Application of alfalfa msnf-y c4 gene to improve plant stress tolerance

Abstract

The application provides a Medicago sativa MsNF-YC4 gene and an encoded protein in improving plant stress tolerance. The research of the application shows that overexpression of the MsNF-YC4 gene can promote root growth, regeneration and increase root density of Medicago sativa, and improve drought tolerance, cold tolerance and other stress tolerance of Medicago sativa.

Description

Technical Field

[0001] This application belongs to the fields of agricultural genetic engineering and plant breeding. Specifically, this application provides the application of the MsNF-YC4 gene in alfalfa in improving plant stress resistance. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa (L.) is an important perennial leguminous forage crop, known as the "King of Forage," and plays a vital role in ensuring the development of grassland animal husbandry. Alfalfa is a water-intensive plant, requiring approximately 1000g of water to form every gram of dry matter. However, alfalfa cultivation areas in my country are mainly distributed in the arid and semi-arid regions of the north. Influenced by the temperate continental climate, rainfall is unevenly distributed in time and space. Furthermore, the soil is mostly sandy with poor water retention, creating a cold and dry environment in winter, leading to alfalfa overwintering failure and severely impacting yield and quality. Genetic improvement to create more resilient alfalfa varieties for development in cold, dry regions aligns with both the national strategy of protecting arable land and the practical need to expand alfalfa cultivation in my country.

[0003] NF-Y (Nuclear Factor-Y) is a class of transcription factors widely distributed in eukaryotes, also known as HAP (Heme-activator Protein) or CBF (CCAAT-binding Factor), and is an evolutionarily conserved transcription factor. NF-Y transcription factors can be divided into three subunits: NF-YA (CBF-B / HAP2), NF-YB (CBF-A / HAP3), and NF-YC (CBF-C / HAP5). NF-YB and NF-YC bind in the cytoplasm to form a dimer, which then translocates to the nucleus. There, they bind with NF-YA to form an NF-Y heterotrimeric complex, which targets and binds to the cis-acting element CCAAT-box before the transcription start site of the regulated gene, thereby regulating the expression of downstream genes. NF-YA is responsible for recognizing and binding to the CCAAT-box motif, while NF-YB and YC participate in gene expression activation. NF-YC plays an important role in plant growth and development, metabolic regulation, and responses to abiotic stress. Modern biological methods are used to study alfalfa... MsNF-YC4 The gene function of this gene may provide a possibility for improving the stress resistance of alfalfa. Summary of the Invention

[0004] On the one hand, this application provides the application of the alfalfa MsNF-YC4 gene in improving plant stress resistance, and the nucleotide sequence of the alfalfa MsNF-YC4 gene is shown in SEQ ID NO.1.

[0005] In another aspect, the present application provides the use of the protein encoded by the Medicago sativa MsNF-YC4 gene in improving the stress tolerance of a plant, wherein the amino acid sequence of the protein encoded by the Medicago sativa MsNF-YC4 gene is shown as SEQ ID NO. 2.

[0006] In another aspect, the present application provides the use of the Medicago sativa MsNF-YC4 gene in promoting the growth, regeneration or increasing the density of the root system of a plant, wherein the nucleotide sequence of the Medicago sativa MsNF-YC4 gene is shown as SEQ ID NO. 1.

[0007] In another aspect, the present application provides the use of the protein encoded by the Medicago sativa MsNF-YC4 gene in promoting the growth, regeneration or increasing the density of the root system of a plant, wherein the amino acid sequence of the protein encoded by the Medicago sativa MsNF-YC4 gene is shown as SEQ ID NO. 2.

[0008] Further, the plant is a legume.

[0009] Further, the plant is a Medicago plant.

[0010] Further, the plant is Medicago sativa.

[0011] Further, the stress tolerance is drought resistance.

[0012] Further, the stress tolerance is cold resistance.

[0013] Further, the stress tolerance is resistance to abiotic stress, such as wind resistance, etc.

[0014] Further, the use involves overexpression of the Medicago sativa MsNF-YC4 gene or the protein encoded by the Medicago sativa MsNF-YC4 gene in the plant.

[0015] Further, the overexpression is achieved by using a pCGW vector to construct a Medicago sativa MsNF-YC4 gene overexpression vector and transforming the plant.

[0016] Further, the plant is transformed using Agrobacterium-mediated transformation. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 pCGW- MsNF-YC4 Vector schematic diagram;

[0018] Figure 2 Overexpression MsNF-YC4 Figure of PCR detection results of hygromycin-resistant plant;

[0019] Figure 3 Figure of 2-month-old MsNF-YC4 -OE plantMsNF-YC4 Relative expression detection result figure;

[0020] Figure 4 Wild type and MsNF-YC4 OE transgenic plant root phenotype figure;

[0021] Figure 5 Wild type and MsNF-YC4 OE transgenic plant drought tolerance evaluation result figure;

[0022] Figure 6 Wild type and MsNF-YC4 OE transgenic plant physiological index comparison figure before and after drought treatment;

[0023] Figure 7 Wild type and MsNF-YC4 OE transgenic plant cold tolerance evaluation result figure. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application.

[0025] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The reagents and materials used in the following examples are all commercially available products, unless otherwise specified.

[0026] The data in the following examples are subjected to variance analysis and multiple comparisons using the single factor variance analysis program in IBM SPSS 24.0 software. The “*” indicates the degree of significance. The treatment marked with “***” is significantly different from the control group at the level of <0.01. The treatment marked with “**” is significantly different from the control group at the level of <0.05. The treatment marked with “*” is significantly different from the control group at the level of <0.1. P <0.01 level, the treatment marked with “**” is significantly different from the control group at the level of <0.05, and the treatment marked with “*” is significantly different from the control group at the level of <0.1. P <0.05 level, and the treatment marked with “*” is significantly different from the control group at the level of <0.1. P <0.1 level.

[0027] Example 1 MsNF-YC4 Gene cloning and expression vector construction

[0028] The previous experimental results of the research group showed that heterologous expression of AtQQS can significantly improve the stress resistance of Medicago polymorpha plants. Using pGBKT7-AtQQS as a bait protein, the Medicago polymorpha cDNA library was screened by yeast two-hybrid experiment, and the endogenous interaction gene MsNF-YC4 of Medicago polymorpha was identified. MsNF-YC4 The coding sequence of the gene is SEQ ID No. 1, which is derived from Medicago polymorpha, and the encoded amino acid sequence is the protein MsNF-YC4 of SEQ ID No. 2.

[0029] MsNF-YC4Nucleotide sequence (SEQ ID No. 1)

[0030] ATGGAGAACAACAACCAACAACAACAATATCCAACACCTTCACCCACGGCAGTTACACCACCACCATTCCAACATCTTCTCCAACAACAACAACAACAGCTTCAAATGTTCTGGTCCTACCAACGTCAAGAAATTGAACACGTCAACGATTTCAAAAACCATCAACTTCCATTAGCACGCATTAAGAAAATCATGAAAGCTGATGAAGACGTTCGAATGATTTCTGCAGAAGCACCAATTCTCTTTGCCAAAGCTTGTGAGCTTTTCATTCTTGAACTCACCATTCGTTCTTGGCTTCATGCTGAAGAGAATAAACGACGAACTCTTCAGAAAAATGATATTGCTGCTGCTATTACTAGGACTGATATTTTTGATTTTTTGGTTGATATTGTTCCGAGAGACGAGATTAAAGATGAAGCTGCTGCTGCTGCTATTGTTGGTGCTGCTGCTAGTGGTGTTCCTTATTATTATCCTCCTATGGGACAACCTGCTGGAATGATGATTGGTCGTCCCGCTGTTGATCCGGCTACCGGTGTTTATGTTCAGCCGCCTTCTCAGGCTTGGCAGTCTGTTTGGCAGACGGGAGCGGATGATGGTTCTTATGCTGGTGGTGGTGTTGGCAGTGGTCAGCACAATGGCGATGGCCAGAGCTGA

[0031] MsNF-YC4 protein sequence (SEQ ID No. 2)

[0032] MENNNQQQQYPTPSPTAVTPPPFQHLLQQQQQQLQMFWSYQRQEIEHVNDFKNHQLPLARIKKIMKADEDVRMISAEAPILFAKACELFILELTIRSWLHAEENKRRTLQKNDIAAAITRTDIFDFLVDIVPRDEIKDEAAAAAIVGAAASGVPYYYPPMGQPAGMMIGRPAVDPATGVYVQPPSQAWQSVWQTGADDGSYAGGGVGSGQHNGDGQS*

[0033] The RNA of Medicago sativa (Zhongmo No. 1) was extracted, and cDNA was obtained by reverse transcription. The sequence cloning primers covering the entire cDNA were designed according to the data of the third generation database as follows:

[0034] MsNF-YC4-Xba I-F: 5'- ATGGAGAACAACAACCAACAACAAC-3' (SEQ ID No. 3, the underlined region is an Xba I recognition site) TCTAGA MsNF-YC4-Xba I-F: 5'- ATGGAGAACAACAACCAACAACAAC-3' (SEQ ID No. 3, the underlined region is an Xba I recognition site)

[0035] MsNF-YC4-Sal I-R: 5'- TCAGCTCTGGCCATCGCCATTG-3' (SEQ ID No. 4, the underlined region is a Sal I recognition site) GGTACC MsNF-YC4-Sal I-R: 5'- TCAGCTCTGGCCATCGCCATTG-3' (SEQ ID No. 4, the underlined region is a Sal I recognition site)

[0036] PCR was performed with cDNA as a template and MsNF-YC4-Xba I-F and MsNF-YC4-Sal I-R as primers, and the cloned PCR product was a DNA molecule encoding a sequence of a coding region with enzyme digestion recognition sites at both ends. MsNF-YC4 The PCR product and the pCGW vector were double-digested by restriction enzymes Xba I and Sal I respectively, the obtained enzyme digestion products were ligated, the obtained ligation product was transformed into DH5α strain, positive clones were selected and sent for sequencing, and the sequencing results showed that the structure of the recombinant vector was MsNF-YC4 The nucleotide sequence SEQ ID No. 1 replaced the nucleotide fragment between the restriction enzyme Xba I and Sal I recognition sites of the pCGW vector, the other nucleotide sequences of the pCGW were kept unchanged, and the obtained recombinant vector was named as pCGW- MsNF-YC4 , which is a MsNF-YC4 gene overexpression vector (see Figure 1 ).

[0037] Example 2: Obtaining the overexpression vector of Medicago sativa MsNF-YC4

[0038] Agrobacterium-mediated genetic transformation

[0039] The constructed pCGW- MsNF-YC4 overexpression vector was transformed into Agrobacterium tumefaciens EHA105 by heat shock method, positive clones were selected on YEP medium containing 50 mg / L kanamycin (kan) and 50 mg / L rifampicin (Rif), and the vector was successfully transformed into EHA105 Agrobacterium by bacterial liquid PCR identification.

[0040] ​The genetic transformation was carried out by using the Agrobacterium-mediated genetic transformation method published in the previous method literature of the laboratory. The receptor plant used was the alfalfa variety "Zhongmo No. 1", the transformation site was the young leaf, and the transformation vector was pCGW-

[0041] MsNF-YC4 The method reference is (Zhang Wanjun, Gao Yanan, Wang Zhijie, et al. An Agrobacterium-mediated efficient genetic transformation method of alfalfa [P]. Beijing: CN202310623918.0, 2023-09-08).

[0042] Through hygromycin screening, 16 resistant plants were obtained.

[0043] Transgenic positive identification of hygromycin-resistant plants

[0044] In order to detect whether the T-DNA fragment containing the target gene in the vector is integrated into the plant genome, the CTAB method was used to extract the DNA of the leaves of wild type and hygromycin-resistant regenerated seedlings. With the DNA of the transgenic resistant plant as the template, the wild type alfalfa Zhongmo No. 1 variety plant (WT, i.e. the receptor plant) as the negative control, and the plant expression vector pCGW- MsNF-YC4 as the positive control, PCR identification was carried out.

[0045] Primer

[0046] 35S-F: 5'-CGCACAATCCCACTATCCTTC-3' (SEQ ID No. 5)

[0047] MsNF-YC4-Sal I-R: 5'-GGTACCTCAGCTCTGGCCATCGCCATTG-3' (SEQ ID No. 4)

[0048] PCR system

[0049]

[0050] Reaction program:

[0051]

[0052] MsNF-YC4 The identification and detection results of overexpression alfalfa are shown in Figure 2 The transgenic positive plant was amplified to obtain a band with the same size as the positive control (+), and the negative control (-) did not amplify the target band, indicating that the vector fragment had been integrated into the plant genome. The overexpression plant was obtained by PCR detection MsNF-YC4 16 plants, labeled as OE plants, such as No. 2 plant labeled as OE2.

[0053] MsNF-YC4qRT-PCR detection of transgenic plants

[0054] To detect genetically modified plants MsNF-YC4 Expression level: Using cDNA from wild-type and transgenic positive plants as templates, the expression level of MsNF-YC4 in transgenic plants was detected by qRT-PCR, with Actin as the internal control gene.

[0055] Primers

[0056] qRTt-MsNFYC3-F: TATTGTTGGTGCTGCTGCTAGTGG (SEQ ID No. 7)

[0057] qRTt-MsNFYC3-R:AACACCACCACCAGCATAAGAACC (SEQ ID No. 8)

[0058] actin-F CAAAAGATGGCAGATGCTGAGGAT (SEQ ID No.9)

[0059] actin-R CATGACACCAGTATGACGAGGTCG (SEQ ID No. 10)

[0060] qRT-PCR system

[0061]

[0062] Reaction procedure:

[0063]

[0064] The results showed that, compared with the wild type, MsNF-YC4 Overexpression in alfalfa plants, MsNF-YC4 The expression level ranged from 1 to 46 times. Figure 3 ).Will MsNF-YC4 OE plants with expression levels below 10-fold were classified as low expression group (LE), OE plants with expression levels above 10-fold but below 20-fold were classified as medium expression group (ME), and OE plants with expression levels above 20-fold were classified as high expression group (HE).

[0065] Example 3 Overexpression MsNF-YC4 Phenotypic observation of alfalfa plant roots

[0066] T0 generation transgenic plants were propagated by stem cuttings, with the same lineage number as the mother plant. Based on previous expression level detection, this study selected plants with different expression levels... MsNF-YC4 We conducted in-depth research on the overexpression levels of plants OE3, OE2, OE12, and OE11.

[0067] 7-day-old wild-type (WT) plants and MsNF-YC4 Observation of stem segments from -OE transgenic plants revealed that transgenic plants exhibited faster root regeneration rates compared to WT plants. Figure 4 Part A). Root phenotype of plants grown from cuttings for 2 months is as follows. Figure 4 As shown in sections B and E, compared to WT, MsNF-YC4 -OE plants have larger root systems. Root scanning analysis of WT and transgenic plants showed that... MsNF-YC4 -OE plants had significantly higher root tip number and branch number than wild-type plants ( P <0.01)( Figure 4 (CD section).

[0068] Example 4 Overexpression MsNF-YC4 Evaluation of stress tolerance of alfalfa plants

[0069] This study selected those with different MsNF-YC4 We conducted in-depth research on the overexpression levels of plants OE3, OE2, OE12, and OE11.

[0070] MsNF-YC4 Evaluation of drought tolerance of transgenic plants

[0071] Wild-type (WT) plants and T0 generation transgenic plants were propagated by stem cuttings, with the same lineage number as the mother plant. After survival, the plants were transplanted into 10 cm × 10 cm pots containing the same weight of soil. After 3 months of growth, four healthy plants were selected from each line, pruned to a 10 cm stubble, and allowed to grow for another month before a drought treatment experiment. Before the experiment, the pots were filled with water and weighed (100% saturated water weight), then placed in a constant temperature incubator (25±2℃) to begin the controlled water experiment. The weight of the pots was measured daily, and when the weight of the pots plus the plant reached 50% of the saturated water weight, this was recorded as drought day 0. The drought treatment continued for 4 days. Leaves were collected on days 0, 3, and 4 of the drought treatment for physiological index measurements.

[0072] The results are as follows Figure 5 As shown, before drought treatment, the plants grew uniformly. After 4 days of drought treatment, the leaves of wild-type (WT) plants withered due to water loss, and the top branches of transgenic plants began to wilt and lose chlorophyll. However, the young leaves of secondary branches at the nodes showed less wilting. After 2 days of rehydration, the survival rate was calculated, and dead branches were pruned. The survival rate of branches of transgenic plants was significantly higher than that of wild-type plants. P <0.05) Figure 6 Nine days after rehydration (part A), all transgenic plants rapidly grew new shoots and survived, while only two wild-type (WT) plants showed signs of growth, and the other two died. Nine days after rehydration, it was found... MsNF-YC4-OE plants have larger root systems than wild-type plants.

[0073] The drought tolerance of wild-type and transgenic plants was quantitatively evaluated by measuring the relative water content, electrolyte permeability, malondialdehyde content, hydrogen peroxide content, and soluble sugar content of the terminal leaves at different stages of drought treatment. Figure 6 As shown in the BF section, after 3 days of drought treatment, except for the OE12 line, the relative water content of the leaves of transgenic plants was significantly higher than that of WT plants. P <0.1), electrolyte permeability and soluble sugar content were significantly lower than those of wild-type (WT) plants ( P <0.01), the H2O2 content of the OE3 strain was significantly lower than that of the WT plant ( P <0.05%, and there was no significant difference in MDA content between other lines and WT plants. After 4 days of drought treatment, the electrolyte permeability and soluble sugar accumulation in the leaves of the OE11 line increased rapidly, while the increases in the other lines were smaller and still significantly lower than those in the leaves of WT plants ( P <0.01). The above results indicate that overexpression MsNF-YC4 The gene significantly enhances the drought resistance of alfalfa.

[0074] MsNF-YC4 Evaluation of cold tolerance of transgenic plants

[0075] Wild-type (WT) plants and T0 generation transgenic plants were propagated by stem cuttings, with the same lineage number as the mother plant. After surviving, the plants were transplanted into 10 cm × 10 cm pots containing the same weight of soil. After 3 months of growth, four healthy plants from each lineage were selected. Leaves were removed and placed in pre-iced glass tubes, kept on ice for 1 hour. The tubes were then transferred to a freezer and equilibrated at 0 °C for 1 hour. The temperature was decreased at a rate of 2 °C per hour, maintained at each temperature for 1 hour, until reaching -8 °C. Samples were taken at five fixed points between 0 °C and -8 °C, thawed overnight at 4 °C, and then the ion permeability was measured. The 50% electrolyte leakage temperature (TEL) was calculated based on the results. 50 The flowerpot was then filled with water, trimmed to 10cm, and placed in a constant temperature incubator (16 / 12 ℃, light / dark 12 / 12 h) for 1 week. The results are as follows: Figure 7 As shown in section AC. MsNF-YC4 -OE plants exhibit greater frost resistance than wild-type leaves, and after harvesting, under cultivation conditions of 16 / 12 ℃ with 12 / 12 h light / dark cycles, they show improved resistance. MsNF-YC4-OE plants grew rapidly. Subsequently, they were acclimated in a constant temperature incubator (4 / 0 ℃ light / dark 12 / 12 h) for 2 w, and subjected to a gradient temperature treatment, starting from 4 ℃, decreasing 1 ℃ per hour to -12 ℃, and then increasing 2 ℃ per hour to 4 ℃ after 3 h at -12 ℃, and keeping at 4 ℃ for 24 h, and then placed in a greenhouse for recovery for 3 w. The results are shown in Figure 7 As shown in D-F parts of Fig. 2, there was no significant difference between the lines after the freezing treatment, and the OE2 line showed a higher survival rate than the WT after 3 w of recovery.

[0076] In summary, overexpression of the gene can promote alfalfa root regeneration, increase root density, and improve alfalfa stress tolerance. MsNF-YC4 In summary, overexpression of the gene can promote alfalfa root regeneration, increase root density, and improve alfalfa stress tolerance.

Claims

1. Application of Medicago sativa MsNF-YC4 gene in improving drought resistance or cold resistance of Medicago sativa, characterized in that, The nucleotide sequence of the Medicago sativa MsNF-YC4 gene is shown as SEQ ID NO. 1; the application overexpresses the Medicago sativa MsNF-YC4 gene in Medicago sativa.

2. The application according to claim 1, wherein a pCGW vector is used to construct a Medicago sativa MsNF-YC4 gene overexpression vector and transform Medicago sativa to overexpress the Medicago sativa MsNF-YC4 gene.

3. The application according to claim 2, wherein Agrobacterium-mediated transformation is used to transform Medicago sativa.

4. The application of Medicago sativa MsNF-YC4 gene in promoting the growth of Medicago sativa root system, promoting the regeneration of Medicago sativa root system or increasing the density of Medicago sativa root system, characterized in that, The nucleotide sequence of the Medicago sativa MsNF-YC4 gene is shown as SEQ ID NO. 1; the application overexpresses the Medicago sativa MsNF-YC4 gene in Medicago sativa.

5. The application according to claim 4, wherein a pCGW vector is used to construct a Medicago sativa MsNF-YC4 gene overexpression vector and transform Medicago sativa to overexpress the Medicago sativa MsNF-YC4 gene.

6. The application according to claim 5, wherein Agrobacterium-mediated transformation is used to transform Medicago sativa.

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