Application of soybean GmZG3 gene in regulating soybean plant height

Abstract

The application discloses application of a soybean GmZG3 gene in regulation of soybean plant height and belongs to the technical field of genetic engineering. The application constructs a Cas9-sgRNA and a PTF-GmZG3 recombinant vector of the soybean GmZG3 gene, and after the recombinant vector is transferred into agrobacterium, EHA-GmZG3-sgRNA and EHA101 / PTF101-GmZG3 recombinant strains are obtained, then the constructed recombinant strains are transformed into wild-type soybean through an agrobacterium-mediated method, and thus mutant and overexpression plants of the GmZG3 gene are obtained. Phenotypes of the mutant and overexpression plants are identified, and it is found that the plant height of the mutant plant is significantly lower than that of the wild-type soybean, and the plant height of the overexpression plant is significantly higher than that of the wild-type soybean. Therefore, the application provides a method for genetically improving the plant type of soybean, and has potential market value and wide application prospect in the agricultural field.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the soybean GmZG3 gene in regulating soybean plant height. Background Technology

[0002] Soybeans are an important grain, oil, and feed crop in my country, playing an indispensable role in ensuring food security and agricultural trade. However, given my country's limited soybean planting area, how to efficiently and rapidly improve soybean varieties and increase yield per unit area using modern bio-breeding technology has become an urgent production problem and technical bottleneck to be solved.

[0003] Crop plant architecture plays a decisive role in the formation of individual plant and plant population morphology, and is a crucial factor influencing plant yield, production level, and economic benefits. Plant architecture comprises plant height, branching (tillering), leaf shape, and spike type (pod-setting habit). In-depth research and exploration of genes regulating plant architecture will not only help identify superior soybean plant architecture genes and cultivate high-yielding ideal plant architectures, but also enable the systematic evaluation and optimization of ideal plant architectures under actual production conditions through the development of specific materials. Therefore, there is an urgent need to develop a new method to improve soybean plant height, thereby promoting the application of soybean breeding and increasing soybean yield. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the purpose of this invention is to provide an application of the soybean GmZG3 gene in regulating soybean plant height, thereby providing a new method for improving soybean plant height.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: providing an application of the soybean GmZG3 gene in regulating soybean plant height, the nucleotide sequence of the GmZG3 gene is shown in SEQ ID NO.1.

[0006] Furthermore, regulating soybean plant architecture by knocking out the soybean GmZG3 gene can reduce soybean plant height.

[0007] Furthermore, regulating soybean plant architecture by overexpressing the soybean GmZG3 gene can increase soybean plant height.

[0008] Furthermore, mutations in the soybean GmZG3 gene can lead to the loss of gene function by inserting or deleting bases at any position in the GmZG3 gene.

[0009] Furthermore, the specific mutations in the soybean GmZG3 gene are as follows: adding an A base between positions 764 and 765 of the soybean GmZG3 gene nucleotide sequence; or adding a T base between positions 764 and 765 of the soybean GmZG3 gene nucleotide sequence; or deleting bases from positions 758 to 764 of the soybean GmZG3 gene nucleotide sequence.

[0010] This invention provides a formulation that can regulate soybean plant architecture, comprising the soybean GmZG3 gene or the protein encoded by the soybean GmZG3 gene.

[0011] This invention provides a formulation that can regulate soybean plant architecture, the formulation comprising a reagent that can induce mutations in the soybean GmZG3 gene.

[0012] This invention provides a formulation that can regulate soybean plant architecture, the formulation comprising a reagent capable of overexpressing the soybean GmZG3 gene.

[0013] This invention provides a method for preparing dwarf soybean varieties by causing mutations in the soybean GmZG3 gene.

[0014] The present invention also provides a method for preparing tall soybean varieties by overexpressing the soybean GmZG3 gene.

[0015] This invention offers the following advantages: It constructs Cas9-sgRNA and PTF-GmZG3 recombinant vectors of the soybean GmZG3 gene. These vectors are transformed into Agrobacterium tumefaciens to obtain EHA-GmZG3-sgRNA and EHA101 / PTF101-GmZG3 recombinant strains. These recombinant strains are then transformed into wild-type soybean using Agrobacterium-mediated transformation, resulting in GmZG3 gene mutants and overexpressing plants. Phenotypic analysis of the mutants and overexpressing plants revealed that the mutant plants were significantly shorter than wild-type soybeans, while the overexpressing plants were significantly taller than wild-type soybeans. Therefore, this invention provides a method for genetically improving soybean plant type, possessing potential market value and broad application prospects in the agricultural field. Attached Figure Description

[0016] Figure 1 The mutation type is GmZG3 gene mutant;

[0017] Figure 2 The plant height phenotype of the GmZG3 gene mutant;

[0018] Figure 3 The plant height of the GmZG3 gene mutant;

[0019] Figure 4 The number of nodes in plants of the GmZG3 gene mutant;

[0020] Figure 5 The plant height is the height of plants overexpressing the GmZG3 gene. Detailed Implementation

[0021] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1: Construction of a CRISPR vector for GmZG3 gene editing

[0023] (1) Obtaining sgRNA

[0024] The nucleotide sequence of the soybean GmZG3 genome coding region was obtained from the Phytozome database (as shown in SEQ ID NO.1). The GmZG3 gene is located on soybean chromosome 18. The target site sequence for GmMBR1 sgRNA was selected using the CRISPR-P online web tool (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). The target site is located in the first exon region of the GmZG3 gene. After the target site was selected, it needed to be integrated into the vector. First, the target site primers for sgRNA were synthesized. 5 μL of GmZG3-F and 5 μL of GmZG3-R primers were added to a 25 μL reaction system, along with 15 μL of water. The mixture was annealed at 95°C for 3 min, then annealed at 0.1°C / s to 16°C, and held at 16°C for 10 min to complete the annealing process, yielding the gRNA annealed product with sticky ends.

[0025] The target site sequence is as follows:

[0026] 5'-GTTCCTCAATGCCGTCGAAC-3' (SEQ ID NO. 2);

[0027] The sequence of the target site primer is as follows:

[0028] GmZG3-F: 5'-TTGGTTCCTCAATGCCGTCGAAC-3' (SEQ ID NO. 3);

[0029] GmZG3-R: 5'-AACGTTCGACGGCATTGAGGAAC-3' (SEQ ID NO. 4).

[0030] (2) Preparation of vectors for expressing sgRNA

[0031] Take 1 μL of the gRNA annealing product with sticky ends obtained above and perform homologous recombination ligation with the Cas9 / gRNA vector (Beijing Weishang Lide Biotechnology Co., Ltd., catalog number: VK005-15, which contains Cas9 protein expression units) to obtain the recombinant vector Cas9-sgRNA, which expresses sgRNA.

[0032] Example 2: Obtaining and identifying the GmZG3 mutant

[0033] (1) Preparation of recombinant bacteria

[0034] The recombinant vector Cas9-sgRNA prepared in Example 1 was transformed into Escherichia coli DH5α, plated on LB+Kan (LB medium + kanamycin) solid medium, and cultured overnight at 37°C. Then, single clones were picked, plasmids were extracted and sequenced. The plasmid with the correct inserted fragment was named recombinant plasmid GmZG3-sgRNA. The recombinant plasmid GmZG3-sgRNA was transformed into Agrobacterium tumefaciens EHA105 by electroporation. The plasmid was extracted and sequenced for verification. The recombinant strain with the correct sequence was named EHA-GmZG3-sgRNA, and the sequence of the sequencing primer SQ was: 5'-GATGAAGTGGACGGAAGGAAGGAG-3' (SEQ ID NO.5).

[0035] (2) Agrobacterium-mediated transformation

[0036] The constructed EHA-GmZG3-sgRNA was transformed into the soybean variety Jack (hereinafter referred to as wild-type soybean) using Agrobacterium-mediated transformation. The specific method is as follows:

[0037] 1. Seed sterilization

[0038] ① Take healthy, plump, uniform, and dry Jack soybean seeds that are free from pests, diseases, and spots, spread them evenly in a petri dish, and then place the petri dish in a desiccator;

[0039] ②After completing step ①, place a 100mL beaker in the desiccator, pour 80mL of 12M sodium hypochlorite aqueous solution into the beaker, then slowly add 4mL of concentrated hydrochloric acid, and then quickly cover the desiccator, seal it with petroleum jelly, and let it stand for 16 hours for chlorine sterilization.

[0040] 2. Preparation of infecting bacterial suspension

[0041] ① The EHA-GmZG3-sgRNA bacterial culture obtained above was cultured at 28℃ and resuspended in liquid culture medium to obtain an infected bacterial culture with OD600nm=0.6;

[0042] ② Place the treated seeds in a clean bench and, under a microscope, peel off the seed coat, separate the two cotyledons along the long axis, and keep the cotyledon with the complete hypocotyl. Make a scratch at the junction of the hypocotyl and cotyledon, usually 3 scratches per cotyledon. Then, soak the seeds in a 28℃ incubator for 2 hours.

[0043] ③ Place the cotyledons with the inner (smooth) side up on a co-culture medium lined with sterile filter paper and incubate in the dark at 22°C for 5 days;

[0044] ④ After 5 days of co-culture, the hypocotyl of the explants elongated to 2 cm. Part of the hypocotyl was cut off, leaving 0.5 cm. The treated explants were then placed in recovery medium and cultured for 7 days at 28℃ under 16h light / 8h dark conditions.

[0045] ⑤ Remove the explants from the recovery medium, remove the new shoots, cut off part of the hypocotyl, leaving 0.5 cm of hypocotyl, and then transfer the trimmed explants into the selection medium and culture them for 21 days under 28℃ 16h light / 8h dark conditions.

[0046] ⑥ After 21 days of selection and induction, the explants produced a large number of adventitious buds. The cotyledons and brown leaves were removed, and the remaining parts were transferred to elongation medium for culture at 28℃ under 16h light / 8h darkness conditions.

[0047] ⑦ In the elongation medium, when the clustered buds produce 8cm young stems, cut them off from the base of the adventitious buds; dip the stem base in 1mg / L IBA solution for 1min, and then transfer it to the rooting medium for culture. Culture for one week at 28℃ under 16h light / 8h darkness conditions. After a large number of roots are produced at the base of the stem, transplant it into pots. The resulting plants are T0 generation transformed soybeans.

[0048] (3) Molecular detection of GmZG3 gene mutant plants

[0049] DNA was extracted from T0 generation transformed soybean leaves as a template for PCR molecular detection, with wild-type soybean as a control. PCR primers were designed near the GmZG3 gene target site for PCR amplification and sequencing. The PCR reaction system consisted of: 12.5 μL 2×PhantaMax Buffer, 0.5 μL dNTP Mix (10 mM), 1 μL DNA (200 ng / μL), 1 μL ZG3-F (10 pmol / μL), 1 μL ZG3-R (10 pmol / μL), 0.5 μL Super-FideLity DNA PoLymerase, and 8.5 μL ddH2O, for a total volume of 25 μL. Amplification reaction system: 95℃ for 3 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 1 min, 35 cycles; 72℃ for 5 min; Amplification primers for GmZG3 gene are: ZG3-F: 5'-CTTTCATGCCCCTGCCCCTGC-3' (SEQ ID NO.6); ZG3-R: 5'-AAGAGAGAAGATGGACCGAGGA-3' (SEQ ID NO.7).

[0050] Plants exhibiting overlapping peaks near the target site were identified as heterozygous edited plants and named T0 generation GmZG3 soybeans. After sowing T0 generation GmZG3 soybeans, seeds of T1 generation GmZG3 soybeans were harvested and cultured to obtain T1 generation GmZG3 soybeans.

[0051] Using the above-mentioned PCR molecular detection method to detect T1 generation GmZG3 soybeans, sequencing results showed that in T1 generation GmZG3 soybeans, the mutant plants (GmZG3) produced any one of the following mutations near the target site, causing premature termination of protein translation:

[0052] (1) Add an A base between positions 764 and 765 of the soybean GmZG3 gene nucleotide sequence (see details). Figure 1 Mutation type 1);

[0053] (2) Add one T base between positions 764 and 765 of the soybean GmZG3 gene nucleotide sequence (see details). Figure 1 Mutation type 2);

[0054] (3) Delete bases 758-764 of the soybean GmZG3 gene nucleotide sequence (see details) Figure 1 Mutation type 3).

[0055] T1 generation soybean mutants with the above-mentioned GmZG3 gene mutation type were further cultured and screened to obtain T2 generation homozygous soybean mutants without transgenic elements, and phenotypic identification was performed.

[0056] (4) Mutant strain type identification

[0057] Wild-type control plants and homozygous mutants of the GmZG3 gene were planted in a greenhouse under a 16-hour long-day condition for 30 days. The plant height and number of nodes of the wild-type control plants and homozygous mutants were counted respectively.

[0058] The results are as follows Figure 3 and Figure 4 As shown, compared with the control plant height of 30.8 cm, the mutant plant height averaged 15.6 cm. The height of the GmZG3 gene homozygous mutant was significantly lower than that of the wild-type control plant. In terms of the number of plant nodes, the control plant had an average of 9 nodes, while the mutant plant had an average of 8.8 nodes. There was no significant change in the number of plant nodes between the mutant and control plants.

[0059] Example 3: Obtaining and phenotypic identification of GmZG3 overexpressing plants

[0060] (1) Cloning of the GmZG3 gene

[0061] RNA was extracted from leaves of the cultivated soybean variety Jack and synthesized into cDNA via reverse transcription. Using the cDNA as a template, PCR amplification was performed using a novel primer pair consisting of primers F and R, and the PCR products were recovered. The nucleotide sequences of primers F and R are shown below:

[0062] F: 5'-ATGAAGAGGGATCACCGAG-3' (SEQ ID NO.8);

[0063] R: 5'-CTACAGTTTACAGTGCAAG-3' (SEQ ID NO. 9).

[0064] (2) Construction of GmTZG3 overexpression vector

[0065] ① Using the PCR amplification product obtained above as a template, PCR amplification was performed using a primer pair consisting of primers F-inf and R-inf, and the PCR amplification product was recovered. The nucleotide sequences of primers F-inf and R-inf are shown below:

[0066] F-inf: 5'-GAGAACACGGGGGACTCTAGAATGAAGAGGGATCACCGAG-3' (SEQ ID NO. 10);

[0067] R-inf: 5'-CCCTTGCTCACCATTCTAGACAGTTTACAGTGCAAG-3' (SEQ ID NO. 11).

[0068] ② The PTF101-GFP vector was digested with the restriction endonuclease XbaⅠ, and the vector backbone was recovered. (The PTF101-GFP vector was constructed by double digesting the PTF101 vector with restriction endonucleases XbaⅠ and SacⅠ and then linking the GFP gene to the PTF101 vector backbone.)

[0069] ③ Ligate the PCR product obtained in step ① and the vector backbone obtained in step ②, and perform recombinant expression to obtain the recombinant expression vector PTF-GmZG3 (the recombinant expression vector PTF-GmZG3 is obtained by inserting the GmZG3 gene shown in sequence 1 of the sequence listing into the PTF101-GFP vector).

[0070] (3) Agrobacterium-mediated transformation

[0071] The recombinant expression vector PTF-GmTZG3 was transformed into Agrobacterium tumefaciens EHA101 to obtain the recombinant strain EHA101 / PTF101-GmZG3. Then, the constructed EHA101 / PTF101-GmZG3 was transformed into the soybean variety Jack using the Agrobacterium-mediated transformation method, including the following steps:

[0072] ① Take healthy, plump, uniform, and dry Jack soybean seeds (hereinafter referred to as wild-type soybeans) that are free from pests, diseases, and spots. Spread them completely flat in a petri dish, then place the petri dish in a desiccator. Place a 100mL beaker in the desiccator, pour 80mL of 12M sodium hypochlorite solution into the beaker, and then slowly add 4mL of concentrated hydrochloric acid. Then quickly cover the desiccator, seal it with petroleum jelly, and sterilize it with chlorine gas after 12-16 hours.

[0073] ②Preparation of infecting bacterial solution

[0074] 1) Incubate EHA101 / PTF101-GmZG3 Agrobacterium tumefaciens in a 28℃ incubator, then resuspend it in liquid culture medium to obtain an infected bacterial suspension with OD600nm=0.6;

[0075] 2) Place the germinated seeds in a clean bench and, under a microscope, peel off the seed coat, separate the two cotyledons along the long axis, and keep the cotyledon with the complete hypocotyl. Make 3-5 cuts at the junction of the hypocotyl and cotyledon. Then incubate at 28°C for 2 hours.

[0076] 3) Place the infected cotyledons with the inner surface (smooth side) facing up on a co-culture medium lined with sterile filter paper, and incubate in the dark at 22°C for 5 days.

[0077] 4) After co-culturing for 5 days, the hypocotyl of the explants elongated to 1-2 cm. Part of the hypocotyl was cut off, leaving 0.5 cm. The treated explants were then placed in recovery medium and cultured for 7 days at 28℃ under 16h light / 8h dark conditions.

[0078] 5) Remove the explants from the recovery medium, remove the new shoots, cut off part of the hypocotyl, leaving 0.5 cm of the hypocotyl, and then transfer the trimmed explants into the selection medium and culture them for 21 days under 28℃ 16h light / 8h dark conditions.

[0079] 6) After 21 days of selection and induction, the explants produced a large number of adventitious buds. The cotyledons and brown leaves were removed, and the remaining parts were transferred to elongation medium for culture. The explants were cultured at 28℃ under 16h light / 8h dark conditions. When the clustered buds produced 5-8cm young stems, they were cut off from the base of the adventitious buds. The stem base was soaked in 1mg / LIBA solution for 1min, and then transferred to rooting medium for culture. The explants were cultured at 28℃ under 16h light / 8h dark conditions for one week. After a large number of roots were produced at the base of the stem, the explants were transplanted into pots. The resulting plants were T0 generation transformed soybean plants.

[0080] (4) Identification of transgenic plants

[0081] Leaves were taken from the transformed soybean plants and placed in a 1.5 mL centrifuge tube containing 200 μL of sterile water. The leaves were then homogenized. A PAT test strip (containing the bar gene as the selection gene on the vector used for transformation) was placed in the tube, and the number of bands appearing on the strip was recorded within 15 minutes. Two bands indicate a positive transformation of the soybean, while one band indicates a negative transformation.

[0082] (5) Phenotypic identification of transgenic plants

[0083] T2 generation transgenic soybean plants were grown in pots under greenhouse conditions, and plant height was recorded. Results showed that the wild-type control plant height was 70.14 cm, while overexpression plant 1 and overexpression plant 2 had heights of 124.14 cm and 122.29 cm, respectively, indicating a significant increase in plant height for the overexpression plants (see...). Figure 5 ).

[0084] The nucleotide sequence of the soybean GmZG3 gene in this invention is shown below:

[0085]

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Soybeans GmZG3 The application of genes in regulating soybean plant height is characterized by, The GmZG3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The soybean according to claim 1 GmZG3 The application of genes in regulating soybean plant height is characterized by, The soybean plant height regulation method is based on overexpression of soybean. GmZG3 Genes can increase the plant height of soybeans.

3. A method for reducing soybean plant height, characterized in that, By making soybeans GmZG3 The gene is mutated to reduce the plant height of soybeans; the nucleotide sequence of the GmZG3 gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, The soybean GmZG3 The specific mutation of the gene is: in soybeans GmZG3 Add an A base between positions 764 and 765 of the gene's nucleotide sequence; or add a soybean base between positions 764 and 765. GmZG3 Add a T base between positions 764 and 765 of the gene's nucleotide sequence; or add a soybean base between positions 764 and 765. GmZG3 The nucleotide sequence of the gene is missing bases 758-764.

5. A method for preparing a dwarf soybean variety, characterized in that, By making soybeans GmZG3 Gene mutations are used to prepare dwarf soybean varieties; wherein, the... GmZG3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The preparation method according to claim 5, characterized in that, The specific mutations in the soybean GmZG3 gene are as follows: adding an A base between positions 764 and 765 of the soybean GmZG3 gene nucleotide sequence; or adding a T base between positions 764 and 765 of the soybean GmZG3 gene nucleotide sequence; or deleting bases from positions 758 to 764 of the soybean GmZG3 gene nucleotide sequence.

7. A method for preparing a tall soybean variety, characterized in that, By overexpressing soybean GmZG3 Genes are used to prepare tall soybean varieties; wherein, the GmZG3 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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