Gmsgt2 gene and mutant and application thereof related to plant height and branch development

Abstract

This invention belongs to the field of biotechnology, specifically relating to the GmSGT2 gene and its mutants related to plant height and branching development, and their applications. Specifically, this invention obtains a soybean mutant with fewer branches and reduced plant height through EMS mutagenesis, and locates the target gene as the GmSGT2 gene of soybean variety 'He Dou 12' using map-based cloning technology. Searching reveals that this gene encodes soybean saponin B glucuronide galactosyltransferase, which can galactosylate soybean saponin B monoglucuronide, thus affecting soybean plant height and reducing branching. Therefore, the gmsgt2 mutant can be used to breed dwarf, high-yielding soybean varieties, which is of profound significance in breeding ideal soybean plant architecture and research on important agronomic traits. It has broad application prospects and high research value for understanding the regulation mechanism of soybean plant height and branching, improving the soybean breeding process, and can provide excellent germplasm resources for regulating soybean dense planting and high-yield breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the GmSGT2 gene and its mutants and applications, which are related to plant height and branching development. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Soybeans (Glycine max) originated in China and are an important dual-purpose crop for grain, oil, and feed in my country and the world, supplying more than half of the global oilseed production and nearly a quarter of the plant protein. Currently, China is the world's largest importer of soybeans, with a high dependence on imports. Increasing my country's soybean production capacity has become an urgent and crucial task to ensure national food security. However, in recent years, with the advancement of industrialization and urbanization, my country's arable land area has been decreasing year by year, while the planting area of ​​crops such as rice and corn has continued to increase due to market influences. This has led to a continuous decline in soybean planting area, resulting in a supply shortage of domestically produced soybeans and a rapid increase in imported soybeans. Therefore, relying on expanding planting area to increase total soybean production is no longer realistic. The only way is to rely on technological means to cultivate more new soybean varieties with ideal plant types, high quality, and high yield to meet the increasing demand. This is also the main way to improve the overall competitiveness of my country's soybean industry.

[0004] Plant height and branch number are both important agronomic traits of crops and are closely related to crop yield. Shorter plants enhance lodging resistance, and fewer branches allow for better planting density and increased light energy utilization, thus increasing crop yield. Therefore, plant height and branch number are key factors determining soybean plant architecture and directly affecting soybean yield. Identifying key genes for soybean plant height and branch number and elucidating their regulatory mechanisms are of great significance for developing high-yielding soybean varieties using molecular design breeding.

[0005] In recent years, research on the regulatory mechanisms of plant height and branch number has gradually increased. A series of key genes regulating plant height and branch number have been isolated from model plants Arabidopsis thaliana and rice (Oryza sativa), establishing a molecular regulatory network. The most extensive research on plant height focuses on rice dwarf mutants. Parnell et al. first reported dwarf mutants generated under natural variation conditions, followed by Oryoji et al. who first reported dwarf mutants generated by artificial mutagenesis and conducted extensive genetic studies on them. The emergence of these dwarf mutants has provided abundant resources for rice breeding. Plant dwarfing has multiple causes. In 1981, Kamijma et al., through research on near-isogenic rice lines, discovered that dwarfing genes affect the size of plant embryonic organs and cell morphology, suggesting that plant dwarfing may be caused by a reduction in the number of internode cells. Plant hormones play a crucial role in plant growth and development. Studies of numerous height and branching mutants in rice and Arabidopsis thaliana have revealed that the biosynthesis, transport, and signal transduction of plant hormones such as auxin, gibberellin, and brassinolide are significant in regulating plant height and branch number. In 2011, Burkhard Schulza et al. found that NA1, a homolog of Arabidopsis DE-ETIOLATED2 (DET2), is a gene in the BR biosynthesis pathway in the maize dwarf mutant nana plant1 (na1). In 2017, Xu Yunyuan et al. reported that overexpression of the OsMIR396d gene in rice leads to a semi-dwarf phenotype and increased leaf angle, a typical BR-enhancing mutant phenotype. The OsmiR396d gene regulates GA biosynthesis and signal transduction, as well as the BR response, by modulating the expression of different downstream target genes, thereby affecting plant architecture development. Hsieh et al. found that applying exogenous GA3 could restore the dwarf phenotype in tomato plants transgenic with the AtCBF1 / DREB1A gene. In 2017, Li Laigeng et al. discovered a gene SBI encoding GA2 oxidase in rice and proved that this gene caused the dwarf phenotype in rice. In 2022, Tian Zhixi and Kong Fanjiang's research group conducted phenotypic identification of branch number in more than 2,400 soybean natural germplasm resources over two consecutive years. Using genome-wide association analysis, they identified the major controlling gene Dt2 for soybean branch number. Genetic analysis revealed that Dt2 negatively regulates soybean branch number. CRISPR / Cas9 gene knockout lines showed a significant increase in branch number and a significant increase in plot yield; overexpression lines showed a significant decrease in branch number and a significant decrease in plot yield. This study found that the Dt2 gene is a key gene controlling soybean branch number in natural soybean populations and elucidated the molecular mechanism by which Dt2 regulates soybean branching, providing an important theoretical basis for molecular design breeding of high-yield soybeans by regulating branching.In 2023, the team led by Liu Bin and Qiu Lijuan at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, located the key gene PH13 that regulates soybean plant height, revealing the important role and molecular mechanism of its superior haplotype in variety breeding in high-latitude regions. This study confirmed that PH13 is a soybean main stem elongation promoting factor by constructing NILs, CRISPR-Cas9 mutants, and overexpression materials. The PH13 gene encodes a WD40 protein that interacts with GmCOP1s to degrade STF1 / 2 transcription factors, thereby promoting stem elongation. At the same time, this study not only elucidated the molecular mechanism by which PH13 regulates soybean plant height to adapt to high latitudes, but also created the phd mutant with significant shade tolerance and yield potential, providing important new genes and materials for breeding high-yielding new varieties suitable for dense planting in high-latitude regions. In 2023, our research group also published a research paper entitled "UV-B irradiation-activated E3 ligase GmILPA1 modulates gibberellin catabolism to increase plant height in soybean" online in Nature Communications. This paper revealed a new mechanism by which the UV-B-dependent E3 ligase GmILPA1 mediates gibberellin metabolism to regulate soybean plant height, providing new clues for a deeper understanding of the adaptive growth mechanism of soybean to UV-B radiation. This study discovered a key module GmILPA1-GmUBL1-GmGA2ox-like that regulates soybean plant height under UV-B conditions. This module inhibits the decrease in active GA content by ubiquitinizing and degrading UV-B-dependent GmGA2ox-like molecules, thereby improving soybean's tolerance to UV-B and maintaining normal growth. This discovery provides a theoretical reference for a deeper understanding of the molecular mechanisms by which plants adapt to UV-B stress, offers insights for further elucidating the molecular mechanisms regulating soybean plant height, and provides a theoretical basis for breeding new crop varieties adapted to strong ultraviolet radiation. In 2024, Li Hongyu et al. from the Institute of Crop Science, Chinese Academy of Agricultural Sciences, revealed the biological function of soybean DELLA protein GmRGAs in regulating soybean plant height, and discovered a new mechanism by which GmRGAs regulate plant height by mediating light signals to inhibit the degradation of GmSTF1 / 2 proteins.

[0006] In summary, plant height and branching, as key factors determining plant type, are complex quantitative traits regulated by multiple genes and influenced by the external environment. The molecular genetic mechanisms of plant height and branching number are complex, involving multiple genetic regulatory pathways, each regulated by numerous unknown genes. Furthermore, soybean dwarf and branching mutants are relatively rare, limiting research on the molecular mechanisms regulating soybean plant type. Exploring soybean resources for plant height and branching number, and isolating key genes regulating these traits, will accelerate the process of molecular breeding for soybean plant type, leading to new soybean germplasm and varieties with ideal plant types. In addition, fewer branches mean less space occupied by the plant, which facilitates efficient use of light, good ventilation, and can increase average yield per acre through denser planting. Therefore, isolating and locating soybean plant height and branching number mutants is of great significance for soybean variety improvement. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a GmSGT2 gene mutant that causes dwarfing and reduced branching in soybeans, and its application in improving soybean and related plant architecture traits and yield. Specifically, the present invention obtains a soybean mutant with fewer branches and reduced plant height through EMS mutagenesis, and locates the target gene as the GmSGT2 gene of soybean variety 'He Dou 12' using map-based cloning technology. The gene is found to encode soybean saponin B glucuronide galactosyltransferase, which can galactosylate soybean saponin B monoglucuronide, thus affecting soybean plant height and causing reduced branching. Based on the above research results, the present invention is thus completed.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a GmSGT2 gene having any of the nucleotide sequences described in (a1)-(a5):

[0010] (a1) The nucleotide sequence shown in SEQ ID NO.1;

[0011] (a2) and (a1) encode proteins with the same amino acid sequence, but are different nucleotide sequences due to the degeneracy of the genetic code.

[0012] (a3) is a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes the same or similar functional protein;

[0013] The nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides according to any of (a4) and (a1)-(a3);

[0014] A nucleotide sequence that is complementary to either (a5) or (a1)-(a4).

[0015] Furthermore, in (a4), the nucleotide sequence of the GmSGT2 gene is as shown in SEQ ID NO.1. In the nucleotide sequence of the soybean gene GmSGT2, G at position 259 is replaced by A, and G at position 910 is replaced by A. Mutations occur at these two sites, and the nucleotide sequence of this mutant is shown in SEQ ID NO.2.

[0016] In a second aspect, the present invention provides a protein encoded by the aforementioned GmSGT2 gene.

[0017] More specifically, the amino acid sequence of the protein is selected from:

[0018] (b1) The amino acid sequence shown in SEQ ID NO.3;

[0019] (b2) The amino acid sequence shown in (b1) has ≥90% identity with the amino acid sequence and has the same or similar biological activity;

[0020] (b3) is an amino acid sequence formed by substitution and / or deletion and / or addition of one or more amino acid residues as shown in either (b1) or (b2).

[0021] Furthermore, in (b3), the amino acid sequence of the protein is mutated based on the amino acid sequence shown in SEQ ID NO.3, resulting in a mutation of valine at position 87 to methionine and alanine at position 304 to threonine. The amino acid sequence of this mutant is shown in SEQ ID NO.4.

[0022] A third aspect of the present invention provides a recombinant expression vector, host cell, or recombinant bacteria containing a nucleic acid molecule containing the above-mentioned GmSGT2 gene and / or a nucleic acid molecule containing an inhibitory or depressant GmSGT2 gene.

[0023] The nucleic acid molecules that inhibit or reduce the expression level of the GmSGT2 gene can be nucleic acid molecules that reduce the expression level of the GmSGT2 gene, including but not limited to sgRNA, microRNA, siRNA, shRNA and / or antisense oligonucleotides.

[0024] As is well known to those skilled in the art, in addition to using gene editing technology to inhibit the expression of the GmSGT2 gene, gene knockdown technology can also be used to inactivate or silence the GmSGT2 gene at the post-transcriptional or translational level. The gene knockdown techniques include RNA interference, Morpholino interference, antisense nucleic acid, ribozymes, or dominant-negative repressive mutations. Furthermore, using shRNA or siRNA expressed by viruses (such as lentiviruses or adeno-associated viruses) to inhibit the expression of the GmSGT2 gene and thus silence it is also well known to those skilled in the art, and is not specifically limited here.

[0025] In this invention, a vector refers to a vector that can carry exogenous DNA or target gene into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages, phages, Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0026] In this invention, the host cell can be a plant cell or an animal cell, with plant cells being preferred. The host cell can be understood to refer not only to a specific recipient cell but also to its offspring. Suitable host cells are those known in the art, and the plant cell can be Arabidopsis thaliana, soybean, tobacco, corn, rice, wheat, etc., with soybean being preferred.

[0027] The recombinant bacteria described in this article refer to recombinant bacteria whose genes have been manipulated and modified to obtain functionally altered recombinant bacteria. This includes recombinant bacteria obtained by introducing a foreign target gene or recombinant expression vector into the target bacteria, or recombinant bacteria obtained by directly editing the endogenous genes of the target bacteria. The term "recombinant bacteria" can be understood not only to a specific recombinant bacterium, but also to its offspring. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cells, but are still included within the scope of recombinant bacteria.

[0028] The target bacteria can be bacteria, fungi, actinomycetes, etc. Specifically, the bacteria can be from genera such as *Escherichia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*, and more specifically, *Escherichia coli*, *Agrobacterium tumefaciens*, *Bacillus subtilis*, or *Bacillus pumilus*. The fungi can be yeasts. The fungi can be from genera such as *Fusarium*, *Rhizoctonia*, *Verticillium*, *Penicillium*, *Aspergillus*, and *Cephalosporium*. The actinomycetes can be from genera such as *Streptomyces*, *Nocardia*, *Micromonospora*, and *Streptomyces*.

[0029] A fourth aspect of the present invention provides the use of the above-mentioned GmSGT2 gene, protein, nucleic acid molecule containing the above-mentioned GmSGT2 gene and / or recombinant expression vector containing nucleic acid molecule that inhibits or reduces the above-mentioned GmSGT2 gene, host cell or recombinant bacteria in any one or more of the following:

[0030] (c1) Regulating plant architecture;

[0031] (c2) Improve and cultivate plants.

[0032] In this invention, the plant is any plant at any developmental stage. In particular, the plant can be a crop, such as a food crop and a cash crop, such as soybean, tobacco, corn, rice, wheat, etc., with soybean being preferred.

[0033] Specifically, (c1) regulates plant architecture by regulating soybean plant height and the number of branches.

[0034] The (c2) improvement and breeding of plants refers to the improvement and breeding of soybeans with fewer branches and / or reduced plant height, thereby obtaining dwarf and high-yielding soybean varieties.

[0035] A fifth aspect of the present invention provides a method for improving and cultivating plants, the method comprising: reducing the expression level and / or activity of the GmSGT2 gene in a target plant.

[0036] In the above method, reducing the expression level and / or activity of the GmSGT2 gene in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce or inactivate the activity of the GmSGT2 gene in the genome of the target plant.

[0037] The method for improving and cultivating plants according to the present invention may include the following steps: suppressing the expression of the nucleic acid molecule of the GmSGT2 gene in the target plant to obtain a transgenic plant; the transgenic plant has at least one of the following changes compared with the target plant: reduced plant height and reduced number of plant branches, thereby promoting high-yield and dense planting and crop yield.

[0038] As described above, in this invention, the plant can be a crop, such as a food crop and a cash crop, and further such as soybean, tobacco, corn, rice, wheat, etc., wherein soybean is preferred.

[0039] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0040] The above technical solution discloses a soybean dwarf mutant, gmsgt2, of the GmSGT2 gene, which regulates soybean plant height and branching, and its applications. After years of screening, a stably heritable dwarf and branching-reducing mutant, gmsgt2, was selected. Controlled by a single recessive nuclear gene, it exhibits reduced branching and lower plant height. For the first time, the soybean plant height and branching-regulating gene GmSGT2 was cloned using map-based cloning and BSA-Seq methods. To date, no studies have reported on EMS mutagenesis leading to base mutations in the soybean GmSGT2 gene that affect yield traits such as plant height and branch number. The gmsgt2 mutant contains two single-base (SNP) mutations in the GmSGT2 gene; this mutant gene is named mGmSGT2. The mGmSGT2 gene has the biological function of inhibiting plant height and branching, and can be used for research on plant branching and height regulation, possessing significant breeding value. The gmsgt2 mutant can be used to breed dwarf and high-yield soybean varieties. It has broad application prospects and high application value in soybean breeding, and can also provide excellent germplasm resources for regulating high-density planting and high-yield soybean breeding. Attached Figure Description

[0041] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0042] Figure 1 Plant morphology at seedling and mature stages of wild-type He Dou 12 (left) and gmsgt2 mutant (right).

[0043] Figure 2 : Map-based cloning of the gmsgt2 mutant gene.

[0044] Figure 3 Schematic diagram of the distribution of ΔSNP-index on chromosomes by BSA-seq of the gmsgt2 mutant.

[0045] Figure 4 Schematic diagram of the distribution of ED in chromosomes of the offspring of the gmsgt2 mutant BSA-seq.

[0046] Figure 5Sequencing analysis of mutation site 1 of the mutant gmsgt2 gene; (a) Sequencing results of candidate gene mutation site 1 (N1) in wild-type phenotype single plants in parental W82, H12 and BC1F2 populations (homozygous, all with base G); (b) Sequencing results of candidate gene mutation site 1 (N1) in mutant, hybrid F2 and BC1F2 populations (all with base A); (ch) Sequencing peak diagram of mutation site N1 of GmSGT2 gene (mutation site is single peak (base A) in mutant phenotype plants, and double peak (heterozygous) and single peak (base G) (hybridgous) in wild-type phenotype plants).

[0047] Figure 6 Sequencing analysis of mutation site 2 of the mutant gmsgt2 gene; (a) Sequencing results of candidate gene mutation site 2 (N2) in wild-type phenotype single plants in parental W82, H12 and BC1F2 populations (homozygous, all with base G); (b) Sequencing results of candidate gene mutation site 2 (N2) in mutant phenotype single plants in gmsgt2 mutant, hybrid F2 and BC1F2 populations (all with base A); (ch) Sequencing peak diagram of mutation site N2 of GmSGT2 gene (mutation site in mutant phenotype plants is a single peak (base A), mutation site in wild-type phenotype plants is a double peak in heterozygotes and a single peak (base G) in homozygotes).

[0048] Figure 7 Plant morphology of wild-type Williams 82 (left) and GmSGT2-crispr knockout line (right). Detailed Implementation

[0049] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. This invention utilizes techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can employ other conventional techniques, methods, and reagents in the art based on the embodiments provided in this invention, without being limited to the specific embodiments of this invention.

[0051] Specifically, this invention used map-based cloning of the F2 generation produced by crossing gmsgt2 with William 82, and BSA sequencing of the F2 generation produced by crossing gmsgt2 with He Dou 12. The GmSGT2 gene was successfully cloned, and its accuracy was verified using gene editing technology. Experiments confirmed that the GmSGT2 gene has the function of regulating soybean plant height and branching development, a novel function of the GmSGT2 gene discovered for the first time. Compared to wild-type He Dou 12, mutant and knockout transgenic plants of the GmSGT2 gene showed reduced plant height and fewer branches, a phenotype significant for the study of soybean plant architecture mechanisms.

[0052] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0053] In the following examples, all materials, reagents, strains, plasmids, enzymes, kits, etc., used were obtained commercially unless otherwise specified. The EMS mutagen (ethyl methanolate) was manufactured by Sigma-Aldrich, USA.

[0054] Example 1: Screening of soybean dwarf mutant gmsgt2

[0055] Wild-type He Dou 12 seeds were mutagenized using a 0.6% EMS mutagen. The specific method was as follows: Approximately 5000 uniformly sized, plump, and intact wild-type He Dou 12 seeds were selected. After soaking at room temperature for about 4 hours, the water was drained, and the seeds were then soaked in a 0.6% EMS mutagen for about 8 hours. 5% sodium thiosulfate was added as a terminator and antidote. The seeds were rinsed under running water for about 1 hour, dried, and then used for sowing. After maturity, individual plants were harvested for seed production, yielding M1 generation material 1231. The following year, M1 generation seeds were sown, with 30 seeds sown per plant, resulting in M2 generation material. Through field phenotypic observation, a soybean dwarf mutant plant was selected from the M2 generation population and named gmsgt2 (see...). Figure 1 The left image shows the seedling phenotypes of He Dou 12 and gmsgt2 mutants. The plant height of the gmsgt2 mutant is significantly reduced. The right image shows the mature phenotypes of He Dou 12 and gmsgt2 mutants. The plant height of the gmsgt2 mutant is significantly reduced, and the branching is significantly decreased.

[0056] Example 2: Genetic analysis of soybean dwarf mutant gmsgt2

[0057] Using soybean mutant gmsgt2 as the female parent and sequencing variety Williams 82 as the male parent, hybridization was performed to obtain F1 hybrid seeds. F1 plants were then self-crossed to obtain the F2 population. Phenotypic analysis of the F2 population revealed 140 dwarf mutants out of 584 F2 plants. A chi-square test was performed on the F2 population, and the calculated chi-square value was 0.3287. With df = 1 and a significance level of α = 0.05, the chi-square value was found to be 0.3287 < 3.841, indicating that the soybean mutant gmsgt2 conforms to a Mendelian segregation ratio of 3:1. Therefore, it was determined that this mutant trait is controlled by a single recessive nuclear gene.

[0058] Example 3: Cloning and transgenic verification of GmSGT2, a gene regulating soybean plant height and branching.

[0059] 1) Use the map-based cloning method for localization.

[0060] During the V4 stage of soybean growth (when the fourth trifoliate leaf is fully expanded), when plant height phenotypic differences are most pronounced, leaves from wild-type He Dou 12, Williams 82, F1 hybrids, and dwarf mutants isolated from F2 populations were collected, and leaf DNA was extracted using the CTAB method. InDel molecular marker primers, designed in the laboratory earlier and uniformly distributed on the 20 soybean chromosomes, exhibiting polymorphism in the parents He Dou 12 and Williams 82, were used to search for molecular markers linked to the target genes. A total of 223 pairs of InDel molecular markers and 5 pairs of SSR molecular markers were selected. After PCR amplification and polyacrylamide gel electrophoresis of the DNA from the above materials, statistical analysis of the electrophoretic banding was performed. The recombination rate was calculated using the formula:

[0061] =(h+2b) / 2(a+b+h) to calculate the recombination rate at each molecular marker location. Initial mapping results showed that the recombination rate of each marker on all 20 chromosomes was greater than 30%, with the lowest recombination rate (26.19%) at the GM11-5 molecular marker on chromosome 11. It was speculated that the mutant trait was likely closely linked to the GM11-5 marker, and the candidate gene was preliminarily identified as being located on chromosome 11 of the soybean genome. More polymorphic molecular markers were designed upstream and downstream of this location to search for sites linked to the dwarfing trait and differentiating among all recombinants. The initial mapping interval was narrowed down to the GM11-3 and GM11-5 molecular markers, with a size of 4.2 Mb. To further narrow down the candidate interval and determine the specific location of the mutation site, new polymorphic molecular markers were designed within the initial mapping interval, and PCR analysis was performed on 199 mutant plants in the selected F2 hybrid population. The mutation site was ultimately located between the SSR11-2 and SSR11-3 markers on chromosome 11, with a range size of 1.2555 Mb (see [link to relevant documentation]). Figure 2 ).

[0062] Plant DNA extraction method: Take about 0.8g of soybean leaf material and add it to a 1.5mL centrifuge tube. Add magnetic beads, cap the tube, and quickly place it in liquid nitrogen for pre-cooling. Grind the sample into powder using a grinder. After the sample is ground into powder, quickly add 1mL of CTAB extraction solution preheated at 65℃. Gently invert the tube in an "∞" shape to mix. Incubate in a 65℃ water bath for 0.5-2h, inverting the tube to mix every 5min. Centrifuge at 12000rpm for 10min and take 600μL of the supernatant into a new 1.5mL centrifuge tube. Add an equal volume of phenol / chloroform / isoamyl alcohol, gently invert in an "∞" shape to mix, and centrifuge at 12000 rpm for 10 min. Transfer 400 μL of the supernatant to a new 1.5 mL centrifuge tube, add 400 μL of pre-chilled isopropanol at -20°C, gently invert in an "∞" shape to mix, and let stand at -20°C for at least 30 min. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, wash twice with 75% ethanol, and dry in a clean bench. Dissolve the precipitate in 200 μL of double-distilled water and store in a -20°C refrigerator for later use.

[0063] PCR amplification system: PCR amplification reaction using standard EasyTaq enzyme (20 μL):

[0064]

[0065] The amplification conditions are as follows:

[0066]

[0067] 2) Use the BSA-seq method for localization.

[0068] Using soybean dwarf mutant gmsgt2 as the female parent and wild-type He Dou 12 as the male parent, backcrossing was performed to obtain BC1F1 generation hybrid seeds. The BC1F1 generation was then self-crossed to obtain the BC1F2 population. Mutants (58 plants), normal plants (68 plants), and wild-type parents (20 plants) isolated from the BC1F2 population were randomly selected for DNA extraction and quality inspection. BSA sequencing was performed by Beijing Berry Genomics Co., Ltd., and the sequencing results were analyzed.

[0069] To minimize the impact of sequencing and alignment errors, polymorphic sites need to be filtered. The filtering criteria in this study are as follows: sites with an SNP-index less than 0.2 and an SNP depth less than 7 in the offspring were filtered out; sites with a parental depth less than 7 and a heterozygous genotype were filtered out; sites with a GQ less than 20 were filtered out. After the above screening, the ΔSNP-index values ​​were calculated using the selected polymorphic marker sites, and the distribution of ΔSNP-index on the chromosomes was plotted to observe the differences in each site between the dwarf mutant pool and the normal wild-type pool. The closer the ΔSNP-index value is to 1, the stronger the association between the marker SNP and the target trait. The Manhattan plot of ΔSNP-index on the 20 chromosomes shows that only the SNP sites on the short arm of chromosome 11 exhibit a unimodal distribution (see...). Figure 3 This indicates that the mutation site is linked to chromosome 11, consistent with the localization results of previous map-based cloning. Simultaneously, based on the SNP frequency distribution analysis, the distribution of ED values ​​on the chromosome was plotted. A 1Mb window was selected, and the average ED value within each window was calculated to reflect the ED distribution in the offspring. A 95% confidence level was selected as the screening threshold. The distribution of ED on the chromosome in the offspring, as well as the Manhattan distribution plot, also showed that the highest peak occurred on the short arm of chromosome 11, further demonstrating the close linkage between the mutation site and chromosome 11 (see...). Figure 4 ).

[0070] 3) The mutant gene was identified by combining map-based cloning and BSA-Seq.

[0071] Based on the map-based cloning localization interval (between SSR11-2 and SSR11-3 markers on chromosome 11) and BSA-Seq data analysis, combined with database annotations and mutant plant phenotypic characteristics, this study continued to screen for possible mutation sites. After PCR amplification, sequencing, and sequence alignment, the target gene and its mutation site were identified. First, candidate SNP sites in the BSA data underwent preliminary screening. Only two sites, N1 and N2, were found to be located between the map-based cloning molecular markers SSR11-2 and SSR11-3. Furthermore, only these two sites had an SNP-index of 1 in the mutant pool. These two SNP sites were homozygous (all G) in *Heze Bean 12*, heterozygous (G or A) in the wild-type pool, and homozygous (all A) in the mutant pool. They also conformed to EMS-induced mutation patterns (GA or CT), with a sequencing depth of at least 23. Further random selection of individual plants from the gmsgt2 mutant, the wild-type parent He Dou 12, hybrid populations, and backcross populations, showing both mutant and wild-type phenotypes, led to sequencing comparisons of the N1 and N2 candidate SNPs. It was found that these two SNPs co-segregated with the mutant phenotype; both loci were mutated in all dwarf mutant plants, while in all plants with normal plant height, this gene locus was either wild-type or heterozygous. Figure 5 and Figure 6 Sequencing results confirmed that EMS mutagenesis resulted in G-to-A base substitutions at both sites. These two SNP mutations caused changes in the amino acids encoded by both genes, and both were located in the exon region of the GmSGT2 gene. Therefore, GmSGT2 is considered a possible gene causing the mutant trait.

[0072] Sequencing confirmed that the nucleotide sequence of the GmSGT2 gene in soybean variety 12, which is related to soybean plant height and branch number development, is shown in SEQ ID NO.1. The nucleotide sequence of the mutated GmSGT2 gene in soybean variety 12, which is related to soybean plant height and branch number development, is shown in SEQ ID NO.2; the mutation is characterized by the substitution of A for G at position 259 and position 910 in the GmSGT2 gene nucleotide sequence shown in SEQ ID NO.1. The amino acid sequence encoded by the aforementioned GmSGT2 gene in soybean variety 12, which is related to soybean plant height and branch number development, is shown in SEQ ID NO.3.

[0073] 4) Transgenic validation and phenotypic analysis of the knockout line of the mutant gene GmSGT2

[0074] Soybean seeds were disinfected with 70% ethanol for 5 minutes, rinsed off the ethanol, and then sterilized with chlorine for 16 hours. They were then placed in a pre-culture medium and cultured under light at 24°C for 1 day. Wild-type Williams spp. were then infected with Agrobacterium containing the target gene using a vacuum-assisted permeation method. 82 germinated embryos; after infection, soybeans were removed, excess bacterial solution was absorbed with sterile filter paper, and the cut surface was placed on a co-culture medium and cultured in the dark at 25℃-28℃ for 3-4 days; the soybean embryos after dark culture were washed 3-4 times with sterile water, then dried with sterile filter paper, and transferred to a shoot induction medium. The medium was changed every two weeks at 25℃-28℃ with 14 hours of light per day for four to five weeks to differentiate shoots. These shoots were then transferred to a shoot elongation medium and cultured until seedlings (resistant seedlings) emerged; shoots that grew to 2-4 cm were selected, and the shoots were cut off individually and transferred to a rooting medium. The plants were cultured for 14 days at 25℃-28℃ with 14±1 hours of light per day to obtain heterozygous edited seedlings (T0 generation). T0 generation seeds were planted, and in the T1 generation, GmSGT2 edited plants were finally obtained, exhibiting reduced plant height and branching (see...). Figure 7 The left image shows the phenotype of Williams 82 plants, and the right image shows the phenotype of GmSGT2-crispr plants. This further demonstrates that the GmSGT2 gene can regulate soybean plant height and branch number.

[0075] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can make modifications or equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for improving and cultivating plants, characterized in that, The method comprises: reducing the expression amount and / or activity of a gene in a plant of interest GmSGT2 The nucleotide sequence of the gene is shown as SEQ ID NO.

1. GmSGT2 The nucleotide sequence of the gene is shown as SEQ ID NO.

1. The plant in question is soybean; said reducing the expression amount and / or activity of said GmSGT2 gene in the plant of interest is achieved by using a gene mutation, gene knockout or gene knockdown technology to reduce or inactivate the activity of said GmSGT2 gene in the genome of the plant of interest; The method for improving and cultivating plants includes the following steps: [The text abruptly ends here, so the translation stops.] GmSGT2 The expression of the gene's nucleic acid molecules is suppressed to obtain a transgenic plant; the transgenic plant has at least one of the following changes compared with the target plant: reduced plant height and reduced number of plant branches, thereby promoting high-yield and dense planting and crop yield.

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