Gene GmHB21 participating in development of vascular bundles of soybean root nodules and application of gene GmHB21

Abstract

The invention relates to a gene GmHB21 participating in development of vascular bundles of soybean root nodules and application of the gene GmHB21. Specifically, the invention relates to a separated gene participating in development of vascular bundles of soybean root nodules or regulation and control of the number of soybean nodules, and a coding region sequence of the gene is shown as SEQ ID NO: 1 or a homologous sequence of the gene. The invention also provides a method for regulating and controlling the nodulation number of the plant, the method comprises the following steps: introducing the gene or a sequence for inhibiting the expression of the gene into a target plant cell or tissue to obtain a transgenic plant, the nodulation number of the transgenic plant is changed compared with that of the target plant, and the plant is a leguminous plant, preferably soybean. In addition, the invention also provides application of the gene or a sequence for inhibiting expression of the gene in cultivation of leguminous plants with changed nodulation number, such as soybean.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and botany, and specifically relates to a gene GmHB21 which participates in the development of soybean nodule vascular bundle and then regulates nodule symbiotic nitrogen fixation and an application thereof. Background Art

[0002] Nodule symbiosis refers to the symbiotic relationship between the roots of leguminous plants and rhizobia, which form nodules in the plant roots and convert atmospheric nitrogen into ammonia (NH3) that can be used by plants. This process is called nitrogen fixation and is essential for plant growth and the nitrogen cycle of the ecosystem. The formation of vascular bundles is not only essential for the material transport and structural support of plants, but also plays a key role in nodule symbiosis. The vascular bundle is involved in the transport of nutrients in the nodule symbiosis. The phloem in the vascular bundle is responsible for transporting photosynthetic products (such as sucrose) from the leaves to the nodules. These photosynthetic products are the main source of energy required for rhizobia to carry out nitrogen fixation. The xylem is responsible for transporting minerals and water from the roots to other parts of the plant, including the nodules. Nitrogen fixation requires the participation of water and minerals, so the function of the xylem is also essential for nodule symbiosis. The vascular bundle provides the necessary structural support for the nodules, allowing them to develop normally and maintain their functions. The combination of lignin and cellulose gives the nodules sufficient strength to support the nitrogen fixation process. The vascular system is also involved in the transport of plant hormones such as auxin and cytokinin, which play a key role in regulating the formation and development of nodules. For example, auxin can promote the formation of nodules, while cytokinin promotes cell division and nodule growth. The vascular system is also involved in the plant's immune response. Once rhizobia enter the plant's root system, the plant needs to recognize and establish a mutually beneficial symbiotic relationship. The vascular system helps transmit immune signals during this process to ensure that the rhizobia are not recognized as potential pathogens.

[0003] The nodule vascular bundle plays an indispensable role in the symbiotic relationship between legumes and rhizobia. Through its multiple functions, it ensures that legumes can efficiently utilize nitrogen resources and maintain a mutually beneficial symbiotic relationship with rhizobia. Research on the nodule vascular bundle helps to improve the productivity of legumes, optimize the symbiotic relationship, increase the efficiency of nitrogen fixation, and reduce dependence on chemical fertilizers. In addition, by studying the functions of the nodule vascular bundle, new biological nitrogen fixation technologies can be developed and applied to non-legume crops, thereby further promoting the sustainable development of agriculture. Summary of the invention

[0004] In-depth research on the symbiotic mechanism between legumes and rhizobia is crucial to improving nitrogen fixation efficiency and expanding its application range. For example, non-legumes can be modified by genetic engineering so that they can establish a similar symbiotic relationship with rhizobia, thereby achieving a wider range of agricultural applications. The application prospects of nodule nitrogen fixation technology in agricultural production are broad. Through continuous basic research and technological innovation, it is expected to achieve more efficient nitrogen cycles and more sustainable agricultural development in the future. This knowledge provides a theoretical basis for the transformation of non-legumes. The purpose of the present invention is to provide a gene GmHB21 that participates in the development of soybean nodule vascular bundles and thus regulates nodule symbiotic nitrogen fixation and its application.

[0005] The inventors used molecular biology and transcriptomics to clone a gene related to the regulation of soybean (Glycine max) vascular bundle development (also referred to as GmHB21 in this article) from the Chinese soybean germplasm Zhonghuang 13. The location of the GmHB21 encoding gene in the sequenced genome of Gmax_ZH13_V2.0 version is Chr11: 4910619..4913061. GmHB21 is specifically expressed in the nodule vascular bundle. The cloning of GmHB21 provides a theoretical basis and gene resources for the subsequent transformation of non-leguminous plants to form nodules through genetic engineering.

[0006] Wherein, in a specific embodiment of the present invention, the coding region sequence of the GmHB21 gene is shown as SEQ ID NO: 1. In a specific embodiment of the present invention, the amino acid sequence of the GmHB21 gene is shown as SEQ ID NO: 2.

[0007] The inventors found that the GmHB21 promoter contains multiple sites (CTCTT and AAAGAT) that can be recognized by rhizobia in 2000bp. Further GUS staining localization experiments driven by the GmHB21 promoter showed that the GmHB21 gene was mainly expressed in the nodule vascular cells. When the GmHB21 protein was subjected to RNAi using a plant expression vector such as Agrobacterium rhizogenes, the number of nodules in the obtained transgenic root system was significantly reduced compared with the source strain. Further USDA110 GUS infection experiments using transgenic root systems and control root systems showed that the reduction in the number of nodules was due to the blocking of the early FOCI infection process of rhizobia, indicating that after rhizobia infection, whether the genes related to the establishment of the vascular bundle can be properly expressed is crucial to nodule formation. Such an effect is unexpected based on the existing technology. Therefore, the present application provides a separated protein, which is a soybean nodule vascular bundle development-related protein, and annotates the function of its coding sequence in the soybean genome 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 objectives, the present invention also provides a method for obtaining transgenic hairy roots, which comprises introducing the aforementioned gene or a vector or host cell containing the aforementioned gene into target soybeans, thereby obtaining a legume plant such as soybean hairy roots that exhibits an altered number of nodules compared to the target plant soybean.

[0010] To achieve the above object, the present invention also provides a use of the above-mentioned protein or the above-mentioned gene or a vector or host cell comprising the above-mentioned gene in genetic engineering of leguminous plants such as soybean.

[0011] The functions of the soybean nodule vascular bundle development-related protein and its encoding nucleic acid provided by the present invention in regulating the number of soybean nodules are the first discovery of the applicant, and the phenotypic analysis of transgenic hairy roots and empty vector hairy root plants shows that inhibiting the expression of the soybean nodule vascular bundle development-related protein of the present invention can reduce the number of soybean transgenic hairy root nodules. The present invention will have great theoretical and application value for studying soybean nodule development 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 involved in soybean nodule vascular bundle development or regulating soybean nodule number, wherein the coding region sequence of the gene is shown in SEQ ID NO: 1 or a homologous sequence thereof.

[0014] In another aspect, the present invention provides a protein involved in the development of soybean nodule vascular bundle or regulating the number of soybean nodules, wherein the amino acid sequence of the protein is shown in SEQ ID NO: 2 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 interfering sequence is as shown in SEQ ID NO:3.

[0017] 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 a red fluorescent protein and a green fluorescent protein, the antibiotic marker is selected from 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, and the anti-chemical marker is an anti-herbicide marker.

[0018] In another aspect, the present invention provides a host cell comprising the expression vector as described above.

[0019] 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 sequence inhibiting the expression of the gene or protein as described above, or the expression vector or host cell as described above into a leguminous plant such as soybean cells or tissues and cultivating transgenic hairy roots, optionally, the nodulation number of the transgenic hairy roots is changed.

[0020] 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.

[0021] In some embodiments, the host cell is selected from Escherichia coli cells, Agrobacterium cells (eg, Agrobacterium rhizogenes cells), and plant cells.

[0022] In some embodiments, the Agrobacterium rhizogenes is selected from K599, AR1193, C58cl, Arqual, MSU440, LBA9402, and R1601.

[0023] 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 sequence inhibiting the expression of the gene or protein as described above, or the expression vector or host cell as described above in cultivating leguminous plants such as soybeans with altered nodulation number.

[0024] In some embodiments, the change in nodule number is manifested as an increase in the number of nodules and / or the number of individual nodules.

[0025] In some embodiments, the change in nodule number is manifested as a decrease in the number of nodules and / or the number of individual nodules.

[0026] On the other hand, the present invention provides a method for regulating the number of plant nodules, which comprises introducing the gene as described above or the nucleotide sequence encoding the protein as described above, or the sequence inhibiting the expression of the gene or 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 leguminous plant, preferably soybean, and 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 single root, and 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 single root.

[0027] definition

[0028] Coding region sequence: The coding region 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. The coding region usually does not include introns because introns are spliced ​​out during post-transcriptional processing, leaving only the exon sequence that encodes the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The functional analysis and verification of the gene GmHB21 involved in the development of soybean nodule vascular bundle are shown.

[0030] A: Location of two Rhizobium consensus motifs on the GmHB21 promoter. The 2,000 bp sequence preceding the gene transcription start site was identified as the promoter.

[0031] B: Expression pattern of GmHB21 pro::GUS during nodule formation. Transverse sections of roots infected with rhizobia are shown. Bar = 100 µm.

[0032] C: USDA110-GUS staining shows four infection events, characterizing the infection stages. Foci: infection focus; IT: infection thread that completely penetrates the root hair; cIT: infection thread that penetrates the root hair and branches to the cortex; NP: nodule primordium. Bar = 50 µm.

[0033] Comparison of the number of four infection events between the control group and the GmHB21 RNAi strain and the GmHB21 RNAi strain. The phenotype was scored on day 5 after inoculation with USDA110-GUS. The three lines in the violin plot represent the 75th percentile, median, and 25th percentile, respectively. Unpaired two-tailed t-test; ns, non-significant; ****p < 0.0001.

[0034] D: Nodulation phenotypes of the control group and the GmHB21 RNAi line.

[0035] E: Comparison of the number of root nodules between the control group and the GmHB21 RNAi line. Data are presented as mean ± standard error. Unpaired two-tailed t-test; ns, non-significant; ****p < 0.0001.

[0036] F: Expression values ​​of GmHB21 gene in the control group and GmHB21 RNAi strain. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] In the following examples, the soybean hairy root transformation receptor is Zhonghuang 13. Zhonghuang 13 is bred by sexual hybridization of Yudou No. 8 and Zhongzuo 90052-76 from the Chinese Academy of Agricultural Sciences. The variety has a growth period of 100 to 105 days, 125 to 127 days for spring sowing, limited pod-setting habit, purple flowers, gray hairs, elliptical leaves, 3 to 5 effective branches, 100-grain weight of 24 to 26 grams, elliptical seeds, yellow color, brown navel, high protein content, lodging resistance, strong disease resistance, good commerciality, and strong adaptability. pDonor221 vector, pK7GWⅡRR vector, pKGWFS7-GUS-DsRed vector, Agrobacterium rhizogenes strain K599, Bradyrhizobium diazoefficiens strain USDA110 were purchased from China Plasmid Vector Strain Cell Gene Collection Center (Biovector Science Lab, Inc).

[0040] Gateway, recovery kits and other consumables were purchased from Thermo Fisher Scientific and Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0041] Table 1 Gene name and gene number

[0042]

[0043] Example 1 Localization of GmHB21 gene

[0044] 1. Construction of pKGWFS7-GUS-DsRed recombinant plasmid

[0045] 1. Separate the leaf tissue from soybean variety Zhonghuang 13 and extract DNA.

[0046] 2. Using the DNA extracted in step 1 as a template, perform PCR amplification using a primer pair consisting of F2 and R2 to obtain a PCR amplification product (SEQ ID NO: 4).

[0047] F2: 5'-caaaatcctctctcctctagaGGACAGGGTGAGAGAAAGTTGT-3' (SEQ ID NO: 5);

[0048] R2: 5'-gcccttgctcaccatccgcggAGTGTATGCATCAGCAGGGAAT-3' (SEQ ID NO: 6).

[0049] 3. Recover PCR products from gel.

[0050] 4. The PCR product of step 3 and pKGWFS7-GUS-DsRed (linearized by double restriction enzyme digestion with XbaI and BsPTI) were connected by pEASY-Uni Seamless Cloning and Assembly Kit (TRAN CU101-03) to obtain the recombinant plasmid GmHB21-pro-pKGWFS7-GUS-DsRed. According to the sequencing results, it was confirmed that the recombinant plasmid GmHB21-pro-pKGWFS7-GUS-DsRed vector was inserted with a double-stranded DNA molecule as shown in SEQ ID NO: 4 in the sequence table. According to the sequencing results, the structure of the recombinant plasmid GmHB21-pro-pKGWFS7-GUS-DsRed was described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 4 in the sequence table was inserted between XbaI and BsPTI of the pK7GWⅡRR vector, which verified that the expression cassette construction was completed.

[0051] II. Obtaining transgenic hairy roots of GmHB21-pro-pKGWFS7-GUS-DsRed

[0052] 1. Introduce the recombinant plasmid GmHB21-pro-pKGWFS7-GUS-DsRed into the Agrobacterium rhizogenes strain K599 to obtain the recombinant Agrobacterium, which was frozen at -80°C and stored in glycerol.

[0053] 2. The Agrobacterium rhizogenes obtained in step 1 is transformed into the recipient plant Zhonghuang 13 using the hairy root transformation method. The specific operation steps are as follows:

[0054] (1) Select Zhonghuang 13 soybean seeds that are large, plump, and free of disease spots.

[0055] (2) The selected seeds were sown in vermiculite and germinated in a greenhouse for 3 days. The soybean seeds were transformed when their cotyledons were about to open.

[0056] (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 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.

[0057] (5) When the OD600 of the bacterial solution was about 0.8, the cells were collected by centrifugation at 5000 rpm for 6 min and resuspended in infection solution (10 MM MES (2-morpholineethanesulfonic acid) and 10 MM MgCl2) for infecting soybean hypocotyls.

[0058] (6) Use the puncture injection method to infect the hypocotyl of the seedling. The injection site is selected to penetrate the middle of the hypocotyl below the cotyledon, leaving two drops of injection solution on both sides of the stem.

[0059] (7) After the injection is completed, fill the black tray with water, cover it with a transparent lid, and secure it with tape (rooting in a high humidity environment). Cultivate at 28°C for 9 to 14 days to induce transgenic hairy roots on the hypocotyl. Cut the hairy roots and soak them in a disposable plastic cup filled with water to continue culturing until the roots elongate.

[0060] (8) dsRED screening label, using green fluorescence observation to screen positive roots.

[0061] (9) Transplant the positive plants into pure vermiculite for cultivation.

[0062] 3. GUS staining of GmHB21-pro-pKGWFS7-GUS-DsRed transgenic positive roots

[0063] 1. Rhizobium inoculation treatment

[0064] (1) Take 1 mL of Bradyrhizobium diazoefficiens strain USDA110 stored at -80°C and add it 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. Incubate at 28°C with a shaker at 200 rpm for about 4 days until the OD600 is between 0.8 and 1.

[0065] (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 OD600 of about 0.04 for inoculation.

[0066] (3) Each seedling was inoculated with 50 mL of resuspended bacterial solution.

[0067] (4) Five days after inoculation, the roots were subjected to GUS staining.

[0068] 2. GUS staining

[0069] (1) Place the positive roots in 90% acetone and fix them under vacuum for 30 min (acetone has strong permeability and can precipitate and solidify proteins, but does not affect the functional groups of proteins and preserves the activity of proteins. It is effective in fixing phosphatases and oxidases, so it can be used for fixation before GUS staining to prevent the spread of GUS signals. It is characterized by fast fixation, strong permeability, and easy contraction of tissue cells; but its ability to maintain cell structure is poor).

[0070] (2) Add GUS washing solution to wash away acetone, and wash twice.

[0071] (3) Add GUS staining solution (a solution of 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferrocyanide, 0.5 mg / mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-glucose), 0.1% (v / v) Triton X-100, and 0.1 M sodium phosphate buffer (pH = 7.0)) and vacuum on ice for 15-20 min.

[0072] (4) Incubate at 37°C overnight and observe the staining.

[0073] (5) Aspirate the staining solution and add 1 mL of 70% ethanol to stop the staining reaction and decolorization.

[0074] (6) Replace the ethanol several times until decolorization is complete.

[0075] (7) Embed with 4% agarose, vibrate and slice, collect nodule slices and observe and photograph them under an optical microscope. The GUS staining site shows that the gene promoter is expressed in the nodule vascular bundle ( Figure 1 B).

[0076] Example 2 Establishment of GmHB21 gene-silenced transgenic plants

[0077] Based on a large number of sequence analyses and functional verifications, a transcription factor of HD-ZIP I regulating nodule development was found in soybean variety Zhonghuang 13, named GmHB21, the coding region sequence of which is shown in SEQ ID NO: 1, and the protein sequence encoded by it is shown in SEQ ID NO: 2. Given that soybean plants have the GmHB21 gene, the present invention studies the function of the gene by gene silencing.

[0078] 1. Construction of GmHB21-pK7GWⅡRR recombinant plasmid

[0079] 1. Separate the nodule tissue of soybean variety Zhonghuang 13 from the root, extract RNA and perform reverse transcription to obtain cDNA of the nodule tissue of soybean variety Zhonghuang 13.

[0080] 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: 3).

[0081] F1: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATTGGACCCACCATTCTTTGGG-3' (SEQID NO: 7);

[0082] R1: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTACACGGCACACTATTGGTCT-3' (SEQ ID NO: 8).

[0083] 3. Recover PCR products from gel.

[0084] 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 GmHB21-pDonor221. According to the sequencing results, it was confirmed that the recombinant plasmid GmHB21-pDonor221 was a double-stranded DNA molecule as shown in SEQ ID NO: 3 in the sequence list inserted into the pDonor221 vector.

[0085] 5. The plasmid GmHB21-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 GmHB21-pK7GWⅡRR. According to the sequencing results, the structure of the recombinant plasmid GmHB21-pK7GWⅡRR was described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 3 in the sequence table was inserted between attR1 and attR2 of the pK7GWⅡRR vector, which verified that the expression cassette was constructed.

[0086] 2. Obtaining transgenic hairy roots with GmHB21 gene silencing

[0087] 1. Introduce the recombinant plasmid GmHB21-pK7GWⅡRR into the Agrobacterium rhizogenes strain K599 to obtain the recombinant Agrobacterium, which was frozen at -80°C and stored in glycerol.

[0088] 2. The Agrobacterium rhizogenes obtained in step 1 is transformed into the recipient plant Zhonghuang 13 using the hairy root transformation method. The specific operation steps are as follows:

[0089] (1) Select Zhonghuang 13 soybean seeds that are large, plump, and free of disease spots.

[0090] (2) The selected seeds were sown in vermiculite and germinated in a greenhouse for 3 days. The soybean seeds were transformed when their cotyledons were about to open.

[0091] (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 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.

[0092] (5) When the OD600 of the bacterial solution was about 0.8, the cells were collected by centrifugation at 5000 rpm for 6 min and resuspended in infection solution (10 MM MES and 10 MM MgCl2) for infecting soybean hypocotyls.

[0093] (6) Use the puncture injection method to infect the hypocotyl of the seedling. The injection site is selected to penetrate the middle of the hypocotyl below the cotyledon, leaving two drops of injection solution on both sides of the stem.

[0094] (7) After the injection is completed, fill the black tray with water, cover it with a transparent lid, and secure it with tape (rooting in a high humidity environment). Cultivate at 28°C for 9 to 14 days to induce transgenic hairy roots on the hypocotyl. Cut the hairy roots and soak them in a disposable plastic cup filled with water to continue culturing until the roots elongate.

[0095] (8) dsRED screening label, using green fluorescence observation to screen positive roots.

[0096] (9) Transplant the positive plants into pure vermiculite for cultivation.

[0097] Example 3 Changes in the number of nodules during GmHB21 gene silencing

[0098] 1. Rhizobium inoculation treatment

[0099] (1) Take 1 mL of Bradyrhizobium diazoefficiens strain USDA110 stored at -80°C and add it 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. Incubate the culture at 28°C with a shaker at 200 rpm for about 4 days until the OD600 is between 0.8 and 1.

[0100] (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 OD600 of about 0.04 for inoculation.

[0101] (3) Each seedling was inoculated with 50 mL of resuspended bacterial solution.

[0102] (4) GUS staining was performed 5 days after inoculation, and nodulation phenotype was observed 21 days after inoculation.

[0103] 2 GUS staining

[0104] (1) Place the positive roots in 90% acetone and fix them under vacuum for 30 min (acetone has strong permeability and can precipitate and solidify proteins, but does not affect the functional groups of proteins and preserves the activity of proteins. It is effective in fixing phosphatases and oxidases, so it can be used for fixation before GUS staining to prevent the spread of GUS signals. It is characterized by fast fixation, strong permeability, and easy contraction of tissue cells; but its ability to maintain cell structure is poor).

[0105] (2) Add GUS washing solution to wash away acetone, and wash twice.

[0106] (3) Add GUS staining solution (a solution of 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferrocyanide, 0.5 mg / mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-glucose), 0.1% (v / v) Triton X-100, and 0.1 M sodium phosphate buffer (pH = 7.0)) and vacuum on ice for 15-20 min.

[0107] (4) Incubate at 37°C overnight and observe the staining.

[0108] (5) Aspirate the staining solution and add 1 mL of 70% ethanol to stop the staining reaction and decolorization.

[0109] (6) Replace the ethanol several times until decolorization is complete.

[0110] (7) The stained roots were observed under an optical microscope to determine the period of rhizobium infection and to make statistics. Figure 1 C. From Figure 1 As can be seen from C, the number of rhizobia infected in the FOCI stage in the GmHB21-RNAi soybean hairy roots was significantly higher than that in the empty vector control roots, and the number in the NP stage was significantly lower than that in the empty vector control roots. This indicates that the GmHB21 gene is involved in the formation of nodules at an early stage.

[0111] 3. Nodule number phenotype statistics and analysis

[0112] 21 days after inoculation, the vermiculite was washed off with tap water and dried with absorbent paper. Green fluorescent light was used to confirm the positive soybean hairy roots of empty vector and GmHB21-RNAi. It was observed that the number of nodules in the hairy roots of GmHB21-RNAi was significantly lower than that in the control of empty vector ( Figure 1 D). The inventors counted the number of nodules in the hairy roots transfected with GmHB21-RNAi and the hairy roots transfected with an empty vector. Figure 1 E. From Figure 1 D and Figure 1 As can be seen in E, the average number of hairy root nodules (number of nodules per root) of the GmHB21-RNAi transgenic line was only 28.3% of that of the empty vector control, which indicates that the GmHB21 gene is involved in the development of soybean nodules.

[0113] 3. Identification of gene expression

[0114] The hairy roots of the empty vector and the hairy roots of the GmHB21 gene silenced were identified as follows:

[0115] (1) Roots and nodules were collected, total RNA was extracted and reverse transcribed into cDNA.

[0116] (2) Using the cDNA extracted in step (1) as a template, the primer pair consisting of F and R was used to identify the expression level of the GmHB21 gene, and the primer pair consisting of F2 and R2 was used to identify the expression level of the internal reference gene (Actin gene).

[0117] 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).

[0118] The expression levels of the GmHB21 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 F. From Figure 1 As can be seen from Figure F, in the gene silencing strain GmHB21-RNAi, the gene expression level of GmHB21 was significantly reduced, with a P value of <0.0001.

[0119] Table 2 Quantitative identification primers:

[0120]

[0121] sequence

[0122] SEQ ID NO:1 GmHB21 coding region sequence

[0123] ATGAATCATCGACCACCTTTCCAAGACCACATGATGCTCATGTCTCAGTTATTCCCTGCTGATGCATACACTCAAATTATTTCTCAACAAGGAGAGACTAATAAGAAGCCAAGACGCCGTCGTAACAAGAAGAACAAAGGAGGAGAAAACGGTGCCTCGGAAGCCAACAAGAAGAGGAAGCTTAGTGAGGTGCAAGTTAATTTACTTGAACAAAACTTTGGAAATGAACGCAAACTTGACTCCGAAAGAAAGGATAGGCTGGCAATGGAGCTTGGTTTGGACCCTCGACAAGTTGCTGTGTGGTTTCAAAACAGAAGAGCCCGTTGGAAGAACAAAAAGTTGGAAGAAGAGTACTCCAGCCTTAAAAAAAATCATGAAGCCACCTTGCTTGAGAAATGTTGCCTGGAGAGTGAGGTGTTGAAGCTCAAAGAGCAACTTTCTGAGGCAGAGAAAGAGATTCAGAGGCTGCTAGAGAGTGCCGAGAGAGTCCCAAGCAACAGTTCTAGTTCGTCACAGTCACAATCAATGGAAGCGGTGGACCCACCATTCTTTGGGGAATTTGGAGTTGATGGATATGAGGATGATGTGTTTTACGTGCCTGAGACCCATTACATCAACGGCATGGAATGGATTAATCTGTATATGTAA

[0124] SEQ ID NO:2 Protein sequence encoded by GmHB21

[0125] MNHRPPFQDHMMLMSQLFPADAYTQIISQQGETNKKPRRRRNKKNKGGENGASEANKKRKLSEVQVNLLEQNFGNERKLDSERKDRLAMELGLDPRQVAVWFQNRRARWKNKKLEEEYSSLKKNHEATLLEKCCLESEVLKLKEQLSEAEKEIQRLLESAERVPSNSSSSSQSQSMEAVDPPFFGEFGVDGYEDDVFYVPETHYINGMEWINLYM

[0126] SEQ ID NO:3 GmHB21 RNAi fragment

[0127] TGGACCCACCATTCTTTGGGGAATTTGGAGTTGATGGATATGAGGATGATGTGTTTTACGTGCCTGAGACCCATTACATCAACGGCATGGAATGGATTAATCTGTATATGTAAAACTACCAATAGGAATTTAGAATTTTGTAGATAATGTGAATGGTCCAATGTATATTAATTGTTCCTAGCTAGCTAGGTAGCTAAGTGTGTTTGTTTCACTATGGTTTGGAAATTGGAAAGTATCCATTAACATAATAATTTGCAATTTGACCCGAAATGGTAGTAGTATCTTCGTTTGTGTGCAATGTCAGCTTATGTCAGCTATGAAGACCAATAGTGTGCCGTGT

[0128] SEQ ID NO:4 GmHB21 promoter fragment

[0129]

[0130] SEQ ID NO:5 Primer F2

[0131] 5'-caaaatcctctctcctctagaGGACAGGGTGAGAGAAAGTTGT-3'

[0132] SEQ ID NO:6 Primer R2

[0133] 5'-gcccttgctcaccatccgcggAGTGTATGCATCAGCAGGGAAT-3'

[0134] SEQ ID NO:7 Primer F1

[0135] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATTGGACCCAC

[0136] CATTCTTTGGG-3'

[0137] SEQ ID NO:8 Primer R1

[0138] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTACACGGCAC

[0139] ACTATTGGTCT-3'

[0140] SEQ ID NO:9 Primer GmHB21-F

[0141] ATGGAGCTTGGTTTGGACCC

[0142] SEQ ID NO: 10 Primer GmHB21-R

[0143] CCTCACTCTCCAGGCAACATT

[0144] SEQ ID NO: 11 Primer GmActin11-F

[0145] GAGCTATGAATTGCCTGATGG

[0146] SEQ ID NO: 12 Primer GmActin11-R

[0147] CGTTTCATGAATTCCAGTAGC

[0148] 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. An isolated gene involved in the development of soybean nodule vascular bundle or regulating the number of soybean nodules, characterized in that: The coding region sequence of the gene is shown in SEQ ID NO: 1 or its homologous sequence.

2. A protein involved in the development of soybean nodule vascular bundle or regulating the number of soybean nodules, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO: 2 or a homologous sequence thereof.

3. An expression vector, characterized in that The expression vector comprises the gene according to claim 1 or the nucleotide sequence encoding the protein according to claim 2, or comprises a sequence that inhibits the expression of the gene according to claim 1 or the protein according to claim 2, such as an interfering sequence of the gene or an interfering sequence of the nucleotide sequence encoding the protein.

4. The expression vector according to claim 3, characterized in that 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 a red fluorescent protein and a green fluorescent protein, the antibiotic marker is selected from 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, and the anti-chemical marker is an anti-herbicide marker.

5. The expression vector according to claim 3 or 4, characterized in that 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.

6. A host cell comprising the expression vector according to any one of claims 3 to 5.

7. The host cell according to claim 6, characterized in that The host cell is selected from Escherichia coli cells, Agrobacterium cells (such as Agrobacterium rhizogenes cells) and plant cells. Optionally, the Agrobacterium rhizogenes is selected from K599, AR1193, C58cl, Arqual, MSU440, LBA9402 and R1601.

8. A method for obtaining soybean hairy roots, comprising transforming the gene according to claim 1 or the nucleotide sequence encoding the protein according to claim 2, or the sequence inhibiting the expression of the gene according to claim 1 or the protein according to claim 2, or the expression vector according to any one of claims 3 to 5 or the host cell according to claim 6 or 7 into a leguminous plant such as soybean cells or tissues and cultivating transgenic hairy roots, optionally, the nodulation number of the transgenic hairy roots is changed.

9. Use of the gene according to claim 1 or the nucleotide sequence encoding the protein according to claim 2, or the sequence inhibiting the expression of the gene according to claim 1 or the protein according to claim 2, or the expression vector according to any one of claims 3 to 5, or the host cell according to claim 6 or 7 in cultivating leguminous plants such as soybeans with altered nodulation number, optionally, the alteration in nodulation number is manifested as an increase in the number of nodules and / or the number of nodules per root, optionally, the alteration in nodulation number is manifested as a decrease in the number of nodules and / or the number of nodules per root.

10. A method for regulating the number of plant nodules, comprising introducing the gene according to claim 1 or the nucleotide sequence encoding the protein according to claim 2, or the sequence inhibiting the expression of the gene according to claim 1 or the protein according to claim 2, or the expression vector according to any one of claims 3 to 5 or the host cell according to claim 6 or 7 into a target plant cell or tissue to obtain a transgenic plant, wherein the number of nodules in the transgenic plant is changed compared with the target plant, and the plant is a leguminous plant, preferably soybean, and 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 root, and 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 root.

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