Alfalfa myb transcription factor, coding gene and application thereof
By cloning and expressing the alfalfa MYB transcription factor gene MsMYB206, the lack of research on flavonoid metabolism regulation and leaf development in alfalfa was addressed, resulting in increased total flavonoid content and the formation of palmate compound leaves, thereby improving plant yield and nutritional value.
Patent Information
- Application Number
- CN202310389793.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-12
- Publication Date
- 2025-12-12
- Estimated Expiration
- 2043-04-12
AI Technical Summary
There is insufficient research on the role of MYB transcription factors in the regulation of flavonoid metabolism and leaf development in alfalfa, which makes it difficult to study its leaf development.
The alfalfa MYB transcription factor gene MsMYB206 was cloned and expressed. Gene editing was performed in alfalfa using plant overexpression vectors and RNA interference vectors to increase total flavonoid content and promote the formation of palmate compound leaves.
It increased the total flavonoid content and leaf nutritional value of alfalfa, enhanced its stress response under adverse conditions, promoted the formation of palmate compound leaves, improved plant quality, and increased yield.
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Figure CN116286872B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a Medicago sativa MYB transcription factor, a coding gene and application thereof. BACKGROUND
[0002] Medicago sativa L. of Leguminosae Medicago genus, also known as alfalfa, is a perennial herb, which is known as the "king of forage" for its six advantages: first, high yield; second, good quality; third, an important energy feed; fourth, Medicago sativa also contains some substances very beneficial to livestock, such as flavonoids; fifth, high ecological benefit; sixth, Medicago sativa can be processed deeply, and leaf protein can be extracted therefrom to be used as a food additive. High leaf stem ratio is an important agronomic trait of cultivated alfalfa, because it is positively correlated with the nutritional value of alfalfa products. Cultivating varieties with more small leaves per leaf can increase the leaf stem ratio of cultivated alfalfa, thereby increasing its yield and nutritional value. Therefore, the study on the leaf of Medicago sativa can provide theoretical and technical guidance for the improvement and breeding of high-quality strains. However, due to the fact that Medicago sativa is a homologous tetraploid with cross-pollination and high heterozygosity, there are many difficulties in the study of leaf development thereof.
[0003] MYB transcription factors are the most numerous, most complex and most diversified proteins in higher plants, which play an important role in plant secondary metabolism (such as flavonoid biosynthesis), stress response, growth and development, disease resistance and the like. MYB transcription factors have been reported in many species such as Arabidopsis thaliana, soybean, corn and rice. However, in Medicago sativa, the research on MYB transcription factor genes is very limited, and there is no report on the participation of MYB transcription factors in flavonoid metabolism regulation and leaf development.
[0004] Therefore, the skilled in the art is committed to developing a MYB transcription factor capable of participating in flavonoid metabolism regulation or palmate compound leaf formation of Medicago sativa and its application in flavonoid metabolism regulation and leaf development of Medicago sativa. SUMMARY
[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present application is to develop a MYB transcription factor capable of participating in flavonoid metabolism regulation or palmate compound leaf formation of Medicago sativa and its application in flavonoid metabolism regulation and leaf development of Medicago sativa.
[0006] To achieve the above-mentioned purpose, the present application provides a MYB transcription factor gene for increasing the total flavonoid content or palmate compound leaf formation of Medicago sativa, and the nucleotide sequence is shown in SEQ ID NO. 1.
[0007] Further, the MYB transcription factor gene for increasing the content of total flavonoids or promoting the formation of palmate compound leaves of alfalfa provided by the application is derived from alfalfa variety "WL525", and is named as MsMYB206.
[0008] The application further provides a plant expression vector of the MYB transcription factor gene for increasing the content of total flavonoids or promoting the formation of palmate compound leaves.
[0009] Further, the plant expression vector includes a plant overexpression vector and an RNA interference expression vector.
[0010] Further, the plant overexpression vector is constructed by homologous recombination technology, and the RNA interference expression vector is constructed by gateway technology.
[0011] Further, the coding gene of MsMYB206 provided by the application is introduced into plant cells by using any kind of vector that can guide the expression of foreign genes in plants, so that transgenic plants with high content of total flavonoids and palmate compound leaves are obtained.
[0012] Further, when the plant overexpression vector is constructed by using the gene of the application, any kind of enhancer or inducible promoter can be added before the transcription initiation nucleotide.
[0013] Further, the expression vector carrying MsMYB206 of the application can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the like, and the transformed plant tissues are cultivated into plants.
[0014] Further, the transformed host can be a monocotyledonous plant or a dicotyledonous plant.
[0015] The application further provides an application of the MYB transcription factor gene for increasing the content of total flavonoids or promoting the formation of palmate compound leaves in the flavonoid metabolism regulation or / and leaf development of alfalfa.
[0016] Further, MsMYB206 of alfalfa is up-regulated under high-salt abiotic stress and hormone treatment.
[0017] The application further provides an application of the plant expression vector of the MYB transcription factor gene for increasing the content of total flavonoids or promoting the formation of palmate compound leaves in the flavonoid metabolism regulation or / and leaf development of alfalfa.
[0018] Further, the plant expression vector is transformed into plant cells or tissues by using conventional biological methods, and the transformed plant tissues are cultivated into plants.
[0019] Further, the application of the plant overexpression vector in increasing the total flavonoid content in alfalfa.
[0020] Further, the application of the RNA interference expression vector in promoting the formation of palmate compound leaves in alfalfa.
[0021] Further, the application comprises increasing the total flavonoid content in transgenic alfalfa.
[0022] Further, the flavonoid metabolism regulation is for increasing the total flavonoid content in transgenic alfalfa.
[0023] Further, the leaf development comprises promoting the formation of palmate compound leaves.
[0024] In a preferred embodiment 1 of the application, the cloning and sequence analysis process of the MsMYB206 gene are described in detail;
[0025] In another preferred embodiment 2 of the application, the tissue expression pattern of the MsMYB206 gene is described in detail;
[0026] In another preferred embodiment 3 of the application, the tissue expression pattern of the MsMYB206 gene under stress and hormone induction is described in detail;
[0027] In another preferred embodiment 4 of the application, the construction of the expression vector and the process of obtaining the transgenic alfalfa plant are described in detail;
[0028] In another preferred embodiment 5 of the application, the process of subcellular localization analysis of the MsMYB206 in tobacco leaves is described in detail;
[0029] In another preferred embodiment 6 of the application, the process of identifying the MsMYB206 overexpression alfalfa and determining the total flavonoid content is described in detail;
[0030] In another preferred embodiment 7 of the application, the process of identifying the MsMYB206 interference alfalfa and the leaf phenotype is described in detail.
[0031] The application has the following beneficial technical effects:
[0032] The application clones a MsMYB206 transcription factor gene involved in the flavonoid metabolism regulation or the formation of palmate compound leaves in alfalfa, studies the expression pattern of the gene in alfalfa, compares the total flavonoid content and leaf morphology of wild type and MsMYB206 gene transgenic alfalfa, and provides a basis for effective application, which has important significance for improving plant quality and increasing alfalfa yield.
[0033] The concept, specific structure and generated technical effects of the present application will be further described below with reference to the drawings, so as to fully understand the purposes, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 is a MsMYB206 tissue expression pattern chart of a preferred embodiment 2 of the present application;
[0035] Figure 2 is a MsMYB206 expression analysis result chart under stress and hormone induction of a preferred embodiment 3 of the present application;
[0036] Figure 3 is a MsMYB206 subcellular localization chart in tobacco epidermal cells of a preferred embodiment 5 of the present application;
[0037] Figure 4 is a fluorescence quantitative PCR identification result chart of a MsMYB206 overexpression alfalfa plant of a preferred embodiment 6 of the present application;
[0038] Figure 5 is a total flavonoid content result chart of a MsMYB206 overexpression alfalfa plant of a preferred embodiment 6 of the present application;
[0039] Figure 6 is a fluorescence quantitative PCR identification result chart of a MsMYB206 interference expression alfalfa plant of a preferred embodiment 7 of the present application;
[0040] Figure 7 is a leaf phenotype chart of a MsMYB206 interference expression alfalfa plant of a preferred embodiment 7 of the present application. DETAILED DESCRIPTION
[0041] The following reference to the drawings of the specification introduces a plurality of preferred embodiments of the present application, so that its technical content is more clear and convenient to understand. The present application can be embodied by many different forms of embodiments, and the protection scope of the present application is not limited to the embodiments mentioned in the text.
[0042] Example 1: Cloning and sequence analysis of MsMYB206 gene
[0043] Extraction of alfalfa RNA and synthesis of cDNA: total RNA of alfalfa "WL525" seedlings was extracted by EasyPure Plant RNA Kit (purchased from Beijing Zison), and reverse transcription was performed by TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (purchased from Beijing Zison) to synthesize cDNA.
[0044] Design and synthesis of primers: The MsMYB206 gene was significantly up-regulated in the transcriptome sequencing analysis of Medicago truncatula seedlings under salt (NaCl) stress. The cDNA sequence of this gene was obtained by searching the database of Medicago truncatula, and primers were designed and synthesized, F: 5'-ATGGCGGAGATTCACATACC-3', R: 5'-TTATTGCCAAATGTTTGAA-3'. The above-mentioned cDNA of Medicago truncatula was used as a template, and PCR amplification was performed according to the following reaction system and conditions: 50 μL system, containing LA Taq Mix 25 μL (purchased from Takara), 2 μL of 10 μM primer F and primer R, 2 μL of cDNA, and deionized water to 50 μL. Reaction conditions: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 1 min 30 s, 30 cycles; 72°C for 5 min after extension. The above-mentioned amplified fragments were electrophoresed on 1% agarose gel, cut and recovered, and the recovered products were ligated with the cloning vector pMD18-T (purchased from Takara). After identification, the positive clones were sent to Huada Gene for sequencing, and the recombinant plasmid with correct sequence was named pMD18-T-MsMYB206.
[0045] The sequence containing the full-length ORF of the MsMYB206 gene was obtained by PCR amplification, which encoded a protein composed of 218 amino acid residues, 33 bp (11 amino acid residues) more than the ORF of the gene in Medicago truncatula, with a similarity of 84.55%. MsMYB206 contains a myb_SHAQKYF domain and belongs to MYB-like transcription factors.
[0046] Example 2: Tissue expression pattern of MsMYB206 gene
[0047] Seeds of Medicago sativa 'WL525' were germinated for 7 days and then transferred to modified 1 / 2-Hoagland solution (Ca(NO3)2.4H2O 945 mg / L, KNO3 506 mg / L, NH4NO3 80 mg / L, KH2PO4 136 mg / L, MgSO4.7H2O 493 mg / L, EDTA-Na2 18.65 mg / L, FeSO4.7H2O 13.9 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4.H2O 16.9 mg / L, ZnSO4.7H2O 8.6 mg / L, Na2MoO4.2H2O 0.25 mg / L, CuSO4.5H2O 0.025 mg / L, CoCl2.6H2O 0.025 mg / L, pH 5.7-5.8) and cultured at 28°C under 16 h light / 8 h dark conditions for 14 days. The apical buds, young leaves, mature leaves, old leaves, stipules, petioles, stems, stem nodes, and roots were quickly placed in liquid nitrogen and stored at -80°C for later use. The results of MsMYB206 tissue expression pattern are shown in Figure 1. Figure 1
[0048] Example 3: Expression pattern of MsMYB206 gene under stress and hormone induction
[0049] Treatment of Medicago sativa 'WL525': Seeds of Medicago sativa 'WL525' were germinated for 7 days and then transferred to the above modified 1 / 2-Hoagland solution and cultured at 28°C under 16 h light / 8 h dark conditions for 7 days. The hydroponic seedlings were then treated as follows:
[0050] High salt treatment: Medicago sativa seedlings were placed in 1 / 2-Hoagland solution containing 100 mmol / L NaCl. Samples were taken at 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h and quickly placed in liquid nitrogen and stored at -80°C for later use.
[0051] Hormone treatment: Medicago sativa seedlings were treated by adding abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) to 1 / 2-Hoagland solution to a final concentration of 100 μmol / L. Samples were taken at 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h and quickly placed in liquid nitrogen and stored at -80°C for later use.
[0052] The method for extracting total RNA and synthesizing cDNA is the same as that in Example 1. The real-time fluorescent quantitative PCR primer is designed according to the cDNA sequence of MsMYB206 (F: 5'-ATGCCTAGGCTTCGTTGGAC-3'; R: 5'-ACCAACTTGGGTGTTGCTCT-3'). The constitutively expressed gene MsEF-a of Medicago sativa is used as an internal reference (F: 5'-GCACCAGTGCTCGATTGC-3'; R: 5'-TCGCCTGTCAATCTTGGTAACAA-3'). The cDNA of each sampling point of Medicago sativa under treatment is used as a template to perform real-time RT-PCR analysis by using a Bio-Rad CFX96 real-time quantitative PCR instrument. The reaction system contains 2x RealStar Fast SYBR qPCR Mix (purchased from Beijing Kangrunchengye Biotechnology Co., Ltd.) 10 μL, cDNA 2 μL, Primer F 0.4 μL, Primer R 0.4 μL, and water to a total volume of 20 μL. The reaction program is 95 °C for 2 min; 95 °C for 15 s, 60 °C for 15 s, 72 °C for 30 s, 40 cycles. The data is analyzed by using the 2-ΔΔCT method to determine the relative expression amount of the gene. Each sampling point is set for 3 technical repeats, and the test is set for 3 biological repeats. The MsMYB206 expression analysis under adversity and hormone induction is shown in FIG. 1, wherein parts A, B, C and D of the figure respectively represent the MsMYB206 expression analysis results under salt, ABA, SA and MeJA treatment. It can be seen that under various abiotic stresses and hormone treatments, the expression of MsMYB206 of Medicago sativa is up-regulated to different degrees. Figure 2
[0053] Example 4: Construction of an expression vector and obtaining of a transgenic Medicago sativa plant
[0054] 1) Constructing MsMYB206 plant overexpression vector by homologous recombination (In-fusion) technology, designing primers according to the sequence of the target gene, designing specific primers from the start and stop codons, introducing Bam HI and Spe I restriction sites into the upstream and downstream primers, respectively, i.e. upstream pHB-MYB206 In-F: 5'-CTTGATATCGAATTCCTGCAGATGGCGGAGATTCACATAC-3', downstream pHB-MYB206 In-R: 5'-TTATCGATACCGTCGGATCCTTATTGCCAAATGTTTGAA-3', and performing PCR amplification with pMD18-T-MsMYB206 plasmid as the template. After amplification, the PCR product was recovered, and the recovered product was ligated to the plant binary expression vector pHB-Flag using a homologous recombination enzyme (ClonExpress II One Step Cloning Kit, purchased from Novagen). After 30 min of reaction at 25C, E. coli DH5α competent cells (purchased from Shanghai Uptide Biotechnology) were transformed. Positive clones were selected on solid LB plates supplemented with ampicillin (purchased from Shanghai Yixing Biotechnology) and sent to Huada Gene for sequencing verification. The positive recombinant plasmid with correct sequencing was named pHB-MsMYB206-Flag. The same method was used to connect the ORF of MsMYB206 to the plant binary expression vector pHB-YFP, so that the target gene was fused and expressed with the yellow fluorescent protein (YFP) gene. The above positive recombinant plasmids (pHB-MsMYB206-Flag and pHB-MsMYB206-YFP) were respectively transformed into Agrobacterium tumefaciens GV3101 competent cells (purchased from Shanghai Uptide Biotechnology).
[0055] 2) Constructing MsMYB206 RNA interference (RNAi) expression vector using gateway technology. Selecting 200-300 bp as the interference fragment in the non-conserved region of MsMYB206, connecting the interference fragment to the entry vector pTOPO-ENTR / D using BP recombination reaction, extracting the recombinant plasmid after sequencing, and connecting the interference fragment to the interference expression vector pHellsgate12 using LR recombination reaction, the recombinant interference plasmid was named pHellsgate12-MsMYB206. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells.
[0056] 3) Transforming Medicago sativa: referring to the Medicago sativa leaf disc transformation method (Chunxiang Fu, Timothy Hernandez, Chuanen Zhou, and Zeng-Yu Wang, Chapter 17) to transform Medicago sativa.
[0057] Example 5: Subcellular localization analysis of MsMYB206 in tobacco leaves
[0058] 1) Take 2.5 μL of Agrobacterium containing pHB-MsMYB206-YFP recombinant plasmid into 5 mL of liquid YEP medium supplemented with Rif and Kan, and small shake at 180 rpm, 28°C for 24 h. Meanwhile, take Agrobacterium containing helper plasmid P19 (P19 can inhibit the RNA silencing effect of the host on foreign genes, improve the stability of heterologous gene transcripts, and thus promote the expression of heterologous proteins) into YEP liquid medium supplemented with Kan only, and small shake for 24 h;
[0059] 2) Take 1 mL of each small shake bacterial solution in 1) into 50 mL of YEP liquid medium supplemented with the same antibiotics, and large shake at 180 rpm, 28°C overnight;
[0060] 3) Transfer the large shake bacterial solution to a 50 mL centrifuge tube, centrifuge at 4500 rpm for 15 min at room temperature, discard the supernatant, and resuspend the bacterial cells with 1 mL of liquid MS medium (MS + 2% sucrose, pH 5.8);
[0061] 4) Take another new 50 mL centrifuge tube, add 5 mL of MS liquid culture, and take 200-300 μL of the resuspended bacterial solution in 3) above, and adjust the OD600 to about 0.6-0.8;
[0062] 5) Add 100 μL of 0.5 M MES and 10 μL of 20 mg / mL As to each bacterial solution per 5 mL of bacterial solution;
[0063] 6) Place at room temperature in the dark for more than 3 h;
[0064] 7) Mix the target bacterial solution and P19 bacterial solution at a volume ratio of 1:1, and inject the bacterial solution from the back of the tobacco leaf with a 1 mL needle-free syringe;
[0065] 8) Irrigate the tobacco injected with bacterial solution with water, and prepare a slice after dark culture at 25°C for 48 h, and observe the green fluorescent signal by laser confocal microscopy (Leica Microsystems, Wetzlar, Germany). The results of the localization of MsMYB206 in the epidermal cells of tobacco leaves are shown in Figure 3 , which indicates that MsMYB206 is localized in the nucleus of tobacco leaf cells.
[0066] Example 6: Identification of MsMYB206 overexpressing alfalfa and determination of total flavonoid content
[0067] Extract RNA from the hygromycin positive seedlings according to Example 1 and reverse transcribe it into cDNA, and determine the expression amount of MsMYB206 in the transgenic alfalfa by qPCR, and the results are shown inFigure 4 The three strains (OE42, OE68 and OE72) with higher expression were selected as the subsequent experimental materials. The transgenic materials were continuously cultured under the following conditions: 16h light, 8h darkness, 22℃, and 70% relative humidity. After 35 days, the leaves were dried and the total flavonoids in the leaves were determined according to the plant flavonoid content detection kit (purchased from Beijing Solabio Biotechnology Co., Ltd.), and the results are shown in Table 2. Figure 5 As shown in Table 2, the total flavonoid content of the three overexpression strains (OE42, OE68 and OE72) was significantly higher than that of the wild type (WT) alfalfa, indicating that overexpression of MsMYB206 can increase the total flavonoid content of transgenic alfalfa.
[0068] Example 7: Identification of MsMYB206-interfered alfalfa and leaf phenotype
[0069] The kanamycin-positive seedlings were extracted for RNA and reverse transcribed into cDNA according to the method of Example 1, and the expression amount of MsMYB206 in the transgenic alfalfa was determined by qPCR. The results of the fluorescent quantitative PCR identification of MsMYB206-interfered alfalfa plants are shown in Table 3. Figure 6 As shown in Table 3, the expression amount of MsMYB206 was significantly down-regulated. Five strains (#R1, #R4, #R6, #R7 and #R8) with better MsMYB206 interference effect were selected as the subsequent experimental materials, and the leaf phenotype was observed and photographed. The leaf phenotype is shown in Table 4. Figure 7 As shown in Table 4, compared with the wild type (WT), the formation of palmate compound leaves of alfalfa was significantly improved.
[0070] The above describes the preferred embodiments of the present application in detail. It should be understood that those skilled in the art can make many modifications and changes to the embodiments of the present application without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application should be within the protection scope defined by the claims.
Claims
1. The use of MYB transcription factor gene to increase the content of total flavonoids in alfalfa under salt stress, characterized in that, The nucleotide sequence of the MYB transcription factor is shown as SEQ ID NO.
1.
2. Use of a MYB transcription factor for increasing the formation of palmate compound leaves in Medicago sativa, characterized in that, The application is to improve the formation of palmate compound leaves by RNA interference of the expression of the MYB transcription factor in Medicago sativa; the nucleotide sequence of the MYB transcription factor is shown as SEQ ID NO.
1.
3. The use of a plant expression vector of MYB transcription factor to increase the content of total flavonoids in Medicago sativa under salt stress, characterized in that, The nucleotide sequence of the MYB transcription factor is shown as SEQ ID NO.
1.
4. Use of a plant expression vector of a MYB transcription factor for increasing the formation of palmate compound leaves in alfalfa, characterized in that, The application is to improve the formation of palmate compound leaves by RNA interference of the expression of the MYB transcription factor in Medicago sativa; the nucleotide sequence of the MYB transcription factor is shown as SEQ ID NO.
1.
5. Use according to claim 4, characterized in that, The application also comprises transforming plant cells or tissues by the plant expression vector through conventional biological methods, and cultivating the transformed plant tissues into plants.