Application of OsMPK4 gene in enhancing low-temperature stress resistance of rice in seedling stage

Abstract

The invention relates to application of an OsMPK4 gene in enhancing low-temperature stress resistance in a rice seedling stage. Specifically, the rice OsMPK4 gene participating in positive regulation and control of low-temperature stress resistance is cloned in rice, so that the rice OsMPK4 gene is overexpressed, and compared with a wild plant, the rice OsMPK4 gene overexpressed plant shows the low-temperature-resistant characteristic, and the survival rate is remarkably increased. The discovery of the new function of the rice OsMPK4 gene provides a new gene target and resource for improving rice low-temperature stress resistance genetic breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more particularly to... OsMPK4 Application of genes in enhancing rice seedling resistance to low temperature stress. Background Technology

[0002] Rice, as one of the world's most important food crops, is extremely sensitive to low temperatures. Cold stress can severely affect its seed growth and development, leading to a significant drop in yield. Statistics show that global rice yield losses due to cold stress amount to tens of billions of US dollars annually (Lou et al., 2007). In China, the annual loss of rice yield due to cold stress is approximately 3 to 5 million tons (Zhu et al., 2015; Sun et al., 2022). Therefore, improving the cold resistance of rice seedlings is crucial for mitigating cold stress and promoting direct-seeded rice cultivation.

[0003] To address the sensitivity of rice seedlings to low temperatures, researchers have explored various strategies to enhance their cold resistance. Among these, improving seed vigor is crucial. Studies have shown that pretreating seeds with chemicals such as melatonin before sowing (Li et al., 2021) can effectively mitigate the negative effects of cold stress and promote seed germination and seedling growth. Furthermore, spraying optimized formulations of liquid chemicals offers another solution for improving the low-temperature adaptability of rice seedlings (Sun et al., 2020; Back, 2021), but its high cost limits its widespread application.

[0004] Besides seed treatment, introducing cold-resistance genes into rice varieties through genetic engineering is another important way to mitigate the damage caused by low temperatures. Currently, several genes related to cold resistance in rice have been identified, such as... OsCTB4a (Related to cold resistance during the booting stage) OsMAPK3 (mitogen-activated protein kinase 3) OsCBL7 (calcineurin B-like protein 7) and OsSAPK6 (Directed stress / ABA-activated protein kinase 6). These genes participate in cold adaptation or cold resistance in rice by regulating the phosphorylation of cold-induced target genes (Zhang et al., 2017; Li et al., 2022; Jia et al., 2022; Guo et al., 2022; Lou et al., 2022). However, cold resistance in rice is a complex quantitative trait controlled by multiple genes (Chen et al., 2018; Zu et al., 2023; Ding et al., 2023), and the number of cold resistance genes cloned and functionally identified is still limited. Therefore, discovering new cold resistance-related genes and elucidating their mechanisms of action is of great significance for improving cold resistance breeding strategies.

[0005] OsMPK4Expression was induced by mechanical damage, invasion by rice stem borer (SSB), and jasmonic acid (JA) treatment, but not by salicylic acid (SA) treatment. Overexpression OsMPK4 It enhanced the constitutive and / or SSB-induced levels of JA, jasmonicyl-l-isoleucine (JA-Ile), ethylene (ET), and SA, as well as the activity of trypsin inhibitors (TrypPIs), thereby reducing SSB behavior (Liu et al. 2018). OsMPK4 acts as a positive regulator of rice resistance to rice stem borer and a negative regulator of growth by modulating JA, ET, and SA-mediated signaling pathways. Overexpression of OsMPK4 in plants with oe-MPK4 reduced rice stem borer feeding, but compared with the wild type, the oe-MPK4 line in the greenhouse showed stunted growth and reduced aboveground and root biomass (Liu et al. 2018). OsMPK4 interacts with IPA1 protein; under salt stress, OsMPK4 is activated and phosphorylates the Thr180 site of IPA1, thereby promoting the ubiquitination and degradation of IPA1, reducing IPA1 protein levels, and ultimately improving rice salt tolerance. This was achieved by targeting IPA1 loss-of-function mutants. ipa1-10 Salt treatment of rice seedlings with gain-of-function IPA1-3D revealed that IPA1 negatively regulates salt tolerance in rice; after 30 minutes of salt stress treatment, IPA1 phosphorylation levels increased but protein levels decreased (Jia et al. 2022). MKKK62-MKK3-MAPK7 / 14 inhibits... OsMFT Transcription negatively regulates rice seed dormancy. OsMFT Homologous to the gene controlling wheat seed dormancy. Overexpression MKKK62 The transgenic rice seeds lost dormancy and showed reduced sensitivity to abscisic acid (ABA). MKKK62 interacts with MKK3, and MKK3 interacts with MAPK7 and MAPK14. MKK3, MAPK7, and MAPK14 are involved in regulating seed dormancy (Mao et al. 2019). The mitogen-activated protein kinase module OsMKK3-OsMPK7-OsWRK30 mediates rice resistance to bacterial blight (Jalmi and Sinha, 2016). Rice ( Oryza Sativa L. is an important food crop, and OsMPK4 is an important phosphorylated kinase in rice. However, there are no research reports on its effects on low temperature stress. Summary of the Invention

[0006] Based on existing technologies, this invention has discovered a method for overexpressing [the gene] in rice plants. OsMPK4 Genes can enhance the resistance of rice plants to low-temperature stress during the seedling stage; therefore, in one aspect, this invention provides rice... OsMPK4Application of genes in improving rice seedling resistance to low-temperature stress. On the other hand, this invention also provides a method for improving rice seedling resistance to low-temperature stress, comprising: overexpressing rice... OsMPK4 Genes. Furthermore, this invention also provides rice. OsMPK4 Application of genes in genetic breeding to improve rice seedling resistance to low temperature stress.

[0007] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In one aspect, the present invention provides rice OsMPK4 The application of the gene in improving rice seedling low-temperature stress resistance and / or rice variety improvement genetic breeding is characterized by overexpression of the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0008] In another aspect, the present invention provides a rice-containing... OsMPK4 The application of recombinant gene vectors in genetic breeding for improving rice seedling resistance to low-temperature stress and / or rice variety improvement is characterized by overexpression of the aforementioned rice... OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0009] In one aspect, the recombinant vector of the present invention comprises an overexpression promoter Ubi.

[0010] Preferably, the recombinant vector of the present invention is a recombinant pCAMBIA1301 vector containing the overexpression promoter Ubi.

[0011] In one aspect, the present invention provides the application of genetically engineered bacteria in improving rice seedling resistance to low-temperature stress and / or in genetic breeding for rice variety improvement, characterized in that the genetically engineered bacteria contains rice... OsMPK4 Gene recombinant vector, overexpressing the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO: 3. Preferably, the genetically engineered bacteria of the present invention are genetically engineered Escherichia coli or Agrobacterium.

[0012] In one aspect, the genetically engineered bacteria of the present invention are genetically engineered Agrobacterium.

[0013] In one aspect, the genetically engineered bacterium described in this invention is a genetically engineered Agrobacterium GV3101.

[0014] In another aspect, the present invention provides a method for improving the resistance of rice seedlings to low-temperature stress and / or for genetic breeding to improve rice varieties, characterized in that the method comprises: overexpressing rice... OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0015] In one aspect, the rice variety improvement described in this invention aims to enhance the resistance of rice seedlings to low-temperature stress.

[0016] In another aspect, the present invention provides a method for obtaining rice plants with improved traits, characterized by comprising the following processing steps: (1) Infecting rice callus tissue with genetically engineered bacteria; and (2) Infected rice callus tissue was cultured into rice plants; The genetically engineered bacteria contain rice OsMPK4 Gene recombinant vector, overexpressing the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0017] In another aspect, the present invention provides the application of rice OsMPK4 protein in improving rice seedling resistance to low temperature stress and / or in genetic breeding for rice variety improvement.

[0018] In one aspect, the amino acid sequence of the rice OsMPK4 protein of the present invention is shown in SEQ ID NO: 3.

[0019] In one aspect, the improved trait described in this invention is enhanced resistance to low-temperature stress during the rice seedling stage.

[0020] In one aspect, the present invention provides for constructing overexpression OsMPK4 The method / steps for gene vectors include: [the following steps are described in the original text, which are not directly related to the gene vector method / steps described in the original text.] OsMPK4 The nucleotide sequence of the gene coding region was ligated into the vector pCAMBIA1301, with the primers designed as follows: OsMPK4-ox -F: 5'-ACTAGGGTCTCGCACCATGGCGATGATGGTGGA-3'; (SEQ ID NO: 4) OsMPK4-ox -R: 5'-ACTAGGGTCTCTCGCCCATATTCACTCCTGCAACA-3'; (SEQ ID NO: 5).

[0021] In one aspect, the present invention also provides for constructing overexpression OsMPK4The method / steps for genetically engineered bacteria include: overexpressing the strain described in this invention... OsMPK4 The gene vector was transformed into the Agrobacterium strain.

[0022] In one aspect, the present invention also provides for obtaining overexpression OsMPK4 The method / steps for producing gene-modified rice plants include: using the overexpression method described in this invention. OsMPK4 Genetically engineered bacteria infect rice callus tissue, which is then differentiated and rooted.

[0023] In one aspect, the rice of the present invention OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0024] In one aspect, the rice of the present invention OsMPK4 The nucleotide sequence of the gene coding region is shown in SEQ ID NO: 1.

[0025] In one aspect, the rice described in this invention is the japonica rice variety Zhonghua 11 ( Oryza.Sativa L.spp. japonica , var. Zhonghua11 ).

[0026] Preferably, the improved low-temperature stress resistance of rice described in this invention is manifested in that, compared to control ordinary rice plants, OsMPK4 Rice seedlings with overexpressed genes showed significantly higher survival rates after low-temperature treatment during the seedling stage.

[0027] In one aspect, the low temperature described in this invention is below 12°C. In another aspect, the low temperature described in this invention is below 11°C, below 10°C, below 9°C, below 8°C, below 7°C, below 6°C, below 5°C, or below 4°C. In one aspect, the low temperature described in this invention is 4°C.

[0028] In one aspect, the present invention will OsMPK4 The gene was introduced as the target gene into the japonica rice variety Zhonghua 11 ( Oryza.Sativa L. spp. japonica , var. Zhonghua11 (obtained from) OsMPK4 The T0 generation overexpressing plants of the gene were continuously self-crossed to obtain homozygous high-expressing T2 generation lines, which were named […]. OsMPK4-ox1 and OsMPK4-ox2 .

[0029] In one aspect, the present invention utilizes CRISPR / Cas9 gene editing technology to modify the rice variety Hua 11 ( Oryza.Sativa L. spp. japonica , var. Zhonghua11 ) OsMPK4Genes were de-functionalized to prepare... OsMPK4 Plants with missing gene function osmpk4-1 and osmpk4-2 .

[0030] In one aspect, in this invention, OsMPK4 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO: 1. OsMPK4 The full-length sequence of the gene is shown in SEQ ID NO: 2. OsMPK4 The gene encodes a protein belonging to the bZIP family of transcription factors, consisting of 369 amino acids. Its amino acid sequence is shown in SEQ ID NO.3, specifically: SEQ ID NO:3: MAMMVDPPNGMGNQGKHYYTMWQTLFEIDTKYVPIKPIGRGAYGIVCSSINRATNEKVAIKKINNVFDNRVDALRTLRELKLLRHLRHENVIALKDIMMPVHRRSFKDVYLVYELMDTDLHQIIKSSQPLSNDHCQYFLFQLLRGLKYLHSAGILHRDLKPGNLLVNANCDLKICDFGLARTNNT KGQFMTEYVVTRWYRAPELLLCCDNYGTSIDVWSVGCIFAELLGRKPIFPGTECLNQLKLIVNVLGTMSEADIEFIDNPKARKYIKTLPYTPGIPLTSMYPQAHPLAIDLLQKMLVFDPSKRISVTEALEHPYMSPLYDPSANPPAQVPIDLDIDENLGVDMIREMMWQEMLHYHPEVVAGVNM* Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves the effect of overexpressing the gene in ordinary rice plants by overexpressing the gene in ordinary rice plants. OsMPK4 The gene was discovered to have a novel use in enhancing rice seedling resistance to low-temperature stress, and it provides an important genetic resource for breeding rice varieties resistant to low-temperature stress.

[0031] (2) This invention utilizes transgenic technology to provide a method for breeding rice germplasm resistant to low-temperature stress, and obtains rice germplasm resistant to low-temperature stress. OsMPK4 Homozygous plants with gene overexpression. Attached Figure Description

[0032] Figure 1 Rice in Example 2 OsMPK4 Gene loss-of-function editing target sites and mutation types (A), and OsMPK4 Plants with missing gene function osmpk4-1 and osmpk4-2 Images showing the growth of wild-type rice plants (ZH11) after low-temperature stress (B), plant survival rate (C), ion leakage rate (D), and hydrogen peroxide content (E).

[0033] Figure 2 Rice in Example 2 OsMPK4 Gene overexpression plants OsMPK4-oxl and OsMPK4-ox2 Compared with wild-type rice plants (ZH11) OsMPK4 Schematic diagram of gene expression levels (A), rice in Example 3 OsMPK4 Gene overexpression plants OsMPK4-ox1 andOsMPK4-ox2 Images showing the growth of wild-type rice plants (ZH11) after low-temperature stress (B), plant survival rate (C), ion leakage rate (D), and hydrogen peroxide content (E). Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0035] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0037] Example 1 Rice OsMPK4 Gene cloning, construction of gene overexpression engineered bacteria and gene editing engineered bacteria 1. Total RNA extraction from rice Total RNA was extracted from young rice leaves using the Tiangen Plant total RNA extraction kit according to the instructions. The extracted RNA was then reverse transcribed using the Thermo Fisher reverse transcription kit according to the instructions to obtain cDNA (SEQ ID NO: 1), which was stored at -20℃ for later use.

[0038] 2. OsMPK4 Construction of gene overexpression engineered bacteria A Design rice OsMPK4 Primers for specific amplification of gene coding region sequences are as follows: OsMPK4-ox -F: 5'-ACTAGGGTCTCGCACCATGGCGATGATGGTGGA-3'; (SEQ ID NO: 4) OsMPK4-ox -R: 5'-ACTAGGGTCTCTCGCCCATATTCACTCCTGCAACA-3'; (SEQ ID NO: 5).

[0039] Using the cDNA (complementary DNA) obtained by reverse transcription of total RNA from the leaves of common rice plant 11 as a template, the cDNA was amplified by polymerase chain reaction (PCR). OsMPK4 The nucleotide sequence of the gene exons was ligated into the vector pCAMBIA1301 containing the overexpression promoter Ubi, and transformed into competent DH5α Escherichia coli by heat shock at 42°C and then plated.

[0040] Successfully transformed monoclonal DH5α Escherichia coli colonies were selected, and the culture solution after shaking was sent to a sequencing company for sequencing. The sequencing results showed that the vector contained... OsMPK4 The nucleotide sequences of the gene exons were obtained, and plasmids were extracted using a plasmid extraction kit from TransGen Biotech. The plasmids were electroporated into GV3101 Agrobacterium competent cells, incubated at 28°C for two days, and then plasmids were picked for PCR verification, yielding the product containing the overexpression vector pCAMBIA1301- OsMPK4 Agrobacterium engineered strain A.

[0041] 3. OsMPK4 Construction of gene-edited engineered bacteria B Based on the genome sequence (SEQ ID NO: 1), a specific target sequence was searched online (http: / / crispr.hzau.edu.cn / CRISPR / ), and the target sequence "GGATGCACTGAGGACCCTAAGGG (SEQ ID NO: 6)" was selected. Complementary primers were synthesized based on this target sequence. Bsa The vector pHun4c12 was digested with restriction endonuclease I, and the linearized vector was obtained by gel extraction. Subsequently, the fusion fragment was ligated into the linearized pHun4c12 vector (Jiang et al., 2019, Mutation of Inositol 1,3,4-trisphosphate 5 / 6-kinase 6 Impairs Plant Growth and Phytic Acid Synthesis in Rice. Plants 8(5): 114). Enzyme digestion confirmed the correct vector, and further sequencing confirmed that the target sequence had been incorporated into the vector. The correct vector was named pHun4c12- OsMPK4 Simultaneously, the thermal shock conversion method was used to convert pHun4c12- OsMPK4 When introduced into Agrobacterium strain EHA105, the gene-editing vector pHun4c12- is obtained. OsMPK4 Agrobacterium engineered strain B was used for subsequent genetic transformation.

[0042] Example 2 Rice OsMPK^ Obtaining homozygous plants with gene overexpression and homozygous plants with gene loss of function 1. Agrobacterium-mediated genetic transformation of rice Using pCAMBIA1301- OsMPK4 Agrobacterium engineered strain A overexpression vector and strain containing pHun4c12- OsMPK4 Gene-editing Agrobacterium B engineered strain B was used to infect rice callus tissue, and after screening on differentiation and rooting media, the corresponding genetically transformed plants were obtained.

[0043] 2. Obtaining homozygous overexpression lines A small amount of leaves and roots from the T0 generation overexpression seedlings were taken, and total RNA was extracted from the plant materials (roots and leaves) using the RNeasy Plant RNA Mini Kit (Qiagen, Hilden, Germany). cDNA was then reverse transcribed using 1 μg of total RNA, oligo-dT18 primers, and the GoScript™ reverse transcription system (Promega). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a SYBR Green GoTaq qPCRMaster Mix (Promega, WI, USA). Rice OsACTIN Genes were used as internal controls, and 2 were used. -ΔΔCt Method calculation OsMPK4 Relative expression level.

[0044] Table 1 Primer list for quantitative real-time polymerase chain reaction (qRT-PCR)

[0045] T0 generation transgenic overexpression plants were self-pollinated to obtain T1 generation seeds. From each T0 generation overexpression plant, 6-9 positive plants were taken and self-pollinated again to produce T2 generation seeds, which were then segregated and analyzed. When all T2 generation seedlings produced from the positive T1 generation plants tested positive, the T1 generation plant was considered a homozygous overexpression plant, and thus the T1 generation was obtained. OsMPK4 Transgenic pure lines with overexpression of the gene are named OsMPK4-ox1 and OsMPK4-ox2 Two plants OsMPK4 See gene expression levels Figure 2 A; otherwise, it is a heterozygous plant.

[0046] 3. Obtaining homozygous plants with gene function loss Young leaves were selected from T0 generation transgenic rice at the 3-4 leaf stage, and genomic DNA was extracted using the CTAB method. Sequencing was then used to verify the PCR products. The results showed... OsMPK4 The gene has undergone a single base deletion, i.e., acquired OsMPK4 Transgenic pure lines with gene function loss are named osmpk4-1 and osmpk4-2 (See)Figure l A).

[0047] Example 3 OsMPK4 Experiment on the resistance of rice to low temperature stress by gene overexpression The rice obtained in Example 2 OsMPK4 Overexpression of T2 generation homozygous lines: OsMPK4-ox-1 and OsMPK4-ox-2 , [[ID=10^]]OsMPK4 Transgenic pure lines with gene function loss: osmpk4-1 and osmpk4-2 Compared with ordinary rice plants, low temperature stress was applied to the plants, and the survival rate of the plants was counted, as well as the ion leakage rate and hydrogen peroxide content were detected.

[0048] To assess the cold resistance of rice plants, rice seedlings soaked for 10 days were transferred to either 4℃ (experimental group) or 30℃ (control group) for 6 days, followed by a 6-day recovery period at 30℃, and survival rates were analyzed. All treatments included three biological replicates of 30 to 40 plants for survival determination. All tests were conducted in an artificial indoor environment under the following conditions (relative humidity: 65–75%; 16 hours light / 8 hours darkness).

[0049] By overexpressing homozygous lines in the T2 generation OsMPK4-ox1 and OsMPK4-ox2 , OsMPK^ Plants with missing gene function osmpk4-1 and osmpk4-2 After cold treatment for 6 days and then culturing under normal conditions for 6 days, the survival rate of plants of the control variety Zhonghua 11 (10-day-old rice) was calculated. The results showed that... OsMPK4 The survival rate of overexpressing rice was significantly higher than that of the control after low-temperature treatment. Figure 2 (B and 2C); and OsMPK4 Plants with missing gene function [[ID=11^]]osmpk4-1 and osmpk4-2 The survival rate of rice after low-temperature treatment was significantly lower than that of the control. Figure l B and 1C).

[0050] Ion leakage was quantified according to Ding et al. (Ding, Y., Li, H., Zhang, X., Xie, Q., Gong, Z., Yang, S. (2015). OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis. Dev. Cell 32: 278–289). Specifically, approximately 0.1 g of leaf tissue from cryogenically treated seedlings was placed in a 15 mL test tube, and 5 mL of deionized water was added. The sample was gently shaken at 200 rpm for 1 hour at room temperature, and the initial conductivity (A1) was recorded. The sample was then heated in boiling water for 20 minutes, and then incubated again at room temperature for 1 hour, and the final conductivity (A2) was measured. The conductivity of deionized water (A0) was used as the baseline. The ion leakage rate was calculated as: Ion leakage (%) = (A1−A0) / (A2−A0). Hydrogen peroxide (H2O2) was measured using a hydrogen peroxide assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Approximately 10 mg of plant material (roots and leaves) was pulverized in liquid nitrogen, and 500 μL of lysate was added. The mixture was centrifuged at 8,000 × g for 10 minutes at 4 °C. 125 μL of the supernatant was mixed with an equal volume of hydrogen peroxide detection reagent and incubated at room temperature for 5 minutes. The absorbance was then measured spectrophotometrically at 415 nm, and the hydrogen peroxide concentration was calculated from the standard curve.

[0051] turn out, [[ID=12^]]OsMPK4 Overexpression rice plants OsMPK4-ox1 and OsMPK4-ox2 After low-temperature treatment, the ion leakage rate was significantly lower than that of the control (see...). Figure 2 D); and OsMPK^ Plants with missing gene function osmpk4-1 and osmpk4-2 The ion leakage rate was significantly higher than that of the control (see Figure l D). Additionally, hydrogen peroxide test results showed that... OsMPK^ Overexpression rice plants OsMPK4-ox1 and OsMPK4-ox2 After low-temperature treatment, the hydrogen peroxide content was significantly lower than that of the control (see...). Figure 2 E); and OsMPK4 Plants with missing gene function osmpk4-1 and osmpk4-2 The hydrogen peroxide content was significantly higher than that of the control (see Figure l E), which indicates that under cold stress treatment... OsMPK4 Loss of gene function increases the content of reactive oxygen species (ROS) in rice plants, thereby reducing the resistance of rice to cold stress.

[0052] The above experiments demonstrate that overexpression OsMPK4 Genes can enhance rice's resistance to low-temperature stress. OsMPK4 Loss of gene function reduces rice’s resistance to low temperature stress.

Claims

1. Rice OsMPK4 The application of genes in genetic breeding for improving rice seedling resistance to low temperature stress and / or rice variety improvement is characterized by, Overexpression of the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO:

3.

2. Contains rice OsMPK4 The application of gene recombinant vectors in genetic breeding for improving rice seedling stage low-temperature stress resistance and / or rice variety improvement is characterized by, Overexpression of the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO:

3.

3. The application of a genetically engineered bacterium in improving rice seedling resistance to low-temperature stress and / or in genetic breeding for rice variety improvement, characterized in that... The genetically engineered bacteria contain rice. OsMPK4 Recombinant vectors of the gene, overexpressing the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO:

3.

4. A method for improving rice seedling resistance to low-temperature stress and / or for genetic breeding of rice varieties, characterized in that, The method includes: overexpressing rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO:

3.

5. A method for obtaining rice plants with improved traits, characterized in that, It includes the following processing steps: (1) Infecting rice callus tissue with genetically engineered bacteria; and (2) Infected rice callus tissue was cultured into rice plants; The genetically engineered bacteria contain rice OsMPK4 Recombinant vectors of the gene, overexpressing the rice OsMPK4 Genes, the rice OsMPK4 The gene encodes the amino acid sequence shown in SEQ ID NO:

3.

6. The application according to any one of claims 1-3 or the method according to 4 or 5, characterized in that, The rice OsMPK4 The nucleotide sequence of the gene coding region is shown in SEQ ID NO:

1.

7. Application of rice OsMPK4 protein in improving rice seedling resistance to low temperature stress and / or in genetic breeding for rice variety improvement.

8. The application according to claim 7, characterized in that, The amino acid sequence of the rice OsMPK4 protein is shown in SEQ ID NO: 3.

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