Alfalfa r2-r3 type transcription factor mfmyb4 and application thereof

Abstract

The application discloses an alfalfa R2-R3 type transcription factor MfMYB4 and application thereof. The amino acid sequence of the alfalfa R2-R3 type transcription factor MfMYB4 is shown as SEQ ID NO. 1. The application clones a cDNA sequence MfMYB4 of the R2-R3 type transcription factor from alfalfa, connects the obtained MfMYB4 gene with a plant expression vector, constructs a recombinant expression vector, transforms plant tissues with the constructed recombinant expression vector, and then cultivates into transgenic plants. The MfMYB4 gene is induced by dehydration stimulation; the application provides a method for cultivating drought-resistant plants by using the MfMYB4 gene, and the drought resistance of the transgenic plants is improved after overexpression of the gene in the plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the R2-R3 type transcription factor MfMYB4 of alfalfa and its application. Background Technology

[0002] Abiotic stresses in the environment, such as drought, salinity, chilling injury, and heat injury, affect plant growth and development, leading to reduced crop yields and impacting the normal growth and quality of forest trees, fruit trees, flowers, and ornamental horticultural plants. Therefore, cultivating stress-tolerant plant varieties is one of the main goals of agriculture. Thus, isolating stress-tolerant genes from plants with strong stress resistance is essential.

[0003] MYB transcription factors can regulate plant drought tolerance through different pathways. For example, MYB proteins can regulate plant drought tolerance by modulating the synthesis of flavonoids and the cuticle. In Arabidopsis, AtMYB12 and AtMYB75 promote the accumulation of antioxidant flavonoids and improve drought tolerance by regulating the expression of flavonoid synthesis genes (Li et al., 2016; Nakabayashi et al., 2014). The cuticle protects plant organs from drought stress. In Arabidopsis, AtMYB41 reduces drought tolerance by negatively regulating the expression of cuticle synthesis genes, while AtMYB94 and AtMYB96 improve drought tolerance by positively regulating the expression of wax synthesis genes (Cominelli et al., 2008; Lee et al., 2015; Seo et al., 2011). In addition, MYB transcription factors can also participate in drought response by regulating stomatal opening and closing through ABA signaling. For example, Arabidopsis thaliana AtMYB15 and AtMYB37 improve the drought resistance of plants by promoting ABA-induced stomatal closure, while AtMYB20 is a negative regulator of ABA-mediated stomatal closure (Ding et al., 2009; Gao et al., 2014; Yue et al., 2016).

[0004] Plants can also cope with salt stress through the accumulation of osmotic regulatory substances, ion balance, and enhanced antioxidant capacity regulated by MYB transcription factors. Arabidopsis thaliana AtMYB49 positively regulates salt tolerance by promoting the expression of cuticle synthesis genes AtKCS2, AtGPAT5, and AtCYP96A9, and by activating antioxidant defenses (Zhang et al., 2020). AtMYB42 enhances salt tolerance by regulating the expression of the ion regulation gene AtSOS2 (Sun et al., 2020). Wang et al. (2016) found that AtMYB12 enhances salt tolerance by promoting flavonoid synthesis and ROS scavenging, while simultaneously inducing the expression of ABA and proline synthesis-related genes (Wang et al., 2016). Additionally, Alfalfa MtMYBS1 can also enhance salt tolerance by activating the expression of stress-resistance genes (Dong et al., 2017).

[0005] Besides regulating drought and salt tolerance in plants, MYB transcription factors have also been shown to participate in the regulation of low-temperature and high-temperature stress. Arabidopsis thaliana AtMYB96 enhances cold tolerance by activating the expression of the low-temperature signal transduction genes AtCBFs and the lipid transfer protein AtLTP3, while AtMYB14 and AtMYB15 inhibit AtCBF expression, reducing tolerance to low-temperature stress (Agarwal et al., 2006; Chen et al., 2013; Guo et al., 2013; Lee et al., 2015). However, the tomato homolog SlMYB15 positively regulates SlCBF expression under low-temperature stress, enhancing cold tolerance (Kim et al., 2017; Zhang et al., 2020). Furthermore, the high-temperature sensitive phenotype of Arabidopsis thaliana AtMYB30 is due to the suppression of the expression of the atlantoxin gene AtANNs (Liao et al., 2017).

[0006] Transcription factors, as a class of proteins that regulate gene expression, participate in almost all physiological and biochemical processes within cells and play an indispensable role in plant growth and development. Among them, MYB transcription factors are the largest family of transcription factors in plants. Based on the number of MYB domains they contain, MYB family members can be divided into four types: 1R type, R2R3 type, 3R type, and 4R type (Dubos et al., 2010).

[0007] The MYB domain of the R2R3-MYB transcription factor is located at the N-terminus of the protein and is highly conserved, while its transcriptional regulatory region is located at the C-terminus. It has the most members in the MYB family, with 109, 126, and 186 members identified in rice, Arabidopsis thaliana, and alfalfa, respectively (Dubos et al., 2010; Zhou et al., 2019).

[0008] Alfalfa is a high-quality legume forage crop, mainly grown in the arid and semi-arid Northwest and North China regions. Drought is the most significant adverse factor in alfalfa production, severely reducing its yield and quality. Yellow alfalfa, in particular, is a highly drought-resistant species. Cloning drought-resistance regulatory genes from yellow alfalfa and elucidating their molecular mechanisms of drought resistance provides an important genetic resource for alfalfa breeding. This research lays a solid foundation for molecular breeding of stress-resistant agricultural and forestry plants and offers significant guidance for research on alfalfa drought resistance mechanisms, production utilization, and the cultivation of new drought-resistant varieties. Summary of the Invention

[0009] To overcome the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a *Alfalfa* R2-R3 type transcription factor MfMYB4, its encoding gene, and its application in order to improve the drought resistance of plants.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention seeks protection for an alfalfa R2-R3 type transcription factor MfMYB4, the amino acid sequence of which is (a) or (b):

[0012] (a) The amino acid sequence as shown in SEQ ID NO.1;

[0013] (b) A sequence with equivalent function formed by substituting, deleting or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO.1.

[0014] Secondly, the present invention seeks protection for the MfMYB4 gene encoding the above-mentioned alfalfa R2-R3 type transcription factor MfMYB4, the nucleotide sequence of which is (1) or (2):

[0015] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0016] (2) A nucleotide sequence that has more than 90% homology with and has the same function as the nucleotide sequence described in (1).

[0017] Thirdly, the present invention seeks protection for biological materials containing the above-mentioned MfMYB4 gene, characterized in that the biological material is an expression cassette, a recombinant vector, a transgenic cell line, or a recombinant microorganism.

[0018] Furthermore, the recombinant vector is a recombinant expression vector, which is obtained by ligating the above-mentioned MfMYB4 gene with a plant expression vector.

[0019] The plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors for monocotyledonous gene gun transformation; the binary Agrobacterium vectors include pBI121 and pCAMBIA series vectors; the plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., the DNA fragment containing polyadenylate signals and any other effector mRNA processing or gene expression.

[0020] When constructing a recombinant expression vector containing the MfMYB4 gene, any strong or inducible promoter can be used before its transcription initiation nucleotide. These strong or inducible promoters include, but are not limited to, the ubiquitin (35Squtin) promoter and the cauliflower mosaic virus (CaMV) 35S promoter, which can be used alone or in combination with other plant promoters. Furthermore, enhancers can also be used when constructing a recombinant expression vector containing the MfMYB4 gene. These enhancer regions include, but are not limited to, the ATG start codon and adjacent regions. The start codon must be identical to the reading frame of the coding sequence to ensure translation of the entire sequence. Translation control signals and start codons can come from various sources, including natural and synthetic ones. The translation initiation region can originate from the transcription initiation region or from structural genes.

[0021] To facilitate the identification and screening of transgenic plants, the plant expression vectors used can be processed, including the addition or replacement of plant-selective markers. Usable selective markers include genes encoding herbicide-resistant enzymes or antibiotic resistance markers; herbicides include glyphosate and glufosinate, and antibiotics include kanamycin sulfate, hygromycin, and gentamicin. From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0022] In a specific embodiment of the present invention, the plant expression vector selected is pCAMBIA3301.

[0023] The method for constructing the recombinant expression vector specifically includes the following steps:

[0024] (1) Primer design

[0025] Primer ① is the upstream primer MfMYB4-F for amplifying MfMYB4:

[0026] 5'-ATGGGAAGATCACCTTGTT-3';

[0027] Primer ② is the downstream primer MfMYB4-R for MfMYB4 amplification:

[0028] 5'-TCATTTCATTTCTAAGCCT-3';

[0029] Primer ③ is the upstream primer MfMYB4-3301-F that introduces the Nco I restriction site:

[0030] 5'-CACGGGGGACTCTTGA CCATGG ACATGGGAAGATCACCTTGTT-3';

[0031] Primer ④ is the downstream primer MfMYB4-3301-R that introduces the BstEIⅠ restriction site:

[0032] 5'-CGGGGAAATTCGAGCT GGTCACC TCATTTCATTTCTAAGCCT-3';

[0033] (2) Obtaining the MfMYB4 gene fragment:

[0034] Using alfalfa cDNA as a template, the MfMYB4 gene fragment was amplified using primers ① and ②, and then... Cloning Kit vector ligation was used to construct the pEASY-MfMYB4 vector;

[0035] (3) Construction of the recombinant expression vector pCAMBIA3301-MfMYB4 containing the MfMYB4 gene:

[0036] Using the pEASY-MfMYB4 vector as a template, primers ③ and ④ were used to introduce the MfMYB4 gene fragment into the Nco I and BstEI I restriction sites, and then ligated it with the plant expression vector pCAMBIA3301 to construct the recombinant expression vector pCAMBIA3301-MfMYB4 containing the MfMYB4 gene.

[0037] The preferred variety of alfalfa is Hailar alfalfa (Medicago falcata L.).

[0038] In a specific embodiment of the present invention, transgenic plants expressing the R2-R3 type transcription factor MfMYB4 of alfalfa are cultivated using the above-mentioned recombinant expression vector pCAMBIA3301-MfMYB44. This is achieved by transforming plant tissues with the above-mentioned recombinant expression vector containing the MfMYB4 gene and then cultivating the transformed plant tissues into transgenic plants.

[0039] The recombinant expression vector containing the MfMYB4 gene can be used to transform plant tissues through various conventional or specific genetic transformation methods, such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation. The transformed plant tissues are then cultured into plants. The transformed plant host can be a monocotyledonous or dicotyledonous plant. Alfalfa is used as an example plant. The method for preparing transgenic plants expressing the yellow alfalfa R2-R3 transcription factor MfMYB4 specifically includes the following steps:

[0040] (1) The recombinant expression vector pCAMBIA3301-MfMYB4 was introduced into Agrobacterium rhizogenes EHA105 by electroporation to obtain Agrobacterium-positive colonies containing the recombinant expression vector pCAMBIA3301-MfMYB4;

[0041] (2) Rice callus tissue was infected with activated Agrobacterium EHA105 containing the recombinant expression vector pCAMBIA3301-MfMYB4, and transgenic alfalfa was obtained after co-culture and antibiotic screening.

[0042] Fourthly, the present invention seeks protection for the above-mentioned alfalfa R2-R3 type transcription factor MfMYB4, the above-mentioned MfMYB4 gene, or the above-mentioned biological material in the application of improving plant drought resistance or cultivating transgenic plants with improved drought resistance.

[0043] Furthermore, the above applications include overexpressing the MfMYB4 gene in target plants or increasing the expression level of the transcription factor MfMYB4 protein to improve the drought resistance of plants.

[0044] Furthermore, the process of overexpressing the MfMYB4 gene in the target plant is as follows: construct the overexpression vector of the MfMYB4 gene as described above, and transfer the overexpression vector into the target plant through Agrobacterium-mediated transformation to obtain transgenic plants with improved drought resistance.

[0045] Fifthly, the present invention claims protection for a method for improving plant drought resistance by overexpressing the MfMYB4 gene in a target plant or increasing the expression level of the transcription factor MfMYB4 protein to improve plant drought resistance.

[0046] The plants mentioned above are monocotyledonous or dicotyledonous plants, such as alfalfa, but are not limited to these.

[0047] The present invention has the following advantages and effects compared with the prior art:

[0048] (1) The present invention cloned the cDNA sequence MfMYB4 of the R2-R3 type transcription factor from alfalfa. The expression of the MfMYB4 gene is induced by drought.

[0049] (2) The present invention connects the obtained MfMYB4 gene with a plant expression vector to construct a recombinant expression vector suitable for dicotyledonous plants, and transforms the dicotyledonous model plant alfalfa. The transgenic plants significantly improve drought resistance.

[0050] (3) This invention provides a method for cultivating drought-resistant plants using the MfMYB4 gene. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the expression cassette of the recombinant expression vector pCAMBIA3301-MfMYB4.

[0052] Figure 2 This is an agarose gel electrophoresis image of the PCR amplification of the open reading frame sequence of the MfMYB4 gene, where M is a standard DNA molecule; lane 1 is the amplified fragment of the MfMYB4 gene.

[0053] Figure 3 This is an agarose gel electrophoresis image of the PCR identification of the recombinant expression vector pCAMBIA3301-MfMYB4, where M is the standard DNA molecule; lanes 1-4 are positive recombinants.

[0054] Figure 4 This is a bar chart analyzing the results of drought-induced MfMYB4 gene expression, i.e., the effect of drought on MfMYB4 gene transcription. The letters a, b, c, d, and e above the bars indicate significant differences between different treatments (P≤0.05). That is, when the letters above the data bars are the same, it means there is no significant difference, and when the letters above the data bars are different, it means there is a significant difference.

[0055] Figure 5 This is an agarose gel electrophoresis image showing the PCR identification results of transgenic alfalfa with the MfMYB4 gene introduced. WT represents wild type; numbers 1 to 7 represent different transgenic alfalfa plants to be identified.

[0056] Figure 6This is a graph showing the real-time quantitative PCR results analysis of three transgenic alfalfas with the MfMYB4 gene introduced. WT represents wild type; OE2, 3, and 4 represent different transgenic alfalfas; the letters a and b above the columns indicate significant differences between different materials (P≤0.05) in the real-time quantitative PCR detection.

[0057] Figure 7 This is an analysis of the drought resistance test results of transgenic alfalfa with the MfMYB4 gene introduced. In this figure, (A) is the survival rate of each plant line 10 days after rehydration following drought stress; (B) is the relative electrical conductivity of the leaves of each plant line after drought treatment; the letters a, b and c above the column indicate significant differences between different materials (P≤0.05).

[0058] Figure 8 This is an example of planting genetically modified alfalfa and its wild-type alfalfa plants. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0060] The yellow alfalfa (Medicago falcata L.) variety Hailar was provided by the laboratory of Professor Guo Zhenfei's research group at the College of Grassland Science, Nanjing Agricultural University.

[0061] Agrobacterium tumefaciens EHA105: This strain is a strain preserved in the laboratory of Professor Guo Zhenfei's research group at the College of Grassland Science, Nanjing Agricultural University.

[0062] Medicago truncatula seeds: variety R108, provided by the laboratory of Professor Guo Zhenfei's research group at the College of Grassland Science, Nanjing Agricultural University.

[0063] pCAMBIA3301 overexpression vector: provided by Professor Guo Zhenfei's laboratory, College of Grassland Science, Nanjing Agricultural University

[0064] Escherichia coli DH5α: purchased from Beijing Qingke Biotechnology Co., Ltd.

[0065] The aforementioned biomaterials are all biomaterials disclosed in the prior art.

[0066] Example 1: Cloning of the MfMYB4 gene

[0067] I. Preparation of alfalfa cDNA template

[0068] Take a small amount of alfalfa (Medicago falcata L.) 'Hailar' seeds and place them in a 1.5 mL sterile centrifuge tube. Add 1 mL of 70% ethanol and treat for 30 seconds, then wash three times with sterile water. Add 10% sodium hypochlorite solution, invert for 10 minutes, and wash 3-5 times with sterile water. Then place the seeds on 1 / 2 MS medium. After germination and rooting, transplant them into a mixed culture medium (vermiculite: nutrient soil = 1:1) and cultivate them in a greenhouse at a temperature of 22℃ / 16℃ (day / night), a photoperiod of 16h / 8h (light / dark), a humidity of 60%, and a light intensity of 800 μmol·m⁻¹. -2 ·s -1 Regularly prune the plants and apply an appropriate amount of compound fertilizer (N:P:K = 15:15:15) solution. After the materials have grown uniformly, they are used for experimental treatment. Mature leaves of alfalfa are taken, and total RNA is extracted using the RNA Prep Pure Plant kit (Tiangen Biotech). cDNA templates are obtained by reverse transcription using the EasyScript First-Strand cDNA Synthesis SuperMix reverse transcriptase kit (TransGen Biotech) and stored at -20℃ for later use.

[0069] II. Primers for amplifying the MfMYB4 gene

[0070] Based on the cDNA sequence of the MtMYB4 gene from the *Medicago truncatula* genome data (http: / / www.medicagohapmap.org / ), homology comparison was performed. Primers for amplifying the open reading frame (OPG) of the *Medicago truncatula* MfMYB4 cDNA (synthesized by Beijing Qingke) were designed accordingly. The upstream primer for amplifying MfMYB4, MfMYB4-F, has the sequence: 5'-ATGGGAAGATCACCTTGTT-3'; the downstream primer for amplifying MfMYB4, MfMYB4-R, has the sequence: 5'-TCATTTCATTTCTAAGCCT-3'.

[0071] III. Amplification of the MfMYB4 gene and construction of the vector

[0072] PCR amplification was performed using the template prepared in step one and the primers designed in step two:

[0073] PCR reaction system (50 μL): KOD-Plus-DNA polymerase (TOYOBO, 1 U / μL) -1 )1μL, 10×buffer 5μL, dNTPs (10mmol·L -1 5 μL, MgSO4 (25 mmol·L) -12 μL of upstream primer (10 μmol·L⁻¹) -1 1.5 μL of downstream primer (10 μmol·L⁻¹) -1 1.5 μL, reverse transcribe first-strand cDNA 2 μL, and add ddH2O to make up to 50 μL;

[0074] PCR reaction program: 94℃ for 3 min; 94℃ for 0.5 min, 55℃ for 0.5 min, 72℃ for 0.5 min, 35 cycles; 72℃ for 10 min; amplification products were detected by 1% agarose gel electrophoresis. Figure 2 );

[0075] A sequence of approximately 900 bp was obtained. Figure 2 The obtained PCR products were recovered using a DNA gel recovery kit (Nanjing Novizan Co., Ltd.);

[0076] PCR-recovered fragments and The Cloning Kit (TaKaRa) is connected in a molar ratio of 3:1, and the reaction system is as follows: 1 μL of Cloning Vector (0.03 pmol) and 0.1 pmol to 0.3 pmol of the recovered target fragment were mixed gently and ligated at 37 °C for 5 min to obtain the ligation product.

[0077] Escherichia coli competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd. and stored at -80℃ for later use.

[0078] Heat shock transformation of E. coli with ligation product: The ligation product was added to 100 μL of DH5α competent cells and placed on ice for 5 min; after heat shock at 42℃ for 45 s, it was placed on ice for another 2 min; the heat-shocked cells were transferred to 1 mL of LB broth and cultured on a shaker at 37℃ with shaking at 200 rpm for 1 h. 100 μL of the bacterial culture was spread onto LB solid medium (containing 100 μg / mL Kana) and incubated upside down overnight at 37℃.

[0079] Screening, purification, and sequencing of recombinant plasmid pEASY-MfMYB4: PCR-positive colonies were picked and inoculated into 2 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate. The cultures were incubated overnight at 37°C with shaking at 200 rpm. 2 mL of the bacterial culture was then used to extract the plasmid using a plasmid DNA purification kit (Qiagen). The purified plasmid was stored at 4°C for later use. The purified recombinant plasmid was named pEASY-MfMYB4 and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO. 3.

[0080] Sequencing results were compared with the library sequence, confirming that the amplified PCR product was the MfMYB4 gene. The gene encoding the stress-induced nuclear transcription factor MfMYB4 consists of 870 bases, of which the protein-coding region (Sequence coding for amino acids in protein, CDS) of MfMYB4 consists of 870 bases from position 30 to 899, encoding 289 amino acids.

[0081] Example 2: Construction of the recombinant expression vector pCAMBIA3301-MfMYB4

[0082] Based on the MfMYB4 open reading frame sequence obtained in Example 1, amplification primers with NcoⅠ and BstEⅡ restriction sites were designed:

[0083] The upstream primer MfMYB4-3301-F, which introduces the Nco I restriction site, is as follows:

[0084] 5'-CACGGGGGACTCTTGA CCATGG ACATGGGAAGATCACCTTGTT-3';

[0085] Introducing the downstream primer MfMYB4-3301-R with the BstE II restriction site:

[0086] 5'-CGGGGAAATTCGAGCT GGTCACC TCATTTCATTTCTAAGCCT-3';

[0087] Using the cloning vector pEASY-MfMYB4 constructed in Example 1 as a template, and MfMYB4-3301-F and MfMYB4-3301-R as upstream and downstream primers, the MfMYB4 gene was amplified by PCR, and the amplified MfMYB4 gene fragment was purified and recovered.

[0088] The obtained MfMYB4 gene fragment and pCAMBIA3301 expression vector were digested with restriction endonucleases NcoⅠ (NEB) and BstEⅡ (NEB), respectively. The target MfMYB4 gene fragment and the digested vector fragment were recovered. After ligation, the MfMYB4 gene was inserted between NcoⅠ and BstEⅡ at the multiple cloning site downstream of the 35S promoter of the expression plasmid pCAMBIA3301 to construct the recombinant expression vector, which was named pCAMBIA3301-MfMYB4. Figure 1 This is a schematic diagram of the expression cassette of the recombinant expression vector pCAMBIA3301-MfMYB4.

[0089] PCR testing confirmed the acquisition of recombinants. Figure 3 Further sequencing of the insert fragment showed that its sequence was completely identical to the coding region of MfMYB4, and the restriction enzyme sites at both ends of the insert fragment were also correct, thus proving the successful construction of the recombinant expression vector pCAMBIA3301-MfMYB4. A schematic diagram of the expression cassette of the recombinant expression vector pCAMBIA3301-MfMYB4 is shown below. Figure 1 .

[0090] Example 3: Stress-induced expression of MfMYB4

[0091] I. Obtaining alfalfa templates under adverse conditions

[0092] Take a small amount of alfalfa seeds and place them in a 1.5 mL sterile centrifuge tube. Add 1 mL of 70% ethanol and treat for 30 seconds, then wash three times with sterile water. Add 10% sodium hypochlorite solution, invert for 10 minutes, and wash 3-5 times with sterile water. Then place the seeds on 1 / 2 MS medium. After germination and rooting, transplant them into a mixed culture medium (vermiculite: nutrient soil = 1:1, w / w) and cultivate them in a greenhouse at a temperature of 22℃ / 16℃ (day / night), a photoperiod of 16h / 8h (light / dark), a humidity of 60%, and a light intensity of 800 μmol·m⁻¹. -2 ·s -1 Apply an appropriate amount of compound fertilizer (N:P:K = 15:15:15) solution at the appropriate time, and use it for experimental treatment after the material grows uniformly;

[0093] Dehydration

[0094] Dehydration treatment of alfalfa leaves: Alfalfa leaves were taken and placed in a clean bench (Shanghai Jinping Instrument Co., Ltd., model: SW-CJ-2). The transformer was set to 75V and the room temperature was 28℃. The samples were subjected to simulated drought treatment by blowing air (Yang J, Guo Z. Cloning of a 9-cis-epoxycarotenoid dioxygenase gene (SgNCED1) from Stylosanthes guianensis and its expression in response to abiotic stresses[J]. Plant cell reports,2007,26(8):1383-1390). Samples were taken at 0h, 1h, 2h, 6h and 12h. The leaves were cut off during sampling, wrapped in tin foil and quickly frozen in liquid nitrogen. They were then stored in a -80℃ freezer for later use.

[0095] The samples stored at -80℃ were taken out and placed in liquid nitrogen. Liquid nitrogen was added to grind the samples. Total RNA was extracted from the leaves using the RNA Prep Pure Plant kit (Tiangen Biotech). The cDNA template was prepared by reverse transcription using the EasyScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech) reverse transcriptase kit and stored at -20℃ for later use.

[0096] II. Design of Specific Detection Primers

[0097] Based on the MfMYB4 gene cDNA sequence and the alfalfa β-actin sequence, quantitative primers for detection were designed using Beacon Designer 7.0 software:

[0098] The upstream quantitative primer for the MfMYB4 gene is qMfMYB4-F: 5'-CGTCTCCGGTGGATTAACTATC-3';

[0099] The downstream quantitative primer for the MfMYB4 gene is qMfMYB4-R: 5'-CTTCCAGCTATCAAAGACCATTTG-3';

[0100] The upstream quantitative primer for the β-actin gene is qActin-F: 5'-CCCACTGGATGTCTGTAGGTT-3';

[0101] The downstream quantitative primer for the β-actin gene is qActin-R: 5'-AGAATTAAGTAGCAGCGCAAA-3';

[0102] III. Quantitative PCR detection of expression differences

[0103] The template cDNA prepared in step one was diluted 50-fold and used as the template for quantitative PCR. The reaction system was 10 μL: 5 μL SYBR Green Supermix (2×), 0.5 μL each of upstream and downstream primers (10 μM), and 4 μL cDNA template. The instrument used was a TaKaRa Thermal Cycler Dice Real Time System II. The PCR reaction conditions were set as follows: 95℃ for 10 s; 94℃ for 5 s, 59℃ for 25 s, for 40 cycles. No cDNA template was used as a negative control. Each sample was set up in 3 replicates, with the housekeeping gene β-actin as the internal reference gene. Melting curve was plotted: 95℃ denaturation for 15 s, 60℃ annealing for 1 min, and 95℃ denaturation for 15 s. After the reaction, the melting curve was analyzed. The PCR amplification efficiency was above 95%.-ΔΔCT The method calculates the relative expression levels of genes;

[0104] Real-time quantitative PCR results showed that MfMYB4 expression was induced starting 1 h after drought treatment, peaked at 2 h, and then decreased. Figure 4 The results showed that drought can induce the expression of the MfMYB4 gene.

[0105] Example 4: Production and Molecular Detection of Transgenic Alfalfa

[0106] I. The Development of Genetically Modified Alfalfa

[0107] 1. The recombinant expression vector pCAMBIA3301-MfMYB4 was introduced into Agrobacterium tumefaciens EHA105.

[0108] The preparation of EHA105 competent cells was based on the method of J. Sambrook (Huang Peitang, Wang Jiaxi, Zhu Houchu. J. Sambrook, D.W. Russell, author [J]. Molecular Cloning: A Laboratory Manual, 2002: 27-30) with modifications: EHA105 bacterial culture was streaked on YEP plates (containing 25 mg / L rifampin) and incubated at 28°C for 48 h. Single colonies were picked and inoculated into 50 mL of liquid LB and incubated overnight at 28°C. 0.5 mL of the bacterial culture was then inoculated into 500 mL of liquid YEP and incubated at 28°C for 8 h until OD reached the target value. 600The solution was set to 0.6, cooled in an ice bath for 10 min, poured into a sterile 200 mL centrifuge tube, and centrifuged at 4000 rpm for 10 min at 4°C after equilibration. The cells were collected, the LB solution was discarded, and the tube was placed upside down on a sterile paper towel to drain. 50 mL of pre-chilled 10% glycerol (glycerol:YEP liquid medium) was added, and the cells were shaken on ice to suspend them. The cells were centrifuged at 4000 rpm for 15 min at 4°C to collect them. The cells were washed once more, and 2 mL of pre-chilled 10% glycerol (glycerol:YEP liquid medium) was added to suspend them. The cells were aliquoted into 25 μL / tube, flash-frozen in liquid nitrogen, and stored at -80°C. Thaw competent cells on ice. Pre-cool 0.2 cm inner diameter electroporation cuvettes on ice. In a clean bench, add 1.5 μL pCAMBIA3301-MfMYB4 plasmid (20 ng / μL) to 20 μL of thawed competent cells, gently tap the tube wall to mix, incubate on ice for 1 min, then transfer to an electroporation cuvette and place it between the electrodes of an electroporator (MicroPulser, Bio-RAD). Select the Agr program and perform electroporation. After electroporation, quickly pour 1 mL of YEP liquid culture medium into the electroporation cuvette in the clean bench, then transfer it to a shaker tube using a pipette. Incubate at 28°C with gentle shaking for 2 h. Take 0.3 mL of the bacterial culture and spread it on a YEP plate (containing 25 mg / L rifampin and 50 mg / L kanamycin sulfate). Incubate upside down in a 28°C incubator for 48 h.

[0109] 2. Identification of Agrobacterium-positive colonies containing the recombinant expression vector pCAMBIA3301-MfMYB4

[0110] Single colonies were picked from the plate for colony PCR detection and labeled. PCR-positive colonies were then picked and added to 3 mL of YEB liquid medium (containing 25 mg / L rifampin and 50 mg / L kanamycin sulfate) and cultured at 28°C with shaking for 40 h. 2 mL of bacterial culture was used to extract plasmids using the FastPure Plasmid Mini Kit (Novizan). Restriction endonucleases NcoⅠ (NEB) and BstEⅡ (NEB) were used for digestion to confirm the presence of the plant expression vector pCAMBIA3301-MfMYB4 in the positive clones. 0.8 mL of Agrobacterium culture was added to 0.2 mL of 80% glycerol (glycerol: YEP liquid medium), mixed well, and stored at -80°C for later use.

[0111] 3. Obtaining transgenic alfalfa plants

[0112] (1) Culture medium preparation

[0113] Mother liquor of macroelements (10×): Weigh 28.30g KNO3, 4.63g (NH4)2SO4, 4.00g KH2PO4, 1.85g MgSO4•7H2O, and 1.66g CaCl2•2H2O and dissolve them in 1L of deionized water;

[0114] Trace element stock solution (1000×): Weigh 10g MnSO4•H2O, 5g H3BO3, 1g KI, 1g ZnSO4•7H2O, 0.2g CuSO4·5H2O, 0.1g CoCl2·6H2O, and 0.1g Na2MoO4•2H2O and dissolve them in 1L of deionized water;

[0115] SH medium organic stock solution (1000×): Weigh 5g thiamine hydrochloride, 5g pyridoxine hydrochloride, and 5g nicotinic acid and dissolve them in 1L deionized water;

[0116] SH medium EDFS stock solution (50×): Weigh 6.97g Na2FeEDTA and dissolve it in 1L deionized water;

[0117] SH3α medium: Add 100 mL of macro-element stock solution, 20 mL of EDFS stock solution, 1 mL of micro-element stock solution, 1 mL of organic matter stock solution, 4 mL of 2,4-D stock solution (1 mg / mL), 0.5 mL of 6-BA stock solution (1 mg / mL), 30 g of sucrose, 0.1 g of inositol, and deionized water to a final volume of 1 L. Adjust the pH to 5.85. Add 8 g of agar powder to the solid medium and autoclave at 121 °C for 20 min.

[0118] SH9 medium: Add 100mL macro-element stock solution, 20mL EDFS stock solution, 1mL micro-element stock solution, 1mL organic matter stock solution, 20g sucrose, 0.1g inositol, and deionized water to a final volume of 1L. Adjust the pH to 5.85, add 8g agar powder, and autoclave at 121℃ for 20min.

[0119] Plant rooting medium: Weigh 2.2g MS medium powder and 15g sucrose, dissolve them, add 0.5mL IBA stock solution (1mg / mL), bring the volume to 1L with deionized water, adjust the pH to 5.85, add 8g agar powder, and autoclave at 121℃ for 20min.

[0120] 1 / 2MS medium: Weigh 2.2g MS medium powder, 15g sucrose, and deionized water to a final volume of 1L. Adjust the pH to 5.85, add 8g agar powder, and autoclave at 121℃ for 20min.

[0121] (2) Cultivation of Alfalfa 'R108'

[0122] Select plump seeds of Alfalfa 'R108', gently grind notches on sandpaper, then place a layer of qualitative filter paper in a large petri dish, moisten with distilled water, and vernalize at 4℃ for 2-3 days. Place in the dark at 24-28℃ overnight. Once the roots have grown to about 2cm, transfer to a substrate of nutrient soil (nutrient soil: vermiculite = 1:1) for cultivation. Growth conditions: 16h light / 8h darkness, 60% humidity, daytime temperature 22℃, nighttime temperature 16℃. After 4-6 weeks of growth, it can be used for infection.

[0123] (3) Activation and suspension of Agrobacterium

[0124] Take Agrobacterium EHA105 stock solution containing the recombinant expression vector pCAMBIA3301-MfMYB4, streak it on YEB plates (containing 25 mg / L rifampin and 50 mg / L kanamycin sulfate), and incubate at 28°C; pick well-isolated single colonies and inoculate them into 2 mL of liquid YEB (containing 25 mg / L rifampin and 50 mg / L kanamycin sulfate), and incubate in the dark with shaking at 28°C for 24 h; one day before transformation, add the overnight bacterial culture containing positive clones to 5-7 mL of YEB containing the same antibiotics at a ratio of 1:50 and shake overnight;

[0125] On the day of transformation, the overnight bacterial culture containing positive clones was added to 50 ml of YEB containing the same antibiotic at a ratio of 1:50, and cultured at 28°C with shaking until the OD600 was 0.6-0.8; centrifuged at 22°C, 3000 rpm for 13 min, and the precipitate was resuspended in 100 ml of SH3α (containing 100 μM acetylsyl syringone) to prepare the infection solution.

[0126] (4) Infection, co-culture and the production of transgenic seedlings

[0127] Cut the leaves with sterilized scissors, rinse the surface with tap water to remove impurities, disinfect the surface with 6.25% NaClO for 30 minutes, rinse 2-3 times with sterile distilled water in a clean bench to remove NaClO, cut off the petioles and leaf margins, and cut the leaves into 5mm × 5mm pieces for transformation experiments. Thoroughly mix the pre-cut leaves with Agrobacterium SH3α infection solution (containing 100 μM acetylsyl syringone, ensuring the Agrobacterium infection solution completely submerges the callus tissue), apply vacuum at -0.1 pka (>0.09 pka) for 30 minutes; then incubate on a shaker at 80 rpm and 28℃ for 1.5 hours. Inside the clean bench, pour out the infection solution, place the leaves on a small petri dish with multiple layers of sterile filter paper to dry for about 10 minutes and remove as much Agrobacterium as possible from the surface of the slices. Then, place the leaves face up onto a solid co-culture medium SH3α (containing 100 μM acetylsyl syringone) with a fresh sterile filter paper, seal and incubate in the dark at 25°C for two days. After that, inoculate them into the selection medium SH3α (containing 2 mg / L glufosinate, 200 mg / L cephalosporin and 200 mg / L termethin), seal and incubate in the dark at 25°C for 2 weeks.

[0128] Differentiation of resistant callus: Select resistant callus with good growth status and inoculate it into differentiation medium SH9 medium (containing 2 mg / L glufosinate, 200 mg / L cephalosporin and 200 mg / L termethin) and culture it at 25℃ under alternating light and dark conditions (16h / 8h). Rotate the plate every 3 weeks. Callus tissue with green spots continues to differentiate into resistant shoots.

[0129] (5) Growth of transgenic alfalfa

[0130] When the differentiated seedlings reach 3-5 cm in length, transfer them from the differentiation medium to 100 mL of 1 / 2 MS medium in a tissue culture flask. Culture them at 25°C with alternating light and dark cycles (16h / 8h) for 4 weeks until new roots develop. If the seedlings grow well and the new roots develop quickly, they can be transplanted earlier to harden them off. When new roots have grown from the seedlings, remove them from the medium, rinse the roots thoroughly, and transplant them individually into vermiculite. After the seedlings have established themselves, transplant them into a 2:1 (V / V) vermiculite:nutrient soil substrate and place them in an incubator for growth. Growth conditions: 16h light / 8h darkness, 60% humidity, daytime temperature 22°C, nighttime temperature 16°C. Finally, cultivate them under natural conditions outdoors in a greenhouse.

[0131] PCR detection of transgenic alfalfa

[0132] 1. Extraction of plant genomic DNA using the CTAB method

[0133] Take one compound leaf into a 2 mL centrifuge tube, freeze it rapidly in liquid nitrogen, grind the material into powder using a tissue homogenizer, add 500 μL of CTAB extraction buffer preheated at 65 °C, invert and mix well, incubate at 65 °C for 30 min, invert and mix every 5-10 min, add an equal volume of chloroform / isoamyl alcohol (24 / 1, V / V), invert and mix well, centrifuge at 12,000 rpm for 15 min, transfer the supernatant to a 1.5 mL centrifuge tube; add 350 μL of isopropanol, invert and mix well, incubate at -20 °C for 30 min; centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash with 1 mL of 75% ethanol; centrifuge at 12,000 rpm for 5 min, discard the supernatant, and air dry in a clean bench; add 100 μL of sterile water to dissolve the DNA.

[0134] 2. PCR detection of transgenic alfalfa

[0135] Since the vector pCAMBIA3301, which infects plants, also carries the bialaphos (Bar) resistance gene in its left and right boundary regions, primers Bar-F (5'-ATGAGCCCAGAACGACGC-3') and Bar-R (5'-TCAAATCTCGGTGACGGG-3'), which specifically recognize this gene, were used to detect whether the target gene was successfully transferred into the plant genome. The DNA obtained from the micro-extraction in the previous step was used as a template for PCR amplification, with the pCAMBIA3301-MfMYB4 recombinant plasmid as a positive control. The reaction system (20 μL) consisted of: Taq DNA polymerase 0.1 μL, PCR 10× buffer 2 μL, dNTPs (10 mM) 1.6 μL, Bar-F (10 μM) 0.5 μL, Bar-R (10 μM) 0.5 μL, DNA 1 μL (pCAMBIA3301-MfMYB4 plasmid 20 ng), and ddH2O 14 μL. The PCR reaction program was: 94℃ for 2 min; 94℃ for 0.5 min, 57℃ for 0.5 min, 72℃ for 30 s, 35 cycles; 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis (e.g., […]). Figure 5 (As shown).

[0136] Example 4: Identification of drought resistance in transgenic alfalfa

[0137] I. Drought Resistance Test

[0138] The determination method is as follows:

[0139] Ten plants each of the transgenic alfalfa and the wild-type alfalfa 'R108' were planted in the same pot. See the example planting diagram below. Figure 8After 30 days of normal growth in a greenhouse, the plants were subjected to a 25-day drought. The second compound leaf of the plant was then taken to measure the relative conductivity (Ionleakage), and the plants were rehydrated. The survival rate was calculated 10 days after rehydration.

[0140] The results are as follows Figure 7 As shown in A and B, the relative electrical conductivity of leaves of the overexpression lines was significantly lower than that of the wild type after drought stress. Meanwhile, the survival rates of the overexpression lines OE-2, OE-3, and OE-4 after rehydration were 50.0±5.8%, 56.7±6.7%, and 70±5.8%, respectively, all significantly higher than the survival rate of the wild type (23.3±6.7%). This indicates that under drought stress, the overexpression of MfMYB4 transgenic alfalfa significantly improved drought resistance compared to the wild type.

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0142] sequence list

[0143] SEQ ID NO.1

[0144] MGRSPCCEKAHTNKGAWTKEEDDRLISYIRSHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFTEEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHIRRKLLNRGIDPATHRPLNEVSHSHSHSQSQSQSQ TLHLQNQEAVTIAVAASTSAPTATKTLPTTISFASSIKQEQYHHHHQEMNTNMVKGLVLERCPDLNLELTISPPRVQEHDEQFRNRERNNLCFVCSLGLQNSKDCTCDEIVGNSSSGNGSTAPAYDFLGLKGGVWDYKGLEMKSEQ IDNO.2

[0145] ATGGGAAGATCACCTTGTTGTGAAAAAGCTCATACAAACAAAGGAGCATGGACAAAAGAAGAAGATGATAGACTTATATCATATATTAGGTCACATGGTGAAGGTTGTTGGAGATCTCTCCCTAAAGCAGCTGGCTTACTCCGATGTGGTAAAAGTTGCCGTCTCCGGTGGATTAACTATCTCAGGCCAGACCTTAAACGGTGGTAACTTCACAGAAGAAGAAGATGAACTCATCATCAAACTCCATAGTCTTCTTGGTAACAAATGGTCTTTGATAGCTGGAAGATTACCTGGAAGAACAGATAATGAGATAAAGAATTATTGGAACACTCATATAAGAAGAAAGCTTTTGAATAGAGGAATTGACCCTGCTACTCATAGGCCTTTAAACGAAGTTTCTCATTCTCATTCTCAATCTCAATCACAATCT CAAACTCTTCATCTTCAAAATCAAGAAGCTGTTACTATAGCTGTAGCAGCATCTACATCAGCTCCCACTGCTACAAAAACCCTACCAACAACTATATCTTTTGCATCATCCATCAAACAAGAACAATATCATCATCATCATCAAGAAATGAACACAAACATGGTTAAAGGGTTGGTTTTTTAGAACGTTGTCCTGATTTGAATCTTGAGCTAACAATTAGTCCACCACGTGTTCAAGAACATGATGAACAATTCAGAAACAGAGAGAGGAACAATCTCTGTTTTGTTTGTAGTTTGGGTTTGCAGAATAGTAAGGATTGTACCTGTGATGAAATTGTTGGAAATTCTAGCAGTGGAAATGGTTCTACTGCACCTGCTTATGATTTCTTGGGTTTGAAAGGTGTGTTTGGGATTACAAAGGCTTAGAAATGAAATGA

[0146] SEQ ID NO.3

[0147] GTAACGGCCGCCAGTCTGGAATTGCCCTT ATGGGAAGATCACCTTGTTGTGAAAAAGCTCATACAAACA AAGGAGCATGGACAAAAGAAGAAGATGATAGACTTATATCATATATTAGGTCACATGGTGAAGGTTGTTGGAGATCT CTCCCTAAAGCAGCTGGCTTACTCCGATGTGGTAAAAGTTGCCGTCTCCGGTGGATTAACTATCTCAGGCCAGACCTTAAACGTGGTAACTTCACAGAAGAAGAAGATGAACTCATCATCAAACTCCATAGTCTTCTTGGTAACAAATGGTCTT TGATAGCTGGAAGATTACCTGGAAGAACAGATAATGAGATAAAGAATTATTGGAACACTCATATAAGAAGAAAGCTT TTGAATAGAGGAATTGACCCTGCTACTCATAGGCCTTTAAACGAAGTTTCTCATTCTCATTCTCATTCTCAATCTCA ATCACAATCTCAAACTCTTCATCTTCAAAATCAAGAAGCTGTTACTATAGCTGTAGCAGCATCTACATCAGCTCCCA CTGCTACAAAAACCCTACCAACAACTATATCTTTTGCATCATCCATCAAACAAGAACAATATCATCATCATCATCAA GAAATGAACACAAACATGGTTAAAGGGTTGGTTTTAGAACGTTGTCCTGATTTGAATCTTGAGCTAACAATTAGTCC ACCACGTGTTCAAGAACATGATGAACAATTCAGAAACAGAGAGAGGAACAATCTCTGTTTTGTTTGTAGTTTGGGTT TGCAGAATAGTAAGGATTGTACCTGTGATGAAATTGTTGGAAATTCTAGCAGTGGAAATGGTTCTACTGCACCTGCT TATGATTTCTTGGGTTTGAAAGGTGGTGTTTGGGATTACAAAGGCTTAGAAATGAAATGA AAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCAT

[0148] SEQ ID NO.4 (MfMYB4 amplification upstream primer MfMYB4-F):

[0149] 5'-ATGGGAAGATCACCTTGTT-3';

[0150] SEQ ID NO.5 (MfMYB4 amplification downstream primer MfMYB4-R):

[0151] 5'-TCATTTCATTTCTAAGCCT-3';

[0152] SEQ ID NO.6 (Upstream primer MfMYB4-3301-F introducing the Nco I restriction site):

[0153] 5'-CACGGGGGACTCTTGA CCATGG ACATGGGAAGATCACCTTGTT-3';

[0154] SEQ ID NO.7 (Downstream primer MfMYB4-3301-R with BstEIⅠ restriction site):

[0155] 5'-CGGGGAAATTCGAGCT GGTCACC TCATTTCATTTCTAAGCCT-3'.

Claims

1. An alfalfa R2-R3 type transcription factor MfMYB4, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. Encoding the alfalfa R2-R3 type transcription factor MfMYB4 as described in claim 1 MfMYB4 Genes, the MfMYB4 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. Containing the contents of claim 2 MfMYB4 Gene-based biomaterials, characterized in that, The biomaterial is an expression cassette, a recombinant vector, or a recombinant microorganism.

4. The biomaterial according to claim 3, characterized in that, The recombinant vector is a recombinant expression vector, which is generated by using the method described in claim 2. MfMYB4 It is obtained by linking genes with plant expression vectors.

5. The biomaterial according to claim 4, characterized in that, The plant expression vector is pCAMBIA3301.

6. The biomaterial according to claim 5, characterized in that, The method for constructing the recombinant expression vector specifically includes the following steps: (1) Primer design Primer ① is MfMYB4 Amplification of upstream primer MfMYB4-F: 5'-ATGGGAAGATCACCTTGTT-3'; Primer ② is MfMYB4 Amplification of downstream primer MfMYB4-R: 5'-TCATTTCATTTCTAAGCCT-3'; Primer ③ is for introduction Nco The upstream primer for the I restriction site is MfMYB4-3301-F: 5'-CACGGGGGACTCTTGACCATGGACATGGGAAGATCACCTTGTT-3'; Primer ④ is for introduction Bst The downstream primer for the EIⅠ restriction site is MfMYB4-3301-R: 5'-CGGGGAAATTCGAGCTGGTCACCTCATTTCATTTCTAAGCCT-3'; (2) MfMYB4 Obtaining gene fragments: Using alfalfa cDNA as a template, primers ① and ② were used for amplification. MfMYB4 Gene fragments were ligated into the pEASY®-Blunt Cloning Kit vector to construct pEASY- MfMYB4 carrier; (3) Contains MfMYB4 The recombinant gene expression vector pCAMBIA3301- MfMYB4 Construction: pEASY- MfMYB4 Using the vector as a template, primers ③ and ④ are used to... MfMYB4 Gene fragment introduction Nco I and BstEI I. Two restriction enzyme sites were ligated to the plant expression vector pCAMBIA3301 to construct a sample containing... MfMYB4 The recombinant gene expression vector pCAMBIA3301- MfMYB4 .

7. The alfalfa R2-R3 type transcription factor MfMYB4 as described in claim 1, and the [missing information] as described in claim 2. MfMYB4 The use of genes, or the biological materials of claim 3, in improving the drought resistance of alfalfa or in breeding transgenic alfalfa with improved drought resistance.

8. The application according to claim 7, characterized in that: Overexpression of the substance described in claim 2 in target alfalfa MfMYB4 Genes, or increasing the expression level of the transcription factor MfMYB4 protein as described in claim 1, can improve the drought resistance of alfalfa.

9. A method for improving the drought resistance of alfalfa, characterized in that, Overexpression of the substance described in claim 2 in target alfalfa MfMYB4 Genes, or increasing the expression level of the transcription factor MfMYB4 protein as described in claim 1, can improve the drought resistance of alfalfa.

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