A gene LS regulating symbiotic nitrogen fixation in leguminous plant root nodules and its application

Abstract

The present invention provides an isolated gene for regulating the number of nodules in leguminous plants, wherein the coding region sequence of the gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or a homologous sequence thereof. The present invention will have great theoretical and application value for leguminous plant breeding and related application research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a gene LS for regulating the number of symbiotic nitrogen fixation or nodules of leguminous plants and an application thereof. Background Art

[0002] GRAS are plant-specific transcription factors that can be divided into different subfamilies. [1] . It plays an important role in the symbiotic nitrogen fixation process of leguminous root nodules. NODULATION SIGNALING PATHWAY 1 (NSP1) [2] ,NSP2 [3] ,DELLA [4,5] ,Hairy Meristem 4(HAM4) [6] ,SHORTROOT-SCARECROW(SHR-SCR)module [7] and Scarecrowlike 13(SCL13)Involved in Nodulation(SIN1) [8] We found that LS has no homologous genes in Leguminosae species, but has homologous genes in non-leguminous species, indicating that it is a gene lost in Leguminosae. [9] ,LATERALSUPPRESSOR (LAS) in Arabidopsis

[10] and the MOC1 gene in rice

[11] All are genes of the LS subfamily, responsible for branching or tillering development. The regulatory mechanism of MOC1 in rice has been reported in detail. MOC1 can be degraded by Tillering and Dwarf 1 (TAD1) [12,13] TAD1 is a homologous gene of CELL CYCLE SWITCH52 (CCS52A) in Medicago truncatula. CCS52A is involved in the differentiation of symbiotic cells.

[14] In addition, MOC1 binds to the DELLA protein SLENDER RICE 1 (SLR1) to prevent it from being degraded.

[15] In Medicago truncatula, DELLA forms a complex with DMI3 (IPD3, orthologue of Lotus japonicus CYCLOPS) and NSP2 to regulate nodule symbiosis. [4,5] MOC1 and MONOCULM 3 (MOC3) regulate tiller bud growth by upregulating the expression of FLORAL ORGAN NUMBER1 (FON1)

[16] FON1 is a homologous gene of CLAVATA1. Homologous genes of CLAVATA1 in Leguminosae, such as NODULE AUTOREGULATION RECEPTOR KINASE (NARK) in soybean, HYPERNODULATIONAND ABERRANT ROOT 1 (HAR1) in lotus root, and SUNN in Medicago truncatula, are all involved in the autoregulation of nodulation (AON) pathway. [17-19] Therefore, the loss of LS gene may be related to symbiotic nitrogen fixation in legume nodules. Studying the role of LS in symbiotic nitrogen fixation in legume nodules will provide clues for the creation of non-legume crops with autonomous nitrogen fixation. Summary of the invention

[0003] In crop breeding, too many or too few nodules will affect crop yields, so the number of nodules needs to be regulated.

[20] The purpose of the present invention is to provide a gene for regulating symbiotic nitrogen fixation in root nodules of leguminous plants and its application.

[0004] The inventors used molecular biology and comparative genomics to clone two genes related to regulating the nodulation number of soybean (Glycine max) and Medicago truncatula (Medicago truncatula) LOC_Os06g40780 (also referred to as MONOCULM 1, MOC1 in this article) and LOC_Os02g10360 (also referred to as Os7 in this article) from Oryza sativa L.ssp. japonica, and cloned a homologous gene Solyc07g066250.1 (also referred to as Lateral suppressor, LS) of MOC1 and Os7 that regulates the nodulation number of soybean and Medicago truncatula from the tomato (Solanum lycopersicum) germplasm Heinz 1706. The location of MOC1 encoding gene in the MSU V7.0 version sequenced genome is Chr6:24311420..24316382, and the location of Os7 encoding gene in the MSU V7.0 version sequenced genome is Chr2:5451819..5453090. The location of LS encoding gene in the ITAG2.4 version sequenced genome is SL2.50ch07:67734548..67735834. The cloning of MOC1, Os7 and LS provides a theoretical basis and gene resources for subsequent molecular assisted breeding and molecular design breeding.

[0005] Wherein, in a specific embodiment of the present invention, the cDNA sequence of the MOC1 gene is shown as SEQ ID NO: 1, the cDNA sequence of the Os7 gene is shown as SEQ ID NO: 2, and the cDNA sequence of the LS gene is shown as SEQ ID NO: 3. In a specific embodiment of the present invention, the amino acid sequence of the MOC1 gene is shown as SEQ ID NO: 4, the amino acid sequence of the Os7 gene is shown as SEQ ID NO: 5, and the amino acid sequence of the LS gene is shown as SEQ ID NO: 6.

[0006] The inventors found that when MOC1 protein, Os7 protein and LS protein were overexpressed using plant expression vectors such as Agrobacterium rhizogenes, the number of nodules in the obtained plants was significantly reduced compared with the source strains. Such an effect was unexpected based on the existing technology, and thus the inventors found an isolated protein that inhibits the symbiotic nitrogen fixation related protein of soybean and Medicago truncatula rhizobia.

[0007] In the present invention, existing plant expression vectors can be used to construct recombinant vectors containing target genes. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes, antibiotic markers with resistance, or chemical agent resistance marker genes. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity. The plant expression vector can also contain enhancers to increase the expression of the inserted nucleotide fragments.

[0008] To achieve the above object, the present invention also provides a method for obtaining transgenic hairy roots, which comprises introducing the aforementioned nucleic acid or a vector or host cell containing the aforementioned gene into a target legume plant such as soybean or Medicago truncatula to obtain hairy roots of the legume plant such as soybean or Medicago truncatula showing an altered number of nodules compared with the target legume plant such as soybean or Medicago truncatula.

[0009] To achieve the above object, the present invention also provides a use of the above-mentioned protein or nucleic acid or a vector or host cell comprising the above-mentioned gene in genetic engineering of leguminous plants such as soybean or Medicago truncatula.

[0010] The functions of the soybean and Medicago truncatula nodule number-related proteins and their encoding nucleic acids provided by the present invention in regulating the number of soybean and Medicago truncatula nodules are the first discovery of the applicant, and the phenotypic analysis of transgenic hairy roots and empty vector hairy root plants has been verified to show that the expression of the soybean and Medicago truncatula nodule number-related proteins of the present invention can reduce the number of nodules of transgenic hairy roots of soybean or Medicago truncatula. The present invention will have great theoretical and application value for regulating the number of soybean or Medicago truncatula nodules and related application research.

[0011] Specifically, the present invention provides the following technical solutions:

[0012] In one aspect, the present invention provides an isolated gene for regulating the nodulation number of soybean or Medicago truncatula, wherein the coding region sequence of the gene is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a homologous sequence thereof.

[0013] In another aspect, the present invention provides a protein for regulating the nodulation number of soybean or Medicago truncatula, wherein the amino acid sequence of the protein is shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or a homologous sequence thereof.

[0014] On the other hand, the present invention provides an expression vector, which comprises the gene as described above or the nucleotide sequence encoding the protein as described above, or comprises a sequence that inhibits the expression of the gene as described above or the protein as described above, such as an interfering sequence of the gene or an interfering sequence of the nucleotide sequence encoding the protein.

[0015] In some embodiments, the expression vector comprises a marker, optionally, the marker is selected from a luminescent marker, an antibiotic marker and an anti-chemical marker, optionally, the luminescent marker is selected from red fluorescent protein and green fluorescent protein, the antibiotic marker is selected from ampicillin, chloramphenicol, kanamycin, neomycin, rifampicin, spectinomycin, hygromycin, streptomycin and tetracycline, the anti-chemical marker is such as an anti-herbicide marker.

[0016] In another aspect, the present invention provides a host cell comprising the expression vector as described above.

[0017] On the other hand, the present invention provides a method for obtaining soybean hairy roots, which comprises transforming the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector or host cell as described above into cells or tissues of leguminous plants such as soybean or Medicago truncatula and cultivating transgenic hairy roots.

[0018] In some embodiments, the vector is a plant expression vector, including a binary Agrobacterium rhizogenes vector and / or a vector that can be used for plant microprojectile bombardment.

[0019] In some embodiments, the host cell is selected from an E. coli cell, an Agrobacterium cell (eg, an Agrobacterium rhizogenes cell), or a plant cell.

[0020] In some embodiments, the Agrobacterium rhizogenes is selected from K599, AR1193, C58cl, Arqual, MSU440, LBA9402, and R1601.

[0021] In another aspect, the present invention provides use of the gene or nucleotide sequence encoding the protein or expression vector or host cell described above in cultivating legumes such as soybean or alfalfa with altered nodulation number.

[0022] In some embodiments, the change in nodule number is manifested as an increase in the number of nodules and / or the number of nodules per unit root length.

[0023] In some embodiments, the change in nodule number is manifested as a decrease in the number of nodules and / or the number of nodules per unit root length.

[0024] On the other hand, the present invention provides a method for cultivating transgenic plants with improved yield or increased nodule number and / or number of nodules per unit root length, which comprises introducing the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector or host cell as described above into the target plant cell or tissue to obtain a transgenic plant, wherein the nodule number of the transgenic plant is changed compared with the target plant, and the plant is a leguminous plant, preferably soybean and Medicago truncatula, optionally, the change in nodule number is manifested as an increase in the nodule number and / or the number of nodules per unit root length, optionally, the change in nodule number is manifested as a decrease in the nodule number and / or the number of nodules per unit root length. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of the nodulation phenotype analysis of the hairy roots of soybeans transfected with an empty vector (EV) and transfected with an LS overexpression vector in Example 2 are shown. Among them, a: Nodulation phenotype of hairy roots transfected with an empty vector, 35S:Os7 and 35S:MOC1. b: Statistics of the number of nodules of hairy roots transfected with an empty vector, 35S:Os7 and 35S:MOC1. c: Statistics of the number of nodules per unit root length of hairy roots transfected with an empty vector, 35S:Os7 and 35S:MOC1. d: Nodulation phenotype of hairy roots transfected with an empty vector and 35S:LS. e: Statistics of the number of nodules of hairy roots transfected with an empty vector and 35S:LS. f: Statistics of the number of nodules per unit root length of hairy roots transfected with an empty vector and 35S:LS.

[0026] Figure 2The nodulation phenotype results of the hairy roots of Medicago truncatula transfected with an empty vector and transfected with an LS overexpression vector in Example 3 are shown. Among them, a: the nodulation phenotype of the hairy roots transfected with an empty vector, 35S:Os7 and 35S:MOC1. b: the nodulation number statistics of the hairy roots transfected with an empty vector, 35S:Os7 and 35S:MOC1. c: the nodulation number statistics of the hairy roots transfected with an empty vector, 35S:Os7 and 35S:MOC1 per unit root length. d: the nodulation phenotype of the hairy roots transfected with an empty vector and 35S:LS. e: the nodulation number statistics of the hairy roots transfected with an empty vector and 35S:LS. f: the nodulation number statistics of the hairy roots transfected with an empty vector and 35S:LS per unit root length.

[0027] Figure 3 The expression analysis results of the hairy roots of the empty vector and the LS overexpression vector in soybean and Medicago truncatula in Example 2 and Example 3 are shown. Among them, a: the gene expression analysis results of the empty vector and the 35S:Os7, 35S:MOC1, 35S:LS in soybean. b: the gene expression analysis results of the empty vector and the 35S:Os7, 35S:MOC1, 35S:LS in Medicago truncatula. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0029] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods or are selected according to the product specifications. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged.

[0030] In the following examples, the soybean hairy root transformation receptor was Williams 82, purchased from the U.S. Department of Agriculture, whose website is http: / / www.ars-grin.gov / npgs / acc / acc_queries.html Williams 82 is a soybean variety (sown in spring), with an indefinite pod-bearing habit. The stems, leaves and pods of the aboveground tissues are all (gray) hairy, with a normal hair density. The receptor for hairy root transformation of Medicago truncatula is A17. pDonor221 vector, pBWG2D.1 vector, Agrobacterium rhizogenes strains K599 and AR1193, Bradyrhizobium diazoefficiens strain USDA110, and Sinorhizobium meliloti 1021 were purchased from Biovector Science Lab, Inc., China.

[0031] Gateway, recovery kits and other consumables were purchased from Thermo Fisher Scientific and Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0032] Example 1 Establishment of LS gene overexpressing transgenic plants

[0033] The homologous genes of the LS gene lost in Leguminosae in rice are MOC1 and Os7, the cds sequence of MOC1 is shown in SEQ ID NO: 1, and the protein sequence encoded by it is shown in SEQ ID NO: 4. The cds sequence of Os7 is shown in SEQ ID NO: 2, and the protein sequence encoded by it is shown in SEQ ID NO: 5. The homologous gene of the LS gene lost in Leguminosae in tomato is LS, the cds sequence of LS is shown in SEQ ID NO: 3, and the protein sequence encoded by it is shown in SEQ ID NO: 6. In view of the fact that the LS gene is a gene lost in Leguminosae plants, the present invention studies the function of the gene by overexpression.

[0034] 1. Construction of recombinant plasmid

[0035] 1. Separate leaf tissue from rice (Oryza sativa) L.ssp.japonica and extract DNA.

[0036] 2. Using the DNA extracted in step 1 as a template, perform PCR amplification using the primer pair consisting of F1 and R1 to obtain a PCR amplification product (SEQ ID NO: 1). Perform PCR amplification using the primer pair consisting of F2 and R2 to obtain a PCR amplification product (SEQ ID NO: 2).

[0037] F1: 5'-ATGCTCCGGTCACTCCAC-3' (SEQ ID NO: 7);

[0038] R1: 5'-CTACGACGACGACGGCTGC-3' (SEQ ID NO: 8).

[0039] F2: 5'-ATGCTCGGCTCATCACCAGC-3' (SEQ ID NO: 9);

[0040] R2: 5'-CTATGGCTGCGGCTGCC-3' (SEQ ID NO: 10).

[0041] 3. Separate the leaf tissue of tomato (Solanum lycopersicum) Heinz 1706 and extract DNA.

[0042] 4. Using the DNA extracted in step 3 as a template, perform PCR amplification using a primer pair consisting of F3 and R3 to obtain a PCR amplification product (SEQ ID NO: 3).

[0043] F3: 5'-ATGTTAGGATCCTTTGGTTCTTCATCATC-3' (SEQ ID NO: 11);

[0044] R3: 5'-TCAACGCCAAGACGAGATGG-3' (SEQ ID NO: 12).

[0045] 5. Recover PCR products from gel.

[0046] 6. The PCR product of step 5 and the intermediate vector pDonor221 were connected by BP ClonaseTM II enzyme mix (Invitrogen 11789-020) to obtain recombinant plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221 and 35S:LS-pDonor221. According to the sequencing results, it was confirmed that the recombinant plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221 and 35S:LS-pDonor221 were double-stranded DNA molecules as shown in the sequence list as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 were inserted into the pDonor221 vector.

[0047] 7. The plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221 and 35S:LS-pDonor221 obtained in step 6 were connected with the final vector pB7WG2D.1 through LR CLONASE II ENZYME MIX (Invitrogen 11791-020) to obtain recombinant plasmids 35S:MOC1-pB7WG2D.1, 35S:Os7-pB7WG2D.1 and 35S:LS-pB7WG2D.1. According to the sequencing results, the recombinant plasmids 35S:MOC1-pB7WG2D.1, 35S:Os7-pB7WG2D.1 and 35S:LS-pB7WG2D.1 were sequenced. The structure is described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the sequence table is inserted between attR1 and attR2 of the pB7WG2D.1 vector, thus verifying that the expression cassette is constructed.

[0048] 2. Obtaining transgenic hairy roots of soybeans with LS gene overexpression

[0049] 1. Introduce the recombinant plasmids 35S:Os7-pB7WG2D.1, 35S:MOC1-pB7WG2D.1 and 35S:LS-pB7WG2D.1 into Agrobacterium rhizogenes strain K599 to obtain recombinant Agrobacterium, which were frozen at -80°C and stored in glycerol.

[0050] 2. The Agrobacterium rhizogenes obtained in step 1 was transformed by the hairy root method [21,22] Transform the recipient plant Williams82. The specific steps are as follows:

[0051] (1) Select Williams 82 soybean seeds that are large, plump, and free of disease spots.

[0052] (2) The selected seeds were spread in a medium-sized culture dish, placed in a desiccator, and treated for 7-11 hours using chlorine disinfection (75 mL of 10% sodium hypochlorite plus 3 mL of 12 M concentrated hydrochloric acid).

[0053] (3) After chlorine disinfection, the seeds were blown away in a clean bench until there was no obvious chlorine smell, and then sown in vermiculite. They were germinated in a greenhouse for 3 days and transformed when the soybean cotyledons were about to open.

[0054] (4) When sowing seeds, streak the Agrobacterium rhizogenes K599 containing the target vector on solid LB medium and culture it in a 28°C incubator for about 2 days. One day before infection, pick a single clone and inoculate it into 3-5 mL of LB liquid medium containing rifampicin (50 μg / mL), streptomycin (10 μg / mL) and spectinomycin (100 μg / mL), and culture it in a shaking incubator at 28°C overnight.

[0055] (5) The activated bacterial suspension was inoculated into 200 mL of LB liquid medium containing spectinomycin (100 μg / mL) and acetosyringone (200 μmol / L) at a ratio of 1:1000 for expansion and culture until OD 600 When the value is about 0.6-0.8, the cotyledon nodes are infected.

[0056] (6) Select seeds that are not contaminated with bacteria and have intact cotyledons. Use a scalpel to cut the germinated seeds from the root system, cut the hypocotyl 1 cm below the cotyledon, and gently cut the lower end of the hypocotyl to create a wound. Soak the scratched explants in the bacterial solution for about 30 minutes, shaking gently at 100 rpm. Place 2-3 sterilized filter papers in an empty dish, pour out the bacterial solution from the infected explants, place them on the filter paper, cover them with a layer of filter paper, and absorb the excess bacterial solution.

[0057] (7) Place sterile filter paper on co-culture medium (CCM), transfer the explants to the co-culture medium with sterile forceps, and culture at 22°C in the dark for 3 days.

[0058] (8) After co-cultivation, the plants can be directly transplanted into vermiculite to induce rooting. Pay attention to moisture retention and temperature. Rooting will be completed in about a week.

[0059] 3. Obtaining transgenic hairy roots of Medicago truncatula with LS gene overexpression

[0060] 1. Introduce the recombinant plasmids 35S:Os7-pB7WG2D.1, 35S:MOC1-pB7WG2D.1 and 35S:LS-pB7WG2D.1 into Agrobacterium rhizogenes strain K599 to obtain recombinant Agrobacterium, which were frozen at -80°C and stored in glycerol.

[0061] 2. The Agrobacterium rhizogenes obtained in step 1 is transformed into the recipient plant A17 using the hairy root transformation method. The specific steps are as follows:

[0062] 1) Preparation

[0063] ① The seeds germinate after imbibition. The day before rooting transformation, the seeds are placed upside down in the dark at 22℃ and germinated overnight.

[0064] ② Resuscitate the frozen recombinant Agrobacterium by streaking, pick a single clone and culture it in LB liquid medium for 1 day, take 200 μL of the bacterial solution and spread it on LB solid medium containing rifampicin (50 μg / mL) and spectinomycin (100 μg / mL), and continue to culture inverted for 1 day.

[0065] 2) Hair root transformation:

[0066] ① In the clean bench, sterilize the required scalpels, tweezers and other tools with alcohol and high temperature or ultraviolet light, wear gloves and disinfect with alcohol;

[0067] ② Use a scalpel to cut the hypocotyl of Medicago truncatula seeds, collect the bacteria on the flat culture medium with a blade, carefully pick up the seedlings with wounds on the hypocotyls with tweezers, dip the collected Agrobacterium on the wounds, and place them on MFP culture medium (the recipe comes from JIC Media Preparation lab recipes). Seal the culture dish with sealing film and place it in a 22℃ incubator. Carefully cover the culture dish, culture in the dark for 24 hours, and then culture in the light for one week.

[0068] 3) Non-transformed root excision: Use a scalpel to remove the non-transformed roots that have grown for one week in a clean bench, and transfer the treated plants (with transformed roots) to HRE medium.

[23] Place in a 22°C incubator and culture for 1 to 2 weeks;

[0069] 4) Before transplanting the transformed seedlings, it is necessary to observe the plant fluorescence under a fluorescent microscope (the transformation plasmid carries a fluorescent screening marker), remove the roots that do not express fluorescence, and obtain the root-forming transformed material for subsequent experiments.

[0070] Example 2 Changes in the number of nodules in soybeans with overexpression of LS gene

[0071] 1. Rhizobium inoculation treatment

[0072] (1) Take 1 mL of Bradyrhizobium diazoefficiens strain USDA110 stored at -80°C and add it to 200 mL of TY liquid medium (tryptone 5 g / L, yeast extract 3 g / L, CaCl2·2H2O 0.7 g / L) containing 50 mg / L spectinomycin. Incubate at 28°C in a shaker at 200 rpm for about 4 days until the OD 600 Between 0.8-1.

[0073] (2) Collect the bacterial suspension in a 500 mL centrifuge bottle and centrifuge at 6000 rpm for 10 min. Resuspend the bacterial cells in sterilized ddH2O to an OD of 600 Between 0.8-1, used for inoculation treatment.

[0074] (3) About 13 days after the hairy root transformation, the seedlings were inoculated with 30 mL of the resuspended bacterial solution.

[0075] (4) Observe the nodulation phenotype 21 days after inoculation.

[0076] 2. Nodule number phenotype statistics and analysis

[0077] 21 days after inoculation, the plants were washed with tap water to clean the vermiculite and dried with absorbent paper. The hairy roots of soybeans transfected with empty vector and 35S:Os7, 35S:MOC1 and 35S:LS were observed with naked eyes. It can be seen that the number of nodules in the hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS was significantly lower than that of the control transfected with empty vector ( Figure 1 a and 1d).

[0078] The inventors counted the number of nodules and root length of hairy roots transfected with 35S:Os7, 35S:MOC1 and 35S:LS and hairy roots transfected with an empty vector. Figure 1 b, 1c, 1e and 1f. Figure 1 As can be seen from b, 1c, 1e and 1f, the average number of nodules in 35S:Os7, 35S:MOC1 and 35S:LS transgenic hairy roots was only 5.6%, 4.7% and 36.6% of the empty vector control; the number of nodules per unit root length in 35S:Os7, 35S:MOC1 and 35S:LS transgenic hairy roots was significantly only 13.2%, 3.1% and 7.2% of the empty vector control, which indicates that the LS gene inhibits the production of soybean nodules.

[0079] 3. Identification of gene expression

[0080] The hairy roots of the empty vector, 35S:Os7, 35S:MOC1 and 35S:LS were identified as follows:

[0081] (1) Take roots and nodules, extract total RNA and reverse transcribe it into cDNA.

[0082] (2) Using the cDNA extracted in step (1) as a template, the primer pair consisting of F4 and R4 was used to identify the expression level of the MOC1 gene, the primer pair consisting of F5 and R5 was used to identify the expression level of the Os7 gene, the primer pair consisting of F6 and R6 was used to identify the expression level of the LS gene, and the primer pair consisting of F7 and R7 was used to identify the expression level of the soybean internal reference gene (Actin gene).

[0083] F4: 5'-ATGCTCCGGTCACTCCAC-3' (SEQ ID NO: 13);

[0084] R4: 5'-GTGCAAGAACATGACGCAGTTCA-3' (SEQ ID NO: 14).

[0085] F5: 5'-ATGCTCGGCTCATCACCAG-3' (SEQ ID NO: 15);

[0086] R5: 5'-GGCGAGCACGGCTTC-3' (SEQ ID NO: 16).

[0087] F6: 5'-TGTTTTCTACCTCCACCGCC-3' (SEQ ID NO: 17);

[0088] R6: 5'-TCAACGCCAAGACGAGATGG-3' (SEQ ID NO: 18).

[0089] F7: 5'-CGGTGGTTTCTATCTTGGCATC-3' (SEQ ID NO: 19);

[0090] R7: 5'-GTCTTTCGCTTCAATAACCCTA-3' (SEQ ID NO: 20).

[0091] The conditions of PCR amplification were as follows: pre-denaturation at 98°C for 30s, amplification (25 cycles for soybean Actin gene, 28 cycles for LS gene) at 98°C for 10s, 59°C for 30s, and 72°C for 40s, and final extension at 72°C for 10min.

[0092] The expression levels of LS genes in different materials obtained by semi-quantitative PCR amplification using the cDNA as a template and various specific primer pairs are shown in Figure 3 a. From Figure 3 As can be seen in a, the gene expression of LS in the gene overexpression strain.

[0093] Example 3 Changes in the number of nodules in Medicago truncatula after LS gene overexpression

[0094] 1. Rhizobium inoculation treatment

[0095] 1) Activation of rhizobia: Use a sterile inoculation loop (or a sterile pipette tip) to streak Sinorhizobium meliloti 1021 stored in a -80°C refrigerator onto a TY plate containing tetracycline (10 μg / mL) and culture at 28°C for 2 to 3 days;

[0096] 2) Shake the bacterial solution: Pick a single clone of activated rhizobia and place it in 3 mL of TY liquid culture medium with corresponding antibiotics, and culture it in a shaking incubator at 28°C for 1 day;

[0097] 3) Shake the bacterial solution: Add the small shake bacterial solution into the new TY liquid resistance medium at a ratio of 1:100, and continue to culture at 28°C in a shaker for one day;

[0098] 4) Take a large shake culture and test OD 600 Value, waiting for OD 600 =0.8-1, transfer the rhizobium solution to a centrifuge tube and centrifuge at 3000-4000 rpm for 5 min;

[0099] 5) Remove the supernatant, resuspend the cells in double distilled water, and measure the OD again. 600 Value, dilute to OD 600 = 0.03. Keep the soil appropriately dry before inoculation, and use an atomizer to evenly spray the diluted bacterial solution on the plant roots and soil.

[0100] 2. Nodule number phenotype statistics and analysis

[0101] The nodulation phenotype was observed 21 days after inoculation. The hairy roots of Medicago truncatula transformed with empty vector, 35S:Os7, 35S:MOC1 and 35S:LS were observed with naked eyes. It can be seen that the number of nodules in the hairy roots transformed with 35S:Os7, 35S:MOC1 and 35S:LS was significantly lower than that in the control transformed with empty vector ( Figure 2 a and 2d).

[0102] The inventors counted the number of nodules and root length of hairy roots transfected with 35S:Os7, 35S:MOC1 and 35S:LS and hairy roots transfected with an empty vector. Figure 2 b, 2c, 2e and 2f. Figure 2 As can be seen from 2b, 2c, 2e and 2f, the average number of nodules in 35S:Os7, 35S:MOC1 and 35S:LS transgenic hairy roots was only 11.1%, 22.6% and 16.5% of that in the empty vector control; the number of nodules per unit root length in 35S:Os7, 35S:MOC1 and 35S:LS transgenic hairy roots was only 9.2%, 20.4% and 26.1% of that in the empty vector control, which indicates that the LS gene inhibits the production of root nodules in Medicago truncatula.

[0103] 3. Identification of gene expression

[0104] The hairy roots of the empty vector, 35S:Os7, 35S:MOC1 and 35S:LS were identified as follows:

[0105] (1) Take roots and nodules, extract total RNA and reverse transcribe it into cDNA.

[0106] (2) Using the cDNA extracted in step (1) as a template, the expression level of the MOC1 gene was identified using a primer pair consisting of F4 and R4, the expression level of the Os7 gene was identified using a primer pair consisting of F5 and R5, the expression level of the LS gene was identified using a primer pair consisting of F6 and R6, and the expression level of the Medicago truncatula internal reference gene (the expression level of the Actin gene) was identified using F8 and R8.

[0107] F8: 5'-AACTTGTTGCATGGGTCTTGA-3' (SEQ ID NO: 21);

[0108] R8: 5'-CATTAAGTTTGACAAAGAGAAAGAGACAGA-3' (SEQ ID NO: 22).

[0109] The conditions for PCR amplification were: pre-denaturation at 98°C for 30 s, amplification (35 cycles) at 98°C for 10 s, 59°C for 30 s, and 72°C for 40 s, and final extension at 72°C for 10 min.

[0110] The expression levels of LS genes in different materials obtained by semi-quantitative PCR amplification using the cDNA as a template and various specific primer pairs are shown in Figure 3 b. From Figure 3 As can be seen in b, the gene expression of LS in the gene overexpression strain.

[0111] References:

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[0114] 3.Kalo,P.,Gleason,C.,Edwards,A.,Marsh,J.,Mitra,R.M.,Hirsch,S.,Jakab,J.,Sims,S.,Long,S.R.,Rogers,J.,et al.(2005).Nodulation signaling in legumesrequires NSP2,amember of the GRAS family of transcriptionalregulators.Science 308,1786-1789.https: / / doi.org / 10.1126 / science.1110951.

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[0133] Entertainment

[0134] SEQ ID NO:1MOC1 1326bp Oryza sativa cDNA

[0135]

[0136] SEQ ID NO:2Os7 1272 bp cDNA Oryza sativa (rice)

[0137]

[0138] SEQ ID NO:3LS1287 bp cDNA Solanum lycopersicum (tomato)

[0139]

[0140] SEQ ID NO:4 MOC1 441aa protein, Oryza sativa (rice)

[0141] MLRSLHSSSSSDTDNNSGGCKNNGGGGGEAAAAVEGGGDQRAVAAAAPSTRDLLLACADLLQRGDLPAARRAAEIVLAAAASPRGDAADRLAYHFARALALRVDAKAGHGHVVVGGGAARPASSGAYLAFNQIAPFLRFAHLTANQAILEAVDGARRVHILDLDAVHGVQWPPLLQAIAERADPALGPPEVRVTGAGADRDTLLRTGNRLRAFARSIHLPFHFTPLLLSCATTAPHHVAGTSTGAAAAASTAAAATGLEFHPDETLAVNCVMFLHNLAGHDELAAFLKWVKAMSPAVVTIAEREAGGGGGGGDHIDDLPRRVGVAMDHYSAVFEALEATVPPGSRERLAVEQEVLGREIEAAVGPSGGRWWRGIERWGGAARAAGFAARPLSAFAVSQARLLLRLHYPSEGYLVQEARGACFLGWQTRPLLSVSAWQPSSS*

[0142] SEQ ID NO:5 Os7 423aa protein, Oryza sativa (rice)

[0143] MLGSSPARDRGGGDDAEASEQPQPQPQPPLSPRAGGGEARGLVLACADLVHRGDLDGARRVAEAVLAAADPRGEAGDRLAHHFARALLALRGGGKGGHGGGGGGVVPSSAAYLAYIKIAPFLRFAHLTANQAILEAAAADAGGAHRRVLHIVDLDAAHGVQWPPLLQAIADRADPAVGPPPEVRLTGAGTDRDVLLRTGDRLRAFSSSLNLPFRFHPLILPCTAELAADPTAALELHPDETLAVNCVLFLHKLGGDGELAAFLRWVKSMNPAVVTIAEREGVLGGDVDDDNVPDELPRRVAAAMDYYSSVFDALEATVPPASADRLAVEQEILSREIDAAVAAPGAGGGGRARDFDAWASAARAAGLAPRPLSAFAASQARLLLRLHYPSEGYKADDDGGRGACFLRWQQRPLMSVSSWQPQP*

[0144] SEQ ID NO:6 LS 428aa protein Solanum lycopersicum (tomato)

[0145] MLGSFGSSSSQSHPHHDEESSDHHQQRRFTATATTITTTTITTSPAIQIRQLLISCAELISQSDFSAAKRLLTILSTNSSPFGDSTERLVHQFTRALSLRLNRYISSTTNHFMTPVETTPTDSSSSSSLALIQSSYLSLNQVTPFIRFTQLTANQAILEAINGNHQAIHIVDFDINHGVQWPPLMQALADRYPAPTLRITGTGNDLDTLRRTGDRLAKFAHSLGLRFQFHPLYIANNNHDHDEDPSIISSIVLLPDETLAINCVFYLHRLLKDREKLRIFLHRVKSMNPKIVTIAEKEANHNHPLFLQRFIEALDYYTAVFDSLEATLPPGSRERMTVEQVWFGREIVDIVAMEGDKRKERHERFRSWEVMLRSCGFSNVALSPFALSQAKLLLRLHYPSEGYQLGVSSNSFFLGWQNQPLFSISSWR*

[0146] SEQ ID NO: 7 F1 primer

[0147] 5'-ATGCTCCGGTCACTCCAC-3'

[0148] SEQ ID NO: 8R1 Primer

[0149] 5'-CTACGACGACGACGGCTGC-3'

[0150] SEQ ID NO: 9F2 Primer

[0151] 5'-ATGCTCGGCTCATCACCAGC-3'

[0152] SEQ ID NO: 10R2 Primer

[0153] 5'-CTATGGCTGCGGCTGCC-3'

[0154] SEQ ID NO: 11F3 Primer

[0155] 5'-ATGTTAGGATCCTTTGGTTCTTCATCATC-3'

[0156] SEQ ID NO: 12R3 Primer

[0157] 5'-TCAACGCCAAGACGAGATGG-3'

[0158] SEQ ID NO: 13F4 Primer

[0159] 5'-ATGCTCCGGTCACTCCAC-3'

[0160] SEQ ID NO: 14R4 Primer

[0161] 5'-GTGCAAGAACATGACGCAGTTCA-3'

[0162] SEQ ID NO: 15F5 Primer

[0163] 5'-ATGCTCGGCTCATCACCAG-3'

[0164] SEQ ID NO: 16R5 Primer

[0165] 5'-GGCGAGCACGGCTTC-3'

[0166] SEQ ID NO: 17F6 Primer

[0167] 5'-TGTTTTCTACCTCCACCGCC-3'

[0168] SEQ ID NO: 18R6 Primer

[0169] 5'-TCAACGCCAAGACGAGATGG-3'

[0170] SEQ ID NO: 19F7 Primer

[0171] 5'-CGGTGGTTCTATCTTGGCATC-3'

[0172] SEQ ID NO: 20R7 Primer

[0173] 5'-GTCTTTCGCTTCAATAACCCTA-3'

[0174] SEQ ID NO: 21F8 Primer

[0175] 5'-AACTTGTTGCATGGGTCTTGA-3'

[0176] SEQ ID NO: 22R8 Primer

[0177] 5'-CATTAAGTTTGACAAAGAGAAAGAGACAGA-3'

[0178] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for cultivating soybean or Medicago truncatula with a changed nodule number, characterized in that: The method comprises introducing a separated gene for regulating the number of nodules of leguminous plants into cells or tissues of soybean or Medicago truncatula for cultivation to obtain plants with altered number of nodules, wherein the coding region sequence of the gene is shown in SEQ ID NO: 1, and the altered number of nodules is manifested as: a. The number of nodules decreases; b. A decrease in the number of nodules per root; or c. The number of nodules and the number of nodules per unit root length decreased.

2. The method according to claim 1, characterized in that The gene encodes the protein shown in SEQ ID NO:

4.

3. A method for cultivating soybean or Medicago truncatula with a changed nodule number, characterized in that: The method comprises introducing an expression vector into cells or tissues of soybean or Medicago truncatula for cultivation to obtain a plant with a changed nodule number, wherein the expression vector comprises a gene according to claim 1 or a nucleotide sequence encoding a protein according to claim 2, and the changed nodule number is manifested as: a. The number of nodules decreases; b. A decrease in the number of nodules per root; or c. The number of nodules and the number of nodules per unit root length decreased.

4. The method according to claim 3, characterized in that The expression vector comprises a marker.

5. The method according to claim 4, characterized in that The label is selected from the group consisting of a luminescent label, an antibiotic label and an anti-chemical agent label.

6. The method according to claim 5, characterized in that The luminescent marker is selected from red fluorescent protein and green fluorescent protein.

7. The method according to claim 5, characterized in that The antibiotic marker is selected from the group consisting of an ampicillin marker, a chloramphenicol marker, a kanamycin marker, a neomycin marker, a rifampicin marker, a spectinomycin marker, a hygromycin marker, a streptomycin marker and a tetracycline marker.

8. The method according to claim 5, characterized in that The chemical resistance marker is a herbicide resistance marker.

9. The method according to any one of claims 3 to 8, characterized in that: The vector is a plant expression vector, including a binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

10. A method for cultivating soybean or Medicago truncatula with a changed nodule number, characterized in that: The method comprises introducing a host cell into a cell or tissue of soybean or Medicago truncatula for cultivation to obtain a plant with a changed nodule number, wherein the host cell comprises an expression vector according to any one of claims 3 to 9, and the changed nodule number is expressed as: a. The number of nodules decreases; b. A decrease in the number of nodules per root; or c. The number of nodules and the number of nodules per unit root length decreased.

11. The method according to claim 10, characterized in that The host cell is selected from the group consisting of Escherichia coli cells, Agrobacterium cells and plant cells.

12. The method according to claim 11, characterized in that The Agrobacterium cells are Agrobacterium rhizogenes cells.

13. The method according to claim 12, characterized in that The Agrobacterium rhizogenes is selected from K599, AR1193, C58cl, Arqual, MSU440, LBA9402 and R1601.

14. Use of the gene according to claim 1 or the nucleotide sequence encoding the protein according to claim 2 or the expression vector according to any one of claims 3 to 9 or the host cell according to any one of claims 10 to 13 in cultivating soybean or Medicago truncatula with altered nodule number, wherein the altered nodule number is manifested as: a. The number of nodules decreases; b. A decrease in the number of nodules per root; or c. The number of nodules and the number of nodules per unit root length decreased.

15. A method for obtaining soybean or Medicago truncatula hairy roots, comprising transforming the expression vector according to any one of claims 3 to 9 or the host cell according to any one of claims 10 to 13 into soybean or Medicago truncatula plant cells or tissues and cultivating them to obtain transgenic hairy roots with altered nodule number, wherein the altered nodule number is manifested as: a. The number of nodules decreases; b. A decrease in the number of nodules per root; or c. The number of nodules and the number of nodules per unit root length decreased.

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