Application of protein DRW1 or substances regulating its expression in regulating heat stress resistance in rice

Abstract

This invention discloses the application of the protein DRW1 or substances regulating its expression in regulating rice heat stress resistance. This invention belongs to the field of biotechnology, specifically relating to the application of the protein DRW1 or substances regulating its expression in regulating rice heat stress resistance. The protein, gene expression regulator, or substance regulating the activity or content of the protein provided by this invention can be applied to regulate plant heat stress resistance or plant breeding. By mutating, knocking out, or inserting transposons at specific target sites in the rice heat stress resistance encoding gene DRW1, new materials are provided for the breeding of heat stress-resistant rice varieties, which plays a positive role in accelerating the improvement of rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of protein DRW1 or substances that regulate its expression in regulating the heat stress resistance of rice. Background Technology

[0002] Rice (Oryza sativa), an important food crop, has suffered yield losses in recent years due to frequent extreme heat waves caused by global warming, resulting in a reduction of rice yield across 150 million mu (approximately 10 million hectares). Studies have shown that every 1°C increase in global average temperature leads to a 3.2% reduction in rice yield. High-temperature stress mainly regulates rice seed setting rate, grain size, and rice quality by reducing pollen viability and inhibiting endosperm development. Against this backdrop, the development of high-yielding and heat-resistant new rice varieties is urgently needed. The key to synergistically improving yield and heat resistance lies in identifying the important genes that regulate rice yield and heat resistance, and creating new high-yielding and heat-resistant rice materials. Summary of the Invention

[0003] The technical problem to be solved by this invention is to improve the resistance of rice to high temperature stress.

[0004] To address the problems existing in the prior art, the present invention provides the application of protein or gene expression substances or substances that regulate the activity or content of said proteins in regulating plant resistance to high temperature stress.

[0005] The use of the protein or gene expression regulator or substance regulating the activity or content of said protein provided by this invention in any of the following:

[0006] 1) Application in regulating plant resistance to high temperature stress;

[0007] 2) Application in the preparation of products that regulate plant resistance to high temperature stress;

[0008] 3) Application in cultivating plants with altered resistance to high-temperature stress;

[0009] 4) Application in the preparation of products from plants with altered resistance to high-temperature stress;

[0010] 5) Applications in plant breeding;

[0011] The protein may be any of the following proteins:

[0012] a1) A protein with the amino acid sequence SEQ ID No. 4;

[0013] Proteins that share more than 75% identity with the amino acid sequences defined by a2)a1) and have the same function;

[0014] The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a3), (a1), or (a2).

[0015] The protein described in a1) above is named DRW1.

[0016] 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. 4 in the sequence listing.

[0017] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0018] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0019] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein DRW1 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the protein DRW1 isolated in this invention, as long as they encode and function as protein DRW1, are derived from and equivalent to the nucleotide sequence of this invention.

[0020] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0021] 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.

[0022] In this document, the 80% or more 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.

[0023] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0024] In the above applications, the protein is derived from rice (Oryza sativa L.).

[0025] 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 DRW1.

[0026] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation:

[0027] 1) Regulation occurring at the transcriptional level of the aforementioned gene;

[0028] 2) Regulation that occurs after the gene is transcribed (i.e., regulation of the splicing or processing of the primary transcript of the gene);

[0029] 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);

[0030] 4) Regulation of the translation of the aforementioned genes;

[0031] 5) Regulation of mRNA degradation of the aforementioned gene;

[0032] 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0033] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0034] In this article, the enhancement, increase or upregulation 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.

[0035] In this article, regulating the expression of the gene encoding the protein can 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.

[0036] 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:

[0037] c1) The nucleic acid molecule that encodes the protein described above;

[0038] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0039] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0040] 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);

[0041] 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);

[0042] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0043] 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);

[0044] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0045] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0046] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0047] 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);

[0048] 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);

[0049] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0050] 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).

[0051] In the above applications, the nucleic acid molecule described in c1) can be any of the following DNA molecules:

[0052] d1) The nucleotide sequence is a DNA molecule composed of SEQ ID No. 1 and SEQ ID No. 2;

[0053] d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 3 of the sequence listing;

[0054] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above;

[0055] d4) Hybridizes under strict conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above.

[0056] 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.

[0057] 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 pCAMBIA1307.

[0058] Expression vectors carrying the DRW1 gene 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.

[0059] The present invention also provides a method for improving plant resistance to high temperature stress, the method comprising step M, wherein step M is to inhibit or reduce or silence 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, thereby improving plant resistance to high temperature stress.

[0060] In the above method, the expression level and / or activity of the gene encoding the protein DRW1 in the target plant can be inhibited, reduced, or silenced by using gene mutation, gene knockout, gene editing, or gene knockdown techniques to reduce or inactivate the activity of the gene encoding the protein DRW1 in the genome of the target plant.

[0061] This invention provides a method for cultivating plants with enhanced resistance to high-temperature stress, comprising inhibiting or reducing or silencing the expression of the coding gene of the aforementioned protein and / or the content and / or activity of the aforementioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the aforementioned protein, thereby obtaining a plant with enhanced resistance to high-temperature stress.

[0062] In one embodiment of the present invention, the breeding method for cultivating plants with enhanced resistance to high-temperature stress includes the following steps:

[0063] (1) Construct recombinant expression vectors that suppress, reduce, or silence the proteins described above;

[0064] (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with stronger resistance to high temperature stress than the recipient plant.

[0065] In this invention, the purpose of plant breeding may include cultivating plants with enhanced resistance to high-temperature stress.

[0066] In one specific embodiment, the plant with enhanced resistance to high temperature stress may be a rice DRW1 knockout mutant, or simply the drw1 mutant.

[0067] In this invention, the high-temperature stress condition can specifically be: cultivating rice grains under high-temperature conditions until they are fully mature. The high-temperature condition can be 37°C.

[0068] In this invention, the enhanced resistance to high-temperature stress can specifically manifest as follows:

[0069] 1) The DRW1 knockout mutant exhibits increased seed setting rate under high temperature stress;

[0070] 2) Compared to DRW1 knockout mutants cultured at room temperature, DRW1 knockout mutants cultured at high temperature until the grains are fully mature showed increased grain width and thousand-grain weight under high temperature stress.

[0071] In this invention, the plant may be one of the following:

[0072] E1) Monocotyledonous or dicotyledonous plants;

[0073] E2) Plants of the order Poales;

[0074] E3) Gramineae plants;

[0075] E4) Plants of the genus *Oryza*;

[0076] E5) Rice.

[0077] This invention relates to a regulatory protein DRW1 related to rice heat tolerance. Inhibiting the accumulation of DRW1 protein can improve rice heat tolerance and can be applied to the regulation of rice heat tolerance. Attached Figure Description

[0078] Figure 1 This image shows the phenotypes and statistical data of rice panicle development after high-temperature stress. Image A shows panicle shapes of ZH11, DRW1-OE, and drw1 mutants after high-temperature stress; image B shows the seed setting rate statistics of ZH11, DRW1-OE, and drw1 mutants after normal conditions and high-temperature stress.

[0079] Figure 2 Images and statistical results of rice grain morphology after high-temperature stress are shown. A represents the grain length phenotype of ZH11 and drw1 mutants under normal and high-temperature stress conditions; B represents the grain width phenotype of ZH11 and drw1 mutants under normal and high-temperature stress conditions; C represents the grain length statistics of ZH11 and drw1 mutants under normal and high-temperature stress conditions; D represents the grain width statistics of ZH11 and drw1 mutants under normal and high-temperature stress conditions; and E represents the thousand-grain weight statistics of ZH11 and drw1 mutants under normal and high-temperature stress conditions.

[0080] Figure 3 For the detection of RNA m by UHPLC-MS / MS 5 The result of C-modification abundance. Detailed Implementation

[0081] 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.

[0082] 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.

[0083] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0084] The drw1 mutant and DRW1-OE overexpression material in the following examples have been described in: Zhang P, Zhu C, Geng Y, Wang Y, Yang Y, Liu Q, Guo W, Chachar S, Riaz A, Yan S, Yang L, Yi K*, Wu C*, GuX*. 2021. Rice and Arabidopsis homologs of yeast CHROMOSOME TRANSMISSIONFIDELITY PROTEIN 4 commonly interact with Polycomb complexes but exert divergent regulatory functions. The Plant Cell, 33:1417-1429. This biological material is available to the public from the applicant and is intended solely for the replication of experiments related to this invention and may not be used for any other purpose.

[0085] The pCAMBIA1307-Flag vector in the following examples has been described in Cui X, Zhang Z, Wang Y, Wu J, Han X, Gu X, Lu T. TWI 1 regulates cell-to-cell movement of OSH15 to control leaf cell fate. The New Phytologist, 2018, 221(1):326-340. It is available to the public from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biomaterial is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0086] Use Excel software for statistical analysis of the data, and use SPASS software for multiple variance comparisons of the data.

[0087] Example 1: Comparison of panicle traits in rice under high temperature stress

[0088] The plants to be tested were homozygous lines of rice ZH11, drw1 mutants and DRW1-OE overexpression material.

[0089] The DRW1 gene (full length 18609 bp) in the rice genomic DNA is a DNA molecule composed of SEQ ID No. 1 (positions 1-15044 of the DRW1 gene genomic sequence) and SEQ ID No. 2 (positions 15045-18609 of the DRW1 gene genomic sequence). The coding sequence of the DRW1 gene is SEQ ID No. 3, and the protein DRW1 is encoded by the amino acid sequence SEQ ID No. 4.

[0090] The drw1 mutant is a Tos17 transposon insertion mutant with the insertion site located in the third exon, specifically positions 99 to 275 of SEQ ID No. 2 (corresponding to positions 1747-1923 of SEQ ID No. 3 (coding sequence)).

[0091] The DRW1-OE overexpression material was obtained by introducing the recombinant vector pCAMBIA1307-Flag-DRW1 into the callus tissue of rice plants.

[0092] The structure of the recombinant vector pCAMBIA1307-Flag-DRW1 is described as follows: The recombinant vector is obtained by replacing the fragment between the XBaI and BamHI recognition sites of pCAMBIA1307-Flag with the DNA molecule shown in SEQ ID No. 3, while keeping the other nucleotides of the pCAMBIA1307-Flag vector unchanged.

[0093] The rice plants to be tested were placed in a greenhouse (28℃) and then transferred to a high-temperature condition (37℃) before flowering. Rice plants grown at 28℃ served as a control. The changes in seed setting rate of ZH11, drw1 mutants, and DRW1-OE overexpression materials under the two culture conditions were statistically analyzed. At least 15 individual plant data were collected for each material.

[0094] The spikelets of the tested plants after high temperature stress were observed Figure 1 After high-temperature stress, the seed setting rate of DRW1-OE did not change significantly compared with ZH11, but the seed setting rate of drw1 mutant increased by more than 4 times. Figure 1 (A and B in the middle).

[0095] Example 2: Comparison of rice grain shape traits under high temperature stress

[0096] The plants to be tested were homozygous lines of rice mutants ZH11 and drw1.

[0097] Rice was grown in a greenhouse (28℃), and before flowering, it was transferred to a high-temperature condition (37℃) for cultivation until the grains were fully mature. Rice cultivated at 28℃ served as a control. Grain length, grain width, and thousand-grain weight of ZH11, drw1 mutants, and DRW1-OE overexpression materials under both conditions were statistically analyzed. At least 15 data points were collected for each material.

[0098] The results are as follows Figure 2As shown, Figures A and B show the grain length and grain width phenotypes of the ZH11 and drw1 mutants after high temperature stress, respectively; Figures C and D show the statistical data of grain length and grain width of the ZH11 and drw1 mutants after high temperature stress; and Figure E shows the thousand-grain weight data of the ZH11 and drw1 mutants after high temperature stress.

[0099] After high-temperature stress at 37℃, the particle length and width of ZH11 decreased significantly, while the particle shape of the drw1 mutant did not change significantly. Figure 2 (A, B, C, and D); meanwhile, the thousand-grain weight of ZH11 decreased by 25.2% after high-temperature stress, while the thousand-grain weight of the drw1 mutant increased by about 8.7%. Figure 2 (E).

[0100] Example 3, RNA m 5 C modification level

[0101] The plants to be tested were homozygous lines of rice mutants ZH11 and drw1.

[0102] Total RNA was extracted from spikelets of each test line and detected using triple quadrupole liquid chromatography-mass spectrometry (UHPLC-MS / MS). 5 The abundance of C-modification was determined. An Agilent 6400 triple quadrupole liquid chromatography-mass spectrometry (LC-MS / MS) instrument was used, with Watson distilled water (0.1% formic acid) and acetonitrile (0.1% formic acid) as the mobile phase. A GOLDAQ column (100 mm × 2.1 mm) with a pore size of 1.9 μm was used, and ion pairing was set for detection. RNA m-modification was also performed. 5 C modifies the level.

[0103] The results are as follows Figure 3 As shown, compared with ZH11, the RNA m of the drw1 mutant is... 5 The level of C modification is reduced.

[0104] 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 application of substances that regulate gene expression or protein activity or content in any of the following: 1) Application in enhancing plant resistance to high temperature stress; 2) Application in the preparation of products that enhance plant resistance to high-temperature stress; 3) Application in cultivating plants with enhanced resistance to high-temperature stress; 4) Application in the preparation of products that cultivate plants with enhanced resistance to high-temperature stress; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No. 4; a2) A fusion protein obtained by attaching a tag to the end of the protein defined in a1); The substance that regulates gene expression or protein activity or content is a substance that downregulates, weakens, or reduces gene expression or protein activity or content. The purpose of the breeding is to select rice varieties with enhanced resistance to high-temperature stress; The plant in question is rice.

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

3. The application according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: 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. A breeding method for cultivating plants with enhanced resistance to high-temperature stress, characterized in that: The invention includes inhibiting, reducing, or silencing the expression of the gene encoding the protein described in claim 1 or 2 in a target plant to obtain a plant with enhanced resistance to high temperature stress, wherein the plant with enhanced resistance to high temperature stress has stronger resistance to high temperature stress than the recipient plant, and the plant is rice.

5. The method according to claim 4, characterized in that: The steps include: (1) Construct a recombinant expression vector that suppresses, reduces, or silences the protein encoding the protein described in claim 1 or 2; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with stronger resistance to high temperature stress than the recipient plant.

6. A method for improving plant resistance to high-temperature stress, characterized in that: The method is to inhibit or reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit or reduce or silence the expression of the gene encoding the protein described in claim 1 or 2, in order to improve the plant's resistance to high temperature stress, wherein the plant is rice.

7. The method according to any one of claims 4-6, characterized in that, The expression of the gene encoding the protein of claim 1 or 2 in the target plant that is inhibited, reduced, or silenced is the gene encoding the protein of claim 1 in the target rice that is knocked out.

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