Application of rice OsREIN1 (T219I) protein and coding gene thereof in drought-tolerant breeding

Abstract

The invention discloses application of rice OsREIN1 (T219I) protein and a coding gene thereof in drought-tolerant breeding, and belongs to the technical field of biological breeding. The invention discloses application of a protein or an expression substance for regulating and controlling a protein coding gene or a substance for regulating and controlling the activity or content of the protein in regulating and controlling the drought resistance of plants, the protein is OsREIN1 (T219I), and the OsREIN1 (T219I) is a protein of which the amino acid sequence is a sequence 2 in a sequence table. Experiments prove that the OsREIN1 (T219I) protein has the function of improving the drought resistance of the rice. The OsREIN1 (T219I) protein and the coding gene thereof have important significance for cultivating drought-resistant rice varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and in particular to application of rice OsREIN1 (T219I) protein and its encoding gene in drought-resistant breeding. Background Art

[0002] Droughts cause massive crop yield losses, making the development of drought-tolerant crops a major goal of the agricultural industry. Numerous genes associated with plant drought tolerance have been reported, including both effector and regulatory genes, from crops such as rice, wheat, maize, and soybean, as well as model plants like Arabidopsis thaliana and halophytes. Several of these genes have been used as target genes for crop stress tolerance engineering, resulting in the successful development of drought- and salt-tolerant rice, maize, and soybean. However, as a staple food crop, the commercialization of genetically engineered transgenic rice remains challenging. Therefore, the use of genetically engineered material with superior alleles, supplemented by molecular marker-assisted breeding, can accelerate the acquisition of the desired high-quality drought-tolerant germplasm.

[0003] Alleles refer to a pair of genes occupying the same position on a pair of homologous chromosomes, which control a pair of relative traits. Different alleles will lead to changes in some genetic characteristics. Allelic variation makes it possible for people to use superior alleles to cultivate superior varieties. At present, many effective molecular biological techniques have been developed for the discovery of superior alleles, such as SSR markers, association analysis of natural populations, etc. Using the above technologies, many superior alleles and their carriers related to traits such as stress tolerance, disease resistance, high quality and high yield have been identified, and are widely used in hybrid breeding, the most commonly used and effective method for breeding varieties. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve the drought resistance of rice or how to cultivate highly drought-resistant rice.

[0005] To solve the above technical problems, in a first aspect, the present invention provides an application of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, wherein the application can be an application of the protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following Q1) to Q6):

[0006] Q1) Regulate plant drought resistance;

[0007] Q2) preparing products that increase plant drought resistance;

[0008] Q3) Cultivating drought-resistant plants;

[0009] Q4) preparing products for cultivating drought-resistant plants;

[0010] Q5) Plant assisted breeding;

[0011] Q6) preparing products for plant-assisted breeding;

[0012] The protein is OsREIN1 (T219I) protein) (hereinafter referred to as OsREIN1 T219I ), the OsREIN1 T219I The protein may be any of the following:

[0013] A1) The amino acid sequence is the protein of sequence 2 in the sequence listing;

[0014] A2) a protein having at least 80% identity to the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of A1) and having the function of regulating plant drought resistance;

[0015] A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0016] In the present invention, the plant breeding assessment index may include drought resistance.

[0017] In the present invention, the purpose of plant breeding includes cultivating plants with improved drought resistance.

[0018] The plant assisted breeding described in Q5) can specifically be the assisted breeding of highly drought-resistant plants. The product produced by the plant assisted breeding described in Q6) can specifically be the product produced by the assisted breeding of highly drought-resistant plants.

[0019] The protein described in A2) above may be a protein derived from A1) or having an identity of 80% or more to the protein described in A1) and having the same function, except that the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is unchanged at amino acid position 219, and the other amino acid residues may be substituted and / or deleted and / or added.

[0020] In the present invention, SEQ ID No. 1 consists of 981 amino acid residues.

[0021] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0022] The connection mentioned in A3) can be via a peptide bond.

[0023] A protein tag is a polypeptide or protein that is fused with a target protein using in vitro DNA recombination techniques to facilitate expression, detection, tracing, and / or purification of the target protein. Examples of protein tags include Flag, His, MBP, HA, myc, GST, and / or SUMO tags.

[0024] In the above applications, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0025] The aforementioned 80% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0026] The 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0027] Furthermore, in the above application, the protein may be derived from rice.

[0028] In the above application, the substance that regulates the activity or content of the protein may be a substance that regulates the expression of the gene encoding the protein.

[0029] In the above application, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0030] Furthermore, in the above application, the substance that regulates the expression of the protein encoding gene or the substance that regulates the activity or content of the protein may be a biological material related to the protein, and the biological material may be any one of the following B1) to B7):

[0031] B1) a nucleic acid molecule encoding the above protein;

[0032] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0033] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0034] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0035] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0036] B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0037] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0038] Furthermore, in the above application, the expression cassette described in B2) is capable of expressing protein OsREIN1 in the host cell. T219I The DNA may include not only a promoter for initiating transcription of the MsSPL12 gene, but also a promoter for terminating OsREIN1 T219IThe terminator of gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include but are not limited to: constitutive promoters, tissue, organ and development-specific promoters and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP," Chao et al. (1999) Plant Physiol 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007)). 10099169.7)), seed storage protein-specific promoters (e.g., phaseolin, napin, oleosin, and soybean beta-conglycin promoters (Beachy et al. (1985) EMBO J. 4: 3047-3053). These can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1996) EMBO J. 4: 3047-3053). 985 ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0039] Furthermore, in the above application, the recombinant vector B3) may contain the protein encoding OsREIN1 shown in SEQ ID No. 1. T219I DNA molecules.

[0040] Plant expression vectors can be used to construct vectors containing the OsREIN1 T219I Recombinant vector containing protein-coding gene expression cassette.

[0041] Furthermore, in the above application, the recombinant microorganism in B4) can specifically be yeast, bacteria, algae and fungi.

[0042] Furthermore, in the above application, the plant tissue in B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0043] Furthermore, in the above application, the transgenic plant organ in B7) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0044] Furthermore, in the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation materials.

[0045] Furthermore, in the above application, the nucleic acid molecule may be the gene shown in C1) or C2) below:

[0046] C1) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of Sequence 1 in the sequence listing;

[0047] C2) The nucleotide of the coding chain is the cDNA molecule or DNA molecule of sequence 1 in the sequence list.

[0048] Furthermore, in the above application, the regulating plant drought resistance may be improving plant drought resistance.

[0049] Furthermore, in the above application, the plant may be any of the following:

[0050] D1) Dicotyledons;

[0051] D1) Monocotyledons,

[0052] D3) Gramineae,

[0053] D4) Grasses,

[0054] D5) Oryza plants,

[0055] D6) Rice.

[0056] In order to solve the above technical problems, in a second aspect, the present invention provides a method for cultivating drought-resistant plants, which may include increasing the protein OsREIN1 in the target plant. T219I Content and / or protein OsREIN1 T219I OsREIN1, the encoding geneT219I The expression level of (the 656th base in OsREIN1 is mutated from C to T (C656T)) is increased to obtain a drought-resistant plant; the drought resistance of the drought-resistant plant is higher than that of the target plant.

[0057] The protein OsREIN1 T219I A protein that is any of the following:

[0058] A1) The amino acid sequence is the protein of sequence 2 in the sequence listing;

[0059] A2) a protein having at least 80% identity to the protein of A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of A1) and having the function of regulating plant drought resistance;

[0060] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2);

[0061] The protein OsREIN1 T219I The coding gene may be the gene shown in C1) or C2) below:

[0062] C1) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of Sequence 1 in the sequence listing;

[0063] C2) The nucleotide of the coding chain is the cDNA molecule or DNA molecule of sequence 1 in the sequence list.

[0064] Furthermore, in the above method, the protein OsREIN1 in the target plant is increased T219I Activity and / or protein of OsREIN1 T219I The expression of the gene encoding the protein OsREIN1 can be T219I OsREIN1, the encoding gene T219I This is achieved by introducing the target plant.

[0065] Furthermore, in the above method, the protein OsREIN1 T219I The introduction of the coding gene into the target plant may include using the target plant and containing OsREIN1 T219I OsREIN1 was obtained by hybridization with germplasm encoding the gene T219I Plants homozygous for the encoding gene or plants genetically engineered to have OsREIN1 T219I The coding gene is introduced into the target plant.

[0066] Furthermore, in the above method, the hybridization may include the following steps:

[0067] S1) the target plant is treated with a mixture containing OsREIN1 T219I hybridizing germplasms encoding genes to obtain an F1 generation, and self-pollinating the F1 generation to obtain an F2 generation;

[0068] S2) Select OsREIN1 T219I Homozygous genotype (both homologous chromosomes contain OsREIN1 T219I ) as the male parent and the target plant (as the female parent) for continuous backcrossing, and each backcross progeny was genotyped and OsREIN1 was selected. T219I Homozygous genotype (both homologous chromosomes contain OsREIN1 T219I ).

[0069] Furthermore, in the above method, the number of backcrossing is not less than 5 times.

[0070] Furthermore, in the above method, the number of backcrossing can be 5 times.

[0071] In this application, the protein OsREIN1 T219I The coding gene belongs to the REIN1 gene of the NBS-LRR family. The genomic sequence of this coding gene differs from the sequence of LOC_Os07g40810 in the rice genome reference sequence. The coding gene undergoes a single base change based on the rice genome reference sequence LOC_Os07g40810, and the genomic sequence is LOC_Os07g40810C1491T, that is, the 1491st base in the rice genome reference sequence LOC_Os07g40810 is mutated from C to T. After comparison, the 656th base in the P-loop (ATP / ADP binding) of the NB-ARC domain in the coding region is mutated from C to T (C656T), resulting in a single amino acid change at position 219 in the encoded protein OsREIN1, from threonine to isoleucine (T219I).

[0072] In one embodiment of the present invention, the target plant may be a rice variety Nipponbare (Nip), and the OsREIN1 T219I The germplasm encoding the gene of may be MC41, which is a rice variety Thang10 from Vietnam. Specifically, MC41 (OsREIN1) in the Nipponbare (Nip) background T219I The construction of a near-isogenic line of MC41 was carried out according to conventional methods. Specifically, MC41 (as the male parent) and Nip (as the female parent) were hybridized to obtain the F1 generation. The F1 generation was self-pollinated to obtain the F2 generation. From the F2 generation, individual plants with roots insensitive to ethylene, i.e., those that grew roots under ethylene treatment, were selected for genotyping. OsREIN1 was selected. T219IHomozygous genotype (both homologous chromosomes contain OsREIN1 T219I The F2 plant of α-Hydroxyproline (Hydroxyproline) was used as the male parent and backcrossed with Nip (as the female parent) for 5 generations. After each backcross generation, the genotype of the hybrid plants was identified by molecular markers to exclude possible mixed self-pollinated individuals. Finally, the mutant OsREIN1 was introduced into the Nip background. T219I The homozygous strain is MC41 (OsREIN1 T219I ), hereinafter referred to as NIL.

[0073] In the above-mentioned genetic engineering method, the protein encoding gene can be modified as follows before being introduced into the target plant to achieve better expression effect:

[0074] 1) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression and also depends on the target species; for example, a tissue- or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although many promoters derived from dicots have been shown to function in monocots and vice versa, ideally, dicot promoters are selected for expression in dicots and monocot promoters are selected for expression in monocots;

[0075] 2) Linking to a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked to the gene of the present invention;

[0076] 3) Introducing enhancer sequences, such as intron sequences (e.g., from Adhl and bronze) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0077] In the above method, the drought-resistant plant may be a transgenic plant or a plant obtained by conventional breeding techniques such as hybridization.

[0078] In the above methods, the transgenic plants are understood to include not only the current transgenic plants but also their progeny. For transgenic plants, the gene can be propagated within the species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic plants include seeds, callus, whole plants, and cells.

[0079] Furthermore, in the above method, the plant may be any of the following:

[0080] D1) Dicotyledons;

[0081] D1) Monocotyledons,

[0082] D3) Gramineae,

[0083] D4) Grasses,

[0084] D5) Oryza plants,

[0085] D6) Rice.

[0086] The target plant can be Nipponbare, and the plant containing OsREIN1 T219I The germplasm / material encoding the gene may be the Vietnamese rice variety Thang 10 (Oryza sativa L. Indica, Thang 10, MC41).

[0087] In a third aspect, the present invention provides the above-mentioned protein.

[0088] In a fourth aspect, the present invention provides biological materials related to the above-mentioned protein.

[0089] In a fifth aspect, the present invention also provides any of the following uses of a substance for detecting the polymorphism of SNP1 in the rice genome or the genotype of SNP1:

[0090] F1), application in identification or auxiliary identification of rice drought resistance;

[0091] F2) Application in the preparation and identification or auxiliary identification of rice drought resistance products;

[0092] F3) Application in detecting or assisting in detecting drought resistance of rice;

[0093] F4) Application in the preparation of products for detecting or assisting in detecting rice drought resistance;

[0094] F6) Application in rice drought-resistant breeding;

[0095] The SNP1 is a SNP site in the rice genome, corresponding to the 656th nucleotide of sequence 1 in the sequence list, which is T or C. The genotype of the SNP1 is genotype CC, TT or CT. Genotype TT is a homozygous type of the SNP1 being T, genotype CC is a homozygous type of the SNP1 being C, and genotype CT is a heterozygous type of the SNP1 being C and T.

[0096] In the above application, the drought resistance of rice of genotype TT is higher or can be higher than that of rice of genotype CC and / or genotype TC.

[0097] In the above application, the substance for detecting the polymorphism of SNP1 or the genotype of SNP1 in the rice genome can be a reagent and / or instrument required for determining the polymorphism of SNP1 or the genotype of SNP1 by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, and SNP chip. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction, and a chip based on fluorescent molecule DNA binding reaction.

[0098] In the above application, the substance for detecting the polymorphism of SNP1 in the rice genome or the genotype of SNP1 can be the following D1), D2) or D3):

[0099] D1) containing PCR primers for amplifying a rice genomic DNA fragment including the SNP1;

[0100] D2) is a PCR reagent containing the PCR primers;

[0101] D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

[0102] This application screened approximately 1,500 representative rice germplasm resources for ethylene response and identified an ethylene-insensitive material, MC41. MC41 is a root-specific ethylene-insensitive material, and therefore, the gene that causes the ethylene-insensitive phenotype in MC41 was named REIN1 ( R oot E thylene I sensitive caused by N The REIN1 variant gene, OsREIN1, was obtained by map-based cloning. T219I Compared with the OsREIN1 gene in Nipponbare (NIP), the C at position 656 in its coding region was changed to T, resulting in the conversion of threonine at position 219 of the OsREIN1 protein to isoleucine. Therefore, this gene was named OsREIN1. T219I , encoding the protein OsREIN1 T219I OsREIN1 T219I The experiment of the present invention proves that OsREIN1 T219I The protein has the function of improving rice drought resistance. T219IThe growth of MC41 carrying OsREIN1 under drought stress was significantly better than that of NIP carrying OsREIN1. T219I The protein and its encoding gene are of great significance for breeding drought-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 This is a dominant / recessive analysis of OsREIN1: The ethylene-insensitive phenotype of MC41 is caused by a recessive mutation. Air indicates no ethylene treatment, and ET indicates ethylene treatment.

[0104] Figure 2 This is the map-based cloning of rice OsREIN1.

[0105] Figure 3 OsREIN1 T219I Identification of ethylene response in near-isogenic lines. (A) OsREIN1 T219I Ethylene-responsive phenotypes of near-isogenic lines. Experimental materials were grown in the dark for 3 days under 10 ppm ethylene treatment. Scale bar is 10 mm. (B) Root length measurements of near-isogenic lines in response to ethylene. Values are mean ± SD (n = 30). (C) Phenotypes of NILs during vegetative growth in the field. Field-grown plants were transferred to pots for photographic documentation. Scale bar is 10 cm.

[0106] T219I

[0107] Figure 4 is the survival rate of OsREIN1-containing materials and controls after drought treatment. Nip represents the Nip group, NIL represents the NIL group, MC41 represents the MC41 group, T219I Rein1 T219I Group.

[0108] T219I T219I

[0109] Figure 5 The mutation type of rein1 was obtained by knocking out the OsREIN1 gene in MC41.

[0110] T219I

[0111] Figure 6 represents the transcription level of OsREIN1 in MC41 and gene-edited materials. DETAILED DESCRIPTION

[0112] The present invention is described in further detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods and are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0113] The rice variety Nipponbare (O. Sativa L. spp. japonica, var. nipponbare, AA genome, Nip) used in the following examples is preserved in our laboratory. The public can obtain the above-mentioned biological material from the applicant in the literature "Yang C, Zhang JS, et al., MHZ6 / OsEIL1 and OsEIL2 Regulate Ethylene Response of Roots and Coleoptiles and Negatively Affect Salt Tolerance in Rice., Plant Physiology, 2015, Vol. 169, pp. 148–165." The obtained biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0114] MC41 in the following examples is the Vietnamese rice variety Thang 10 (Oryza sativa L. Indica, Thang 10 GROS No. 310420), which is from the core germplasm collection of the USDA rice germplasm collection (USDA-ARS Genetic Stock Oryza Collection, http: / / www.ars.usda.gov / Main / Docs.htm?docid=23695, accessed March 7, 2016).

[0115] Example 1: Screening and genetic analysis of rice ethylene response-deficient materials

[0116] During a screening of 1,500 rice accessions for ethylene response defects, researchers discovered that the etiolated seedlings of Thang10, designated MC41, showed no shortening of their roots when treated with ethylene, compared to the control, Oryza sativa L. cv. Nipponbare (Nip). Furthermore, the F1 generation obtained by hybridizing MC41 with Nip exhibited a similar ethylene response to Nip, with significantly shortened roots under ethylene treatment. Therefore, it is hypothesized that the abnormal ethylene response of MC41 may be caused by a single recessive gene.

[0117] Further analysis was conducted to determine the dominance and recessiveness of the abnormal ethylene response variation in MC41. MC41 was used as the female parent and hybridized with rice variety Minghui 63 (MH63) (Oryza sativa L. MH63) as the male parent. MH63 and Nip exhibited the same normal ethylene response. The F1 generation of etiolated seedlings obtained from the hybridization of MH63 and Nip exhibited the same phenotype as MH63. Specifically, under ethylene treatment, their root length was significantly shortened ( Figure 1 ), indicating that the abnormal ethylene response of MC41 is a recessive mutation. Phenotypic analysis of the F2 generation etiolated seedlings revealed a 3:1 (MH63 phenotype: MC41 phenotype) segregation, which was consistent with the χ 2

[0118] The above results indicate that the variation of MC41 is a recessive mutation controlled by a single gene.

[0119] Table 1 Dominance and recessiveness analysis of abnormal ethylene response phenotype in MC41

[0120]

[0121] Note: "+" indicates MC41 ethylene-insensitive phenotype; "-" indicates MH63 normal ethylene-sensitive phenotype; critical value (0.05, 1) = 3.84.

[0122] Example 2: Identification of MC41 variant alleles

[0123] Map-based cloning pinpointed the MC41 mutation site within a 73k interval on the long arm of chromosome 7. A specific single nucleotide polymorphism (SNP1) was identified within one of the genes, located at LOC_Os07g40810 in the rice gene, resulting in a nonsynonymous amino acid substitution. This gene was subsequently designated as a candidate gene. The protein encoded by this gene is REIN1, a member of the NBS-LRR family, and was subsequently designated OsREIN1. Primers were designed based on the rice genome reference gene sequence:

[0124] REIN1F: 5-'ATGGAACATGCTGTTGTTAGTGC-3';

[0125] REIN1R:5-'TCAGTCTTCGTCGAAAGATGAAGATAGT-3'.

[0126] Using MC41 total mRNA as a template, the aforementioned primers amplified a DNA band of approximately 3 kb. Sequencing confirmed that it was the REIN1 gene belonging to the NBS-LRR family, but differed from the sequence of LOC_Os07g40810 in the rice genome reference sequence. OsREIN1 in MC41 underwent a single base change, with the genomic sequence being C1491T. Comparison revealed that the 656th base in the P-loop (ATP / ADP binding) of the NB-ARC domain in the coding region had a C to T mutation (C656T), resulting in a single amino acid change at position 219 in the encoded protein, OsREIN1, from threonine to isoleucine (T219I) ( Figure 2 Therefore, the mutated protein in rice MC41 was named OsREIN1 T219I , OsREIN1 T219I The amino acid sequence of the protein is shown in Sequence 2 in the sequence table. T219I Protein gene (OsREIN1 T219I ) is a DNA molecule shown in Sequence 1 in the sequence table. T219I The nucleotide at the SNP1 specific mutation site in the gene (position 656 of sequence 1 in the sequence list) is T, corresponding to OsREIN1 T219I The amino acid at this position of the protein (position 219 of sequence 2 in the sequence listing) is isoleucine (I).

[0127] In rice varieties MH63 and Nip, the nucleotide at the SNP1 site is C (corresponding to position 656 of SEQ ID NO: 1 in the sequence listing), and the corresponding amino acid is threonine (T). Therefore, in the rice genome, the nucleotide at the SNP1 site is C or T, and the corresponding amino acid is threonine (T) or isoleucine (I). This site has three genotypes: CC, TT, or CT. Genotype TT is homozygous for SNP1 with T, genotype CC is homozygous for SNP1 with C, and genotype CT is heterozygous for SNP1 with both C and T.

[0128] Next, the alleles of OsREIN1 were detected in 5518 rice materials, and it was found that only the OsREIN1 gene in MC41 had a point mutation at the SNP1 site, so this mutation is a rare mutation.

[0129] Drought tolerance testing of 261 individual plants from the F2 generation of the MC41×MH63 combination showed that 62 plants had significantly higher drought tolerance than the other 199 plants, a ratio of approximately 1:3, indicating that the MC41 mutation is a recessive mutation controlled by a single gene. Rice plants with the TT genotype at the SNP marker site had higher, or potentially higher, drought tolerance than rice plants with the CC and / or TC genotypes at the SNP marker site.

[0130] Table 2 OsREIN1 T219I Protein amino acid sequence and its coding sequence

[0131]

[0132]

[0133]

[0134] Example 3, OsREIN1 T219I Participate in regulating plant drought tolerance

[0135] 3.1 Preparation of experimental materials

[0136] 3.1.1、MC41(OsREIN1) with Nipponbare(Nip) as the background T219I ) is a near-isogenic line of MC41, a Vietnamese indica rice variety with a significant genetic difference from Nip. Its plant type and height, among other agronomic traits, are significantly different from those of Nip. T219I (LOC_Os07g40810) function, and constructed a near-isogenic line (NIL). T219I The construction of a near-isogenic line of MC41 was carried out according to conventional methods. Specifically, MC41 (as the male parent) and Nip (as the female parent) were hybridized to obtain the F1 generation. The F1 generation was self-pollinated to obtain the F2 generation. From the F2 generation, individual plants with roots insensitive to ethylene, i.e., those that grew roots under ethylene treatment, were selected for genotyping. OsREIN1 was selected. T219I Homozygous genotype (both homologous chromosomes contain OsREIN1 T219I The F2 plants of α-Hydroxyproline (as the male parent) were backcrossed with Nip (as the female parent) for 5 generations. After each backcross generation, the genotypes of the hybrid plants were identified by molecular markers to exclude possible contaminants from self-pollinated individuals. Finally, the mutant OsREIN1 was introduced into the Nip background. T219I The homozygous strain is MC41 (OsREIN1T219I ) isogenic line, referred to as NIL. Ethylene response assays showed that NIL had similar phenotypes to MC41. Ethylene treatment inhibited root growth in the control Nip, while root length in NIL remained unchanged. NIL roots were insensitive to exogenous ethylene treatment. Like NIL, they contained a variant form of OsREIN1. T219I MC41 is also insensitive to ethylene treatment ( Figure 3 Therefore, the mutant form of OsREIN1 was introduced into the Nip background. T219I This also leads to an ethylene-insensitive phenotype similar to that of MC41, further proving that the ethylene-insensitive phenotype of MC41 is indeed due to OsREIN1. T219I due to the variation of .

[0137] Field phenotypic observations of the plants revealed that the plant height of MC41 was significantly higher than that of Nip. However, after multiple generations of backcrossing, the plant height of NIL had returned to the level of Nip, indicating that the genetic background of NIL was similar to that of Nip ( Figure 3 In conclusion, OsREIN1 in the MC41 genome T219I The mutation of the gene (LOC_Os07g40810) is the reason why it is insensitive to ethylene, so this gene is OsREIN1 T219I .

[0138] 3.2 Drought treatment experiment of seedlings

[0139] The experimental materials are Nipponbare (Nip) and MC41 (OsREIN1) with Nipponbare (Nip) as the background. T219I The experiment was repeated three times, and Nip and NIL (Osrein1 T219I Thirty plump seeds of each species were placed in a conical flask filled with distilled water and placed in a dark incubator at 37°C for germination. The water was changed daily. After two days, white seeds were selected and planted in flower nutrient soil, 5 plants per pot. The seedlings were grown normally in the soil for 10 days. Watering was then stopped to implement drought treatment, and the seedlings were observed daily. Nip seedlings in the Nip group and NIL seedlings in the NIL group were drought-treated for 5 days each. After the sixth day of drought treatment, the seedlings were rewatered to resume growth. Survival rates were calculated on the 10th day after the seedlings resumed growth.

[0140] The results showed that there was no significant difference in the growth of Nip and NIL under hydroponic conditions, and both showed no wilting. NiP wilted significantly after 5 days of drought treatment, while NIL wilted to a lesser extent. After 10 days of rehydration, the survival rates of Nip and NIL were 0% and 74%, respectively. Figure 4 The survival rates of Nip and NIL after drought treatment showed extremely significant differences.

[0141] Example 4: MC41 gene knockout of OsREIN1 T219I Material rein1 T219I preparation

[0142] The gene OsREIN1 in rice MC41 was expressed T219I The knockout was performed using the CRISPR-Cas9 system, which was manufactured by Biotech. The vectors used for gene editing were SK-gRNA and pC1300-Cas9. A total of two gene editing materials were obtained, named rein1 and MC41 -1 and rein1 MC41 -2. Gene editing sites such as Figure 5 As shown:

[0143] rein1 MC41 -1 relative to wild-type MC41, for OsREIN1 T219I Gene, located on one chromosome, OsREIN1 T219I Gene mutation OsREIN1 T219I -1 gene, OsREIN1 T219I -1 gene is a deletion of 97 bases from position 21 to 117 of the DNA molecule shown in SEQ ID NO.1, thereby replacing OsREIN1 T219I gene knockout;

[0144] rein1 MC41 -2 relative to wild-type MC41, for OsREIN1 T219I Gene, on both chromosomes, OsREIN1 T219I Gene mutation OsREIN1 T219I -2 gene, OsREIN1 T219I -2 gene is a deletion of 4 bases from 5-'AGTG-3' at positions 19-22 of the DNA molecule shown in SEQ ID NO.1, thereby T219I Gene knockout.

[0145] Respectively detect the rein1 provided by Biotech MC41 -1 and rein1 MC41 -2 in OsREIN1 T219I The results showed that OsREIN1 was not detected in either material. T219I The transcript ( Figure 6 ), indicating rein1 MC41 -1 and rein1 MC41 -2 in OsREIN1 T219I In the drought tolerance experiment, rein1 MC41-1 and rein1 MC41 -2 marked as rein1 T219I .

[0146] 3.2 Drought treatment experiment of seedlings

[0147] The experimental materials were MC41 and MC41 with OsREIN1 knockout gene. T219I Non-expressed material rein1 T219I The experiment was repeated three times, with MC41 and rein1 T219I Thirty full-bodied seeds were placed in a conical flask filled with distilled water and placed in a dark incubator at 37°C for germination. The water was changed daily. After two days, white seeds were selected and planted in flower nutrient soil, 5 seeds per pot. The seedlings grew normally in the soil for 10 days, then stopped watering to implement drought treatment. The seedling status was observed daily. T219I Rein1 of the group T219I After 6 days of drought treatment, the seedlings were rewatered to resume growth, and the survival rate was calculated after 7 days of recovery.

[0148] The results showed that after drought treatment and rewatering, MC41 and rein1 T219I The survival rates of OsREIN1 in MC41 were 100% and 11%, respectively. T219I It will make rice seedlings more sensitive to drought treatment and the survival rate will be significantly reduced ( Figure 4 ).

[0149] The above experimental results show that OsREIN1 T219I It can increase the drought resistance of rice seedlings.

[0150] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Application, characterized in that, The application is the application of the protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein in any one of the following Q1) to Q6): Q1) Regulate plant drought resistance; Q2) preparing products that increase plant drought resistance; Q3) Cultivating drought-resistant plants; Q4) preparing products for cultivating drought-resistant plants; Q5) Plant assisted breeding; Q6) preparing products for plant-assisted breeding; The protein is OsREIN1 T219I , the OsREIN1 T219I A protein that is any of the following: A1) The amino acid sequence is the protein of sequence 2 in the sequence listing; A2) a protein having at least 80% identity to the protein of A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues from the amino acid sequence of A1) and having the function of regulating plant drought resistance; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The use according to claim 1, characterized in that The protein is derived from rice.

3. The use according to claim 1 or 2, characterized in that The substance that regulates the expression of the protein encoding gene or the substance that regulates the activity or content of the protein is a biological material related to the protein, and the biological material is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

4. The use according to claim 3, characterized in that The coding sequence of the nucleic acid molecule as the coding chain is the cDNA molecule or DNA molecule of sequence 1 in the sequence table.

5. The use according to any one of claims 1 to 4, characterized in that The regulating plant drought resistance is to improve the plant drought resistance.

6. A method for cultivating drought-resistant plants, characterized in that: The method comprises increasing the protein OsREIN1 in the target plant T219I Activity and / or protein of OsREIN1 T219I The expression level of the coding gene is increased to obtain a drought-resistant plant; the drought resistance of the drought-resistant plant is higher than the drought resistance of the target plant; The protein OsREIN1 T219I A protein that is any of the following: A1) The amino acid sequence is the protein of sequence 2 in the sequence listing; A2) a protein having at least 80% identity to the protein of A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues from the amino acid sequence of A1) and having the function of regulating plant drought resistance; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

7. The method according to claim 6, characterized in that The method for increasing the protein OsREIN1 in target plants T219I Content and / or protein OsREIN1 T219I The expression level of the coding gene of protein OsREIN1 was determined by T219I This is achieved by introducing the encoding gene into the target plant.

8. The method according to claim 7, characterized in that The protein OsREIN1 T219I The coding gene of the target plant is introduced into the target plant, including using the target plant and the gene containing OsREIN1 T219I The germplasm containing the coding gene of OsREIN1 was hybridized T219I Plants encoding the gene or genetically engineered to T219I The coding gene is introduced into the target plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. Any of the following uses of a substance for detecting the polymorphism of SNP1 in the rice genome or the genotype of SNP1: F1), application in identification or auxiliary identification of rice drought resistance; F2) Application in the preparation and identification or auxiliary identification of rice drought resistance products; F3) Application in detecting or assisting in detecting drought resistance of rice; F4) Application in the preparation of products for detecting or assisting in detecting rice drought resistance; F6) Application in rice drought-resistant breeding; The SNP1 is a SNP site in the rice genome, corresponding to the 659th nucleotide of sequence 1 in the sequence list, which is T or C.

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