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 legumes, wherein the coding 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. The present invention will have significant theoretical and practical value for legume breeding and related application research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly 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] NODULATION SIGNALING PATHWAY 1 (NSP1) plays an important role in the symbiotic nitrogen fixation process of legume root nodules. [2] , NSP2 [3] , DELLA [4,5] ,Hairy Meristem 4 (HAM4) [6] , SHORTROOT-SCARECROW (SHR-SCR) module [7] andScarecrow like 13 (SCL13) Involved in Nodulation (SIN1) [8] We found that LS has no homologous genes in legume species, but has homologous genes in non-legume species, indicating that it is a gene lost in the legume family. [9] , LATERAL SUPPRESSOR (LAS) in Arabidopsis

[10] and the MOC1 gene in rice

[11] Both 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] Furthermore, MOC1 binds to the DELLA protein SLENDER RICE 1 (SLR1) to prevent its degradation.

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

[16] FON1 is a homologous gene of CLAVATA1. Homologous genes of CLAVATA1 in leguminous plants, such as NODULE AUTOREGULATION RECEPTOR KINASE (NARK) in soybean, HYPERNODULATION AND ABERRANT ROOT 1 (HAR1) in lotus roots, and SUNN in Medicago truncatula, are all involved in the autoregulation of nodulation (AON) pathway. [17-19] Therefore, the loss of the LS gene may be related to symbiotic nitrogen fixation in legume nodules. Studying the role of LS in symbiotic nitrogen fixation in legume nodules may provide clues for the creation of non-legume crops that independently fix nitrogen. 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 leguminous plant root nodules and its application.

[0004] Using molecular biology and comparative genomics, the inventors cloned two genes, LOC_Os06g40780 (also referred to herein as MONOCULM 1, MOC1) and LOC_Os02g10360 (also referred to herein as Os7), from Oryza sativa L. ssp. japonica, which regulate nodule number in soybean (Glycine max) and Medicago truncatula, respectively. They also cloned a homologous gene, Solyc07g066250.1 (also referred to herein as Lateral suppressor, LS), to MOC1 and Os7, which regulate nodule number in soybean and Medicago truncatula, from the tomato (Solanum lycopersicum) line Heinz 1706. The MOC1 encoding gene is located at Chr6:24311420..24316382 in the MSU V7.0 genome, and the Os7 encoding gene is located at Chr2:5451819..5453090 in the MSU V7.0 genome. The LS encoding gene is located at SL2.50ch07:67734548..67735834 in the ITAG V2.4 genome. The cloning of MOC1, Os7, and LS provides a theoretical foundation and genetic resources for subsequent molecular-assisted breeding and molecular design breeding.

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

[0006] The inventors discovered that when MOC1, Os7, and LS proteins were overexpressed using plant expression vectors such as Agrobacterium rhizogenes, the resulting plants produced significantly fewer nodules than the source strains. This effect was unexpected based on existing technology, leading the inventors to identify an isolated protein that inhibits nitrogen fixation in the symbiotic relationship between soybean and Medicago truncatula rhizobia.

[0007] In the present invention, existing plant expression vectors can be used to construct recombinant vectors containing the target gene. These include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment. To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors can be modified, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants and produce color changes, antibiotic resistance markers, or chemical resistance marker genes. For safety reasons, the transgenic plants can be screened directly for transformed plants using stress without the addition of any selectable marker genes. The plant expression vectors can also contain enhancers to increase expression of the inserted nucleotide fragments.

[0008] To achieve the above objectives, 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, which exhibit an altered number of nodules compared to the target legume plant, such as soybean or Medicago truncatula.

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

[0010] The soybean and Medicago truncatula nodule number-related proteins and their encoding nucleic acids provided by the present invention are the first to be discovered by the applicants in terms of their functions in regulating soybean and Medicago truncatula nodule number. Phenotypic analysis of transgenic hairy roots and vector-free hairy root plants has demonstrated that expression of the soybean and Medicago truncatula nodule number-related proteins of the present invention can reduce the number of nodules in transgenic hairy roots of soybean or Medicago truncatula. This invention will have significant theoretical and applied value for research on regulating soybean or Medicago truncatula nodule number and related applications.

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

[0012] In one aspect, the present invention provides an isolated gene for regulating the nodule 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 number of nodules in 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 comprising the gene as described above or the nucleotide sequence encoding the protein as described above, or comprising 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 a chemical resistance 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 chemical resistance marker is such as a herbicide resistance marker.

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

[0017] On the other hand, the present invention provides a method for obtaining soybean hairy roots, which comprises transforming the gene 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 a legume plant 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 Escherichia 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 above-mentioned gene or nucleotide sequence encoding the above-mentioned protein or the above-mentioned expression vector or host cell in cultivating legumes with altered nodule number, such as soybean or alfalfa.

[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 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 root length.

[0024] On the other hand, the present invention provides a method for cultivating transgenic plants with improved yield or increased number of nodules 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 number of nodules of the transgenic plant is changed compared with the target plant, and the plant is a legume, preferably soybean and Medicago truncatula, optionally, the change in the number of nodules is manifested as an increase in the number of nodules and / or the number of nodules per unit root length, optionally, the change in the number of nodules is manifested as a decrease in the number of nodules 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 soybean hairy roots transfected with an empty vector (EV) and 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 in 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 in 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 hairy roots of Medicago truncatula transfected with an empty vector and LS overexpression vector in Example 3 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 in 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 in 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.

[0027] Figure 3 The results of gene expression analysis in soybean and Medicago truncatula hairy roots expressing an empty vector and an LS overexpression vector, as described in Examples 2 and 3, are shown. (a) Gene expression analysis results for soybeans expressing an empty vector and those expressing 35S:Os7, 35S:MOC1, and 35S:LS. (b) Gene expression analysis results for Medicago truncatula expressing an empty vector and those expressing 35S:Os7, 35S:MOC1, and 35S:LS. 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, were 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 recipient was Williams 82, purchased from the U.S. Department of Agriculture (http: / / www.ars-grin.gov / npgs / acc / acc_queries.html). Williams 82 is a spring-sown soybean variety with an indefinite pod-bearing habit. Aboveground stems, leaves, and pods are all grayish hairs with a normal hair density. The Medicago truncatula hairy root transformation recipient was A17. The pDonor221 vector, pBWG2D.1 vector, Agrobacterium rhizogenes strains K599 and AR1193, Bradyrhizobium diazoefficiens strain USDA110, and Sinorhizobium meliloti 1021 were purchased from the China Plasmid Vector, Strain, and Cell Gene Collection (Biovector Science Lab, Inc.).

[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 rice homologs of the lost LS gene from the Leguminosae family are MOC1 and Os7. The cds sequence of MOC1 is shown in SEQ ID NO:1, and the protein sequence it encodes is shown in SEQ ID NO:4. The cds sequence of Os7 is shown in SEQ ID NO:2, and the protein sequence it encodes is shown in SEQ ID NO:5. The tomato homolog of the lost LS gene from the Leguminosae family is LS. The cds sequence of LS is shown in SEQ ID NO:3, and the protein sequence it encodes is shown in SEQ ID NO:6. Given that the LS gene is lost in Leguminosae plants, the present invention investigates its function by overexpression.

[0034] 1. Construction of recombinant plasmid

[0035] 1. Isolate 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 with the primer pair consisting of F1 and R1 to obtain a PCR amplification product (SEQ ID NO: 1). Perform PCR amplification with 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'-CTACGACGACGGCTGC-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 leaf tissue from tomato (Solanum lycopersicum) Heinz 1706 and extract DNA.

[0042] 4. Using the DNA extracted in step 3 as a template, perform PCR amplification with the 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 from step 5 was ligated with the intermediate vector pDonor221 using BP Clonase™ II enzyme mix (Invitrogen 11789-020) to obtain recombinant plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221, and 35S:LS-pDonor221. Sequencing results confirmed that the recombinant plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221, and 35S:LS-pDonor221 were double-stranded DNA molecules with the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 inserted into the pDonor221 vector.

[0047] 7. The plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221, and 35S:LS-pDonor221 obtained in step 6 were ligated with the final vector pB7WG2D.1 using 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. Based on 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 was inserted between attR1 and attR2 of the pB7WG2D.1 vector, thus verifying that the expression cassette was constructed.

[0048] 2. Obtaining transgenic hairy roots of soybeans overexpressing the LS gene

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

[0050] 2. The Agrobacterium rhizogenes obtained in step 1 was transformed into hairy roots [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) Spread the selected seeds in a medium-sized culture dish, place it in a desiccator, and treat the seeds with chlorine disinfection (add 3 mL of 12 M concentrated hydrochloric acid to 75 mL of 10% sodium hypochlorite) for 7-11 hours.

[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 Agrobacterium rhizogenes K599 containing the target vector on solid LB medium and culture in a 28°C incubator for about 2 days. One day before infection, pick a single colony 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 Cotyledonary nodes are infected when the value is about 0.6-0.8.

[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. Gently cut the lower end of the hypocotyl to create a wound. Soak the scratched explant in the bacterial solution for about 30 minutes, shaking it gently at 100 rpm. Place 2-3 sterile filter papers in an empty dish, drain the bacterial solution from the infected explant, and place it on the filter paper. Cover it with a layer of filter paper and absorb the excess bacterial solution.

[0057] (7) Sterile filter paper was placed on the co-culture medium (CCM). The explants were transferred to the co-culture medium with sterilized forceps and cultured in the dark at 22°C 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 overexpression of LS gene

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

[0061] 2. Transform the recipient plant A17 with the Agrobacterium rhizogenes obtained in step 1 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, place the seeds upside down in the dark at 22°C and germinate overnight.

[0064] ② Resuscitate the frozen recombinant Agrobacterium by streaking, pick a single colony and culture it in LB liquid medium for 1 day. Take 200 μL of the bacterial liquid 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 necessary scalpels, tweezers and other tools with alcohol and high temperature or ultraviolet radiation, wear gloves and disinfect with alcohol;

[0067] ② Use a scalpel to cut the hypocotyl of a Medicago truncatula seed. Use a razor blade to collect the bacteria on the plate culture medium. Carefully use tweezers to remove the seedling with the wound on the hypocotyl. Apply the collected Agrobacterium to the wound and place it on MFP medium (recipe from JIC Media Preparation lab recipes). Seal the culture dish with parafilm and place it in a 22°C incubator. Carefully cover the dish and incubate in the dark for 24 hours, then 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, observe the plant fluorescence under a fluorescent microscope (the transformed plasmid contains a fluorescent selection marker). Remove the roots that do not express fluorescence to obtain the root-forming transformed material for subsequent experiments.

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

[0071] 1. Rhizobium inoculation treatment

[0072] (1) 1 mL of Bradyrhizobium diazoefficiens strain USDA110 stored at -80°C was added to 200 mL of TY liquid medium (5 g / L tryptone, 3 g / L yeast extract, 0.7 g / L CaCl2·2H2O) containing 50 mg / L spectinomycin, and cultured at 28°C with 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 suspension in sterilized ddH2O to an OD of 0. 600 Between 0.8-1, used for inoculation treatment.

[0074] (3) About 13 days after the hairy root transformation, the inoculation treatment was carried out, and each seedling was inoculated with 30 mL of resuspended bacterial solution.

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

[0076] 2. Nodule number phenotypic statistics and analysis

[0077] 21 days after inoculation, the vermiculite was washed off with tap water and dried with absorbent paper. Visual observation of soybean hairy roots transfected with empty vector and 35S:Os7, 35S:MOC1 and 35S:LS showed that the number of nodules in hairy roots transfected with 35S:Os7, 35S:MOC1 and 35S:LS was significantly lower than that in 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 those transfected with an empty vector. Figure 1 b, 1c, 1e and 1f. Figure 1 As can be seen from Figures 1b, 1c, 1e and 1f, the average number of nodules in the hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS transgenic plants was only 5.6%, 4.7% and 36.6% of that in the empty vector control group; the number of nodules per root unit in the hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS transgenic plants was significantly only 13.2%, 3.1% and 7.2% of that in the empty vector control group, indicating that the LS gene inhibits the formation 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) Roots and nodules were collected, total RNA was extracted, and reverse transcribed 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'-CGGGTGGTTCTATCTTGGCATC-3' (SEQ ID NO: 19);

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

[0091] PCR amplification conditions were as follows: initial denaturation at 98°C for 30 seconds, amplification (25 cycles for soybean Actin gene and 28 cycles for LS gene) at 98°C for 10 seconds, 59°C for 30 seconds, and 72°C for 40 seconds, and final extension at 72°C for 10 minutes.

[0092] The expression levels of LS genes in different materials were obtained by semi-quantitative PCR amplification using the cDNA as a template and various specific primer pairs. 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) Activate rhizobia: Use a sterile inoculating loop (or a sterile pipette tip) to streak Sinorhizobium meliloti 1021 stored at -80°C onto a TY plate containing tetracycline (10 μg / mL) and incubate at 28°C for 2-3 days.

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

[0097] 3) Shake the culture medium: Add the small shaker culture medium to 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 shaker culture and measure the 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 = about 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 phenotypic 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 was found that the number of nodules in the hairy roots of 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 those transfected with an empty vector. Figure 2 b, 2c, 2e and 2f. Figure 2 As can be seen from Figures 2b, 2c, 2e and 2f, the average number of nodules in the hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS transgenic plants was only 11.1%, 22.6% and 16.5% of that in the empty vector control; the number of nodules per root length in the hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS transgenic plants was only 9.2%, 20.4% and 26.1% of that in the empty vector control, indicating that the LS gene inhibits the formation 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) Roots and nodules were collected, total RNA was extracted, and reverse transcribed into cDNA.

[0106] (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 F8 and R8 was used to identify the expression level of the Medicago truncatula internal reference gene (Actin gene).

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

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

[0109] PCR amplification conditions were as follows: initial 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 were obtained by semi-quantitative PCR amplification using the cDNA as a template and various specific primer pairs. 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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[0115] 4.Fonouni-Farde, C., Tan, S., Baudin, M., Brault, M., Wen, J.,Mysore, K.S., Niebel, A., Frugier, F., and Diet, A. (2016). DELLA-mediatedgibberellin signalling regulates Nod factor signalling and rhizobialinfection. Nat. Commun. 7, 12636. https: / / doi.org / 10.1038 / ncomms12636.

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[0120] 9.Schumacher, K., Schmitt, T., Rossberg, M., Schmitz, G., and Theres,K. (1999). The Lateral suppressor (Ls) gene of tomato encodes a new member ofthe VHIID protein family. Proc. Natl. Acad. Sci. USA 96, 290-295. https: / / doi.org / 10.1073 / pnas.96.1.290.

[0121] 10.Greb, T., Clarenz, O., Schafer, E., Muller, D., Herrero, R.,Schmitz, G., and Theres, K. (2003). Molecular analysis of the LATERALSUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism foraxillary meristem formation. Genes Dev. 17, 1175-1187. https: / / doi.org / 10.1101 / gad.260703.

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[0135] Entertainment

[0136] SEQ ID NO:1 MOC1 1326bp Oryza sativa cDNA

[0137]

[0138] SEQ ID NO:2 Os7 1272 bp cDNA Oryza sativa (Rice)

[0139]

[0140] SEQ ID NO:3 LS 1287 bp cDNA Solanum lycopersicum (Tomato)

[0141]

[0142] SEQ ID NO:4 MOC1 441aa protein Oryza sativa (rice)

[0143] MLRSLHSSSSSDTDNNSGGCKNNGGGGGEAAAAVEGGGDQRAVAAAAPSTRDLLLACADLLQRGDLPAARRAAEIVLAAAASPRGDAADRLAYHFARALALRVDAKAGHGHVVVGGGAARPASSGAYLAFNQIAPFLRFAHLTANQAILEAVDGARRVHILDLDAVHGVQWPPLLQAIAERADPALGPPEVRVTGAGADRDTLLRTGNRLRAFARSIHLPFHFTPLLLSCATTAPHHVAGTSTGAAAAASTAAAATGLEFHPDETLAVNCVMFLHNLAGHDELAAFLKWVKAMSPAVVTIAEREAGGGGGGGDHIDDLPRRVGVAMDHYSAVFEALEATVPPGSRERLAVEQEVLGREIEAAVGPSGGRWWRGIERWGGAARAAGFAARPLSAFAVSQARLLLRLHYPSEGYLVQEARGACFLGWQTRPLLSVSAWQPSSS*

[0144] SEQ ID NO:5 Os7 423aa protein Oryza sativa (rice)

[0145] MLGSSPARDRGGGDDAEASEQPQPQPQPPLSPRAGGGEARGLVLACADLVHRGDLDGARRVAEAVLAAADPRGEAGDRLAHHFARALLALRGGGKGGHGGGGGGVVPSSAAYLAYIKIAPFLRFAHLTANQAILEAAAADAGGAHRRVLHIVDLDAAHGVQWPPLLQAIADRADPAVGPPPEVRLTGAGTDRDVLLRTGDRLRAFSSSLNLPFRFHPLILPCTAELAADPTAALELHPDETLAVNCVLFLHKLGGDGELAAFLRWVKSMNPAVVTIAEREGVLGGDVDDDNVPDELPRRVAAAMDYYSSVFDALEATVPPASADRLAVEQEILSREIDAAVAAPGAGGGGRARDFDAWASAARAAGLAPRPLSAFAASQARLLLRLHYPSEGYKADDDGGRGACFLRWQQRPLMSVSSWQPQP*

[0146] SEQ ID NO:6 LS 428aa protein Solanum lycopersicum (tomato)

[0147] MLGSFGSSSSQSHPHHDEESSDHHQQRRFTATATTITTTTITTSPAIQIRQLLISCAELISQSDFSAAKRLLTILSTNSSPFGDSTERLVHQFTRALSLRLNRYISSTTNHFMTPVETTPTDSSSSSSLALIQSSYLSLNQVTPFIRFTQLTANQAILEAINGNHQAIHIVDFDINHGVQWPPLMQALADRYPAPTLRITGTGNDLDTLRRTGDRLAKFAHSLGLRFQFHPLYIANNNHDHDEDPSIISSIVLLPDETLAINCVFYLHRLLKDREKLRIFLHRVKSMNPKIVTIAEKEANHNHPLFLQRFIEALDYYTAVFDSLEATLPPGSRERMTVEQVWFGREIVDIVAMEGDKRKERHERFRSWEVMLRSCGFSNVALSPFALSQAKLLLRLHYPSEGYQLGVSSNSFFLGWQNQPLFSISSWR*

[0148] SEQ ID NO:7 F1 primer

[0149] 5'-ATGCTCCGGTCACTCCAC-3'

[0150] SEQ ID NO: 8 R1 primer

[0151] 5'-CTACGACGACGACGGCTGC-3'

[0152] SEQ ID NO: 9 F2 primer

[0153] 5'-ATGCTCGGCTCATCACCAGC-3'

[0154] SEQ ID NO: 10 R2 primer

[0155] 5'-CTATGGCTGCGGCTGCC-3'

[0156] SEQ ID NO: 11 F3 primer

[0157] 5'- ATGTTAGGATCCTTTGGTTCTTCATCATC-3'

[0158] SEQ ID NO: 12 R3 primer

[0159] 5'-TCAACGCCAAGACGAGATGG-3'

[0160] SEQ ID NO: 13 F4 primer

[0161] 5'-ATGCTCCGGTCACTCCAC-3'

[0162] SEQ ID NO: 14 R4 primer

[0163] 5'-GTGCAAGAACATGACGCAGTTCA-3'

[0164] SEQ ID NO: 15 F5 primer

[0165] 5'-ATGCTCGGCTCATCACCAG-3'

[0166] SEQ ID NO: 16 R5 primer

[0167] 5'-GGCGAGCACGGCTTC-3'

[0168] SEQ ID NO: 17 F6 primer

[0169] 5'-TGTTTTCTACCTCCACCGCC-3'

[0170] SEQ ID NO: 18 R6 primer

[0171] 5'-TCAACGCCAAGACGAGATGG-3'

[0172] SEQ ID NO: 19 F7 primer

[0173] 5'-CGGTGGTTCTATCTTGGCATC-3'

[0174] SEQ ID NO: 20 R7 primer

[0175] 5'-GTCTTTCGCTTCAATAACCCTA-3'

[0176] SEQ ID NO: 21 F8 primer

[0177] 5'-AACTTGTTGCATGGGTCTTGA-3'

[0178] SEQ ID NO: 22 R8 primer

[0179] 5'-CATTAAGTTTTGACAAAGAGAAAGAGACAGA-3'

[0180] 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 are only specific embodiments of the present invention and are 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 altered nodule number, characterized in that: The method comprises introducing an isolated gene for regulating the number of nodules in legumes into cells or tissues of soybean or Medicago truncatula and cultivating the cells to obtain plants with altered nodule number, wherein the gene encodes a protein as shown in SEQ ID NO: 6, and the altered nodule number is manifested as: a. The number of nodules decreased; 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 coding region sequence of the gene is shown in SEQ ID NO:

3.

3. A method for cultivating soybean or Medicago truncatula with altered 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 an altered nodule number, wherein the expression vector comprises a gene encoding the protein according to claim 1 or a gene according to claim 2, and the altered nodule number is manifested as: a. The number of nodules decreased; 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 a chemical-resistant 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 label is selected from the group consisting of an ampicillin label, a chloramphenicol label, a kanamycin label, a neomycin label, a rifampicin label, a spectinomycin label, a hygromycin label, a streptomycin label and a tetracycline label.

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 altered nodule number, characterized in that: The method comprises introducing a host cell into cells or tissues of soybean or Medicago truncatula for cultivation to obtain a plant with an altered nodule number, wherein the host cell comprises an expression vector according to any one of claims 3 to 9, and the altered nodule number is characterized by: a. The number of nodules decreased; 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 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 a gene encoding the protein according to claim 1 or a gene according to claim 2 or an expression vector according to any one of claims 3 to 9 or a 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 decreased; 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 the cells to obtain transgenic hairy roots with altered nodule number, wherein the altered nodule number is manifested as: a. The number of nodules decreased; 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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