CN111087458B

CN111087458B - Alfalfa MYB transcription factor and aluminum toxin resistant application thereof

Info

Publication number: CN111087458B
Application number: CN201911426217.8A
Authority: CN
Inventors: 安渊; 苏连泰; 周鹏; 于晨; 张钰靖; 王如月; 赵恩华
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2021-09-03
Anticipated expiration: 2039-12-30
Also published as: CN111087458A

Abstract

The invention discloses an alfalfa MYB transcription factor and aluminum-toxin-resistant application thereof, and relates to the field of plant genetic engineering, wherein the MYB transcription factor is derived from an alfalfa variety WL525 and is named as an MsMYB741 transcription factor, and the amino acid sequence of the MsMYB741 transcription factor is as shown in a sequence SEQ ID NO: 2, or the sequence SEQ ID NO: 2 and the protein encoded by said derived amino acid sequence has MYB transcription factor properties; also provides a coding gene for coding the MYB transcription factor, wherein the nucleotide sequence of the coding gene is shown as a sequence SEQ ID NO: 1, or the sequence SEQ ID NO: 1, derived nucleotide sequence. The MsMYB741 transcription factor coding gene provided by the invention has obvious effects on constructing an expression vector and cultivating aluminum toxicity-resistant transgenic plants, provides a basis for improving the stress resistance of plants, especially cultivating aluminum toxicity-resistant alfalfa varieties, and has very important application value.

Description

Alfalfa MYB transcription factor and aluminum toxin resistant application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to an alfalfa MYB transcription factor and aluminum toxin resistant application thereof.

Background

Alfalfa (Medicago sativa L.) genus, alfalfa, is a perennial herb, known as the king of pasture, and has six advantages: firstly, the yield is high; secondly, the quality is good; thirdly, the feed is an important energy feed; the alfalfa also contains some substances which are very beneficial to livestock; fifthly, the ecological benefit is high; and the alfalfa can be deeply processed to extract leaf protein from the alfalfa, and can be used as an additive of food. However, biotic and abiotic stresses have a very adverse effect on the growth and development of alfalfa, such as aluminum poisoning.

Acid soils with a pH below 5.0 account for 30% -40% of the total tillerable soil area and are one of the important abiotic factors limiting crop growth. Aluminum (Al) is present in the earth's crust in an amount of about 7% and is very widespread, usually in the form of silicates or other precipitates, and is not toxic to plants. Under acidic conditions (pH)<5) Can promote the dissolution of aluminum in soil to form an ionic state (Al) with toxic effect on plants³⁺) Acting on plant root to inhibit root elongation and growth by destroying the structure and function of plant root cells The effect can generally occur at micromolar concentrations. Therefore, it is necessary to clarify the response mechanism of alfalfa to aluminum toxicity, identify important genes related to aluminum toxicity resistance and utilize the genes to improve the tolerance of alfalfa to aluminum toxicity, so that it is particularly important to greatly improve the yield of alfalfa.

When stimulated by external environment, plants can produce a large amount of related proteins to adapt to environmental changes, wherein the plant transcription factors can often regulate and control the expression of a series of functional genes related to stress at the downstream, and play an increasingly important role in the stress-resistant physiology of plants, thereby becoming a hotspot of research of people. MYB (v-MYB avian myeloblastosis viral oncogene homolog) class of transcription factors are named because they all structurally have a conserved DNA binding region, the MYB domain. The DNA binding domain of this transcription factor is most conserved, and typically comprises 1-3 incomplete repeats (R), each repeat R consisting of 51-52 conserved amino acid residues and spacer sequences, regularly spaced at 1 tryptophan residue every about 18 amino acids, which participate in the formation of the hydrophobic core and stabilize the helix-turn-helix spatial configuration. Sometimes a tryptophan residue is replaced by an aromatic or hydrophobic amino acid, especially in the case of plant R2R3-MYB transcription factors, the first tryptophan of the R3 domain is often replaced by leucine, isoleucine or phenylalanine. MYB transcription factors are widely present in plants and participate in various physiological and biochemical processes. Such as controlling cell morphology and pattern establishment, responding to external environment and stimulation of plant hormones and the like, regulating plant secondary metabolism and the like.

MYB is the largest transcription factor family in plants, and MYB genes have been cloned from different plants such as corn, snapdragon, cotton, Arabidopsis, cabbage, apple and soybean, wherein about 180 members exist in Arabidopsis and about 200 members exist in corn. Through analyzing promoter regions of arabidopsis drought, high-salt and ABA inducible genes, a core sequence of MYB binding sites is identified to be TAACTG; these transcription factors are present in ABA dependent pathway of high salt, low temperature and drought response, and after being combined with TAACTG core sequence of downstream target gene promoter, induce the expression of stress-resistant gene. James et al (2016) studied MYB transcription factors MYB115 and MYB134 in poplar, and the results showed that after MYB115 is overexpressed in poplar, proanthocyanidin content in transgenic plants is obviously increased, and salicide accumulation is reduced, which is similar to the function of MYB134, and transient expression studies showed that MYB115 and MYB134 can activate flavonoid gene promoters in the presence of bHLH auxiliary factors, thereby regulating the expression of related genes. Hu et al (2016) studied MdMYB1 in apples and found that MdMYB1 can alter anthocyanin accumulation and alter vacuolar acidification by directly activating vacuolar transporter genes, including MdVHA-B1, MdVHA-E, MdVHA-P1, and MdtDT, among others. After Hu et al (2017) find that MdMYB73 can be directly combined with gene promoters such as MdALMT9, MdVHA-A and MdVHP1 to activate the expression of the genes so as to improve the activity of the genes, and research in transgenic apple healing finds that MdMYB73 influences the accumulation of malic acid and the pH value of vacuoles. In alfalfa, research on MYB transcription factor family genes is very limited, and although the sequencing of the alfalfa genome of the tribulus is completed, related gene annotation is incomplete, so far, no related report for overall description of the MYB transcription factor family in the alfalfa is found. In the currently published researches, the functional research on MYB transcription factors in alfalfa mainly focuses on regulating the synthesis of Proanthocyanidin (PA), PAs and monomer catechol and epicatechin forming the PAs are antioxidants which are very beneficial to human health, can also resist cancers, have anti-inflammatory activity and the like, and recently, researches show that phenols and flavonoids can reduce the aluminum toxicity of plants, but further research data are not reported. In addition, the study of Zhang et al (2016) in Medicago truncatula shows that MtCBF4 (low temperature resistance-related transcription factor) can be directly combined with the promoter of the downstream low temperature resistance-related gene MtCAS15, and MtMYB3 can be combined with the MtCBF4 promoter to inhibit the expression of MtCBF4, so that the low temperature resistance of plants is reduced, which indicates that MtMYB3 is a negative regulator. In order to clarify the regulation effect of MtMYB3-MtCBF4 in low-temperature domestication, MtMYB3 is used as bait to be subjected to yeast double-hybrid screening to finally screen a protein MtMYB61 capable of interacting with MtMYB3, and MtMYB61 can relieve the transcription inhibition effect of MtMYB3 on MtCBF4, so that the expression of a downstream gene MtCAS15 is increased, and the low-temperature resistance of a plant is improved. The above examples illustrate the important role played by MYB transcription factors in stress response of plants, and further research on the functions of the MYB transcription factors of alfalfa is beneficial to improvement of stress resistance quality of alfalfa.

Therefore, those skilled in the art are devoted to develop a new alfalfa MYB transcription factor which can effectively improve the aluminum toxicity resistance of plants, and realize the improvement of the stress resistance of plants, especially the successful cultivation of the aluminum toxicity resistance alfalfa.

Disclosure of Invention

In view of the above defects in the prior art, the technical problem to be solved by the present invention is how to provide a MYB transcription factor capable of effectively improving the aluminum toxicity resistance of plants, and to realize improvement of plant stress resistance, especially cultivation of aluminum toxicity resistant medicago sativa varieties.

In order to achieve the aim, the invention provides an alfalfa MYB transcription factor, wherein the MYB transcription factor is derived from alfalfa variety WL525 and is named as MsMYB741 transcription factor, and the amino acid sequence of the MsMYB741 transcription factor is shown as a sequence SEQ ID NO: 2, or the sequence SEQ ID NO: 2 and the protein encoded by said derived amino acid sequence has MYB transcription factor properties.

Further, the derivative amino acid sequence is the sequence SEQ ID NO: 2 by deletion, insertion and/or substitution of 1 to 50 amino acids, or addition of 1 to 20 amino acids at the C-terminus and/or N-terminus.

The invention also provides a coding gene of the alfalfa MYB transcription factor, and the nucleotide sequence of the coding gene is shown as a sequence SEQ ID NO: 1, or the sequence SEQ ID NO: 1, derived nucleotide sequence.

Further, the homologous sequence is identical to the sequence SEQ ID NO: 1 has at least 70% homology.

Further, the derivative nucleotide sequence is the sequence SEQ ID NO: 1 by deletion, insertion and/or substitution of 1-90 nucleotides, or adding 1-60 nucleotides at the 5 'and/or 3' end.

The invention also provides an application of the encoding gene of the alfalfa MYB transcription factor in aluminum toxicity resistance, which specifically comprises the following steps: and constructing an expression vector by using the encoding gene of the MsMYB741 transcription factor, introducing the expression vector into a host, and culturing to obtain a transgenic plant with improved aluminum stress tolerance.

Further, the mode for constructing the expression vector comprises an enzyme digestion connection technology and a gateway technology.

Further, when an expression vector is constructed by adopting the enzyme digestion ligation technology, an upstream primer and a downstream primer are designed according to a nucleotide sequence of a coding gene of the MsMYB741 transcription factor, and Pst I and Bam HI enzyme digestion sites are respectively introduced into the upstream and downstream of the nucleotide sequence of the coding gene of the MsMYB741 transcription factor.

Further, the means for introducing the expression vector into the host include Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation.

Further, the host includes monocotyledons and dicotyledons.

Compared with the prior art, the invention at least has the following beneficial technical effects:

(1) the MsMYB741 gene and the encoding protein thereof provided by the invention have obvious up-regulation expression performance in environments with aluminum stress, high salt content, drought and excessive multiple hormones, and can provide a basis for culturing transgenic plants with excellent characteristics such as aluminum toxin resistance;

(2) according to the MsMYB741 gene provided by the invention, an expression vector is successfully constructed, transgenic arabidopsis thaliana and alfalfa hairy roots with obviously improved aluminum toxicity resistance are cultivated, and an important basis is provided for further cultivating an aluminum toxicity resistance alfalfa variety.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 shows the expression pattern of MsMYB741 gene under different stresses and hormone treatments, wherein A is Al stress, B is high salt stress, C is drought stress, D is abscisic acid (ABA) treatment, E is Salicylic Acid (SA) treatment, and F is methyl jasmonate (MeJA) treatment;

FIG. 2 is a schematic diagram of the structure of a plant expression vector according to a preferred embodiment of the present invention;

FIG. 3 is a photograph of a transgenic alfalfa hairy root obtained in accordance with a preferred embodiment of the present invention;

FIG. 4 shows a transgenic T according to a preferred embodiment of the present invention₃Phenotypic photographs of Arabidopsis thaliana under Al stress, wherein A represents pH4.5, and B represents pH4.5+1.2mM AlCl₃；

FIG. 5 shows a transgenic T according to a preferred embodiment of the present invention₃Root length statistics of generation Arabidopsis under Al stress, wherein Vector represents a transgenic empty Vector plant, and N17, N18, N19, N25 and N27 represent transgenic MsMYB741 strains;

FIG. 6 is a photograph showing the phenotype of alfalfa hairy roots overexpressing MsMYB741 under Al stress, wherein A represents pH4.5 and B represents pH4.5+1.2mM AlCl₃；

FIG. 7 is a statistics of root length of alfalfa hairy roots overexpressing MsMYB741 under Al stress according to a preferred embodiment of the present invention, wherein Vector represents trans-empty Vector hairy roots, and 741-11 and 741-12 represent trans-MsMYB 741 strains;

FIG. 8 is a photograph showing the phenotype of alfalfa hairy roots interfering with expression of MsMYB741 under Al stress, wherein A represents pH4.5, and B represents pH4.5+1.2mM AlCl ₃；

FIG. 9 is a root length statistics of alfalfa hairy roots of interference-expressed MsMYB741 under Al stress according to a preferred embodiment of the present invention, wherein Vector represents trans-empty Vector hairy roots, and R28 and R34 represent interference-expressed strains.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1: MsMYB741 gene cloning and sequence analysis

Extracting alfalfa RNA and synthesizing cDNA: total RNA from the roots of alfalfa WL525 was extracted with the EasyPure Plant RNA Kit (from King Konjac), and reverse transcribed with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (from King Konjac), to synthesize cDNA.

Design and synthesis of primers: germinating seeds of the alfalfa WL525 variety for 7 days, then carrying out aluminum stress treatment, sampling the whole plant after 60 hours, sending the sample to an Invitrogen company for gene chip analysis, wherein the gene chip adopts a Medicago truncatula (Medicago truncatula) gene chip of Agilent company. And (3) finding that an expression probe is obviously up-regulated compared with a control under aluminum stress through analysis, searching in a medicago truncatula database to obtain the cDNA sequence of the section, and designing a synthetic primer F: 5'-ATGTATTCAGGAATGATGGA-3', R: 5'-TTAACAAAAGGGAGCAACT-3' are provided. And (3) performing PCR amplification by using the alfalfa cDNA as a template according to the following reaction system and conditions: a50. mu.L system containing Ex Taq Mix 25. mu.L (purchased from Takara), 10. mu.M of each of primer F and primer R1. mu.L, cDNA 1. mu.L, and deionized water to 50. mu.L. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s,58 ℃ for 30s, and 72 ℃ for 1 min; extension was carried out for 8min after 72 ℃. And (3) performing 1% agarose electrophoresis on the amplified fragment, cutting and recovering gel, connecting the recovered product with a cloning vector pMD18-T (purchased from Takara), identifying the positive clone, sequencing the Huada gene, and identifying the recombinant plasmid with the correct sequence, wherein the recombinant plasmid is named as pMD18-T-MsMYB 741.

PCR amplification is carried out to obtain a sequence containing the full-length ORF of the MsMYB741 gene, a protein consisting of 264 amino acid residues is encoded, 84bp (28 amino acid residues) is less than the ORF of the gene in medicago truncatula, the similarity is 81.2%, and the MsMYB741 gene contains two conserved MYB binding domains and belongs to R2R3-MYB transcription factors.

Example 2: expression mode of MsMYB741 gene under stress and hormone induction

Alfalfa variety WL525 treatment: using modified 1/2Hoagland medium (Ca (NO)₃)₂·4H₂O 945mg/L、KNO₃ 506mg/L、NH₄NO₃ 80mg/L、KH₂PO₄ 136mg/L、MgSO₄·7H₂O 493mg/L、EDTA-Na₂18.65mg/L、FeSO₄·7H₂O 13.9mg/L、KI 0.83mg/L、H₃BO₃ 6.2mg/L、MnSO₄·H₂O 16.9mg/L、ZnSO₄·7H₂O 8.6mg/L、Na₂MoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.025mg/L、CoCl₂·6H₂O0.025 mg/L, pH 5.7-5.8), culturing the water culture seedlings of the alfalfa at the temperature of 28 ℃ and under the conditions of 16h of illumination/8 h of darkness, and taking the water culture seedlings at the four-leaf stage for the following treatment:

aluminum stress treatment: placing alfalfa seedlings in a container containing 100 mu mol/L AlCl₃·6H₂Treating the culture solution of 1/2Hoagland of O for 0h, lh, 3h, 6h, 9h, 12h and 24h, respectively sampling, and rapidly placing in liquid nitrogen to store at-80 ℃ for later use;

high-salt treatment: placing the seedling of the alfalfa into 1/2Hoagland culture solution containing 200mmol/L NaCl, processing for 0h, lh, 3h, 6h, 9h, 12h and 24h, respectively sampling, and rapidly placing in liquid nitrogen for storage at-80 ℃ for later use;

drought treatment: placing the seedling of alfalfa in 1/2Hoagland culture solution containing 15% PEG8000, processing for 0h, lh, 3h, 6h, 9h, 12h and 24h, respectively sampling, rapidly placing in liquid nitrogen, and storing at-80 deg.C;

Hormone treatment: treating alfalfa seedlings according to the following method, respectively adding abscisic acid (ABA), Salicylic Acid (SA) and methyl jasmonate (MeJA) into 1/2Hoagland culture solution until the final concentration is 100 mu mol/L, respectively sampling after treating for 0h, lh, 3h, 6h, 9h, 12h and 24h, and quickly placing in liquid nitrogen for storage at-80 ℃ for later use.

The total RNA extraction and cDNA synthesis were performed as in example 1. Design of real-time fluorescent quantitative PCR primer (F: 5' -ACACAAGA) according to cDNA sequence of MsMYB741ACAACAGGGTTGC-3'; r: 5'-ACATTGGCTGGCTTTGCTTG-3'). The alfalfa constitutive expression gene EF-a is taken as an internal reference (F: 5'-GCACCAGTGCTCGATTGC-3'; R5'-TCGCCTGTCAATCTTGGTAACAA-3'). And (3) performing real-time RT-PCR analysis by using a Bio-Rad CFX96 real-time quantitative PCR instrument and cDNA of each processing sampling point of the alfalfa as a template. The reaction system contained 10. mu. L, cDNA 2. mu.L of 2 XStart Top Green qPCR SuperMix (purchased from Beijing Quanyu-jin biosystems), 0.4. mu.L of Primer F and 0.4. mu.L of Primer R, and water was added to a total volume of 20. mu.L. The reaction program is 94 ℃ for 30 s; 94 ℃ for 5s,60 ℃ for 15s,72 ℃ for 10s,40 cycles. By using 2^-ΔΔCTThe method analyzes data and determines the relative expression amount of the gene. Each sampling point was assigned 3 technical replicates and the experiment was assigned a total of 3 biological replicates. Statistics of relative expression of MsMYB741 in roots under stress and hormone treatment are shown in table 1. Meanwhile, the relative expression amounts of MsMYB741 in leaves and roots under each stress or hormone treatment were compared, as shown in FIG. 1 (wherein A is Al stress, B is high salt stress, C is drought stress, D is abscisic acid (ABA) treatment, E is Salicylic Acid (SA) treatment, and F is methyl jasmonate (MeJA) treatment)

TABLE 1 relative expression levels (mean) of MsMYB741 in roots under stress and hormone treatment

The quantitative results show that: the MsMYB741 gene has up-regulated expression to different degrees under various stress conditions.

Example 3: construction of expression vectors

In this example, a plant expression vector of MsMYB741 was constructed according to the expression vector structure diagram shown in fig. 2. The following methods can be specifically adopted:

1) constructing a plant overexpression vector of MsMYB741 by using an enzyme digestion connection technology, designing primers according to a target gene sequence, respectively introducing Pst I and Bam HI enzyme digestion sites into an upstream primer and a downstream primer, and respectively introducing an upstream pHB-MsMYB 741-F: 5'-AACTGCAGATGTATTCAGGAATGATG-3', downstream pHB-MsMYB 741-R: 5'-CGCGGATCCACAAAAGGGAGCAACTAC-3', and PCR amplification is carried out by taking pMD18-T-MsMYB741 plasmid as a template. After the amplification, the PCR product was recovered in gel, and the recovered product was ligated with the cloning vector pMD18-T (purchased from Takara). Escherichia coli (e.coli) DH5 α competent cells (purchased from shanghai vironly) were transformed after overnight incubation at 16 ℃. Positive clones were selected on solid LB plates supplemented with ampicillin (purchased from Shanghai assist in san-biosome) and sent to Huada for sequencing validation. The positive recombinant plasmid with correct sequencing is named as pMD18T-pHB-MsMYB741, the positive recombinant plasmid and the expression vector plasmid pHB-Flag are subjected to double enzyme digestion by Pst I and Bam HI (purchased from Takara), enzyme digestion products are respectively subjected to gel recovery, the recovered products are connected and transformed into escherichia coli (E.coli) DH5 alpha competent cells, positive clones are screened on an LB solid plate with kanamycin, the positive clones are cultured, recombinant plasmids are extracted, the recombinant plasmids are the final plant expression vector connected with a target gene MsMYB741, and the recombinant plasmid is named as pHB-MsMYB741-Flag and transformed into agrobacterium tumefaciens GV3101 competent cells (purchased from Shanghai Weiji organisms).

2) Constructing an interference expression vector of MsMYB741 by using gateway technology. 300-400BP is selected from a non-conserved region of MsMYB741 as an interference fragment, the interference fragment is connected into a portal vector pTOPO-ENTR/D by using BP recombination reaction, recombinant plasmid is extracted after the sequencing is correct, the interference fragment is connected into an interference expression vector pHellsgate12 by using LR recombination reaction, and the recombinant interference plasmid is named as pHellsgate12-MsMYB 741. The recombinant plasmid was transferred into Agrobacterium rhizogenes LBA 9402.

Example 4: obtaining transgenic Material

(1) Acquisition of transgenic Arabidopsis

Arabidopsis thaliana (ecotype Columbia) is infected by using an Agrobacterium-mediated flower dipping method. The specific method comprises the following steps:

1) seed disinfection: placing wild type Arabidopsis seeds in a 1.5ml centrifuge tube, sterilizing with 75% alcohol for 5min, then sterilizing with anhydrous alcohol for 3 times, each time for 2min, and finally placing the seeds on sterile filter paper for air drying;

2) sowing the dried Arabidopsis seeds on an MS solid culture medium (the pH value of the MS culture medium is adjusted to 5.7-5.8 by KOH), and culturing for 7-10d under the conditions of 16h of illumination (25 ℃)/8h of darkness (22 ℃) and 80% of humidity;

3) sterilizing turfy soil and vermiculite, mixing according to a ratio of 1:1, placing into a nutrition pot, irrigating with a flower-free water soluble fertilizer solution, transferring the arabidopsis thaliana plantlets in a culture medium into the nutrition pot, covering with a preservative film for 5-7d, and placing into an incubator to culture under the conditions of 2);

4) About 3 weeks later, arabidopsis begins to flower, the flowers which have already opened are cut off, and the remaining buds are ready for infection;

5) selecting an agrobacterium tumefaciens monoclonal containing a pHB-MsMYB741-Flag vector, and culturing the agrobacterium tumefaciens monoclonal in 5ml of liquid YEP culture medium added with 50mg/L Rif and 50mg/L Kan at 28 ℃ and 180rpm for 24 h;

6) 2ml of the above culture was poured into 500ml of YEP containing the same final concentration of Rif and Kan, and cultured at 28 ℃ and 150rpm to OD₆₀₀＝1.0-1.2；

7) Placing the bacterial liquid in a 50ml centrifuge tube, centrifuging at room temperature and 5000rpm for 15min, discarding the supernatant, and suspending the thallus in a transformation medium MS liquid culture medium to OD₆₀₀Adding a few drops of surface adsorbent Tween into the mixture before infection, and uniformly mixing to improve the conversion efficiency;

8) completely immersing the upper part of the arabidopsis into a transformation medium, and carrying out vacuum infection for 10 min;

9) after infection, absorbing excess bacteria liquid by using absorbent paper, covering the plants with a black plastic bag, removing the plastic bag after dark culture for 16-24h, pouring enough nutrient solution, and normally culturing;

10) after normal cultivation for one month, gradually reducing the watering amount, and stopping watering when most siliques are mature;

11) harvesting seeds, i.e. T₀Seed generation;

12) will T₀Sowing seeds in a matrix, uniformly spraying 1/1000 Basta on the seedlings after about 15 days to screen transgenic seedlings, transferring the surviving seedlings into a new matrix to culture, extracting DNA of leaves to perform PCR identification, and harvesting positive plants, namely T ₁Seed generation;

13) will T₁Culturing the generation seeds according to the method in 12), and performing PCR identification to obtain seeds T₂The seed is used as the substitute of the seed,continuing to use the method in 12) until T is obtained₃Seed generation, using T₃The seeds were used for subsequent experiments.

(2) Obtaining the hairy root of the transgenic alfalfa, operating according to the methods of Paracetamol, etc

1) Preparing a genetic engineering strain: agrobacterium rhizogenes containing pHellsgate12-MsMYB741 was streaked on a solid medium containing YEP medium supplemented with 50mg/L of Rif (rifampicin) and 50mg/L of Sp (spectinomycin), and cultured overnight at 28 ℃. Selecting single colony, inoculating to YEP liquid culture medium containing Rif and Sp, culturing overnight, and performing amplification culture with 1% inoculum size the next day to OD₆₀₀Centrifuging at 18 deg.C and 3500rpm for 15min at 0.6-0.8, discarding supernatant, and diluting with transformation solution to OD₆₀₀0.2-0.4 for standby;

2) preparation of explants: the whole plant of the sterile seedling of the alfalfa (WL525) can be used as an explant, the root part of the alfalfa can be directly used for infection and transformation, a stem or a petiole is cut into small sections of about 1.5cm, and the edge of a leaf is slightly cut to generate a wound;

3) infection with Agrobacterium rhizogenes: putting the explant obtained in the step 2) into the bacterial liquid prepared in the step 1), and slightly shaking for 20min on a decoloring shaking table;

4) Co-culturing: after the infection liquid in the step 3) is completely absorbed, putting the explant on sterile filter paper, airing the explant in an ultra-clean workbench, then putting the explant on a co-culture medium, and carrying out dark culture at 25 ℃ for 24-48 h;

5) induction of hairy roots: transferring the explants co-cultured in the step 4) to a hairy root induction screening medium (SHS), and culturing in the dark to grow hairy roots in about 15-25 days. The temperature of the incubator is 25 +/-2 ℃, and dark culture is carried out.

The main state changes during the culture are shown in FIG. 3.

Example 5: t is₃Aluminum-tolerant phenotypic analysis of transgenic Arabidopsis thaliana

Will T₃Sterilizing Arabidopsis seeds with 75% ethanol for 5min, sterilizing with anhydrous ethanol for 3 times (2 min each time), air drying the sterilized seeds on sterile filter paper, and sowing the seeds in a container with 1.2mM AlCl₃(pH4.5) MS culture medium, with no AlCl addition as control₃(pH4.5) MS medium, which was observed and photographed at any time after 10d germination (as shown in FIG. 4), and root length statistics (as shown in FIG. 5) were performed after 15d germination, and culture conditions: the photoperiod is 16h of illumination, 8h of darkness, the temperature is 22 ℃, and the relative humidity is 70%.

Comparing the results of the photographs of FIG. 4 with the statistics of the data of FIG. 5 shows that AlCl is not added ₃In the case of (1), the control (vector) and transgenic lines (N18, N27) were of comparable root length, with no significant difference when added with AlCl₃In the case of (2), the root length of the transgenic lines (N18, N27) is significantly better than that of the control (vector), indicating that the transgenic lines have good aluminum toxicity resistance under aluminum stress.

Example 6: aluminum toxin-resistant phenotype analysis of transgenic alfalfa hairy roots

Cutting transgenic alfalfa hairy root into 1.5cm segments (1.5 cm from root tip to top), adding 1.2mM AlCl₃(pH4.5) in SH9 medium, as a control, without addition of AlCl₃SH9 culture medium (pH4.5), observed and photographed at any time (as shown in FIG. 6), cultured for about 7d to perform root length statistics (statistical results are shown in FIG. 7), the SH9 culture medium formula mentioned above refers to Fuchunxiang and the like, and the culture conditions are as follows: dark culture at 22 deg.C and 50% relative humidity.

Comparative analysis of the photographic results of FIG. 6 and the data statistics of FIG. 7 shows that transgenic hairy root lines overexpressing MsMYB741(MsMYB741-OE) have no AlCl added₃In the case of (1), the control (vector) and the transgenic line (741-11, 741-12) had equivalent root lengths, and no significant difference was observed when AlCl was added₃In the case of (2), the root length of the transgenic line (741-11, 741-12) is significantly longer than that of the control (vector), wherein the root length of the 741-11 line is different from that of the control to a significant degree.

Similarly, photographing observation (shown in FIG. 8) and root length statistics (shown in FIG. 9) were performed on transgenic hairy root lines interfering with expression of MsMYB741(MsMYB741-RNAi), and analysis revealed that AlCl was not added₃In the case of (1), the control (vector) and the transgenic lines (R28, R34) had comparable root lengths, with no significant difference when AlCl was added₃In this case, the root length of the transgenic lines (R28, R34) after 6d of culture was significantly shorter than that of the control (vector), and reached a very significant level of difference compared to the control.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

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gcaagaagct tgccggggag aacagacaat gagataaaga actattggag aactcatttc 360

aagaaaaaga ccaaaaaccc ctctgatgct gctgaaaagg cgaaaaatcg ttctttcaag 420

aggcagcagc aacaacaatt gaagaaacaa cagcaacaag ttcagatgca gcaacaacaa 480

ctgcaataca acatggatat gaaagggatc atagacttgt tgcttgagga aaatgactac 540

tgtactagtg tgccttctac ttctcaagag acacaagaaa tggtttccat gtatgctgat 600

acacaagaac aacagggttg cttttattct atgctcaatg ataatagtgg taatgtctat 660

gcacaagagt cttcaaatga agagaatttg tgggatgaac tttggaactt ggatgatgct 720

cttggaaatt caatgcagct aatgcttcaa gcaaagccag ccaatgtgca caatgtagtt 780

gctccctttt gttaa 795

<210> 2

<211> 264

<212> PRT

<213> alfalfa (Medicago sativa)

<400> 2

Met Tyr Ser Gly Met Met Glu Gly Asn Thr Gly Trp Ser Val Met Glu

1 5 10 15

Glu Asp Arg Trp Arg Lys Gly Pro Trp Thr Ser Glu Glu Asp Lys Leu

20 25 30

Leu Ile Glu Tyr Val Lys Leu His Gly Glu Gly Arg Trp Asn Ser Val

35 40 45

Ser Arg Leu Ala Gly Leu Arg Arg Asn Gly Lys Ser Cys Arg Leu Arg

50 55 60

Trp Val Asn Tyr Leu Arg Pro Asp Leu Lys Arg Gly Gln Ile Thr Gln

65 70 75 80

Gln Glu Glu Ser Ile Ile Leu Glu Leu His Ala Arg Trp Gly Asn Arg

85 90 95

Trp Ser Thr Ile Ala Arg Ser Leu Pro Gly Arg Thr Asp Asn Glu Ile

100 105 110

Lys Asn Tyr Trp Arg Thr His Phe Lys Lys Lys Thr Lys Asn Pro Ser

115 120 125

Asp Ala Ala Glu Lys Ala Lys Asn Arg Ser Phe Lys Arg Gln Gln Gln

130 135 140

Gln Gln Leu Lys Lys Gln Gln Gln Gln Val Gln Met Gln Gln Gln Gln

145 150 155 160

Leu Gln Tyr Asn Met Asp Met Lys Gly Ile Ile Asp Leu Leu Leu Glu

165 170 175

Glu Asn Asp Tyr Cys Thr Ser Val Pro Ser Thr Ser Gln Glu Thr Gln

180 185 190

Glu Met Val Ser Met Tyr Ala Asp Thr Gln Glu Gln Gln Gly Cys Phe

195 200 205

Tyr Ser Met Leu Asn Asp Asn Ser Gly Asn Val Tyr Ala Gln Glu Ser

210 215 220

Ser Asn Glu Glu Asn Leu Trp Asp Glu Leu Trp Asn Leu Asp Asp Ala

225 230 235 240

Leu Gly Asn Ser Met Gln Leu Met Leu Gln Ala Lys Pro Ala Asn Val

245 250 255

His Asn Val Val Ala Pro Phe Cys

260

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atgtattcag gaatgatgga 20

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ttaacaaaag ggagcaact 19

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

acacaagaac aacagggttg c 21

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

acattggctg gctttgcttg 20

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gcaccagtgc tcgattgc 18

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

tcgcctgtca atcttggtaa caa 23

<210> 9

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

aactgcagat gtattcagga atgatg 26

<210> 10

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

cgcggatcca caaaagggag caactac 27

Claims

1. An alfalfa MYB transcription factor, wherein the MYB transcription factor is derived from alfalfa variety WL525 and is named as MsMYB741 transcription factor, and the amino acid sequence of the MsMYB741 transcription factor is shown as the sequence SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

2. A coding gene encoding the alfalfa MYB transcription factor of claim 1, wherein the nucleotide sequence of the coding gene is as set forth in sequence SEQ ID NO: 1.

3. The application of the encoding gene of the alfalfa MYB transcription factor as claimed in claim 2 in aluminum toxicity resistance, wherein the application is specifically as follows: and constructing an expression vector by using the encoding gene of the MsMYB741 transcription factor, introducing the expression vector into a host, and culturing to obtain a transgenic plant with improved aluminum stress tolerance.

4. The use of the encoding gene of the alfalfa MYB transcription factor as claimed in claim 3 for aluminum toxicity resistance, wherein the expression vector is constructed by a method selected from the group consisting of enzyme ligation and gateway technology.

5. The application of the coding gene of the alfalfa MYB transcription factor in aluminum toxin resistance according to claim 4, wherein when the enzyme digestion ligation technology is adopted to construct an expression vector, an upstream primer and a downstream primer are designed according to a nucleotide sequence of the coding gene of the MsMYB741 transcription factor, and Pst I and BamHI enzyme digestion sites are respectively introduced into the upstream and downstream of the nucleotide sequence of the coding gene of the MsMYB741 transcription factor.

6. The use of a gene encoding an alfalfa MYB transcription factor as claimed in claim 3 for aluminum toxicity resistance, wherein the expression vector is introduced into the host in a manner selected from the group consisting of Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, and Agrobacterium mediation.

7. The use of a gene encoding an alfalfa MYB transcription factor as claimed in claim 3 for aluminum toxicity resistance, wherein the host is a monocot or a dicot.