Application of CKX gene in regulation and control of alfalfa root system

Abstract

The invention discloses an application of a CKX gene in regulation and control of an alfalfa root system, which comprises the following steps of: 1, preparing a transgenic body of wild alfalfa, and over-expressing an MtCKX1 gene in the alfalfa to obtain a transgenic plant; the cDNA sequence and the amino acid sequence of the MtCKX1 gene are a sequence 1 and a sequence 2 in a sequence table; 2, comparing the root system of the alfalfa transgenic plant with the root system of a wild type plant, wherein the root system of the alfalfa transgenic plant is larger than that of the wild type plant; the cytokinin content of the transgenic plant is inhibited, and the root system of the transgenic plant is enhanced and enlarged; the cytokinin has a negative regulation effect in the aspect of root system growth of plants, after overexpression of the MtCKX1 gene, the cytokinin is inhibited, the root system of the plants is enhanced and enlarged, and the effects of well fixing soil and preventing soil loss are achieved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of CKX gene in regulating alfalfa root system. Background Art

[0002] Alfalfa is a perennial leguminous forage grass widely planted around the world, with the characteristics of high yield, good feeding quality and strong adaptability. Alfalfa can coexist with nitrogen-fixing rhizobia, and also has the characteristics of a well-developed root system, strong regeneration ability and long service life. It plays a significant role in intercepting runoff, preventing erosion and reducing soil erosion.

[0003] Cytokinin is a plant hormone with a variable molecular structure. It participates in regulating many processes of plant growth and development, including the growth of aerial parts and roots, leaf senescence, nutrient absorption, vascular system and flower development. Cytokinin also regulates the response of plants to biotic and abiotic stresses. The de novo synthesis of cytokinin in plants is catalyzed by isopentenyl transferase (IPT), while its catabolism is catalyzed by cytokinin dehydrogenase (CKX). Direct exogenous addition and endogenous increase or decrease of cytokinin content are commonly used methods to study the physiological functions of cytokinin. Among them, overexpressing CKX gene in plants through transgenic technology to increase CKX activity and reduce endogenous cytokinin content can provide more accurate results.

[0004] Alfalfa is mostly grown in grassland areas in northern my country. How to regulate the root growth of alfalfa is a very important research topic. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of a CKX gene capable of improving alfalfa root system in alfalfa root system regulation.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: application of CKX gene in alfalfa root regulation, comprising the following steps:

[0007] Step 1, preparing a transgenic wild-type alfalfa, overexpressing the MtCKX1 gene in the alfalfa, and obtaining a transgenic plant;

[0008] The cDNA sequence and amino acid sequence of the MtCKX1 gene are sequence 1 and sequence 2 in the sequence table;

[0009] Step 2: Compare the root system of the transgenic alfalfa plant with the root system of the wild-type plant. The root system of the transgenic alfalfa plant is larger than that of the wild-type plant.

[0010] As a preferred technical solution, in the step 1, the MtCKX1 gene is transformed into detached alfalfa leaves by Agrobacterium transformation, forming callus in a culture dish, which is then redifferentiated into seedlings.

[0011] As a preferred technical solution, the Agrobacterium transformation method comprises the following steps:

[0012] Step 1: construct the full-length MtCKX1 cDNA into a plant expression vector, transform the constructed vector into Agrobacterium EHA105 competent cells by electroporation, screen the positive clones, shake them at low and high speed until the OD is 0.4-0.6, and resuspend them in the infection buffer;

[0013] Step 2: After disinfecting the alfalfa leaves, cut off the edges in a clean bench, put them into the resuspended bacteria, and culture them on the co-culture medium for three days after vacuum pump treatment;

[0014] Step three, wash off the excess bacteria, culture on the culture medium until callus tissue is formed; move to light, grow seedlings on the differentiation medium, identify positive seedlings, and harvest.

[0015] As a preferred technical solution, among the differentiated seedlings, the expression level of the target gene in the positive seedlings is identified by real-time fluorescence quantitative PCR, and plants with different expression multiples are selected and transplanted outdoors to obtain transgenic plants.

[0016] As a preferred technical solution, the leaves of transgenic alfalfa plants and leaves of wild-type plants were taken to measure the cytokinin content. Overexpression of the MtCKX1 gene inhibited the overall cytokinin content of the transgenic plants.

[0017] As a preferred technical solution, the cytokinins measured include isopentenyl adenine iP, isopentenyl adenine riboside iPR, trans-zeatin tZ, trans-zeatin riboside tZR and dihydrozeatin DZ.

[0018] As a preferred technical solution, compared with wild-type plants, the transgenic plants have reduced contents of isopentenyl adenine iP, isopentenyl adenine nucleoside iPR and trans-zeatin nucleoside tZR, increased content of dihydrozeatin DZ, and trans-zeatin tZ is undetectable in the transgenic plants.

[0019] As a preferred technical solution, the wild alfalfa is alfalfa.

[0020] As a preferred technical solution, the MtCKX1 gene comes from Medicago truncatula.

[0021] As a preferred technical solution, the indexes for root comparison include main root length, total lateral root length, number of lateral roots, root surface area and root dry weight; the main root length, total lateral root length, number of lateral roots, root surface area and root dry weight of the root system of transgenic alfalfa plants are larger than those of the wild type.

[0022] Due to the adoption of the above technical scheme, the application of CKX gene in the regulation of alfalfa root system includes the following steps: step 1, preparing a transgenic body of wild-type alfalfa, overexpressing MtCKX1 gene in alfalfa, and obtaining transgenic plants; the cDNA sequence and amino acid sequence of the MtCKX1 gene are sequence 1 and sequence 2 in the sequence table; step 2, comparing the root system of the transgenic alfalfa plant with the root system of the wild-type plant, and the root system of the transgenic alfalfa plant is larger than that of the wild-type; after the MtCKX1 gene is overexpressed, the cytokinin content of the transgenic plant is inhibited, and the root system of the transgenic plant is strengthened and enlarged; cytokinin has a negative regulatory effect on the root growth of plants, and cytokinin can inhibit the growth of roots by inhibiting the division of plant root meristem cells and the proliferation of root elongation zone cells. After the MtCKX1 gene is overexpressed, the cytokinin is inhibited, and the root system of the plant is strengthened and enlarged, which plays a good role in fixing the soil and preventing soil loss. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the embodiments. In the following detailed description, only certain exemplary embodiments of the present invention are described by way of illustration. Needless to say, those of ordinary skill in the art will appreciate that the described embodiments may be modified in a variety of ways without departing from the spirit and scope of the present invention. Therefore, the description is illustrative in nature and is not intended to limit the scope of protection of the claims.

[0024] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0025] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0026] In the following examples involving quantitative detection, the experiment was repeated three times and the results were averaged.

[0027] The application of CKX gene in regulating alfalfa root system includes the following steps:

[0028] Step 1, preparing a transgenic wild-type alfalfa, overexpressing the MtCKX1 gene in the alfalfa, and obtaining a transgenic plant; the cDNA sequence and amino acid sequence of the MtCKX1 gene are sequence 1 and sequence 2 in the sequence table;

[0029] Step 2: Compare the root system of the transgenic alfalfa plant with the root system of the wild-type plant. The root system of the transgenic alfalfa plant is larger than that of the wild-type plant.

[0030] Furthermore, in the step 1, the MtCKX1 gene is transformed into detached leaves of alfalfa by Agrobacterium transformation, and callus tissue is formed in a culture dish, which is then redifferentiated into seedlings.

[0031] Furthermore, the Agrobacterium transformation method comprises the following steps:

[0032] Step 1: construct the full-length MtCKX1 cDNA into a plant expression vector, transform the constructed vector into Agrobacterium EHA105 competent cells by electroporation, screen the positive clones, shake them at low and high speed until the OD is 0.4-0.6, and resuspend them in the infection buffer;

[0033] Step 2: After disinfecting the alfalfa leaves, cut off the edges on a clean bench, put them into the resuspended bacteria, and culture them on a co-culture medium for three days after vacuum pump treatment; the culture medium is a differentiation medium containing 20 mg / mL hygromycin, 4 mg / L 2,4-D, and 0.5 mg / L BAP;

[0034] Step three, wash off the excess bacteria, culture on the culture medium until callus tissue is formed; move to light, grow seedlings on the differentiation medium, identify positive seedlings, and harvest.

[0035] Sequence 1:

[0036]

[0037] Sequence 2:

[0038] MVLKLVSFSKHVIFSFTKTIIILILSFILYKADSGCNNSITTSLVQISPYDVIISLQSQSFDGHLSLKDNEDAAKDFGNIHHFPPLAVLHPKTVSDISRTVKHIFEKGSDSELKVAARGHGHSLQGQAQAHQGLVI KMESLQSPEMKIQTGEFPFVDVSGGELWINILHETLKHGLAPKSWTDYLHLTVGGTLSNAGISGQAFRHGPQINNIFQLEIVTGKGEVVTCSENRNADLFHGVLGGLGQFGIITRARISLEPAPKMVKWIRVLYSD FSKFTRDQEYLISLKDKIDYIEGFVIINRTGILNGWRLSFDPKDPLQASQFNSDGKTFYCLEMAKYFNPDEADVMNQDVDHLLSQLSYIPPTLFLSEVSYVEFLDRVHVSEKKLRAQGLWEVHHPWLNLLIPRSEI HDFAKEVFGNILKDTSNGPILIYPVNQTRWNSKTSFVTPEEDVFYLVAFLTSAVPFSTGENSLEYILNQNKRILDFCTHAQLNVKQYLAHYDTQEEWQVHFGSQWGAFVERKRTYDPLALLAPGHRIFQKAVSSSI

[0039] Furthermore, among the differentiated seedlings, the expression level of the target gene in the positive seedlings was identified by real-time fluorescence quantitative PCR, and plants with different expression multiples were selected and transplanted outdoors to obtain transgenic plants.

[0040] Furthermore, the cytokinin content of leaves of alfalfa transgenic plants and leaves of wild-type plants was measured, and the overexpression of the MtCKX1 gene inhibited the overall cytokinin content of the transgenic plants.

[0041] The cytokinins measured include isopentenyl adenine iP, isopentenyl adenine nucleoside iPR, trans-zeatin tZ, trans-zeatin riboside tZR and dihydrozeatin DZ. Compared with wild-type plants, the transgenic plants have reduced contents of isopentenyl adenine iP, isopentenyl adenine nucleoside iPR and trans-zeatin riboside tZR, increased contents of dihydrozeatin DZ, and trans-zeatin tZ is not detected in transgenic plants. The specific test results are shown in the following table:

[0042] Table 1.1 Comparison of cytokinin content in leaves of wild-type and overexpression plants

[0043]

[0044] Furthermore, the wild type alfalfa is selected from alfalfa. The MtCKX1 gene is from alfalfa truncatula.

[0045] Furthermore, in the third step, the indexes for root system comparison include main root length, total lateral root length, number of lateral roots and root surface area.

[0046] The plant roots were laid flat in a transparent plastic culture dish (40 cm × 40 cm), and the entire root system was scanned using the Express ion 1000XL scanner. The WinRHIZO root / leaf analysis system was used to analyze and obtain root morphological indicators such as root length, number of lateral roots, and root surface area. The measured root data are shown in Table 1.2 below.

[0047] Table 1.2 Comparison of root systems of wild-type and transgenic plants

[0048]

[0049] Note: ** in the above table indicates extremely significant difference (P<0.01).

[0050] Experiment to determine the root development of overexpression plants: Each plastic flower pot contains 300g of dried garden soil, and the pot mouth is trimmed so that the soil surface is just at the edge of the pot. Six wild-type or overexpression plants are planted in each pot, with ten replicates. Cultivate normally for three months, and then place it in a vent for one month. During this period, regularly trim the pot mouth so that the pot mouth is flush with the soil surface. Then, collect the soil and alfalfa roots of each pot, dry and weigh them separately. The data are shown in Table 1.3 below.

[0051] Table 1.3 Comparison of root and soil dry weight between wild-type and transgenic plants

[0052]

[0053] Note: ** in the above table indicates extremely significant difference (P<0.01).

[0054] From the experimental data in Table 1.3, we can see that after the overexpression plants were placed in a wind outlet for one month, about 285g of the original 300g of garden soil remained, which is a great improvement compared to the experimental data of 238g of the wild type. The overexpression plants are more effective in preventing wind and fixing sand.

[0055] Through transgenic technology, the cytokinin dehydrogenase gene MtCKX1 was overexpressed in alfalfa, which reduced the endogenous content of alfalfa cytokinin, changed the properties of alfalfa, and increased the root system of alfalfa; alfalfa also limits soil erosion and improves soil tillage and structure due to its deep root system and perennial characteristics. Alfalfa can form a symbiotic relationship with rhizobia in the soil to fix nitrogen in the atmosphere, provide nitrogen for plants, and increase nitrogen fertilizer in the soil for the next round of crops; therefore, the transgenic alfalfa has a very beneficial effect in improving soil shape and preventing soil erosion.

[0056] Alfalfa, as the most widely cultivated plant in the genus Alfalfa, can be used as a representative of the genus Alfalfa to characterize the characteristics of all plants in the genus Alfalfa.

[0057] The above technical solution involves experimental methods, which are described in detail as follows:

[0058] 1. DNA extraction (CTAB method)

[0059] 1) Take 100 mg of fresh and mature plant material and place it in a mortar. Quickly add liquid nitrogen and grind it with a grinding rod. Transfer it to a 1.5 mL centrifuge tube.

[0060] 2) Add 200 μL of 2% (w / v) CTAB lysis buffer (as shown in Table 2.1) into the centrifuge tube and mix thoroughly.

[0061] Table 2.1 CTAB lysis buffer formula

[0062]

[0063] 3) Place in a 65°C water bath for 1 hour, inverting every 20 minutes.

[0064] 4) After the water bath, cool naturally and add 200 μL of chloroform to the centrifuge tube to mix well. This process should be gentle to prevent DNA breakage. After mixing, centrifuge at 12000 rpm for 5 minutes at room temperature.

[0065] 5) Transfer the supernatant to a clean centrifuge tube, add an equal volume of isopropanol, mix thoroughly, and centrifuge at 12000 rpm for 5 minutes at room temperature to precipitate DNA.

[0066] 6) Carefully remove the supernatant, add 600 mL of 70% ethanol to rinse the precipitate twice, and centrifuge at 12,000 rpm for 3 min at room temperature.

[0067] 7) Remove the ethanol, blow dry the precipitate, and add 30-50 μL ddH2O to dissolve the DNA.

[0068] 2. RNA extraction (TRIzol method)

[0069] 1) Take an appropriate amount of fresh and mature plant material and place it in a 2 mL centrifuge tube containing steel balls, add liquid nitrogen for quick freezing, grind it with a vibration grinder at 65 Hz for 2 min, add 1 mL of TRIzol to the centrifuge tube, shake it vigorously for 15 s, and let it stand at room temperature for 5 min.

[0070] 2) Add 200 μL of chloroform, mix by inversion, shake vigorously for 15 seconds, and let stand at room temperature for 2 minutes.

[0071] 3) Centrifuge at 12000 rpm for 15 min at 4°C, transfer the supernatant to a clean 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix slowly, and let stand at room temperature for 10 min.

[0072] 4) Centrifuge at 12000 rpm for 10 min at 4°C, remove the supernatant, wash the precipitate twice with 500 μL 75% ethanol, centrifuge at 7000 rpm for 5 min at 4°C, and blow dry the ethanol in a clean bench.

[0073] 5) Add 20-30 μL RNase-free water to dissolve the precipitate.

[0074] 3. RNA Reverse Transcription

[0075] 1) Reverse transcription system 1 (Table 2.2).

[0076] Table 2.2 RNA reverse transcription system 1 reagent composition table

[0077]

[0078] 2) Mix the above system and place in a 42°C water bath for 2 min, then immediately place in an ice bath.

[0079] 3) Add reaction system 2 (Table 2.3).

[0080] Table 2.3 RNA reverse transcription system 2 reagent composition table

[0081]

[0082] 4) Mix the above system and place in a 37°C water bath for 30 min and a 85°C high temperature water bath for 15 s to inactivate the enzyme. Then dilute the cDNA obtained by reverse transcription 10 times and store it in a -20°C refrigerator.

[0083] 4. Real-time quantitative PCR (RT-qPCR)

[0084] To ensure the accuracy of fluorescence quantification, 10 μL of the reaction system was divided into two mixed systems and added, with 8.8 μL of enzyme system and 1.2 μL of primer system added to each reaction well. Each sample was repeated 3 times.

[0085] 1) 10 μL reaction system (Table 2.4).

[0086] Table 2.4 RT-qPCR system

[0087]

[0088]

[0089] 2) Real-Time system program was performed according to the SYBR green mix instruction manual.

[0090] 5. Alfalfa seed germination treatment

[0091] 1) Place Medicago truncatula or Alfalfa seeds in a centrifuge tube, add concentrated sulfuric acid to submerge the seeds, shake upside down for 6 minutes, aspirate the concentrated sulfuric acid, and wash with tap water for 4 to 5 times.

[0092] 2) Spread the cleaned seeds on a 0.8% agar plate and place in a 4°C refrigerator away from light for 48 hours (vernalization).

[0093] 3) Take out the seeds after vernalization and germinate them at 26℃ in the dark for about 24 hours. After the roots grow to 2-3 cm, move them to a mixture of vermiculite and black soil at a ratio of 2:1 (v / v) for cultivation. The cultivation conditions are 14 hours of light and a light intensity of 200 μmol m -2 s -1 , temperature 26℃, dark 10h, temperature 20℃.

[0094] 6. Expression vector construction

[0095] 1) Design primers for target gene Medtr7g090920 (Medicago truncatula MtCKX1) (Table 2.5). The transformation vector is pMDC32 preserved in the laboratory.

[0096] 2) Using Medicago truncatula cDNA as template, configure the PCR amplification system as shown in Table 2.6, and use the high-fidelity amplification enzyme HSDNA polymerase.

[0097] 3) Perform PCR amplification. The reaction procedure is shown in Table 2.7.

[0098] 4) The obtained PCR amplification products were separated and identified by agarose gel electrophoresis. The preparation of 50×TAE nucleic acid electrophoresis buffer is as follows: 242g Tris base and 37.2g Na2EDTA·H2O were weighed and dissolved in deionized water. After complete dissolution, about 40mL acetic acid was added, and the volume was adjusted to 1L after sufficient stirring, and the pH was adjusted to 8.8.

[0099] Table 2.5 Target gene primers

[0100]

[0101] Table 2.6 PCR amplification system

[0102]

[0103]

[0104] Table 2.7 PCR reaction program

[0105]

[0106] 5) Cut the gel containing the target fragment and use a DNA gel recovery kit to recover the PCR product.

[0107] 6) Use the corresponding restriction endonuclease to cut the corresponding large vector, and the cutting system is shown in Table 2.8. After cutting, place in a 37°C water bath for 3 hours, and then perform agarose gel electrophoresis on the enzyme cutting product, and use a kit to recover the product.

[0108] Table 2.8 Enzyme digestion reaction system

[0109]

[0110] 7) Connect the recovered vector fragment to the target fragment. The connection system is shown in Table 2.9. Incubate in a 50°C water bath for 15 minutes to obtain the connection product.

[0111] Table 2.9 Ligation reaction system

[0112]

[0113] 8) The ligation product was transformed into Escherichia coli, and the strain was the Escherichia coli Trans-T1 strain purchased from Transgen. The transformation method is as follows:

[0114] a) Freeze-thaw E. coli competent cells on ice, add 10 μL of the above ligation product to 50 μL of frozen-thawed E. coli competent cells, mix gently, avoid violent shaking, and place on ice for 30 min.

[0115] b) After bathing in 42℃ water for 1 min, quickly remove the tube and place it in an ice box for 2 min.

[0116] c) Add YEB liquid culture medium without screening antibiotics to the centrifuge tube, prepare the concentration as shown in Table 2.10, sterilize at high temperature and high pressure after preparation, and store at room temperature. Place the mixed YEB culture medium in a shaker at 37°C and 120 rpm for 1 hour.

[0117] d) Spread the recovered product on a screening medium containing the antibiotic corresponding to the vector, and after the plate is air-dried, seal it with plastic wrap and incubate it upside down in a 37°C incubator.

[0118] Table 2.10YEB medium components

[0119]

[0120] e) The next day, pick the monoclonal colonies grown on the culture medium and place them in a liquid culture medium containing the selection antibiotics, and place them in a shaker at 37°C and 220rpm. Perform PCR identification of the bacterial solution 3 hours later. The PCR system is shown in Table 2.11, and the procedure is shown in Table 2.7, but the first step of pre-denaturation time is extended to 10 minutes to allow the bacteria to fully lyse and release the genome.

[0121] Table 2.11 Bacterial liquid PCR reaction system

[0122]

[0123] f) Select positive clones and send them to a gene company for sequencing. The sequencing results are compared using DNAMAN software. Add 15% glycerol to the correct bacterial solution and store it in a -80°C refrigerator.

[0124] 7. Plasmid extraction

[0125] 1) Take 50 μL of the bacterial solution with the correct sequence alignment and place it in a sterilized 10 mL centrifuge tube, add 7 mL of YEB liquid culture medium with the corresponding resistance, place it in a shaker, and culture it at 37°C and 200 rpm for 8 hours.

[0126] 2) Pour the bacterial solution into a 2 mL centrifuge tube and centrifuge at 12000 rpm for 1 min.

[0127] 3) Pour off the supernatant, add 250 μL of Solution 1 in the plasmid extraction kit, and use a pipette to suspend the cells.

[0128] 4) Add 250 μL of solution 2, invert 6 to 8 times, and gently mix until clear.

[0129] 5) Add 250 μL of solution 3, invert 6 to 8 times, and gently mix until flocculent precipitation is produced. Centrifuge at 12000 rpm for 10 min, and transfer the supernatant to the adsorption column.

[0130] 6) Place the adsorption column in a centrifuge and centrifuge at 12000 rpm for 1 min. Pour away the waste liquid and place it in a new collection tube.

[0131] 7) Add 700 μL of rinse solution W2 to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid and place it in a new collection tube.

[0132] 8) Repeat the previous step, centrifuge at 12000 rpm for 2 min, pour out the waste liquid, and let it stand at room temperature until the remaining rinse liquid is completely dried.

[0133] 9) Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 μL of ddH2O (previously placed in a 65°C water bath) to the middle of the adsorption film, place at room temperature for 10 min, centrifuge at 12000 rpm for 2 min, and collect the solution in a centrifuge tube.

[0134] 8. Preparation of competent cells of Agrobacterium

[0135] 1) Dip a small amount of GV3101 / EHA105 Agrobacterium culture liquid on solid YEB medium containing 100 μg / mL rifampicin. After constant temperature cultivation at 28°C for 48 hours, pick out Agrobacterium monoclonal plaques and place them in 5 mL liquid YEB medium containing 100 μg / mL rifampicin. Place them in a shaker and culture them overnight at 28°C and 220 rpm.

[0136] 2) Add the above bacterial solution into 50 mL of liquid YEB medium containing 100 μg / mL rifampicin, place in a shaker, and culture at 28°C and 220 rpm until the OD value reaches 0.5.

[0137] 3) Transfer the above bacterial solution to a 50 mL centrifuge tube and place it on ice for 30 min.

[0138] 4) Place in a centrifuge, centrifuge at 4°C, 4000 rpm for 10 min, discard the supernatant, retain the bacterial pellet, and resuspend the bacteria in 10 mL of pre-cooled ddH2O.

[0139] 5) Place in a centrifuge, centrifuge at 4°C, 4000 rpm for 10 min, discard the supernatant, retain the bacterial pellet, resuspend the bacteria in 10 mL of pre-cooled 10% glycerol, divide into 50 μL / centrifuge tubes and store in a -80°C refrigerator.

[0140] 9. Electroporation of Agrobacterium

[0141] 1) Soak the cuvette in 75% alcohol to clean it. Use a 1mL pipette to absorb the alcohol and focus on blowing and cleaning the gaps in the cuvette.

[0142] 2) Place the electric shock cup on the filter paper and place it in a clean bench to blow dry, and sterilize it under ultraviolet light for 30 minutes.

[0143] 3) Cover the sterilized electric shock cup with a lid and place it in an ice box for precooling.

[0144] 4) Add 500 μL of liquid YEB medium without antibiotics to a sterilized 1.5 mL centrifuge tube.

[0145] 5) Take 50 μL of Agrobacterium EHA105 competent cells, add 1-2 μL of plasmid, mix gently and slowly inject it along the gap of the electroporation cup to prevent bubbles, cover the lid, and electroporate at 2000V for about 50ms.

[0146] 6) Rapidly transfer the electroporated bacterial solution to the culture medium in step 4 and place it in a shaker at 28°C and 120 rpm for at least 1 hour.

[0147] 7) Spread the transformed and recovered bacterial solution on a solid YEB medium containing 50 μg / mL kanamycin and 100 μg / mL rifampicin, place in an incubator, and culture inverted at 28°C for 48 hours. Pick a single clone plaque for bacterial solution PCR and save the positive clone bacterial solution.

[0148] 10. Agrobacterium transformation of alfalfa leaves

[0149] 1) Except for the culture medium, all items required for the experiment were sterilized twice by high temperature and high pressure.

[0150] 2) Pick Agrobacterium-positive monoclonal plaques onto 5 mL of solid YEB medium containing 50 μg / mL kanamycin and 100 μg / mL rifampicin, place in a shaker, and culture at 28°C and 220 rpm for 1 day.

[0151] 3) Perform bacterial liquid PCR to verify the target gene.

[0152] 4) Add the positive bacteria to 50 mL of liquid YEB medium containing 50 μg / mL kanamycin and 100 μg / mL rifampicin, place in a shaker, and culture at 28°C and 220 rpm until the OD value reaches 0.5.

[0153] 5) Use a 50 mL centrifuge tube to precipitate the bacteria, place it in a centrifuge, and centrifuge at room temperature, 4000-5000 rpm for 10 minutes.

[0154] 6) Place the leaves in a 50 mL screw-top flat-bottom plastic tube, soak in 75% alcohol for 1 min, wash with ddH2O 3 times, soak in 10% sodium hypochlorite for 10 min, shake constantly, and wash with ddH2O 3 to 5 times. Cut off the edges of the leaves and transfer to a conical flask.

[0155] 7) After resuspending the bacterial pellet in liquid transformation medium, transfer it to a conical flask with leaves, evacuate the medium for 10 minutes, and then place it in a shaker at 25°C and 100 rpm for 1 hour.

[0156] 8) Take out the leaves and spread them with the front side facing up on solid transformation medium. Culture them in the dark at 25°C. The composition of the medium is shown in Table 2.12.

[0157] 9) After 3 days, take out the leaves one by one and place them in a conical flask. Wash off the Agrobacterium attached to the outside of the leaves with sterile ddH2O, and then spread them flat on new transformation medium again to continue dark culture.

[0158] 10) The culture medium was replaced every two weeks on average. When the callus tissue grew to a certain extent, it was moved to the light culture medium for light culture. The composition of the culture medium is shown in Table 2.12, and the composition of the storage solution for preparing the culture medium is shown in Table 2.13.

[0159] 11) After the callus turns green, hygromycin is no longer added to the culture medium, and the callus is transferred to the rooting medium for cultivation. After rooting, it is removed from the triangular bottle and first hydroponically cultivated to adapt to the external environment. The pH of the hydroponic nutrient solution is 6.0. Then it is moved to a mixture of vermiculite and black soil. The components of the hydroponic nutrient solution and storage solution are shown in Table 2.14 and Table 2.15.

[0160] Table 2.12 Transformation-related culture medium components

[0161]

[0162]

[0163] Table 2.13 Composition of transformation medium stock solution

[0164]

[0165] Table 2.14 Alfalfa culture medium components

[0166]

[0167]

[0168] Table 2.15 Alfalfa culture medium storage solution composition

[0169]

[0170] 11. Identification of positive transgenic alfalfa seedlings

[0171] 1) Extract DNA from leaves of transgenic plants and use Hyg-S / A shown in Table 2.16 as primers for PCR and hygromycin identification. The PCR product of positive plants has bands in electrophoresis, while the wild type has no bands.

[0172] Table 2.16 Primers for identification of transgenic positive plants

[0173]

[0174] 2) RNA was extracted from leaves of transgenic plants and RT-qPCR was performed to identify the expression level of the target gene. The primers used are shown in Table 2.17.

[0175] Table 2.17 RT-qPCR primers

[0176]

[0177]

[0178] 12. Cutting propagation of positive transgenic alfalfa plants

[0179] Select transgenic line plants with different expression multiples, and select upright and strong branches as mother branches at the budding stage. Cut a small section of about 5 cm long with one leaf node from the middle and upper part of the mother branch at a certain angle to the branch as a cutting. Wrap it with a sponge strip as soon as possible after cutting and fix it in a hydroponic box containing alfalfa nutrient solution. After new roots and stems are generated, transplant it into a soil pot.

[0180] 13. Transgenic alfalfa positive seedlings are transplanted to the greenhouse for soil culture

[0181] Transgenic alfalfa plants of different lines were selected and transplanted to the greenhouse of this group for soil culture. To avoid the error of nutrients in different plots, single-factor random blocks were selected for transplantation. A total of 9 blocks were set up, marked as A to I; each block was divided into 3 small plots, marked as 1 to 3; 3 cuttings from the same line plant were transplanted in each small plot.

[0182] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of CKX gene in regulating alfalfa root system, characterized in that: The steps include: Step 1, preparing a transgenic wild-type alfalfa, overexpressing the MtCKX1 gene in the alfalfa, and obtaining a transgenic plant; The cDNA sequence and amino acid sequence of the MtCKX1 gene are sequence 1 and sequence 2 in the sequence table; Step 2: Compare the root system of the transgenic alfalfa plant with the root system of the wild-type plant. The root system of the transgenic alfalfa plant is larger than that of the wild-type plant.

2. The use of the CKX gene in alfalfa root regulation as claimed in claim 1, characterized in that: In the step 1, the MtCKX1 gene is transformed into detached leaves of alfalfa by Agrobacterium transformation, callus is formed in a culture dish, and then redifferentiated into seedlings.

3. The use of the CKX gene in regulating alfalfa root system as claimed in claim 2, characterized in that: The Agrobacterium transformation method comprises the following steps: Step 1: construct the full-length MtCKX1 cDNA into a plant expression vector, transform the constructed vector into Agrobacterium EHA105 competent cells by electroporation, screen the positive clones, shake them at low and high speed until the OD is 0.4-0.6, and resuspend them in the infection buffer; Step 2: After disinfecting the alfalfa leaves, cut off the edges in a clean bench, put them into the resuspended bacteria, and culture them on the co-culture medium for three days after vacuum pump treatment; Step three, wash off the excess bacteria, culture on the culture medium until callus tissue is formed; move to light, grow seedlings on the differentiation medium, identify positive seedlings, and harvest.

4. The use of the CKX gene in regulating alfalfa root system as claimed in claim 2, characterized in that: Among the differentiated seedlings, the expression level of the target gene in the positive seedlings was identified by real-time fluorescence quantitative PCR, and plants with different expression multiples were selected and transplanted outdoors to obtain transgenic plants.

5. The use of the CKX gene in regulating alfalfa root system according to claim 1, characterized in that: The cytokinin content in the leaves of transgenic alfalfa plants and wild-type plants was determined. Overexpression of the MtCKX1 gene inhibited the overall cytokinin content in the transgenic plants.

6. The use of the CKX gene in regulating alfalfa root system according to claim 5, characterized in that: The cytokinins tested included isopentenyl adenine iP, isopentenyl adenine riboside iPR, trans-zeatin tZ, trans-zeatin riboside tZR and dihydrozeatin DZ.

7. The use of the CKX gene in regulating alfalfa root system according to claim 6, characterized in that: Compared with wild-type plants, the contents of isopentenyl adenine iP, isopentenyl adenine nucleoside iPR and trans-zeatin riboside tZR in transgenic plants are reduced, the content of dihydrozeatin DZ is increased, and trans-zeatin tZ is undetectable in transgenic plants.

8. The use of the CKX gene in regulating alfalfa root system according to claim 1, characterized in that: The wild type alfalfa is selected from alfalfa.

9. The use of the CKX gene in regulating alfalfa root system according to claim 1, characterized in that: The MtCKX1 gene is from Medicago truncatula.

10. Use of the CKX gene according to any one of claims 1 to 9 in regulating alfalfa root system, characterized in that: The indexes of root system comparison included main root length, total lateral root length, number of lateral roots, root surface area and root dry weight. The main root length, total lateral root length, number of lateral roots, root surface area and root dry weight of transgenic alfalfa plants were larger than those of wild-type roots.

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