CN117625638A - Alfalfa MsTIFY10a gene and application thereof - Google Patents

Abstract

The invention discloses an alfalfa MsTIFY10a gene and application thereof. The sequences of the alfalfa MsTIFY10a gene and the coded protein are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. The invention clones MsTIFY10a gene from alfalfa for the first time and through a special genetic transformation method, the biological function of the gene is verified by over-expressing the gene in various alfalfa. The MsTIFY10a gene has the functions of regulating and controlling the seed coat color and improving the size and weight of seeds, can provide a powerful tool for alfalfa transgenic research and provides a valuable candidate gene for molecular breeding. Meanwhile, the invention obtains a high-efficiency alfalfa genetic transformation system by improving the formula of the culture medium and optimizing the transformation steps, the system has stronger stability in various alfalfa varieties, the genetic transformation time is greatly shortened, and a powerful means is provided for alfalfa through genetic engineering breeding.

Description

Alfalfa MsTIFY10a gene and its application

Technical Field

The invention belongs to the technical field of plant gene engineering, and in particular relates to the application of a gene for deepening seed coat color and increasing alfalfa seed size and weight.

Background Art

The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

The seed industry is the "chip" of the grass industry, and the breeding of excellent alfalfa varieties has become a bottleneck problem restricting the development of my country's alfalfa industry. At present, the development of the alfalfa industry is mainly concentrated in the fields of cultivation and planting, while the breeding of high-quality alfalfa varieties is relatively lagging behind. Improving plants through genetic transformation to obtain transgenic plants has received increasing attention. The temporary expression of transgenics can increase crop yields and improve tolerance to abiotic stresses. Seed size is one of the most important agronomic traits in agricultural production and an important factor in determining yield. However, there are currently few reports on regulating seed size.

Transcription factors (TFs) have a special structure and regulate the transcription process of genes by specifically binding to the DNA sequence of the regulatory region. Transcription factors participate in regulating plant growth and development by activating or inhibiting gene expression, and also play a key role in plant stress response. TIFY is a type of transcription factor unique to plants, and members of the TIFY gene family play an important role in regulating grain size in plant growth and development. However, there is little research on this transcription factor in alfalfa, and its function is still unclear.

At present, genetic transformation is often used to obtain excellent alfalfa varieties. However, genetic transformation methods are often not universal for different varieties. The same gene transformation of different alfalfa varieties requires a new system, which consumes manpower and material resources. In addition, the existing genetic transformation methods have a long transformation cycle, are prone to callus browning and vitrification, and have low transformation efficiency.

Summary of the invention

In order to overcome the above-mentioned deficiencies of the prior art, the object of the present invention is to provide alfalfa MsTIFY10a gene and application thereof.

To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

In a first aspect, a clover MsTIFY10a gene is provided, the polynucleotide sequence of which is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

In a second aspect, a biological material containing the gene of the first aspect is provided, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a non-renewable plant part.

In a third aspect, there is provided an application of the gene described in the first aspect or the biological material described in the second aspect in regulating the seed coat color of a plant. Specifically, the regulation is manifested as overexpression of the MsTIFY10a gene to deepen the seed coat color; the plant is preferably a dicotyledonous plant, and the dicotyledonous plant includes alfalfa and soybean; preferably, the alfalfa is Medicago truncatula or Medicago sativa.

In a fourth aspect, there is provided use of the gene described in the first aspect or the biological material described in the second aspect in regulating plant seed size or regulating plant seed weight.

In a fifth aspect, a method for regulating the color of a plant seed coat is provided, comprising: using genetic engineering means to overexpress the gene of the first aspect in a plant;

The overexpression method is selected from the following 1) to 5), or an optional combination:

1) by introducing a plasmid carrying the gene;

2) by increasing the copy number of the gene on the plant chromosome;

3) by changing the promoter sequence of the gene on the plant chromosome;

4) by operably linking a strong promoter to the gene;

5) by introducing enhancers;

The plant is preferably a dicotyledonous plant, and the dicotyledonous plant includes alfalfa and soybean; preferably, the alfalfa is Medicago truncatula or Medicago sativa;

Preferably, the exogenous gene MsTIFY10a is transferred into Medicago truncatula and Medicago sativa plants by Agrobacterium-mediated method to obtain transgenic plants overexpressing the gene;

Preferably, the MsTIFY10a gene is constructed into the plant expression vector pEarleyGate 100, and Agrobacterium is transformed, and then Medicago truncatula and Medicago sativa transformants carrying the MsTIFY10a gene are obtained by genetic transformation methods, and transgenic plants are screened.

In a sixth aspect, a method for increasing the size and weight of plant seeds is provided, comprising: using genetic engineering means to overexpress the gene described in the first aspect in the plant;

The overexpression method is selected from the following 1) to 5), or an optional combination:

1) by introducing a plasmid carrying the gene;

2) by increasing the copy number of the gene on the plant chromosome;

3) by changing the promoter sequence of the gene on the plant chromosome;

4) by operably linking a strong promoter to the gene;

5) by introducing enhancers;

The plant is preferably a dicotyledonous plant, and the dicotyledonous plant includes alfalfa and soybean; preferably, the alfalfa is Medicago truncatula or Medicago sativa;

Preferably, the exogenous gene MsTIFY10a is transferred into Medicago truncatula plants by Agrobacterium-mediated method to obtain transgenic plants overexpressing the gene;

Preferably, the MsTIFY10a gene is constructed into the plant expression vector pEarleyGate 100, and Agrobacterium is transformed, and then Medicago truncatula and Medicago sativa transformants carrying the MsTIFY10a gene are obtained by genetic transformation methods, and transgenic plants are screened.

In a seventh aspect, the present invention provides the use of the transgenic plant obtained by the method described in the fifth aspect in plant breeding.

In a specific embodiment of the present invention, the breeding method includes transgenic, hybridization, backcrossing, selfing or asexual reproduction.

In an eighth aspect, the present invention provides an efficient method for genetic transformation of alfalfa, the method comprising the following steps:

1) mixing the Agrobacterium infection solution containing the gene described in the first aspect or the biological material described in the second aspect with the transformed explant, pouring the mixture into a silage bag, and obtaining the infected explant by vacuuming, ultrasonicating, and then vacuuming;

2) transferring the infected explants to a co-culture solid medium and culturing until the explants become hard and the edges slightly curl up;

3) transferring the explants cultured in step 2) to a screening medium and then subculturing them in a callus medium to obtain callus tissue;

4) placing the callus tissue in a differentiation medium for culture;

5) placing the callus obtained in step 4) in a bud-forming medium containing a selection agent for induction culture to obtain explants with clustered buds;

6) Placing the clustered bud explants in step 5) in a rooting medium for rooting culture to obtain regenerated alfalfa plants, thereby achieving genetic transformation of alfalfa.

In a specific embodiment of the present invention, the explant is alfalfa trifoliate compound leaf; a wound is lightly scratched on the surface of the explant; the vacuum condition is: evacuation for 75 seconds, the temperature is medium temperature; the ultrasonic condition is: 40kHz, 5min;

Preferably, the formula of the callus culture medium is: weigh 4.43 g MS powder, add 30 g sucrose, 7.5 g agar, 2 mL 2,4-D, 0.1 mL 6-BAP, adjust the pH to 5.8, and add water to make up to 1 L.

One or more of the above technical solutions have the following beneficial effects:

The present invention reveals for the first time the biological function of the alfalfa TIFY10a gene, and transforms Medicago truncatula and Medicago sativa by constructing a TIFY10a gene expression vector and combining it with Agrobacterium-mediated genetic transformation to investigate the effect of TIFY10a on the performance of transgenic alfalfa seeds, and simultaneously detects the effect of TIFY10a on the seed coat color, seed size and weight of Medicago truncatula, thereby providing a powerful tool for transgenic research on Medicago sativa and a valuable candidate gene for molecular breeding of Medicago sativa.

Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

FIG1 is a gel image of a cloned fragment of the MsTIFY10a gene according to Example 1 of the present invention, right: MARKER: DL2000 Plus (2000, 1500, 1000, 750, 500, 250, 100), left: MsTIFY10a gene (537 bp).

Figure 2pEarleyGate 100-MsTIFY10a spectrum.

Figure 3 Schematic diagram of the construction process of vector pEarleyGate 100-MsTIFY10a.

Figure 4 Silage bags and vacuum packaging machine.

Figure 5 shows the genetic transformation process and phenotype of alfalfa 'Caoyuan No. 4' in Example 3 of the present invention.

FIG. 6 shows the seed coat color phenotype of transgenic Medicago truncatula carrying the MsTIFY10a gene in Example 5 of the present invention.

FIG. 7 shows the seed size of transgenic Medicago truncatula carrying the MsTIFY10a gene in Example 5 of the present invention.

FIG8 shows the 100-seed weight of transgenic Medicago truncatula carrying the MsTIFY10a gene in Example 5 of the present invention.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If not otherwise specified, the examples are all based on conventional experimental conditions, such as Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

Identification of positive plant seedlings in the present invention:

1. Bar gene rapid test strip identification (purchased from Arton)

2. Bar gene PCR

bar F1:AGTCGACCGTGTACGTCTCC;

bar R1:GAAGTCCAGCTGCCAGAAAC

3. PCR identification of target gene and vector

Primer F2: TGAATCATCACAACATGACACCTATGAA

PEG100-SeR:AAAACCGGCGGTAAGGATCTG

The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

Example 1: Genetic transformation vector and strain acquisition of alfalfa TIFY10a

1. Cloning of TIFY10a gene from alfalfa

(1) RNA extraction and synthesis of the first strand of cDNA

The Trizol method was used to extract RNA from the leaves of ‘Grassland No. 4’ alfalfa grown for 45 days. The RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit from Fermentas according to the instructions and stored at -20°C for later use.

(2) Gene amplification

① Primer design

Two specific primers were designed based on the known sequence (accession number: MZ457926) using Premier 5.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Table 1: Primer names and sequences

②Amplification system and reaction conditions

Table 2: Reaction system

Table 3: Reaction conditions

(3) Electrophoresis gel cutting and recovery

The PCR products were separated by electrophoresis using 1% agarose gel, photographed and cut using a gel imaging system ( FIG. 1 ), and amplified fragments were recovered using a DNA agarose gel recovery kit (Tian Gen, DP210831) according to the instructions.

(4) Obtaining positive strains with target fragments

The vector and the target fragment in step (3) were connected using Takara's T4 ligase kit (TaKaRa, e0881) according to the instructions, and then transferred into Escherichia coli competent cells (Kangwei, CW0808S). After identification, the positive clone culture was sent to Sangon Biotech Co., Ltd. for sequencing.

The sequencing results were accurate after sequence comparison. The gene sequence of TIFY10a is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2.

SEQ ID NO.1:

ATGAATCATCACAACATGACACCTATGAATTTTCAACAACTTCCTCATCCTCTCCTA

CAAGAGATTCCTATTTTGGGAAACTCCAGTGTAATGAAGGCTAATGTTAAGAAGGA

AGAGGCTAGATGTGCACAAATGACAATCTTCTATGATGGGAAAGTAATTGTGTTTG

ATGAGTTCCAGCTGATAAAGCCAAAGACATCATGGACTTTTCCACCAAAGGAATT

ACTAGTACTTCACAAAACCACAATAACAACTATGCTTACTCTTCATTTCTTGCTAGA

AATTCTCTTCAAGACTACCCTCAAGTACCATCTATACCTGTTATTTATGATTTACCAA

TGACTAGGAAAGCTTCACTTCATCGGTTCTTGGAGAAAAGGAAGGATAGAATTGC

TGCCAAGGCACCATATCAAACAAGCAATCCAAGAACCATTAATAAACCAATTGATG

AATCGATGACATGGCTCTCTTTGGCACCACAATCACCACAACATAAATCTGAGTGT

AGCAGCTCCTCCACCCATGTGTTACTTTAA

SEQ ID NO.2:

MNHHNMTPMNFQQLPHLFLQEIPILGNSSVMKANVKKEEARCAQMTIFYDGKVIVF

DDVPADKAKDIMDFSTKGITSTSQNHNNNYAYSSFLARNSLQDYPQVPSIPVIYDLPM

TRKASLHRFLEKRKDRIAAKAPYQTSNPRTINKPIDESMTWLSLAPQSPQHKSECSSSSS

THVLL

2. Construction of plant overexpression vector of MsTIFY10a gene

1. Target gene cloning

When cloning the target gene and constructing the vector, use PrimerStar to clone to ensure the accuracy of the cloned fragment. The target gene cloning system is as follows:

Set up the required parallel reactions and perform PCR amplification as follows:

Recovery of target gene: After adding PCR product to DNA loading buffer, perform agarose gel electrophoresis at the required concentration to detect its size. After electrophoresis, use agarose gel DNA recovery kit (Tiangen) to recover the target fragment. Use NANO DROP 2000c to determine RNA concentration and purity, and run agarose gel electrophoresis to detect integrity.

The pEarleyGate 100 vector was originally constructed by the Picard Laboratory of Indiana University. The vector map and vector sequence can be found on the laboratory website, specifically at: https://pikweb.sitehost.iu.edu/Vectors%20homepage.html. The pEarleyGate 100 vector used in the present invention was purchased directly from: Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences.

2. Construction of intermediate vector-related vectors

①In-fusion reaction vector construction process

The Gateway-T (PGWC) vector map and vector sequence can be found on the website, specific URL: https://www.novopro.cn/vector/Vgy4timq (Figure 2). The Gateway-T vector used in the present invention was directly purchased from: Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences.

After the cloned target fragment is tapped and recovered, refer to HD kit (TAKaLa, 639648) instructions Prepare the following system in a 200 μl EP tube:

Table 4

*The target fragment and the intermediate vector react at a molar ratio of 2:1, and the formula is as follows:

Optimal vector dosage = "0.002 × number of base pairs of cloning vector" ng (0.03pmol)

Optimal amount of insert fragment = "0.04 × number of base pairs of insert fragment" ng (0.06 pmol)

Incubate at 50℃ for 30min, then quickly place on ice for 2min. (Incubate at 37℃ for 30min, add to competent E. coli, mix, incubate on ice for 30min, heat shock at 42℃ for 60-90s, on ice for 2min, add 600μL empty LB liquid, shake and culture at 37℃ for 1h, and apply LB solid plate with response resistance) Transform E. coli DH5α and select positive clones. Use TIFY10a-PGWC-F+T7 primer, TIFY10a-PGWC-R primer+SP6 primer (Table 5) for PCR detection and then send for sequencing. After culturing the colonies with correct sequencing, extract the plasmid and store it at -20℃ for subsequent experiments.

Table 5 Full-length primers with restriction endonuclease linker of intermediate vector PGWC:

Primer name Sequence (5′ to 3′) TIFY10a-PGWC-F ATGAATCATCACAACATGACA T7 Primer AAAGCAGGCTTTGACTTT SP6 Primers GCTGGGTCTAGAGACTT TIFY10a-PGWC-R TTAAAGTAACACATGGGTGG

3. Connect the final vector (pEarleyGate 100)

①LR reaction vector construction process:

Target fragment connected to the final vector: the fragment reference LR Clonase ^TM II Enzyme Mix Kit (Thermo Fisher Scientific, 11791020) instructions, connected to the final vector (pEarleyGate 100), the connection system is as follows:

Table 6:

After reacting at 25°C for 6 hours, the vector was transformed into E. coli DH5α to obtain DH5α/pEarleyGate 100-MsTIFY10a, that is, the MsTIFY10a fragment was inserted into the vector pEarleyGate 100 to obtain the vector pEarleyGate 100-MsTIFY10a; then the vector pEarleyGate 100-MsTIFY10a was transformed into E. coli DH5α to obtain DH5α/pEarleyGate 100-MsTIFY10a. The vector map of pEarleyGate 100-MsTIFY10a is shown in Figure 2, and the schematic diagram of the construction process is shown in Figure 3.

Positive clones were selected and detected using target gene F primers B816F1+PEG100seR (Table 7) to extract recombinant plasmid DNA, which was then detected using 1% agarose gel (*Agarose detection requires approximately 10 kb).

Table 7

4. Heat shock method to transform Agrobacterium

The plasmid was extracted from the positive clone strain DH5α/pEarleyGate 100-MsTIFY10a obtained in step 3 and transformed into the strain GV3101 (purchased from Bomade Biotechnology, BC304), and 10 μL (1 μg) was added to 100 μl of competent state. Use a pipette to gently mix, place on ice for 30 minutes, freeze in liquid nitrogen for 1 minute, heat shock at 37°C for 3 minutes, and place on ice for 2 minutes. Then, 600 μL YEB liquid culture medium was added to the clean bench and cultured on a shaker at 28°C~200pm for 2 hours. After centrifugation at 4000rpm~2min, part of the supernatant was removed, and the bacterial cell was resuspended and then coated on the YEB solid culture medium containing Kan (50 mg/ml) and Rif (50 mg/ml). After culturing at 28°C for 2 days, a single clone of the growing colony was picked and PCR detection was performed. The positive bacteria were added to a final concentration of 40% sterile glycerol and stored at -80°C.

The vector carries a plant resistance screening marker bialaphos resistance gene (bar), and can be positively screened using PPT. The constructed pEarleyGate 100 overexpression vector is transferred into the Agrobacterium GV3101 strain by heat shock method to obtain the strain GV3101/pEarleyGate 100-MsTIFY10a, and the gene is transferred into Medicago truncatula by Agrobacterium-mediated genetic transformation method.

Example 2. Genetic transformation method of Medicago truncatula

1. Seed disinfection

Treat the Medicago truncatula R108 seeds with concentrated sulfuric acid for (5-10) minutes to break dormancy, pour out all the sulfuric acid, immediately suspend the seeds with a large amount of clean water, rinse with distilled water for more than 3 times, place in a petri dish containing sterilized filter paper and culture in the dark for 3 days, then plant the germinated seedlings in a 10×10 flower pot filled with vermiculite and volcanic stone (2:1) at 25℃, 16/8, and use the leaves grown for 2-3 weeks as explants for infection. This step is performed in a clean bench.

2. Preparation of infection solution

100ul of successfully transformed Agrobacterium culture was pipetted into 150mL YEB liquid containing kanamycin (50mg/ml) and rifampicin (50mg/ml), placed in a 250mL conical flask, and cultured overnight at 28°C and 200rpm until OD600 = 0.8.

The shaken bacterial liquid was dispensed into 50mL centrifuge tubes in the clean bench, centrifuged at 4000r for 10min, the supernatant was poured out, and diluted with liquid culture medium to OD600=0.4 to obtain a bacterial suspension. This step was performed in the clean bench. The strains stored in the refrigerator can also be activated in a solid culture medium first, and then the activated bacteria are picked and placed in a fresh YEB liquid culture medium containing kanamycin and rifampicin for expansion culture. The subsequent culture conditions are as above. This step was performed in the clean bench.

3. Explant Sterilization

Place the leaves in a 50ml centrifuge tube for disinfection, 20-30 leaves per tube. Gently stir with 1/1000 Tween 20 for 15 minutes, rinse with sterile water 5 times until there is no foam; then gently shake with 6% sodium hypochlorite solution, disinfect for about 7 minutes, and rinse with sterile water 5 times. This step is carried out in a clean bench.

4. Leaf infection

Place the cleaned leaves on sterile filter paper to absorb the surface moisture, then use a scalpel to gently scratch the surface of the leaves, and place the treated leaves in the infection solution prepared in step 1; then place the centrifuge tube in a vacuum pump, evacuate for about 20 minutes, and then slowly release the vacuum. The purpose of evacuation is to allow Agrobacterium to better invade the leaves. After the vacuum is released, place the bacterial solution at room temperature and shake at 50-60r for 1-2 hours to allow the bacteria to invade. This step is performed in a clean bench.

5. Tissue Culture

(1) Co-cultivation:

In a clean bench, transfer the explant to a sterilized 9 cm culture dish and remove as much bacterial liquid as possible from the explant. Transfer the explant to solid Mt-CO medium (without antibiotics), with the abaxial end (back) in contact with the medium. Then seal the culture dish with sealing film and culture in the dark at 25°C for 1-2 days. The sealing film should be air-permeable and be careful not to allow excessive growth of Agrobacterium.

(2) Callus formation

Remove the leaf explants from the co-cultivation medium and gently wipe them on a new solid medium (Mt-YD1) to remove excess bacteria from the explants. Then transfer the explants to MtYD2 solid medium containing PPT. Incubate the plates in the dark at 25°C for 5-6 weeks, checking for contamination frequently. Resistant calli form approximately two weeks after infection, and transfer the resistant calli to new MtYD2 medium every 2-3 weeks.

(3) Embryoid formation

20-30 days after transfer to MtYD2 medium, the proembryo begins to transform into embryoids. Callus tissue is transferred to Ms-SY medium every 3 weeks until the proembryo appears (3-6 weeks). The embryoids at this stage are fluffy and begin to turn green. Each callus will form about 5-20 embryoids, and each explant will retain 5-10 embryoids and then form a single transgenic seedling.

(4) Plant formation

After about 2-3 weeks, the embryoids began to form plantlets. Once the plantlets were formed, they were transferred to Ms-SG medium for rooting induction.

(5) Hardening of seedlings:

Transfer the plantlets with a few leaves and roots from the Ms-SG medium to sterilized sandy soil and cover with a transparent plastic cover. Water the plants with tap water for the first two weeks, then change to nutrient solution. Keep the transparent plastic cover. Keep the soil moist, seal the lid for the first 5 days, then slowly open it to allow it to slowly adapt to the humidity. In the second week, remove the plastic cover and water the plants with nutrient solution. When the plants grow new plants in the greenhouse, they can be transplanted into pots. This is the T0 generation plant. In about 2-3 months, the inoculated seeds are the T1 generation plants.

6. Culture medium formulation

Preparation method of 1L co-culture medium (Mt-Co): Weigh 4.43g MS powder, add 30g sucrose, 4g agar, 5mg/L 2,4-D, 0.2mg/L KT, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, divide 25mL into culture medium, and store at 25℃ for later use.

Preparation method of 1L screening medium (Mt-YD1): Weigh 4.43g MS powder, add 30g sucrose, 4g agar, 5mg/L 2,4-D, 0.2mg/L KT, PPT, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 700mg/L cephalosporin, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L screening medium (Mt-YD2): Weigh 4.43g MS powder, add 30g sucrose, 4g agar, 5mg/L 2,4-D, 0.2mg/L KT, PPT, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 700mg/L cephalosporin, 3mg/L PPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L callus medium (Mt-SY): Weigh 4.43g MS powder, add 30g sucrose, 4g agar, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 700mg/L cephalosporin, 3mg/L PPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L germination medium: weigh 4.43g MS powder, add 30g sucrose, 7.5g agar, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1mL cephalosporin, 400μL PPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L rooting medium (Mt-SG): weigh 2.22g MS powder, add 15g sucrose, 3.5g agar, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, divide 25mL into culture medium, and store at 24℃ for later use.

Example 3. Efficient and rapid genetic transformation method of alfalfa

1. Preparation of infection solution

100ul of successfully transformed Agrobacterium culture was pipetted into 150mL YEB liquid containing kanamycin (50mg/ml) and rifampicin (50mg/ml), placed in a 250mL conical flask, and cultured overnight at 28°C and 200rpm until OD600 = 0.8.

The shaken bacterial solution was dispensed into 50 mL centrifuge tubes in a clean bench, centrifuged at 4000 r for 10 min, the supernatant was poured out, and diluted with liquid culture medium to OD600 = 0.4 to obtain a bacterial suspension. This step was performed in a clean bench.

Alternatively, the strains stored in the refrigerator can be activated in a solid culture medium first, and then the activated bacteria can be selected and cultured in a fresh YEB liquid culture medium containing kanamycin and rifampicin. The subsequent culture conditions are the same as above. This step is performed in a clean bench.

The Agrobacterium culture liquid was GV3101/pEarleyGate 100-MsTIFY10a or GV3101/pEarleyGate 100, GV3101/pEarleyGate 100-MsTIFY10a was a recombinant bacterium obtained by inserting a gene into a pEarleyGate 100 vector and then transforming it into GV3101; GV3101/pEarleyGate 100 was a recombinant bacterium obtained by transforming a pEarleyGate100 vector without an exogenous gene inserted into GV31015. The pEarleyGate 100 vector was purchased from the Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences, and the gene Bar was screened; GV3101 was purchased from Biomed Biotechnology, product catalog number BC304.

2. Explant Preparation

Take healthy alfalfa (variety: Zhongyuan No. 4) trifoliate leaves and put them into a culture bottle, stir them with 1/1000 Tween 20 for 15 minutes, rinse them with sterile water 5 times until there is no foam; add 75% alcohol to soak for 30 seconds, wash them with sterile water 3 times; add 40% sodium hypochlorite and stir gently for 10 minutes, and wash them with sterile water 5 times. This step is carried out in a clean bench.

3. Leaf infection

Place the cleaned leaves on sterile filter paper to absorb the surface moisture, then use a scalpel to gently cut several wounds on the leaf surface, and place the treated leaves into the infection solution prepared in step 1; then pour the infection solution and leaves into a silage bag (Figure 4A), use a vacuum packaging machine (manufacturer: Wenzhou Dafeng Machinery Co., Ltd., item number: DZ400/2D) (Figure 4B) to evacuate for 75 seconds when the heat sealing temperature is set to medium temperature, and use 40kHz ultrasonic waves for 5 minutes, and then cut the seal with sterile scissors in the clean bench, and then use the DZ400/2D vacuum packaging machine to evacuate for 75 seconds.

4. Tissue culture

The leaves were gently removed with tweezers in a clean bench, placed on sterile filter paper, and the leaf surface was air-dried. The leaves were transferred to the co-cultivation solid medium (Figure 5A) and co-cultivated for 3-5 days at 25°C in the dark.

When the leaves become hard (slightly curled at the edges), cut them in half and place them in the screening medium (Figure 5B). After being placed in the screening medium, subculture them to the callus medium 14 days later, and then place them in the differentiation medium for 7-10 days (Figure 5C). After obvious buds are formed, place them in the budding medium (Figure 5D). Subculture them every 14 days, and after 3-4 weeks, place them in the rooting medium (Figure 5E) when needles grow out. Rooting and hardening the seedlings will occur in about 2 weeks (Figure 5F) (the culture temperature is 25°C and the light cycle is 16/8h).

5. Statistics of genetic transformation cycle, see Table 8 for details.

The above culture medium formula is as follows:

Dichlorophenoxyacetic acid (2,4-Dichlorophenoxyacetic acid, referred to as 2,4-D) Manufacturer: Sigma Product No.: BB0615

6-Benzylaminopurine (6-BAP) Manufacturer: Sigma Product No.: BB0615

MS medium (without agar and sucrose) Manufacturer: Solarbio Item No.: M8521

Agar powder Agar Manufacturer: biosharp Product number: BS195

Sugarcane manufacturer: Tianjin Damao Chemical Reagent Factory AR99%

Cefotaxime sodium salt Manufacturer: Sigma Product No.: TC6515

Glyphosate (PPT Glyphosate) Manufacturer: Solarbio Item No.: G9130

Kinetin KT Manufacturer: Sigma Item No.: K3378

Naphthaleneacetic acid NAA Manufacturer: Sigma Product number: N0640

Preparation method of 1L infection liquid medium: weigh 4.43g MS powder, 30g sucrose, 4mL 2,4-D, 0.2mL 6-BAP, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, and store at 25℃ for later use.

Preparation method of 1L co-culture medium: weigh 4.43g MS powder, add 30g sucrose, 7.5g agar, 4mL 2,4-D, 0.2mL 6-BAP, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, divide 25mL into culture medium, and store at 25℃ for later use.

Preparation method of 1L screening medium: weigh 4.43g MS powder, add 30g sucrose, 7.5g agar, 4mL 2,4-D, 0.2mL 6-BAP, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1mL cephalosporin, 400μLPPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L callus medium: weigh 4.43g MS powder, add 30g sucrose, 7.5g agar, 2mL 2,4-D, 0.1mL 6-BAP, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1mL cephalosporin, 400μLPPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L differentiation medium: weigh 4.43g MS powder, add 30g sucrose, 7.5g agar, 1mL kinetin, 0.5mL 6-BAP, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1mL cephalosporin, 400μL PPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L germination medium: weigh 4.43g MS powder, add 30g sucrose, 7.5g agar, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1mL cephalosporin, 400μL PPT, 25mL each aliquot to the medium, store at 24℃ for later use.

Preparation method of 1L rooting medium: weigh 2.22g MS powder, add 15g sucrose, 3.5g agar, 1ml NAA, adjust pH to 5.8, add water to 1L. Sterilize at 121℃ high pressure steam for 15min, cool to 50℃, divide 25mL into culture medium, and store at 24℃ for later use.

Embodiment 4:

The difference from Example 3 is that the transformed alfalfa variety is different, Medicago truncatula R108 is used in this example, and the other method steps are the same as in Example 3.

Table 8

Comparative Example 1:

The difference from Example 2 is that the formulas of callus, bud and rooting medium are different. Specifically, the preparation method of 1L callus medium is as follows: 1L co-cultivation medium: weigh 4.43g MS powder, dissolve in 800ml ddH2O, add 30g sucrose, 4ml 2,4-D, 0.2ml 6-BAP, make up to 1000ml, adjust pH to 5.8, add 3.5g plant gel, sterilize at 121℃ high pressure steam for 15min, cool to 50℃, divide 25ml into medium, and store at 24℃ for later use.

1L screening medium: weigh 4.43g MS powder, dissolve in 800ml ddH2O, add 30g sucrose, 4ml 2,4-D, 0.2ml 6-BAP, dilute to 1000ml, adjust pH to 5.8, add 3.5g phytogenin, sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1ml cephalosporin, 1ml timentin, 400μl PPT, divide 25ml into medium, store at 24℃ for later use.

1L differentiation medium: weigh 4.43g MS powder, dissolve in 800ml ddH2O, add 30g sucrose, 1ml kinetin, 0.5ml 6-BAP, dilute to 1000ml, adjust pH to 5.8, add 3.5g phytagel, sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1ml cephalexin, 1ml timentin, 400μl PPT, divide 25ml into medium, store at 24℃ for later use.

1L rooting medium: weigh 2.22g MS powder, dissolve in 800ml ddH2O, add 15g sucrose, dilute to 1000ml, adjust pH to 5.8, add 3.5g phytagel, sterilize at 121℃ high pressure steam for 15min, cool to 50℃, add 1ml cephalosporin, 1ml timentin, 400μl PPT, divide 25ml into each aliquot into the medium, and store at 24℃ for later use.

As can be seen from the results in Table 1, the present invention provides a method for genetic transformation of alfalfa, which can be efficiently and stably transformed between different alfalfa varieties, with high transformation efficiency and short transformation time. As can be seen from the results of the embodiments of different alfalfa varieties of the present invention and the same comparative example, the configuration of the culture medium and the method of tissue culture are crucial to the efficiency of genetic transformation. Changing the formula of the callus culture medium obviously reduces the degree of browning and vitrification of callus tissue, increases the amount that can be used for callus, and greatly improves the efficiency of genetic transformation.

Example 5 Phenotypic Identification of MsTIFY10a Transgenic Plants

The leaves of the transformed plants in Example 2 were taken, and positive pure plants were identified by using Bar test paper and PCR to obtain seeds (Example 2).

Transgenic Medicago truncatula seeds overexpressing the MsTIFY10a gene showed darker seed coat color (Figure 6) and increased seed size (Figure 7) and weight (Figure 8) (Table 9). This indicates that the MsTIFY10a gene can participate in the regulation of plant seed coat color, seed size and weight, and can accelerate the breeding process of high-quality and high-yield varieties in plant genetic engineering.

Table 9 Seed size and 100-grain weight of MsTIFY10a transgenic Medicago truncatula

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. The alfalfa MsTIFY10a gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1; the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

2. A biological material comprising the gene of claim 1, said biological material being a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a non-regenerable plant part.

3. Use of the gene of claim 1 or the biomaterial of claim 2 for modulating the colour of the seed coat of a plant, in particular, by over-expression of the MsTIFY10a gene to darken the colour of the seed coat; the plant is preferably dicotyledonous plant, including alfalfa, soybean; preferably, the alfalfa is medicago truncatula or medicago sativa.

4. Use of the gene of claim 1 or the biological material of claim 2 for regulating the size of plant seeds or for regulating the weight of plant seeds.

5. A method for controlling the color of a plant seed coat, comprising: overexpressing the gene of claim 1 in plants by genetic engineering means;

the manner of overexpression is selected from the following 1) to 5), or an optional combination:

1) By introducing a plasmid having the gene;

2) By increasing the copy number of the gene on the plant chromosome;

3) By altering the promoter sequence of said gene on the plant chromosome;

4) By operably linking a strong promoter to the gene;

5) By introducing enhancers;

the plant is preferably dicotyledonous plant, including alfalfa, soybean; preferably, the alfalfa is medicago truncatula or medicago sativa;

preferably, an agrobacterium-mediated method is adopted to transfer the exogenous gene MsTIFY10a into the alfalfa plants and the alfalfa plants so as to obtain transgenic plants with over-expressed genes;

preferably, the MsTIFY10a gene is constructed on a plant expression vector pEarley gate 100, agrobacterium is transformed, then the alfalfa and alfalfa transformants carrying the MsTIFY10a gene are obtained by genetic transformation methods, and transgenic plants are selected.

6. A method of increasing the size and weight of a plant seed comprising: overexpressing the gene of claim 1 or 2 in a plant by genetic engineering means;

the manner of overexpression is selected from the following 1) to 5), or an optional combination:

1) By introducing a plasmid having the gene;

2) By increasing the copy number of the gene on the plant chromosome;

3) By altering the promoter sequence of said gene on the plant chromosome;

4) By operably linking a strong promoter to the gene;

5) By introducing enhancers;

the plant is preferably dicotyledonous plant, including alfalfa, soybean; preferably, the alfalfa is medicago truncatula or medicago sativa;

preferably, an agrobacterium-mediated method is adopted to transfer the exogenous gene MsTIFY10a into the alfalfa plants and the alfalfa plants so as to obtain transgenic plants with over-expressed genes;

preferably, the MsTIFY10a gene is constructed on a plant expression vector pEarley gate 100, agrobacterium is transformed, then the alfalfa and alfalfa transformants carrying the MsTIFY10a gene are obtained by genetic transformation methods, and transgenic plants are selected.

7. Use of a transgenic plant obtained according to the method of claim 6 in plant breeding.

8. The use according to claim 9, wherein the breeding method comprises transgenesis, crossing, backcrossing, selfing or asexual reproduction.

9. An efficient alfalfa genetic transformation method, the method comprising the steps of:

1) Mixing agrobacterium infection solution containing the gene of claim 1 or the biological material of claim 2 with the explant to be transformed, pouring into a silage bag, and obtaining the infected explant by adopting a vacuumizing and ultrasonic re-vacuumizing mode;

2) Transferring the infected explant to a co-culture solid culture medium, and culturing until the explant becomes hard and the edge slightly warps;

3) Transferring the explant cultured in the step 2) to a screening culture medium for subsequent generation to a callus culture medium for culture to obtain a callus;

4) Placing the callus in a differentiation culture medium for culture;

5) Placing the callus obtained in the step 4) in a bud culture medium containing a screening agent for induction culture to obtain an explant with cluster buds;

6) Placing the cluster bud explant in the step 5) in a rooting culture medium for rooting culture to obtain regenerated alfalfa plants, and realizing genetic transformation of alfalfa.

10. The genetic transformation method according to claim 9, wherein the explant is alfalfa three-out complex leaf; lightly scratching the surface of the explant; the conditions of vacuum were: pumping for 75s; the ultrasonic conditions are as follows: 40kHz,5min;

preferably, the formula of the callus culture medium is as follows: 4.43g MS powder was weighed, 30g sucrose, 7.5g agar, 2mL2,4-D,0.1mL6-BAP was added, the pH was adjusted to 5.8, and water was added to a volume of 1L.

Preferably, the rooting medium comprises the following formula: 4.43g MS powder was weighed, 15g sucrose, 3.5g agar, 1ml NAA was added, the pH was adjusted to 5.8, and water was added to a constant volume of 1L.