RGAb function acquired soybean mutant and application thereof

Abstract

The invention belongs to the technical field of plant biology, and discloses an RGAb function acquired soybean mutant and application thereof. According to the soybean RGAb gene functional acquisition mutant rgab 66bp, RGAb is knocked out in a targeted mode through a specific target point by means of a CRISPR / Cas9 technology, and the soybean RGAb gene functional acquisition mutant rgab 66bp is obtained and shows phenotypes with the constant node number, the shortened node spacing, the shortened plant height and the enhanced lodging-resistant capacity. The invention provides a new method for soybean plant height regulation and control, and the mutant can be applied to plant height regulation and control and lodging resistance. The invention also develops an efficient and convenient molecular marker of the RGAb function acquired soybean mutant. The invention provides a DELLA function acquired mutant editing idea for other plants.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a soybean mutant with RGAb gain of function and application thereof. Background Art

[0002] Soybean [Glycine max (L.) Merr.] is an important grain, oil and feed crop in the world and is closely related to social economy. Plant height is the most important factor affecting yield traits in soybean plant type, because plant height determines the number of main stem nodes, lodging resistance, number of plants per unit area, number of pods per plant and harvest index. How to regulate soybean plant height to ultimately achieve high yield without lodging is an urgent problem to be solved in production practice.

[0003] The production of crops such as rice and wheat has increased several times due to the breeding of dwarf and semi-dwarf varieties, as well as the use of fertilizers, irrigation, pesticides, and farming methods, which has greatly alleviated the huge food pressure caused by population growth. The control of the semi-dwarf trait in wheat is caused by the Reduced height (Rht)-B1b and Rht-D1b mutations. However, since the pods for harvesting grains of soybean plants are located on the internodes, the soybean plant type is different from that of crops such as rice, wheat, and corn. Therefore, we cannot blindly borrow improvement measures from other crops. How to quickly obtain functional mutants that regulate the height of soybean plants through gene editing has become the key. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a soybean mutant with RGAb gain-of-function and its application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a RGAb gain-of-function soybean mutant, wherein the mutant gene of the RGAb gain-of-function soybean mutant is the RGAb gene, the wild-type nucleotide sequence of the RGAb gene is shown in SEQ ID NO.2, and the corresponding nucleotide sequence of the RGAb gene of the RGAb gain-of-function soybean mutant is shown in SEQ ID NO.4.

[0007] The present invention utilizes CRISPR / Cas9 gene editing technology to design the editing site of the RGAb gene (Glyma.08G095800), which is a 23bp DNA sequence, and the sequence is shown in SEQ ID NO: 1. The recombinant expression vector pYLCRISPR / Cas9 of the above gene editing site is transformed into Agrobacterium EHA101. The soybean cultivar Williams 82 (W82) is transformed using the cotyledon node method to obtain a stably transformed mutant strain. It was found that in one of the positive transgenic strains, after high-generation reproduction and homozygosity, the RGAb gene was missing 66bp. The mutant strain was expressed as "rgab△66bp". Phenotypic identification found that rgab△66bp showed a phenotype of shortened internode spacing and shorter plant height; the number of nodes of the mutant soybean remained unchanged, the internode spacing was shortened, the plant height was shortened, and the lodging resistance of the soybean was enhanced. To this end, an Indel molecular marker was developed for this missing sequence, and the effect diagram is shown in the figure. Figure 3 The base sequence of RGAb in W82 is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO:3; the base sequence of rgab△66bp created after CRISPR / Cas9 editing is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO:5; the base sequence of rgab△66bp missing from the normal gene is shown in SEQ ID NO.6, and the corresponding missing amino acid sequence is shown in SEQ ID NO:7. Compared with SEQ ID NO.2, SEQ ID NO.4 lacks 66bp at the 89th bp of the coding region; compared with SEQ ID NO.3, SEQ ID NO.5 lacks 22 amino acids at the 30th position.

[0008] In a second aspect, the present invention provides a method for constructing a RGAb gain-of-function soybean mutant, using gene editing tools to construct the RGAb gain-of-function soybean mutant in wild-type soybeans, or using soybeans containing the RGAb gain-of-function mutant to hybridize with wild-type soybeans, and then screening the offspring for plants with shortened internode distance and shorter plant height.

[0009] In a third aspect, the present invention provides another method for constructing a soybean mutant with a gain of function of RGAb, using the CRISPR / Cas9 gene editing tool to construct a gene knockout vector expressing a gene editing site, which is transferred into wild-type soybeans, and from the offspring plants of the transformation event, plants showing a phenotype of shortened internode distance and shorter plant height are screened to obtain a soybean mutant with a gain of function of RGAb;

[0010] The nucleotide sequence of the wild soybean RGAb gene is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the gene editing site is shown in SEQ ID NO: 1.

[0012] As a preferred embodiment of the construction method of the present invention, the wild soybean variety or line is Williams 82.

[0013] In a fourth aspect, the present invention provides a protein for regulating soybean internode distance and plant height, and its amino acid sequence is shown in SEQ ID NO:5.

[0014] In a fifth aspect, the present invention provides a nucleic acid encoding a protein that regulates soybean internode spacing and plant height, and the nucleotide sequence thereof is shown in SEQ ID NO:4.

[0015] In a sixth aspect, the present invention provides a gene editing site for constructing soybean internode spacing and plant height mutants, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0016] In a seventh aspect, the present invention provides a vector tool for expressing the above-mentioned protein regulating soybean internode spacing and plant height; the vector tool is any one of an expression plasmid, a microorganism, a cell, a plant organelle, a plant tissue, and a plant organ.

[0017] In an eighth aspect, the present invention uses the RGAb gain-of-function soybean mutant, the protein, the nucleic acid, the gene editing site, and the vector tool in any of the following fields, including:

[0018] i. Regulate soybean internode spacing and plant height;

[0019] ii. Regulate the yield of soybean per plant;

[0020] iii. Breeding for short internode spacing and / or short stems in soybeans;

[0021] iv.Soybean variety improvement;

[0022] V. Resistant to lodging.

[0023] In a ninth aspect, the present invention provides a molecular marker for detecting a soybean mutant with gain-of-function RGAb, the nucleotide sequence of which is shown in SEQ ID NO:8 and / or SEQ ID NO:9.

[0024] In the tenth aspect, the present invention provides a method for identifying or detecting a soybean mutant with a gain-of-function RGAb, wherein the RGAb gene of the soybean sample to be identified or detected is amplified using the molecular marker, and the genotype of the soybean sample is determined based on the base length of the amplified product; the base length of the wild type of the genotype is 166bp, the base length of the RGAb gain-of-function soybean mutant is 100bp, and the amplified product of the heterozygous type is two, and the base lengths thereof are 100bp and 166bp, respectively.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention uses CRISPR / Cas9 technology to specifically target and knock out RGAb, and obtains the soybean RGAb gene gain-of-function mutant rgab△66bp, which presents a phenotype of unchanged number of nodes, shortened internode distance, shorter plant height and enhanced lodging resistance. The present invention provides a new method for soybean plant height regulation.

[0027] 2. The present invention reduces the internode length by utilizing the sequence gene lacking in the RGAb gene, thereby achieving the purpose of increasing soybean yield by regulating the height of soybean plants and enhancing lodging resistance.

[0028] 3. The present invention knocks out RGAb through a specific target, and there is a certain probability of causing a gain-of-function mutation. This can provide a reference for the design of gain-of-function mutants of RGAb genes in other plants; the amino acid information of the rgab△66bp deletion can also provide ideas for editing RGAb gain-of-function mutants in other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the editing type and phenotype of RGAb; Figure 1 In the figure, A is the gene structure diagram of RGAb and the locations of the two designed targets; B is the editing method of the homozygous mutant rgab#1; C is the editing method of the homozygous mutant rgab#2; D is the editing method of the homozygous mutant rgab△66bp; E is a phenotypic photo of the wild-type W82 and three mutant strains of the RGAb gene in the greenhouse, with a bar length of 10 cm; F is the plant height (cm) of the wild-type W82 and three mutant strains of the RGAb gene in the incubator.

[0030] Figure 2 The rgab△66bp mutant showed a shorter plant height in the greenhouse and field. Figure 2In the figure, A is a phenotypic photo of the wild-type W82 and the rgab△66bp mutant in the greenhouse, with a bar length of 10 cm; B is the plant height (cm) of the wild-type W82 and the rgab△66bp mutant in the greenhouse; C is the number of main stem nodes of the wild-type W82 and the rgab△66bp mutant in the greenhouse; D is the average internode distance (cm) of the wild-type W82 and the rgab△66bp mutant in the greenhouse; E is a phenotypic photo of the wild-type W82 and the rgab△66bp mutant in the field, with a bar length of 10 cm; F is the plant height (cm) of the wild-type W82 and the rgab△66bp mutant in the field; G is the average internode distance (cm) of the wild-type W82 and the rgab△66bp mutant in the field. The number of main stem nodes of the ab△66bp mutant in the field; H is the average internode distance (cm) of the wild-type W82 and the rgab△66bp mutant in the field; I is the number of branches of the wild-type W82 and the rgab△66bp mutant in the field; J is the main stem cross-section of the wild-type W82 and the rgab△66bp mutant in the field, and the Bar length is 1 cm; K is the diameter of the first node of the wild-type W82 and the rgab△66bp mutant in the field (mm); L is the diameter of the second node of the wild-type W82 and the rgab△66bp mutant in the field (mm); M is the total number of pods per plant of the wild-type W82 and the rgab△66bp mutant in the field.

[0031] Figure 3 Electrophoresis verification of the molecular marker of the gain-of-function mutant rgab△66bp. DETAILED DESCRIPTION

[0032] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0034] Example 1: Construction of knockout vector

[0035] (1) Main reagents and sources

[0036] The competent Escherichia coli strain DH5α and the competent Agrobacterium tumefaciens strain EHA101 were purchased from Quanshijin Company. pYLgRNA-AtU3d, pYLgRNA-AtU3b and pYLCRISPR / Cas9 vectors (all published in "Arobust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants") were provided by Professor Liu Yaoguang's laboratory at South China Agricultural University. BsaⅠ restriction endonuclease and T4 DNA ligase were purchased from NEB Company; PCR product recovery kit and Master Taq mix were purchased from Kangwei Century Company; plasmid extraction kit was purchased from Quanshijin Company; other chemical reagents such as tryptone, agar powder, sodium chloride, kanamycin (Kan, 50μg / mL), spectinomycin (Spec, 100μg / mL) were all domestic analytical reagents; primer synthesis and sequencing were completed by Tianyi Huiyuan Company.

[0037] Target adapter primer:

[0038] T1-F: 5'-gtcaAATCTTTGGGAGGAAGAAGG-3' (SEQ ID NO: 10)

[0039] T1-R: 5'-aaacCCTTCTTCCTCCCAAAGATT-3' (SEQ ID NO: 11)

[0040] T2-F: 5'-gtcaGCCATGTCGGAGGTTTACTT-3' (SEQ ID NO: 12)

[0041] T2-R: 5'-aaacAAGTAAACCTCCGACATGGC-3' (SEQ ID NO: 13)

[0042] The primers for the first round of PCR were:

[0043] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO: 14)

[0044] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID NO: 15)

[0045] The primers for the second round of PCR were:

[0046] B1-F: 5'-TTCAGAGGTCTCTctcgACTAGTGGAATCGGCAGCAAAGG-3' (SEQ ID NO: 16)

[0047] B1-R: 5'-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3' (SEQ ID NO: 17)

[0048] B2-F: 5'-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3' (SEQ ID NO: 18)

[0049] BL-R: 5'-AGCGTGGGTCTCGaccgACGCGTCCATCCACTCCAAGCTC-3' (SEQ ID NO: 19)

[0050] Vector sequencing primers:

[0051] SP-L2: 5'-GTCGTGTCCACATGTTGACC-3' (SEQ ID NO: 20)

[0052] SP-R: 5'-TGCAATAACTTCGTATAGGCT-3' (SEQ ID NO: 21)

[0053] (2) Operation steps

[0054] 1) Target adapter and primer annealing.

[0055] Take 10 μL (10 μM) of each forward and reverse primer (T1-F, T1-R, T2-F and T2-R) and add them to 80 μL H2O, incubate at 90°C for 30 seconds, and then transfer to room temperature.

[0056] 2) The double-stranded adapter is digested and ligated with the gRNA expression cassette.

[0057] gRNA vector connection (cut and connect): add pYLgRNA-AtU3d / 3b vector 10ng, T4 DNAligase 0.1μL, T4 DNAligase buffer 1μL, gDNA (10ug / ul) 1μL, annealed linker primer 1μL, BsaⅠ 0.5μL, H2O 6.4μL, PCR, 5 cycles of 37℃5min, 20℃5min. Adapter primers T1-F and T1-R correspond to pYLgRNA-AtU3d vector, adapter primers T2-F and T2-R correspond to pYLgRNA-AtU3b vector.

[0058] 3) The first round of PCR amplifies the gRNA expression cassette.

[0059] Add Master Taq mix 5μL, UF 0.15μL, gRNA-R 0.15μL, H2O 3.3μL, and 1.4μL of the PCR product from the previous step, and perform PCR at 94℃ for 3min, 10 cycles (94℃ for 10s, 55℃ for 20s, 72℃ for 10s), and 25 cycles (94℃ for 10s, 60℃ for 20s, 72℃ for 10s). The product containing the T1 target sequence was named T1-gRNA, and the product containing the T2 target sequence was named T2-gRNA. Take 3μL of each PCR product for 1% agarose gel electrophoresis, and use a gel imager to check whether there is a 500bp band.

[0060] 4) Second round of PCR amplification.

[0061] In one tube, add Master Taq mix 10μL, B1-F 0.15μL, B1-R 0.15μL, H2O 8.7μL, and dilute T1-gRNA tenfold to take 1μL; in another tube, add Master Taq mix 10μL, B2-F 0.15μL, BL-R 0.15μL, H2O8.7μL, and dilute T2-gRNA tenfold to take 1μL, and perform PCR at 94℃ for 3min, 35 cycles (94℃ for 10s, 60℃ for 20s, 72℃ for 30s), 72℃ for 5min, and keep warm at 4℃. Take 3μL of PCR product for 1% agarose gel electrophoresis, and use a gel imager to check whether there is a 500bp band. After confirming the presence of a 500bp band, use a PCR product recovery kit to recover the remaining product that was not subjected to agarose gel electrophoresis, and measure the concentration after the product is recovered.

[0062] 5) The sgRNA expression cassette was connected to the pYLCRISPR / Cas9 vector.

[0063] 15μL reaction system: Cut Smart Buffer 1.5μL, pYLCRISPR / Cas9 plasmid 0.5μL, Bsa I 1μL, 4μL of the purified product in the previous step (concentration 60ng / μL-70ng / μL), T4 DNA ligase 0.2μL, 10×NEB T4DNAligase buffer 1.5μL, 10×NEB Cut Smart Buffer 1.5μL, add sterile water to 15μL. The PCR program was set to: 37℃5min, 10℃5min, 20℃5min, 37℃5min, 15 cycles to obtain the connected vector product.

[0064] 6) The ligation product is used to transform E. coli.

[0065] Thaw the E.coli DH5α competent cells on ice for 5 minutes, then add 10 μL of the previous ligation reaction solution and gently flick to mix. After ice bathing for 30 minutes, heat shock in a 42°C water bath for 1 minute and immediately ice bath for 2 minutes. Add 500 μL of LB culture medium without antibiotics to the clean bench, incubate at 37°C 220rpm for 1 hour, centrifuge at 5000rpm for 1 minute, pour out part of the supernatant, resuspend the bacterial solution, aspirate it, evenly spread it on the LB solid medium containing spectinomycin, and place it in a 37°C incubator overnight. Pick bacterial monoclonal clones, use SP-L2 and SP-R for PCR detection, and pick monoclonal colonies with 1000bp bands to send to Tianyi Huiyuan for sequencing.

[0066] 7) EHA101 Agrobacterium transformation.

[0067] The sequenced single clone containing the target pYLCRISPR / Cas9 was cultured overnight at 37° in liquid LB for plasmid extraction. Take 50μL of Agrobacterium competent cells EHA101 and add 0.1μg of positive plasmid, mix gently, ice bath for 30min, freeze in liquid nitrogen for 1min, heat shock at 37℃ water bath for 5min, and cool on ice for 2min. Inoculate the bacterial liquid into 500μL LB liquid culture medium, culture on a shaker at 28℃ and 220rpm for 2h, centrifuge for 1min, and evenly spread the suspended cells on LB solid culture medium with kanamycin, spectinomycin and rifampicin antibiotics, and culture inverted at 28℃ for 36h. Perform colony PCR with primers SP-L2 and SP-R to select positive single clones.

[0068] Example 2: Obtaining transgenic homozygous mutants

[0069] The transformation in this example adopts the cotyledon node method to transform soybean, and the recipient material is the cultivar W82. The operation steps are as follows:

[0070] (1) Seed germination

[0071] Soybean seeds with smooth and undamaged surfaces were dried and surface disinfected in chlorine for 16 hours, taken out, placed in a clean bench and blown for 30 minutes to remove the chlorine, and then sown on germination medium and cultured at 28°C for 3 days.

[0072] (2) Preparation of Agrobacterium culture solution

[0073] From the LB plate containing kanamycin, spectinomycin and rifampicin antibiotics prepared in Example 1, the EHA101 monoclonal clone containing the dual target sites of the knockout RGAb gene was selected and inoculated into 2 mL of LB liquid medium supplemented with kanamycin, spectinomycin and rifampicin antibiotics, and shaken at 28°C overnight, the colonies were collected by centrifugation, and the bacterial solution was suspended in CCM liquid to an OD of 650 It is 0.58-0.61.

[0074] (3) Co-culture

[0075] Pour the bacterial solution into a culture dish, cut the germinated seeds with a scalpel, split the beans vertically along the hypocotyl of the cotyledons, remove the young shoots on the hypocotyl, and make 7-8 incisions perpendicular to the axis in the cotyledon node area; put the cut explants into a culture dish with suspended bacterial solution and infect for 30 minutes, transfer the explants to the co-culture nutrient medium with tweezers, with the incision facing down, seal it and culture it in the dark in an incubator for 3 days.

[0076] (4) Shoot induction

[0077] After 3 days of co-cultivation, the explants were placed on a bud induction medium and grown for 14 days. The hypocotyls of the cotyledonary nodes were cut flush with the cotyledonary nodes, and the newly exposed wounds were inserted into new bud induction medium and cultured for another 14 days.

[0078] (5) Bud growth

[0079] The cotyledons on the differentiated explants were removed and a new incision was made at the base of the node. The explants were then transferred to stem elongation medium and grown in a culture chamber for 2-8 weeks, with the medium replaced every two weeks and a fresh horizontal incision made at the base of the explant each time.

[0080] (6) Rooting

[0081] When the shoots grow to 3 cm in length, they are cut from the tissue and transferred to a culture bottle containing rooting medium for cultivation. After 2 weeks, when the seedlings have grown enough roots, they are transferred to a substrate and cultured in an incubator for 4 weeks. The seedlings are then transferred to a greenhouse for cultivation until they set pods.

[0082] (7) Detection of transgenic plants

[0083] Herbicide screening: Select one of the fully expanded trifoliate leaves on the transgenic plant, mark one half with a marker, and apply the diluted herbicide on the other half with a cotton swab to the front of the leaf for 2-3 days to observe the changes in the leaves. If the leaves lose green color, turn yellow, wilt, or have colored spots, it means that the plant is not resistant to herbicides and is a non-transgenic material; if the leaves do not change, it means that the plant has herbicide resistance and may be a positive plant, that is, a T0 generation plant resistant to herbicides.

[0084] Identification of transgenic plants: After the seeds of the herbicide-resistant T0 plants are harvested, the T1 generation is propagated. The T1 seedlings continue to be treated with herbicides on the leaves, and the editing of the T1 plants without herbicide resistance is mainly detected. DNA is extracted from new leaves, and PCR identification is performed using SP-L2 and SP-R primers. Plants that test negative are subjected to PCR using target detection primers, and the PCR products are sent for sequencing to detect target editing. The test results show that three types of rgab were obtained, among which rgab#1 and rgab#2 are edited as follows: Figure 1 B and Figure 1 As shown in C, there is no significant difference in plant height compared with W82 ( Figure 1 E), while the functional mutant rgab△66bp was significantly shorter than W82 ( Figure 1 E). Further observation of the phenotype of rgab△66bp under greenhouse conditions and natural fields revealed that the number of nodes of rgab△66bp remained unchanged, the internode distance became shorter, the plant height became shorter, the stem thickness became thicker, and the grain weight per plant increased ( Figure 2 ). rgab△66bp Its DNA sequence lacks 66bp compared to the wild type W82 ( Figure 1 C), and molecular markers were designed (SEQ ID NO: 8 and 9), wherein the normal type is 166 bp, the mutant type is 100 bp, and the heterozygous type is two 100 bp and 166 bp ( Figure 3 ).

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A soybean mutant with RGAb gain-of-function, characterized in that: The mutant gene of the RGAb gain-of-function soybean mutant is the RGAb gene, the wild-type nucleotide sequence of the RGAb gene is shown in SEQ ID NO.2, and the corresponding nucleotide sequence of the RGAb gene of the RGAb gain-of-function soybean mutant is shown in SEQ ID NO.

4.

2. A method for constructing a soybean mutant with RGAb gain-of-function, characterized in that: Gene editing tools are used to construct the RGAb gain-of-function soybean mutant as claimed in claim 1 in wild-type soybeans, or soybeans containing the RGAb gain-of-function soybean mutant as claimed in claim 1 are hybridized with wild-type soybeans, and then the offspring are screened for plants with shortened internode distance and shorter plant height.

3. A method for constructing a soybean mutant with RGAb gain-of-function, characterized in that: Using CRISPR / Cas9 gene editing tools, a gene knockout vector expressing the gene editing site was constructed and transferred into wild-type soybeans. From the offspring plants of the transformation event, plants showing a phenotype of shortened internodes and shorter plant height were screened to obtain RGAb gain-of-function soybean mutants. The nucleotide sequence of the wild soybean RGAb gene is shown in SEQ ID NO.2; The nucleotide sequence of the gene editing site is shown in SEQ ID NO:

1.

4. A protein for regulating soybean internode spacing and plant height, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

5.

5. A nucleic acid encoding a protein that regulates soybean internode spacing and plant height, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:

4.

6. A gene editing site for constructing soybean internode spacing and plant height mutants, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:

1.

7. A carrier tool, characterized in that: Expressing the protein of claim 4; the vector tool is any one of an expression plasmid, a microorganism, a cell, a plant organelle, a plant tissue, and a plant organ.

8. Use of the RGAb gain-of-function soybean mutant of claim 1, the protein of claim 4, the nucleic acid of claim 5, the gene editing site of claim 6, and the vector tool of claim 7 in any of the following fields, characterized in that: include: i. Regulate soybean internode spacing and plant height; ii. Regulate the yield of soybean per plant; iii. Breeding for short internode spacing and / or short stems in soybeans; iv.Soybean variety improvement; V. Resistant to lodging.

9. A molecular marker for detecting a soybean mutant with gain-of-function RGAb, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:8 and / or SEQ ID NO:

9.

10. A method for identifying or detecting a soybean mutant with RGAb gain-of-function, characterized in that: The molecular marker described in claim 9 is used to amplify the RGAb gene of the soybean sample to be identified or detected, and the genotype of the soybean sample is determined based on the base length of the amplified product; the base length of the wild-type genotype is 166bp, the base length of the RGAb functional soybean mutant is 100bp, and the amplified product of the heterozygous type is two, and the base lengths thereof are 100bp and 166bp, respectively.

Images