Application of SDG711 protein or substances regulating its expression in regulating stress resistance in rice.

Abstract

This invention discloses the application of SDG711 protein or substances regulating its expression in regulating the stress resistance of rice. This invention belongs to the field of biotechnology. The protein SDG711, or substances regulating the expression of the gene encoding said protein, or substances regulating the activity or content of said protein, can be applied in any of the following ways: 1) in regulating plant stress resistance; 2) in preparing products that regulate plant stress resistance; 3) in cultivating plants with altered stress resistance; 4) in preparing products that cultivate plants with altered stress resistance; 5) in plant breeding. The SDG711 protein and its encoding gene play an important role in the salt and heat tolerance of plants, and will have broad application prospects and market potential in the agricultural field.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of SDG711 protein or substances that regulate its expression in regulating the stress resistance of rice. Background Technology

[0002] Rice ( Oryza sativa As the staple food of more than half the world's population, rice production directly affects the livelihoods of hundreds of millions. Rising sea levels lead to seawater intrusion, and improper irrigation causes secondary soil salinization, resulting in the salinization of large areas of arable land worldwide. Cultivating salt-tolerant rice can revitalize land that was previously uncultivable. Meanwhile, global warming is an undeniable fact, and high-temperature heat damage has become one of the major abiotic stresses threatening agricultural production. Cultivating heat-resistant rice can stabilize yields and ensure safety during critical growth periods. Rice production is facing various threats from adverse environments, and identifying important stress-resistant genetic resources and then cultivating new stress-resistant and high-yielding rice varieties is an effective way to solve these problems. Summary of the Invention

[0003] The main problem this invention aims to solve is how to improve the stress resistance of plants and increase their survival rate and yield under extreme conditions.

[0004] To address the aforementioned problems, this invention provides the application of proteins or substances that regulate the expression of genes encoding said proteins, or substances that regulate the activity or content of said proteins, in regulating plant stress resistance.

[0005] The use of the protein or substance regulating the expression of the gene encoding the protein, or substance regulating the activity or content of the protein provided by this invention, in any of the following: 1) Application in regulating plant stress resistance; 2) Application in the preparation of products that regulate plant stress resistance; 3) Application in cultivating plants with altered stress resistance; 4) Application in the preparation of products containing plants with altered stress resistance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:1; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:1, but with one or more amino acid residues substituted and / or deleted and / or added. Proteins that have more than 75% identity with the amino acid sequence defined in (a3), (a1), or (a2) and have the same function; The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), (a3).

[0006] The protein described in a1) above is named SDG711.

[0007] To facilitate the purification or detection of the protein in a1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:1 in the sequence listing.

[0008] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0009] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein SDG711 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the protein SDG711 isolated in this invention, provided they encode and function as protein SDG711, are derived from and equivalent to the nucleotide sequence of this invention.

[0011] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0012] In this article, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid or nucleotide sequences, then the identity value (%) can be obtained.

[0013] In this document, the 80% or more of identity can 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.

[0014] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0015] In the above applications, the protein is derived from rice ( Oryza sativa L.).

[0016] In this article, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) Regulation occurring at the transcriptional level of the aforementioned gene; 2) Regulation that occurs after the gene is transcribed (i.e., 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 mRNA of the gene from the nucleus to the cytoplasm). 4) Regulation of the translation of the aforementioned genes; 5) Regulation of mRNA degradation of the aforementioned gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0017] In this article, the regulation can be upregulation, enhancement, or increase. The regulation can also be inhibition, reduction, or downregulation.

[0018] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein SDG711.

[0019] In this paper, the upregulation, enhancement, or increase of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase, or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0020] In this article, regulating the expression of the gene encoding the protein can also be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0021] In the above applications, the substance regulating gene expression or the substance regulating protein activity or content can be a biological material related to the protein described above, and the biological material can be any of the following: c1) The nucleic acid molecule that encodes the protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-coding genes mentioned above; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0022] In the above applications, c1) the nucleic acid molecule can be any of the following DNA molecules: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No:2; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above; d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.

[0023] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0024] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the vector pC1305-3HA.

[0025] Existing plant expression vectors can be used to construct structures containing... SDG711 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0026] use SDG711 When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0027] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0028] Using any vector capable of guiding the expression of exogenous genes in plants, the present invention can be used to... SDG711 Introducing genes or gene fragments into plant cells or recipient plants can yield transgenic cell lines and transgenic plants with altered stress resistance. (Carrying...) SDG711 Gene expression vectors can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0029] In one specific embodiment, the nucleic acid molecule described in c3) may be a DNA molecule with the nucleotide sequence shown in SEQ ID No:4.

[0030] The present invention also provides a method for improving plant stress resistance, wherein the method is to enhance, increase or upregulate the activity and / or content of the proteins described above in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the proteins described above, so as to improve plant stress resistance.

[0031] The present invention also provides a method for reducing plant stress resistance, wherein the method involves inhibiting or reducing or silencing the activity and / or content of the proteins described above in the target plant, or / and inhibiting or reducing or silencing the expression level of the encoding genes of the proteins described above, thereby reducing plant stress resistance.

[0032] The present invention provides a method for cultivating plants with enhanced stress resistance, comprising enhancing, increasing or upregulating the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and enhancing, increasing or upregulating the activity and / or content of the coding gene of the above-mentioned protein, thereby obtaining plants with enhanced stress resistance.

[0033] In one embodiment of the present invention, the breeding method for cultivating plants with enhanced stress resistance includes the following steps: (1) Construct recombinant expression vectors that enhance, improve, or upregulate the coding genes of the proteins described above; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with stronger stress resistance than the recipient plant.

[0034] The proteins and / or the biological materials mentioned above are also within the scope of protection claimed in this invention.

[0035] In this invention, the purpose of plant breeding may include cultivating plants with improved stress resistance.

[0036] In this invention, the improved stress resistance is specifically manifested in the following ways: the expression level of the protein encoding gene in the target plant is increased and / or increased, the activity and / or content of the protein encoding gene is increased and / or increased, salt tolerance is improved, high temperature tolerance is improved, the survival rate of the plant under high temperature or high salt environment is improved, and the H3K27me3 modification level is improved.

[0037] In this article, the plant may be: N1) Monocotyledonous or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Rice plants; N5) rice.

[0038] This invention obtains overexpressed SDG711 protein-coding genes by introducing them into rice. SDG711 Rice plants with the overexpressed gene. Experiments on the stress resistance of transgenic rice revealed that, compared to wild-type rice, the overexpressed lines exhibited enhanced salt tolerance and heat tolerance. SDG711 Genes and the proteins they encode play an important role in the salt and heat tolerance of plants, and will have broad application prospects and market potential in the agricultural field. Attached Figure Description

[0039] Picture 1 This is a schematic diagram of the structure of the recombinant plasmid pC1305-3HA-SDG711.

[0040] Picture 2 The results are from PCR identification of genetically modified rice.

[0041] Picture 3 These are the results of RT-qPCR testing of transgenic rice.

[0042] Picture 4The statistical results of rice salt tolerance phenotype and survival rate are as follows: A, before 150mM NaCl treatment (time point A); B, during 150mM NaCl treatment (time point B); C, after 150mM NaCl treatment (time point C); D, rice salt tolerance survival rate; E, before 42℃ high-temperature treatment (time point A); F, during 42℃ high-temperature treatment (time point B); G, after 42℃ high-temperature treatment (time point C); H, rice high-temperature tolerance survival rate.

[0043] Picture 5 To detect changes in histone methylation H3K27me3 modification using Western blot. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0047] The rice variety Nippobare (Nip) described in the following examples is described in: Zhang Q, Liang Z, Cui X, et al. N6-methyladenine DNA methylation in Japonica and Indica ricegenomes and its association with gene expression, plant development, and stress responses. Molecular plant, 2018, 11(12): 1492-1508. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention; it may not be used for any other purpose.

[0048] The vector backbone (pC1305-3HA vector) in the following examples is modified from pCAMBIA1300-3×Flag, which has been described in Niu J, Wang F, Yang C, et al. Identification of Increased GrainLength 1 (IGL1), a novel gene encoded by a major QTL for modulating grain length in rice. Theor Appl Genet. 2024;137(1):24. This biomaterial is available to the public from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. It is intended solely for repeating experiments related to this invention and should not be used for other purposes.

[0049] The following examples use statistical software to process the data. The experimental results are expressed as mean ± standard deviation. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0050] Example 1 SDG711 Creation of gene overexpression plants 1. Rice SDG711 Acquisition of genes RNA was extracted from leaves of rice variety NIP and reverse transcribed into cDNA. Using this cDNA as a template, primers were then used... SDG711- F: 5'-AGAAGAAAATCACATCGGACCGC-3'; SDG711- R: 5'- GGTCTTCTTGCATCCGCTGG-3' was amplified by PCR using Phanta® Max Super-Fidelity DNA Polymerase (catalog number: P505-d1, Vazyme) to obtain the amplified product (i.e. SDG711 The coding region of a gene.

[0051] SDG711The coding sequence of the gene in the rice variety NIP is shown in SEQ ID No:2 (nucleotide sequence), and the amino acid sequence encoding the protein is shown in SEQ ID No:1. In the genomic DNA of rice Nip, the gene encoding the SDG711 protein is shown in SEQ ID No:3 of the sequence listing. In SEQ ID No:3, positions 2294-2317 are exon 1, positions 2418-2519 are exon 2, positions 26282-947 are exon 3, positions 3738-3899 are exon 4, positions 4622-4706 are exon 5, positions 4823-4972 are exon 6, positions 5059-5106 are exon 7, positions 5176-5244 are exon 8, and positions 9059-5106 are exon 8. Exon 9 is located at positions 5368-6071, exon 10 is located at positions 6897-7032, exon 11 is located at positions 7107-7327, exon 12 is located at positions 7826-7957, exon 13 is located at positions 8787-8877, exon 14 is located at positions 9349-9396, exon 15 is located at positions 9475-9603, and exon 16 is located at positions 9828-10097.

[0052] 2. Construction of recombinant plasmid pC1305-3HA- SDG711 Using NIP cDNA as a template, primers were designed ( SDG711- F: 5'-AGAAGAAAATCACATCGGACCGC-3'; SDG711- R: 5'-GGTCTTCTTGCATCCGCTGG-3') was subjected to PCR amplification. SDG711 The coding region sequence, glue recovery SDG711 Target gene fragment.

[0053] The target gene and vector pC1305-3HA were digested and ligated with the same restriction endonucleases (KpnI and SalI), respectively, and then transformed into E. coli. After verification by Sanger sequencing and sequence alignment, pC1305-3HA was obtained. SDG711 Overexpression vector. Recombinant plasmid pC1305-3HA- SDG711 See the structural diagram of the skeleton. Picture 1 Whole plasmid sequencing revealed the recombinant plasmid pC1305-3HA- SDG711 As shown in SEQ ID No:4.

[0054] 3. Obtaining genetically modified rice The recombinant plasmid pC1305-3HA- obtained in step 2 was used... SDG711Recombinant Agrobacterium tumefaciens EHA105 was introduced to obtain Agrobacterium. The recombinant Agrobacterium was then used to genetically transform embryogenic callus tissue of rice NIP using the Agrobacterium tumefaciens infiltration method. Resistant callus tissue was then screened (resistance screening used 100 mg / L hygromycin), followed by differentiation and regeneration culture, and then rooting culture to obtain regenerated plants.

[0055] The specific steps are as follows: (1) Take out the mature seeds of the plant, remove the shells, and select plump, clean seeds without sterile spots for disinfection.

[0056] (2) Inoculate the sterilized seeds onto the induction medium and culture them in the dark at 28°C for about 14 days. Select callus tissue with good appearance and good growth.

[0057] (3) Take the recombinant vector pC1305-3HA- constructed in step 2. SDG711 By introducing Agrobacterium tumefaciens EHA105, a recombinant bacterium was obtained and named EHA105 / pC1305-3HA- SDG711 .

[0058] (4) Take the recombinant bacteria obtained in step (3), resuspend the bacterial cells in infection medium, and obtain EHA105 / pC1305-3HA- SDG711 bacterial suspension.

[0059] (5) Immerse the NIP callus from step (2) in the EHA105 / pC1305-3HA prepared in step (4). -SDG711 Infect the bacterial suspension for 20 min. After infection, discard the bacterial suspension, take the callus tissue, blot dry with sterile filter paper, and then place it on a co-culture medium containing acetylsuccinone and glucose, and incubate in the dark at 28°C for 50-55 h.

[0060] (6) After completing step (5), select callus tissues without obvious Agrobacterium on the surface and transfer them to antibacterial culture medium with added cephalosporin, and incubate in the dark at 28°C for 3-4 days.

[0061] (7) The above-cultured callus tissue was transferred to a selection medium containing hygromycin and cephalosporin and cultured in the dark at 28°C for 30 days, and subcultured every 10 days.

[0062] (8) After completing step (7), take fresh hygromycin-resistant callus tissue, inoculate it into pre-regeneration medium, and culture it in the dark at 28°C for 7 days. Then place it in a light culture room (12h light / 12h dark) and continue to culture for 7 days. Then transfer it to regeneration medium and continue to culture in the light until regenerated plants grow, and obtain transgenic plants.

[0063] Using the recombinant vector pC1305-3HA -SDG711The resulting transgenic plants are denoted as SDG711 Genetically modified plants.

[0064] Culture media and formulations used for genetic transformation: The formulations for both induction and differentiation media are MS media.

[0065] 4. SDG711 Identification of genetically modified rice Plants tested: Nippobare (Nip) and the plants obtained in step 3 SDG711 Genetically modified plants.

[0066] 1) PCR identification Genomic DNA was extracted from the plants to be tested. Using the genomic DNA as a template, primers were designed across introns, and upstream primers were designed on exon 2. SDG711- F, design downstream primers on exon 4. SDG711-R .

[0067] SDG711- F: 5'- AGAAGAAAATCACATCGGACCGC-3'; SDG711- R: 5'-GGTCTTCTTGCATCCGCTGG-3' PCR amplification was performed using this primer pair, and the PCR amplification products were obtained. The agarose gel electrophoresis image is shown below. Picture 2 . Picture 2 In the sequence, the marker sizes from top to bottom are 1000bp and 250bp, respectively, and the amplified product band sizes are approximately 1279bp and 381bp. The products were recovered and sequenced; the sequencing results are shown in sequence 2 of the sequence listing. SDG711 It refers to positions 540 to 3227 in sequence 4.

[0068] Of the 7 extracted OsSDG711-OE Genomic DNA was collected from the strains, and six individual plants were randomly selected from each strain for positive plant identification. Through this identification process, 30 overexpressing strains were obtained. SDG711 T2 generation plants of the gene ( Picture 2 OE1 contains 6 positive single plants, OE2 and OE4 each contain 5, OE5 and OE6 each contain 4, and OE7 and OE8 each contain 3. OsSDG711-OE1 It contains 6 positive single plants; OsSDG711-OE2 , OsSDG711-OE4 It contains 5 positive single plants; OsSDG711-OE5 , OsSDG711-OE6 It contains 4 positive single plants; OsSDG711-OE7 , OsSDG711-OE8 It contains 3 positive single plants.

[0069] 2) SDG711 -HA gene expression level detection Detection SDG711 The specific steps for determining HA gene expression are as follows: Dilute the cDNA from step 1 10-fold. Use this as a template, and the primers are... SDG711- F: 5'-AGAAGAAAATCACATCGGACCGC-3'; SDG711- R: 5'- GGTCTTCTTGCATCCGCTGG-3', detected by RT-qPCR using 2×TaqPro Universal SYBR qPCR Master Mix.

[0070] Through testing SDG711 -HA gene expression levels, the results showed: all transgenic lines SDG711 Expression levels were all upregulated. Picture 3 ), indicating that it is imported from abroad. SDG711 The gene was successfully and correctly expressed in transgenic rice.

[0071] SDG711-OE#1 Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0072] SDG711-OE#2 Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0073] SDG711-OE#4 Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0074] SDG711-OE#5 Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0075] SDG711-OE#6 Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0076] SDG711-OE#7Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0077] SDG711-OE#8 Hygromycin-resistant plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. T2 generation plants are self-pollinated and seeds are harvested, which are the T3 generation seeds.

[0078] Example 2 SDG711 Research on stress resistance of transgenic rice with overexpression 1. Salt tolerance test The plants to be tested were: rice NIP and SDG711-OE#7 as well as SDG711-OE#8 The T3 generation homozygous strain.

[0079] Salt tolerance test: Seeds of each test line were germinated in a greenhouse and cultured to the three-leaf stage (time point A, photographed). They were then treated with 150mM NaCl solution for 6 days (time point B, photographed), and subsequently transferred to a nutrient solution without NaCl for another 7 days (time point C, photographed). Survival rate was calculated (at least 30 plants of each test line were counted). Survival rate (%) = (number of recovered plants / total number of plants) × 100%. Greenhouse conditions were: 28℃, 10 hours light / 14 hours darkness.

[0080] The growth status of the plants to be tested can be seen in the following figures. Picture 4 In diagram A and D, figure A corresponds to time point A before treatment with 150mM NaCl, figure B corresponds to time point B during treatment with 150mM NaCl, and figure C corresponds to time point C after treatment with 150mM NaCl. Picture 4 It can be seen that: before treatment with 150mM NaCl, NIP and SDG711-OE#7 as well as SDG711-OE#8 Uniform growth Picture 4 After treatment with 150mM NaCl for 6 days (A), SDG711-OE#7 as well as SDG711-OE#8 Its growth is better than that of NIP ( Picture 4 (B) after recovery culture SDG711-OE#7 as well as SDG711-OE#8 The number of surviving plants was greater than that of NIP ( Picture 4 (C)

[0081] Survival rate results are shown in Picture 4 The survival rate of D. NIP is approximately 50%. SDG711-OE#7 The survival rate of the plants is approximately 80%. SDG711-OE#8 The plant survival rate was approximately 85%. Compared to NIP, SDG711-OE#7 as well as SDG711-OE#8 Its salt tolerance is significantly enhanced.

[0082] 2. High temperature resistance test The plants to be tested were: rice NIP and SDG711-OE#1, SDG711-OE#2 as well as SDG711-OE#4 The T3 generation homozygous strain.

[0083] High-temperature tolerance test: Seeds of each test strain were germinated in a greenhouse and cultured to the three-leaf stage (time point A, photographed). They were then treated in a 42℃ high-temperature incubator for 3 days (time point B, photographed), and subsequently transferred to a greenhouse for another 2 days (time point C, photographed). Survival rate was calculated (at least 30 plants of each test strain were counted). Survival rate (%) = (number of recovered plants / total number of plants) × 100. Greenhouse conditions were: 28℃, 10 hours light / 14 hours darkness.

[0084] The growth status of the plants to be tested can be seen in the following figures. Picture 4 (EH), Figure E corresponds to before the 42℃ high temperature treatment (time point A), Figure F corresponds to during the 42℃ high temperature treatment (time point B), and Figure G corresponds to after the 42℃ high temperature treatment (time point C).

[0085] Depend on Picture 4 It can be seen that NIP and SDG711-OE#1, SDG711-OE#2 as well as SDG711-OE#4 Uniform growth Picture 4 (E), treated at 42℃ for 3 days SDG711-OE#1, SDG711-OE#2 as well as SDG711-OE#4 Its growth is better than that of NIP ( Picture 4 (F), after recovery culture SSDG711-OE#1, SDG711-OE#2 as well as SDG711-OE#4 The number of surviving plants was greater than that of NIP ( Picture 4 (G).

[0086] Survival rate results are shown in Picture 4 The survival rate of H. NIP is approximately 5%. SDG711-OE#1 The survival rate of the plants is approximately 28%. SDG711-OE#2 The survival rate of the plants is approximately 20%. SDG711-OE#4 The survival rate of the plants was approximately 15%. Compared to NIP, SDG711-OE#1, SDG711-OE#2 as well as SDG711-OE#4 Its high-temperature resistance is significantly enhanced.

[0087] 3. Histone modification level The plants to be tested were: rice Nip and SDG711 - OE#1, SDG711 - OE#2, SDG711 - OE#3, SDG711 - OE#4 T3 generation seeds of the strain.

[0088] Seeds of each test line were germinated in a greenhouse and cultured until the three-leaf stage. The above-ground parts were harvested, and histones were extracted from the test lines to determine the histone modification levels. The specific experimental steps are as follows: 1) Histone extraction: Grind the rice leaves to be tested into powder. Take about 200 μL and put it into a 1.5 mL centrifuge tube. Add 200 μL of 1×pre-lysis buffer, mix thoroughly, and centrifuge at 12,000 rpm for 5 min. Discard the supernatant, add 240 μL of lysis buffer, mix thoroughly, and incubate on ice for 30 min. Then, centrifuge repeatedly at 12,000 rpm for 5 min until no precipitate is found. Take the supernatant, add 0.3V balance buffer (with 1 / 500 of 1M DDT added), determine the concentration and quantify, and then perform SDS-PAGE electrophoresis. The extracted total protein or histones need to be aliquoted and stored at -80℃ for long-term storage.

[0089] 2) SDS-PAGE gel electrophoresis: using LabPAGE TM For pre-prepared protein gels, be sure to select the gel concentration according to the molecular weight of the proteins. Electrophoresis settings: 80 V, 30 min; 120 V, 2 h.

[0090] Transfer was performed using a semi-dry transfer apparatus. After incubation with IgG secondary antibody, the membrane was transferred via Clarity. TM Visualization of electrophoretic bands was performed using the Western ECL Substrate chromogenic kit (BIO-RAD).

[0091] The results showed that, under the premise of consistent input histone H3 expression abundance, SDG711-OE The H3K27me3 modification level was higher than that of Nip in different strains. Picture 5 ).

[0092] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein, in any of the following; 1) Application in regulating plant stress resistance; 2) Application in the preparation of products that regulate plant stress resistance; 3) Application in cultivating plants with altered stress resistance; 4) Application in the preparation of products from plants with altered stress resistance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:1; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:1, but with one or more amino acid residues substituted and / or deleted and / or added. a3) Proteins that share more than 75% identity with the amino acid sequence defined by a1) or a2) and have the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

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

3. Use according to claim 1 or 2, characterized in that, The substance is a biomaterial related to the protein in the application of claim 1 or 2, and the biomaterial is any of the following: c1) The nucleic acid molecule encoding the protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. Use according to claim 3, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:3; d2) The coding sequence is the DNA molecule shown in SEQ ID No:2; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein of claim 1; d4) Hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein of claim 1.

5. A method for improving the stress resistance of plants, characterized in that: The method is to improve the plant's stress resistance by increasing or upregulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhancing, increasing or upregulating the expression level of the gene encoding the protein described in claim 1 or 2.

6. A method for reducing plant stress resistance, characterized in that: The method is to inhibit, reduce, or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit, reduce, or downregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to reduce the plant's stress resistance.

7. A method for cultivating plants with enhanced stress resistance, characterized in that, This includes enhancing, increasing, or upregulating the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or the activity and / or content of the protein, to obtain a plant with enhanced stress resistance, wherein the plant with enhanced stress resistance has stronger stress resistance than the target plant.

8. The method according to claim 7, characterized in that, The enhancement, improvement, or upregulation of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule of claim 4c1), the expression cassette of claim 4c2), or the recombinant vector of claim 4c3) into the target plant to obtain a plant with enhanced stress resistance.

9. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.

10. The method according to any one of claims 5-8, characterized in that, The plant is any one of the following: N1) Monocotyledonous or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Rice plants; N5) rice.

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