned after the 35S promoter in the pKGW vector. The vector contained a combined promoter / coding region fragment of SEQ ID: 14 ​​(35S: OsRePRP2.1). Transgenic Arabidopsis thaliana plants were produced using this construct via Agrobacterium-mediated vacuum infiltration transformation in a transgenic plant laboratory.

[0096] The OsRePRP-RNAi knockout line was generated as follows. The coding regions of the OsRePRP1.1 and 2.1 genes were amplified by genomic PCR and fused together in pCR8 / GW / TOPO (Invitrogen). The combined fragment was excised and cloned into the pANDA binary vector described in Miki et al., 2004, via LR recombination (LRClonase, Invitrogen). Transgenic lines were obtained from this construct as described above.

[0097] A stress-inducible OsRePRP2.1 construct was obtained by fusing the OsRePRP2.1 coding region with the 3XABRC321 promoter (SEQ ID NO: 11) (Chen et al., 2015) in the pENTR vector (Invitrogen). The combined promoter / coding region fragment was cloned into the pZP200 binary vector via LR recombination. This vector contained the combined promoter / coding region fragment of SEQ ID NO: 13 (3XABRC321: OsRePRP2.1). Similarly, transgenic plants were generated by Agrobacterium-mediated transformation.

[0098] 1.3 RT-PCR Analysis

[0099] To determine OsRePRP transcript levels, total RNA was isolated from rice tissues using TRIzol reagent (Invitrogen) as recommended by the supplier. First-strand cDNA was synthesized using the SuperScript II first-strand synthesis system (Invitrogen). The gene-specific primer sets used for RT-PCR quantification of OsRePRP gene expression are provided in Table 1 below. The rice OsActin gene was used as an internal control.

[0100] Table 1: RT-PCR primers

[0101]

[0102] 1.4 Adversity Management

[0103] For hydroponic growth, wild-type (TNG67) and transgenic plants (10 plants per row) were grown in the same pots in a normal half-strength Kimura B solution for 2 weeks. Two-week-old seedlings (three-leaf stage) were exposed to (i) a 30% aqueous PEG solution (PEG 6000; Merck) for 18–20 hours, or (ii) 250 mM NaCl in a half-strength Kimura B solution for 5 days. Seedlings recovered in the normal half-strength Kimura B solution for 10–12 days, after which plant survival was assessed. For soil growth, seven 2-day-germinated seeds per pot were grown for two weeks in a 1:1 (v / v) clay and vermiculite soil mixture. Two-week-old seedlings (three-leaf stage) were withheld from water for 12–14 days, then watered again for 12 days, after which photographs were taken and survival was measured.

[0104] 2. Results

[0105] 2.1 OsRePRP 2.1 Overexpression enhances tolerance to high salinity and dehydration.

[0106] Multiple stable transforming lines were generated that overexpressed the OsRePRP2.1 gene (“OsRePRP2.1OX”). Ectopic expression of this gene is controlled by the maize ubiquitin promoter. These stable transforming lines were already present in T4 generation homozygous plants. Using the primers shown in Table 1 above, the transcriptional levels of several OsRePRP genes in the roots of these lines were quantified by RT-PCR.

[0107] The results are shown in Table 2.

[0108] Table 2: Relative mRNA expression of OsRePRP transgenic rice lines.

[0109]

[0110] Under normal conditions, the OsRePRP2.1 transcript levels were significantly higher in the overexpression lines compared to wild-type TNG67 plants. The OsRePRP2.1 expression levels induced by salt treatment in TNG67 plants were similar to those observed in the untreated OsRePRP2.1 overexpression lines. The expression levels of OsRePRP2.2, OsRePRP 1.1, and OsRePRP 1.2 were not significantly affected by OsRePRP2.1 overexpression.

[0111] 2.3 OsRePRP2.1 Overexpression enhances high salt tolerance

[0112] The response of the aforementioned transgenic rice plants to high salinity conditions was tested to determine whether OsRePRP-mediated root structure modification is beneficial to rice plants under stress. Ten individual plants from each transgenic line were grown side-by-side with TNG67 plants in a hydroponic culture chamber. Three-leaf-aged plants were treated with 250 mM NaCl for 5 days, followed by a 12-day recovery period on normal culture medium. After the recovery period, the survival rates of each line were compared with those of TNG67. The results showed that the three independent OsRePRP2.1OX transgenic rice plants had a survival rate of 25–40%, while the survival rate of TNG67 was less than 5%. See also Figure 1 Clearly, overexpression of OsRePRP2.1 enhances the rice's adaptability to salinity conditions.

[0113] 2.4 OsRePRP2.1 Overexpression enhances dehydration tolerance

[0114] Besides salinity, water deficiency (i.e., dehydration) is another significant environmental stress that inhibits rice plant growth. Dehydration was simulated by treating rice with 30% PEG for 20 hours. More specifically, three-leaf-aged plants were treated with 30% PEG for 20 hours and then switched to normal culture medium for recovery. Survival rates were determined 10 days after recovery. The results showed that the survival rate of the three independent OsRePRP2.1OX transgenic rice plants was as high as 75%, while that of the TNG67 wild-type plants was only 15%. See also Figure 2 Furthermore, when the PEG treatment time was shortened to 16 hours, the withered leaves of OsRePRP2.1OX transgenic rice plants were able to fully swell within one day after recovery, and the survival rate increased to 95%.

[0115] 2.5 RNA interference (“RNAi”)-mediated knockdown of OsRePRP expression reduced the stress tolerance of rice.

[0116] OsRePRP RNAi transgenic rice lines were generated to reduce the expression of all four OsRePRP genes. Rice OsRePRP family transcripts were measured in salt-treated roots of the OsRePRP RNAi lines by RT-PCR as described above. The results are shown in Table 3 below.

[0117] Table 3. Relative RePRP gene expression in RNAi knockdown lines

[0118]

[0119] Even after salt treatment, all four OsRePRP RNAi lines tested showed significantly lower levels of RNA transcripts from the endogenous OsRePRP1.1, OsRePRP1.2, OsRePRP2.1, and OsRePRP2.2 genes.

[0120] 2.6 OsRePRP knockdown of transgenic rice is sensitive to salinity and drought conditions.

[0121] As described above, the sensitivity of OsRePRP RNAi transgenic rice plants to salt and PEG was tested. In the salinity test (250 mM NaCl), the average survival rate of the RNAi lines was 3.2%, less than half of the 9.1% shown by wild-type TNG67 plants. Under the same conditions, the survival rate of OsRePRP2.1OX transgenic plants was as high as 80%.

[0122] In the drought test (30% PEG), the average survival rate of the RNAi lines was 2.7%, lower than the 4.2% survival rate of the TNG67 plants. Under the same conditions, 45% and 83% of the OsRePRP2.1OX transgenic rice survived. See also Figure 4Based on these results, knocking down the expression of the OsRePRP gene in rice leads to greater sensitivity to salinity and drought treatments.

[0123] 2.7 Transgenic rice plants overexpressing OsRePRP2.1 were tolerant of field drought conditions.

[0124] Field drought tests were conducted to assess the tolerance levels of TNG67 wild-type and OsRePRP2.1OX transgenic plants. Field tests were conducted twice, during the first and second growing seasons of 2015. In both seasons, the OsRePRP2.1OX line was grown alongside TNG67 plants, with 24 plants per line, spaced 25 x 25 cm apart, simultaneously in irrigated and unirrigated fields. Testing began by transplanting 25-day-old transgenic seedlings into fields certified by the genetically modified organism.

[0125] Normal irrigation involves flooding the field with 1-5 cm of water until the end of the active tillage stage, i.e., 60-70 days after swelling, at which point the water is drained. The soil is kept moist until the end of the tillering stage. Then, the field is flooded with 3-10 cm of water until the grain-filling stage, and then drained again.

[0126] In non-irrigated fields, the soil was kept moist rather than flooded throughout the growing season. Plant growth and grain yield were observed. The results are summarized in Table 4 below.

[0127] Table 4. Results of reduced plant growth and grain yield in plants overexpressing OsRePRP.

[0128]

[0129] 1. Plant height, spike length, and number of spikes were measured for each individual of each line in both seasons of 2015 and are shown as mean ± SD (n = 24).

[0130] 2. Grain yield was measured for each individual plant of each line in both seasons of 2015. Grain yield loss (%) is shown as the average (%) of the two seasons' loss for each line, where the loss for each season for each line was calculated as follows: (average grain yield under irrigated conditions – average grain yield under non-irrigated conditions) / average grain yield under irrigated conditions × 100 (%).

[0131] The results showed that under normal irrigation conditions, the plant height, spike length, and number of spikes were similar for both OsRePRP2.1OX and TNG67 plants. Furthermore, transgenic Arabidopsis plants overexpressing OsRePRP2.1 also exhibited similar growth patterns to non-transgenic Arabidopsis plants under normal conditions. See also Figures 6A to 6CThis shows that overexpression of OsRePRP2.1 does not affect plant growth, including plant height, spike length, number of spikes, and root length.

[0132] On the other hand, TNG67 plants grown in non-irrigated fields resulted in a grain yield loss of approximately 15% compared to TNG67 plants grown under irrigated conditions. In contrast, OsRePRP2.1OX transgenic rice plants grown in non-irrigated fields showed a rice yield reduction of less than 15% compared to OsRePRP2.1OX transgenic rice plants grown under irrigated conditions. Surprisingly, some of these lines (such as OX#2 and OX#10) exhibited very low grain loss, with a loss of only about 5%. These observations suggest that overexpression of OsRePRP2.1 in rice helps plants survive under drought conditions and maintain near-normal seed yields.

[0133] 2.8 Transgenic rice plants with stress-induced OsRePRP2.1 overexpression

[0134] Although constitutive expression of OsRPRP2.1 in rice improves drought tolerance, overexpression also leads to yield loss under non-stress conditions.

[0135] The rice plants produced contain the OsRePRP2.1 transgene controlled by the synthetic ABA / stress-inducible promoter 3XABRC321, which enables the gene to be expressed at high levels only under stress conditions (see Chen et al., 2015).

[0136] As mentioned above, drought tolerance of first-generation T1 and second-generation T2 plants was tested in a PEG stress mode. After PEG treatment in a hydroponic culture system, three independent T2 rice lines carrying the inducible ABRC321:OsRePRP2.1 construct showed significantly higher survival rates compared to wild-type plants.

[0137] Furthermore, no root growth inhibition was observed in the induced expression lines, as was observed in the overexpression lines. See also Figure 5A and Figure 5B The plant growth of ABRC321:OsRePRP2.1 transgenic plants was not affected. Under normal conditions, ABRC321:OsRePRP2.1 transgenic rice showed similar plant height, panicle length, and panicle number to wild-type plants. See Table 5.

[0138] Table 5. Plant growth results of ABRC321: OsRePRP2.1 transgenic plants

[0139] Plant height (cm) Ear length (cm) number of ears TNG67 95.9±3.3 18.5±2.7 8.3±1.4 ABRC:P2.1#2 86.3±3.3 16.6±2.5 10.5±2.0 ABRC:P2.1#10 90.7±3.8 17.8±2.5 7.5±1.9 ABRC:P2.1#12 91.6±4.8 17.5±2.7 10.9±1.6

[0140] 1. Plant height, spike length and number of spikes for each individual of each line were measured in the first season of 2017 and are shown as mean ± SD (n = 15).

[0141] These results indicate that ABRC321:OsRePRP2.1-induced transgenic rice plants exhibit drought tolerance due to OsRePRP2.1 expression. However, these plants do not suffer from any growth retardation effects due to OsRePRP2.1 overexpression.

[0142] In summary, the technology provided by this invention enhances stress tolerance and / or prevents reduced plant growth by introducing polynucleotides encoding repeating proline-rich proteins (RePRPs) into plants. This not only helps plants survive in adverse conditions but also enables them to maintain growth and productivity, which is beneficial to agricultural development.

[0143] The following references can be used to better understand the background technology of this application:

[0144] Boyer,JS(1982),Science 218,443-448.

[0145] Chen et al(2015),Plant Biotechnology J.13,105-116.

[0146] Hong et al.(2004),Transgenic Research 13,29-39.

[0147] Hsu et al.(2003),Plant,Cell Environment 26,867-874.

[0148] Kar(2011),Plant Signaling Behavior 6,1741-1745.

[0149] Miki et al. (2004), Plant Cell Physiology 45, 490-495.

[0150] Saab et al. (1990), Plant Physiology 93, 1329-1336.

[0151] Sharp et al. (2004), J. Experimental Botany 55, 2343-2351.

[0152] Tilman et al.(2002),Nature 418,671-677.

[0153] Tseng et al.(2013),Plant Physiology 163,118-134.

[0154] Xu et al.(2013),The New Phytologist 197,139-150.

[0155] The contents of the above references are incorporated into this paper in their entirety through citation.

[0156] Other specific embodiments

[0157] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise expressly stated, each disclosed feature is merely one example of a series of equivalent or similar features.

[0158] From the above description, those skilled in the art can readily identify the basic features of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

[0159] Sequence information

[0160] >OsRePRP1.1 (SEQ ID NO:1)

[0161] MARRSPCLTAAVLLLGALAVASALVDEAAAAGQGLGHGARFMSKQGRAMYEKPPELEPKPKPKPHPKHESKPEPKPEPKPEPKPYPEPKPETKPELKPEPKPNPEPKPEPKPEPKPEPKPYPEPKPKPEPKPEPKPEHKPEPKPEPKPYPKPKPEPKPGPKPEPKPEPKPHPEPK PEPKPKPVPHPEPKPEPKPEPKPHPEPKPEPKPEPKLHPKPEPKPHPEPEPKLKPEPKPEPKPEPEPKPEPKPEPKPYPKPKPEPKPVPKPKPIPHPGPKPKPDPKLEPKPHPEPKPHPMPEPEPKPKPEPKPEPKPYPEPKPKLKPEPKPGPKPIAPPNKHKPPHMPPATNQ

[0162] >OsRePRP1.2(SEQ ID NO:2)

[0163] MARRSPCLAVAMLLLGALAVASAFIDEAAAAGRGLGHGARFMSKQGRVTYEKLPEPEPKPKPKPHPKPTPKPEPKPEPEPKPVPEPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEP EPKPEPKPEPKPEPKPKPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPYPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEP

[0164] >OsRePRP2.1(SEQ ID NO:3)

[0165] MRSILSLCFHLALAAIALAANVPDHIANGRVIEAKSDPKPADPNPKPDPTPKPQPETKPSPQPNPQPNPQPDPKPPSQPDPKPTPQPEPKQDPKPNPQPDPKPTPQPDPKPTPQPDPKQDPQPNPQPDPKPTPQPNPKQDPQPNPQPDPKPTPQPDPKQDPQPNPQSPKADPKPNPKPKPQPESPNPKPEPKPEPKPEPSPNPKPNPKPEPQPDPKPEPKPQPEPSLPKPPLSPAIAIIVPGN

[0166] >OsRePRP2.2(SEQ ID NO:4)

[0167] MRRSILSLCFHLALVIALAANVPDIANGRVIEAKSDPKPADPKPKPDPTPKPQPETKPSPQPNPQPNPQPDPKPSPQPDPKPTPQPEPKQDPQPNPQPDPKQSPQPDPKPTPQPNPKQDPQPNPQPDPKPTLQPNPKQDPQPNPQPNPKPTPQLDPKQDPQPNPQPSPKADPKPNPKPKPQPEPSPNPKPEPKPEPKPEPSPNPKPNPNPKPEPQPDPKPEPKPQPEPSQPKLPPLSPAIAIIVPGN

[0168] >OsRePRP1.1(SEQ ID NO:5)

[0169]

[0170]

[0171] >OsRePRP1.2(SEQ ID NO:6)

[0172]

[0173] OsRePRP2.1(SEQ ID NO:7)

[0174]

[0175]

[0176] >OsRePRP2.2(SEQ ID NO:8)

[0177]

[0178] >1XABRC321(SEQ ID NO:9)

[0179]

[0180] >2XABRC321(SEQ ID NO:10)

[0181]

[0182] >3XABRC321(SEQ ID NO:11)

[0183]

[0184]

[0185] >Ubi:OsRePRP2.1(SEQ ID NO:12)

[0186]

[0187]

[0188] >3xABRC321i:OsRePRP2.1(SEQ ID NO:13)

[0189]

[0190] >35S:OsRePRP2.1 (SEQ ID NO:14)

[0191]

[0192] sequence list <110> Academia Sinica, Taiwan <120> Methods to enhance plant stress tolerance <130> IA0001 / ACA0128US <150> 62 / 472,669 <151> 2017-03-17 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 358 <212> PRT <213> Rice (Oryza sativa) <400> 1 Met Ala Arg Arg Ser Pro Cys Leu Thr Ala Ala Val Leu Leu Leu Gly 1 5 10 15 Ala Leu Ala Val Ala Ser Ala Leu Val Asp Glu Ala Ala Ala Ala Gly 20 25 30 Gln Gly Leu Gly His Gly Ala Arg Phe Met Ser Lys Gln Gly Arg Ala 35 40 45 Met Tyr Glu Lys Pro Pro Glu Leu Glu Pro Lys Pro Lys Pro Lys Pro 50 55 60 His Pro Lys His Glu Ser Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 65 70 75 80 Glu Pro Lys Pro Tyr Pro Glu Pro Lys Pro Glu Thr Lys Pro Glu Leu 85 90 95 Lys Pro Glu Pro Lys Pro Asn Pro Glu Pro Lys Pro Glu Pro Lys Pro 100 105 110 Glu Pro Lys Pro Glu Pro Lys Pro Tyr Pro Glu Pro Lys Pro Lys Pro 115 120 125 Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu His Lys Pro Glu Pro 130 135 140 Lys Pro Glu Pro Glu Pro Lys Pro Tyr Pro Lys Pro Lys Pro Glu Pro 145 150 155 160 Lys Pro Gly Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro His Pro 165 170 175 Glu Pro Lys Pro Glu Pro Lys Pro Lys Pro Val Pro His Pro Glu Pro 180 185 190 Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro His Pro Glu Pro Lys Pro 195 200 205 Glu Pro Lys Pro Glu Pro Lys Leu His Pro Lys Pro Glu Pro Lys Pro 210 215 220 His Pro Glu Pro Glu Pro Lys Leu Lys Pro Glu Pro Lys Pro Glu Pro 225 230 235 240 Lys Pro Glu Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 245 250 255 Glu Pro Lys Pro Tyr Pro Lys Pro Lys Pro Glu Pro Lys Pro Val Pro 260 265 270 Lys Pro Lys Pro Ile Pro His Pro Gly Pro Lys Pro Lys Pro Lys Pro 275 280 285 Asp Pro Lys Leu Glu Pro Lys Pro His Pro Glu Pro Lys Pro His Pro 290 295 300 Met Pro Glu Pro Glu Pro Lys Pro Lys Pro Glu Pro Lys Pro Glu Pro 305 310 315 320 Lys Pro Tyr Pro Glu Pro Lys Pro Lys Leu Lys Pro Glu Pro Lys Pro 325 330 335 Gly Pro Lys Pro Ile Ala Pro Pro Asn Lys His Lys Pro Pro His Met 340 345 350 Pro Pro Ala Thr Asn Gln 355 <210> 2 <211> 416 <212> PRT <213> Rice (Oryza sativa) <400> 2 Met Ala Arg Arg Ser Pro Cys Leu Ala Val Ala Met Leu Leu Leu Gly 1 5 10 15 Ala Leu Ala Val Ala Ser Ala Phe Ile Asp Glu Ala Ala Ala Ala Gly 20 25 30 Arg Gly Leu Gly His Gly Ala Arg Phe Met Ser Lys Gln Gly Arg Val 35 40 45 Thr Tyr Glu Lys Leu Pro Glu Pro Glu Pro Lys Pro Lys Pro Lys Pro 50 55 60 His Pro Lys Pro Thr Pro Lys Pro Glu Pro Lys Pro Glu Pro Glu Pro 65 70 75 80 Lys Pro Val Pro Glu Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 85 90 95 Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Tyr Pro Glu Pro Lys Pro 100 105 110 Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Glu Pro Lys Pro Glu Pro 115 120 125 Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Tyr Pro Glu Pro Lys Pro 130 135 140 Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 145 150 155 160 Lys Pro Glu Pro Lys Pro His Pro Glu Pro Lys Pro Asp Pro Lys Pro 165 170 175 Glu Pro Lys Pro His Pro Glu Pro Glu Pro Lys Pro Glu Pro Lys Pro 180 185 190 Glu Pro Lys Pro His Pro Glu Pro Glu Pro Lys Pro Glu Pro Lys Pro 195 200 205 Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 210 215 220 Lys Pro Lys Pro Glu Pro Lys Pro Lys Pro Glu Pro Lys Pro Tyr Pro 225 230 235 240 Glu Pro Lys Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 245 250 255 Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 260 265 270 Lys Pro Glu Pro Lys Pro Lys Pro Glu Pro Lys Pro His Pro Lys Pro 275 280 285 Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 290 295 300 Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Glu Pro Lys Pro Glu Pro 305 310 315 320 Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Tyr Pro Glu Pro Lys Pro 325 330 335 Asp Pro Lys Pro Glu Pro Lys Pro His Pro Glu Pro Lys Pro Glu Pro 340 345 350 Lys Pro Gln Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 355 360 365 Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Tyr Pro Glu Pro 370 375 380 Lys Pro Glu Pro Lys Pro Lys Pro Lys Pro Glu Pro Lys Pro Glu Ala 385 390 395 400 Pro Pro Lys Lys His Lys Pro Pro His Ile Pro Pro Ala Thr Asp Gln 405 410 415 <210> 3 <211> 247 <212> PRT <213> Rice (Oryza sativa) <400> 3 Met Arg Ser Ile Leu Ser Leu Cys Phe His Leu Ala Leu Ala Ile Ala 1 5 10 15 Leu Ala Ala Asn Val Pro Asp His Ile Ala Asn Gly Arg Val Ile Glu 20 25 30 Ala Lys Ser Asp Pro Lys Pro Ala Asp Pro Asn Pro Lys Pro Asp Pro 35 40 45 Thr Pro Lys Pro Gln Pro Glu Thr Lys Pro Ser Pro Gln Pro Asn Pro 50 55 60 Gln Pro Asn Pro Gln Pro Asp Pro Lys Pro Ser Pro Gln Pro Asp Pro 65 70 75 80 Lys Pro Thr Pro Gln Pro Glu Pro Lys Gln Asp Pro Lys Pro Asn Pro 85 90 95 Gln Pro Asp Pro Lys Pro Ser Pro Gln Pro Asp Pro Lys Pro Thr Pro 100 105 110 Gln Pro Asp Pro Lys Gln Asp Pro Gln Pro Asn Pro Gln Pro Asp Pro 115 120 125 Lys Pro Thr Pro Gln Pro Asn Pro Lys Gln Asp Pro Gln Pro Asn Pro 130 135 140 Gln Pro Asp Pro Lys Pro Thr Pro Gln Pro Asp Pro Lys Gln Asp Pro 145 150 155 160 Gln Pro Asn Pro Gln Pro Ser Pro Lys Ala Asp Pro Lys Pro Asn Pro 165 170 175 Lys Pro Lys Pro Gln Pro Glu Pro Ser Pro Asn Pro Lys Pro Glu Pro 180 185 190 Lys Pro Glu Pro Lys Pro Glu Pro Ser Pro Asn Pro Lys Pro Asn Pro 195 200 205 Asn Pro Lys Pro Glu Pro Gln Pro Asp Pro Lys Pro Glu Pro Lys Pro 210 215 220 Gln Pro Glu Pro Ser Leu Pro Lys Pro Pro Pro Leu Ser Pro Ala Ile 225 230 235 240 Ala Ile Ile Val Pro Gly Asn 245 <210> 4 <211> 247 <212> PRT <213> Rice (Oryza sativa) <400> 4 Met Arg Arg Ser Ile Leu Ser Leu Cys Phe His Leu Ala Leu Val Ile 1 5 10 15 Ala Leu Ala Ala Asn Val Pro Asp Ile Ala Asn Gly Arg Val Ile Glu 20 25 30 Ala Lys Ser Asp Pro Lys Pro Ala Asp Pro Lys Pro Lys Pro Asp Pro 35 40 45 Thr Pro Lys Pro Gln Pro Glu Thr Lys Pro Ser Pro Gln Pro Asn Pro 50 55 60 Gln Pro Asn Pro Gln Pro Asp Pro Lys Pro Ser Pro Gln Pro Asp Pro 65 70 75 80 Lys Pro Thr Pro Gln Pro Glu Pro Lys Gln Asp Pro Gln Pro Asn Pro 85 90 95 Gln Pro Asp Pro Lys Gln Ser Pro Gln Pro Asp Pro Lys Pro Thr Pro 100 105 110 Gln Pro Asn Pro Lys Gln Asp Pro Gln Pro Asn Pro Gln Pro Asp Pro 115 120 125 Lys Pro Thr Leu Gln Pro Asn Pro Lys Gln Asp Pro Gln Pro Asn Pro 130 135 140 Gln Pro Asn Pro Lys Pro Thr Pro Gln Leu Asp Pro Lys Gln Asp Pro 145 150 155 160 Gln Pro Asn Pro Gln Pro Ser Pro Lys Ala Asp Pro Lys Pro Asn Pro 165 170 175 Lys Pro Lys Pro Gln Pro Glu Pro Ser Pro Asn Pro Lys Pro Glu Pro 180 185 190 Lys Pro Glu Pro Lys Pro Glu Pro Ser Pro Asn Pro Lys Pro Asn Pro 195 200 205 Asn Pro Lys Pro Glu Pro Gln Pro Asp Pro Lys Pro Glu Pro Lys Pro 210 215 220 Gln Pro Glu Pro Ser Gln Pro Lys Leu Pro Pro Leu Ser Pro Ala Ile 225 230 235 240 Ala Ile Ile Val Pro Gly Asn 245 <210> 5 <211> 1493 <212> DNA <213> Rice (Oryza sativa) <400> 5 caacagcaga agtgagagag ggagaagaag ataagcgaag aggaggagct tagcttgcca 60 gccatggcta ggcgctctcc ttgcctcact gccgccgtgc tcctgcttgg ggcattggcg 120 gtggcgagcg ctttagttga tgaagcggcg gcagctggcc agggactcgg ccatggcgcc 180 cgcttcatga gcaagcaggg ccgtgcgatg tacgagaagc cgccagagct ggagccgaag 240 ccaaagccaa agcctcatcc taagcatgaa tcaaaaccgg agccaaagcc agaacctaag 300 ccggagccaa agccataccc agagccgaag ccagagacga aaccggagct aaagccagaa 360 ccaaaaccta atccagaacc taaacctgag cctaagcctg aaccaaaacc agaaccaaag 420 ccatacccag agccgaagcc aaagcccaaa ccggagccaa agccagaacc aaaacctgag 480 cataaacctg aaccaaaacc agaaccagaa ccaaagccat acccaaagcc aaagccagag 540 ccaaaaccgg ggcccaaacc cgagccgaag ccagagccta agccacaccc agaaccgaaa 600 ccggagccca aaccaaagcc agtgccacac cctgaacca aaccggacc aaagccggag cccaaaccac acccagaacc aaagcctgag ccgaaacccg agcctaagct acacccgaag cctgagccaa agccacaccc agagcctgag cctaagctta aacctgaacc aaaaccagag ccaaagccag agcctgaacc gaagcccgag ccaaagcctg aaccaaacc agagcctaaa ccatatccaa agccaaaacc ggacctaa ccggtgccga agccgaagcc cattccacac ccaggacca aaccaaagcc taaacctgac ccaaagctag agcccaagcc acacccggag 1020. ccaaaaccac atccgatgcc tgaacctgaa ccaaagccta agcccgaacc aaagccagag cctaaccat acccagaacc aaagcctaaa ctgaaacctg aacctaagcc tggaccgaaa cctatagcac cgccgaacaa gcacaagccg ccgcacatgc caccagcgac aaaccagtga cggcgatcgc tggagaccga gcatttgctg gctgcacggt tgaggcaccg acgacattat ttcacccgag gaaggagcgc tagcgagtca ctacactgta ccgtttctgg aataaagtga tgagctagct ttctgcttgc cttttctttt cctctcttat tttcctttta tttcatgttg 1320 gtttttcgga tgtgccactg ctagctagtg taattaaatt atttattatg tgcctaccgt 1380 catttttatt accgtgtctg tgacattcta ttgtctattg gcattattct cattgtaaaa 1440 tcttttggta atattatttg tcatcatttt tacccagctt ctaaaaaaaa aaa 1493 <210> 6 <211> 1251 <212> DNA <213> Rice (Oryza sativa) <400> 6 atggcgaggc gctctccttg cctcgccgtc gccatgctcc tgcttggggc gttggcggtg 60 gcgagcgcct tcattgatga agcggcggct gctggccggg ggctcggcca tggcgcccgc 120 ttcatgagca agcagggtcg tgtgacatac gagaagctgc cggagccgga gccgaagcca 180 aagccaaagc ctcatcctaa acccacgcca aaacctgagc ccaagccaga gccggagcca 240 aaaccagtac ctgagcctga gcctaaaccg gaaccaaagc cagaaccaaa acctgagcct 300 aagcctgaac ctaaaccata cccagagcca aaaccggagc cgaagccaga gccaaaacct 360 gagccggagc ctaaacctga gcctaagcca gaaccaaaac cagaaccaaa gccgtaccca 420 gagccgaagc cagagccaaa accggaaccg aagccggaac caaaaccgga gcccaaacca 480 aagccagagc ccaaaccaca cccagaacca aagcctgatc cgaaacctga gcctaagcca 540 cacccagagc ctgagcctaa gcctgaacct aagcctgagc ccaagccaca ccctgagcct 600 gaaccaaagc ctgagcctaa gcctgagcca aagccagaac caaagccgga gccaaaacct 660 gaaccaaaac caaagccaaa gccagagcca aagccaaagc ctgagcccaa gccataccct 720 gagcctaagc ctaagcctga accaaagcct gagcctaagc ctgagccaaa gccagaacca 780 aagccggagc caaaacctga accaaaacca gagccaaagc cagagccaaa gccaaagcct 840 gagcccaagc cacaccctaa gcctgagcct aagcctgagc ccaagccaga accaaagcca 900 gagccaaaac ctgaaccaaa accagagcca aaaccagagc ctgaaccgaa gcctgagcca 960 aagcctgaac caaaaccaga gcccaaacca tatccagagc ctaaaccgga tcccaaacca 1020 gaacccaaac cacacccaga accaaagcca gagcccaagc cacagccgga gccaaaacca 1080 gagccgaagc ctgaacctaa accagagcct aagcccgaac caaaaccgga gcctaaacca 1140 tacccagagc caaagcctga accgaaacct aagcctaagc ctgagccaaa acctgaagca 1200 cctccgaaga agcacaagcc gccgcacata ccgccagcga ccgaccagtg a 1251 <210> 7 <211> 1088 <212> DNA <213> Oryza sativa <400> 7 aacacaccta actaccacag cttgtgaact atcaagagtg agtagtagag tttgcagtga 60 caacgagatg aggagatcaa tcctctcact gtgcttccat ttggcgcttg tcattgcatt 120 ggcagcaaat gttcctgaca ttgccaatgg acgcgtgatt gaagctaaat ctgatccaaa 180 gccagcagat cccaagccta aacctgaccc aacaccaaaa ccacaaccag agacaaagcc 240 cagtccacag cctaaccctc aacctaaccc acagccagat ccaaaaccat caccgcagcc 300 tgatccaaaa cctacaccac agcctgaacc aaaacaagat cctcaaccaa acccacagcc 360 ggatccaaaa caatcgccgc agcctgaccc aaaacctaca ccacagccta acccaaaaca 420 agatcctcaa ccgaacccac aacctgaccc aaaaccaacg ctgcaaccta acccaaaaca 480 agatcctcag ccgaacccac agcctaaccc gaaaccaacg ccacagcttg acccgaaaca 540 agatcctcaa ccgaacccac aacctagccc caaagctgac ccaaaaccaa atccaaagcc 600 agatcctcaa ccgaacccac aacctagccc caaagctgac ccaaaaccaa atccaaagcc 600 taagccacaa ccggagccga gcccaaatcc taagccggag ccaaaacctg aacccaaacc 660 taagccacaa ccggagccga gcccaaatcc taagccggag ccaaaacctg aacccaaacc 660 tgagccgagt cctaacccca agccaaatcc taatcccaag ccggagccac agcctgatcc 720 tgagccgagt cctaacccca agccaaatcc taatcccaag ccggagccac agcctgatcc 720 taagccagaa cccaagcctc agccagagcc gtctcaacca aagctgccac cactttcacc 780 taagccagaa cccaagcctc agccagagcc gtctcaacca aagctgccac cactttcacc 780 agcaatagct ataattgtgc ccgggaactg agtagacttg gttgtttgct acgtatgatc 840 agcaatagct ataattgtgc ccgggaactg agtagacttg gttgtttgct acgtatgatc 840 ccgcatactt ttggtatgta ctattgctct agtgactatt tgtgtgtttt tcgtgtgttg 900 ccgcatactt ttggtatgta ctattgctct agtgactatt tgtgtgtttt tcgtgtgttg 900 ttcactagtg tgtccatgtg gctatctatg tgttttctta atgccgttgc atatgagcag 960 ttcactagtg tgtccatgtg gctatctatg tgttttctta atgccgttgc atatgagcag 960 gcgtgcttct tataataaag catacataca tacatacata catacataca tacatatata 1020 gcgtgcttct tataataaag catacataca tacatacata catacataca tacatatata 1020 tatacacgtg tgttatgtat gtgcgtacat accatcaata aaaagagcat gtatccctgt 1080 tatacacgtg tgttatgtat gtgcgtacat accatcaata aaaagagcat gtatccctgt 1080 gtgtcaat 1088 gtgtcaat 1088 <210> 8<210> 8 <211> 1057<211> 1057 <212> DNA<212> DNA <213> 水稻(Oryza sativa)<213> Rice (Oryza sativa) <400> 8 <400> 8 aacacaccta gctaccacag cttgtgtact gtcaagagtg agtagtagag tttgtagtga 60 aacacaccta gctaccacag cttgtgtact gtcaagagtg agtagtagag tttgtagtga 60 caacgagatg agatcaatcc tctcactgtg cttccatttg gcgcttgcca ttgcattggc 120 ggcaaatgtt cctgatcaca ttgccaatgg acgcgtgatt gaagctaaat ctgatccaaa 180 gccagcagat cccaatccta aacctgaccc aacaccaaaa ccacaaccag agacaaagcc 240 cagtccacag cctaaccctc aacctaaccc acagccagat ccaaaaccat caccgcagcc 300 tgacccaaaa cctacaccac agcctgaacc aaaacaagat cctaaaccaa acccacaacc 360 ggatccaaaa ccatctccgc agcctgaccc gaaacctaca ccacagcctg acccaaaaca 420 agatcctcaa ccgaacccac aacctgaccc aaaaccaacg ccgcaaccta acccaaaaca 480 agatcctcag ccgaacccac agcctgaccc aaaaccaacg ccacagcctg acccgaaaca 540 agatcctcaa ccgaacccgc aacctagccc caaagctgac ccaaaaccaa atccaaagcc 600 taagccacaa ccggagccga gcccaaatcc taagccggag ccaaagcctg aacccaaacc 660 tgagccaagt cctaacccca agccaaatcc taatcctaag ccggagccac agcctgatcc 720 taagccagaa cccaagcctc agccagagcc atctctgcca aagccaccac ctctttcacc 780 agcaatagct ataattgtgc ccgggaactg agcatagctt ttgctacgta tgattccgca 840. tagttttggt atgtactatt gctctagtga ctatctatgt gtttgtcgtg tgttgttcac 900 tggtgtatgt gtccatgtgg ctatctatgt gttttcttaa tgctgttgca tctgagcagg 960 cgtgcttctt fatheragc fathertatgc acgtgtgtta tgtatgtgcg tacatatata ccatgaataa aaagagcatg tatccctgtg tgtcact <210> 9 <211> 166 <212> DNA <213> The snowstorm <220> <223> 1XABRC321 <400> 9 60. ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg actgcagcaa ttccggcatg ccgcagcaca ctaaatac ctggccagac acacaagctg aatgcatcag ttctccatcg tactcttcga gagcacagca agagag <210> 10 <211> 216 <212> DNA <213> The snowstorm <220> <223> 2XABRC321 <400> 10 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg actgcagcaa 120 ttccggcatg ccgcagcaca ctataaatac ctggccagac acacaagctg aatgcatcag 180 ttctccatcg tactcttcga gagcacagca agagag 216 <210> 11 <211> 266 <212> DNA <213> Artificial sequence <220> <223> 3XABRC321 <400> 11 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa 60 cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa cgcgtgtcct 120 ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg actgcagcaa ttccggcatg 180 ccgcagcaca ctataaatac ctggccagac acacaagctg aatgcatcag ttctccatcg 240 tactcttcga gagcacagca agagag 266 <210> 12 <211> 2735 <212> DNA <213> Artificial sequence <220> <223> Ubi:OsRePRP2.1 <400> 12 ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60 agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120 tctttataca tatatttaaa ctttactcta cgaataat aatctatagt actacaataa 180 tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240 gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300 ttttttgcaa atagcttcac ctatatata cttcatccat tttattagta catccattta 360 gggtttaggg ttaatggttt ttagacta atttttttag tacatctatt ttattctatt 420 ttagcctcta attaagaaa actaaaactc tattttagtt tttttattta ataatttaga 480 tataaatag aataaaataa agtgactaaa attaaaaa atacccttta agaaattaaa 540 aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt aaacgccgtc 600 gacgcagtct aacggacacc aaccagcgaa ccagcagcgt cgcgtcgggc caagcgaagc 660 agacggcacg gcatctctgt cgctgcctct ggacccctct cgagagttc gctccaccgt 720 tggacttcgt ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg 780 cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc 840 accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc 900 tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat 960 ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ctctctacct 1020 tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080 tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140 tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200 atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260 gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320 tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380 agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440 gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500 ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg cttttgttcg 1560 cttggttgtg atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga 1620 atactgtttc aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac 1680 atcttcatag ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt 1740 tgatgtgggt tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc 1800 taaccttgag tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg 1860 atatacttgg atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca 1920 tacgctattt atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt 1980 tacttctgca gatgaggaga tcaatcctct cactgtgctt ccatttggcg cttgtcattg 2040 cattggcagc aaatgttcct gacattgcca atggacgcgt gattgaagct aaatctgatc 2100 caaagccagc agatcccaag cctaaacctg acccaacacc aaaaccacaa ccagagacaa 2160 agcccagtcc acagcctaac cctcaaccta acccacagcc agatccaaaa ccatcaccgc 2220 agcctgatcc aaaacctaca ccacagcctg aaccaaaaca agatcctcaa ccaaacccac 2280 agccggatcc aaaacaatcg ccgcagcctg acccaaaacc tacaccacag cctaacccaa 2340 aacaagatcc tcaaccgaac ccacaacctg acccaaaacc aacgctgcaa cctaacccaa 2400 aacaagatcc tcagccgaac ccacagccta acccgaaacc aacgccacag cttgacccga 2460 aacaagatcc tcaaccgaac ccacaaccta gccccaaagc tgacccaaaa ccaaatccaa 2520 agcctaagcc acaaccggag ccgagcccaa atcctaagcc ggagccaaaa cctgaaccca 2580 aacctgagcc gagtcctaac cccaagccaa atcctaatcc caagccggag ccacagcctg 2640 atcctaagcc agaacccaag cctcagccag agccgtctca accaaagctg ccaccacttt 2700 caccagcaat agctataatt gtgcccggga actga 2735 <210> 13 <211> 1331 <212> DNA <213> Artificial sequence <220> <223> 3xABRC321:OsRePRP2.1 <400> 13 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa 60 cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa cgcgtgtcct 120 ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg acctgcagca attccggcat 180 gccgcagcac actataaata cctggccaga cacacaagct gaatgcatca gttctccatc 240 gtactcttcg agagcacagc aagagagtga tcatttcagg taagatctag agtcgacctg 300 caggcgaccg tatgtatatt accctatctc taccttgcaa atcgcgtgtg tacggatctt 360 ctccgtggtc gagccgagtg attgctgatc tgatatccta tctgctgctt cgtttccttg 420 cgcaggccaa gcatcacgct gctgtaccct ctgtaagttg atcagtcgct tgtggtactt 480 tttagtacgt ggggaagtaa tccttgtgct ggatgtgacc ctggcggatc tgtataatac 540 aggtatgcgg atcccccggg ctgcaggaat tcgatatcaa gctcaccatg aggagatcaa 600 tcctctcact gtgcttccat ttggcgcttg tcattgcatt ggcagcaaat gttcctgaca 660 ttgccaatgg acgcgtgatt gaagctaaat ctgatccaaa gccagcagat cccaagccta 720 aacctgaccc aacaccaaaa ccacaaccag agacaaagcc cagtccacag cctaaccctc 780 aacctaaccc acagccagat ccaaaaccat caccgcagcc tgatccaaaa cctacaccac 840 agcctgaacc aaaacaagat cctcaaccaa acccacagcc ggatccaaaa caatcgccgc 900 agcctgaccc aaaacctaca ccacagccta acccaaaaca agatcctcaa ccgaacccac 960 aacctgaccc aaaaccaacg ctgcaaccta acccaaaaca agatcctcag ccgaacccac 1020 agcctaaccc gaaaccaacg ccacagcttg acccgaaaca agatcctcaa ccgaacccac 1080 aacctagccc caaagctgac ccaaaaccaa atccaaagcc taagccacaa ccggagccga 1140 gcccaaatcc taagccggag ccaaaacctg aacccaaacc tgagccgagt cctaacccca 1200 agccaaatcc taatcccaag ccggagccac agcctgatcc taagccagaa cccaagcctc 1260 agccagagcc gtctcaacca aagctgccac cactttcacc agcaatagct ataattgtgc 1320 ccgggaactg a 1331 <210> 14 <211> 1193 <212> DNA <213> Artificial Sequence <220> <223> 35S:OsRePRP2.1 <400> 14 tcgagggatc cgtcccccgt gttctctcca aatgaaatga acttccttat atagaggaag 60 ggtcttgcga aggatagtgg gattgtgcgt catcccttac gtcagtggag attccagata 120 ggcctaacgc ttgtccaaga tctattcagg attccagata ggcctaacgc ttgtccaaga 180 tctattcagg atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 240 ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 300 ttcaacgatg gcctttcctt tatcgcaatg atggcatttg taggagccac cttccttttc 360 cactatcttc acaataaagt gacagatagc tgggcaatgg aatccgagga ggtttccgga 420 taatgaggag atcaatcctc tcactgtgct tccatttggc gcttgtcatt gcattggcag 480 caaatgttcc tgacattgcc aatggacgcg tgattgaagc taaatctgat ccaaagccag 540 cagatcccaa gcctaaacct gacccaacac caaaaccaca accagagaca aagcccagtc 600 cacagcctaa ccctcaacct aacccacagc cagatccaaa accatcaccg cagcctgatc 660 caaaacctac accacagcct gaaccaaaac aagatcctca accaaaccca cagccggatc 720 caaaacaatc gccgcagcct gacccaaaac ctacaccaca gcctaaccca aaacaagatc 780 ctcaaccgaa cccacaacct gacccaaaac caacgctgca acctaaccca aaacaagatc 840 ctcagccgaa cccacagcct aacccgaaac caacgccaca gcttgacccg aaacaagatc 900 ctcaaccgaa cccacaacct agccccaaag ctgacccaaa accaaatcca aagcctaagc 960 cacaaccgga gccgagccca aatcctaagc cggagccaaa acctgaaccc aaacctgagc 1020 cgagtcctaa ccccaagcca aatcctaatc ccaagccgga gccacagcct gatcctaagc 1080 cagaacccaa gcctcagcca gagccgtctc aaccaaagct gccaccactt tcaccagcaa 1140 tagctataat tgtgcccggg aactacccat acgatgttcc agattacgct tga 1193 <210> 15 <211> 28 <212> DNA <213> Artificial sequence <220> <223> OsRePRP1.1 primer <400> 15 acaagctcac agttcagtta cgtacaac 28 <210> 16 <211> 16 <212> DNA <213> Artificial sequence <220> <223> OsRePRP1.1 primer <400> 16 gcgctccttc ctcgggt 16 <210> 17 <211> 25 <212> DNA <213> Artificial sequence <220> <223> OsRePRP1.2 Introduction <400> 17 gatcacagaa gctcacagtt cagtt 25 <210> 18 <211> 18 <212> DNA <213> Artificial sequence <220> <223> OsRePRP1.2 Introduction <400> 18 tgactcgctc gctcctcc 18 <210> 19 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> OsRePRP2.1 Introduction <400> 19 atgaggagat caatcctctc actg 24 <210> 20 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> OsRePRP2.1 Introduction <400> 20 tcagttcccg ggcacaatta tag 23 <210> twenty one <211> twenty four <212> DNA <213> Artificial sequence <220> <223> OsRePRP2.2 Introduction <400> twenty one aatgttcctg atcacattgc caat 24 <210> twenty two <211> twenty four <212> DNA <213> Artificial sequence <220> <223> OsRePRP2.2 Introduction <400> twenty two cataccaaaa ctatgcggaa tcat 24 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> OsActin introduction <400> twenty three ctgatggaca ggttatcacc 20 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <223> OsActin introduction <400> twenty four caggtagcaa taggtattac ag 22 <210> 25 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Proline enrichment motif <220> <221> Miscellaneous features <222> (2)..(2) <220> <221> Miscellaneous features <222> (4)..(4) <223> Xaa can be any naturally occurring amino acid other than proline. <400> 25 Pro Xaa Pro Xaa 1

Claims

1. A method for enhancing plant stress tolerance while preventing plant growth loss, comprising: (a) Transform plant cells with a vector containing a nucleic acid operatively linked to a promoter to obtain recombinant plant cells expressing a repeat proline enrichment protein (RePRP), wherein the nucleic acid encodes the RePRP protein; (b) Culturing the recombinant plant cells obtained in (a) to produce a majority of transgenic plants; and (c) Select transgenic plants from the majority of transgenic plants produced in (b) that exhibit enhanced tolerance to adversity and no reduction in growth, when compared with their non-transgenic counterparts grown under the same conditions. The REPRP protein described therein is composed of the amino acid sequence of SEQ ID NO: 3; and The aforementioned adversity is an abiotic adversity selected from a group consisting of drought adversity, salt adversity, or combinations thereof.

2. The method of claim 1, wherein the transgenic plant exhibits less yield reduction when compared with its non-transgenic counterpart.

3. The method of claim 1, wherein the promoter is heterologous to a naturally occurring gene encoding the RePRP protein.

4. The method of claim 1, wherein the promoter is a constitutive promoter or an inducible promoter.

5. The method of claim 4, wherein the promoter is a constitutive promoter selected from the group consisting of the maize ubiquitin (Ubi) promoter, the rice actin (Act1) promoter, and the cauliflower mosaic virus 35S (CaMV35S) promoter.

6. The method of claim 4, wherein the promoter is an inducible promoter selected from the group consisting of the Arabidopsis corl SA promoter, the Arabidopsis heat shock factor (HSF) promoter, the Arabidopsis kin1 promoter, the Arabidopsis rd29A promoter, the α-amylase promoter, and the synthetic ABRC321 promoter.

7. The method of claim 6, wherein the inducible promoter is 3XABRC321.

8. The method of claim 1, wherein the carrier comprises SEQ ID NO: 12, 13 or 14.

9. The method of claim 1, wherein the transgenic plant is a monocotyledonous plant.

10. The method of claim 9, wherein the monocotyledonous plant is rice, barley, wheat, rye, oats, or corn.

11. The method of claim 1, wherein the transgenic plant is a dicotyledonous plant.

12. The method of claim 11, wherein the transgenic plant is Arabidopsis thaliana, soybean, or peanut.

Images