Gmsgt2 gene related to plant height and branch development, and mutant thereof and use thereof

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a GmSGT2 gene related to plant height and branch development, and a mutant thereof and the use thereof. A Glycine max mutant having fewer branches and decreased plant height is obtained by means of EMS mutagenesis, and a target gene thereof that is located by means of a map-based cloning technique is a GmSGT2 gene of Hedou 12. It is found through searching that the gene encodes soyasapogenol B glucuronide galactosyltransferase, and can galactosylate soyasapogenol B monoglucuronide, thereby affecting the plant height of Glycine max and reducing the branches of Glycine max. Therefore, the GmSGT2 mutant can be used for cultivating dwarf high-yield Glycine max varieties, and is of great significance in the breeding of ideal plant types of Glycine max and research on important agronomic traits of plants. The mutant has broad application prospects and great research value with regard to understanding the plant height and branch regulation mechanism of Glycine max and improving the process of Glycine max breeding; and can provide an excellent germplasm resource stock for regulating the close planting and high-yield breeding of Glycine max.

Description

GmSGT2 gene and mutant thereof related to plant height and branch development and application

[0001] Cross-reference to related applications

[0002] The present application claims priority to the Chinese patent application No. 202410731149.0 filed on June 06, 2024 to the State Intellectual Property Office of China and entitled "GmSGT2 gene and mutant thereof related to plant height and branch development and application", the entire contents of which are incorporated herein by reference and form a part of the present application for all purposes. TECHNICAL FIELD

[0003] The present application belongs to the field of biotechnology, and particularly relates to GmSGT2 gene and mutant thereof related to plant height and branch development and application. BACKGROUND

[0004] The information disclosed in this Background section is for the purpose of increasing the understanding of the general context of the present application and it may not necessarily be accepted as prior art with respect to this application.

[0005] Soybean (Glycine max) originated in China, is an important food and oil crop in China and even in the world, and provides more than half of the world's oil production and nearly one-fourth of the world's plant protein. At present, China is the world's largest soybean importer, with high dependence on foreign countries. Improving China's soybean production capacity has become an urgent task to ensure China's food security. However, in recent years, with the advancement of industrialization and urbanization, China's arable land area has been decreasing year by year, and the cultivation area of crops such as rice and corn has been continuously increasing due to market influence, resulting in a continuous decline in soybean planting area, a shortage of domestic soybeans, and a rapid increase in imported soybeans. Therefore, it is not very realistic to increase the total yield of soybeans by expanding the planting area. The only way is to rely on technical means to cultivate more soybean varieties with ideal plant type, high quality and high yield to meet the increasing demand of people, which is also the main way to improve the comprehensive competitiveness of China's soybeans.

[0006] Plant height and branch number are important agronomic traits of crops, and are closely related to crop yield. Dwarf plants can enhance resistance to lodging, and plants with fewer branches can improve planting density and improve light energy utilization, thereby improving crop yield. Therefore, plant height and branch number are key factors in determining soybean plant type formation and directly affect soybean yield. Identification of key genes of soybean plant height and branch number and analysis of their regulation mechanisms are of great significance for cultivating high-yield soybean varieties through molecular design breeding.

[0007] In recent years, the research on the regulation mechanism of plant height and branch number has gradually increased. A series of key genes that regulate plant height and branch number have been isolated in model plants Arabidopsis thaliana and Oryza sativa, and a molecular regulation network has been established. The most studied plant height is the study of rice dwarf mutants. Parnell et al. first reported the natural variation of dwarf mutants, and Oryoji et al. first reported the artificial mutation of dwarf mutants and conducted a large number of genetic studies. The production of these dwarf mutants provides a rich resource for rice breeding. There are many reasons for the production of plant dwarfism. In 1981, Kamijma et al. found that dwarf genes affect plant embryonic organ size and cell morphology by studying near-isogenic lines of rice. Plant dwarfism may be caused by a decrease in the number of internode cells. Plant hormones play an important role in plant growth and development. Through the study of a large number of height and branch mutants in plants such as rice and Arabidopsis, it was found that the biosynthesis, transport, and signal transduction of plant hormones such as auxin, gibberellin, and brassinosteroid are important for regulating plant height and branch number. In 2011, Burkhard Schulza et al. found that NA1, as 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 semi-dwarf and leaf angle phenotypes, which is a typical BR-enhanced mutant phenotype. The gene OsmiR396d regulates GA biosynthesis and signal transduction and BR response processes by regulating the expression of different target genes downstream, thereby affecting plant development. Hsieh et al. found that applying exogenous GA3 can restore the dwarf phenotype of tomato plants transformed with the AtCBF1 / DREB1A gene. In 2017, Li Laigeng et al. found a gene SBI encoding GA2 oxidase in rice and proved that the gene causes rice dwarfism. In 2022, Tian Zhixi and Kong Fajiang's team phenotyped the branch number of more than 2,400 soybean natural germplasm resources over two years. Using genome-wide association analysis, they identified the Dt2 gene, which controls soybean branch number. Genetic analysis found that Dt2 negatively regulates soybean branch number, and CRISPR / Cas9 gene knockout lines have significantly increased branch number and yield in small plots. Overexpression lines have significantly reduced branch number and yield in small plots. The study found that Dt2 is a key gene that controls soybean branch number in natural populations and elucidated the molecular mechanism of Dt2 regulating soybean branch number, providing an important theoretical basis for molecular design breeding for high-yield soybeans through branch regulation.In 2023, Liu Bin's team from the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and Qiu Lijuan et al. located the key gene PH13 that regulates soybean plant height, revealing the important role of its excellent haplotype in variety breeding in high-latitude areas and its molecular mechanism. The study confirmed that PH13 is a soybean main stem elongation promoter through the construction of NILs, CRISPR-Cas9 mutants, and overexpression materials. PH13 gene encodes a WD40 protein that interacts with GmCOP1s to degrade STF1 / 2 transcription factors, thereby promoting stem elongation. This study not only analyzes the molecular mechanism of PH13 regulating soybean height adaptation to high latitudes, but also creates phd mutants with significant shade tolerance and yield potential, providing important new genes and materials for breeding new varieties suitable for high-density planting in high-latitude areas. In 2023, our research group also published a research paper titled UV-B irradiation-activated E3 ligase GmILPA1 modulates gibberellin catabolism to increase plant height in soybean in Nature Communications, revealing a new mechanism of UV-B-dependent E3 ligase GmILPA1-mediated gibberellin metabolism regulating soybean height. This study provides new clues for understanding the adaptive growth mechanism of soybean to UV-B radiation. The study discovered a key module GmILPA1-GmUBL1-GmGA2ox-like that regulates soybean height under UV-B, which inhibits the degradation of active GA content through ubiquitination of UV-B-dependent GmGA2ox-like, thereby improving soybean tolerance to UV-B and maintaining normal growth. This finding provides a theoretical reference for understanding the molecular mechanism of plant adaptation to UV-B stress, further analyzing the molecular mechanism of regulating soybean height, and selecting new crop varieties that adapt 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 GmRGAs, a DELLA protein in soybean, in regulating plant height, and discovered a new mechanism of GmRGAs regulating plant height by mediating light signals to inhibit the degradation of GmSTF1 / 2 protein.

[0008] In summary, as one of the important factors determining plant architecture, plant height and branch number are controlled by multiple genes and affected by external environment, and are complex quantitative traits. The molecular genetic mechanism of plant height and branch number is complex, involving multiple genetic regulatory pathways, and different pathways are also regulated by many unknown genes. In addition, there are few soybean mutants with short stem and less branches, and the molecular mechanism of soybean plant architecture regulation is limited. Mining soybean resources with high plant height and branch number and isolating key genes regulating soybean plant height and branch number will help to accelerate the process of soybean plant architecture molecular breeding and obtain new soybean germplasm and varieties with ideal plant architecture. In addition, the whole plant with less branches occupies less space, which is conducive to the rational use of light source and good ventilation, and can also increase the average yield per mu by close planting. Therefore, isolating and locating soybean mutants with high plant height and branch number is of great significance for soybean variety improvement. SUMMARY

[0009] In view of the deficiencies in the prior art, the present application aims to provide a GmSGT2 gene mutant causing soybean dwarfing and branch reduction and its application in improving the traits and yield of soybean and related plant architecture. Specifically, the present application obtains a soybean mutant with less branches and lower plant height by EMS mutagenesis, and locates the target gene to GmSGT2 gene of Heidou 12 by map-based cloning technology. It is found by searching that the gene encodes soybean saponin B glucuronide galactosyltransferase, which can galactosidize soybean saponin B monoglucuronide, and can affect soybean plant height and cause soybean branch reduction. Based on the above research results, the present application is completed.

[0010] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

[0011] In a first aspect of the present application, a GmSGT2 gene is provided, which has any one of the nucleotide sequences (a1)-(a5):

[0012] (a1) the nucleotide sequence represented by SEQ ID NO. 1;

[0013] (a2) a nucleotide sequence encoding the same amino acid sequence as the nucleotide sequence of (a1), but different in sequence due to the degeneracy of genetic code;

[0014] (a3) a nucleotide sequence having ≥90% identity with the nucleotide sequence represented by (a1) or (a2) and encoding the same or similar functional protein;

[0015] (a4) a nucleotide sequence obtained by substitution and / or deletion and / or addition of one or more nucleotides of the nucleotide sequence represented by any one of (a1)-(a3);

[0016] (a5) a nucleotide sequence complementary to any one of (a1)-(a4).

[0017] Further, in the (a4), the GmSGT2 gene is a nucleotide sequence of a soybean gene GmSGT2 as shown in SEQ ID NO. 1, wherein the G at position 259 is substituted by A, and the G at position 910 is substituted by A, and the mutant is formed by the mutations at the two sites, and the nucleotide sequence of the mutant is as shown in SEQ ID NO. 2.

[0018] In a second aspect of the present application, a protein encoded by the GmSGT2 gene is provided.

[0019] More specifically, the amino acid sequence of the protein is selected from:

[0020] (b1) the amino acid sequence as shown in SEQ ID NO. 3;

[0021] (b2) an amino acid sequence having ≥ 90% identity to the amino acid sequence as shown in (b1) and having the same or similar biological activity;

[0022] (b3) an amino acid sequence obtained by substitution and / or deletion and / or addition of one or more amino acid residues in any one of (b1) or (b2).

[0023] Further, in the (b3), the amino acid sequence of the protein is a mutant of the amino acid sequence as shown in SEQ ID NO. 3, wherein the valine at position 87 is mutated to methionine, and the alanine at position 304 is mutated to threonine, and the amino acid sequence of the mutant is as shown in SEQ ID NO. 4.

[0024] In a third aspect of the present application, a nucleic acid molecule containing the GmSGT2 gene and / or a recombinant expression vector, a host cell or a recombinant bacteria containing a nucleic acid molecule inhibiting or reducing the GmSGT2 gene are provided.

[0025] The nucleic acid molecule inhibiting or reducing the GmSGT2 gene can be a nucleic acid molecule reducing the expression amount of the GmSGT2 gene, including but not limited to sgRNA, microRNA, siRNA, shRNA and / or antisense oligonucleotide.

[0026] It is well known to those skilled in the art that, in addition to using gene editing technology to inhibit the expression of the GmSGT2 gene, gene knock-down technology can also be used to inactivate or silence the GmSGT2 gene at the post-transcriptional level or the translation level. The gene knock-down technology includes RNA interference, Morpholino interference, antisense nucleic acids, ribozymes, or dominant negative inhibition mutations, etc. In addition, it is also well known to those skilled in the art that shRNA or siRNA expressed by viruses (such as lentivirus, adeno-associated virus) can be used to inhibit the expression of the GmSGT2 gene and silence the GmSGT2 gene, which is not specifically limited herein.

[0027] In the present application, the vector refers to a vector capable of carrying exogenous DNA or a 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, cosmids, Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0028] In the present application, the host cell can be a plant cell or an animal cell, wherein the plant cell is preferred. The host cell can be understood not only as a specific recipient cell, but also as the progeny of such a cell. Suitable host cells are known in the art, wherein the plant cell can be an Arabidopsis, soybean, tobacco, corn, rice, wheat, etc. plant cell, and the soybean is preferred.

[0029] The recombinant bacteria described herein refer to the recombinant bacteria obtained by operating and modifying the genes of the target bacteria. For example, the recombinant bacteria obtained by introducing an exogenous target gene or a recombinant expression vector into the target bacteria, or the recombinant bacteria obtained by directly gene editing the endogenous genes of the target bacteria. The recombinant bacteria can be understood not only as a specific recombinant bacteria, but also as the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny can not necessarily be completely consistent with the original parent cell, but is still included in the scope of the recombinant bacteria.

[0030] The target bacteria can be bacteria, fungi, actinomycetes, etc. The bacteria can be from the genus Escherichia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc., and further be, for example, Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis, or Bacillus pumilus. The fungi can be yeast. The fungi can be from the genus Fusarium, Naematospora, Verticillium, Penicillium, Aspergillus, Cephalosporium, etc. The actinomycetes can be from the genus Streptomyces, Nocardia, Micromonospora, Streptosporangium, etc.

[0031] In a fourth aspect of the present application, the GmSGT2 gene, the protein, the nucleic acid molecule containing the GmSGT2 gene, and / or the recombinant expression vector, the host cell or the recombinant bacteria containing the nucleic acid molecule for inhibiting or reducing the GmSGT2 gene are used in any one or more of the following aspects:

[0032] (c1) regulating plant architecture;

[0033] (c2) improving and cultivating plants.

[0034] In the present application, the plants are any plants at any developmental stage, and in particular, the plants can be crops, such as food crops and economic crops, and further such as soybeans, tobacco, corn, rice, wheat, etc., among which soybeans are preferred.

[0035] The (c1) regulating plant architecture specifically refers to regulating the plant height and the number of branches of soybeans.

[0036] The (c2) improving and cultivating plants refers to improving and cultivating soybeans with less branching and / or reduced plant height, and further obtaining dwarf high-yield soybean varieties.

[0037] In a fifth aspect of the present application, a method for improving and cultivating plants is provided, which comprises reducing the expression amount and / or activity of the GmSGT2 gene in the target plants.

[0038] In the above method, the reduction of the expression amount and / or activity of the GmSGT2 gene in the target plants can be achieved by reducing or inactivating the activity of the GmSGT2 gene in the genome of the target plants using gene mutation, gene knockout, gene editing or gene knockdown technology, etc.

[0039] The method for improving and cultivating plants according to the present application can comprise the following steps: inhibiting the expression of the nucleic acid molecule of the GmSGT2 gene in the target plants to obtain transgenic plants; and the transgenic plants have at least one of the following changes compared with the target plants: reduced plant height and reduced number of branches, and further promoting high-yield and high-density planting of plants and crop yield.

[0040] As described above, in the present application, the plants can be crops, such as food crops and economic crops, and further such as soybeans, tobacco, corn, rice, wheat, etc., among which soybeans are preferred.

[0041] The one or more technical solutions have the following beneficial technical effects:

[0042] The technical scheme discloses a soybean plant height and branch regulation gene GmSGT2 gene mutant, namely a soybean dwarf mutant gmsgt2 and application thereof. After years of screening work, a stable hereditary dwarf and branch reduction mutant gmsgt2 is bred, which is controlled by a single recessive nuclear gene, has reduced branches and reduced plant height. The soybean plant height and branch regulation gene GmSGT2 is cloned by a method combining map-based cloning and BSA-Seq for the first time. So far, no research report has been made on the base mutation of the soybean GmSGT2 gene caused by EMS mutagenesis, which further affects the plant height, branch number and yield traits of soybean. Two single base (SNP) mutations occur in the GmSGT2 gene in the gmsgt2 mutant, and the mutant gene is named as mGmSGT2. The mGmSGT2 gene has the biological function of inhibiting the generation of plant height and branches, and can be used for the research on the regulation of plant branches and height, and has important breeding value. The gmsgt2 mutant can be used for cultivating a dwarf high-yield soybean variety, has a wide application prospect and high application value in soybean breeding, and can also provide an excellent germplasm resource reserve for the regulation of soybean close planting and high yield breeding. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0044] Figure 1: Plant morphology of wild type Hebean 12 (left) and gmsgt2 mutant (right) at seedling stage and mature stage.

[0045] Figure 2: Schematic diagram of gene location of gmsgt2 mutant map-based cloning.

[0046] Figure 3: Distribution diagram of gmsgt2 mutant BSA-seq ΔSNP-index on chromosomes.

[0047] Figure 4: Distribution diagram of gmsgt2 mutant BSA-seq ED on chromosomes.

[0048] Figure 5: Sequencing analysis of mutation site 1 of the mutant gmsgt2 gene; where (a) the 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) the sequencing results of candidate gene mutation site 1 (N1) in mutant, hybrid F2 and BC1F2 populations (all with base A), (ch) is the sequencing peak diagram of mutation site N1 of GmSGT2 gene (the mutation site in mutant phenotype plants is a single peak (base A), the mutation site in wild-type phenotype plants is a double peak in heterozygotes and a single peak (base G) in homozygotes).

[0049] Figure 6: Sequencing analysis of mutation site 2 of the mutant gmsgt2 gene; where (a) the sequencing results of candidate gene mutation site 2 (N2) in wild-type phenotype single plants in parental W82, H12 and BC1F2 populations (homozygous all have base G), (b) the sequencing results of candidate gene mutation site 2 (N2) in mutant phenotype single plants in gmsgt2 mutant, hybrid F2 and BC1F2 populations (all have base A), (ch) is the sequencing peak diagram of mutation site N2 of GmSGT2 gene (the mutation site in mutant phenotype plants is a single peak (base A), the mutation site in wild-type phenotype plants is a double peak in heterozygotes and a single peak (base G) in homozygotes).

[0050] Figure 7: Plant morphology of wild-type Williams 82 (left) and GmSGT2-crispr knockout line (right). Detailed Implementation

[0051] 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.

[0052] 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.

[0053] Specifically, the F2 generation generated by crossing gmsgt2 and Williams 82 is used for map-based cloning, the F2 generation generated by crossing gmsgt2 and HeDou 12 is used for BSA sequencing, the GmSGT2 gene is successfully cloned, and the accuracy of the gene is verified and determined through gene editing technology. Experiments prove that the GmSGT2 gene has the functions of regulating the height and branch development of soybean, which is the first time to find the new function of the GmSGT2 gene. Compared with the wild type HeDou 12, the mutant and knockout lines of the GmSGT2 gene transgenic plants all have the traits of reduced plant height and reduced branch number, which is of great significance to the research on the mechanism of soybean plant type.

[0054] The content of the present application will be described in detail below in combination with the drawings and specific embodiments.

[0055] In the following examples, the materials, reagents, strains, plasmids, enzymes, kits, etc. are obtained from commercial channels unless otherwise specified. Among them, the EMS mutagen (ethyl methan sulfonate) is produced by Sigma Company in the United States.

[0056] Example 1: Screening of soybean dwarf mutant gmsgt2

[0057] The seeds of wild type HeDou 12 were treated with 0.6% EMS mutagen. The specific method is: about 5000 seeds of wild type HeDou 12 with uniform size, fullness and sound seed coat were selected, soaked at room temperature for about 4 hours, then the water was poured out, and then the seeds were soaked with 0.6% EMS mutagen for about 8 hours, 5% sodium thiosulfate was added as a terminating agent and detoxifying agent, and then the seeds were washed with water for about 1 hour, and then dried for sowing. After maturation, single plant seeds were collected to obtain M1 generation material 1231. The next year, the M1 generation seeds were sown, 30 seeds per plant, which is M2 generation material. Through field phenotype observation, a soybean dwarf mutant plant named gmsgt2 was screened from the M2 generation population (see Figure 1). The left figure is the phenotype of HeDou 12 and gmsgt2 mutant at seedling stage, the gmsgt2 mutant has significantly reduced plant height, and the right figure is the phenotype of HeDou 12 and gmsgt2 mutant at mature stage, the gmsgt2 mutant has significantly reduced plant height and reduced branches.

[0058] Example 2: Genetic analysis of soybean dwarf mutant gmsgt2

[0059] The soybean mutant gmsgt2 plant was used as the female parent, the sequencing variety Williams 82 was used as the male parent, F1 hybrid seeds were obtained by hybridization, and F2 population was obtained by selfing F1 generation. The F2 population was phenotyped, 140 dwarf mutants were separated from 584 F2 population, χ2 test was performed on the F2 population, χ2=0.3287 was calculated, df=1, the significant level α=0.05, and χ2=0.3287<3.841 was obtained by table lookup, which proved that the soybean mutant gmsgt2 was in accordance with the Mendelian genetic segregation ratio of 3:1, and thus it was determined that the mutant trait was controlled by a single recessive nuclear gene.

[0060] Example 3: Cloning and transgenic verification of soybean plant height and branch regulation gene GmSGT2

[0061] 1) Positioning by using the method of map-based cloning

[0062] At the stage of the most obvious difference in plant height phenotype of soybean growth to V4 (the 4th trifoliate leaf fully expanded), the leaves of wild type Heidou 12, Williams 82, hybrid F1 generation, and F2 population separated out dwarf mutants were taken, and the leaf DNA was extracted by CTAB method. The InDel molecular marker primers evenly distributed on 20 soybean chromosomes and showing polymorphism in the parents Heidou 12 and Williams 82 designed in the early stage of the laboratory were used to find molecular markers linked to the target gene. A total of 223 InDel molecular markers and 5 SSR molecular markers were selected. After PCR amplification and polyacrylamide gel electrophoresis of the DNA of the above materials, the electrophoretic band type was statistically analyzed. According to the formula: recombination rate=(h+2b) / 2(a+b+h), the recombination rate of each molecular marker was calculated. The initial positioning result showed that the recombination rates of the markers on the 20 chromosomes were all greater than 30%, and the recombination rate of the marker GM11-5 located on chromosome 11 was the lowest, which was 26.19%, it was speculated that the mutant trait was likely to be closely linked to the GM11-5 marker, and the candidate gene was preliminarily determined to be located on chromosome 11 of the soybean genome. More polymorphic molecular markers were designed upstream and downstream of the position, to find a site linked to the dwarf trait and different between all recombinants, and the initial positioning interval was locked between the molecular markers GM11-3 and GM11-5, with an interval size of 4.2 Mb. In order to further narrow the candidate interval and determine the specific location of the mutation site, new polymorphic molecular markers were designed within the initial positioning interval, and PCR analysis was performed on 199 mutant single plants in the screened hybrid F2 population. Finally, the mutation site was determined to be between the markers SSR11-2 and SSR11-3 on chromosome 11, with an interval size of 1.2555 Mb (see Figure 2).

[0063] Plant DNA extraction method: take about 0.8g soybean leaf material into 1.5mL centrifuge tube, add magnetic beads, buckle, quickly pre-cool in liquid nitrogen, use sample grinder to grind the sample into powder; after the sample is ground into powder, quickly add 1mL 65℃ preheated CTAB extraction solution, mix gently according to the "∞" shape, 65℃ water bath for 0.5-2h, mix gently every 5min during the period; 12000rpm centrifugation for 10min, take 600μL supernatant into a new 1.5mL centrifuge tube. Add equal volume of phenol / chloroform / isoamyl alcohol, mix gently according to the "∞" shape, centrifuge at 12000rpm for 10min; take 400μL supernatant into a new 1.5mL centrifuge tube, add -20° pre-cooled isopropanol 400μL, mix gently according to the "∞" shape, stand at -20℃ for more than 30min; 4℃, 12000rpm, centrifuge for 10min, discard the supernatant, wash twice with 75% ethanol, blow dry in a clean bench; dissolve the precipitate with 200μL double distilled water, store in a -20℃ refrigerator for standby.

[0064] PCR amplification system: ordinary EasyTaq enzyme for PCR amplification reaction (20μL):

[0065] The amplification conditions are as follows:

[0066] 2) Using BSA-seq method for positioning

[0067] Take soybean dwarf mutant gmsgt2 plant as female parent, wild type Hebean No. 12 as male parent, backcross to obtain BC1F1 hybrid seeds, self-cross BC1F1 generation to obtain BC1F2 population, randomly select the mutants (58 strains), normal plants (68 strains) and wild type parents (20 strains) separated from BC1F2 population for DNA extraction and quality inspection, then perform BSA sequencing by Beijing Baire He Kang Biotechnology Co., Ltd., and analyze the sequencing results.

[0068] To minimize the impact of sequencing errors and alignment errors, it is necessary to filter polymorphic sites. The filtering criteria in this study are as follows: sites with SNP-index less than 0.2 and depth less than 7 in the offspring are filtered out; sites with depth less than 7 and heterozygous genotype in the parents are filtered out; sites with GQ less than 20 are filtered out. After the above screening, the filtered polymorphic marker sites are used to calculate the ΔSNP-index value, and the distribution of ΔSNP-index on the chromosome is plotted to observe the difference of each site between the dwarf mutant pool and the normal wild type pool. The closer the ΔSNP-index value is to 1, the greater the correlation between the marker SNP and the target trait. The Manhattan plot of ΔSNP-index on 20 chromosomes shows that only the SNP sites on the short arm of chromosome 11 show a unimodal distribution (see Figure 3), indicating that the mutation site is linked to chromosome 11, which is consistent with the previous mapping results. At the same time, according to the analysis results of SNP frequency distribution, the distribution of ED on the chromosome is plotted. Selecting 1 Mb as the window, the average value of ED in each window is calculated to reflect the distribution of ED in the offspring. Selecting 95% confidence level as the threshold for screening. The distribution of offspring ED on the chromosome and the Manhattan distribution map also show that the highest peak appears on the short arm of chromosome 11, and this result further indicates that the mutation site is tightly linked to chromosome 11 (see Figure 4).

[0069] 3) Combined with map-based cloning and BSA-Seq to determine the mutant gene

[0070] According to the interval located by map-based cloning (between SSR11-2 and SSR11-3 markers on chromosome 11) and BSA-Seq data analysis, combined with database annotation and mutant plant phenotype characteristics, the possible mutation sites were further screened, and the target gene and mutation site were determined by PCR amplification, sequencing and sequence alignment. First, the candidate SNP sites in the BSA data were preliminarily screened, and it was found that only N1 and N2 sites were located between the molecular markers of SSR11-2 and SSR11-3, and only these two sites had SNP-index = 1 in the mutant pool. The bases at these two SNP sites in Heidou 12 were homozygous (all G), and in the wild type pool, they were heterozygous (G or A), and in the mutant pool, they were homozygous (all A), which was consistent with the mutation rule of EMS mutagenesis (G-A or C-T), and the sequencing depth was at least greater than 23. Further, single plants with mutant phenotype and wild type phenotype were randomly selected from gmsgt2 mutants, wild type parent Heidou 12, hybrid population and backcross population, and the N1 and N2 candidate sites were sequenced and compared. It was found that the two SNP sites were co-segregated with the mutant phenotype, and the two sites were mutated in all dwarf mutant plants, and the gene sites were wild type or heterozygous in all normal plant height plants (Fig. 5 and Fig. 6). The sequencing results confirmed that EMS mutagenesis caused G to A base substitution at the two sites. The two SNP mutations caused changes in the encoded amino acids, and were located in the exon region of GmSGT2 gene, so the GmSGT2 was considered as a possible gene causing mutant traits.

[0071] The nucleotide sequence of the GmSGT2 gene associated with soybean plant height and branch number development in Heidou 12 is shown in SEQ ID NO. 1. The nucleotide sequence of the GmSGT2 gene associated with soybean plant height and branch number development in Heidou 12 after mutation is shown in SEQ ID NO. 2; the mutation is that the G at position 259 and the G at position 910 in the GmSGT2 gene nucleotide sequence shown in SEQ ID NO. 1 are replaced by A. The amino acid sequence of the GmSGT2 gene associated with soybean plant height and branch number development in Heidou 12 is shown in SEQ ID NO. 3.

[0072] 4) Knockout transgenic verification and phenotype analysis of mutant gene GmSGT2

[0073] The soybean seeds are sterilized with 70% ethanol for 5 minutes, washed with ethanol and sterilized with chlorine for 16 hours, then placed in pre-culture medium, cultured under light at 24°C for 1 day; the Agrobacterium containing the target gene is used to infect the wild type Williams 82 germinating embryo by vacuum infiltration assisted method; the infected soybean is taken out, the excess bacterial solution is absorbed with sterile filter paper, and the cut surface is placed on the co-culture medium, dark cultured at 25-28°C for 3-4 days; the dark cultured soybean embryo is washed with sterile water for 3-4 times, then dried with sterile filter paper, transferred to the cluster bud induction medium, cultured at 25-28°C with 14 hours of light per day, replaced with new medium every two weeks, continuously cultured for four to five weeks, and the cluster buds are differentiated, then the cluster buds are transferred to the bud elongation medium to culture to the small seedlings (resistant seedlings); the 2-4 cm long cluster buds after screening are cut off and transferred to the rooting medium, continuously cultured at 25-28°C with 14±1 hours of light per day for 14 days, to obtain the hybrid edited seedlings T0 generation, the T0 generation seeds are planted, and the GmSGT2 edited plants are finally obtained in T1 generation, with the phenotype of reduced plant height and reduced branching (see FIG. 7). The left of the figure is the phenotype of Williams 82 plant, and the right is the phenotype of GmSGT2-crispr plant, which further proves that the GmSGT2 gene can regulate the plant height and branching number of soybean.

[0074] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit it. Although the present application is explained in detail with reference to the examples given, the technical solutions of the present application can be modified or replaced equivalently according to the needs without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A GmSGT2 gene, wherein the GmSGT2 gene has any one of the nucleotide sequences in (a1)-(a4) : (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence encoding the same amino acid sequence as the nucleotide sequence in (a1) but differing in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence having ≥ 90% identity to the nucleotide sequence shown in (a1) or (a2) and encoding the same or a similar functional protein; (a4) a nucleotide sequence obtained by substitution and / or deletion and / or addition of one or more nucleotides from any one of the nucleotide sequences shown in (a1)-(a3) ; (a5) a nucleotide sequence complementary to any one of (a1)-(a4) ; Further, in the (a4), the nucleotide sequence of the GmSGT2 gene is shown in SEQ ID NO.

2. 2.A protein encoded by the GmSGT2 gene of claim 1. The amino acid sequence of the protein is selected from: (b1) the amino acid sequence shown in SEQ ID NO. 3; (b2) an amino acid sequence having ≥ 90% identity to the amino acid sequence shown in (b1) and having the same or similar biological activity; (b3) an amino acid sequence obtained by substitution and / or deletion and / or addition of one or more amino acid residues from any one of the amino acid sequences shown in (b1) or (b2) ; Further, in the (b3), the amino acid sequence of the protein is shown in SEQ ID NO.

4. 4.A recombinant expression vector, a host cell or a recombinant bacterium containing the nucleic acid molecule of the GmSGT2 gene of claim 1 and / or containing a nucleic acid molecule inhibiting or reducing the GmSGT2 gene of claim 1. The nucleic acid molecule inhibiting or reducing the GmSGT2 gene of claim 1 is a nucleic acid molecule reducing the expression amount of the GmSGT2 gene, including sgRNA, microRNA, siRNA, shRNA and / or antisense oligonucleotide. The host cell is a plant cell or an animal cell, wherein the plant cell is preferred; the plant cell is Arabidopsis thaliana, soybean, tobacco, corn, rice and wheat, preferably soybean. 7.Use of the GmSGT2 gene of claim 1, the protein of claim 2, the nucleic acid molecule containing the GmSGT2 gene of any one of claims 5-6 and / or the recombinant expression vector, the host cell or the recombinant bacterium containing a nucleic acid molecule inhibiting or reducing the GmSGT2 gene in any one or more of the following: (c1) regulating plant architecture; (c2) improving and breeding plants.

3. The protein of claim 2, wherein The plant is a crop, including food crops and economic crops, further including soybean, tobacco, corn, rice and wheat. The (c1) regulating plant architecture specifically refers to regulating the plant height and branch number of soybean. The (c2) improving and breeding plants refers to improving and breeding soybean with less branching and / or reduced plant height, thereby obtaining dwarf high-yield soybean varieties. ​ ​ ​ 5. The recombinant expression vector, host cell or recombinant bacteria of claim 4, wherein, ​ 6. The recombinant expression vector, host cell or recombinant bacteria of claim 4, wherein, ​ ​ ​ ​ 8. The use according to claim 7, characterized in that, ​ 9. The use according to claim 8, wherein the compound is ###0002### ​ ​ 10. A method of improving and cultivating plants, characterized by, The method comprises: reducing the expression amount and / or activity of the GmSGT2 gene in the plant; The reduction of the expression amount and / or activity of the GmSGT2 gene in the plant is 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 plant genome; Further, the method for improving and cultivating plants comprises the following steps: inhibiting the expression of the nucleic acid molecule of the GmSGT2 gene in the plant to obtain a transgenic plant; and the transgenic plant has at least one of the following changes compared with the plant: the plant height is reduced, and the number of branches of the plant is reduced, thereby promoting high yield and high density planting of the plant and crop yield. Further, the plant is a crop, including food crops and economic crops, further including soybeans, tobacco, corn, rice and wheat; preferably, the plant is soybeans.

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