Application of CKX genes in regulating the root system of alfalfa

Abstract

The invention discloses an application of a CKX gene in regulating alfalfa root systems, comprising the following steps: 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, comparing the root systems of the transgenic alfalfa plant with those of the wild-type plant, and the root systems of the transgenic alfalfa plant are larger than those of the wild-type plant; the cytokinin content of the transgenic plant is suppressed, and the root systems of the transgenic plant are strengthened and enlarged; cytokinin has a negative regulatory effect on the root growth of the plant, and after the MtCKX1 gene is overexpressed, the cytokinin is suppressed, and the root system of the plant is strengthened and enlarged, thereby effectively fixing the soil and preventing soil loss.

Description

Technical Field

[0001] The present 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 legume forage widely cultivated worldwide, characterized by high yield, excellent forage quality, and strong adaptability. Alfalfa can coexist with nitrogen-fixing rhizobia, and its well-developed root system, strong regeneration capacity, and long lifespan make it particularly effective in intercepting runoff, preventing erosion, and reducing soil erosion.

[0003] Cytokinins are plant hormones with diverse molecular structures that regulate numerous processes in plant growth and development, including aerial and root growth, leaf senescence, nutrient absorption, vascular system development, and flower development. Cytokinins also regulate plant responses to biotic and abiotic stresses. Cytokinins are synthesized de novo in plants by isopentenyl transferase (IPT), while their catabolism is catalyzed by cytokinin dehydrogenase (CKX). Direct exogenous addition and endogenous enhancement or reduction of cytokinin levels are commonly used methods to study the physiological functions of cytokinins. Transgenic overexpression of the CKX gene in plants to enhance CKX activity and reduce endogenous cytokinin levels offers more precise 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] 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: producing a transgenic wild-type alfalfa, overexpressing the MtCKX1 gene in the alfalfa to obtain 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: Comparing the root system of the transgenic alfalfa plant with that 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 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 includes 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 for positive clones, shake gently and vigorously until the OD = 0.4-0.6, and resuspend in 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-cultivation medium for three days after vacuum pump treatment;

[0014] Step 3: Clean the excess bacteria and culture on the culture medium until callus tissue is formed; move to the 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 cytokinin content of leaves of transgenic alfalfa plants and leaves of wild-type plants was measured. Overexpression of the MtCKX1 gene suppressed the overall cytokinin content of the transgenic plants.

[0017] As a preferred technical solution, the cytokinins measured include isopentenyl adenine iP, isopentenyl adenine nucleoside iPR, trans-zeatin tZ, trans-zeatin nucleoside 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 is from Medicago truncatula.

[0021] As a preferred technical solution, the root system comparison indicators 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 the alfalfa transgenic plant are larger than those of the wild type.

[0022] Due to the adoption of the above technical solution, the application of the CKX gene in alfalfa root regulation includes the following steps: 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, comparing the root system of the transgenic alfalfa plant with that of the wild-type plant, and the root system of the transgenic alfalfa plant is larger than that of the wild-type plant; after overexpressing the MtCKX1 gene, the cytokinin content of the transgenic plant is suppressed, and the root system of the transgenic plant is strengthened and enlarged; cytokinin has a negative regulatory effect on plant root growth. Cytokinin can inhibit root growth by inhibiting the division of plant root meristem cells and the proliferation of root elongation zone cells. After overexpressing the MtCKX1 gene, cytokinin is suppressed, and the plant root system is strengthened and enlarged, which effectively fixes the soil and prevents soil erosion. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the following examples. In the following detailed description, certain exemplary embodiments of the present invention are described by way of illustration only. It is understood that those skilled in the art will recognize that the described embodiments may be modified in various 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 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 commercially available.

[0026] In the following examples involving quantitative detection, the experiments were 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 listing;

[0029] Step 2: Comparing the root system of the transgenic alfalfa plant with that 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 alfalfa leaves by Agrobacterium transformation, forming callus tissue 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 for positive clones, shake gently and vigorously until the OD = 0.4-0.6, and resuspend in infection buffer;

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

[0034] Step 3: Clean the excess bacteria and culture on the culture medium until callus tissue is formed; move to the 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 levels of the target gene in the positive seedlings were 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 in leaves of transgenic alfalfa plants and leaves of wild-type plants was measured. Overexpression of the MtCKX1 gene suppressed the overall cytokinin content in the transgenic plants.

[0041] The cytokinins tested included isopentenyladenine iP, isopentenyladenine riboside iPR, trans-zeatin tZ, trans-zeatin riboside tZR, and dihydrozeatin DZ. Compared to wild-type plants, the transgenic plants showed decreased levels of isopentenyladenine iP, isopentenyladenine riboside iPR, and trans-zeatin riboside tZR, increased levels of dihydrozeatin DZ, and undetectable trans-zeatin tZ. The specific test results are shown in the table below:

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

[0043]

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

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

[0046] Plant roots were spread flat in a transparent plastic Petri dish (40 cm × 40 cm). The entire root system was scanned using an Express ion 1000XL scanner. Root morphology metrics such as root length, lateral root number, and root surface area were analyzed using the WinRHIZO root / leaf analysis system. 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 differences (P<0.01).

[0050] To measure the root development of overexpressing plants, each plastic pot was filled with 300 g of dried garden soil. The pot rims were trimmed so that the soil surface was just above the pot edge. Six wild-type or overexpressing plants were planted in each pot, with ten replicates. The plants were cultured normally for three months and then placed in a well-ventilated area for one month. During this period, the pot rims were periodically trimmed to level the pot rims with the soil surface. The soil and alfalfa roots from each pot were then harvested, dried, and weighed. 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 vent 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 genetic engineering, the cytokinin dehydrogenase gene MtCKX1 was overexpressed in alfalfa, reducing endogenous cytokinin levels and altering the plant's characteristics, resulting in a larger root system. Alfalfa's deep root system and perennial nature also limit soil erosion and improve soil tillage and structure. Alfalfa forms a symbiotic relationship with soil rhizobia, fixing atmospheric nitrogen, providing nitrogen to plants and increasing nitrogen fertilizer in the soil for the next crop cycle. Therefore, genetically modified alfalfa has a significant impact on improving soil quality 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 introduced 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) to 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 cooling naturally in the water bath, add 200 μL of chloroform to the centrifuge tube and mix thoroughly. Be gentle to prevent DNA breakage. Centrifuge at 12,000 rpm for 5 minutes at room temperature.

[0065] 5) Transfer the supernatant to a clean centrifuge tube, add an equal volume of isopropanol, and mix thoroughly. Centrifuge at 12,000 rpm for 5 minutes at room temperature to precipitate the 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 minutes at room temperature.

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

[0068] 2. RNA extraction (TRIzol method)

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

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

[0071] 3) Centrifuge at 12,000 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 gently, 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 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

[0077]

[0078] 2) Mix the above system and place in a 42°C water bath for 2 minutes, 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

[0081]

[0082] 4) Mix the above system and incubate in a 37°C water bath for 30 minutes and then in an 85°C water bath for 15 seconds to inactivate the enzyme. Then dilute the cDNA obtained by reverse transcription 10-fold and store in a -20°C refrigerator.

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

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

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

[0086] Table 2.4 RT-qPCR system

[0087]

[0088]

[0089] 2) The Real-Time system procedure 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 them upside down for 6 minutes, aspirate the concentrated sulfuric acid, and rinse with tap water 4 to 5 times.

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

[0093] 3) Remove the vernalized seeds and germinate them in the dark at 26°C for about 24 hours. After the roots grow to 2-3 cm, transfer 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 the target gene Medtr7g090920 (Medicago truncatula MtCKX1) (Table 2.5). The transformation vector was pMDC32 stored 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 PCR amplification products were separated and identified by agarose gel electrophoresis. To prepare 50×TAE nucleic acid electrophoresis buffer, weigh 242 g of Tris base and 37.2 g of Na₂EDTA·H₂O and dissolve them in deionized water. Once completely dissolved, add approximately 40 mL of acetic acid, stir thoroughly, and dilute to 1 L. Adjust the pH 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. The cutting system is shown in Table 2.8. After cutting, incubate in a 37°C water bath for 3 hours. Then, perform agarose gel electrophoresis on the enzyme-digested products and recover them using a kit.

[0108] Table 2.8 Enzyme digestion reaction system

[0109]

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

[0111] Table 2.9 Ligation reaction system

[0112]

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

[0114] a) Thaw the E. coli competent cells on ice. Add 10 μL of the ligation product to 50 μL of the frozen-thawed E. coli competent cells. Mix gently, avoiding violent shaking. Incubate on ice for 30 minutes.

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

[0116] c) Add YEB liquid medium without the selected antibiotic to the centrifuge tube at the concentrations shown in Table 2.10. Sterilize by high-temperature and high-pressure sterilization after preparation and store at room temperature. Place the mixed YEB medium in a shaker at 37°C, 120 rpm, and recover for 1 hour.

[0117] d) Spread the recovered product onto a selection medium containing the corresponding antibiotic for the vector. 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 a single colony growing on the culture medium and place it in liquid medium containing the selection antibiotic. Incubate on a shaker at 37°C, 220 rpm. After 3 hours, perform PCR analysis on the culture medium. The PCR system is as shown in Table 2.11, and the procedure is as shown in Table 2.7, but extend the first pre-denaturation time to 10 minutes to allow for complete bacterial lysis and release of the genome.

[0121] Table 2.11 Bacterial liquid PCR reaction system

[0122]

[0123] f) Select positive clones and send them to a genetic company for sequencing. The sequencing results are compared using DNAMAN software. The correct bacterial solution is added with 15% glycerol and stored in a -80°C refrigerator.

[0124] 7. Plasmid Extraction

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

[0126] 2) Pour the bacterial solution into a 2 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min.

[0127] 3) Discard the supernatant, add 250 μL of Solution 1 from 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 mix gently until clear.

[0129] 5) Add 250 μL of Solution 3 and gently mix by inverting 6-8 times until a flocculent precipitate forms. Centrifuge at 12,000 rpm for 10 minutes and transfer the supernatant to an adsorption column.

[0130] 6) Place the adsorption column in a centrifuge and centrifuge at 12,000 rpm for 1 minute. Discard 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 12,000 rpm for 1 min, discard the waste liquid, and place it in a new collection tube.

[0132] 8) Repeat the previous step, centrifuge at 12,000 rpm for 2 minutes, discard 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 and drop 50 μL of ddH2O (previously placed in a 65°C water bath) onto the center of the adsorption film. Allow to stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 2 minutes and collect the solution in a centrifuge tube.

[0134] 8. Preparation of competent Agrobacterium

[0135] 1) Apply a small amount of GV3101 / EHA105 Agrobacterium culture liquid to solid YEB medium containing 100 μg / mL rifampicin. After incubation at 28°C for 48 h, pick out a single colony of Agrobacterium and transfer it to 5 mL of liquid YEB medium containing 100 μg / mL rifampicin. Incubate the culture in a shaker at 28°C, 220 rpm, and incubate overnight.

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

[0137] 3) Transfer the bacterial suspension to a 50 mL centrifuge tube and place on ice for 30 minutes.

[0138] 4) Centrifuge the tube at 4000 rpm for 10 min at 4°C. Discard the supernatant, retain the bacterial pellet, and resuspend the cells in 10 mL of pre-chilled ddH2O.

[0139] 5) Centrifuge at 4000 rpm for 10 min at 4°C. Discard the supernatant and retain the bacterial pellet. Resuspend the cells in 10 mL of pre-chilled 10% glycerol. Aliquot 50 μL / tube and store in a -80°C refrigerator.

[0140] 9. Electroporation of Agrobacterium

[0141] 1) Soak and clean the cuvette with 75% alcohol. Use a 1 mL pipette to draw up the alcohol, focusing on cleaning the gaps in the cuvette.

[0142] 2) Place the electric shock cup on filter paper and place it in a clean bench to air 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 antibiotic-free liquid YEB medium to a sterilized 1.5 mL centrifuge tube.

[0145] 5) Take 50 μL of Agrobacterium tumefaciens EHA105 competent cells, add 1-2 μL of plasmid, mix gently, and slowly inject the cells through the slit of the electroporation cuvette to prevent bubbles. Close the lid and electroporate at 2000 V for approximately 50 ms.

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

[0147] 7) Spread the transformed and recovered bacterial suspension onto 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 suspension PCR and save the positive clone bacterial suspension.

[0148] 10. Agrobacterium transformation of alfalfa leaves

[0149] 1) Except for the culture medium, all items required for the experimental process 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, and culture in a shaker at 28°C and 220 rpm for 1 day.

[0151] 3) Perform PCR on the bacterial solution 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, and culture in a shaker at 28°C and 220 rpm until the OD value reaches 0.5.

[0153] 5) Use a 50 mL centrifuge tube to pellet the cells, place in a centrifuge, and centrifuge at 4000-5000 rpm for 10 min at room temperature.

[0154] 6) Place the leaf in a 50 mL screw-top, flat-bottomed plastic tube. Soak in 75% ethanol for 1 minute. Rinse three times with ddH2O. Soak in 10% sodium hypochlorite for 10 minutes, shaking constantly. Rinse three to five times with ddH2O. Trim the leaf edges and transfer to a conical flask.

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

[0156] 8) Remove the leaves and spread them face up on solid transformation medium. Incubate in the dark at 25°C. The medium composition is shown in Table 2.12.

[0157] 9) After 3 days, remove 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 and continue dark cultivation.

[0158] 10) Change the culture medium every two weeks. Once the callus reaches a certain size, transfer it to a light-treated culture medium for light culture. See Table 2.12 for the composition of the culture medium and Table 2.13 for the composition of the stock solution for preparing the culture medium.

[0159] 11) After the callus turns green, remove hygromycin from the culture medium and transfer to rooting medium. After rooting, remove from the flask and acclimate to the external environment by hydroponics in a nutrient solution at pH 6.0. Then transfer to a mixture of vermiculite and black soil. See Tables 2.14 and 2.15 for the composition of the hydroponics nutrient solution and storage solution.

[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 stock solution composition

[0169]

[0170] 11. Identification of positive transgenic alfalfa seedlings

[0171] 1) Extract DNA from leaves of transgenic plants and perform PCR with primers Hyg-S / A shown in Table 2.16 for hygromycin identification. The PCR product of positive plants will show bands on electrophoresis, while the wild type will not.

[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 varying expression levels. Select upright, robust branches at the budding stage as mother branches. Cut small, approximately 5 cm long, segments with one leaf node from the upper midsection of the mother branch at an angle to the branch as cuttings. Wrap them with sponge strips as soon as possible after cutting and secure them in a hydroponic box containing alfalfa nutrient solution. Once new roots and stems have formed, transplant them into a soil pot.

[0180] 13. Transplant the positive transgenic alfalfa seedlings to the greenhouse for soil culture

[0181] Transgenic alfalfa plants of different lines were transplanted to the greenhouse of this group. To minimize nutrient variation across plots, single-factor randomized blocks were used for transplanting. Nine blocks were set up, labeled A through I; each block was divided into three plots, labeled 1 through 3. Three cuttings from a single line were transplanted into each plot.

[0182] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. CKX The application of the gene in regulating the root system of alfalfa is characterized in that: The steps include: Step 1: Create transgenic plants of wild-type alfalfa and overexpress MtCKX1 Gene, to obtain transgenic alfalfa plants; described MtCKX1 The cDNA sequence of the gene is shown in SEQ ID NO:

1. MtCKX1 The amino acid sequence of the gene is shown in SEQ ID NO: 2; Step 2: comparing the root system of the transgenic alfalfa plant with the root system of the wild-type alfalfa plant, and finding that the root system of the transgenic alfalfa plant is larger than that of the wild-type alfalfa plant; The root system comparison indicators 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 the alfalfa transgenic plant are larger than those of the root system of the wild-type alfalfa plant.

2. The method according to claim 1 CKX The application of the gene in regulating the root system of alfalfa is characterized in that: In the step 1, the MtCKX1 The gene was transformed into detached leaves of wild-type alfalfa, which formed callus in culture dishes and then redifferentiated into plantlets.

3. The method according to claim 2 CKX The application of the gene in regulating the root system of alfalfa is characterized in that: The Agrobacterium transformation method comprises the following steps: Step 1: MtCKX1 cDNA was constructed into a plant expression vector, and the constructed vector was transformed into Agrobacterium EHA105 competent cells by electroporation. After screening positive clones, the cells were shaken at low and high speed until OD = 0.4-0.6, and resuspended in infection buffer; Step 2: After disinfecting the wild-type 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 3: Clean the excess bacteria and culture on the culture medium until callus tissue is formed; move to the light, grow seedlings on the differentiation medium, identify positive seedlings, and harvest.

4. The method according to claim 2 CKX The application of the gene in regulating the root system of alfalfa is 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 alfalfa transgenic plants.

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

6. The method according to claim 5 CKX The application of the gene in regulating the root system of alfalfa is 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 method according to claim 6 CKX The application of the gene in regulating the root system of alfalfa is characterized in that: Compared with wild-type alfalfa plants, the contents of isopentenyl adenine iP, isopentenyl adenine nucleoside iPR and trans-zeatin riboside tZR in transgenic alfalfa plants are reduced, the content of dihydrozeatin DZ is increased, and trans-zeatin tZ is undetectable in transgenic alfalfa plants.

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

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

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