Soybean dominant negative effect allele gmspp-d and application thereof

Abstract

This invention discloses the soybean dominant negative-effect allele Gmspp-D and its application. Two dominant negative-effect alleles Gmspp-D (Gmspp-D-CDS-1 and Gmspp-D-CDS-2) regulating soybean pubescence density were cloned and overexpression vectors were constructed. Stable overexpression lines were obtained by transforming these vectors into the soybean cultivar Williams82 (W82). Compared to the control material W82, overexpression of both dominant negative-effect alleles significantly reduced soybean pubescence density, thus validating the function of the target genes. This invention provides the application of the soybean dominant negative-effect allele Gmspp-D in regulating plant pubescence density and demonstrates the feasibility and efficiency of constructing dominant negative-effect mutants for rapid gene function studies, showing promising application prospects in the field of breeding technology.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biotechnology, and in particular to the soybean Gmspp-D (Glycine maxsparse pubescence-Dominant) allele and its applications. Background Technology

[0002] Soybeans originated in my country and are an important food and economic crop, providing humans with approximately 60% of their plant protein and 30% of their fat, possessing high nutritional value. With the decreasing soybean planting area and increasing human demand, my country has transformed from a soybean exporter to its largest importer. Therefore, improving the yield and quality of soybeans in my country is urgently needed. Hairs, as a barrier against external biotic and abiotic stresses, play a crucial role in plant stress resistance, and hair traits are closely related to soybean yield.

[0003] When breeding superior soybean varieties, identifying regulatory genes for important traits is crucial. CRISPR / Cas9 gene editing is a common method for gene function verification, which usually requires screening homozygous lines. Given the long growth period of soybeans, this process is extremely time-consuming and labor-intensive. Furthermore, as an ancient tetraploid, soybean exhibits functional redundancy in many genes, posing significant challenges to the verification of soybean functional genes.

[0004] Dominant-negative effect refers to the phenomenon where mutant proteins disrupt the function of wild-type proteins, which is particularly common in transcription factors. Soybean Glyma.07G228600 encodes an R2R3-MYB transcription factor, a regulatory gene for soybean sparse pubescence mutants. This invention constructed an overexpression vector for the dominant-negative effect allele Gmspp-D (Glycine max sparsepubescence-Dominant) of Glyma.07G228600, expressed in soybean material W82 with normal pubescence, obtaining transgenic soybean plants with reduced pubescence density. Furthermore, only 60 days elapsed from the start of soybean tissue culture to the first observation of a positive phenotype. This demonstrates that constructing dominant-negative effect mutants for gene function verification is an efficient, rapid, and feasible method with significant application value in soybean breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a soybean dominant negative effect allele Gmspp-D and its application in gene function research by obtaining dominant negative effect mutants through overexpression of this gene.

[0006] To achieve the objectives of this invention, we first obtained two dominant-negative alleles, named Gmspp-D-CDS-1 and Gmspp-D-CDS-2, by amplifying the Glyma.07G228600 mutant of soybean with sparse pubescence, ligating it into a TA cloning vector, and performing sequencing analysis. We then constructed overexpression vectors for these two alleles and overexpressed them in W82 soybeans using Agrobacterium-mediated soybean genetic transformation, obtaining transgenic soybean lines with sparse pubescence and achieving gene function verification in a relatively short time.

[0007] This invention provides a soybean Gmspp-D-CDS-1 gene, the nucleotide sequence of which is any one of the following (1)-(4):

[0008] (1) The nucleotide sequence as shown in SEQ ID NO.1;

[0009] (2) A nucleotide sequence with equivalent function formed by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1;

[0010] (3) The nucleotide sequence hybridized with SEQ ID NO.1 under strict hybridization conditions;

[0011] (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence described in (1) and has the same function.

[0012] The protein encoded by the soybean Gmspp-D-CDS-1 gene, as described above, also falls within the scope of protection of this invention. The amino acid sequence of this protein is as follows (a) or (b):

[0013] (a) The amino acid sequence as shown in SEQ ID NO.2;

[0014] (b) A sequence with equivalent function formed by substituting, deleting or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO.2.

[0015] This invention provides a soybean Gmspp-D-CDS-2 gene, the nucleotide sequence of which is any one of the following (1)-(4):

[0016] (1) The nucleotide sequence shown in SEQ ID NO.3;

[0017] (2) A nucleotide sequence with equivalent function formed by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.3;

[0018] (3) The nucleotide sequence hybridized with SEQ ID NO.3 under strict hybridization conditions;

[0019] (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence described in (1) and has the same function.

[0020] The protein encoded by the soybean Gmspp-D-CDS-2 gene, as described above, also falls within the scope of protection of this invention. The amino acid sequence of this protein is as follows (a) or (b):

[0021] (a) The amino acid sequence as shown in SEQ ID NO.4;

[0022] (b) A sequence with equivalent function formed by substituting, deleting or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO.4.

[0023] Biological materials containing the soybean Gmspp-D-CDS-1 gene or the soybean Gmspp-D-CDS-2 gene described above are also within the scope of protection of this invention. Such biological materials are at least one of expression vectors, cell lines, and host bacteria.

[0024] This invention also provides the application of the aforementioned soybean Gmspp-D-CDS-1 gene, the aforementioned soybean Gmspp-D-CDS-2 gene, the aforementioned protein, or the aforementioned biological material in regulating plant pubescence density. The regulation of plant pubescence density refers to increasing or decreasing pubescence density. Preferably, overexpression of the aforementioned soybean Gmspp-D-CDS-1 gene or the aforementioned soybean Gmspp-D-CDS-2 gene in plants can reduce plant pubescence density.

[0025] The present invention also provides the use of the above-mentioned soybean Gmspp-D-CDS-1 gene, the above-mentioned soybean Gmspp-D-CDS-2 gene, the above-mentioned protein, or the above-mentioned biological material in any one of the following (1) to (3):

[0026] (1) Prepare dominant-negative effect mutants for gene function research;

[0027] (2) Preparation of transgenic plants with reduced pubescence density;

[0028] (3) Plant molecular design breeding or plant germplasm resource improvement.

[0029] The present invention also provides a method for preparing transgenic plants with reduced pubescence density by overexpressing the above-mentioned soybean Gmspp-D-CDS-1 gene or the above-mentioned soybean Gmspp-D-CDS-2 gene in transgenic plant cells.

[0030] The plants mentioned are monocotyledonous or dicotyledonous plants.

[0031] Overexpression of the Gmspp-D gene can be achieved using various methods, such as optimizing the gene promoter to achieve overexpression, gene overexpression mediated by plant virus vectors, and Agrobacterium-mediated transformation. The method for overexpressing the gene in this invention is not limited to the above methods; any method that can overexpress the Gmspp-D gene is acceptable.

[0032] In constructing the plant expression vector for the Gmspp-D gene, any enhancing or inducible promoter can be used before the transcription initiation nucleotide of Gmspp-D. To facilitate the identification and screening of transgenic plants, the vector can be modified, such as by adding plant-selective markers or antibiotic resistance markers. The former includes the GUS gene and luciferase gene, while the latter includes gentamicin and kanamycin. The transformed plant recipient can be either monocotyledonous or dicotyledonous, including but not limited to soybean, Arabidopsis thaliana, tobacco, maize, rice, wheat, cucumber, tomato, and poplar. The expression vector carrying the Gmspp-D gene of this invention can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0033] The room temperature described in this invention is generally 25±5℃.

[0034] The beneficial effects of this invention are:

[0035] This invention cloned two dominant-negative-effect alleles, Gmspp-D-CDS-1 and Gmspp-D-CDS-2, of the soybean pubescence density regulator. Overexpression of either of these two alleles resulted in a noticeable sparse pubescence-positive phenotype observed in the T0 generation, with the fastest result being only 60 days from the start of tissue culture. Therefore, this invention, using Gmspp-D as an example, provides a solid basis for the feasibility and efficiency of constructing dominant-negative-effect mutants for rapid gene function studies, and shows promising application prospects in the field of plant molecular design breeding. Attached Figure Description

[0036] Figure 1 Analysis of PCR amplification products of Gmspp-D;

[0037] A shows the agarose gel electrophoresis result of the CDS amplification product of Gmspp-D; B shows the sequencing peaks of the CDS amplification product of Gmspp-D. The top and bottom figures represent sequencing from the F and R directions, respectively.

[0038] Figure 2: Sparse pubescence positive phenotype of soybean seedlings during the shoot elongation stage on day 60 of soybean tissue culture.

[0039] Figure 3 Phenotypes of T0 generation soybean plants, leaves, and pods overexpressing Gmspp-D.

[0040] Figure 4 Analysis of Gmspp-D expression levels in homozygous overexpression lines of Gmspp-D-CDS-1(A) and Gmspp-D-CDS-2(B).

[0041] Figure 5 Phenotypic identification of homozygous lines overexpressing Gmspp-D-CDS-1(A) and Gmspp-D-CDS-2(B). Scale bar, 1 mm. Detailed Implementation

[0042] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0043] Unless otherwise specified, all experimental materials, reagents and instruments used in the embodiments of the present invention are commercially available. Unless otherwise specified, all technical means used in the embodiments are conventional means well known to those skilled in the art.

[0044] Example 1: Cloning of the soybean Gmspp-D gene

[0045] A pair of near-isogenic lines were constructed using a local soybean variety from Henan Province, “Dahuapi (DHP)”. Specifically, the normal pubescence stable selection line NJD-4 was crossed with the sparse pubescence mutant material DHP. In the F2 generation, pubescence density phenotype segregation occurred. Single plants with sparse pubescence were selected from the segregating rows as candidate residual heterozygous lines. After continuous self-pollination and selection, the F9 generation segregating lines were finally obtained as a pair of near-isogenic lines. The homology between the normal and mutant plants was 99.8%. The normal pubescence density and sparse pubescence phenotype materials were named NIL-WT and NIL-MT, respectively. The Glyma.07G228600(Gmspp-D) transcript was amplified in NIL-MT using a pair of primers located at the 5'UTR and 3'UTR. The specific primer sequences were: F, TGTTCTCTGCACCAAACCCT; R, ACACTCCCAAGAAAACGGGT. Agarose gel electrophoresis analysis of the PCR products revealed that the Gmspp-D transcript was approximately 800 bp in length. Figure 1 A) Sequencing analysis revealed that the PCR product of Gmspp-D consisted of random peaks. Figure 1B) It is speculated that it contains multiple transcripts. Multiple transcripts of Gmspp-D were isolated using a TA vector. 150 single clones were selected for culture and sequencing identification, revealing four different CDS types. The most frequent and least frequent CDS types were named Gmspp-D-CDS-1 (nucleotide sequence shown in SEQ ID NO.1, encoding the amino acid sequence shown in SEQ ID NO.2) and Gmspp-D-CDS-2 (nucleotide sequence shown in SEQ ID NO.3, encoding the amino acid sequence shown in SEQ ID NO.4), respectively. Compared to wild-type Glyma.07G228600, both Gmspp-D-CDS-1 and Gmspp-D-CDS-2 lacked the C-terminal sequence, representing a dominant-negative effect allele of Glyma.07G228600.

[0046] Example 2: Obtaining overexpression lines of the soybean dominant negative-effect allele Gmspp-D

[0047] 1. Plasmid extraction of Gmspp-D-CDS-1 and Gmspp-D-CDS-2

[0048] Plasmids were extracted from the bacterial cultures that yielded single-clone sequencing results of Gmspp-D-CDS-1 and Gmspp-D-CDS-2 using the AxyPrep Plasmid DNA Mini Kit, following the instructions.

[0049] 1) Take 2-5 mL of overnight culture, centrifuge at 12000 rpm for 1 min, and discard the supernatant;

[0050] 2) Add 250 μL of Buffer S1 solution to evenly suspend the precipitate at the bottom of the tube, ensuring no bacterial clumps remain;

[0051] 3) Add 250 μL of Buffer S2 solution and gently and thoroughly invert the container 4-6 times to fully lyse the cells until a clear solution is formed;

[0052] 4) Add 350 μL of Buffer S3, gently and thoroughly invert 6-8 times, then centrifuge at 12000 rpm for 10 min;

[0053] 5) Transfer the supernatant to a preparation tube, place it in a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0054] 6) Place the preparation tube back into the 2mL centrifuge tube, add 500μL of Buffer W1 solution to the preparation tube, centrifuge at 12000rpm for 1min, and discard the filtrate;

[0055] 7) Place the preparation tube back into a 2mL centrifuge tube, add 700μL of Buffer W2 solution, centrifuge at 12000rpm for 1min, and discard the filtrate. Repeat the operation once.

[0056] 8) Place the preparation tube back into the 2mL centrifuge tube and centrifuge at 12000rpm for 1min;

[0057] 9) Transfer the preparation tube to a new 1.5 mL centrifuge tube, add 60-80 μL of ddH2O to the center of the membrane in the preparation tube, let it stand at room temperature for 1 min, and then centrifuge at 12000 rpm for 1 min.

[0058] 2. Construction of overexpression vectors for Gmspp-D-CDS-1 and Gmspp-D-CDS-2

[0059] 1) Using plasmids Gmspp-D-CDS-1 and Gmspp-D-CDS-2 as templates, PCR amplification was performed using primers with the p0641 vector homologous arm added. The specific primer sequence information is as follows:

[0060] Primer-F: GGAGAGCCACCATGCTCGAGATGGGTAGGTCACCATGCTG;

[0061] Primer-R: CCACTAGTCCCGGGCTCGAGTTGTTCACGTTGCACGGTGG.

[0062] PCR reaction system:

[0063]

[0064] PCR reaction procedure:

[0065]

[0066] 2) The above PCR products were ligated into the p0641 vector using homologous recombination. The p0641 vector is also known as pFGC5941. According to the patent published by Liu Bin et al. (2022), recombinant plasmids named p0641-Gmspp-D-CDS-1 and p0641-Gmspp-D-CDS-2 were constructed. The recombinant plasmids were transformed into competent E. coli DH5α cells. The primer sequences on the vector were used to detect positive clones, and bacterial solutions with the correct band size were sent to the company for sequencing.

[0067] The recombinant plasmid with correct sequencing was introduced into Agrobacterium strain EHA105 using the freeze-thaw method. The strain was screened using 50 μg / mL kanamycin and 50 μg / mL rifampin. The bacterial suspension with the correct size as determined by PCR was mixed with 30% glycerol at a 1:1 (v:v) ratio and stored at -80°C.

[0068] 3. Obtaining transgenic positive plants

[0069] Agrobacterium-mediated soybean genetic transformation was used to obtain Gmspp-D-CDS-1 and Gmspp-D-CDS-2 overexpressing positive plants. The soybean genetic transformation procedure followed the method of Li et al. (2017), specifically as follows:

[0070] 1) Preparation of the inoculation solution

[0071] Mix Agrobacterium bacterial suspension with YEP liquid medium containing kanamycin and rifampin at a ratio of 1:1000, incubate overnight until OD600 reaches 0.6-0.8, centrifuge, add an appropriate amount of CCM liquid medium to the bacterial cells to dissolve the bacterial cells in the CCM medium, adjust OD600 to 0.6-0.8, and let stand at room temperature for 30 minutes.

[0072] 2) Seed selection and disinfection

[0073] Select W82 soybean seeds with undamaged, wrinkled, smooth surfaces and no disease spots. Place them in a glass petri dish and put it in a desiccator. Add 100 mL of sodium hypochlorite solution to the conical flask in the center of the desiccator. Add 15 mL of concentrated hydrochloric acid to the separatory funnel of the desiccator, allowing the concentrated hydrochloric acid to slowly drip into the conical flask. The reaction produces chlorine gas, which sterilizes the seed surface for about 2 hours.

[0074] 3) Seed germination

[0075] Soybean seeds were planted with the hilum facing down in GM germination medium and kept in the dark at 25°C overnight to allow the seeds to fully absorb moisture.

[0076] 4) Preparation and infection of explants

[0077] Soybean seeds were placed in a petri dish lid. Using a scalpel, the cotyledons and radicle of the soybean were separated in half along the hilum from the radicle, thus creating two explants. Two-thirds of the radicle of each explant was removed and the plumule was removed. The explants were then immersed in a petri dish containing bacterial solution and gently shaken on a shaker for 30 minutes.

[0078] 5) Co-culture of explants and Agrobacterium

[0079] Remove the seed coat of the infected explant with tweezers, then place the explant with the adaxial surface facing up on CCM solid medium and incubate in the dark at 25°C for 5 days.

[0080] 6) Induction of clustered buds

[0081] The elongated radicle is removed with a scalpel, leaving approximately 2-3 mm. The explants are then immersed in sterile ultrapure water for 30 minutes, gently shaking the container every 5-10 minutes, for a total of two rinses. Next, the explants are rinsed twice with SI liquid medium using the same method. After drying on filter paper, the explants are inserted into SI solid medium with the radicle facing down and the adaxial surface facing up. The explants are then cultured for two weeks in a tissue culture room at 25°C, under 16 hours of light and 8 hours of darkness.

[0082] 7) Elongation culture of clustered buds

[0083] Cut off half of the cotyledons of the explant and remove any brown, dried debris from the surface. Remove any robust buds. After removing a small piece of tissue from the back of the explant, insert it obliquely into bud elongation medium (SE) with the cut side facing down. Culture in a tissue culture room for 2 weeks. After 2 weeks, remove all cotyledons and gently scrape away any blackened, dead buds. Make an incision on the back of the explant with a scalpel and insert it obliquely into fresh SE solid medium with the cut side facing down. Culture in a tissue culture room. Replace the SE solid medium every 2 weeks until the clustered buds stop elongating.

[0084] 8) Rooting culture of clustered buds

[0085] When the elongated bud reaches about 4cm in length, cut it off with scissors in a clean bench. Dip the wound in 1mg / mL IBA solution and transfer it to rooting medium (RM). After about 2 weeks of culture in a tissue culture room, a taproot will grow from the bottom of the bud. Once the taproot and lateral roots have grown, it can be transplanted into the soil.

[0086] 9) Transplanting and hardening off seedlings

[0087] Remove the rooted tissue culture seedlings from the culture medium, wash away any remaining culture medium from the roots, and transfer them to disposable plastic cups. Invert a similarly sized disposable plastic cup over the seedling and secure it to maintain humidity inside the cup. Once roots are visible on the outer wall of the cup, slightly open the top of the cup to allow the seedling to gradually acclimate to its environment. Remove the plastic cup after 3 days. Seedlings identified as positive are then transferred to flowerpots and placed in a greenhouse for further cultivation.

[0088] The culture media involved in Examples 1 and 2, such as YEP liquid medium, GM germination medium, CCM liquid medium, CCM solid medium, SI liquid medium, SI solid medium, SE solid medium, and RM rooting medium, are all well known to those skilled in the art.

[0089] Example 3: Identification of transgenic positive lines

[0090] The soybean overexpression vector used in this invention contains the glufosinate resistance gene bar. Therefore, three methods are used to detect positive seedlings: glufosinate (basta, active ingredient PPT) application, bar test strip detection, and PCR amplification of the target fragment.

[0091] Glufosinate application: Mark one side of a newly emerged, fully unfolded leaf with a marker to indicate no treatment. Apply a 250 mg / L Basta solution to the other side of the leaf with a brush. Observe the leaf growth after about 4 days. If the half of the leaf treated with Basta turns yellow and dries out, the seedling is a false positive. If both sides of the leaf grow equally, the seedling is a positive seedling.

[0092] Bar test strip testing: The bar gene detection kit manufactured by EnviroLogix was used, and the procedure was performed according to the instructions.

[0093] PCR amplification of the target fragment: DNA was extracted from the transgenic plants and PCR amplification was performed using bar gene-specific primers. The results were detected by 1% agarose gel electrophoresis. Plants with the target size band were considered positive, otherwise they were considered wild-type negative plants.

[0094] Example 4: Analysis of Gmspp-D expression levels in transgenic positive lines

[0095] RNA was collected from the shoot tip meristems of transgenic lines and control materials at soybean V3 stage and converted into cDNA. The cDNA was diluted 10-fold and used as a template for quantitative real-time PCR detection. The expression level of Glyma.07G228600 in control material W82 was set as the control (value = 1), and the soybean actin encoding gene GmActin was used as an internal reference. Each sample was subjected to three biological replicates, and the relative expression level was calculated using a 22 ratio. -ΔΔCt The calculation method was used. Statistical analysis was performed using the t-test. Real-time quantitative PCR results showed that the expression levels of the target gene in the four overexpression lines were 10.81-fold, 16.05-fold, 6.27-fold, and 12.51-fold higher than the control, respectively, with all differences reaching a highly significant level. Figure 2 ).

[0096] Example 5: Identification of the trichome phenotype in transgenic plants

[0097] In Example 2, during the elongation of the shoot clusters in the tissue culture, specifically on day 60 from the start of the tissue culture, a sparse pubescent phenotype could be observed. Figure 3 Furthermore, the phenotype was stable, and multiple T0 generation seedlings obtained all exhibited a positive phenotype. Figure 4Phenotypic identification was performed on the obtained T2 generation homozygous overexpression lines Gmspp-D-CDS-1-OE-51, Gmspp-D-CDS-1-OE-70, Gmspp-D-CDS-2-OE-6, and Gmspp-D-CDS-2-OE-39. The results showed that overexpression of both CDSs yielded a phenotype consistent with NIL-MT, and the trichome density of the overexpressing lines was significantly reduced compared to the control material W82. Figure 5 ).

[0098] This invention, using molecular biology and genetic engineering techniques, demonstrates for the first time that the dominant-negative allele Gmspp-D in soybean Glyma.07G228600 negatively regulates soybean pubescence density. It also proves that constructing dominant-negative mutants for gene function studies is feasible and offers advantages such as high efficiency, speed, and avoidance of invalid mutations caused by functional redundancy. This provides a comprehensive approach to molecular design breeding and has significant practical implications for crop variety improvement.

[0099] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0100] SEQ ID NO.1

[0101] ATGGGTAGGTCACCATGCTGTGAAAAAGAGGGCTTGAAGAAAGGGCCCGTGGACTCCA

[0102] GAGGAAGACCAAAAGCTCATGGCGTACATTGAAGAGTTTGGCCACGGAAGCTGGCGT

[0103] GCTTTGCCTGCCAAAGCTGGACTTCAAAGATGTGGGAAGAGCTGTAGGCTAAGGTGGA

[0104] CTAACTACCTCCGACCAGACATAAAAGAGGAAAGTTCAGCTTGCAGGAGGAGCAAA

[0105] CAATCATTCAACTCCATGCCCTTCTTGGGAACAGATGGTCAGCCATAGCAGCTCAACTT

[0106] CCCAAGAGAACCGATAATGAAATCAAGAACTACTGGAACACACACCTGAAGAAGAGG

[0107] CTAACCAGAATGGGGATAGACCCCACCACCCACAAGCCGAAAACCGACGCACTCGGC

[0108] GGCTCCGGTGGTGGCCAAACCAGGTTCGCCGCCACCGTGCAACGTGAACAATAASEQ ID NO.2

[0109] MGRSPCCEKEGLKKGPWTPEEDQKLMAYIEEFGHGSWRALPAKAGLQRCGKSCRLRWTN

[0110] YLRPDIKRGKFSLQEEQTIIQLHALLGNRWSAIAAQLPKRTDNEIKNYWNTHLKKRLTRMGIDPTTHKPKTDALGGSGGGQTRFAATVQREQ*

[0111] SEQ ID NO.3

[0112] ATGGGTAGGTCACCATGCTGTGAAAAAGAGGGCTTGAAGAAAGGGCCGTGGACTCCAG

[0113] AGGAAGACCAAAAGCTCATGGCGTACATTGAAGAGTTTGGCCACGGAAGCTGGCGTGC

[0114] TTTGCCTGCCAAAGCTGGACTTCAAAGATGTGGGAAGAGCTGTAGGCTAAGGTGGACTA

[0115] ACTACCTCCGACCAGACATAAAAAGAGGAAAGTTCAGCTTGCAGGAGGAGCAAACAAT

[0116] CATTCAACTCCATGCCCTTCTTGGGAACAGATGGTCAGCCATAGCAGCTCAACTTCCCAA

[0117] GAGAACCGATAATGAAATCAAGAACTACTGGAACACACACCTGAAGAAGAGGCTAACC

[0118] AGAATGAGTTCGCCGCCACCGTGCAACGTGAACAATAA

[0119] SEQ ID NO.4

[0120] MGRSPCCEKEGLKKGPWTPEEDQKLMAYIEEFGHGSWRALPAKAGLQRCGKSCRLRWTN

[0121] YLRPDIKRGKFSLQEEQTIIQLHALLGNRWSAIAAQLPKRTDNEIKNYWNTHLKKRLTRMRFAATVQREQ*

[0122] References

[0123] Liu Bin, Xu Zhiyong, Zhao Tuanjie, Kong Keke, Li Hongyu, Zhao Tao, et al. Soybean CS1 gene and its application, 2022.05.03, CN 112646017. (Note: The last part, "Li S, Cong Y, Liu Y, Wang T, Shuai Q, Chen N, et al. (2017). Optimization of Agrobacterium-Mediated Transformation in Soybean. Frontiers in Plant Science 8. doi:10.3389 / fpls.2017.00246," appears to be a typo and can be left as is.)

Claims

1. A soybean Gmspp-D-CDS-1 gene, characterized in that, The nucleotide sequence of the soybean Gmspp-D-CDS-1 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the soybean Gmspp-D-CDS-1 gene as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A soybean Gmspp-D-CDS-2 gene, characterized in that, The nucleotide sequence of the soybean Gmspp-D-CDS-2 gene is shown in SEQ ID NO.

3.

4. The protein encoded by the soybean Gmspp-D-CDS-2 gene as described in claim 3, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

4.

5. A biomaterial containing the soybean Gmspp-D-CDS-1 gene of claim 1 or the soybean Gmspp-D-CDS-2 gene of claim 3, characterized in that, The biomaterial is at least one of an expression vector, a cell line, and a host bacterium.

6. The application of the soybean Gmspp-D-CDS-1 gene of claim 1, the soybean Gmspp-D-CDS-2 gene of claim 3, the protein of claim 2 or 4, or the biomaterial of claim 5 in regulating soybean pubescence density, characterized in that, The regulation of soybean pubescence density is to reduce pubescence density.

7. The application according to claim 6, characterized in that, Overexpression of the soybean Gmspp-D-CDS-1 gene of claim 1 or the soybean Gmspp-D-CDS-2 gene of claim 3 in soybeans can reduce soybean pubescence density.

8. The use of the soybean Gmspp-D-CDS-1 gene of claim 1, the soybean Gmspp-D-CDS-2 gene of claim 3, the protein of claim 2 or 4, or the biomaterial of claim 5 in the preparation of transgenic soybeans with reduced pubescence density.

9. A method for preparing transgenic soybeans with reduced pubescence density, characterized in that, Overexpression of the Gmspp-D-CDS-1 gene as described in claim 1 or the Gmspp-D-CDS-2 gene as described in claim 3 in transgenic soybean cells.

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