Use of GmSAUR46b in Regulating Soybean Yield, Light Stress Resistance and Plant Height

Abstract

The application discloses application of GmSAUR46b in regulating soybean yield, light stress resistance and plant height. GmSAUR46b The application discloses that molecular breeding by using the gene can be used for directional improvement of soybean plant type, such as breeding short-stalk anti-lodging varieties, optimizing midrib structure to improve photosynthetic efficiency, regulating trichome density to enhance stress resistance, and has important agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to GmSAUR46b Applications in regulating soybean yield, light stress resistance, and plant height. Background Technology

[0002] Plant architecture is a core agronomic trait that affects photosynthetic efficiency and yield, and its formation depends on the coordinated regulation of cell elongation, differentiation, and pattern building. Small Auxin-Up RNA ( SAUR The auxin gene family, as early auxin response genes, plays a key regulatory role in plant growth, development, and stress response. Although SAUR Genes exhibit functional conservation in promoting cell expansion across different species, but their molecular mechanisms in regulating complex morphological features in soybeans (such as midrib development in leaves and trichome pattern formation in stems) remain unclear.

[0003] Midrib thickness in leaves directly affects mechanical support capacity and vascular transport efficiency, while stem trichomes, as physical barriers against biotic and abiotic stresses, are crucial for understanding SAUR-mediated regulation of soybean stress resistance traits. As a globally important oilseed crop, optimizing soybean plant architecture is a key pathway to improving productivity. Currently identified... GmSAUR The molecular mechanisms by which this family integrates auxin signaling, photosynthesis, and hormone networks are still poorly understood.

[0004] Therefore, in-depth research and exploration of the key factors regulating soybean plant architecture are needed. GmSAUR Genes have significant theoretical value and promising application prospects. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention provides... GmSAUR46b Applications in regulating soybean yield, light stress resistance, and plant height.

[0006] First aspect of protection of the present invention GmSAUR46b The use of genes or proteins or related biological materials in any of the following:

[0007] A1) Regulating soybean yield or preparing products that regulate soybean yield;

[0008] A2) Regulating soybean light stress resistance or preparing products that regulate soybean light stress resistance;

[0009] A3) Regulating soybean plant height or preparing products that regulate soybean plant height;

[0010] A4) Cultivating soybeans with increased or decreased yields;

[0011] A5) Cultivate soybeans with improved resistance to light stress;

[0012] A6) Cultivate soybeans with increased or decreased plant height.

[0013] In some embodiments, the GmSAUR46b The nucleotide sequence of the gene contains the sequence shown in SEQ ID No. 1.

[0014] SEQ ID No.1

[0015] ATGAAGGGAAAGTTTCTAAGAGGGTGCCTTAACAAGTGGAAGAAAATGGGAAGTAGAGTGTTCCATTGTGCTGCCTACGGTTACTGCTGTGAATGGGAACTGGGGTCATCCATGCATGAAGATGAAGGAGACTCCATTCCCAACGATGTGCCAAAGGGTCACTTGGTTGTGTACGTGGGAGAGCACCAT AAGAGATATGTCATCAAGATTACCTTACTCAATCATCCACTCTTCAAGACTCTGCTGGATCAAGCTAAGGATGAGTATGATTTCATTGCAGATTCAAAGCTCTATATTCCTTGCAGTGAGCATCTTTTCCTCACTGTCCTTCGCCGTGCAAGCACTCCACACAACGAACGAGTGTTTGTGCGTGTCTGA.

[0016] In some embodiments, the amino acid sequence of the GmSAUR46b protein comprises the sequence shown in SEQ ID No. 2.

[0017] SEQ ID No.2

[0018] MKGKFLRGCLNKWKKMGSRVFHCAAYGYCCEWELGSSMHEDEGDSIPNDVPKGHLVVYVGEHHKRYVIKITLLNHPLFKTLLDQAKDEYDFIADSKLYIPCSEHLFLTVLRRASTPHNERVFVRV* .

[0019] In some implementations, overexpression GmSAUR46b Genes or proteins can increase soybean plant height.

[0020] In some implementations, knockout GmSAUR46b Genes or proteins can reduce soybean plant height, increase soybean yield, and improve resistance to light stress.

[0021] In some implementations, the plant height also includes the number of internodes.

[0022] In some implementations, the increased yield refers to an increase in the number of pods.

[0023] In some embodiments, improving soybean light stress resistance means improving soybean light stress resistance by increasing the thickness of the midrib center of the leaves and / or the density and number of trichomes in the stems.

[0024] In some embodiments, the biomaterial is one or more of the following:

[0025] B1) includes the above GmSAUR46b Nucleic acid constructs of the gene or nucleic acid constructs expressing the GmSAUR46b protein.

[0026] B2) Genetically engineered bacteria containing the nucleic acid constructs described in B1).

[0027] A second aspect of the present invention protects a method for cultivating soybeans with reduced plant height and / or increased yield and / or increased resistance to light stress, comprising the following steps: reducing the content and / or activity of GmSAUR46b protein in the recipient plant to obtain soybeans with reduced plant height and / or increased yield and / or increased resistance to light stress.

[0028] In some embodiments, the method for reducing the content and / or activity of GmSAUR46b protein in the recipient plant is to introduce a substance that interferes with the expression of the GmSAUR46b protein-encoding gene into the recipient plant, wherein the substance that interferes with the expression of the GmSAUR46b protein-encoding gene contains the sequence shown in SEQ ID No. 3 or SEQ ID No. 4.

[0029] SEQ ID No. 3

[0030] TACTGCTGTGAATGGGAACTGGG

[0031] SEQ ID No. 4

[0032] CTTTGGCACATCGTTGGGAATGG

[0033] A third aspect of the present invention protects a method for reducing soybean plant height and / or increasing soybean yield and / or increasing soybean light stress resistance, comprising the steps of: reducing the content and / or activity of GmSAUR46b protein in recipient plants.

[0034] In some embodiments, the method for reducing the content and / or activity of GmSAUR46b protein in the recipient plant is to introduce a substance that interferes with the expression of the GmSAUR46b protein-encoding gene into the recipient plant, wherein the substance that interferes with the expression of the GmSAUR46b protein-encoding gene contains the sequence shown in SEQ ID No. 3 or SEQ ID No. 4.

[0035] A fourth aspect of the present invention protects a method for cultivating soybeans with increased plant height, comprising: increasing the content and / or sex of GmSAUR46b protein in a recipient plant to obtain soybeans with increased plant height.

[0036] In some embodiments, the method for increasing the content and / or activity of GmSAUR46b protein in the recipient plant is to overexpress GmSAUR46b protein in the recipient plant.

[0037] In some embodiments, the overexpression method involves introducing the gene encoding the GmSAUR46b protein into a recipient plant.

[0038] The fifth aspect of the present invention protects a method for increasing soybean plant height, comprising: increasing the content and / or activity of GmSAUR46b protein in recipient plants.

[0039] In some embodiments, the method for increasing the content and / or activity of GmSAUR46b protein in the recipient plant is to overexpress GmSAUR46b protein in the recipient plant.

[0040] In some embodiments, the overexpression method involves introducing the gene encoding the GmSAUR46b protein into a recipient plant.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1) This invention is the first to clearly define GmSAUR46b The study revealed the key role of genes in regulating soybean plant height, leaf midrib development, and stem trichome density, elucidating the molecular mechanism by which genes regulate plant morphology through the integration of auxin and light signals, and providing a new theory for understanding soybean plant architecture.

[0043] 2) This invention discovers and utilizes GmSAUR46b Molecular breeding using genes can be used to selectively improve soybean plant architecture, such as developing short-statured, lodging-resistant varieties, optimizing midrib structure to improve photosynthetic efficiency, and regulating trichome density to enhance stress resistance, which has significant agricultural application value.

[0044] 3) The gene editing and overexpression technology system provided by this invention can efficiently regulate... GmSAUR46b This provides a practical tool for precision soybean breeding. Attached Figure Description

[0045] Figure 1A For the present invention GmSAUR46b A phylogenetic tree diagram of homologous genes.

[0046] Figure 1B For the present invention GmSAUR46b Structural analysis diagram of homologous genes.

[0047] Figure 1C For the present invention GmSAUR46b Analysis diagram of gene expression patterns in response to light signals.

[0048] Figure 2 For the present invention GmSAUR46b A schematic diagram of the CRISPR-Cas9 gene target design.

[0049] Figure 3 For the present invention GmSAUR46b Atlas of gene editing vectors.

[0050] Figure 4 For the present invention GmSAUR46b Map of gene overexpression vectors.

[0051] Figure 5A In Embodiment 1 of the present invention gRNA Target diagram 。

[0052] Figure 5B This is a schematic diagram of the six stages of the soybean genetic transformation process in Example 1 of the present invention. 。

[0053] Figure 5C In Embodiment 1 of the present invention, Cas9 editing is used. GmSAUR46b The sequencing results are shown in the image.

[0054] Figure 5D The knockout strain in Example 1 of the present invention Gmsaur46b Actual photos of the T1 generation model.

[0055] Figure 5E The knockout strain in Example 1 of the present invention Gmsaur46b The statistical results of plant height, number of internodes, number of pods, and flowering period of the T1 generation are shown in the figure.

[0056] Figure 6 The knockout strain in Example 1 of the present invention Gmsaur46b The plant height phenotype of the T2 generation.

[0057] Figure 7A This is a spectral diagram of normal illumination treatment in Embodiment 1 of the present invention.

[0058] Figure 7BThis is a spectral diagram of the shading treatment in Embodiment 1 of the present invention.

[0059] Figure 8 This is a schematic diagram of the statistical sampling of the midrib center thickness of the leaf and the trichomes of the stem according to the present invention.

[0060] Figure 9A In Example 1 of the present invention, wild-type W82 and knockout strains were subjected to normal light treatment and shading treatment conditions, respectively. Gmsaur46b Optical micrographs of the overexpressing strain 35S-GmSAUR46b.

[0061] Figure 9B In Example 1 of the present invention, wild-type W82 and knockout strains were subjected to normal light treatment and shading treatment conditions, respectively. Gmsaur46b Statistical analysis of midrib center thickness in leaves of the overexpressing strain 35S-GmSAUR46b.

[0062] Figure 9C In Example 1 of the present invention, under shading conditions, wild-type W82 and knockout strains... Gmsaur46b Statistical analysis of midrib center thickness in leaves of the overexpressing strain 35S-GmSAUR46b.

[0063] Figure 10A In Example 1 of the present invention, under normal light treatment conditions, wild-type WT and knockout strains were... Gmsaur46b A photograph of the trichomes on the stems of the 35S-GmSAUR46b overexpression strain.

[0064] Figure 10B In Example 1 of the present invention, under shading conditions, wild-type WT and knockout strains... Gmsaur46b Real photos of the trichomes on the stems of the overexpressing strain 35S-GmSAUR46b.

[0065] Figure 10C In Example 1 of the present invention, wild-type WT and knockout strains were obtained under normal light treatment. Gmsaur46b Statistical analysis of trichomes in the stems of the overexpressing strain 35S-GmSAUR46b.

[0066] Figure 10D In Example 1 of the present invention, under shading conditions, wild-type WT and knockout strains... Gmsaur46b Statistical analysis of trichomes in the stems of the overexpressing strain 35S-GmSAUR46b. Detailed Implementation

[0067] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the described contents. Unless otherwise specified, the methods in this embodiment are operated according to conventional methods, and the reagents used are conventional reagents unless otherwise specified.

[0068] Example 1

[0069] Plant material: Fully mature seeds of the conventional soybean cultivar Williams 82 were selected, which are abbreviated as W82 or WT in the examples.

[0070] Strains and vectors: Agrobacterium ( Agrobacterium tumefaciens L. strain EHA105; CRISPR-Cas9 vector PEG401 for gene editing (vector map as shown) Figure 3 ), overexpression vectors such as PTF101-35S (vector maps are shown in...) Figure 4 ).

[0071] Reagents and instruments: FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China); HiScript II 1st Strand cDNA Synthesis Kit (Vazyme); Premix Ex Taq (Takara); QuantStudio 5 Real-Time PCR system (ThermoFisher Scientific, Waltham, MA, USA); PANNORAMIC (3DHISTECH, Hungary) whole-slice scanner; CaseViewer 2.4 (3DHISTECH, Hungary) software for image viewing; scanning electron microscope (Hitachi SU8100).

[0072] 1.1 Protein and gene structure analysis and light response characteristics

[0073] Total RNA was extracted from the leaves, stems, and shoot tip meristems of wild-type soybean variety Williams 82 using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). One μg of total RNA was used to synthesize complementary DNA via reverse transcription using Oligo (dT) 18 primers and the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme).

[0074] qRT-PCR analysis of gene transcription levels was performed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) system. Using 2 μL of 10-fold diluted cDNA as a template, a 10 μL volume of ChamQ Universal SYBR qPCRMaster Mix (Vazyme) was used for quantitative PCR. The reaction conditions were: 95℃ pre-denaturation for 30 seconds, followed by 40 cycles (95℃ for 10 seconds, 60℃ for 30 seconds). GhTubulin The primers used to standardize the RNA content of the internal reference gene are shown in Table 1.

[0075] Table 1 Primer design for QRT expression level detection

[0076]

[0077] Protein sequences were obtained from the SoyBase database. The GmSAUR46b protein structure was visualized using the Pfam database. Gene sequences were also obtained from the SoyBase database, and their structures were visualized using the SMART online tool. Results are shown below. Figure 1A -C.

[0078] from Figure 1A and 1B It can be seen that the comparison of soybean genome sequences identified 6 genes. GmSAUR46 Homologous genes ( Figure 1A The sequence of each color segment is as follows: Figure 1B As shown in Figure 1A), analysis of their gene structure and conserved functional domains revealed significant differences in gene structure and conserved domains among different GmSAUR46 variants.

[0079] To further verify GmSAUR46 The function of soybeans was determined through a shading treatment experiment (before shading, the soybeans were grown under normal light conditions, and the light spectrum was as follows). Figure 7A And 7B, in the leaves of the wild-type soybean variety Williams 82 at the stage of the third trifoliate compound leaf unfolding ( Figure 8 Genes in leaves GmSAUR46a / b / c The expression patterns were analyzed, among which GmSAUR46a ID is Glyma.03G182100.1 , GmSAUR46b ID is Glyma.19G182600.1 , GmSAUR46c ID is Glyma.10G209700.1 Sampling began 12 hours before the light-blocking procedure, with samples taken every 3 hours. The sample was then blocked from light when the value was marked as 0. Real-time quantitative PCR (QRT-PCR) analysis revealed... GmSAUR46a and GmSAUR46c The expression pattern did not change significantly, but GmSAUR46b Expression levels spiked dramatically immediately after the light-blocking treatment, then rapidly decreased, before stabilizing (Figure 1C). This indicates that the gene... GmSAUR46b It is sensitive to light signals and may be involved in light signal regulation pathways, making it a potential candidate gene for us.

[0080] 1.2 Gene knockout, construction of overexpression vectors, and genetic transformation

[0081] 1.2.1 Construction and transformation of knockout vectors

[0082] 1) Carrier construction

[0083] Gene editing was performed using CRISPR-Cas9 technology. GmSAUR46b Gene sequence design for specific sgRNAs (sequences shown in Table 2 and ...) Figure 2 ), cloned into the BsaI site of the PEG401 vector, to construct the CRISPR-Cas9 vector (see Figure 3 The vector was transferred into Agrobacterium EHA105 via electroporation.

[0084] The method for constructing CRISPR-Cas9 vectors is as follows:

[0085] ①Synthesize the following target gene amplification primers.

[0086] F: cagtGGTCTCatgcatactgctgtgaatgggaactgttttagagc(SEQ ID No.17)

[0087] R: cagtGGTCTCaaaacctttggcacatcgttgggaatgcac(SEQ ID No.18)

[0088] ②PCR amplification

[0089] The PCR amplification system is shown in Table 2.

[0090] Table 2

[0091]

[0092] The PCR amplification procedure is shown in Table 3.

[0093] Table 3

[0094]

[0095] Subsequently, the T1-T2 (193 bp) electrophoretic fragment was excised under UV light using 1.5% agarose gel electrophoresis at 5 V / cm for 20 minutes. The fragment was then recovered by sol-gel extraction, and the DNA was dissolved and recovered using 30 μL of water. After the recovered DNA was confirmed to be correct, it was ligated into the vector.

[0096] ③ Enzyme digestion and ligation

[0097] The enzyme digestion and ligation system is shown in Table 4.

[0098] Table 4

[0099]

[0100] The enzyme digestion reaction conditions are shown in Table 5.

[0101] Table 5

[0102]

[0103] ④ Transform the ligation product into competent cells

[0104] Transform 5-10 μL of the ligation product into competent E. coli cells, plate them on kanamycin-resistant plates, incubate at 37°C for 12 hours, and then perform plaque PCR identification.

[0105] ⑤ Plaque PCR identification

[0106] Twelve bacterial plaques were selected and simultaneously inoculated into 1.5 mL EP tubes for PCR identification. The PEG401 identification primers were:

[0107] F:GCAACGCTCTGTCATCGTTACAAT (203 bp) (SEQ ID No.19)

[0108] R:gcgattaagttgggtaacgccaggg (8026 bp) (SEQ ID No.20)

[0109] The PCR reaction system is shown in Table 6.

[0110] Table 6

[0111]

[0112] The PCR reaction procedure is shown in Table 7.

[0113] Table 7

[0114]

[0115] The target band is a fragment of approximately 7843 bp. Take 100 μL of bacterial culture corresponding to 2-3 positive bands for sequencing. Inoculate the remaining 400 μL of bacterial culture into LB agar containing 5-10 mL of kanamycin-resistant culture. Shake the tubes and wait for the sequencing results. Extract the plasmid from the tube corresponding to the correctly sequenced result. Store the bacterial strain and plasmid at -80℃.

[0116] The specific method of electroporation is as follows: Add 1 μL of plasmid to 50 μL of EHA105 Agrobacterium competent cells, mix thoroughly, and then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate at 30℃ and 180 rpm for 30 min on a shaker. Inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 30℃ for 48 h. (sgRNA target sites) Figure 5A .

[0117] Table 8 GmSAUR46b Gene editing sgRNA sequence

[0118]

[0119] 2) Soybean conversion

[0120] The genetic transformation process of soybean can be divided into six stages according to its different growth and development states: seed germination stage, co-culture stage, induced differentiation stage, redifferentiation stage, elongation stage, and rooting stage (Figure 5B). The culture medium formulations used in different stages are referenced in references 1, 2, and 3 (Reference 1: Li, S. et al., Optimization of...). Agrobacterium -Mediated Transformation in Soybean. Frontiers in PlantScience 2017, 8; Document 2: Zhong, H. et al., A fast and genotype-independentin plantaAgrobacterium -mediated transformation method for soybean. PlantCommunications 2024, 5, (12); Document 3: Hada, A. et al., Refined glufosinateselection and its extent of exposure for improving the Agrobacterium -mediatedtransformation in Indian soybean ( Glycine max(genotype JS-335. PlantBiotechnology 2016, 33, (5), 341-350). The transformation method refers to the soybean transformation technology system of Boyuan Biotechnology, as follows:

[0121] ① Select soybean seeds, disinfect their surface, and then germinate them under sterile conditions. After 3-5 days, obtain uncontaminated cotyledon explants.

[0122] ② Immerse the explants in an inoculum containing recombinant Agrobacterium EHA105 for 2-3 minutes (OD of Agrobacterium solution at infection time). 600 The value is 0.5), and the explants are stirred from time to time. Then the explants are transferred to a co-culture plate lined with filter paper and co-cultured at 25°C in the dark for 3-5 days.

[0123] ③ Afterwards, select healthy and uncontaminated explants, cut off the end of the hypocotyl, transfer them to callus dedifferentiation solid medium, and culture them in a flat layer for 7-10 days under a photoperiod of 16 hours of light / 8 hours of darkness, and then subculture them in fresh medium.

[0124] ④ After the embryoids are formed, they are inoculated onto the redifferentiation medium and cultured for 21 days under a photoperiod of 16 hours of light / 8 hours of darkness, and then subcultured in fresh medium.

[0125] ⑤ After screening, the well-growing embryoids were transferred to a new elongation medium. They were cultured for 21 days under a photoperiod of 16 hours light / 8 hours dark, and then subcultured in fresh medium.

[0126] ⑥ When the young shoots grow to about 5 cm, they are transferred to rooting medium for further selection. Under a photoperiod of 16 hours of light / 8 hours of darkness, they are cultured for 21 days to promote the initiation and differentiation of root and stem meristems.

[0127] ⑦ The presence or absence of Bar / pat protein in T0 generation transgenic plants was directly identified using the Bar test strip method.

[0128] ⑧ Remove the positive seedlings from the culture medium after testing with Bar test strips, wash the roots of the seedlings, and transplant them into seedling trays filled with nutrient soil. Harden the seedlings for 3-4 weeks under a photoperiod of 27℃ and 16 hours of light / 8 hours of darkness.

[0129] ⑨ The received seeds were of generation T1. They were planted in nutrient pots filled with nutrient soil at 26℃, 60% humidity, and a photoperiod of 16 hours of light / 8 hours of darkness.

[0130] ⑩ The T1 generation was tested using the Hi-TOM platform to determine whether the transgenic plants carried the Cas9 protein and whether they had mutated. The mutant genotype was verified by Hi-TOM sequencing. The relevant primers are shown in Table 9, and the sequencing verification results are shown in [Table 9]. Figure 5C .

[0131] Table 9 GmSAUR46b Primer sequences for gene editing detection

[0132]

[0133] from Figure 5C It can be seen that the T1 generation number 14 (i.e. T1-14 in the figure) has an 8-base deletion mutation (8 repeats indicate that there is a specific base sequence change pattern repeated 8 times in this mutant, reflecting that after gene editing, the sequence near the target site shows a consistent repetitive mutation feature in multiple copies or multiple plants, indicating the genetic stability of the mutant).

[0134] 1.2.2 Construction of overexpression vectors and genetic transformation

[0135] PCR amplification GmSAUR46b Complete CDS (primers are shown in Table 10), inserted into the XbaI and SacI sites of the PTF101-35S vector ( Figure 4 Agrobacterium EHA105 was transformed, and soybean explants were also transformed using the same Agrobacterium-mediated method. The subsequent culture process was similar to that of gene editing transformation.

[0136] Table 10 GmSAUR46b overexpression primers

[0137]

[0138] The primer sequences used for plaque PCR identification are:

[0139] 35seq: tTCATTTGGAGAGAACACGGGggac (840 bp) (SEQ ID No.27)

[0140] NOSseq-R: caagaccggcaacaggattcaatc (1297 bp) (SEQ ID No.28)

[0141] 1.3 Phenotypic Identification

[0142] The natural height of the plant, the number of internodes, and the time of pod formation were measured at maturity. The number of pods after pod formation was also counted. The mean and standard deviation of three biological replicates were calculated. Figure 5D and 5E ).

[0143] Built using the CRISPR-Cas9 system GmSAUR46b Gene knockout mutants. From Figure 5D Phenotypic analysis showed that homozygous mutant plants exhibited pleiotropic effects, with shorter plant height and significantly reduced pod formation.

[0144] from Figure 5E Statistical analysis shows that the T1 generation knockout strains Gmsaur46b The average plant height was 23.1 cm, which was significantly shorter than the wild-type WT (35.06 cm) by 34.11%. p =5.59E-08).

[0145] from Figure 5E It can be seen that the T1 generation knockout strain Gmsaur46b The number of internodes was significantly reduced, averaging 8.5 internodes per plant, compared to 9.88 internodes for the wild-type WT, which was significantly less than the wild-type WT. p =0.0035).

[0146] from Figure 5E It can be seen that the T1 generation knockout strain Gmsaur46b The number of pods produced was significantly increased, averaging 30.17 pods per plant, a significant increase of 49.19% compared to wild-type plants (15.33 pods). p =0.00028), and Figure 5D .

[0147] from Figure 5E It can be seen that the T1 generation knockout strain Gmsaur46b The flowering time of the cultivar was significantly earlier, starting 15.20 days after sowing, while that of the wild-type WT was 21.30 days, which was significantly earlier than that of the wild-type WT. p =1.32E-06).

[0148] from Figure 6 It can be seen that, compared with wild-type plants (i.e., wild-type WT), the T2 generation knockout lines Gmsaur46b The plant height was significantly reduced.

[0149] 1.4 Regulation of the thickness of the midrib center in soybean leaves

[0150] Data were collected from plants grown under shaded conditions (photosynthetic photon flux density, PPFD = 149.8 μmol·m⁻¹). -2 .s -1 (simulating shading and lighting conditions) Figure 7A ) and normal light conditions (PPFD = 314.5 μmol·m -2 .s -1 (simulating normal lighting conditions) Figure 7B Wild-type soybean Williams 82 (also known as wild-type WT), knockout strains Gmsaur46b and overexpression line 35S- GmSAUR46b The third trifoliate compound leaf of soybean leaves at the stage of flattening ( Figure 8 The tissue was immersed in 50% FAA (50% anhydrous ethanol, 10% 37% formaldehyde solution, 5% acetic acid) under vacuum for three 15-minute vacuum infiltrations to fix the tissue. After infiltration, the FAA solution was replaced with fresh FAA, and the tissue was post-fixed at 4°C for at least 12 hours. The fixed samples were then dehydrated sequentially with different concentrations of ethanol (30%, 50%, 70%, 95%, and 100%) for 1 hour each. After overnight incubation, the tissue was sequentially immersed in 100% ethanol three times, 3 / 4 ethanol and 1 / 4 xylene, 1 / 2 ethanol and 1 / 2 xylene, 1 / 4 ethanol and 3 / 4 xylene, and 100% xylene twice, and then immersed in paraffin. The samples were then sequentially immersed in xylene and 1 / 4 paraffin for at least 12 hours, and then transferred to refined paraffin three times for 3 hours each time. After embedding and cleaning, the tissue was placed in a microtome holder and cut into 7 μm thick sections using a ThermoScientific microtome (Microm HM 340 E). The sections were then dried on a 37°C hot plate for 2 hours. After mounting with resin, the tissue sections were scanned and imaged using a PANNORAMIC panoramic microtome scanner. The images were then opened with CaseViewer 2.4 software, allowing for magnification from 1x to 400x for observation. Using CaseViewer 2.4, the target area of ​​the tissue was selected for 100x imaging, ensuring the tissue filled the entire field of view and maintaining consistent background lighting in each image. After imaging, Image-Pro Plus 6.0 analysis software was used to uniformly measure the thickness of the midrib center on each section at 100x magnification, using millimeters as the standard unit. Five fields of view were recorded for each tissue. The results of optical microscopy observations are shown below. Figure 9A The statistical results of the thickness of the central vein are shown in [the figure]. Figure 9B and 9C .

[0151] from Figure 9A It can be seen that under normal light treatment (CK) conditions, the overexpression line 35S- GmSAUR46b and knockout strains Gmsaur46b The thickness of the midrib center in the leaves was increased compared to the wild-type WT. However, under shading conditions, the overexpressing line 35S- showed increased thickness compared to the wild-type WT. GmSAUR46b It still maintains a high thickness at the center of the midrib of the leaf, while the knockout strain Gmsaur46b The thickness of the midrib center of the leaf is significantly reduced.

[0152] from Figure 9B and 9CIt can be seen that under normal light treatment (CK) conditions, the overexpression line 35S- GmSUR46b The thickness of the leaf midrib at the center was 0.88 mm, which was 11.36% greater than the wild-type WT (0.78 mm). p =0.081); Knockout strains Gmsaur46b The thickness of the leaf midrib center was 0.84 mm, which was 7.69% greater than that of the wild type. p =0.284), with no significant difference (9B). However, under shading conditions, the overexpressing line 35S- GmSAUR46b The thickness of the midrib at the center of the leaf reached 0.75 mm, which was significantly increased by 15.38% compared to the wild-type WT (0.65 mm). p =0.019), which was significantly reduced by 14.33% compared to the overexpression lines under normal light treatment conditions. p =0.029)(9C); while the knockout strain Gmsaur46b Under shaded conditions (0.545 mm), the difference in WT compared to wild-type was not significant; however, compared to normal light conditions, it was significantly reduced by 35.71%. P =0.0063).

[0153] In summary, GmSAUR46b It specifically regulates the thickness of the midrib center in soybean leaves. Shading treatment further enhances... GmSAUR46b The regulatory effect on the thickness of the central vein.

[0154] 1.5 Regulation of soybean stem trichome density

[0155] Sampling and fixation: Samples were taken from plants grown under shaded conditions (PPFD = 149.8 μmol·m⁻¹). -2 .s -1 (simulating shading and lighting conditions) Figure 7A ) and normal light conditions (PPFD = 314.5 μmol·m -2 .s -1 (simulating normal lighting conditions) Figure 7B The third trifoliate compound leaf of the wild-type soybean Williams 82 was fully expanded at the third node of the main stem. Figure 8To minimize mechanical damage such as pulling, contusion, and compression, samples should be taken within 1-3 minutes. Gently rinse the sample with PBS to remove impurities from the sample surface, protecting and marking the areas to be scanned (e.g., trimming corners on the opposite side). Immediately immerse the sample in electron microscopy fixative at room temperature for 2 hours, then transfer to 4°C for storage. After fixation, rinse the sample three times with 0.1 M phosphate buffer (PB) (pH 7.4), 15 minutes each time. Prepare 1% osmium tetroxide solution with 0.1 M phosphate buffer (PB) (pH 7.4) and fix at room temperature in the dark for 1-2 hours. Rinse three times with 0.1 M phosphate buffer (PB) (pH 7.4), 15 minutes each time. Dehydration treatment: Immerse the tissue sequentially in 30%-50%-70%-80%-90%-95%-100%-100% ethanol for 15 minutes each time, followed by isoamyl acetate for 15 minutes. Drying treatment: The sample was placed in a critical point desiccator for drying. After fixing the sample on the SEM sample stage and sputtering with gold for 30 s, the distribution of stem trichomes was observed under a scanning electron microscope, and density statistical analysis was performed to count the number of trichomes per 3 mm of stem length (n=5). The results of the scanning electron microscope analysis are shown in Figures 10A and 10B, and the results of the density statistical analysis are shown in Figures 10C and 10D.

[0156] from Figure 10A and 10B As can be seen from this, under normal light treatment (CK) conditions, compared with wild-type WT, the knockout strains... Gmsaur46b and overexpression line 35S- GmSAUR46b The trichomes density increased. However, under shading conditions, the overexpressing line 35S- showed increased trichome density compared to wild-type WT. GmSAUR46b It still maintains a high trichome density, while the knockout strains Gmsaur46b The density of trichomes was significantly reduced.

[0157] from Figure 10C and 10D It can be seen that under normal light (CK) treatment conditions, the overexpression line 35S- GmSAUR46b The number of trichomes per 3 mm stem length was 290.5, which was a highly significant increase of 15.46% compared to the wild-type WT (251.6 trichomes). p =2.78E-06); Knockout strains Gmsaur46b The number of trichomes per 3 mm stem length was 361, which was a highly significant increase of 36.23% compared to the wild-type WT. p =1.72E-07). However, under shading conditions, the overexpressing line 35S- GmSAUR46b It still maintains a high trichome density, with 150.9 trichomes per 3 mm stem length, a highly significant increase of 231.03% compared to the wild-type WT (45.6 trichomes). p=5.08E-13); Knockout strains Gmsaur46b The number of trichomes per 3 mm stem length was 28.5, which was a significant decrease of 37.54% compared to the wild-type WT. p =9.31E-08).

[0158] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

1. Use of a knockout GmSAUR46b gene in any one of: A1) increasing pod number or preparing a product for increasing pod number; A2) decreasing soybean plant height or preparing a product for decreasing soybean plant height; A3) breeding soybean with increased pod number; A4) breeding soybean with decreased plant height; the nucleotide sequence of the GmSAUR46b gene is shown as SEQ ID No.

1.

2. The use according to claim 1, characterized in that The use also comprises decreasing the number of internodes of soybean.

3. A method for breeding soybean with reduced plant height and / or increased pod number, characterized by, It comprises: decreasing the content and / or activity of GmSAUR46b protein in soybean, the amino acid sequence of the GmSAUR46b protein is shown as SEQ ID No. 2, to obtain soybean with decreased plant height and / or increased pod number.

4. A method of reducing plant height and / or increasing pod number in soybean, comprising, It comprises: decreasing the content and / or activity of GmSAUR46b protein in soybean, the amino acid sequence of the GmSAUR46b protein is shown as SEQ ID No.

2.

5. The method of claim 3 or 4, wherein, The method for decreasing the content and / or activity of GmSAUR46b protein in soybean is to introduce a substance interfering with the expression of GmSAUR46b protein coding gene into soybean, the nucleotide sequence of the substance interfering with the expression of GmSAUR46b protein coding gene is shown as SEQ ID No. 3 or SEQ ID No.

4.

6. Use of GmSAUR46b gene or protein or biological material related thereto in any one of: A1) increasing the shade stress resistance of soybean or preparing a product for increasing the shade stress resistance of soybean; A2) breeding soybean with increased shade stress resistance; The GmSAUR46b The nucleotide sequence of the gene is shown as SEQ ID No. 1, and the improving the shade stress resistance of soybean refers to improving the shade stress resistance of soybean by increasing the thickness of the midrib center of the leaf and / or the density and number of the hairs of the stem. The biomaterial is: B1) comprising the above GmSAUR46b A nucleic acid construct of the gene or a nucleic acid construct expressing the GmSAUR46b protein; and / or, B2) genetically engineered bacteria comprising the nucleic acid construct of B1).

7. A method of increasing shade stress resistance in soybean, characterized by, It comprises: increasing the content and / or activity of GmSAUR46b protein in soybean, the amino acid sequence of the GmSAUR46b protein is shown as SEQ ID No.

2.

8. A method of breeding soybean with improved shade stress resistance, characterized in that, It comprises: increasing the content and / or activity of GmSAUR46b protein in soybean, the amino acid sequence of the GmSAUR46b protein is shown as SEQ ID No. 2.

Images