Gene gmhb1a involved in soybean root nodule vascular bundle development and application thereof

Abstract

The present application relates to a gene GmHB1a involved in soybean root nodule vascular bundle development and its application. Specifically, the present application relates to an isolated gene involved in soybean root nodule vascular bundle development or regulating the number of soybean nodulation, the coding region sequence of the gene is shown as SEQ ID NO:1 or a homologous sequence thereof. The present application also provides a method for regulating the number of plant nodulation, which comprises introducing the gene as described above or a sequence inhibiting the expression of the gene as described above into a plant cell or tissue of interest to obtain a transgenic plant, compared with the plant of interest, the number of nodulation of the transgenic plant is changed, the plant is a legume plant, preferably soybean, optionally, the change in the number of nodulation is manifested as an increase in the number of nodulation and / or single root nodulation, optionally, the change in the number of nodulation is manifested as a decrease in the number of nodulation and / or single root nodulation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and botany, and specifically relates to a gene GmHB1a involved in soybean nodule vascular bundle development and application thereof. BACKGROUND

[0002] Nodule vascular bundle plays a key role in the symbiotic relationship between legumes and rhizobia, ensuring smooth exchange of materials and energy between the two. The vascular bundle is responsible for the transport of nutrients and metabolites, and the carbohydrates produced by photosynthesis in plants are transported to the nodule through the nodule vascular bundle, providing energy for rhizobia. Rhizobia fix atmospheric nitrogen into ammonia ions, and these nitrogen products are transported back to the plant from the nodule through the nodule vascular bundle for plant growth. The vascular bundle is responsible for the transmission of signal molecules, and the nodule vascular bundle transports plant hormones such as gibberellins and cytokinins, which regulate the growth and development of nodules. Signal molecules produced by rhizobia can also be transmitted through the vascular bundle, affecting the physiology and metabolism of plants. The nodule vascular bundle is involved in the immune response of plants, transporting substances against pathogens, enhancing the resistance of plants to diseases. The nodule vascular bundle ensures the normal development of vascular tissue in the nodule, maintaining the structural stability of the nodule. Through the transport of metabolites by the vascular bundle, the stability of the nodule environment is maintained, promoting efficient nitrogen fixation process.

[0003] Studying nodule vascular bundle helps to improve the productivity of legumes, optimize symbiotic relationship, improve nitrogen fixation efficiency, and reduce dependence on chemical fertilizers; helps to develop new biological nitrogen fixation technology, and apply it to non-legume crops. SUMMARY

[0004] Nodule nitrogen fixation not only significantly improves the yield and quality of legumes, but also helps to reduce the dependence of agricultural production on chemical fertilizers, and promotes the sustainable development of agriculture. Through further basic research and application development, nodule nitrogen fixation technology is expected to play an important role in wider agricultural production. In-depth study of the symbiotic mechanism between legumes and rhizobia, including signal transmission, nodule formation and development, etc. These knowledge provides a theoretical basis for the modification of non-legume plants. The purpose of the present application is to provide a gene involved in soybean nodule vascular bundle development and its application.

[0005] The inventors cloned a gene involved in vascular development in Glycine max from Chinese soybean line Huang 13 by means of molecular biology and transcriptome. The GmHB1a gene encodes a protein with the amino acid sequence as shown in SEQ ID NO: 2. The inventors found that the GmHB1a protein, when used in RNAi with plant expression vectors such as Agrobacterium rhizogenes, resulted in a significant reduction in the number of nodules in the obtained plants compared to the source line. Further GUS experiments driven by the GmHB1a promoter showed that the reduction in the number of nodules was blocked at the early stage of infection by rhizobium, indicating that the establishment of early vascular is crucial for nodule formation. Such an effect is unexpected based on the prior art, and thus the present application provides an isolated protein which is a soybean nodule development related protein, and first annotates the coding sequence of the protein in the soybean genome.

[0006] In specific embodiments of the application, the coding region sequence of the GmHB1a gene is as shown in SEQ ID NO: 1. In specific embodiments of the application, the amino acid sequence of the GmHB1a gene is as shown in SEQ ID NO: 2.

[0007] The inventors found that the GmHB1a protein, when used in RNAi with plant expression vectors such as Agrobacterium rhizogenes, resulted in a significant reduction in the number of nodules in the obtained plants compared to the source line. Further GUS experiments driven by the GmHB1a promoter showed that the reduction in the number of nodules was blocked at the early stage of infection by rhizobium, indicating that the establishment of early vascular is crucial for nodule formation. Such an effect is unexpected based on the prior art, and thus the present application provides an isolated protein which is a soybean nodule development related protein, and first annotates the coding sequence of the protein in the soybean genome.

[0008] In the present application, a plant expression vector containing the gene of interest can be constructed using existing plant expression vectors. The plant expression vector includes 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 vector used can be processed, such as adding a gene that can express an enzyme or a luminescent compound in plants that can produce a color change, a resistant antibiotic marker, or a chemical reagent resistant marker gene, etc. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress. The plant expression vector can also contain enhancers to increase the expression of the inserted nucleotide fragment.

[0009] To achieve the above-mentioned purpose, the present application also provides a method for obtaining a transgenic hairy root, which is to introduce the aforementioned gene or a vector or host cell containing the aforementioned gene into a soybean plant of interest to obtain a legume plant such as a soybean hairy root that exhibits a change in the number of nodules compared to the soybean plant of interest.

[0010] To achieve the above object, the present application also provides an application of the aforementioned protein or the aforementioned gene or a vector or a host cell containing the aforementioned gene in genetic engineering of leguminous plants, such as soybean.

[0011] The soybean nodule vascular bundle development related protein and the coding gene thereof provided by the present application are first discovered by the applicant in terms of the function in regulating the number of soybean nodulation, and the phenotype analysis of the transgenic hairy root and the trans-empty vector hairy root plant verifies that reducing the expression of the soybean nodule vascular bundle development related protein of the present application can reduce the number of soybean transgenic hairy root nodulation. The present application will have great theoretical and application value for the research on the soybean nodule vascular bundle development and the related application research.

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

[0013] In one aspect, the present application provides an isolated gene involved in the soybean nodule vascular bundle development or the regulation of the number of soybean nodulation, wherein the coding region sequence of the gene is shown as SEQ ID NO: 1 or a homologous sequence thereof.

[0014] In another aspect, the present application provides a protein involved in the soybean nodule vascular bundle development or the regulation of the number of soybean nodulation, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2 or a homologous sequence thereof.

[0015] In another aspect, the present application provides an expression vector, wherein the expression vector comprises the gene or the nucleotide sequence encoding the protein as described above, or comprises a sequence inhibiting the expression of the gene or the protein as described above, such as an interference sequence of the gene or an interference sequence of the nucleotide sequence encoding the protein.

[0016] In some embodiments, the interference sequence is shown as 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 reagent 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 reagent marker is an anti-herbicide marker.

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

[0019] In another aspect, the present application provides a method of obtaining a soybean hairy root, comprising transforming a gene or a nucleotide sequence encoding a protein as described above, or a sequence inhibiting the expression of a gene or a protein as described above, or an expression vector or a host cell as described above into a legume plant, for example a soybean cell or tissue, and cultivating the transgenic hairy root, optionally, the number of nodules of the transgenic hairy root is changed.

[0020] In some embodiments, the vector is a plant expression vector, including a binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

[0021] In some embodiments, the host cell is selected from the group consisting of an E. coli cell, an Agrobacterium cell (for example, a Rhizobium cell) and a plant cell.

[0022] In some embodiments, the Rhizobium cell is selected from the group consisting of K599, AR1193, C58cl, Arqual, MSU440, LBA9402 and R1601.

[0023] In another aspect, the present application provides the use of a gene or a nucleotide sequence encoding a protein as described above, or a sequence inhibiting the expression of a gene or a protein as described above, or an expression vector or a host cell as described above in cultivating a legume plant, for example a soybean, with a changed number of nodules.

[0024] In some embodiments, the changed number of nodules is manifested as an increased number of nodules and / or an increased number of single root nodules.

[0025] In some embodiments, the changed number of nodules is manifested as a decreased number of nodules and / or a decreased number of single root nodules.

[0026] In another aspect, the present application provides a method of modulating the number of nodules of a plant, comprising introducing a gene or a nucleotide sequence encoding a protein as described above, or a sequence inhibiting the expression of a gene or a protein as described above, or an expression vector or a host cell as described above into a plant cell or tissue of interest to obtain a transgenic plant, the number of nodules of the transgenic plant is changed as compared with the plant of interest, the plant is a legume plant, preferably a soybean, optionally, the changed number of nodules is manifested as an increased number of nodules and / or an increased number of single root nodules, optionally, the changed number of nodules is manifested as a decreased number of nodules and / or a decreased number of single root nodules.

[0027] Definitions

[0028] Coding sequence: The coding sequence is the portion of a transcript that is actually translated into protein, i.e., the sequence of exons. It represents the sequence in the gene that encodes amino acids and is the direct template for protein synthesis. The coding sequence usually does not include introns, as introns are spliced out during post-transcriptional processing, leaving only the exon sequences that encode protein. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Functional analysis and validation of the gene GmHB1a involved in soybean nodule vascular development are shown. Among them:

[0030] A: The positions of two rhizobial consensus motifs on the GmHB1a and GmHB1b promoters. The sequence located 2,000 bp before the transcription start site of the gene was identified as the promoter.

[0031] B: The bulk RNA-seq tissue expression pattern of GmHB1a, GmHB1b in root nodules and root & nodule tissues.

[0032] C: The expression pattern of GmHB1a pro::GUS during nodule formation. The transverse sections of roots and rhizobial infection are shown. Bar = 100 µm.

[0033] D: Nodulation phenotype of the control and GmHB1s RNAi lines.

[0034] E: Comparison of the number of root nodules between the control and GmHB1s RNAi lines. Data are expressed as mean ± standard error. Unpaired two-tailed t test; ns, not significant; ****p < 0.0001.

[0035] F: Expression values of the GmHB1a gene in the control and GmHB1s RNAi lines.

[0036] G: Expression values of the GmHB1b gene in the control and GmHB1s RNAi lines.

[0037] H: USDA110-GUS staining showing four infection events, characterizing the infection stages. Foci: infection foci; IT: infection thread that completely traverses the root hair; cIT: infection thread that traverses the root hair and branches into the cortex; NP: nodule primordium. Bar = 50 µm.

[0038] Comparison of the four infection events between the control and GmHB1s RNAi line and GmHB2 1 RNAi line. Phenotypes were scored at 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, not significant; ****p < 0.0001. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application with reference to specific embodiments and the accompanying drawings.

[0040] The following examples are intended to better illustrate the present application but not to limit the present application. In the following examples, the experimental methods are routine methods or selected according to the product instructions unless otherwise specified. In the following examples, the test materials are routine biochemical reagents purchased from a reagent store unless otherwise specified. In the following examples, the quantitative tests are set up with at least three repeated experiments, and the results are averaged.

[0041] In the following examples, the soybean hairy root transformation receptor is Zhonghuang 13. Zhonghuang 13 is obtained by sexual hybridization of Yudou 8 as the female parent and Zhongzu 90052-76 as the male parent, and is bred by pedigree method. The growth period of the variety is 100-105 days, the spring sowing period is 125-127 days, the pod setting habit is limited, the flower color is purple, the pubescence is gray, the leaf shape is oval, the effective branch number is 3-5, the hundred seed mass is 24-26 grams, the kernel shape is oval, the kernel color is yellow, the kernel color is brown, the kernel contains high protein, the lodging resistance is strong, the disease resistance is strong, the commodity is good, and the adaptability is strong. The pDonor221 vector, the pK7GWII RR vector, the pKGWFS7-GUS-DsRed vector, the Agrobacterium rhizogenes strain K599, and the Bradyrhizobium diazoefficiens strain USDA110 are purchased from the China Plasmid Vector Strain Cell Gene Preservation Center (Biovector Science Lab, Inc).

[0042] Gateway, recovery kits and other consumables are purchased from Thermo Fisher and Tian Gen Biochemical Technology (Beijing) Co., Ltd.

[0043] Table 1 Gene name and gene number

[0044]

[0045] Example 1 Positioning of GmHB1a gene

[0046] I. Construction of pKGWFS7-GUS-DsRed recombinant plasmid

[0047] 1. Isolate leaf tissue from soybean variety Zhonghuang 13 and extract DNA.

[0048] 2. Use the DNA extracted in step 1 as a template and perform PCR amplification with a primer pair composed of F2 and R2 to obtain a PCR amplification product (SEQ ID NO: 4).

[0049] F2: 5'-caaaatcctctctcctctagaAGATAATGGCATGCGATGTCAAA-3' (SEQ ID NO: 5);

[0050] R2: 5'- gcccttgctcaccatccgcggCAGGTCGCCGGAGAAAGATT-3' (SEQ ID NO: 6).

[0051] 3. Gel recover the PCR product.

[0052] 4. Connect the PCR product of step 3 and pKGWFS7-GUS-DsRed (linearized by XbaI and BsPTI double enzyme digestion) through pEASY-Uni Seamless Cloning and Assembly Kit (TRAN CU101-03) to obtain recombinant plasmid GmHB1a-pro-pKGWFS7-GUS-DsRed. According to the sequencing results, it is confirmed that the recombinant plasmid GmHB1a-pro-pKGWFS7-GUS-DsRed vector inserts a double-stranded DNA molecule as shown in SEQ ID NO: 4 in the sequence listing. According to the sequencing results, the structure of the recombinant plasmid GmHB1a-pro-pKGWFS7-GUS-DsRed is described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 4 in the sequence listing is inserted between XbaI and BsPTI of the pK7GWII RR vector, i.e., it is verified that the expression cassette construction is completed.

[0053] II. Obtaining of transgenic hairy roots of GmHB1a-pro-pKGWFS7-GUS-DsRed

[0054] 1. Introduce the recombinant plasmid GmHB1a-pro-pKGWFS7-GUS-DsRed into Agrobacterium rhizogenes strain K599 to obtain a recombinant Agrobacterium, which is stored at -80°C in glycerol.

[0055] 2. Use the Agrobacterium rhizogenes obtained in step 1 to transform the receptor plant Zhonghuang 13 by the hairy root transformation method. The specific operation steps are as follows:

[0056] (1) Select soybean seeds of Zhonghuang 13 that are large, full, and free of disease spots.

[0057] (2) The selected seeds are sown in vermiculite and germinated in a greenhouse for 3 days, and the soybean cotyledons are transformed when they are about to open.

[0058] (4) The Agrobacterium rhizogenes K599 containing the target vector is streaked on solid LB medium and cultured in a 28°C incubator for about 2 days. On the day before infection, single colonies are inoculated into 3-5 mL of LB liquid medium containing rifampicin (50 μg / mL), streptomycin (10 μg / mL), and spectinomycin (100 μg / mL) and cultured overnight at 28°C on a shaker.

[0059] (5) The bacterial cells are collected by centrifugation at 5000 rpm for 6 min when the OD600 of the bacterial solution is about 0.8, and the bacterial cells are resuspended in an infection solution (10 MM MES (2-morpholinoethanesulfonic acid) and 10 MM MgCl2) for infection of the soybean hypocotyls.

[0060] (6) The hypocotyls of the seedlings are infected by the puncture injection method, and the injection site is selected to penetrate the hypocotyls below the cotyledon. Two drops of injection solution are left on both sides of the stem.

[0061] (7) After injection, the black tray is filled with water, covered with a transparent cover, and fixed with tape (high-humidity environment for rooting). The transgenic hairy roots are induced on the hypocotyls after 9-14 days of culture at 28°C, and the roots are cut and cultured in a disposable plastic cup filled with water.

[0062] (8) dsRED screening tag, observed with green fluorescence, and positive roots are selected.

[0063] (9) The positive plants are transplanted to pure vermiculite for culture.

[0064] III. GUS Staining of GmHB1a-pro-pKGWFS7-GUS-DsRed

[0065] 1. Rhizobium infection treatment

[0066] (1) The preserved slow-growing rhizobium Bradyrhizobium diazoefficiens strain USDA110 1 mL is added 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 and cultured at 28°C on a shaker at 200 rpm for about 4 days until the OD600 is between 0.8 and 1.

[0067] (2) Collect the bacterial solution in 500 mL centrifuge bottles, centrifuge at 6000 rpm for 10 min. Resuspend the bacteria in sterilized ddH2O to OD600 of about 0.04 for inoculation.

[0068] (3) Inoculate each seedling with 50 mL of the resuspended bacterial solution.

[0069] (4) Perform GUS staining on the roots 5 days after inoculation.

[0070] 2. GUS Staining

[0071] (1) Place the positive roots in 90% acetone and fix under vacuum for 30 min (acetone has strong penetration, can make protein precipitate and coagulate, but does not affect the functional groups of protein and preserves the activity of protein, is used for fixing phosphatase and oxidase, and has good effect, thus can be used for fixing before GUS staining, and can prevent the diffusion of GUS signal. The characteristics are fast fixation, strong penetration, and easy to make tissue cells shrink; but the ability to maintain cell structure is poor).

[0072] (2) Wash the acetone with GUS washing solution, wash twice.

[0073] (3) Add GUS staining solution (0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.5 mg / mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc), 0.1% (v / v) Triton X-100 and 0.1 M sodium phosphate buffer (pH = 7.0) solution), vacuum for 15-20 min on ice.

[0074] (4) Place at 37°C overnight, and observe the staining.

[0075] (5) Suck out the staining solution, add 1 mL of 70% ethanol to stop the staining reaction and decolorize.

[0076] (6) Change the ethanol several times until complete decolorization.

[0077] (7) The stained root nodules are embedded with 4% agarose, oscillation sectioned, and the root nodule sections are collected to be observed under an optical microscope and photographed. The GUS-stained sites show that the sites expressed by the promoter of the gene are located in the root nodule vascular bundle (as shown in C). Figure 1

[0078] Example 2 Establishment of GmHB1a gene silencing transgenic plants

[0079] ​On the basis of a large number of sequence analysis and function verification, a HD-ZIP I transcription factor regulating nodule vascular development is found from soybean variety zhonghuang 13, which is named GmHB1a, the coding region sequence of which is shown as SEQ ID NO: 1, and the protein sequence encoded by the same is shown as SEQ ID NO: 2. Since leguminous plants have the GmHB1a gene, the function of the gene is studied by gene silencing in the present application.

[0080] I. Construction of GmHB1a-pK7GWII RR recombinant plasmid

[0081] 1. The nodule tissue of soybean variety zhonghuang 13 is separated from the root, and after RNA extraction and reverse transcription, the cDNA of the nodule tissue of soybean variety zhonghuang 13 is obtained.

[0082] 2. The cDNA synthesized in step 1 is used as a template, and a primer pair composed of F1 and R1 is used for PCR amplification, so as to obtain a PCR amplification product (SEQ ID NO: 3).

[0083] F1: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATTGAAAGCAGGGAGATCGATGA-3' (SEQ ID NO: 7);

[0084] R1: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTTTCCTCAGGCACCTCTTTAGC-3' (SEQ ID NO: 8).

[0085] 3. The PCR product is recovered.

[0086] 4. The PCR product of step 3 and the intermediate vector pDonor221 are connected by BP ClonaseTM II enzyme mix (Invitrogen 11789-020), so as to obtain a recombinant plasmid GmHB1a-pDonor221. According to the sequencing result, it is confirmed that the recombinant plasmid GmHB1a-pDonor221 is a double-stranded DNA molecule shown as SEQ ID NO: 3 inserted into the pDonor221 vector in the sequence table.

[0087] 5. The plasmid GmHB1a-pDonor221 obtained in step 4 was ligated to the final vector pK7GWⅡRR using LR CLONASEII ENZYME MIX (Invitrogen 11791-020) to obtain the recombinant plasmid GmHB1a-pK7GWⅡRR. Based on the sequencing results, the structure of the recombinant plasmid GmHB1a-pK7GWⅡRR is described as follows: A double-stranded DNA molecule, as shown in SEQ ID NO:3, was inserted between attR1 and attR2 of the pK7GWⅡRR vector, thus confirming the completion of the expression cassette construction.

[0088] II. Obtaining hairy roots from GmHB1a gene-silenced transgenic plants

[0089] 1. The recombinant plasmid GmHB1a-pK7GWⅡRR was introduced into Agrobacterium rhizogenes strain K599 to obtain recombinant Agrobacterium, which was then frozen at -80℃ and stored in glycerol.

[0090] 2. The Agrobacterium rhizogenes obtained in step 1 was used to transform the recipient plant Zhonghuang 13 using the hairy root transformation method. The specific operation steps are as follows:

[0091] (1) Select large, plump soybean seeds without disease spots, such as Zhonghuang 13 soybean seeds.

[0092] (2) Sow the selected seeds into vermiculite and germinate them in a greenhouse for 3 days. When the soybean cotyledons are about to open, transform them.

[0093] (4) When sowing seeds, streak Agrobacterium rhizogenes K599 containing the target vector on solid LB medium and incubate in an incubator at 28°C for about 2 days. One day before infection, pick a single colony and inoculate it into 3-5 mL of LB liquid medium containing rifampicin (50 μg / mL), streptomycin (10 μg / mL) and spectinomycin (100 μg / mL) and incubate overnight in a shaker at 28°C.

[0094] (5) The bacterial culture OD600 was about 0.8. The cells were collected by centrifugation at 5000 rpm for 6 min. The infection solution (10 MM MES and 10 MM MgCl2) was resuspended and used to infect soybean hypocotyls.

[0095] (6) The hypocotyl of seedlings was infected by puncture injection. The injection site was selected below the cotyledon, in the middle of the hypocotyl, and two drops of injection solution were left on both sides of the stem.

[0096] (7) After injection, fill the black tray with water, cover it with a transparent lid, and secure it with tape (for rooting in a high-humidity environment). Culture at 28℃ for 9-14 days to induce transgenic hairy roots on the hypocotyl. Cut them off and soak them in a disposable plastic cup filled with water for further culture until the roots elongate.

[0097] (8) dsRED screening tag, observed with green fluorescence, screened positive roots.

[0098] (9) Transplant positive plants to pure vermiculite for culture.

[0099] Example 3 Change in nodule number in GmHB1a gene silencing

[0100] 1. Rhizobium inoculation treatment

[0101] (1) At -80°C, 1 mL of preserved slow-growing rhizobium Bradyrhizobium diazoefficiens strain USDA110 was added to 200 mL of TY liquid medium containing 50 mg / L spectinomycin (tryptone 5 g / L, yeast extract 3 g / L, CaCl2·2H2O 0.7 g / L), and cultured at 28°C on a shaker at 200 rpm for about 4 days until the OD600 was between 0.8-1.

[0102] (2) The bacterial solution was collected in a 500 mL centrifuge bottle and centrifuged at 6000 rpm for 10 min. The bacterial pellet was resuspended with sterilized ddH2O to an OD600 of about 0.04 for inoculation treatment.

[0103] (3) Each seedling was inoculated with 50 mL of resuspended bacterial solution.

[0104] (4) Five days after inoculation, samples were taken for GUS staining, and 21 days later the nodule phenotype was observed.

[0105] 2. Nodule number phenotype statistics and analysis

[0106] The plants 21 days after inoculation were washed with tap water to remove the vermiculite, and the water was absorbed with paper towels. Green fluorescent light was used to confirm the positive empty vector and GmHB1a-RNAi soybean hairy roots, and it was observed that the nodule number of the GmHB1a-RNAi hairy roots was significantly lower than that of the control (empty vector) (D). The inventors counted the nodule number of the GmHB1a-RNAi hairy roots and the empty vector hairy roots, as shown in E. Figure 1 Figure 1 From D and E, it can be seen that the average nodule number (single root nodule number) of the GmHB1a-RNAi transgenic hairy roots was only 33.6% of that of the control (empty vector), which indicates that the GmHB1a gene is involved in the development of soybean nodules. Figure 1 Figure 1 3. Gene expression level identification

[0107] The empty vector hairy roots and the GmHB1a gene silenced hairy roots were identified as follows:

[0108] The empty vector hairy roots and the GmHB1a gene silenced hairy roots were identified as follows: ​​

[0109] (1) Take roots and nodules, extract total RNA and reverse transcribe into cDNA.

[0110] (2) Take the cDNA extracted in step (1) as template, use the primer pair composed of F and R to identify the expression amount of GmHB1a gene, and use F2 and R2 to identify the expression amount of the internal reference gene (Actin gene).

[0111] The conditions for quantitative PCR amplification are: pre-denaturation 95°C for 5 min; amplification (45 cycles) 95°C for 10 s, 59°C for 15 s, 72°C for 20 s. After the end of the amplification cycle, start the melting curve: 95°C for 5 s, 65°C for 1 min, 65-97°C (temperature increases at a rate of 0.11°C / s).

[0112] The expression amount of GmHB1a gene in different materials obtained by quantitative PCR amplification with each specific primer pair using the cDNA as template is shown in Figure 1 F and Figure 1 G. It can be seen from Figure 1 F that the gene expression amount of GmHB1a in the gene silencing line GmHB1a-RNAi is significantly reduced, with a P value less than 0.0001. GmHB1b is a homologous gene of GmHB1a, and its expression amount is significantly reduced, with a P value less than 0.0001.

[0113] Table 2 Quantitative identification primers

[0114]

[0115] 4、GUS staining

[0116] (1) Place the positive roots infected by rhizobium for 5 days in 90% acetone and fix under vacuum for 30 min (acetoue has strong penetration, can make protein precipitate coagulation, but does not affect the functional groups of protein and save the activity of protein, is good for fixing phosphatase and oxidase, and therefore can be used for GUS staining before fixing, which can prevent the diffusion of GUS signal. The characteristics are fast fixation, strong penetration, easy to make tissue cells shrink; but the ability to maintain cell structure is poor).

[0117] (2) Wash away the acetone with GUS washing solution, wash 2 times.

[0118] (3) Add GUS staining solution (0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.5 mg / mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc), 0.1% (v / v) Triton X-100 and 0.1 M sodium phosphate buffer (pH = 7.0) solution), vacuum for 15-20 min on ice.

[0119] (4) Place at 37°C overnight, and observe the staining.

[0120] (5) Remove the staining solution, add 1 mL of 70% ethanol to stop the staining reaction and decolorize.

[0121] (6) Replace the ethanol several times until the decolorization is complete.

[0122] (7) Observe the nodule infection period of the stained roots under an optical microscope and count, Figure 1 H. As can be seen from Figure 1 H, the number of rhizobial infection at FOCI and IT stages in GmHB1a-RNAi soybean hairy roots is significantly higher than that in the control roots transformed with empty vector, and the number of rhizobial infection at cIT and NP stages is significantly lower than that in the control roots transformed with empty vector. This indicates that GmHB1a gene is involved in the early formation of nodules.

[0123] Sequence

[0124] SEQ ID NO: 1 GmHB1a CDS sequence

[0125] ATGAAAGCAGGGAGATCGATGATGAGCATGGAGGAAGCCTCGAAGAGGCGACCTTTCTTCACCTCACCGGATGAACTGTATGATGAAGAGTATTATGAGAAGCAGTCGCCGGAGAAGAAGCACCGCCTCAGTTCCGAACAGGTCCATCTGTTGGAGAAGAGCTTTGAGGAAGAGAACAAATTGGAGCCTGAGAGGAAGACCCAGTTGGCTAAGAAGCTGGGATTGCAGCCCAGGCAGGTGGCTGTGTGGTTTCAGAACCGCAGGGCTCGATGGAAGACCAAGCAACTTGAAAGGGATTTTGATGTTCTCAAGTCTTCATACGATACCCTACTTTCATCCTATGATTCAATTATGAAGGAGAATGAGAAACTCAAATCTGAGGTGGTATCCTTAAATGAAAAGCTTCAAGTTCAAGCTAAAGAGGTGCCTGAGGAACCATTATGTGACAAGAAAGTTGATCCAATTCCAGTAGATGAAGATATGGCTCCGATTTTCGGCACAAGGGTGGAGGACCACCTGAGTAGTGGGAGTGTTGGAAGTGCGGTGGTGGATGAGGGTAGTCCACAGGTGGTTGTTGACAGTGTTGATTCATACATTCTAGCTGACAACTATGGTGGATGTGTGGGCCCGGTCGAGAGGGTTCAGTCGGAGGAGGAGGATGGGAGTGATGATGGGAGGAGTTACTTGGATGTGTTTGTGGTATCTGAAACTGAGCACCAAAACCATGAGGAAGGAGAGACATTGGGTTGGTGGACTAATATGTATTATGTTGGATAA

[0126] SEQ ID NO: 2 Protein sequence encoded by GmHB la

[0127] MESGRIFFGASASSGNNMLFLGNTELAFRAGRSIMSMEEASKRRPFFTSPDELYDEE YYEKQSPEKKHRLSSEQVHLLEKSFEEENKLEPERKTQLAKKLGLQPRQVAVWFQ NRRARWKTKQLERDYDVLKSSYDTLLSSYDSIMKENEKLKSEVVSLNEKLQVQAKE VPEEPLCDKKVDPIPVDEDMAPIFGTRVEDHLSSGSVGSAVVDEGSPQVVVDSVDS YILADNYGGCVGPVERVQSEEEDGSDDGRSYLDVFVVSETEHQNHEEGETLGWWTN MYYVGX

[0128] SEQ ID NO: 3 GmHB la RNAi fragment

[0129] TGAAAGCAGGGAGATCGATGATGAGCATGGAGGAAGCCTCGAAGAGGCGACCTTTCTTCACCTCACCGGATGAACTGTATGATGAAGAGTATTATGAGAAGCAGTCGCCGGAGAAGAAGCACCGCCTCAGTTCCGAACAGGTCCATCTGTTGGAGAAGAGCTTTGAGGAAGAGAACAAATTGGAGCCTGAGAGGAAGACCCAGTTGGCTAAGAAGCTGGGATTGCAGCCCAGGCAGGTGGCTGTGTGGTTTCAGAACCGCAGGGCTCGATGGAAGACCAAGCAACTTGAAAGGGATTTTGATGTTCTCAAGTCTTCATACGATACCCTACTTTCATCCTATGATTCAATTATGAAGGAGAATGAGAAACTCAAATCTGAGGTGGTATCCTTAAATGAAAAGCTTCAAGTTCAAGCTAAAGAGGTGCCTGAGGA

[0130] SEQ ID NO: 4 GmHB la promoter fragment

[0131]

[0132] SEQ ID NO:5 primer F2

[0133] 5'- caaaatcctctctcctctagaAGATAATGGCATGCGATGTCAAA-3'

[0134] SEQ ID NO:6 primer R2

[0135] 5'- gcccttgctcaccatccgcggCAGGTCGCCGGAGAAAGATT-3'

[0136] SEQ ID NO:7 primer Fl

[0137] 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATTGAAAGCA

[0138] GGGAGATCGATGA-3'

[0139] SEQ ID NO:8 primer Rl

[0140] 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTTTCCTCAGGCAC

[0141] CTCTTTAGC-3'

[0142] SEQ ID NO:9 primer GmHB la-F

[0143] GGTGGAGGACCACCTGAGTA

[0144] SEQ ID NO: 10 primer GmHB la-R

[0145] TCCGACTGAACCCTCTCGAC

[0146] SEQ ID NO: 11 primer GmHB lb-F

[0147] GTGGTGGATGAGGGTAGTCC

[0148] SEQ ID NO: 12 primer GmHB lb-R

[0149] ATCACTCCCATCCTCCTGTT

[0150] SEQ ID NO: 13 primer GmActinl l-F

[0151] GAGCTATGAATTGCCTGATGG

[0152] SEQ ID NO: 14 primer GmActinl l-R

[0153] CGTTTCATGAATTCCAGTAGC

[0154] The above-described specific embodiments further illustrate the objects, technical solutions, and advantages of the present application. It should be understood that the above-described specific embodiments are merely for the purpose of illustrating the present application, and are not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for reducing the number of nodules on soybean plants, characterized in that, Introducing a sequence inhibiting expression of a GmHB1a protein sequence into a soybean cell or tissue, wherein the GmHB1a protein sequence is set forth in SEQ ID NO: 2, obtains transgenic soybean with reduced nodule number.

2. The method of claim 1, wherein, The gene sequence encoding the GmHB1a protein is set forth in SEQ ID NO:

1.

3. A method for reducing the number of nodules on soybean plants, characterized in that, Introducing an expression vector comprising a sequence inhibiting expression of a GmHB1a protein sequence into a soybean cell or tissue, wherein the GmHB1a protein sequence is set forth in SEQ ID NO: 2, obtains transgenic soybean with reduced nodule number.

4. The method of claim 3, wherein, The sequence inhibiting expression of the GmHB1a protein is an interfering sequence.

5. The method of claim 3, wherein, The expression vector comprises a marker.

6. The method of claim 5, wherein, The marker is selected from the group consisting of a luminescent marker, an antibiotic marker, and an anti-chemical reagent marker.

7. The method of claim 6, wherein, The luminescent marker is selected from the group consisting of a red fluorescent protein and a green fluorescent protein.

8. The method of claim 6, wherein, 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.

9. The method of claim 6, wherein, The anti-chemical reagent marker is an anti-herbicide marker.

10. The method according to any one of claims 3 to 9, 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.

11. A method of reducing the number of nodules on soybean plants, characterized in that, Introducing an E. coli cell or an Agrobacterium cell containing the expression vector of any one of claims 3 to 10 into a soybean cell or tissue, obtains transgenic soybean with reduced nodule number.

12. The method of claim 11, wherein, The Agrobacterium cell is an Agrobacterium rhizogenes cell.

13. The method of claim 12, wherein, The Agrobacterium rhizogenes is selected from the group consisting of K599, AR1193, C58cl, Arqual, MSU440, LBA9402, and R1601.

14. Use of a GmHBla protein as set forth in SEQ ID NO: 2 in a soybean plant that has a reduced number of nodules, characterized in that, Inhibiting expression of a GmHB1a protein.

Images