Application of GmSAUR46b in regulation and control of soybean yield, light stress resistance and plant height

Abstract

The invention discloses application of GmSAUR46b in regulation and control of soybean yield, light stress resistance and plant height. The invention finds that soybean plant types can be directionally improved by using the GmSAUR46b gene for molecular breeding, such as cultivating dwarf lodging-resistant varieties, optimizing midrib structures to improve photosynthetic efficiency, regulating hairy body density to enhance stress resistance, and the GmSAUR46b gene has important agricultural application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to GmSAUR46b Application in regulating soybean yield, light stress resistance and plant height. Background Art

[0002] Plant architecture is a core agronomic trait that affects photosynthesis efficiency and yield, and its formation depends on the coordinated regulation of cell elongation, differentiation and pattern construction. Small Auxin-Up RNA ( SAUR ) gene family as early auxin response genes, plays a key regulatory role in plant growth and development and stress response. SAUR The gene is functionally conserved in promoting cell expansion in different species, but its molecular mechanism in regulating complex morphological characteristics of soybean (such as leaf midrib development and stem trichome patterning) has not yet been clarified.

[0003] The thickness of the leaf midrib directly affects the mechanical support capacity and vascular material transport efficiency, while the stem trichomes serve as a physical barrier against biotic and abiotic stresses, which is of great significance for understanding the regulation of soybean stress resistance mediated by SAUR. As an important oil crop in the world, the optimization of soybean plant type is a key way to improve productivity. GmSAUR Among the family members, the molecular mechanisms that integrate auxin signaling, photosynthesis and hormone networks still lack systematic research.

[0004] Therefore, in-depth research and exploration of the key to regulating soybean plant type GmSAUR Genes have important theoretical value and application prospects. Summary of the Invention

[0005] In view of the technical problems existing in the prior art, the present invention provides GmSAUR46b Application in regulating soybean yield, light stress resistance and plant height.

[0006] The first aspect of the present invention is to protect GmSAUR46b Use of genes or proteins or biological materials related thereto in any of the following: A1) Regulating soybean yield or preparing products that regulate soybean yield; A2) regulating soybean light stress resistance or preparing a product for regulating soybean light stress resistance; A3) Regulating soybean plant height or preparing products for regulating soybean plant height; A4) Breeding soybeans with increased or decreased yield; A5) Breeding soybeans with improved light stress resistance; A6) Breeding soybeans with increased or decreased plant height.

[0007] In some embodiments, the GmSAUR46bThe nucleotide sequence of the gene comprises the sequence shown in SEQ ID No.1.

[0008] SEQ ID No.1 ATGAAGGGAAAGTTTCTAAGAGGGTGCCTTAACAAGTGGAAGAAAATGGGAAGTAGAGTGTTCCATTGTGCTGCCTACGGTTACTGCTGTGAATGGGAACTGGGGTCATCCATGCATGAAGATGAAGGAGACTCCATTCCCAACGATGTGCCAAAGGGTCACTTGGTTGTGTACGTGGGAGAGCACCAT AAGAGATATGTCATCAAGATTACCTTACTCAATCATCCACTCTTCAAGACTCTGCTGGATCAAGCTAAGGATGAGTATGATTTCATTGCAGATTCAAAGCTCTATATTCCTTGCAGTGAGCATCTTTTCCTCACTGTCCTTCGCCGTGCAAGCACTCCACACAACGAACGAGTGTTTGTGCGTGTCTGA.

[0009] In some embodiments, the amino acid sequence of the GmSAUR46b protein comprises the sequence shown in SEQ ID No.2.

[0010] SEQ ID No. 2 MKGKFLRGCLNKWKKMGSRVFHCAAYGYCCEWELGSSMHEDEGDSIPNDVPKGHLVVYVGEHHKRYVIKITLLNHPLFKTLLDQAKDEYDFIADSKLYIPCSEHLFLTVLRRASTPHNERVFVRV* .

[0011] In some embodiments, overexpression GmSAUR46b Genes or proteins that increase soybean plant height.

[0012] In some embodiments, knockout GmSAUR46b The gene or protein reduces soybean plant height, increases soybean yield, and improves light stress resistance.

[0013] In certain embodiments, the plant height further comprises the number of internodes.

[0014] In certain embodiments, the increased yield refers to an increase in the number of pods.

[0015] In certain embodiments, improving the light stress resistance of soybean refers to improving the light stress resistance of soybean by increasing the center thickness of the midrib of leaves and / or the density and number of trichomes on the stems.

[0016] In certain embodiments, the biological material is one or more of the following: B1) contains the GmSAUR46b A nucleic acid construct of a gene or a nucleic acid construct expressing the GmSAUR46b protein B2) A genetically engineered bacterium comprising the nucleic acid construct described in B1).

[0017] The second aspect of the present invention protects a method for cultivating soybeans with reduced plant height and / or increased yield and / or improved light stress resistance, comprising the following steps: reducing the content and / or activity of GmSAUR46b protein in a recipient plant to obtain soybeans with reduced plant height and / or increased yield and / or improved light stress resistance.

[0018] 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 gene encoding the GmSAUR46b protein into the recipient plant, and the substance that interferes with the expression of the gene encoding the GmSAUR46b protein comprises the sequence shown in SEQ ID No.3 or SEQ ID No.4.

[0019] SEQ ID No. 3 TACTGCTGTGAATGGGAACTGGG SEQ ID No.4 CTTTGGCACATCGTTGGGAATGG The third aspect of the present invention protects a method for reducing soybean plant height and / or increasing soybean yield and / or improving soybean light stress resistance, comprising the steps of reducing the content and / or activity of GmSAUR46b protein in a recipient plant.

[0020] 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 gene encoding the GmSAUR46b protein into the recipient plant, and the substance that interferes with the expression of the gene encoding the GmSAUR46b protein comprises the sequence shown in SEQ ID No.3 or SEQ ID No.4.

[0021] The fourth aspect of the present invention protects a method for cultivating soybeans with increased plant height, comprising: increasing the content and / or activity of GmSAUR46b protein in a recipient plant to obtain soybeans with increased plant height.

[0022] In some embodiments, the method for increasing the content and / or activity of the GmSAUR46b protein in the recipient plant is to overexpress the GmSAUR46b protein in the recipient plant.

[0023] In certain embodiments, the overexpression method is to introduce the gene encoding the GmSAUR46b protein into the recipient plant.

[0024] 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 a recipient plant.

[0025] In some embodiments, the method for increasing the content and / or activity of the GmSAUR46b protein in the recipient plant is to overexpress the GmSAUR46b protein in the recipient plant.

[0026] In certain embodiments, the overexpression method is to introduce the gene encoding the GmSAUR46b protein into the recipient plant.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention clearly states for the first time GmSAUR46b The key role of genes in regulating soybean plant height, leaf midrib development and stem trichome density reveals its molecular mechanism of regulating plant morphology by integrating auxin signals and light signals, providing a new theory for analyzing soybean plant type formation.

[0028] 2) The invention is discovered and utilized GmSAUR46b Molecular breeding of genes can be used to improve soybean plant type in a targeted manner, such as cultivating dwarf and lodging-resistant varieties, optimizing midrib structure to improve photosynthetic efficiency, and regulating trichome density to enhance stress resistance, which has important agricultural application value.

[0029] 3) The gene editing and overexpression technology system provided by the present invention can efficiently regulate GmSAUR46b Expression, providing practical tools for soybean precision breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A For the present invention GmSAUR46b Phylogenetic tree analysis of homologous genes.

[0031] Figure 1B For the present invention GmSAUR46b Structural analysis diagram of the homologous genes of the gene.

[0032] Figure 1C For the present invention GmSAUR46b Analysis of gene expression patterns in response to light signals.

[0033] Figure 2 For the present invention GmSAUR46bSchematic diagram of gene CRISPR-Cas9 target design.

[0034] Figure 3 For the present invention GmSAUR46b Map of gene editing vectors.

[0035] Figure 4 For the present invention GmSAUR46b Map of gene overexpression vectors.

[0036] Figure 5A In Example 1 of the present invention gRNA Schematic diagram of the target 。

[0037] Figure 5B Schematic diagram of the six stages of soybean genetic transformation process in Example 1 of the present invention 。

[0038] Figure 5C Cas9 editing is used in Example 1 of the present invention GmSAUR46b The sequencing results are shown in Figure 3.

[0039] Figure 5D This is the knockout strain in Example 1 of the present invention Gmsaur46b Actual photos of the T1 generation phenotype.

[0040] Figure 5E This is the knockout strain in Example 1 of the present invention Gmsaur46b Statistical results of plant height, number of internodes, number of pods and flowering period of T1 generation.

[0041] Figure 6 This is the knockout strain in Example 1 of the present invention Gmsaur46b Plant height phenotype of the T2 generation.

[0042] Figure 7A This is a spectrum diagram of normal light treatment in Example 1 of the present invention.

[0043] Figure 7B This is a spectrum diagram of the shading treatment in Example 1 of the present invention.

[0044] Figure 8 Schematic diagram of the statistical sampling of the leaf midrib center thickness and stem trichomes of the present invention.

[0045] Figure 9A In Example 1 of the present invention, the wild type W82 and the knockout strains were treated under normal light and shade conditions. Gmsaur46b and optical microscopy observations of the overexpression line 35S-GmSAUR46b.

[0046] Figure 9BIn Example 1 of the present invention, the wild type W82 and the knockout strains were treated under normal light and shade conditions. Gmsaur46b Statistical analysis of the midrib center thickness of leaves of the overexpressing strain 35S-GmSAUR46b.

[0047] Figure 9C In Example 1 of the present invention, under the shading condition, the wild type W82 and the knockout strain Gmsaur46b Statistical analysis of the midrib center thickness of leaves of the overexpressing strain 35S-GmSAUR46b.

[0048] Figure 10A In Example 1 of the present invention, under normal light treatment conditions, the wild type WT and knockout strains Gmsaur46b and actual photos of trichomes in the stem of the overexpressing strain 35S-GmSAUR46b.

[0049] Figure 10B In Example 1 of the present invention, under the condition of shading, the wild type WT and knockout strains Gmsaur46b Actual photos of the trichomes of the stem of the overexpressing strain 35S-GmSAUR46b Figure 10C In Example 1 of the present invention, under normal light treatment, the wild type WT and knockout strains Gmsaur46b Statistical analysis of trichomes in the stems of the overexpressing line 35S-GmSAUR46b.

[0050] Figure 10D In Example 1 of the present invention, under the shade treatment condition, the wild type WT and knockout strains Gmsaur46b Statistical analysis of trichomes in the stems of the overexpressing line 35S-GmSAUR46b. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the scope of protection of the present invention is not limited to the contents described above. In the present examples, the methods are operated according to conventional methods unless otherwise specified, and the reagents used are conventional reagents unless otherwise specified.

[0052] Example 1 Plant material: Fully mature seeds of the conventional soybean cultivar Williams 82 were used, abbreviated as W82 or WT in the examples.

[0053] Strains and vectors: Agrobacterium tumefaciens ( Agrobacterium tumefaciens L.) strain EHA105; CRISPR-Cas9 vector PEG401 for gene editing (vector map as shown in Figure 3 ), overexpression vector PTF101-35S, etc. (vector map such as Figure 4 ).

[0054] Reagents and instruments: RNA extraction kit FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China); reverse transcription kit 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-slide scanner; CaseViewer 2.4 (3DHISTECH, Hungary) software for image viewing; scanning electron microscope (Hitachi SU8100).

[0055] 1.1 Protein and gene structure analysis and photoresponse characteristics Total RNA was extracted from leaves, stems, and shoot apical meristems of the wild-type soybean cultivar 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 using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) with an Oligo (dT) 18 primer.

[0056] qRT-PCR was performed on the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) system to analyze gene transcription levels. Quantitative PCR reactions were performed in a volume of 10 μL using 2 μL of 10-fold diluted cDNA as template and ChamQ Universal SYBR qPCR Master Mix (Vazyme). Reaction conditions were: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. GhTubulin The primers used as internal reference genes to normalize RNA content are shown in Table 1.

[0057] Table 1 Primer design for QRT expression detection

[0058] The protein sequence was obtained from the SoyBase database. The protein structure of GmSAUR46b was visualized using the Pfam database. The gene sequence was also obtained from the SoyBase database, and the gene structure was visualized using the SMART online tool. Figure 1A -C.

[0059] from Figure 1A and 1B It can be seen that the comparison of soybean genome sequences identified 6 GmSAUR46 Homologous genes ( Figure 1A , the sequence of each color segment is as follows Figure 1B Analysis of their gene structures and conserved functional domains revealed that different GmSAUR46s differed greatly in gene structure and conserved domains (Figure 1 A).

[0060] To further verify GmSAUR46 The function in soybean was studied through shading treatment experiment (growth under normal light conditions before shading, light pattern such as Figure 7A and 7 B, in the third trifoliate leaf of the wild-type soybean variety Williams 82 during its flattening period ( Figure 8 ) Genes in leaves GmSAUR46a / b / c The expression patterns of GmSAUR46a The ID is Glyma.03G182100.1 , GmSAUR46b The ID is Glyma.19G182600.1 , GmSAUR46c The ID is Glyma.10G209700.1 Sampling started 12 hours before shading, and samples were taken every 3 hours. The light was shading when the time marked as 0. The results of real-time fluorescence quantitative PCR (QRT-PCR) analysis showed that GmSAUR46a and GmSAUR46c The expression pattern of GmSAUR46b The expression level increased sharply at the moment of shading treatment, then decreased rapidly, and then showed a stable trend (Figure 1 C). GmSAUR46b It is sensitive to light signals and may be involved in the light signal regulation pathway, so it can be used as our candidate gene.

[0061] 1.2 Construction of gene knockout and overexpression vectors and genetic transformation 1.2.1 Construction and transformation of knockout vector 1) Vector construction Gene editing is performed using CRISPR-Cas9 technology. GmSAUR46b Gene sequence-specific sgRNA design (sequences are 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 transformed into Agrobacterium EHA105 by electroporation.

[0062] The method for constructing CRISPR-Cas9 vector is as follows: ①Synthesize the following target gene amplification primers F: cagtGGTCTCatgcatactgctgtgaatgggaactgttttagagc(SEQ ID No.17) R: cagtGGTCTCaaaacctttggcacatcgttgggaatgcac(SEQ ID No.18) ②PCR amplification The PCR amplification system is shown in Table 2.

[0063] Table 2

[0064] The PCR amplification program is shown in Table 3.

[0065] Table 3

[0066] Subsequently, 1.5% agarose gel electrophoresis was performed at 5 v / cm for 20 minutes. The T1-T2 (193 bp) electrophoresis fragment was cut out under ultraviolet light and the sol was recovered. The DNA was dissolved in a total volume of 30 μL of water and the recovered DNA was connected to the vector after testing.

[0067] ③ Enzyme digestion and ligation The enzyme digestion and ligation system is shown in Table 4.

[0068] Table 4

[0069] The enzyme digestion reaction conditions are shown in Table 5.

[0070] Table 5

[0071] ④Transform the ligation product into competent cells 5-10 μL of the ligation product was transformed into competent E. coli, coated with kanamycin-resistant plates, cultured at 37°C for 12 hours, and identified by plaque PCR.

[0072] ⑤ Plaque PCR identification Twelve plaques were picked and inoculated into 1.5 mL EP tubes and identified by PCR. The primers for PEG401 identification were: F:GCAACGCTCTGTCATCGTTACAAT (203 bp) (SEQ ID No.19) R:gcgattaagttgggtaacgccaggg (8026 bp) (SEQ ID No.20) The PCR reaction system is shown in Table 6.

[0073] Table 6

[0074] The PCR reaction program is shown in Table 7.

[0075] Table 7

[0076] The target band is a fragment of approximately 7843 bp. Take 100 μL of the bacterial suspension corresponding to 2-3 positive bands and send it for sequencing. Inoculate the remaining 400 μL of the bacterial suspension into 5-10 mL of LB containing kanamycin-resistant bacteria. Shake the tube and wait for the sequencing results to be released. Extract the plasmid from the tube corresponding to the correct sequence. Store the strain and plasmid at -80°C.

[0077] The specific method of electroporation is as follows: take 1 μL of plasmid and add 50 μL of EHA105 Agrobacterium competent cells, mix thoroughly and then transfer to an electroporation cup. After electroporation, add 1 mL of LB liquid medium, mix thoroughly and then transfer to a 1.5 mL centrifuge tube. Incubate on a shaker at 30°C and 180 rpm for 30 minutes. Take 50 μL of the activated Agrobacterium liquid and inoculate it on LB solid medium. Incubate in the dark at 30°C for 48 hours. Figure 5A .

[0078] Table 8 GmSAUR46b Gene editing sgRNA sequence

[0079] 2) Soybean transformation The genetic transformation process of soybean can be divided into six stages according to its growth and development status: seed germination, co-cultivation, differentiation induction, redifferentiation, elongation, and rooting (Figure 5B). The culture medium formulas used in different stages refer to 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. Plant Biotechnology 2016, 33, (5), 341-350). The transformation method was based on the soybean transformation technology system of Boyuan Biotechnology, as follows: ① Select soybean seeds for surface disinfection, then germinate the seeds under sterile conditions, and obtain uncontaminated cotyledonary node explants after 3-5 days.

[0080] ② Immerse the explant in the inoculum containing recombinant Agrobacterium EHA105 for 2-3 minutes (OD of Agrobacterium solution during infection 600 The explants were stirred occasionally during the culture period and then transferred to a co-culture plate lined with filter paper and co-cultured at 25°C in the dark for 3-5 days.

[0081] ③Afterwards, select explants with good growth and no contamination, cut off the end of the hypocotyl, transfer to a solid culture medium for callus dedifferentiation, culture flatly under a photoperiod of 16 hours light / 8 hours dark for 7-10 days, and subculture in fresh culture medium.

[0082] ④ After embryoids are formed, they are inoculated onto redifferentiation medium, screened and cultured for 21 days under a photoperiod of 16 hours light / 8 hours dark, and subcultured in fresh medium.

[0083] ⑤ After screening, transfer the well-growing embryoids to a fresh elongation medium. Culture them under a 16-hour light / 8-hour dark photoperiod for 21 days and subculture them in fresh medium.

[0084] ⑥ When the young shoots grow to about 5 cm, transfer them to rooting medium for further screening. Under the photoperiod of 16 hours light / 8 hours dark, screen and culture for 21 days to promote the initiation and differentiation of root and shoot meristems.

[0085] ⑦ Use the Bar test paper method to directly identify the presence of Bar / pat protein in T0 generation transgenic plants.

[0086] ⑧ Remove the culture medium of the positive seedlings detected by the Bar test paper, wash the roots of the seedlings, and transplant them into a seedling tray filled with nutrient soil. Under the photoperiod conditions of 27°C, 16 hours of light / 8 hours of darkness, train the seedlings for 3-4 weeks.

[0087] ⑨ The seeds received were of the T1 generation and were planted in nutrient pots filled with nutrient soil at 26°C, 60% humidity, and a photoperiod of 16 hours light / 8 hours dark.

[0088] ⑩ The T1 generation was tested for Cas9 protein and mutations in transgenic plants using the Hi-TOM platform, and the mutant genotypes were verified by Hi-TOM sequencing. The relevant primers are shown in Table 9, and the sequencing verification results are shown in Figure 5C .

[0089] Table 9 GmSAUR46b Gene editing detection primer sequence

[0090] from Figure 5C It can be seen that the strain numbered 14 in the T1 generation (i.e. T1-14 in the figure) underwent a mutation with an 8-base deletion (8repeats means that there is a specific base sequence change pattern repeated 8 times in the mutant, reflecting that after gene editing, the sequence near the target site shows consistent repeated mutation characteristics in multiple copies or multiple plants, indicating the genetic stability of the mutant).

[0091] 1.2.2 Construction of overexpression vector and genetic transformation PCR amplification GmSAUR46b The complete CDS (primers are shown in Table 10) was inserted into the XbaI and SacI sites of the PTF101-35S vector ( Figure 4 ), transformed into Agrobacterium EHA105, and soybean explants were transformed by the Agrobacterium-mediated method. The subsequent culture process was similar to gene editing transformation.

[0092] Table 10 GmSAUR46b Overexpression primers

[0093] The primer sequences used for plaque PCR identification are: 35seq: tTCATTTGGAGAGAACACGGGggac (840 bp) (SEQ ID No.27) NOSseq-R: caagaccggcaacaggattcaatc (1297 bp) (SEQ ID No.28) 1.3 Phenotypic identification At maturity, the natural height of the plant, the number of internodes, and the development time were measured, and the number of pods was counted. The mean and standard deviation of three biological replicates were calculated ( Figure 5D and 5E ).

[0094] Construction via the CRISPR-Cas9 system GmSAUR46b Knockout mutants. Figure 5D Phenotypic analysis showed that the homozygous mutant plants exhibited pleiotropic effects, with shorter plant height and significantly reduced pod production.

[0095] from Figure 5E It can be seen that statistical analysis showed that the T1 generation knockout strain Gmsaur46b The average plant height was 23.1 cm, which was significantly shorter by 34.11% than the wild type WT (35.06 cm). p =5.59E-08).

[0096] from Figure 5E It can be seen that the T1 generation knockout strain Gmsaur46b The number of internodes of the plant was significantly reduced, with an average of 8.5 internodes per plant, while the wild type WT had 9.88 internodes, which was significantly less than the wild type WT ( p =0.0035).

[0097] from Figure 5E It can be seen that the T1 generation knockout strain Gmsaur46b The number of pods produced by the wild-type plants increased significantly, with an average of 30.17 pods per plant, which was a significant increase of 49.19% ( p =0.00028), and Figure 5D .

[0098] from Figure 5E It can be seen that the T1 generation knockout strain Gmsaur46b The flowering time of the WT was significantly advanced, starting at 15.20 days after sowing, while that of the wild type was 21.30 days, which was significantly earlier than that of the wild type ( p =1.32E-06).

[0099] from Figure 6 It can be seen that compared with the wild-type plants (i.e., wild-type WT), the T2 generation knockout lines Gmsaur46bThe plant height was significantly reduced.

[0100] 1.4. Regulation of the thickness of the midrib of soybean leaves The growth conditions were shaded (Photosynthetic Photon Flux Density, PPFD = 149.8 μmol.m -2 .s -1 , simulating shade lighting conditions)( Figure 7A ) and normal light conditions (PPFD=314.5 μmol.m -2 .s -1 , simulating normal lighting conditions) ( Figure 7B ) of wild-type soybean Williams 82 (also known as wild-type WT), knockout strains Gmsaur46b and overexpression lines 35S- GmSAUR46b The third trifoliate leaf of soybean leaves is flattened ( Figure 8 The tissue was fixed by immersion in 50% FAA (50% anhydrous ethanol, 10% 37% formaldehyde solution, 5% acetic acid) and vacuum infiltration three times for 15 minutes each time. After infiltration, the FAA solution was replaced with fresh one and post-fixed at 4°C for at least 12 hours. The fixed samples were dehydrated in different concentrations of ethanol (30%, 50%, 70%, 95%, and 100%) for 1 hour each. After overnight incubation, the tissue was 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, followed by immersion in paraffin. The samples were immersed in xylene and 1 / 4 paraffin for at least 12 hours, then transferred to refined paraffin three times for 3 hours each. After embedding and cleaning, the tissue was placed on a microtome tissue clamp and cut into 7 μm thick slices using a ThermoScientific sliding microtome (Microm HM 340 E), and dried on a 37°C hot plate for 2 hours. After sealing with resin, the information on the imaged tissue slices was scanned with a PANNORAMIC panoramic slice scanner. After opening with CaseViewer2.4 software, it can be magnified at any magnification of 1-400 times for observation. Use CaseViewer2.4 scanning and browsing software to select the target area of ​​the tissue for 100x imaging. When imaging, try to make the tissue fill the entire field of view to ensure that the background light of each photo is consistent. After imaging is completed, use Image-Pro Plus 6.0 analysis software to unify millimeters as the standard unit, select 100 times, and measure the thickness of the center of the midrib of each slice separately. 5 fields of view for each plant are counted. The results of optical microscopy observation are shown in Figure 9A , the statistical results of the midrib center thickness are shown in Figure 9B and 9C .

[0101] 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 leaf midrib center of the WT was increased compared with the wild type. However, under shade treatment, the overexpression line 35S- GmSAUR46b The leaf midrib thickness was still high, while the knockout lines Gmsaur46b The thickness of the leaf midrib center is significantly reduced.

[0102] from Figure 9B and 9C It can be seen that under normal light treatment (CK) conditions, the overexpression line 35S- GmSUR46b The leaf midrib center thickness of the WT was 0.88 mm, which increased by 11.36% ( p =0.081); knockout strains Gmsaur46b The leaf midrib center thickness of the WT was 0.84 mm, which increased by 7.69% compared with the wild type WT ( p =0.284), and there was no significant difference (9B). However, under the shading treatment, the overexpression line 35S- GmSAUR46b The thickness of the leaf midrib reached 0.75 mm, which was significantly increased by 15.38% compared with the wild type WT (0.65 mm). p =0.019), which was significantly reduced by 14.33% compared with the overexpression strain under normal light treatment ( p =0.029)(9C); while the knockout lines Gmsaur46b There was no significant difference in the shade treatment (0.545 mm) compared with the wild type WT, but it was significantly reduced by 35.71% ( P =0.0063).

[0103] Comprehensive description, GmSAUR46b Specifically regulates the thickness of the midrib of soybean leaves. Shading treatment further enhances GmSAUR46b Regulatory effect on midrib thickness.

[0104] 1.5. Regulation of soybean stem trichome density Sampling and fixation: Take the samples grown under shade conditions (PPFD = 149.8 μmol.m -2 .s -1 , simulating shade lighting conditions)( Figure 7A ) and normal light conditions (PPFD=314.5 μmol.m -2 .s -1 , simulating normal lighting conditions) ( Figure 7B) of the wild type soybean Williams 82, the third segment of the main stem during the flattening period of the third trifoliate leaf ( Figure 8 Minimize mechanical damage such as pulling, contusion, and squeezing. Samples should be removed within 1-3 minutes and gently rinsed with PBS to remove surface impurities. Protect the surface to be scanned and mark it (e.g., cut corners on the opposite side). Immediately place the sample in electron microscopy fixative at room temperature for 2 hours, then transfer to 4°C for storage. Fixed samples should be rinsed three times in 0.1 M phosphate buffer (PB) (pH 7.4) for 15 minutes each. Fix with 1% osmium phosphate prepared in 0.1 M phosphate buffer (PB) (pH 7.4) for 1-2 hours at room temperature in the dark. Rinse three times in 0.1 M phosphate buffer (PB) (pH 7.4) for 15 minutes each. Dehydrate the tissue by sequentially placing it in 30%-50%-70%-80%-90%-95%-100%-100% alcohol for 15 minutes each, followed by isoamyl acetate for 15 minutes. Drying: Samples were dried in a critical point dryer and mounted on a SEM stage. After 30 seconds of gold spraying, the distribution of stem trichomes was observed under a scanning electron microscope (SEM). Density analysis was performed, counting the number of trichomes per 3 mm of stem length (n = 5). The results of the SEM analysis are shown in Figures 10A and 10B, and the results of the density analysis are shown in Figures 10C and 10D.

[0105] from Figure 10A and 10B It can be seen that under normal light treatment (CK) conditions, the knockout lines showed a significant difference compared to the wild type WT. Gmsaur46b and overexpression lines 35S- GmSAUR46b However, under shade treatment, the trichome density of the overexpressing strain 35S- GmSAUR46b Still maintain a high trichome density, while the knockout lines Gmsaur46b The trichome density was significantly reduced.

[0106] 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 significant increase of 15.46% compared with the wild type WT (251.6). p =2.78E-06); knockout strains Gmsaur46b The number of trichomes per 3 mm stem length was 361, which was significantly increased by 36.23% compared with the wild type WT ( p =1.72E-07). However, under shade treatment, the overexpression line 35S- GmSAUR46bThe trichome density was still high, with 150.9 trichomes per 3 mm stem length, which was a significant increase of 231.03% compared with 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 significantly reduced by 37.54% compared with the wild type WT ( p =9.31E-08).

[0107] The present invention is not limited by the above specific description. Various changes can be made to the present invention within the scope outlined by the claims, and these changes are all within the scope of the present invention.

Claims

1. Use of the GmSAUR46b gene or protein or biological materials related thereto in any of the following: A1) Regulating soybean yield or preparing products that regulate soybean yield; A2) regulating soybean light stress resistance or preparing a product for regulating soybean light stress resistance; A3) Regulating soybean plant height or preparing products for regulating soybean plant height; A4) Breeding soybeans with increased or decreased yield; A5) Breeding soybeans with improved light stress resistance; A6) Breeding soybeans with increased or decreased plant height.

2. The use according to claim 1, characterized in that described GmSAUR46b The nucleotide sequence of the gene comprises the sequence shown in SEQ ID No. 1; and / or, G The amino acid sequence of the mSAUR46b protein comprises the sequence shown in SEQ ID No. 2; and / or, overexpressing the GmSAUR46b gene or protein to increase soybean plant height; And / or, knocking out the GmSAUR46b gene or protein increases soybean yield, improves light stress resistance, and reduces soybean plant height.

3. The use according to claim 2, characterized in that The plant height also includes the number of internodes; and / or, the increased yield refers to an increase in the number of pods; And / or, the improving the light stress resistance of soybean refers to improving the light stress resistance of soybean by increasing the thickness of the center of the leaf midrib and / or the density and number of trichomes on the stem; And / or, the biological material is B1) comprising GmSAUR46b A nucleic acid construct of the gene or a nucleic acid construct expressing the GmSAUR46b protein; and / or, B2) a genetically engineered bacterium comprising the nucleic acid construct of B1).

4. A method for cultivating soybeans with reduced plant height and / or increased yield and / or improved light stress resistance, characterized in that: include: The content and / or activity of the GmSAUR46b protein in the recipient plant is reduced to obtain soybeans with reduced plant height and / or increased yield and / or increased light stress resistance.

5. A method for reducing soybean plant height and / or increasing soybean yield and / or improving soybean light stress resistance, characterized in that: include: Reducing the content and / or activity of GmSAUR46b protein in recipient plants.

6. The method according to claim 4 or 5, characterized in that 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 gene encoding the GmSAUR46b protein into the recipient plant, wherein the substance that interferes with the expression of the gene encoding the GmSAUR46b protein comprises the sequence shown in SEQ ID No.3 or SEQ ID No.

4.

7. A method for cultivating soybeans with increased plant height, characterized in that: include: The content and / or activity of the GmSAUR46b protein in the recipient plant is increased to obtain soybeans with increased plant height.

8. A method for increasing soybean plant height, characterized in that: include: Increase the content and / or activity of GmSAUR46b protein in the recipient plant.

9. The method according to claim 7 or 8, characterized in that The method for increasing the content and / or activity of the GmSAUR46b protein in the recipient plant is to overexpress the GmSAUR46b protein in the recipient plant.

10. The method according to claim 9, characterized in that: The overexpression method is to introduce the coding gene of GmSAUR46b protein into the recipient plant.

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