A gene LS for regulating nodule symbiotic nitrogen fixation in leguminous plants and its application

Abstract

The present invention provides isolated genes for regulating the nodulation number of leguminous plants, and the coding region sequences of the genes are as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or their homologous sequences. The present invention will have great theoretical and application values for leguminous plant breeding and its related applied research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a gene LS for regulating legume root nodule symbiotic nitrogen fixation or nodule number and its application. Background Art

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

[10] and the MOC1 gene in rice

[11] are all genes of the LS subfamily and are responsible for branch or tiller 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] , and TAD1 is a homologous gene of Medicago truncatula CELL CYCLE SWITCH52 (CCS52A). CCS52A is involved in the differentiation of symbiotic cells

[14] . In addition, MOC1 binds to the DELLA protein SLENDER RICE 1 (SLR1) and thus is not degraded

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

[16] 。FON1 is a homologous gene of CLAVATA1. Homologous genes of CLAVATA1 in legumes, such as NODULE AUTOREGULATION RECEPTOR KINASE (NARK) in soybean, HYPERNODULATION AND ABERRANT ROOT 1 (HAR1) in Lotus japonicus, 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 provides clues for creating non-leguminous crops with autonomous nitrogen fixation Summary of the Invention

[0003] In crop breeding, too many or too few nodules will affect the yield of crops. Therefore, it is necessary to regulate the number of nodules

[20] 。The object of the present invention is to provide a gene for regulating symbiotic nitrogen fixation in legume nodules and its application

[0004] The inventors used molecular biology and comparative genomics methods to clone a gene LOC_Os06g40780 (also called MONOCULM 1, MOC1 in this article) and LOC_Os02g10360 (also called Os7 in this article) related to regulating nodule numbers in soybean (Glycine max) and Medicago truncatula from Oryza sativa L. ssp. japonica, and cloned a homologous gene Solyc07g066250.1 (also called Lateral suppressor, LS in this article) of MOC1 and Os7 that regulates nodule numbers in soybean and Medicago truncatula from the tomato (Solanum lycopersicum) germplasm Heinz 1706. The position of the MOC1 coding gene in the MSU V7.0 version sequenced genome is Chr6:24311420..24316382, the position of the Os7 coding gene in the MSU V7.0 version sequenced genome is Chr2:5451819..5453090. The position of the LS coding 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] Among them, in a specific embodiment of the present invention, the cDNA sequence of the MOC1 gene is as shown in SEQ ID NO:1, the cDNA sequence of the Os7 gene is as shown in SEQ ID NO:2, and the cDNA sequence of the LS gene is as shown in SEQ ID NO:3. In a specific embodiment of the present invention, the amino acid sequence of the MOC1 gene is as shown in SEQ ID NO:4, the amino acid sequence of the Os7 gene is as shown in SEQ ID NO:5, and the amino acid sequence of the LS gene is as shown in SEQ ID NO:6.

[0006] The inventors found that when the MOC1 protein, Os7 protein, and LS protein are overexpressed using a plant expression vector such as Agrobacterium rhizogenes, the number of nodules in the obtained plants is significantly reduced compared to the source germplasm. Such an effect is unexpected based on the existing technology. Thus, the inventors discovered an isolated protein that is a protein related to the inhibition of symbiotic nitrogen fixation between soybean and Medicago truncatula rhizobia.

[0007] In the present invention, a recombinant vector containing the target gene can be constructed using an existing plant expression vector. The plant expression vector includes binary Agrobacterium vectors and vectors suitable for plant particle bombardment, etc. To facilitate the identification and screening of transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants, an antibiotic marker with resistance, or an anti-chemical reagent marker gene, etc. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions. The plant expression vector can also contain an enhancer to increase the expression of the inserted nucleotide fragment.

[0008] To achieve the above object, the present invention also provides a method for obtaining transgenic hairy roots, which is to introduce the aforementioned nucleic acid or the vector or host cell containing the aforementioned gene into a target leguminous plant such as soybean or Medicago truncatula to obtain a leguminous plant such as soybean or Medicago truncatula hairy root that shows a change in the number of nodules compared to the target leguminous plant such as soybean or Medicago truncatula.

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

[0010] The functions of the proteins related to nodulation number in soybean and Medicago truncatula provided by the present invention and their encoding nucleic acids in regulating the nodulation number of soybean and Medicago truncatula were first discovered by the applicant. The phenotypic analysis of transgenic hairy roots and empty vector-transformed hairy root plants verified that expressing the proteins related to nodulation number in soybean and Medicago truncatula of the present invention can reduce the nodulation number of transgenic hairy roots of soybean or Medicago truncatula. The present invention will have great theoretical and application values for regulating the nodulation number of soybean or Medicago truncatula and its related application research.

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

[0012] On the one hand, the present invention provides an isolated gene for regulating the nodulation number of soybean or Medicago truncatula, and the coding region sequence of the gene is shown as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or its homologous sequence.

[0013] On the other hand, the present invention provides a protein for regulating the nodulation number of soybean or Medicago truncatula, and the amino acid sequence of the protein is shown as SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 or its homologous sequence.

[0014] On the other hand, the present invention provides an expression vector, which contains the gene as described above or the nucleotide sequence encoding the protein as described above, or contains a sequence for inhibiting 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 contains a marker. Optionally, the marker is selected from a luminescent marker, an antibiotic marker and an anti-chemical reagent marker. Optionally, the luminescent marker is selected from a red fluorescent protein and a green fluorescent protein, the antibiotic marker is selected from ampicillin, chloramphenicol, kanamycin, neomycin, rifampicin, spectinomycin, hygromycin, streptomycin and tetracycline, and the anti-chemical reagent marker is, for example, an anti-herbicide marker.

[0016] On the other hand, the present invention provides a host cell, which contains the expression vector as described above.

[0017] On the other hand, the present invention provides a method for obtaining soybean hairy roots, which includes 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 leguminous plant cells or tissues such as soybean or Medicago truncatula cells or tissues 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 for plant particle bombardment.

[0019] In some embodiments, the host cell is selected from Escherichia coli cells, Agrobacterium cells (such as Agrobacterium rhizogenes cells), or plant cells.

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

[0021] On the other hand, the present invention provides the use of the gene as described above, or the nucleotide sequence encoding the protein as described above, or the expression vector as described above, or the host cell as described above in cultivating leguminous plants with altered nodule numbers, such as soybean or alfalfa.

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

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

[0024] On the other hand, the present invention provides a method for cultivating transgenic plants with increased yield or increased nodule number and / or nodule number per unit root length, which includes introducing the gene as described above, or the nucleotide sequence encoding the protein as described above, or the expression vector as described above, or the host cell as described above into target plant cells or tissues to obtain transgenic plants. Compared with the target plant, the transgenic plants have altered nodule numbers. The plant is a leguminous plant, preferably soybean and Medicago truncatula. Optionally, the alteration in nodule number is manifested as an increase in the nodule number and / or the nodule number per unit root length. Optionally, the alteration in nodule number is manifested as a decrease in the nodule number and / or the nodule number per unit root length. Description of the Drawings

[0025] Figure 1 Showing the results of nodulation phenotype analysis of hairy roots transformed with empty vector (EV) and LS overexpression vector in soybean in Example 2. Among them, a: Nodulation phenotypes of hairy roots transformed with empty vector, 35S:Os7, and 35S:MOC1. b: Statistical analysis of nodule numbers of hairy roots transformed with empty vector, 35S:Os7, and 35S:MOC1. c: Statistical analysis of nodule numbers per unit root length of hairy roots transformed with empty vector, 35S:Os7, and 35S:MOC1. d: Nodulation phenotypes of hairy roots transformed with empty vector and 35S:LS. e: Statistical analysis of nodule numbers of hairy roots transformed with empty vector and 35S:LS. f: Statistical analysis of nodule numbers per unit root length of hairy roots transformed with empty vector and 35S:LS.

[0026] Figure 2Show the nodulation phenotype results of hairy roots transformed with empty vector and LS overexpression vector in Medicago truncatula in Example 3. Among them, a: Nodulation phenotypes of hairy roots transformed with empty vector, 35S:Os7, and 35S:MOC1. b: Statistical analysis of the number of nodules on hairy roots transformed with empty vector, 35S:Os7, and 35S:MOC1. c: Statistical analysis of the number of nodules per unit root length of hairy roots transformed with empty vector, 35S:Os7, and 35S:MOC1. d: Nodulation phenotypes of hairy roots transformed with empty vector and 35S:LS. e: Statistical analysis of the number of nodules on hairy roots transformed with empty vector and 35S:LS. f: Statistical analysis of the number of nodules per unit root length of hairy roots transformed with empty vector and 35S:LS.

[0027] Figure 3 Show the expression analysis results of hairy roots transformed with empty vector and LS overexpression vector in soybean and Medicago truncatula in Example 2 and Example 3. Among them, a: Gene expression analysis results of hairy roots transformed with empty vector, 35S:Os7, 35S:MOC1, and 35S:LS in soybean. b: Gene expression analysis results of hairy roots transformed with empty vector, 35S:Os7, 35S:MOC1, and 35S:LS in Medicago truncatula. Detailed implementation manners

[0028] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0029] The following embodiments are for better understanding of the present invention, but are not used to limit the present invention. The experimental methods in the following embodiments are all conventional methods or selected according to the product specifications unless otherwise specified. The test materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified. For the quantitative tests in the following embodiments, at least three repeated experiments are set, and the results are averaged.

[0030] In the following examples, the hairy root transformation receptor of soybean was Williams 82, which was purchased from the United States Department of Agriculture, and its website is http: / / www.ars-grin.gov / npgs / acc / acc_queries.html. Williams 82 is a (spring sowing) soybean variety with an indeterminate podding habit. The stems, leaves and pods of the above-ground tissues are all (gray) pubescent, and the pubescence density is (normal). The hairy root transformation receptor of Medicago truncatula was A17. The pDonor221 vector, pBWG2D.1 vector, Agrobacterium rhizogenes strains K599 and AR1193, the slow-growing rhizobium Bradyrhizobium diazoefficiens strain USDA110, and Sinorhizobium meliloti 1021 were purchased from the China Center for Conservation of Plasmid Vectors, Strains and Cells (Biovector Science Lab, Inc).

[0031] Consumables such as Gateway and recovery kits were purchased from Thermo Fisher and Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0032] Example 1 Establishment of Transgenic Plants with Overexpressed LS Gene

[0033] The homologous genes of the lost LS gene in legumes 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 lost LS gene in legumes 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. Since the LS gene is a lost gene in leguminous plants, the function of this gene was studied by overexpression in this invention.

[0034] I. Construction of Recombinant Plasmids

[0035] 1. Isolate the leaf tissues of 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 composed of F1 and R1 to obtain a PCR amplification product (SEQ ID NO: 1). Perform PCR amplification with the primer pair composed 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. Isolate 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 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 the PCR product by gel extraction.

[0046] 6. Ligate the PCR product in step 5 and the intermediate vector pDonor221 with 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, confirm that the recombinant plasmids 35S:MOC1-pDonor221, 35S:Os7-pDonor221, and 35S:LS-pDonor221 are double-stranded DNA molecules inserted with the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in 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 the recombinant plasmids 35S:MOC1-pB7WG2D.1, 35S:Os7-pB7WG2D.1, and 35S:LS-pB7WG2D.1. According to the sequencing results, the structures of the recombinant plasmids 35S:MOC1-pB7WG2D.1, 35S:Os7-pB7WG2D.1, and 35S:LS-pB7WG2D.1 are described as follows: Double-stranded DNA molecules shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sequence listing were inserted between attR1 and attR2 of the pB7WG2D.1 vector, which verified the completion of the expression cassette construction.

[0048] II. Obtaining of LS Gene-Overexpressing Transgenic Hairy Roots of Soybean

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

[0050] 2. The Agrobacterium rhizogenes obtained in step 1 was used to transform the receptor plant Williams82 by the hairy root transformation method [21,22] The specific operation 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 petri dish, place them in a desiccator, and treat the seeds for 7 - 11 hours using the chlorine disinfection method (add 3 mL of 12 M concentrated hydrochloric acid to 75 mL of 10% sodium hypochlorite).

[0053] (3) After the chlorine disinfection is completed, blow the seeds in a laminar flow hood until there is no obvious chlorine smell, then sow them in vermiculite and germinate them in the greenhouse for 3 days. When the soybean cotyledons are about to open, transform them.

[0054] (4) When sowing the seeds, streak the Agrobacterium rhizogenes K599 containing the target vector on a solid LB medium and culture it in an incubator at 28 °C for about 2 days. One day before infection, pick a monoclonal 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 overnight on a shaker at 28 °C.

[0055] (5) Inoculate the activated bacterial liquid 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 culture. When the OD 600 value reaches about 0.6 - 0.8, perform cotyledon node infection.

[0056] (6) Select seeds without bacterial contamination and with intact and undamaged cotyledons. Use a scalpel to cut the germinated seeds from the roots, cut at the hypocotyl 1 cm below the lower end of the cotyledon, and gently make a wound at the lower end of the hypocotyl. Immerse the scratched explants in the bacterial liquid for about 30 min, gently shaking at 100 rpm during this period. Place 2 - 3 pieces of sterilized filter paper in an empty dish, pour off the bacterial liquid from the infected explants and place them on the filter paper, then cover them with another layer of filter paper to absorb the excess bacterial liquid.

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

[0058] (8) After the co - culture, the explants can be directly transplanted into vermiculite to induce root growth. Pay attention to moisture retention and temperature, and rooting is completed in about one week.

[0059] III. Obtaining transgenic hairy roots of Medicago truncatula with over - expressed LS gene

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

[0061] 2. Use the Agrobacterium rhizogenes obtained in step 1 to transform the receptor plant A17 by the hairy root transformation method. The specific operation steps are as follows:

[0062] 1) Preparation work

[0063] ① After the seeds are imbibed and germinated, invert the seeds in the dark at 22 °C one day before hairy root transformation for overnight germination;

[0064] ② Resuscitate the cryopreserved recombinant Agrobacterium by streaking on a plate, pick a monoclonal colony and culture it in LB liquid medium for 1 day. Take 200 μL of the bacterial solution and spread it on an LB solid medium containing rifampicin (50 μg / mL) and spectinomycin (100 μg / mL), and continue to culture it upside down for 1 day.

[0065] 2) Root hair transformation:

[0066] ① In a laminar flow hood, disinfect tools such as scalpels and forceps by alcohol high-temperature disinfection or ultraviolet irradiation sterilization. Wear gloves on both hands and disinfect with alcohol.

[0067] ② Use a scalpel to cut the hypocotyl of Medicago truncatula seeds, collect the bacteria on the plate medium with a blade, carefully pick up the wounded seedlings of the hypocotyl with forceps, dip the collected Agrobacterium at the wound, and place it on MFP medium (formula from JIC Media Preparation lab recipes). Seal the petri dish with parafilm and put it in an incubator at 22 °C. Carefully cover the petri dish and culture it in the dark for 24 h, then culture it under light for one week.

[0068] 3) Removal of non-transformed roots: Remove the non-transformed roots that have grown for one week with a scalpel in a laminar flow hood, and transfer the treated plants (with transformed roots) to HRE medium

[23] and culture them in an incubator at 22 °C for 1 - 2 weeks;

[0069] 4) Before transplanting the transformed seedlings, it is necessary to observe the plant fluorescence under a fluorescence stereomicroscope (the transformed plasmid has a fluorescence screening marker), remove the roots that do not express fluorescence, and use the obtained root hair transformation materials for subsequent experiments.

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

[0071] 1. Rhizobium inoculation treatment

[0072] (1) Take 1 mL of the preserved slow-growing rhizobium Bradyrhizobium diazoefficiens strain USDA110 at -80 °C and add it to 200 mL of TY liquid medium containing 50 mg / L spectinomycin (tryptone 5 g / L, yeast extract 3 g / L, CaCl 2 ·2H 2 O 0.7 g / L), and culture it in a shaker at 28 °C at 200 rpm for about 4 days until the OD 600 is between 0.8 - 1.

[0073] (2) Collect the bacterial solution with a 500 mL centrifuge bottle and centrifuge at 6000 rpm for 10 min. Use sterilized ddH 2O heavy suspension bacteria to OD 600 Between 0.8 and 1, for inoculation treatment.

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

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

[0076] 2. Statistical analysis of nodule number phenotype

[0077] The plants 21 days after inoculation are washed with tap water to remove vermiculite and dried with absorbent paper. Visually observe the hairy roots of soybean transformed with empty vector, 35S:Os7, 35S:MOC1 and 35S:LS. It can be seen that the number of nodules on the hairy roots transformed with 35S:Os7, 35S:MOC1 and 35S:LS is significantly lower than that of the control transformed with empty vector ( Figure 1 a and 1d).

[0078] The inventors counted the number of nodules and root length of the hairy roots transformed with 35S:Os7, 35S:MOC1 and 35S:LS and the hairy roots transformed with empty vector, Figure 1 as shown in b, 1c, 1e and 1f. From Figure 1 b, 1c, 1e and 1f, it can be seen that the average values of the number of nodules on the transgenic hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS only account for 5.6%, 4.7% and 36.6% of the control transformed with empty vector; the number of nodules per unit root length of the transgenic hairy roots of 35S:Os7, 35S:MOC1 and 35S:LS only accounts for 13.2%, 3.1% and 7.2% of the control transformed with empty vector, indicating that the LS gene inhibits the formation of soybean nodules.

[0079] 3. Identification of gene expression level

[0080] The hairy roots transformed with empty vector, 35S:Os7, 35S:MOC1 and 35S:LS are respectively 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, use the primer pair composed of F4 and R4 to identify the expression level of MOC1 gene, use the primer pair composed of F5 and R5 to identify the expression level of Os7 gene, use the primer pair composed of F6 and R6 to identify the expression level of LS gene, and use F7 and R7 to identify the expression level of 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'-CGGTGGTTCTATCTTGGCATC-3' (SEQ ID NO: 19);

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

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

[0092] The expression levels of the LS gene in different materials obtained by semi-quantitative PCR amplification using the respective specific primer pairs with the said cDNA as a template are shown in Figure 3 a. From Figure 3 a, it can be seen that in the gene overexpression lines, the gene expression of LS.

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

[0094] 1. Rhizobium inoculation treatment

[0095] 1) Activate Rhizobium: Streak Sinorhizobium meliloti 1021 stored in an -80°C refrigerator onto a TY plate containing tetracycline (10 μg / mL) using a sterile inoculation loop (or sterile pipette tip), and place it in an incubator at 28°C for 2 - 3 days;

[0096] 2) Small-scale shaking culture of bacterial liquid: Pick a single colony of rhizobia after activation and place it in 3 mL of TY liquid medium with the corresponding antibiotic. Culture it on a shaker at 28 °C for 1 day;

[0097] 3) Large-scale shaking culture of bacterial liquid: Add the small-scale shaken bacterial liquid to a new TY liquid resistant medium at a ratio of 1:100 and continue to culture it on a shaker at 28 °C for one day;

[0098] 4) Take the large-scale shaken bacterial liquid to detect the OD 600 value. When OD 600 = 0.8 - 1, transfer the rhizobia bacterial liquid to a centrifuge tube and centrifuge it at 3000 - 4000 rpm for 5 min;

[0099] 5) Discard the supernatant, resuspend the bacterial cells with double-distilled water, and measure the OD 600 value again. Dilute the concentration to OD 600 ≈ 0.03. Keep the soil appropriately dry before inoculation and evenly spray the diluted bacterial liquid onto the roots and soil of the plants using an atomizing sprayer.

[0100] 2. Statistical analysis of nodule number phenotypes

[0101] Observe the nodulation phenotypes 21 days after inoculation. Visually observe the hairy roots of Medicago truncatula transformed with the empty vector, 35S:Os7, 35S:MOC1, and 35S:LS. It can be seen that the number of nodules on the hairy roots transformed with 35S:Os7, 35S:MOC1, and 35S:LS is significantly lower than that of the control transformed with the empty vector ( Figure 2 a and 2d).

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

[0103] 3. Identification of gene expression levels

[0104] Conduct the following identifications on the hairy roots transformed with the empty vector, 35S:Os7, 35S:MOC1, and 35S:LS respectively:

[0105] (1) Roots and root nodules were taken, total RNA was extracted and reverse transcribed into cDNA.

[0106] (2) Using the cDNA extracted in step (1) as a template, the expression level of the MOC1 gene was identified with the primer pair consisting of F4 and R4, the expression level of the Os7 gene was identified with the primer pair consisting of F5 and R5, the expression level of the LS gene was identified with the primer pair consisting of F6 and R6, and the expression level of the reference gene (Actin gene) in Medicago truncatula was identified with 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, 72°C for 40 s. Final extension at 72°C for 10 min.

[0110] The expression levels of the LS gene in different materials obtained by semi-quantitative PCR amplification using the cDNA as a template and each specific primer pair were shown in Figure 3 b. It can be seen from Figure 3 b that in the gene overexpression lines, the gene expression of LS.

[0111] References:

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[0135] Sequence Listing

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

[0137]

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

[0139]

[0140] SEQ ID NO:3 LS 1287bp 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'- CATTAAGTTTGACAAAGAGAAAGAGACAGA-3'

[0180] The specific embodiments described above further elaborate on the object, technical solution and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope 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 gene encodes a protein as shown in SEQ ID NO: 5, 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 coding region sequence of the gene is shown in SEQ ID NO:

2.

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 encoding the protein according to claim 1 or a gene according to claim 2, and the change in 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 Escherichia coli cells and Agrobacterium 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 a 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 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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