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

Abstract

The present invention relates to a gene CTSH for regulating symbiotic nitrogen fixation of leguminous plant root nodules, wherein the coding region sequence of the gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a homologous sequence thereof. By reducing the expression of CTSH in leguminous nodulation plants, the number of nodules of leguminous plants is significantly regulated, and the invention has a good application prospect in the genetic improvement of plants. 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 botany, and specifically relates to a gene CTSH for regulating symbiotic nitrogen fixation in root nodules of leguminous plants and an application thereof. Background Art

[0002] Nitrogen is a macronutrient essential for plant growth and development and is also one of the factors limiting food production. The lack of nitrogen will cause agricultural production to decrease. The application of nitrogen fertilizer has revolutionized the yield of crops and greatly promoted food production. However, the current utilization rate of chemical fertilizers for crops is generally low. The inefficient use of nitrogen fertilizers not only causes huge economic losses, but also brings serious water pollution and soil acidification, causing serious damage to the ecological environment. Nitrogen in the air is the main form of nitrogen in nature. However, plants cannot directly use nitrogen in the air, but can only use ammonia nitrogen or nitrate nitrogen to convert these inorganic nitrogen into organic nitrogen such as amino acids and nucleotides in plants. Animals directly or indirectly use plants as food, absorb organic nitrogen in plants and assimilate it into organic nitrogen in their own bodies. The process of reducing nitrogen with three high-energy bonds in the atmosphere into ammonia is called nitrogen fixation. Biological nitrogen fixation is the process of converting nitrogen into ammonia by microorganisms, and it is also the main way of nitrogen fixation. Microorganisms that can carry out biological nitrogen fixation are called nitrogen-fixing bacteria, and nitrogen-fixing bacteria that can only fix nitrogen or fix nitrogen efficiently when in symbiosis are called symbiotic nitrogen-fixing bacteria. The symbiotic nitrogen fixation systems of rhizobia and legumes, rhizobia and non-legumes, and actinomycetes and actinomycete-nodulated plants can all form nodules, collectively referred to as root nodule symbiosis (RNS).

[0003] There are 15 cathepsins in humans, including 11 cysteine ​​proteases (Cathepsin B, C, F, H, K, L, O, S, V, X and W); 2 serine proteases (Cathepsin A and G) and 2 aspartic proteases (Cathepsin E and D). [1] Among them, cysteine ​​proteases play an important role in multiple processes of leguminous root nodule development. For example, the cysteine ​​protease gene PsCyp1 in pea [2] , AsNODf32, a nodule-specific cysteine ​​protease gene in Astragalus membranaceus [3,4] , a C13 family CD-type cysteine ​​protease specific to soybean nodules GmCysP1 [5] Two different classes of cysteine ​​proteases, MtCP6 and MtVPE, are expressed in senescent tissues of root nodules in alfalfa [6], which is associated with nodule senescence. [7] Expression in root nodule symbionts and CP genes in soybean [8] and MtCP77 gene in alfalfa [9] Participates in programmed cell death of nodule cells. Therefore, it is of great significance to explore the regulatory mechanism of cysteine ​​proteases in nitrogen fixation in leguminous plant nodules, so as to make it possible to regulate the efficiency of nitrogen fixation in nodule symbiosis by genetic engineering. Summary of the invention

[0004] In crop breeding, too many or too few nodules will affect crop yields, so the number of nodules needs to be regulated.

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

[0005] The inventors used molecular biology and comparative genomics to clone a gene Glyma.15G177800 (also referred to herein as GmCTSH) that regulates the number of nodules in soybean (Glycine max) from the American soybean line Williams82, and cloned a homologous gene Medtr4g125300 (also referred to herein as MtCTSH) that regulates the number of nodules in Medicago truncatula from the Medicago truncatula line A17. The position of the GmCTSH encoding gene in the Williams 82a2.V1 version sequenced genome is Chr15:16892475..16897664, and the position of the MtCTSH encoding gene in the A17 4.0V1 version sequenced genome is chr4:51952476..51956344. The cloning of GmCTSH and MtCTSH provides a theoretical basis and genetic resources for subsequent molecular-assisted breeding and molecular-design breeding.

[0006] Wherein, in a specific embodiment of the present invention, the cDNA sequence of the GmCTSH gene is shown as SEQ ID NO: 1, and the cDNA sequence of the MtCTSH gene is shown as SEQ ID NO: 2. In a specific embodiment of the present invention, the amino acid sequence of the GmCTSH gene is shown as SEQ ID NO: 3, and the amino acid sequence of the MtCTSH gene is shown as SEQ ID NO: 4.

[0007] The inventors found that when GmCTSH protein and MtCTSH protein were used for RNAi 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. Therefore, the present application provides an isolated protein, which is a symbiotic nitrogen fixation-related protein of soybean and Medicago truncatula rhizobia, and annotates the function of its coding sequence in the soybean and Medicago truncatula genomes for the first time.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] Specifically, the present invention provides the following technical solutions:

[0013] 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 or SEQ ID NO: 2 or a homologous sequence thereof.

[0014] 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: 3 or SEQ ID NO: 4 or a homologous sequence thereof.

[0015] 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 or protein as described above, such as an interfering sequence of the gene or an interfering sequence of the nucleotide sequence encoding the protein.

[0016] 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.

[0017] In another aspect, the present invention provides a host cell comprising the expression vector as described above.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In some embodiments, the Agrobacterium rhizogenes is selected from K599, AR1193, C58cl, Arqual, MSU440, LBA9402, and R1601.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] On the other hand, the present invention provides a method for cultivating transgenic plants with increased yield or altered nodulation 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 yield of the transgenic plant is increased or the number of nodules is altered compared with the target plant, and the plant is a leguminous plant, preferably soybean and Medicago truncatula, optionally, the change in the nodulation number 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 nodulation number is manifested as a decrease in the number of nodules and / or the number of nodules per unit root length.

[0026] definition

[0027] CDS sequence: CDS is the part of the transcript that is actually translated into protein, that is, the sequence of exons. It represents the sequence of amino acids encoded in the gene and is the direct template for protein synthesis. CDS usually does not include introns because introns are spliced ​​out during post-transcriptional processing, leaving only the exon sequence encoding the protein.

[0028] Transcript sequence: Transcripts are RNA molecules produced by gene transcription, including mRNA, tRNA and rRNA. Transcripts may contain introns and exons. Introns are spliced ​​out during post-transcriptional processing, while exons are retained and ultimately used to encode proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of nodulation phenotype analysis and expression level analysis of hairy roots transfected with empty vector (EV) and GmCTSH-RNAi in Example 2 are shown. Among them, a: Nodulation phenotype of hairy roots transfected with empty vector and GmCTSH-RNAi. b: Statistics of the number of nodules of hairy roots transfected with empty vector and GmCTSH-RNAi. c: Statistics of the number of nodules per unit root length of hairy roots transfected with empty vector and GmCTSH-RNAi. d: Analysis of the expression level of GmCTSH in hairy roots transfected with empty vector and GmCTSH-RNAi.

[0030] Figure 2The results of nodulation phenotype analysis and expression level analysis of hairy roots transfected with empty vector and MtCTSH-RNAi in Example 3 are shown. Among them, a: Nodulation phenotype of hairy roots transfected with empty vector and MtCTSH-RNAi. b: Statistics of the number of nodules of hairy roots transfected with empty vector and MtCTSH-RNAi. c: Statistics of the number of nodules per unit root length of hairy roots transfected with empty vector and MtCTSH-RNAi. d: Analysis of the expression level of MtCTSH in hairy roots transfected with empty vector and MtCTSH-RNAi. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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 hairy root transformation receptor of Medicago truncatula is A17. pDonor221 vector, pK7GWⅡRR vector, pDONR207 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 Center (Biovector Science Lab, Inc).

[0034] Gateway, recovery kits and other consumables were purchased from Thermo Fisher Scientific and Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0035] Example 1 Establishment of GmCTSH gene silencing transgenic plants

[0036] Based on a large number of sequence analyses and functional verifications, a H-type cysteine ​​protease gene (Cathepsin H) that regulates the number of nodules was found in the soybean variety Williams 82, which was named GmCTSH, and its cds sequence is shown in SEQ ID NO: 1, its transcript sequence is shown in SEQ ID NO: 5, and its encoded protein sequence is shown in SEQ ID NO: 3. In view of the fact that leguminous plants have CTSH genes, the present invention studies the function of this gene through gene silencing.

[0037] 1. Construction of recombinant plasmid

[0038] 1. Separate the nodule tissue of soybean variety Williams 82 from the root, extract RNA and perform reverse transcription to obtain cDNA of the nodule tissue of soybean variety Williams 82.

[0039] 2. Using the cDNA synthesized in step 1 as a template, perform PCR amplification using a primer pair consisting of F1 and R1 to obtain a PCR amplification product (SEQ ID NO: 6).

[0040] F1: 5'-AGCATAACATAACCCATTGAAGATTCC-3' (SEQ ID NO: 8);

[0041] R1: 5'-CCTATCACCCGAACCACCTCC-3' (SEQ ID NO: 9).

[0042] 3. Recover PCR products from gel.

[0043] 4. The PCR product of step 3 and the intermediate vector pDonor221 were connected by BP ClonaseTM II enzyme mix (Invitrogen 11789-020) to obtain the recombinant plasmid GmCTSH-pDonor221. According to the sequencing results, it was confirmed that the recombinant plasmid GmCTSH-pDonor221 was a double-stranded DNA molecule as shown in the sequence list as SEQ ID NO: 6 inserted into the pDonor221 vector.

[0044] 5. The plasmid GmCTSH-pDonor221 obtained in step 4 was connected to the final vector pK7GWⅡRR through LR CLONASEII ENZYME MIX (Invitrogen 11791-020) to obtain the recombinant plasmid GmCTSH-pK7GWⅡRR. According to the sequencing results, the structure of the recombinant plasmid GmCTSH-pK7GWⅡRR was described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 6 in the sequence table was inserted between attR1 and attR2 of the pK7GWⅡRR vector, which verified that the expression cassette was constructed.

[0045] 2. Obtaining transgenic hairy roots with GmCTSH gene silencing

[0046] 1. The recombinant plasmid GmCTSH-pK7GWⅡRR was introduced into the Agrobacterium rhizogenes strain K599 to obtain the recombinant Agrobacterium, which was frozen at -80°C and stored in glycerol.

[0047] 2. The Agrobacterium rhizogenes obtained in step 1 was transformed by the hairy root method [11,12] Transform the recipient plant Williams82. The specific steps are as follows:

[0048] (1) Select Williams 82 soybean seeds that are large, plump, and free of disease spots.

[0049] (2) Spread the selected seeds in a medium-sized culture dish, place it in a desiccator, and treat the seeds for 7-11 hours using chlorine disinfection (add 3 mL of 12 M concentrated hydrochloric acid to 75 mL of 10% sodium hypochlorite).

[0050] (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.

[0051] (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.

[0052] (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.

[0053] (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.

[0054] (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.

[0055] (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.

[0056] Example 2 Changes in the number of nodules during GmCTSH gene silencing

[0057] 1. Rhizobium inoculation treatment

[0058] (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.

[0059] (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.

[0060] (3) About 13 days after the hairy root transformation, the seedlings were inoculated with 30 mL of the resuspended bacterial solution.

[0061] (4) Observe the nodulation phenotype 21 days after inoculation.

[0062] 2. Nodule number phenotype statistics and analysis

[0063] 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 GmCTSH-RNAi were observed with naked eyes. It can be seen that the number of nodules in the hairy roots of GmCTSH-RNAi transfected plants was significantly lower than that of the control plants transfected with empty vector ( Figure 1 a).

[0064] The inventors counted the number of nodules and root length of hairy roots transfected with GmCTSH-RNAi and hairy roots transfected with an empty vector. Figure 1 b and Figure 1 c. Figure 1 b and Figure 1 As can be seen from c, the average number of nodules in GmCTSH-RNAi transgenic hairy roots is only 60.5% of that in the empty vector control; the average number of nodules per unit root length in transgenic hairy roots is only 61.7% of that in the empty vector control, which indicates that the GmCTSH gene promotes the formation of soybean nodules.

[0065] 3. Identification of gene expression

[0066] The hairy roots of the empty vector and the hairy roots of the GmCTSH gene silenced were identified as follows:

[0067] (1) Take roots and nodules, extract total RNA and reverse transcribe it into cDNA.

[0068] (2) Using the cDNA extracted in step (1) as a template, the primer pair consisting of F3 and R3 was used to identify the expression level of the GmCTSH gene, and the primer pair consisting of F2 and R2 was used to identify the expression level of the internal reference gene (Actin gene).

[0069] F2: 5'-CGGTGGTTTCTATCTTGGCATC-3' (SEQ ID NO: 10);

[0070] R2: 5'-GTCTTTCGCTTCAATAACCCTA-3' (SEQ ID NO: 11).

[0071] F3: 5'-GAGTTTACACTAGTGACATTTGC-3' (SEQ ID NO: 12);

[0072] R3: 5'-TTATGCCACAACTGGATAAGAC-3' (SEQ ID NO: 13).

[0073] The conditions for quantitative PCR amplification were: pre-denaturation at 95°C for 5 min; amplification (45 cycles) at 95°C for 10 s, 59°C for 15 s, and 72°C for 20 s. After the amplification cycle, the melting curve was started: 95°C for 5 s, 65°C for 1 min, and 65-97°C (the temperature increased at a rate of 0.11°C / s).

[0074] The expression levels of the GmCTSH gene in different materials obtained by quantitative PCR amplification using the cDNA as a template and each specific primer pair are shown in Figure 1 d. From Figure 1As can be seen in Figure d, in the gene silencing strain GmCTSH-RNAi, the average gene expression level of GmCTSH is only 9.97% of the control with the empty vector.

[0075] Example 3: Establishment of MtCTSH gene-silenced transgenic plants

[0076] Based on a large number of sequence analyses and functional verifications, a homologous gene of the GmCTSH gene that regulates the nodule number was found in the Medicago truncatula variety A17, and was named MtCTSH. Its cds sequence is shown in SEQ ID NO:2, and its encoded protein sequence is shown in SEQ ID NO:4.

[0077] 1. Construction of recombinant plasmid

[0078] 1. Separate the nodule tissue of Medicago truncatula variety A17 from the root, extract RNA and perform reverse transcription to obtain cDNA of the nodule tissue of Medicago truncatula variety A17.

[0079] 2. Using the cDNA synthesized in step 1 as a template, PCR amplification was performed using a primer pair consisting of F4 and R4 to obtain a PCR amplification product (SEQ ID NO: 7).

[0080] F4: 5'-ATGGCACAGTGGACGCTC-3' (SEQ ID NO: 14);

[0081] R4: 5'-TCATCAACGGTATCGTATCTTTTGCC-3' (SEQ ID NO: 15).

[0082] 3. Recover PCR products from gel.

[0083] 4. The PCR product of step 3 and the intermediate vector pDONR207 were connected by BP reaction (Gateway cloning step) to obtain the recombinant plasmid MtCTSH-pDONR207. According to the sequencing results, it was confirmed that the recombinant plasmid MtCTSH-pDONR207 was a double-stranded DNA molecule as shown in SEQ ID NO: 7 in the sequence list inserted into the pDONR207 vector.

[0084] 5. The plasmid MtCTSH-pDonor221 obtained in step 4 was connected with the final vector pUB-GWS-GFP (from John Innes Centre, JIC, UK) through LR reaction (Gateway cloning step) to obtain the recombinant plasmid MtCTSH-pUB-GWS-GFP. According to the sequencing results, the structure of the recombinant plasmid MtCTSH-pUB-GWS-GFP is described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 7 in the sequence table was inserted between attR1 and attR2 of the pUB-GWS-GFP vector, which verified that the expression cassette was constructed.

[0085] 2. Obtaining transgenic hairy roots with MtCTSH gene silencing

[0086] 1. The recombinant plasmid MtCTSH-pUB-GWS-GFP was introduced into Agrobacterium Rhizogenes strain AR1193 to obtain recombinant Agrobacterium, which was frozen at -80°C and stored in glycerol.

[0087] 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:

[0088] 1) Preparation

[0089] 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.

[0090] ② Streak the frozen recombinant Agrobacterium to revive, 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 (15 μg / mL) and kanamycin (50 μg / mL), and continue to culture inverted for 1 day.

[0091] 2) Hair root transformation:

[0092] 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;

[0093] 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 in 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.

[0094] 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.

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

[0095] 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.

[0096] Example 4 Changes in the number of nodules during MtCTSH gene silencing

[0097] 1. Rhizobium inoculation treatment

[0098] 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;

[0099] 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;

[0100] 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;

[0101] 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;

[0102] 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.

[0103] 2. Nodule number phenotype statistics and analysis

[0104] The nodulation phenotype was observed 21 days after inoculation. The hairy roots of Medicago truncatula transfected with empty vector and MtCTSH-RNAi were observed with naked eyes. It can be seen that the number of nodules in the hairy roots transfected with MtCTSH-RNAi was significantly lower than that in the control transfected with empty vector ( Figure 2 a).

[0105] The inventors counted the number of nodules and root length of hairy roots transfected with MtCTSH-RNAi and hairy roots transfected with an empty vector. Figure 2 b and Figure 2 c. Figure 2 b and Figure 2 As can be seen from c, the average number of hairy root nodules of the MtCTSH-RNAi transgenic plants is only 61.2% of that of the empty vector control; the average number of nodules per unit root length is only 68.7% of that of the empty vector control, which indicates that the MtCTSH gene promotes the formation of root nodules in Medicago truncatula.

[0106] 3. Identification of gene expression

[0107] The hairy roots of the empty vector and the hairy roots of the MtCTSH gene silenced were identified as follows:

[0108] (1) Take roots and nodules, extract total RNA and reverse transcribe it into cDNA.

[0109] (2) Using the cDNA extracted in step (1) as a template, the primer pair consisting of F6 and R6 was used to identify the expression level of the MtCTSH gene, and the primer pair consisting of F5 and R5 was used to identify the expression level of the internal reference gene (Actin gene).

[0110] F5: 5'-AACTTGTTGCATGGGTCTTGA-3' (SEQ ID NO: 16);

[0111] R5: 5'-CATTAAGTTTGACAAAGAGAAAGAGACAGA-3' (SEQ ID NO: 17).

[0112] F6: 5'-CTTCCCGCTGAGAAAGACTG-3' (SEQ ID NO: 18);

[0113] R6: 5'-TATTCAAAGGCTTGGGATGG-3' (SEQ ID NO: 19).

[0114] The conditions for quantitative PCR amplification were: pre-denaturation at 95°C for 5 min; amplification (45 cycles) at 95°C for 10 s, 59°C for 15 s, and 72°C for 20 s. After the amplification cycle, the melting curve was started: 95°C for 5 s, 65°C for 1 min, and 65-97°C (the temperature increased at a rate of 0.11°C / s).

[0115] The expression levels of the MtCTSH gene in different materials obtained by quantitative PCR amplification using the cDNA as a template and each specific primer pair are shown in Figure 2 d. From Figure 2As can be seen in Figure d, in the gene silencing strain MtCTSH-RNAi, the average gene expression level of MtCTSH is only 24.9% of that of the control with the empty vector.

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[0124] specific plant cysteine proteinase,AsNODF32,is involved in nodulesenescence andnitrogen fixation activity of the green manure legumeAstragalus sinicus.New Phytol.

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[0135] Transcription factor bHLH2 represses CYSTEINE PROTEASE77 tonegativelyregulate nodule senescence.Plant Physiol.181,1683-1703.

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[0144] Entertainment

[0145] SEQ ID NO:1GmCTSH 1065bp cDNA Glycine max(chain)

[0146]

[0147] SEQ ID NO:2MtCTSH 1053 bp cDNA Medicago truncatula (Medicago truncatula)

[0148]

[0149] SEQ ID NO: 3 Glycine max (soybean) 355 - aa GmCTSH protein

[0150] MARLITVVWCAVAVLLCAAAGASWVEEAANPIRMVAGVEAEVVRVIGQCRRALKFARFMSRFGKSYRSEEEMRERYEIFSQNLRFIRSHNKNRLPYTLSVNHFADWTWEEFKRHRLGAAQNCSATLNGNHKLTDAVLPPTKDWRKEGIVSDVKDQGSCGSCWTFSTTGALEAAYAQAFGKSISLSEQQLVDCAGRFNNFGCNGGLPSQAFEYIKYNGGLETEEAYPYTGKDGVCKFSAENVAVQVIDSVNITLGAENELKHAVAFVRPVSVAFQVVNGFHFYENGVYTSDICGSTSQDVNHAVLAVGYGVENGVPYWLIKNSWGESWGENGYFKMELGKNMCGVATCASYPVVA

[0151] SEQ ID NO: 4 Medicago truncatula 351 - aa MtCTSH protein

[0152] MAQWTLLIVFFCVATAAAGLSFHDSNPIRMVSDMEEQLLQVIGESRHAVSFARFANRYGKRYDTVDEMKRRFKIFSENLQLIKSTNKKRLGYTLGVNHFADWTWEEFRSHRLGAAQNCSATLKGNHRITDVVLPAEKDWRKEGIVSEVKDQGHCGSCWTFSTTGALESAYAQAFGKNISLSEQQLVDCAGAYNNFGCNGGLPSQAFEYIKYNGGLETEEAYPYTGQNGLCKFTSENVAVQVLGSVNITLGAEDELKHAVAFARPVSVAFQVVDDFRLYKKGVYTSTTCGSTPMDVNHAVLAVGYGIEDGVPYWLIKNSWGGEWGDHGYFKMEMGKNMCGVATCSSYPVVA

[0153]

[0154] SEQ ID NO:6 GmCTSH 187bp RNAi Glycine max (soybean)

[0155] AGCATAACATAACCCCATTGAAGATTCCGCAACAGAGAGTAGAAAA

[0156] GATGGCGCGGTTGATCACAGTGGTGTGGTGCGCGGTGGCGGTGCTA

[0157] CTGTGCGCCGCGGCGGGCGCGTCGTGGGTGGAGGAGGCGGCGAAC

[0158] CCGATACGAATGGTGGCGGGGGTGGAGGCGGAGGTGGTTCGGGTGA

[0159] TAGG

[0160] SEQ ID NO:7 MtCTSH 200bp RNAi Medicago truncatula (barrel medic) ATGGCACAGTGGACGCTCCTTATCGTTTTCTTCTGCGTGGCCACCGCCGCCGCCGGATTGAGCTTCCATGACTCCAATCCTATTCGGATGGTCTCCGACATGGAGGAGCAGCTTCTTCAGGTCATCGGAGAATCTCGCCACGCTGTCTCTTTCGCTCGTTTTGCTAACAGATACGGCAAAAGATACGATACCGTTGATGA

[0161] SEQ ID NO:8 F1 primer

[0162] 5'-AGCATAACATAACCCCATTGAAGATTCC-3'

[0163] SEQ ID NO:9 R1 primer

[0164] 5'-CCTATCACCCGAACCACCTCC-3'

[0165] SEQ ID NO:10 F2 primer

[0166] 5'-CGGTGGTTCTATCTTGGCATC-3'

[0167] SEQ ID NO:11 R2 primer

[0168] 5'-GTCTTTCGCTTCAATAACCCTA-3'

[0169] SEQ ID NO: 12F3 Primer

[0170] 5'-GAGTTTACACTAGTGACATTTGC-3'

[0171] SEQ ID NO: 13R3 Primer

[0172] 5'-TTATGCCACAACTGGATAAGAC-3'

[0173] SEQ ID NO: 14F4 Primer

[0174] 5'-ATGGCACAGTGGACGCTC-3'

[0175] SEQ ID NO: 15R4 Primer

[0176] 5'-TCATCAACGGTATCGTATCTTTTGCC-3'

[0177] SEQ ID NO: 16F5 Primer

[0178] 5'-AACTTGTTGCATGGGTCTTGA-3'

[0179] SEQ ID NO: 17R5 Primer

[0180] 5'-CATTAAGTTTGACAAAGAGAAAGAGACAGA-3'

[0181] SEQ ID NO: 18F6 Primer

[0182] 5'-CTTCCCGCTGAGAAAGACTG-3'

[0183] SEQ ID NO: 19R6 Primer

[0184] 5'-TATTCAAAGGCTTGGGATGG-3'

[0185] 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 the RNAi sequence of the gene with the coding region sequence of regulating the symbiotic nitrogen fixation or nodulation number of legume root nodules such as SEQ ID NO: 1 into the cells or tissues of soybean for cultivation, or introducing the RNAi sequence of the gene with the coding region sequence of regulating the symbiotic nitrogen fixation or nodulation number of legume root nodules such as SEQ ID NO: 2 into the cells or tissues of Medicago truncatula for cultivation, to obtain a plant with a changed nodulation number, wherein the changed nodulation 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.

2. The method according to claim 1, characterized in that The RNAi sequence of the gene whose coding region sequence is shown in SEQ ID NO:1 is shown in SEQ ID NO:

6.

3. The method according to claim 1, characterized in that The RNAi sequence of the gene whose coding region sequence is shown in SEQ ID NO:2 is shown in SEQ ID NO:

7.

4. The method according to claim 1, characterized in that: The gene whose coding region sequence is shown in SEQ ID NO:1 encodes the protein shown in SEQ ID NO:3, and the gene whose coding region sequence is shown in SEQ ID NO:2 encodes the protein shown in SEQ ID NO:

4.

5. The method according to claim 1, 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 an RNAi sequence of the gene according to claim 1, 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.

6. The method according to claim 5, characterized in that The expression vector comprises a marker.

7. The method according to claim 6, characterized in that The label is selected from the group consisting of a luminescent label, an antibiotic label and an anti-chemical agent label.

8. The method according to claim 7, characterized in that The luminescent marker is selected from red fluorescent protein and green fluorescent protein.

9. The method according to claim 7, 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.

10. The method according to claim 7, characterized in that The chemical resistance marker is a herbicide resistance marker.

11. The method according to any one of claims 5 to 10, characterized in that: The vector is a plant expression vector, and the plant expression vector is a binary Agrobacterium rhizogenes vector and / or a vector that can be used for plant microprojectile bombardment.

12. A method for cultivating soybeans with a changed number of nodules, characterized in that: The method comprises introducing a host cell into a soybean cell or tissue for cultivation to obtain a plant with a changed nodulation number, wherein the host cell comprises an expression vector, wherein the expression vector comprises an RNAi sequence of a gene as shown in SEQ ID NO: 1, and the host cell is an Agrobacterium cell, and the change in nodulation 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.

13. A method for cultivating Medicago truncatula with a changed nodule number, characterized in that: The method comprises introducing a host cell into a cell or tissue of Medicago truncatula for cultivation to obtain a plant with a changed nodule number, wherein the host cell comprises an expression vector, wherein the expression vector comprises an RNAi sequence of a gene as shown in SEQ ID NO: 2, and the host cell is an Agrobacterium cell, 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.

14. The method according to claim 12 or 13, characterized in that The Agrobacterium cells are Agrobacterium rhizogenes cells.

15. The method according to claim 14, characterized in that The Agrobacterium rhizogenes is selected from K599, AR1193, C58cl, Arqual, MSU440, LBA9402 and R1601.

16. A method for obtaining soybean hairy roots, comprising transforming an expression vector comprising an RNAi sequence of the gene shown in SEQ ID NO: 1 or a host cell comprising the expression vector into soybean plant cells or tissues and culturing the cells 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.

17. A method for obtaining Medicago truncatula hairy roots, comprising transforming an expression vector comprising an RNAi sequence of a gene as shown in SEQ ID NO: 2 or a host cell comprising the expression vector into Medicago truncatula plant cells or tissues and culturing the cells 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.

18. Use of an expression vector comprising an RNAi sequence of the gene as shown in SEQ ID NO: 1 or a host cell comprising the expression vector in cultivating soybeans 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.

19. Use of an expression vector comprising an RNAi sequence of the gene as shown in SEQ ID NO: 2 or a host cell comprising the expression vector in cultivating Medicago truncatula with an 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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