Application of OsMYB4P gene in enhancing low temperature stress resistance of rice seedling stage

Abstract

The present application relates to rice OsMYB4P The application of the gene in enhancing the low-temperature stress resistance of rice seedlings. Specifically, the present application clones a rice OsMYB4P gene involved in positively regulating low-temperature stress resistance in rice, so that the rice OsMYB4P gene overexpression, compared with wild type plants, the rice OsMYB4P gene overexpression plants show low temperature characteristics, the survival rate is significantly increased, the ion leakage rate is significantly reduced. The rice OsMYB4P OsMYB4P gene new function discovery provides important gene resources for breeding low-temperature stress resistant rice varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to OsMYB4P Application of genes in enhancing resistance to low temperature stress in rice seedlings. Background Art

[0002] Rice, a vital component of global food security, is sensitive to cold stress. Low temperatures inhibit normal seed growth and development, resulting in reduced rice yields. Globally, cold damage results in estimated annual rice yield losses of tens of billions of dollars (Lou et al., 2007). In China, cold stress causes an estimated 3–5 million tons of rice yield losses annually (Zhu et al., 2015; Sun et al., 2022). Therefore, enhancing the cold resistance of rice seedlings is crucial for reducing the impact of cold damage and promoting direct-seeding rice cultivation.

[0003] Many methods have been explored to improve the cold resistance of rice seedlings. On the one hand, the vitality of rice seeds must be improved before sowing. For example, promoting seed germination with various chemicals such as melatonin (Li et al., 2021) can greatly alleviate the cold stress effect and improve seed germination and seedling growth. Spraying optimized liquid chemicals is also an alternative solution to improve the cold adaptation of rice seedlings (Sun et al., 2020; Back, 2021). However, the high cost is a limiting factor. On the other hand, the identification of cold-resistant genes and their subsequent introduction in variety breeding are also important ways to mitigate yield losses caused by low temperatures. For example, OsCTB4a (cold resistance at the booting stage), OsMAPK3 (mitogen-activated protein kinase 3), OsCBL7 (calcineurin B-like protein 7) and OsSAPK6 Directed stress / ABA-activated protein kinase 6 (DPS6) has been shown to regulate cold adaptation or cold resistance in rice by phosphorylating cold-induced target genes (Zhang et al., 2017; Li et al., 2022; Jia et al., 2022; Guo et al., 2022; Lou et al., 2022). Despite these reports, the regulatory mechanisms of cold resistance in rice are incomplete. Plant cold resistance 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 characterized in rice is currently very limited. Therefore, discovering new genes involved in cold resistance and revealing their underlying mechanisms will provide essential information for these approaches.

[0004] It is widely believed that dehydration-responsive element-binding factor 1 (DREB1) plays a crucial role in cold adaptation or cold tolerance through its transcriptional regulatory signals on cold-regulated (COR) genes (Thomashow et al., 1999; Shi et al., 2018; Jia et al., 2022; Kidokoro et al., 2022; Yang et al., 2022; Li et al., 2023). Low temperature stress often activates DREB1 expression at different levels, particularly through transcriptional regulation (Ding et al., 2020; Zhang et al., 2022; Li et al., 2022; Song et al., 2021; Jiang et al., 2022; Sun et al., 2022). In rice, some reports have shown that OsDREB1 Genes are transcriptionally induced by cold to improve cold tolerance (Dubouzet et al., 2003; Ito et al., 2006; Gutha et al., 2008; Mao et al., 2019; Zhang et al., 2022). In addition to DREB1, many other transcription factors (TFs) are also involved in the regulation of CORs (Sun et al., 2022; Tang et al., 2022; Li et al., 2022; Jiang et al., 2022; Song et al., 2022; Zhang et al., 2022). For example, bZIP68 is expressed in maize ( Zea mays L.) negatively regulates cold tolerance during the vegetative period (Li et al., 2022d), while bZIP73 coordinates bZIP71 to regulate cold tolerance by regulating the balance between abscisic acid (ABA) levels and reactive oxygen species (ROS) concentrations in rice (Liu et al., 2018). Interestingly, the TF-DREB1-CORs module plays an important role in cold tolerance. For example, the OsWRKY63-OsWRKY76-OsDREB1B module was revealed to regulate cold tolerance in rice (Zhang et al., 2022). Furthermore, the interaction between OsWRKY76 and OsbHLH148 is responsible for the transcriptional activation of OsDREB1B (Zhang et al., 2022). Similarly, the inventors recently reported that the NAC-type TF OsNAC5 interacts with the bZIP-type TF OsABI5 (abscisic acid insensitive 5) to regulate cold tolerance in rice (Li et al., 2023). However, how TF-regulated signaling promotes cold resistance is far from clear; therefore, screening for novel TFs involved in cold resistance will further help establish cold resistance signaling pathways.

[0005] OsMYB4P It encodes a nuclear-localized GRAS family protein that controls axillary meristem formation during both vegetative and reproductive growth stages. OsMYB4P It is expressed in the epidermal and hypodermal cells of the axillary bud before the axillary bud undergoes morphological changes, and is subsequently expressed in the entire axillary bud and later in the leaf axil primordium. OsMYB4P plays an important role in the formation of leaf axillary meristems and axillary buds, and also promotes the outward growth of axillary buds. MOC3 and OsMYB4P are not only key factors for the initiation of tiller buds, but also can regulate the elongation of tiller buds. MOC3 can directly bind to FON1 OsMYB4P can not directly bind to the promoter of FON1 promoter, but acts as a coactivator of MOC3 and is further activated in the presence of MOC3 FON1 Expression, and FON1 It can positively regulate the elongation of tiller buds. FON1 Loss-of-function mutants can form normal buds, but bud elongation is defective, resulting in reduced tiller number (Shao et al., 2019). The tillering regulator OsMYB4P binds to the DELLA protein SLR1 and is not degraded. Gibberellic acid GAs triggers SLR1 degradation, leading to stem elongation and OsMYB4P degradation, thereby reducing tiller number (Liao et al., 2019). Rice ( Oryza Sativa L.) is an important food crop. OsMYB4P has been reported to be involved in controlling rice tillering, but its function in improving resistance to low temperature stress has not been reported. Summary of the Invention

[0006] Based on the prior art, the present invention found that overexpression in rice plants OsMYB4P The gene can improve the low temperature stress resistance of rice plants at the seedling stage. Therefore, in one aspect of the present invention, a rice OsMYB4P On the other hand, the present invention also provides a method for improving the resistance of rice to low temperature stress at the seedling stage, which comprises: overexpressing the rice OsMYB4P Furthermore, the present invention also provides a rice OsMYB4P Application of genes in genetic breeding to improve resistance to low temperature stress in rice seedlings.

[0007] In this regard, the present invention includes but is not limited to the following:

[0008] In one aspect, the present invention provides rice OsMYB4P The application of the gene in improving the resistance of rice seedlings to low temperature stress and / or rice variety improvement and breeding is characterized in that the rice OsMYB4P Gene.

[0009] In another aspect, the present invention provides a method comprising OsMYB4P Application of a recombinant vector of a gene in improving the resistance of rice seedlings to low temperature stress and / or rice variety improvement breeding, characterized in that the rice OsMYB4P Gene.

[0010] In one aspect, the recombinant vector of the present invention comprises the overexpression promoter Ubi.

[0011] Preferably, the recombinant vector of the present invention is a recombinant pCAMBIA1301 vector comprising the overexpression promoter Ubi.

[0012] In one aspect, the present invention provides an application of a genetically engineered bacterium in improving the resistance of rice seedlings to low temperature stress and / or in genetic breeding for rice variety improvement, characterized in that the genetically engineered bacterium contains OsMYB4P Recombinant vector of gene, overexpressing the rice OsMYB4P Preferably, the genetically engineered bacteria of the present invention are genetically engineered Escherichia coli or Agrobacterium.

[0013] In one aspect, the genetically engineered bacteria of the present invention are genetically engineered Agrobacterium.

[0014] In one aspect, the genetically engineered bacteria of the present invention is genetically engineered Agrobacterium GV3101.

[0015] In another aspect, the present invention provides a method for improving the resistance of rice to low temperature stress at the seedling stage and / or improving rice varieties through genetic breeding, characterized in that the method comprises: overexpressing rice OsMYB4P Gene.

[0016] In one aspect, the rice variety improvement of the present invention is to improve the resistance of rice to low temperature stress at the seedling stage.

[0017] In another aspect, the present invention provides a method for obtaining rice plants with improved traits, characterized in that it comprises the following steps:

[0018] (1) using genetically engineered bacteria to infect rice callus; and

[0019] (2) Cultivating the infected rice callus into rice plants;

[0020] The genetically engineered bacteria contain rice OsMYB4P Recombinant vector of the gene, overexpressing the rice OsMYB4P Gene.

[0021] In yet another aspect, the present invention provides a use of rice OsMYB4P protein in improving resistance to low temperature stress in rice seedlings and / or in genetic breeding for rice variety improvement.

[0022] In one aspect, the amino acid sequence of the rice OsMYB4P protein of the present invention is shown in SEQ ID NO: 3.

[0023] In one aspect, the improved trait of the present invention is improved resistance to low temperature stress in rice seedlings.

[0024] In one aspect, the present invention provides a method for constructing an overexpression OsMYB4P The method / step of gene carrier comprises: amplifying the gene carrier obtained by PCR OsMYB4P The nucleotide sequence of the gene coding region was ligated into the vector pCAMBIA1301, where the primers were designed as follows:

[0025] OsMYB4P -OE-F: 5'-CTCGGTACCCGGGGATCCATGGGGAGACCTCCATGCTG-3'; (SEQ ID NO: 4)

[0026] OsMYB4P -OE-R: 5'-GTCGACTCTAGAGGATCCGAACATGATAGGGTCTGCACA-3'; (SEQ ID NO: 5).

[0027] In one aspect, the present invention also provides a method for constructing an overexpression OsMYB4P The method / step of genetic engineering bacteria comprises: overexpressing the OsMYB4P The gene vector was transformed into Agrobacterium strain.

[0028] In one aspect, the present invention also provides a method for obtaining overexpression OsMYB4P Method / step for producing a transgenic rice plant comprising: using the overexpression method of the present invention OsMYB4P Genetically engineered bacteria infect rice callus tissue, which is then differentiated and rooted.

[0029] In one aspect, the rice of the present invention OsMYB4P The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0030] In one aspect, the rice of the present invention OsMYB4P The nucleotide sequence of the gene coding region is shown in SEQ ID NO: 1.

[0031] In one aspect, the rice variety of the present invention is the japonica rice variety Zhonghua 11 ( Oryza.Sativa L.spp. japonica , var. Zhonghua11 ).

[0032] Preferably, the improved resistance to low temperature stress of rice according to the present invention is manifested as follows: compared with the control ordinary rice plants, OsMYB4P The survival rate of gene-overexpressing rice was significantly higher after low temperature treatment at the seedling stage.

[0033] In one aspect, the low temperature of the present invention is 12° C. or less. In one aspect, the low temperature of the present invention is 11° C. or less, 10° C. or less, 9° C. or less, 8° C. or less, 7° C. or less, 6° C. or less, 5° C. or less, or 4° C. or less. Preferably, the low temperature of the present invention is 4° C.

[0034] In the present invention, OsMYB4P The nucleotide sequence of the protein coding region of the gene is shown in SEQ ID NO: 1, OsMYB4P The full-length sequence of the gene is shown in SEQ ID NO.2. OsMYB4P The protein encoded by the gene is a MYB family transcription factor, consisting of 330 amino acids, and its amino acid sequence is shown in SEQ ID NO.3, specifically:

[0035] SEQ ID NO: 1:ATGGGGAGACCTCCATGCTGCGACAATGGCGTCGGCGTCAAGAAAGGGCCATGGACGCCAGAGGAGGACATCATCCTCGTCTCCTACATCCAGCAGCATGGCCCCGGGAACTGGCGCTCCGTGCCCGAGAACACCGGATTGATGAGGTGCAGCAAGAGCTGCAGGCTGCGGTGGACGAACTACTTGAGACCGGGGATCAAGCGTGGCAACTTCACCCCTCATGAGGAGGGGATCATCATCCACCTCCAGGCATTGCTTGGCAACAAGTGGGCAGCAATAGCCTCCTACCTCCCCCAAAGAACAGACAACGACATCAAGAACTACTGGAACACACACCTCAAGAAGAAGGTGAAGAGGCTGCAACAACAACAACAATCACACCCTGATCATCATCACCACCATTCCTTCCAAACCACCCCTTCTTCCTCCAATGCAGCAGCAGTAGCAACAACCAGCCCAAACTACTACAACCCTAACAACAGCAACAGCAACAGCAGCAATTACCTCCATAACAACAACCACAATCTTGAATCCATGCAATCCATGGCCACTGCACCTAGCAATGAGGCCACCACCATCCCCAAGCTCTTCCAGTTCCAGACATGGATGAAGCCATCACCAGCAACAACATCATCAGCAGCAACAGCTGCTGCAGGTAGCTGCTACAAGCAGGCCATGGCCATGCAGGAGCTCCAAGAGGAGCAAGAGGGCTCTGCTGCTGCTGCTGCAATGGCTTCTTCCATTGATGGCGTCTCCAAGGACCAGGATTATCACATGTGTGCTGTGATCAGTGGTGATGACAAGTCGTCGTCGTCGGAGATGATGACGGCTGCGGCAATGGCCGGCCATGGCGAGGCGGCCACGACGACCTTCTCGCTGCTCGAGAACTGGCTGCTCGACGACATGCCGGGGCAGGCGGCCATGAGCGCCGCCATGGATGGGTTCTTGGAGATCTCTGCTGGATACTGCTGTGCAGACCCTATCATGTTCTGA

[0036]

[0037] SEQ ID NO: 3: MGRPPCCDNGVGVKKGPWTPEEDIILVSYIQQHPGGNWRSVPENTGLMRCSKSCRLRWTNYLRPGIKRGNFTPHEEGIIIHLQALLGNKWAAIASYLPQRTDNDIKNYWNTHLKKKVKRLQQQQQSHPDHHHHHSFQTTPSSSNAAAVATTSPNYYNPNNSNSNSS NYLHNNNHNLESMQSMATAPSNEATTIPKLFQFQTWWMKPSPATTSSAATAAAGSCYKQAMAMQELQEEQEGSAAAAAMASSIDGVSKDQDYHMCAVISGDDKSSSSEMMTAAAMAGHGEAATTTFSLLENWLLDDMPGQAAMSAAMDGFLEISAGYCCADPIMF*.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention overexpresses and silences the 11-mercaptoethanol in common rice plants. OsMYB4P The gene was identified and its new use in enhancing the resistance of rice to low temperature stress at the seedling stage was discovered, providing important genetic resources for breeding rice varieties resistant to low temperature stress.

[0040] (2) The present invention provides a method for breeding rice germplasm resistant to low temperature stress by using transgenic technology, and obtains OsMYB4P Gene overexpression homozygous plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 图1 For the rice in Example 2 OsMYB4P Gene editing target site (A), mutated protein sequence (B) and protein structure diagram (C).

[0042] 图2 Example 3 Rice OsMYB4P Gene-deficient plants osmyb4p-1 and osmyb4p-2 Plant survival rate and ion leakage rate after low temperature stress compared with wild-type rice plants (ZH11);

[0043] Among them, A is a photo of plant growth; B is a bar graph of plant survival rate; C is a bar graph of plant leakage rate.

[0044] 图3 The measured values ​​in Example 2 OsMYB4P Gene overexpression materials OsMYB4P Schematic diagram of gene expression (A) and Example 3 Rice OsMYB4P Gene overexpression plants OsMYB4P-OE1 and OsMYB4P-OE2 Plant survival rate and ion leakage rate (BD) after low temperature stress compared with wild-type rice plants (ZH11);

[0045] Among them, B is a photo of plant growth; C is a bar graph of plant survival rate; D is a bar graph of plant leakage rate. DETAILED DESCRIPTION

[0046] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0047] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0049] Example 1 Rice OsMYB4P Gene cloning, construction of gene overexpression engineered bacteria and gene editing engineered bacteria

[0050] 1. Extraction of total RNA from rice

[0051] Total RNA was extracted from young rice leaves using the Tiangen Plant total RNA extraction kit. The extracted RNA was reverse transcribed using a Thermo Fisher reverse transcription kit according to the manufacturer's instructions to obtain cDNA (SEQ ID NO. 1), which was then stored at -20°C for future use.

[0052] 2. OsMYB4P Construction of gene overexpression engineered bacteria A

[0053] Design Rice OsMYB4P Specific amplification primers for gene coding region sequences, the primer sequences are as follows:

[0054] OsMYB4P -OE-F: 5'-CTCGGTACCCGGGGATCCATGGGGAGACCTCCATGCTG-3'; (SEQ IDNO.4)

[0055] OsMYB4P -OE-R: 5'-GTCGACTCTAGAGGATCCGAACATGATAGGGTCTGCACA-3'; (SEQ IDNO.5)

[0056] The cDNA (Complementary DNA) obtained by reverse transcription of total RNA from leaves of common rice Zhonghua 11 plants was used as a template to amplify the OsMYB4P The nucleotide sequence of the gene exon was ligated to the overexpression promoter Ubi vector pCAMBIA1301, transformed into competent DH5α Escherichia coli by heat shock at 42°C, and then plated.

[0057] The successfully transformed monoclonal DH5α E. coli colony was selected and the bacterial solution after shaking was sent to a sequencing company for sequencing. The sequencing results showed that the vector contained OsMYB4P The nucleotide sequence of the gene exon was obtained by using the plasmid extraction kit of Quanshijin Company to extract the plasmid, and the plasmid was electroporated into GV3101 Agrobacterium competent cells. After culturing at 28℃ for two days, the plaque was picked and PCR verification was performed to obtain the overexpression vector pCAMBIA1301- OsMYB4P Agrobacterium engineered bacteria A.

[0058] 3. OsMYB4P Construction of gene-edited engineered bacteria B

[0059] Based on the rice genome OsMYB4P The gene coding region sequence (SEQ ID NO: 1) was used to search for specific target sequences online (http: / / crispr.hzau.edu.cn / CRISPR / ), and the target sequence "AGACCTCCATGCTGCGACAATGG" was selected. Complementary primers were synthesized based on the target sequence. Bsa I restriction endonuclease enzyme digestion vector pHun4c12, by gel recovery to obtain linearized vector. Subsequently, the above fusion fragment was ligated into the linearized pHun4c12 vector (Jiang et al., 2019, Mutation of Inositol 1,3,4-trisphosphate 5 / 6-kinase6Impairs Plant Growth and Phytic Acid Synthesis in Rice. Plants 8(5): 114), enzyme digestion identification to obtain the correct vector, and further sequencing confirmed that the target sequence has been introduced into the vector, the correct vector was named pHun4c12- OsMYB4P At the same time, pHun4c12- OsMYB4P Introduced into Agrobacterium strain EHA105, the gene editing vector pHun4c12- OsMYB4P Agrobacterium engineering bacteria B are used for subsequent genetic transformation.

[0060] Example 2 Rice OsMYB4P Obtaining homozygous plants for gene overexpression and homozygous plants for gene function loss

[0061] 1. Agrobacterium-mediated genetic transformation of rice

[0062] Using pCAMBIA1301- OsMYB4P Agrobacterium engineering strain A containing overexpression vector and pHun4c12- OsMYB4P The gene-edited Agrobacterium engineering bacteria B infected rice callus tissue and after screening with differentiation medium and rooting medium, the corresponding genetically transformed plants were obtained.

[0063] 2. Overexpression OsMYB4P Obtaining genetically homozygous strains

[0064] A small amount of leaves and roots from the T0 generation overexpressing seedlings were collected and total RNA was extracted from the plant material (roots and leaves) using the RNeasy Plant RNA Mini Kit (Qiagen, Hilden, Germany). Reverse transcription of cDNA was performed 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 SYBR Green GoTaq qPCR Master Mix (Promega, WI, USA). ACTIN The gene was used as an internal control and 2 -ΔΔCt Calculation OsMYB4P Relative expression levels.

[0065] Table 1 Quantitative real-time polymerase chain reaction (qRT-PCR) primers

[0066]

[0067] The T0 generation transgenic overexpressing plants were self-pollinated to obtain T1 generation seeds. 6-9 positive plants were taken from each T0 generation overexpressing plant to produce T2 generation and separated and analyzed. If all T2 generation seedlings produced by T1 generation positive plants were positive after testing, the T1 generation plants were homozygous for overexpression. OsMYB4P The transgenic lines with overexpressed genes are named OsMYB4P-OE1 and OsMYB4P-OE2, The relative gene expression results can be found in 图3 A; otherwise, it is a heterozygous strain.

[0068] 3. OsMYB4P Obtaining homozygous plants for gene function loss

[0069] The T0 generation transgenic rice was grown to the 3-4 leaf stage and young leaves were selected. Genomic DNA was extracted by CTAB method and the endogenous gene of transgenic rice was amplified using pHun4c12-OsMYB4P specific primers. OsMYB4P Gene, sequencing verified the PCR product, and the results showed that OsMYB4P The gene has a single base deletion (see 图1 A), that is, to obtain OsMYB4P Transgenic lines with loss of gene function are named osmyb4p-1和osmyb4p-2, The sequence and structure diagram of the mutated OsMYB4P protein are shown in 图1 B and 1C.

[0070] Example 3 OsMYB4P Experiment on resistance of gene overexpression rice to low temperature stress

[0071] The rice obtained in Example 2 OsMYB4P Overexpression T2 generation homozygous strain: OsMYB4P-OE1 and OsMYB4P-OE2 , OsMYB4P Loss of gene function transgenic pure lines: osmyb4p-1 and osmyb4p-2 The control ordinary rice plants were subjected to low temperature stress treatment.

[0072] 1. Low temperature stress treatment of rice

[0073] To assess the cold tolerance of rice plants, 14 days after seed soaking, rice seedlings were transferred to either 4°C (experimental) or 30°C (control) for 7 days, followed by 7 days of recovery at 30°C. Survival was then analyzed. All treatments included three biological replicates, each consisting of 30 to 40 plants, for survival determination. All tests were conducted in an artificial chamber under the following conditions (relative humidity: 65-75%; 16 h light / 8 h dark).

[0074] 2. Determination of gene expression

[0075] A small amount of rice leaves was taken from the above-mentioned rice plants and total RNA was extracted from the plant material using the RNeasy Plant RNA Mini Kit (Qiagen, Hilden, Germany). 1 μg of total RNA was used, oligo-dT18 primers were used, and the GoScript™ Reverse Transcription System (Promega) was used to reverse transcribe cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green GoTaq qPCR Master Mix (Promega, WI, USA). OsACTIN The gene was used as an internal control and 2 -ΔΔCt Calculation OsMYB4P Relative expression levels.

[0076] By overexpressing the homozygous T2 line OsMYB4P-OE1 and OsMYB4P-OE2 、 OsMYB4P Gene-deficient plants osmyb4p-1 and osmyb4p-2 Fourteen-day-old plants of Zhonghua 11 (a common rice strain) and its control were cold-treated for 7 days and then cultured under normal conditions for 7 days. Plant survival and ion leakage rates were measured. The ion leakage assay was performed 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). Approximately 0.1 g of leaf tissue from cold-treated seedlings was placed in a 15 mL 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 incubated again at room temperature for 1 hour, and the final conductivity (A2) was measured. The deionized water conductivity (A0) was used as the baseline. The ion leakage rate was calculated as: ion leakage (%) = (A1 − A0) / (A2 − A0).

[0077] turn out, OsMYB4P The survival rate of overexpressing rice after low temperature treatment was significantly higher than that of the control (see 图3 B and 3C), the ion leakage rate was significantly lower than that of the control (see 图3 D); and OsMYB4P gene knockout plants osmyb4p-1 and osmyb4p- 2 The survival rate of rice after low temperature treatment was significantly lower than that of the control (see 图1 A and 1B), the ion leakage rate was significantly higher than that of the control (see 图1 C).

[0078] The above experiments show that overexpression OsMYB4P Genes can improve rice's resistance to low temperature stress. OsMYB4P The loss of gene function reduced the resistance of rice to low temperature stress, indicating that OsMYB4P Genes have very important application value in plant resistance to low temperature stress genetic engineering.

Claims

1. Rice OsMYB4P The application of the gene in improving the resistance of rice to low temperature stress at the seedling stage is characterized in that: Overexpressing the rice OsMYB4P Gene, the rice OsMYB4P The nucleotide sequence of the gene coding region is shown in SEQ ID NO:

1.

2. Contains rice OsMYB4P The application of a recombinant vector of a gene in improving the resistance of rice to low temperature stress at the seedling stage is characterized in that: Overexpressing the rice OsMYB4P Gene, the rice OsMYB4P The nucleotide sequence of the gene coding region is shown in SEQ ID NO:

1.

3. A method for improving the resistance of rice to low temperature stress at the seedling stage using a genetically engineered bacterium, characterized in that: The genetically engineered bacteria contain rice OsMYB4P Recombinant vector of gene, overexpressing the rice OsMYB4P Gene, the rice OsMYB4P The nucleotide sequence of the gene coding region is shown in SEQ ID NO:

1.

4. A method for improving the resistance of rice to low temperature stress at the seedling stage, characterized in that: The method comprises: overexpressing rice OsMYB4P Gene, the rice OsMYB4P The nucleotide sequence of the gene coding region is shown in SEQ ID NO:

1.

5. A method for obtaining rice plants with improved traits, characterized in that: The following processing steps are included: (1) using genetically engineered bacteria to infect rice callus; and (2) Cultivating the infected rice callus into rice plants; The genetically engineered bacteria contain rice OsMYB4P Recombinant vector of gene, overexpressing the rice OsMYB4P Gene, the rice OsMYB4P The nucleotide sequence of the gene coding region is shown in SEQ ID NO: 1, and the improved trait is improved resistance to low temperature stress in rice seedlings.

6. Use of a rice OsMYB4P protein in improving resistance to low temperature stress in rice seedlings, wherein the amino acid sequence of the rice OsMYB4P protein is shown in SEQ ID NO: 3.

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