OsHox12 protein related to cold resistance of rice at booting stage as well as related biological materials and application of OsHox12 protein

Abstract

The invention relates to the technical field of biology, and particularly discloses OsHox12 protein related to cold resistance of rice at a booting stage as well as a related biological material and application of the OsHox12 protein. The OsHox12 protein is a protein of A1), A2) or A3) as follows: A1) a protein with an amino acid sequence as shown in SEQ ID NO.1; a2) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1); the OsHox12 gene provided by the invention can positively regulate and control the cold resistance of the rice at the booting stage, has important application value, can be used for genetic improvement of the cold resistance of the rice at the booting stage, and has important significance on cultivation of new varieties of low-temperature-resistant rice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to OsHox12 protein related to cold tolerance of rice at the booting stage and related biological materials and applications thereof. Background Art

[0002] Rice originated in tropical and subtropical regions. It is a thermophilic crop and is sensitive to low temperature and chilling damage. Low temperature not only affects the planting season and geographical distribution of rice, but also seriously affects the normal growth and development of rice, resulting in reduced rice production and quality. Rice is widely planted and low temperature and chilling damage occurs frequently.

[0003] Rice is sensitive to low temperature and chilling damage throughout its entire growth period. Among them, chilling damage at the booting stage occurs during the critical period of rice pollen mother cell meiosis and microspore formation, and is most sensitive to low temperatures. Chilling damage at the booting stage causes pollen abortion and a decrease in fruiting rate, resulting in irreversible yield losses, and is the most harmful barrier-type chilling damage.

[0004] Breeding new cold-resistant and high-quality rice varieties is the fundamental way to reduce the harm of low temperatures. However, breeding new cold-resistant and high-quality rice varieties through traditional breeding methods has the problems of long breeding cycles and high randomness. Summary of the invention

[0005] In order to solve the problems of long breeding cycle and high contingency in cultivating new cold-resistant and excellent rice varieties by traditional breeding methods in the prior art, and to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: The invention provides an OsHox12 protein related to cold tolerance of rice at the booting stage.

[0006] The OsHox12 protein is the following protein A1), A2), A3) or A4): A1) The amino acid sequence is the protein shown in SEQ ID NO.1.

[0007] A2) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1).

[0008] A3) A protein obtained by replacing, deleting and / or adding at least one amino acid residue of the protein shown in A1) and having ≥80% homology with the protein shown in A1).

[0009] A4) A protein obtained by substituting, deleting and / or adding at least one amino acid residue of the protein shown in A2) and having ≥80% homology with the protein shown in A1).

[0010] The proteins shown in A3) and A4) are all proteins related to plant cold tolerance.

[0011] The amino acid sequence shown in SEQ ID NO.1 consists of 239 amino acid residues.

[0012] The above protein can be derived from rice; it can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

[0013] In the above proteins, the protein tag refers to a polypeptide or protein fused with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a Myc tag, a GST tag and / or a SUMO tag, etc.

[0014] In the above proteins, homology refers to the homology of amino acid sequences. The homology of amino acid sequences can be detected using homology search sites on the Internet, such as the BLAST page on the NCBI homepage website; the homology of more than 80% can be at least 81%, 85%, 91%, 95% or 100%.

[0015] The OsHox12 protein related to cold tolerance at the rice booting stage provided by the present invention can be used to cultivate new cold-tolerant rice varieties, and has very important theoretical and practical significance for cultivating new cold-tolerant rice varieties. Through the OsHox12 protein related to cold tolerance at the rice booting stage, new cold-tolerant and excellent rice varieties can be cultivated by genetic engineering means, with a short breeding cycle and no chance, which solves the problem of long breeding cycle and high chance in cultivating new cold-tolerant and excellent rice varieties by traditional breeding methods in the prior art.

[0016] At the same time, modern biological breeding methods based on gene and protein resources can greatly improve breeding efficiency and shorten breeding cycles. Discovering and cloning rice cold tolerance-related proteins during the booting stage will provide important gene resources for rice cold tolerance breeding, which has important theoretical significance and application value.

[0017] The present invention also provides a nucleic acid molecule encoding the OsHox12 protein. The nucleic acid molecule is as follows:

[0018] The nucleotides of the coding chain are the cDNA molecule or DNA molecule shown in SEQ ID NO.2.

[0019] The nucleic acid molecule may be DNA, such as genomic DNA, cDNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0020] Among them, SEQ ID NO.2 in the sequence list consists of 720 nucleotides and encodes the protein shown in SEQ ID NO.1 in the sequence list.

[0021] Preferably, the nucleic acid molecule is OsHox12 The gene, whose nucleotide sequence is shown in SEQ ID NO.2. OsHox12 The superior allele of the gene exists in the natural rice population. Combined with molecular marker-assisted selection technology, OsHox12 The gene is expected to be directly applied to improving rice cold-tolerance breeding.

[0022] The biological material related to the above OsHox12 protein or the nucleic acid molecule also belongs to the protection scope of the present invention. Based on this, the present invention also provides a biological material related to the nucleic acid molecule, and the biological material is any one of the following B1) to B5):

[0023] B1) An expression cassette containing the nucleic acid molecule.

[0024] B2) A recombinant vector containing the nucleic acid molecule; the recombinant vector includes a recombinant vector containing the expression cassette described in B1).

[0025] B3) A recombinant microorganism containing the nucleic acid molecule; the recombinant microorganism includes a recombinant microorganism containing the expression cassette described in B1) or containing the recombinant vector described in B2).

[0026] B4) a transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule; the transgenic plant cell line, transgenic plant tissue or transgenic plant organ includes a transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in B1).

[0027] B5) A DNA molecule that reduces the expression of the nucleic acid molecule.

[0028] Preferably, the biological material further comprises an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the DNA molecule described in B5).

[0029] Among the above biological materials, the expression cassette containing the nucleic acid molecule mentioned in B1) refers to a cassette capable of expressing the nucleic acid molecule in a host cell, and the nucleic acid molecule may include a promoter. OsHox12 The promoter of gene transcription may also include the terminator OsHox12 The terminator of transcription may also include enhancer sequences.

[0030] In the above biological materials, the recombinant vector refers to a recombinant vector containing the coding gene or expression cassette of the OsHox12 protein constructed using a plant expression vector; the plant expression vector may be a Gateway system vector or a binary Agrobacterium vector, etc.

[0031] In the above biological materials, the recombinant microorganisms can specifically be bacteria, fungi, yeast and algae; for example, the bacteria can be Agrobacterium EHA105 strain, and the yeast can be yeast Y2HGold strain.

[0032] The application of the OsHox12 protein, the nucleic acid molecule or the biomaterial provided by the present invention includes any one or more of the following applications: Regulating the cold tolerance of plants.

[0033] Preparation of products for regulating plant cold tolerance.

[0034] The above-mentioned regulation of the cold tolerance of plants is to improve the cold tolerance of plants or reduce the cold tolerance of plants. The improvement of the cold tolerance of plants is to improve the fruit setting rate at low temperatures during the booting stage, and the reduction of the cold tolerance of plants is to reduce the fruit setting rate at low temperatures during the booting stage; the improvement of the cold tolerance of plants is achieved by promoting or increasing the expression amount of the OsHox12 protein, or / and the activity or / and content of the OsHox12 protein; the reduction of the cold tolerance of plants is achieved by inhibiting or reducing the expression amount of the nucleic acid molecule, or / and the activity or / and content of the OsHox12 protein.

[0035] Preferably, regulating the cold tolerance of plants is to improve the cold tolerance of plants.

[0036] Improving the cold tolerance of plants refers to improving the fruit setting rate of plants at low temperatures during the booting stage.

[0037] The improvement of the cold tolerance of the plant is achieved by promoting the expression of the nucleic acid molecule, or by promoting the activity and / or content of the OsHox12 protein or the nucleic acid molecule.

[0038] Preferably, regulating the cold tolerance of the plant is reducing the cold tolerance of the plant.

[0039] The reducing the cold tolerance of plants refers to reducing the fruit setting rate of plants at low temperatures during the booting stage.

[0040] The reduction of the cold tolerance of the plant is achieved by reducing the expression level of the nucleic acid molecule, or by reducing the activity and / or content of the OsHox12 protein or the nucleic acid molecule.

[0041] Preferably, the plant is any one of a Gramineae plant, a Rice plant, a Rice plant and a japonica rice variety Towada.

[0042] The present invention also provides a method for improving plant cold tolerance, by increasing or decreasing the expression amount and / or activity of the OsHox12 protein or the nucleic acid molecule in the plant, thereby obtaining a transgenic plant with enhanced cold tolerance at the booting stage.

[0043] Preferably, the plant comprises rice. The nucleic acid molecule is OsHox12 The gene is a gene encoding the above-mentioned OsHox12 protein.

[0044] In the above method, the promoting or increasing OsHox12 The expression level of the gene, and / or the activity and / or content of the OsHox12 protein, is achieved by introducing the nucleic acid molecule into a recipient plant.

[0045] In the above method, the method of introducing the nucleic acid molecule into the recipient plant comprises hybridizing a plant containing a gene encoding the OsHox12 protein with a plant not containing a gene encoding the OsHox12 protein, or / and inserting the nucleic acid molecule into a plant expression vector, and then transferring it into a recipient plant not containing a gene encoding the OsHox12 protein through genetic engineering.

[0046] Knockout rice OsHox12 Gene knockout experiments have shown that OsHox12 The rice lines with the gene had a reduced seed setting rate at low temperature during the booting stage; OsHox12 Genetic experiments have shown that the transgenic rice overexpressing OsHox12 protein has a higher fruit set rate under low temperature during the booting stage compared with the recipient rice; both knockout experiments and overexpression experiments indicate that OsHox12 protein is a protein related to plant cold tolerance, and increasing the amount of OsHox12 protein can enhance the cold tolerance of plants.

[0047] The method of the invention is simple to operate, low in cost, can shorten the breeding cycle, is easy to promote and operate, and has broad application prospects.

[0048] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides an OsHox12 protein related to cold tolerance of rice at the booting stage. The present invention provides an OsHox12 protein related to cold tolerance of rice at the booting stage. OsHox12 The protein can be used to breed new cold-tolerant rice varieties, which has very important theoretical and practical significance for breeding new cold-tolerant rice varieties. Through the OsHox12 protein related to cold tolerance in the rice booting stage, new cold-tolerant and excellent rice varieties can be bred by genetic engineering means, with a short breeding cycle and no chance, solving the problem of long breeding cycle and high chance in breeding new cold-tolerant and excellent rice varieties through traditional breeding methods in the prior art.

[0049] 2. The present invention also provides a gene for encoding the OsHox12 protein related to the cold tolerance of rice at the booting stage, and the superior allele of the gene exists in the natural population of rice. Combined with molecular marker-assisted selection technology, OsHox12The gene is expected to be directly applied to the improvement of rice cold-tolerance breeding and has significant promotion value.

[0050] 3. The application method provided by the present invention is simple to operate, low in cost, can shorten the breeding cycle, is easy to promote and operate, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The cold-tolerant gene of rice at the booting stage in the present invention CTB5 Interacting genes OsHox12 The molecular experiments used are yeast two-hybrid assay (Y2H), luciferase complementation assay (LCI) and protein co-immunoprecipitation assay (Co-IP) from left to right; Figure 1 Figure A in the figure is a yeast two-hybrid experiment, in which two adjacent culture dishes of similar morphology are parallel test results; Figure 1 Panel B in the figure is a luciferase complementation experiment; Figure 1 Panel C shows a protein co-immunoprecipitation experiment.

[0052] Figure 2 For the present invention OsHox12 Identification of the expression level of gene overexpression strains, and identification of cold tolerance phenotypes at the booting stage under two cold treatment conditions: natural low temperature (CS-HAA) and artificial climate chamber (CS-PT); Figure 2 Figure A shows the wild-type Towada and OsHox12 Photos of the panicles of the two gene overexpression lines OsHox12-OE1 and OsHox12-OE2 under natural low temperature conditions. OsHox12 The three samples from the two gene overexpression lines OsHox12-OE1 and OsHox12-OE2 were parallel test samples; the scale bar in the ear morphology photos in the figure is 2 cm; Figure 2 Figure B in the figure is OsHox12 The gene is expressed in wild-type Towada and OsHox12 Expression level detection in two overexpression lines OsHox12-OE1 and OsHox12-OE2; Figure 2 Figure C shows the wild-type Towada and OsHox12 Statistics and comparison of the fruiting rates of two gene overexpression lines OsHox12-OE1 and OsHox12-OE2 under natural low temperature conditions; Figure 2 Figure D shows the wild-type Towada and OsHox12 Statistics and comparison of the fruiting rates of two gene overexpression lines OsHox12-OE1 and OsHox12-OE2 under artificial climate chamber conditions; different letters a, b and c in the figure represent significant differences, which were calculated by one-way analysis of variance (Duncan test).

[0053] Figure 3 For the present invention OsHox12 Sequencing and identification of gene knockout strains, as well as identification of cold-tolerant phenotypes at the booting stage under two cold treatment conditions: natural low temperature (CS-HAA) and artificial climate chamber (CS-PT); Figure 3 Figure A is a near isogenic line NIL CTB5 and OsHox12 Nucleotide mutations at the knockout target site of two knockout strains, OsHox12-cr1 and OsHox12-cr2. The near-isogenic line NIL CTB5 and OsHox12 Three samples from the two knockout lines, OsHox12-cr1 and OsHox12-cr2, were parallel test samples; Figure 3 Figure B shows the near isogenic line NIL CTB5 and OsHox12 The nucleotide sequence sequencing results of the two knockout strains OsHox12-cr1 and OsHox12-cr2 at the knockout target site; Figure 3 Figure C shows the near-isogenic line NIL CTB5 and OsHox12 Photos of the ears of the two gene knockout strains OsHox12-cr1 and OsHox12-cr2 under natural low temperature conditions; the scale bar in the ear morphology photos in the figure is 2 cm; Figure 3 Figure D shows the near isogenic line NIL CTB5 and OsHox12 Statistics and comparison of the fruiting rates of two knockout strains, OsHox12-cr1 and OsHox12-cr2, under natural low temperature conditions; Figure 3 Figure E shows the near isogenic line NIL CTB5 and OsHox12 Statistics and comparison of the fruiting rates of two knockout strains, OsHox12-cr1 and OsHox12-cr2, under artificial climate chamber conditions; different letters a, b, and c in the figure represent significant differences, which were calculated by one-way analysis of variance (Duncan test).

[0054] Figure 4 For the present invention OsHox12 Tissue expression, cold-induced expression, and subcellular localization of genes; Figure 4 Figure A in OsHox12 Tissue expression pattern of the gene; in the figure, P-3cm, P-6cm, P-9cm, P-12cm and P-15cm represent ears with lengths of 3cm, 6cm, 9cm, 12cm and 15cm, respectively; A-1mm and A-1.5mm represent anthers with lengths of 1mm and 1.5mm, respectively; Figure 4Figure B in the figure is OsHox12 Cold-induced expression pattern of genes; 0h, 8h, 16h, 1d, 2d, 3d, 5d and 7d in the figure represent the time points before and after cold treatment at the booting stage; Figure 4 Figure C in the figure is OsHox12 Subcellular localization results of genes; the scale bar in the subcellular localization in the figure is 5μm. DETAILED DESCRIPTION

[0055] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0056] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0057] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0058] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0059] In the cold tolerance identification experiment at the booting stage, the natural low temperature (Cold Stress in a High-altitude Area, CS-HAA) refers to sowing the test materials in Baiyi District, Kunming City, Yunnan Province (1974 meters above sea level, average daily temperature <20℃), and transplanting them to the field after 30 days; after maturity and harvest, at least 10 plants of each material are selected to examine the average fruit setting rate of the main ear.

[0060] The artificial climate chamber treatment (Cold Stress in a Phytotron, CS-PT) refers to sowing the test materials at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing, transplanting them to the field after 30 days, selecting the tillers with the second leaf pulvinus 2 cm above and below the flag leaf pulvinus at the booting stage for hanging, hanging 3 tillers per plant, and treating at least 10 plants for each material; moving the hung single plants to an artificial climate chamber (temperature 17°C) for one week, and then transplanting them to the field to grow under normal conditions until maturity; harvesting the ears corresponding to the hung tillers, and examining the fruit set rate (number of filled grains / total number of grains).

[0061] Embodiment 1: OsHox12 Acquisition of genes In the early stage of this invention, a high-generation backcross population constructed by cold-tolerant highland japonica rice Kunming Xiaobaigu and cold-sensitive japonica rice Towada was cloned to regulate cold tolerance at the booting stage. CTB5 , and constructed CTB5 NIL CTB5 Through yeast library screening, an interacting protein of CTB5 protein, OsHox12, was identified. The molecular verification results of the interaction between OsHox12 and CTB5 protein are as follows Figure 1 As shown, yeast two-hybrid, luciferase complementation and protein immunoprecipitation experiments all demonstrated that OsHox12 protein is an interacting protein of CTB5.

[0062] Among them, the source of the cold-resistant plateau japonica rice Kunming Xiaobaigu was provided by the research team of Professor Li Zichao of China Agricultural University.

[0063] The source of cold-sensitive japonica rice Towada was provided by Professor Li Zichao's research group at China Agricultural University.

[0064] The specific steps of the yeast library screening and yeast two-hybrid experiment are as follows: In order to screen the interacting proteins of CTB5, the CTB5 truncated protein without the activation domain was connected to the pGBKT7 vector as bait protein to screen the cDNA library constructed using the cold-treated ears of Kunming Xiaobaigu at the booting stage; in order to verify the protein interaction, the CTB5 truncated protein without the activation domain was amplified and cloned into the pGBKT7 vector using homologous recombination seamless cloning ligase; at the same time, the full-length OsHox12 protein was cloned into the pGBKT7 vector using homologous recombination seamless cloning ligase. The above two fusion plasmids were transferred into the yeast strain Y2H Gold by PEG-mediated method; the positive transformed clones screened by the two-deficient medium (SD-Trp-Leu) were spread on the four-deficient medium (SD-Trp-Leu-His-Ade), and the interaction was observed after culturing at 30℃ for 3 days.

[0065] Among them, homologous recombination seamless cloning ligase was purchased from Sino-US Taihe Biotechnology Co., Ltd.

[0066] The yeast strain Y2H Gold was provided by Professor Li Zichao's research group at China Agricultural University.

[0067] SD-Trp-Leu medium (SD-Trp-Leu) was purchased from Takara Biotech Co., Ltd.

[0068] The SD-Trp-Leu-His-Ade medium was purchased from Takara Biotech Co., Ltd.

[0069] The specific steps of the above luciferase complementation experiment are as follows: From NILCTB5 Medium Amplification CTB5 and OsHox12 The full-length coding region of the gene was cloned into 35S-nLUC and 35S-cLUC vectors using homologous recombination seamless cloning ligase, respectively, to construct CTB5-nLUC and OsHox12-cLUC fusion vectors, and the fusion vectors were transferred into Agrobacterium by freeze-thaw method to obtain Agrobacterium containing the fusion vector. Agrobacterium containing the fusion vector was transferred into tobacco leaves using suspension; tobacco leaves were harvested 2 days later, incubated with 1mM luciferin, and developed and photographed on a Fusion X7 chemiluminescence detector to detect fluorescence activity.

[0070] Among them, 2-aminoethanesulfonic acid, magnesium chloride and acetosyringone were purchased from OXOID.

[0071] The formula of the suspension is: 10 mM 2-aminoethanesulfonic acid, 10 mM magnesium chloride and 150 μM acetosyringone.

[0072] The Chinese name of luciferin is firefly luciferin, and its source is Nanjing Novozyme Biotechnology Co., Ltd.

[0073] The specific steps of the above protein immunoprecipitation experiment are as follows: 35S: Flag-CTB5 and Super: OsHox12-Myc plasmids were co-transfected into rice protoplasts and incubated overnight. Total protein was extracted using 10 mL IP buffer, the mixture was centrifuged at 13000 rpm and 4°C for 15 minutes, and 100 microliters of supernatant was taken as input; the remaining protein supernatant was incubated with Myc agarose beads at 4°C for 2 hours; and immunoblotting was performed using anti-FLAG and anti-Myc.

[0074] Among them, overnight refers to a time ≥ 12 hours.

[0075] The formula of IP buffer is: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 10mM MgCl2, 0.1% (v / v) NP-40, 3mM DTT and 1×Protease Inhibitor Cocktail).

[0076] Myc agarose beads: LABLEAD, MNA-25-500.

[0077] anti-FLAG: Sigma, F1804, dilution1, 5,000.

[0078] nti-Myc: Sigma, M4439, dilution, 1:5,000.

[0079] The cDNA of panicle of cold-tolerant japonica rice Kunming Xiaobaigu was used as template and amplified by PCR. OsHox12 The genes, primers used and nucleotide sequences are shown below: The nucleotide sequence of OsHox12-F is shown in SEQ ID NO.4: 5'-ATGAGCCGTGAGGAGGATGAG-3'.

[0080] The nucleotide sequence of OsHox12-R is shown in SEQ ID NO.5: 5′-TCAGCTGAATTGGTCGTAGA-3′.

[0081] After sequencing, OsHox12 The coding nucleotide sequence of the gene is shown in SEQ ID NO.2: ATGAGCCGTGAGGAGGATGAGAAGCTGCTGTTCCCTTCGTTCGCCTTCCCGGCGGAGTGCTTCCCGGAAGCCGCCACCTCCGGTGGCGAGCAGAAGAAGGCTCGGCAGCGGCGGAGGAGGAAGGTGAAGCCGGAAGCGGCGGCAGCGTTGGCTGGAGAGAGCGGAGGGGATGAGCAGGCGAAGAAGCGGCGGCTGAGCGACGAGCAGGCGCGGTTTCTGGAGATGAGCTTCAAGAAGGAGCGGAAGCTGGAGACGCCGCGCAAGGTGCAGCTCGCCGCGGAGCTGGGCCTCGACGCCAAGCAGGTCGCCGTCTGGTTCCAGAACCGCCGCGCCCGCCACAAGAGCAAGCTCATGGAGGAGGAGTTCGCCAAGCTCCGCTCCGCCCACGACGCCGTCGTCCTCCAGAACTGCCACCTCGAGACCGAGTTGCTGAAGCTGAAGGAGAGGCTGGCGGATGTAGAGGAGGAGAAGGCGAAGCTAGCAGCTGTCGCCGCGGCGACGACCGGCGGCGGTGGTGGCGGCGGCGGCGGCAGCAGCAGCCCGACCTCGTCGTCGTTCTCCACGGTGACGTACCACCCGGCGCTGGCGGGGCAGTTCGGCGTGGAGGCGGCGGCCGAGGAGGCCGACCTCACCTACATGAGTGAGTACGCGTACAACAGCTACATGCTGGAGTTGGCGGCGGCCGGCTACTGCGGCGGGGTCTACGACCAATTCAGCTGA。

[0082] Encoding the OsHox12 protein, the amino acid sequence of the OsHox12 protein is shown in SEQ ID NO.1: MSREEDEKLLFPSFAFPAECFPEAATSGGEQKKARQRRRRKVKPEAAAALAGESGGDEQAKKRRLSDEQARFLEMSFKKERKLETPRKVQLAAELGLDAKQVAVWFQNRRARHKSKLMEE EFAKLRSAHDAVVLQNCHLETELLKLKERLADVEEEKAKLAAVAAATTGGGGGGGGGSSPTSSSFSTVTYHPALAGQFGVEAAAEEADLTYMSEYAYNSYMLELAAAGYCGGVYDQFS.

[0083] Example 2: Functional verification of OsHox12 protein 1. Construction of overexpression vector The overexpression vector used in this example is named OsHox12-OE, which is a vector derived from Kunming Xiaobai Valley. OsHox12 The CDS of the gene (as shown in SEQ ID NO.2) was linked to the plant expression vector PS1300-MYC with a strong MAS promoter by homologous recombination, and the restriction site was Hind III and Kpn I. The amplification primers used are as follows:

[0084] The nucleotide sequence of OsHox12-OE-F is shown in SEQ ID NO.6: 5'-CACCAAATCGACTCTAGAAAGCTTATGAGCCGTGAGGAGGATGAG-3'.

[0085] The nucleotide sequence of OsHox12-OE-R is shown in SEQ ID NO.7: 5'-AAATGAGCTTTGCTCCATGGTACCGCTGAATTGGTCGTAGACCC-3'.

[0086] The structure of the OsHox12-OE overexpression vector is described as follows: the restriction site of the PS1300-MYC vector Hind III and Kpn The small fragment between I is replaced by OsHox12 The gene coding region sequence is retained, and the other sequences of the PS1300-MYC overexpression vector are kept unchanged to obtain the positive plasmid, which is the recombinant overexpression vector of OsHox12 protein. OsHox12 The gene coding sequence is shown as SEQ ID NO.2.

[0087] 2. Construction of knockout vector (1) OsHox12The genomic nucleotide sequence is shown in SEQ ID NO.3, with 3 exons and 2 introns.

[0088] Wherein, the nucleotide sequence of SEQ ID No.3 is as follows:

[0089] In SEQ ID No. 3, positions 1 to 211 are 5'UTR, positions 212 to 293 are the first exon, positions 294 to 412 are the first intron, positions 413 to 756 are the second exon, positions 757 to 877 are the second intron, positions 878 to 1171 are the third exon, and positions 1172 to 1407 are 3'UTR.

[0090] Log in to the website CRISPR-plant to screen target sites, and evaluate off-target conditions at http: / / www.rgenome.net / cas-offinder / . Select sequences with low off-target rates as target sequences for this study, as follows:

[0091] The nucleotide sequence of the target sequence is shown in SEQ ID NO.8: 5'CCCTTCGTTCGCCTTCCCGG3'; same as positions 33-52 of SEQ ID NO.2, and positions 244-263 of SEQ ID NO.3. OsHox12 The location of the gene knockout target sequence on its genome is as follows Figure 3 As shown, it is located in the first exon.

[0092] Among them, the URL for logging into the website CRISPR-plant is as follows: https: / / www.genome.arizona.edu / crispr / CRISPRsearch.html.

[0093] (2) Design two complementary DNA sequences, add GGCG before the forward target sequence and add AAAC before the reverse complementary target sequence, as follows: The nucleotide sequence of OsHox12-sgRNA-F is shown in SEQ ID NO.9: 5'-GGCGCCCTTCGTTCGCCTTCCCGG-3'.

[0094] The nucleotide sequence of OsHox12-sgRNA-R is shown in SEQ ID NO.10: 5′-AAACCCGGGAAGGCGAACGAAGGG-3′, the underlined sequence is the reverse complement of the underlined sequence in F.

[0095] (3) Construction of knockout vector pBUE411 a. Perform four-primer PCR amplification using 100-fold diluted pCBC-MT1T2 as template.

[0096] b. Purify and recover the PCR product and establish the restriction enzyme digestion-ligation system as shown in Table 1: Table 1 Enzyme digestion-ligation system c. Transform E. coli DH5α to obtain the recombinant plasmid; perform colony PCR identification with OsU3-FD3 and TaU3-RD, and sequence confirmation with OsU3-FD3 and TaU3-FD2. The correct vector is named OsHox12-CR. The primer sequences are as follows:

[0097] The nucleotide sequence of OsU3-FD3 is shown in SEQ ID NO.11: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3'.

[0098] The nucleotide sequence of TaU3-RD is shown in SEQ ID NO.12: 5'-CTCACAAATTATCAGCACGCTAGTC-3'.

[0099] The nucleotide sequence of TaU3-FD is shown in SEQ ID NO.13.

[0100] 5'-TTAGTCCCACCTCGCCAGTTTACAG-3'; The nucleotide sequence of TaU3-FD2 is shown in SEQ ID NO.14: 5'-TTGACTAGCGTGCTGATAATTTGTG-3'.

[0101] The structure of OsHox12-CR vector is described as follows: OsHox12 Insert the gene target sequence fragment into the vector pBUE411 restriction endonuclease Bsa I recognition site, keeping the other sequences of pBUE411 vector unchanged to obtain the recombinant vector, which is OsHox12 Gene knockout vector; knockout vector OsHox12-CR expresses gRNA, and the target sequence of gRNA is located in rice OsHox12 On the first exon of the gene, the specific target sequence is shown at positions 33-52 of SEQ ID NO.2.

[0102] 3. Obtaining genetically modified rice (1) Recombinant bacteria The overexpression vector OsHox12-OE prepared in the above step 1 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain the recombinant bacterium EHA105-OsHox12-OE.

[0103] The knockout vector OsHox12-CR prepared in the above step 2 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain the recombinant bacterium EHA105-OsHox12-CR.

[0104] (2) Transgenic rice Using the Agrobacterium-mediated callus infection method, the recombinant bacteria EHA105-OsHox12-OE was used to infect the callus tissue of the recipient rice variety Towada, and the recombinant bacteria EHA105-OsHox12-CR was used to infect the recipient rice line NIL. CTB5 The specific steps are as follows:

[0105] a. Obtaining embryonic callus: After the mature seeds of the rice variety Towada are shelled, they are disinfected with 75% (v / v) alcohol, then disinfected with 20% (v / v) sodium hypochlorite solution, rinsed with sterile water for 4 times, and air-dried for 6 hours; inoculated on NB solid medium, cultured in the dark at 28°C for 2 weeks, the embryonic callus is peeled off, subcultured to new NB medium, and subcultured for 2 weeks.

[0106] Among them, NB solid culture medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30 g of sucrose and 3 g of plant gel in 1 L of distilled water, and adjust the pH to 5.8.

[0107] b. Prepare infection solution: Take the stored Agrobacterium solution and spread it on YEP solid medium containing 50 mg / mL rifampicin and 50 mg / mL kanamycin. Incubate it in the dark at 28°C for 2 days. Scrape a small amount of Agrobacterium into AAM liquid medium, mix it by pipetting, and measure the concentration of the bacterial solution at OD 600 It is about 0.3, and the prepared infection solution is obtained.

[0108] The preparation method of Agrobacterium liquid is to transfer the constructed fusion vector into Agrobacterium by freeze-thaw method, identify by sequencing, and select positive clones for storage.

[0109] YEP solid culture medium: Dissolve 10 g yeast extract, 5 g NaCl and 10 g tryptone in distilled water, make up to 1 L with distilled water, and add 15 g agar powder.

[0110] AAM liquid culture medium: dissolve 50 ml of AAM macro (20×), 5 ml of MS micro (200×), 10 ml of AAM organic (100×), 10 ml of iron salt (100×), 500 mg of hydrolyzed casein, 68.5 g of sucrose, 36 g of glucose, 75 mg of glycine, 877 mg of glutamine, 266 mg of aspartic acid and 228 mg of arginine in appropriate amount of deionized water, make up to 1 L with deionized water, adjust the pH to 5.5, and sterilize at 115°C for 15 min; finally, add 1 ml of 20 mg / ml acetosyringone solution and mix well.

[0111] c. Co-cultivation: Select naturally dispersed, bright yellow, and 4 mm in diameter granular callus tissues into a triangular flask, add the prepared infection solution, infect for 15 minutes, absorb the excess infection solution with sterile filter paper, place it on a co-cultivation medium covered with a layer of filter paper, and co-cultivate at 20°C for 3 days. Among them, co-cultivation medium: 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin, and 2 mg of 2,4-D are dissolved in 1 L of distilled water, the pH is adjusted to 5.4, and sterilized at 121°C for 15 minutes. When cooled to 50-60°C, 10 g of glucose and 20 mg of acetosyringone that have been sterilized by 0.22 μM filtration are added.

[0112] d. Screening of resistant callus: Take out the callus after co-cultivation, wash it 6 times with sterile water by rapid shaking, then wash it with sterile water containing 250 mg / mL cephalosporin and 250 mg / mL carbenicillin for 20 minutes, and finally place it on sterile filter paper to drain for 3 hours. Then move it to the delayed screening medium. After one week, move it to the first round screening medium, and after two weeks, move it to the second round screening medium, continue to culture for two weeks, and screen the resistant callus.

[0113] Among them, delayed screening culture medium: 20mL of N6 bulk mother liquor (50×), 10mL of B5 trace mother liquor (100×), 1mL of B5 organic mother liquor (1000×), 150mg of inositol, 300mg of hydrolyzed casein, 500mg of glutamine, 600mg of proline, 30.0g of sucrose, 3g of plant gel and 2,4-D 2mg are dissolved in 1L of distilled water, the pH is adjusted to 5.8, sterilized at 121℃ for 15min, and when cooled to 50-60℃, 500mg of cephalosporin sterilized by 0.22μM filtration is added.

[0114] The first round of screening culture medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 500 mg of cephalosporin and 50 mg of hygromycin sterilized by 0.22 μM filtration when cooled to 50-60°C.

[0115] Second round screening culture medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 50 mg of 0.22 μM filtered sterilized hygromycin when cooled to 50-60°C.

[0116] e. Differentiation culture: The resistant callus obtained by screening was inoculated into a pre-differentiation medium, cultured in the dark at 28°C for 2 weeks, then transferred to a differentiation medium, cultured in light for 3 weeks, and regenerated transgenic seedlings were obtained.

[0117] Among them, pre-differentiation culture medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of phytogels, 1 mg of 6-BA, 2 mg of NAA and 5 mg of ABA in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 50 mg of hygromycin sterilized by 0.22 μM filtration when cooled to 50-60°C.

[0118] Differentiation medium: Dissolve 20 mL of N6 macro-mother solution (50×), 10 mL of B5 micro-mother solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of phytogenin, 2 mg of 6-BA, 1 mg of NAA and 1 mg of KT in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 50 mg of 0.22 μM filtered sterilized hygromycin when cooled to 50-60°C.

[0119] f. The regenerated transgenic seedlings were transferred to a seedling culture medium. After the seedlings took root and grew up, they were removed from the culture bottle, the culture medium on the roots was washed off, the seedlings were hardened for 2 weeks, and then transferred to the field for planting until they matured to obtain T0 generation OsHox12-OE transgenic rice plants.

[0120] Among them, seedling culture medium: dissolve 25mL of MS large mother solution (20×), 2.5mL of MS trace mother solution (200×), 2.5mL of MS iron salt mother solution (200×), 2.5mL of MS organic mother solution (200×), 3g of plant gel, 0.5mg of NAA and 0.25mg of paclobutrazol in 1L of distilled water, adjust the pH to 5.8, and sterilize at 121℃ for 15min.

[0121] (3) PCR identification to obtain positive transgenic materials The T0 generation OsHox12-OE transgenic rice plants obtained in step (2) were subjected to PCR identification at the DNA level and expression level identification at the RNA level.

[0122] The PCR primers for identification of OsHox12-OE transgenic plants are OsHox12-OE-check-F and OsHox12-OE-check-R, and their nucleotide sequences are shown below: The nucleotide sequence of OsHox12-OE-check-F is shown in SEQ ID NO.15: 5′-GATCCGCAGCCATTAACGAC-3′.

[0123] The nucleotide sequence of OsHox12-OE-check-R is shown in SEQ ID NO.16: 5′-CGAACTCCTCCTCCATGAGC-3′.

[0124] PCR amplification reaction system (10 μL): 6.5 μL ddH2O, 1 μL 10×Taq buffer, 0.4 μL dNTPs (10 mM), 0.5 μL primer F chain, 0.5 μL primer R chain, 0.1 μL Taq enzyme and 1 μL template DNA.

[0125] PCR amplification program: (1) pre-denaturation, 95°C, 5 min; (2) denaturation (95°C, 30 sec), annealing (55°C, 30 sec), extension (72°C, 1 min), 35 cycles; (3) extension, 72°C, 10 min.

[0126] The target fragment size is 579 bp. Plants containing the target fragment in the PCR amplification product are positive, and plants without the target fragment are negative. Positive plants are T0 generation OsHox12-OE transgenic materials (i.e. OsHox12 gene overexpression materials).

[0127] The T0 generation OsHox12-CR transgenic rice plants obtained in step (2) were sequenced and identified at the DNA level.

[0128] The primers for sequencing identification of OsHox12-CR transgenic plants are OsHox12-CR-check-F and OsHox12-CR-check-R, and their nucleotide sequences are shown below: The nucleotide sequence of OsHox12-CR-check-F is shown in SEQ ID NO.17: 5'-GCCATAGCTTAGTGTTTGGTG-3'.

[0129] The nucleotide sequence of OsHox12-CR-check-R is shown in SEQ ID NO.18: 5′-GAACACATTGCCATCAGCTC-3′.

[0130] After the target band was expanded by PCR, it was sent to Nosai Biotechnology Co., Ltd. for conventional sequencing. In the sequencing identification results, the plants with sequence variation in the target region were positive, and the plants without sequence variation were off-target. The positive plants were T0 generation OsHox12-CR transgenic materials (i.e. OsHox12 gene knockout material).

[0131] IV. Identification of cold tolerance of transgenic materials at the booting stage After the T0 generation transgenic strains of OsHox12-OE were harvested, they were soaked in 20 mg / L hygromycin for germination before sowing the T1 generation. The seedlings with normal rooting were selected and transplanted into the field. One month later, leaves were taken to extract DNA, and the above-mentioned OsHox12-OE-check-F and OsHox12-OE-check-R were used for DNA level identification to obtain positive individual plants.

[0132] The PCR identification primers are OsHox12-OE-check-F and OsHox12-OE-check-R. The nucleotide sequence of OsHox12-OE-check-F is shown in SEQ ID NO.15: 5'-GATCCGCAGCCATTAACGAC-3'.

[0133] The nucleotide sequence of OsHox12-OE-check-R is shown in SEQ ID NO.16: 5'-CGAACTCCTCCTCCATGAGC-3'.

[0134] PCR amplification reaction system (10 μL): 6.5 μL ddH2O, 1 μL 10×Taq buffer, 0.4 μL dNTPs (10 mM), 0.5 μL primer F chain, 0.5 μL primer R chain, 0.1 μL Taq enzyme and 1 μL template DNA.

[0135] PCR amplification program: (1) pre-denaturation, 95°C, 5 min; (2) denaturation (95°C, 30 sec), annealing (55°C, 30 sec), extension (72°C, 1 min), 35 cycles; (3) extension, 72°C, 10 min.

[0136] Before sowing the T2 generation, all positive individual seeds of the T1 generation were treated with 20 mg / L hygromycin solution. All T1 individual seeds that could root normally were homozygous lines, recorded as OsHox12-OE1 and OsHox12-OE2. The homozygous positive T2 generation OsHox12 overexpression lines (OsHox12-OE1 and OsHox12-OE2) were sown at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing and the Experimental Station of Yunnan Academy of Agricultural Sciences in Kunming, Yunnan Province, and transplanted to the field one month later.

[0137] At the booting stage, young ears of OsHox12-OE1 and OsHox12-OE2 were collected at the Shangzhuang Experimental Station of China Agricultural University. Total RNA was extracted using an RNA extraction kit, and reverse transcriptase M-MLV was used to reverse transcribe the first-chain cDNA. The first-chain cDNA was used as a template and primers OsHox12-qRT-F and OsHox12-qRT-R were used to amplify the cDNA. OsHox12 The specific fragment of the rice Actin gene (Os03g0718100) was amplified using primers Actin-F and Actin-R as internal reference for real-time quantitative analysis. Real-time fluorescence quantitative PCR was performed on the real-time fluorescence quantitative PCR instrument Applied Biosystems 7500 RealTime PCR system. Each experiment was repeated three times, and each biological replicate was repeated three times. The method reported by Livak KJ and Schmittgen TD (2001) was used, that is, 2 -ΔΔCT Calculate the relative expression.

[0138] The RNA extraction kit was purchased from Beijing Adelaide Biotechnology Co., Ltd.

[0139] The nucleotide sequence of primer OsHox12-qRT-F is shown in SEQ ID NO.19: 5'-ATGAGCCGTGAGGAGGATGAG-3'.

[0140] The nucleotide sequence of primer OsHox12-qRT-R is shown in SEQ ID NO.20: 5′-GCTGCCGAGCCTTCTTCTG-3′.

[0141] The nucleotide sequence of primer Actin-F is shown in SEQ ID NO.21: 5'-CACAGGTATTGTGTTGGACTCTG-3'.

[0142] The nucleotide sequence of primer Actin-R is shown in SEQ ID NO.22: 5′-AGTAACCACGCTCCGTCAGG-3′.

[0143] The expression detection results are as follows Figure 2 As shown, the expression levels of OsHox12-OE transgenic lines OsHox12-OE1 and OsHox12-OE2 were significantly higher than those of wild-type Towada.

[0144] The artificial climate chamber treatment method was used at the Shangzhuang Experimental Station of China Agricultural University, and the natural low temperature identification method was used at the Experimental Station of Yunnan Academy of Agricultural Sciences to identify the cold tolerance of wild-type Towada and OsHox12-OE transgenic lines at the booting stage, and the fruit set rate was used as the evaluation index for cold tolerance at the booting stage.

[0145] The results of cold tolerance test at the booting stage are as follows Figure 2 As shown, compared with wild-type Towada, OsHox12 The cold tolerance of gene overexpression lines OsHox12-OE1 and OsHox12-OE2 at the booting stage was significantly enhanced.

[0146] The seeds harvested from the T0 generation positive strains of OsHox12-CR were planted in the South China Breeding Base in Sanya City, Hainan Province. After maturity, they were harvested to obtain the T1 generation. Two homozygous positive strains OsHox12-cr1 and OsHox12-cr2 were obtained by sequencing.

[0147] Wild-type NIL CTB5 Compared with OsHox12-cr1 and OsHox12-cr2, OsHox12 The gene has mutated, and the sequencing results are as follows Figure 3As shown: In the two homologous chromosomes, the nucleotide sequence is the sequence shown in SEQ ID NO.3 in the sequence list. OsHox12 The following changes occurred in the genomic genes of the sequence: a nucleotide G and a nucleotide T were inserted after the 260th position of the sequence shown in SEQ ID NO.3 in the sequence list, which caused the amino acid sequence of sequence 1 in the sequence list to undergo a frameshift mutation, thereby causing OsHox12 Gene knockout.

[0148] The artificial climate chamber was used at the Shangzhuang Experimental Station of China Agricultural University, and the natural low temperature identification method was used at the Experimental Station of Yunnan Academy of Agricultural Sciences. CTB5 The cold tolerance phenotype of homozygous positive T2 generation OsHox12-cr1 and OsHox12-cr2 at the booting stage was evaluated, and the fruit setting rate was used as the evaluation index of cold tolerance at the booting stage.

[0149] The results of cold tolerance test at the booting stage are as follows Figure 3 As shown, compared with wild-type NIL CTB5 , OsHox12 The cold tolerance of gene knockout lines OsHox12-cr1 and OsHox12-cr2 at the booting stage was significantly reduced.

[0150] The above results show that low temperature stress at the booting stage affects the rice seed setting rate. OsHox12 The cold tolerance of the gene overexpression lines at the booting stage was significantly stronger than that of Towada; OsHox12 The cold tolerance of the knockout lines at the booting stage was significantly lower than that of the NILs. CTB5 ,illustrate OsHox12 The gene positively regulates the cold tolerance of rice during the heading stage and can reduce the impact of low temperature on the fruit setting rate under cold stress.

[0151] Embodiment 3: OsHox12 Gene expression pattern For analysis OsHox12 To determine the tissue expression pattern of the gene, tissue samples of roots, leaf sheaths, leaves, culms, ears at different developmental stages (lengths of 3 cm, 6 cm, 9 cm, 12 cm, and 15 cm, respectively), and anthers (lengths of 1 mm and 1.5 mm) of Towada were collected at heading.

[0152] For analysis OsHox12 The cold-induced expression pattern of the gene was determined by taking ears at the booting stage before Towada treatment and at 8h, 16h, 1d, 2d, 3d, 5d, and 7d after cold treatment in an artificial climate chamber at 16℃.

[0153] Total RNA of the sample was extracted by TRIZOL method, and the first-chain cDNA was synthesized by reverse transcription using reverse transcriptase M-MLV. The first-chain cDNA was used as a template and amplified using primers OsHox12-qRT-F and OsHox12-qRT-R. OsHox12 The specific fragment of the gene was amplified using primers Actin-F and Actin-R in rice Actin The specific fragment of the gene (Os03g0718100) was used as an internal reference for real-time quantitative analysis. The primer sequences used are as follows:

[0154] The nucleotide sequence of OsHox12-qRT-F is shown in SEQ ID NO.19: 5'-ATGAGCCGTGAGGAGGATGAG-3'.

[0155] The nucleotide sequence of OsHox12-qRT-R is shown in SEQ ID NO.20: 5′-GCTGCCGAGCCTTCTTCTG-3′.

[0156] The nucleotide sequence of Actin-F is shown in SEQ ID NO.21: 5'-CACAGGTATTGTGTTGGACTCTG-3'.

[0157] The nucleotide sequence of Actin-R is shown in SEQ ID NO.22: 5′-AGTAACCACGCTCCGTCAGG-3′.

[0158] Real-time fluorescence quantitative PCR was performed on an Applied Biosystems 7500 Real Time PCR system, with three biological replicates per experiment and three mechanical replicates per biological replicate. -ΔΔCT Calculate the relative expression.

[0159] The results of expression pattern analysis were as follows Figure 4 As shown, OsHox12 The gene was mainly expressed in ears and anthers at different developmental stages, and was significantly induced by low temperature during the booting stage.

[0160] To study the subcellular localization of OsHox12 protein, this study constructed a GFP vector of OsHox12 protein, named OsHox12-GFP; OsHox12The CDS of the gene (as shown in SEQ ID NO.2) was linked to the plant expression vector PS1300-GFP with a strong 35S promoter by homologous recombination, and the restriction site was Hind III and Kpn I. The amplification primers used are as follows:

[0161] The nucleotide sequence of OsHox12-GFP-F is shown in SEQ ID NO.23: 5'-CACCAAATCGACTCTAGAAAGCTTATGAGCCGTGAGGAGGATGAG-3'.

[0162] The nucleotide sequence of OsHox12-GFP-R is shown in SEQ ID NO.24: 5'-CCTCGCCCTTGCTCACCATGGTACCGCTGAATTGGTCGTAGACCC-3'.

[0163] The structure of OsHox12-GFP vector is described as follows: the restriction site of PS1300-GFP vector Hind III and Kpn The small fragment between I is replaced by OsHox12 The positive plasmid obtained by removing the gene coding region sequence and keeping other sequences of the PS1300-GFP vector unchanged is the recombinant overexpression vector of OsHox12 protein.

[0164] The above recombinant plasmid was transformed into Escherichia coli DH5α, and clones were selected for PCR amplification and sequencing identification. The amplification and sequencing primers used were OsHox12-GFP-check-F and OsHox12-GFP-check-R.

[0165] The nucleotide sequence of OsHox12-GFP-check-F is shown in SEQ ID NO.25: 5'-GACGCCATTTCGCCTTTTCAG-3'.

[0166] The nucleotide sequence of OsHox12-GFP-check-R is shown in SEQ ID NO.26: 5'-GAACTTGTGGCCGTTTACGTC-3'.

[0167] The correctly sequenced positive clone plasmid OsHox12-GFP was transferred into rice protoplasts and placed in the dark at 28°C for 16 hours. The fluorescence signal was observed using a laser confocal microscope (Olympus FV1000); RGN1-mcherry was used to observe the fluorescence signal in the cell nucleus.

[0168] The results of subcellular localization are as follows Figure 4 As shown, OsHox12 protein can be specifically localized in the cell nucleus.

[0169] From the above experimental results, we can see that OsHox12 The gene responds to low temperature stress and can positively regulate the cold tolerance of rice at the booting stage. OsHox12 The gene and OsHox12 protein have very important theoretical and practical significance for breeding new cold-tolerant rice varieties. Through this nucleic acid molecule, genetic engineering methods can be used to breed new cold-tolerant and excellent rice varieties, with a short breeding cycle and no chance, solving the problem of long breeding cycles and high chance in the existing technology of breeding new cold-tolerant and excellent rice varieties through traditional breeding methods.

[0170] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0171] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. An OsHox12 protein related to cold tolerance at the booting stage of rice, characterized in that: The OsHox12 protein is the following protein A1), A2), A3) or A4): A1) The amino acid sequence is the protein shown in SEQ ID NO.1; A2) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1); A3) a protein obtained by substituting, deleting and / or adding at least one amino acid residue of the protein shown in A1), and having ≥80% homology with the protein shown in A1); A4) A protein obtained by replacing, deleting and / or adding at least one amino acid residue of the protein shown in A2) and having ≥80% homology with the protein shown in A1); The proteins shown in A3) and A4) are all proteins related to plant cold tolerance.

2. A nucleic acid molecule encoding the OsHox12 protein according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

3. A biological material related to the nucleic acid molecule according to claim 2, characterized in that: The biological material is any one of the following B1) to B5): B1) an expression cassette containing the nucleic acid molecule; B2) a recombinant vector containing the nucleic acid molecule; B3) a recombinant microorganism containing the nucleic acid molecule; B4) transgenic plant cell lines, transgenic plant tissues or transgenic plant organs containing the nucleic acid molecule; B5) A DNA molecule that reduces the expression of the nucleic acid molecule.

4. Use of the OsHox12 protein according to claim 1, the nucleic acid molecule according to claim 2 or the biomaterial according to claim 3, characterized in that: The application includes any one or more of the following applications: Regulate the cold tolerance of plants; Preparation of products for regulating plant cold tolerance.

5. The use according to claim 4, characterized in that: The regulating the cold tolerance of the plant is to improve the cold tolerance of the plant; Improving the cold tolerance of plants refers to improving the fruit setting rate of plants at low temperatures during the booting stage; The improvement of the cold tolerance of the plant is achieved by promoting the expression of the nucleic acid molecule, or by promoting the activity and / or content of the OsHox12 protein.

6. The use according to claim 4, characterized in that: The regulating the cold tolerance of the plant is to reduce the cold tolerance of the plant; The reducing the cold tolerance of plants refers to reducing the fruit setting rate of plants at low temperatures during the booting stage; The reduction of the cold tolerance of the plant is achieved by reducing the expression level of the nucleic acid molecule, or by reducing the activity and / or content of the OsHox12 protein.

7. The use according to claim 4, characterized in that: The plant is any one of a Gramineae plant, a Rice plant, a Rice plant and a japonica rice variety Towada.

8. A method for improving cold tolerance of plants, characterized in that: By increasing or decreasing the expression level and / or activity of the OsHox12 protein according to claim 1 or the nucleic acid molecule according to claim 2 in the plant, a transgenic plant with enhanced cold tolerance at the booting stage is obtained.

9. The method according to claim 8, characterized in that The plants include rice.

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