Application of soybean GmZG3 gene in regulation and control of soybean plant height

Abstract

The invention discloses application of a soybean GmZG3 gene in regulation and control of soybean plant height, and belongs to the technical field of gene engineering. According to the present invention, Cas9-sgRNA and PTF-GmZG3 recombinant vectors of the soybean GmZG3 gene are constructed, the Cas9-sgRNA and PTF-GmZG3 recombinant vectors are transferred into agrobacterium to obtain EHA-GmZG3-sgRNA and EHA101 / PTF101-GmZG3 recombinant strains, and the constructed recombinant strains are converted into wild soybeans through an agrobacterium-mediated method so as to obtain the GmZG3 gene mutant and the overexpression plant. The phenotypes of the mutant and the overexpressed plant are identified, and the plant height of the mutant plant is obviously lower than that of the wild type soybean, and the plant height of the overexpressed plant is obviously higher than that of the wild type soybean. Therefore, the invention provides a method for genetically improving the soybean plant type, and the method has potential market value and wide application prospect in the agricultural field.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an application of soybean GmZG3 gene in regulating soybean strain growth. Background Art

[0002] Soybean is an important grain, oil and feed crop in my country, and plays an indispensable role in ensuring food security and agricultural product trade. However, facing the limited soybean planting area in my country, how to use modern biological breeding technology to efficiently and quickly improve soybean varieties and increase yields has become a production problem and technical bottleneck that needs to be solved urgently.

[0003] Crop plant type plays a decisive role in the formation of individual and group morphology, and is an important factor affecting plant yield, production level and economic benefits. The composition of plant type includes plant height, branching (tillering), leaf shape and ear type (pod-setting habit), etc. In-depth research and exploration of plant type regulatory genes will not only help to identify excellent soybean plant type genes and cultivate high-yield ideal plant types, but also through the development of specific materials, systematically evaluate and optimize the ideal plant type under actual production conditions. Therefore, it is urgent to develop a new method to improve soybean plant height, so as to promote the application of soybean breeding and increase soybean yield. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of soybean GmZG3 gene in regulating soybean plant height, so as to provide a new method for improving soybean plant height.

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

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

[0007] Furthermore, regulating the soybean plant type to overexpress the soybean GmZG3 gene can increase the soybean plant height.

[0008] Furthermore, the soybean GmZG3 gene mutates by inserting or deleting bases at any position of the GmZG3 gene, thereby causing the GmZG3 gene to lose its function.

[0009] Furthermore, the mutation of soybean GmZG3 gene is specifically: adding a base A between the 764th and 765th positions of soybean GmZG3 gene nucleotide sequence; or adding a base T between the 764th and 765th positions of soybean GmZG3 gene nucleotide sequence; or deleting bases at positions 758-764 of soybean GmZG3 gene nucleotide sequence.

[0010] The present invention provides a preparation capable of regulating soybean plant type, comprising the above-mentioned soybean GmZG3 gene or a protein encoded by the above-mentioned soybean GmZG3 gene.

[0011] The invention provides a preparation capable of regulating soybean plant type, the preparation comprising a reagent capable of causing mutation of the soybean GmZG3 gene.

[0012] The invention provides a preparation capable of regulating soybean plant type, wherein the preparation comprises a reagent capable of overexpressing the soybean GmZG3 gene.

[0013] The invention provides a method for preparing a dwarf soybean variety, and the dwarf soybean variety is prepared by mutating the soybean GmZG3 gene.

[0014] The present invention also provides a method for preparing a tall soybean variety, and the tall soybean variety is prepared by overexpressing the soybean GmZG3 gene.

[0015] The present invention has the following beneficial effects: the present invention constructs a Cas9-sgRNA and PTF-GmZG3 recombinant vector of the soybean GmZG3 gene, transfers the vector into Agrobacterium to obtain EHA-GmZG3-sgRNA and EHA101 / PTF101-GmZG3 recombinant strain, and then transforms the constructed recombinant strain into wild-type soybean by Agrobacterium-mediated method, thereby obtaining a mutant and an overexpression plant of the GmZG3 gene. The phenotypes of the mutant and the overexpression plant were identified, and it was found that the plant height of the mutant plant was significantly lower than that of the wild-type soybean, while the plant height of the overexpression plant was significantly higher than that of the wild-type soybean. Therefore, the present invention provides a method for genetically improving soybean plant type, which has potential market value and broad application prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0019] Figure 4 is the number of plant nodes of the GmZG3 gene mutant;

[0020] Figure 5 is the plant height of the GmZG3 gene overexpressing plants. DETAILED DESCRIPTION

[0021] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

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

[0023] (1) Obtaining sgRNA

[0024] The nucleotide sequence of the soybean GmZG3 genome coding region (as shown in SEQ ID NO.1) was obtained from the Phytozome database. The GmZG3 gene is located on soybean chromosome 18. The GmMBR1 sgRNA target site sequence 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 is selected, it needs to be integrated into the vector. First, the target site primers of the sgRNA are synthesized. 5uL of GmZG3-F and GmZG3-R primers are added to the 25uL reaction system, 15uL of water, 95℃3min, 0.1℃ / s annealing to 16℃, 16℃ for 10min, and annealing is completed to obtain gRNA annealing products with sticky ends.

[0025] The target site sequence is:

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

[0027] The sequences of the target site primers are:

[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 expressing sgRNA

[0031] Take 1uL of the gRNA annealing product with sticky ends obtained above and connect it with the cas9 / gRNA vector (Beijing Weishanglide Biotechnology Co., Ltd., catalog number: VK005-15, which contains the Cas9 protein expression unit) for homologous recombination to obtain the recombinant vector Cas9-sgRNA, which expresses sgRNA.

[0032] Example 2: Acquisition and phenotypic identification of GmZG3 mutants

[0033] (1) Preparation of recombinant bacteria

[0034] The recombinant vector Cas9-sgRNA prepared in Example 1 was transferred into Escherichia coli DH5α, spread on a solid culture medium of LB+Kan (LB medium + kanamycin), and cultured overnight at 37°C. Then, a single clone was picked to extract the plasmid and sequenced. The plasmid with the correct insertion fragment was named recombinant plasmid GmZG3-sgRNA. The recombinant plasmid GmZG3-sgRNA was transformed into Agrobacterium EHA105 by electroporation, and the plasmid was extracted for sequencing verification. The recombinant strain with correct sequencing verification was named EHA-GmZG3-sgRNA, wherein 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 soybean variety Jack (hereinafter referred to as wild-type soybean) by Agrobacterium-mediated method. The specific method is as follows:

[0037] 1. Seed sterilization

[0038] ① Take the soybean variety Jack seeds that are free of pests and spots, plump, uniform, and dry, spread them completely flat in a petri dish, and then put the petri dish into 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, then quickly cover the desiccator, seal it with vaseline, and leave it for 16 hours for chlorine sterilization.

[0040] 2. Preparation of infection solution

[0041] ① Cultivate the EHA-GmZG3-sgRNA bacterial solution obtained above at 28°C, resuspend it in liquid culture medium, and obtain an infection bacterial solution with OD600nm=0.6;

[0042] ②Put the treated seeds in a clean bench, peel off the seed coat under a microscope, separate the two cotyledons along the long axis, keep the cotyledon with a complete hypocotyl, and scratch the connection between the hypocotyl and the cotyledon. Generally, one cotyledon is scratched 3 times, and then immersed in a 28℃ incubator for 2 hours;

[0043] ③ Place the inner surface (smooth surface) of the cotyledon upward on the co-cultivation medium covered with sterile filter paper and culture 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, and part of the hypocotyl was cut off, leaving 0.5 cm. The treated explants were placed in recovery medium and cultured for 7 days at 28°C with 16h light / 8h dark conditions.

[0045] ⑤ Remove the explants from the recovery medium, remove the new buds, cut off part of the hypocotyl, retain 0.5 cm of the hypocotyl, and then transfer the trimmed explants to the screening medium and culture them at 28°C with 16 h light / 8 h dark for 21 days.

[0046] ⑥ After 21 days of screening 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 and cultured at 28°C with 16h light / 8h dark conditions.

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

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

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

[0050] The plants with overlapping peaks near the target site were heterozygous edited plants and were named T0 transgenic GmZG3 soybeans. After sowing T0 transgenic GmZG3 soybeans, the seeds of T1 transgenic GmZG3 soybeans were harvested and cultivated to obtain T1 transgenic GmZG3 soybeans.

[0051] The above-mentioned PCR molecular detection method was used to detect T1-generation GmZG3 soybeans. The sequencing results showed that in the T1-generation GmZG3 soybeans, the mutant plants (GmZG3) had any of the following mutations near the target site, causing protein translation to terminate prematurely:

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

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

[0054] (3) The nucleotide sequence of soybean GmZG3 gene was deleted from bases 758 to 764 (see Figure 1 Mutation type 3).

[0055] The T1 generation soybean mutant plants carrying the GmZG3 gene with the above GmZG3 gene mutation type are further cultivated to screen out T2 generation soybean homozygous mutants without transgenic elements, and the phenotypic identification is performed.

[0056] (4) Identification of mutant strains

[0057] The wild-type control plants and the homozygous mutant of the GmZG3 gene were planted in a greenhouse under 16-h long-day conditions for 30 days, and the plant height and number of nodes of the wild-type control plants and the homozygous mutant 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 average plant height of the mutant plants was 15.6 cm, and the plant height of the homozygous mutant of the GmZG3 gene was significantly lower than that of the wild-type control plants; in terms of the number of plant nodes, the control plants had an average of 9 nodes, and the mutant plants had an average of 8.8 nodes, and there was no significant difference in the number of plant nodes between the mutant plants and the control.

[0059] Example 3: Obtaining GmZG3 overexpressing plants and identifying their phenotypes

[0060] (1) Cloning of GmZG3 gene

[0061] RNA was extracted from the leaves of the cultivated soybean variety Jack, and reverse transcribed to synthesize cDNA. Using cDNA as a template, a primer pair consisting of a new primer F and a primer R was used for PCR amplification, and the PCR amplification product was recovered. The nucleotide sequences of primer F and primer R are as follows:

[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, a primer pair consisting of primer F-inf and primer R-inf is used for PCR amplification, and the PCR amplification product is recovered. The nucleotide sequences of primer F-inf and primer R-inf are as follows:

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

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

[0068] ② Use restriction endonuclease XbaⅠ to digest the PTF101-GFP vector and recover the vector backbone (the PTF101-GFP vector is constructed by double digesting the PTF101 vector with restriction endonucleases XbaⅠ and SacⅠ and then connecting the GFP gene to the PTF101 vector backbone).

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

[0070] (3) Agrobacterium-mediated transformation

[0071] The recombinant expression vector PTF-GmTZG3 is transferred into Agrobacterium tumefaciens EHA101 to obtain the recombinant bacteria EHA101 / PTF101-GmZG3, and then the constructed EHA101 / PTF101-GmZG3 is transformed into soybean variety Jack by Agrobacterium-mediated method, comprising the following steps:

[0072] ① Take the soybean variety Jack (hereinafter referred to as wild soybean) seeds that are free of pests and spots, plump, uniform and dry, and spread them completely in a culture dish, then put the culture dish into a desiccator, put a 100 mL beaker in the desiccator, pour 80 mL of 12M sodium hypochlorite aqueous solution into the beaker, then slowly add 4 mL of concentrated hydrochloric acid, then quickly cover the desiccator, seal it with vaseline, and sterilize it with chlorine after leaving it for 12-16 hours.

[0073] ②Preparation of infection solution

[0074] 1) Cultivate EHA101 / PTF101-GmZG3 Agrobacterium culture in a 28°C incubator, and then resuspend it in liquid culture medium to obtain an infection culture with an OD600nm of 0.6;

[0075] 2) Put the germinated seeds in a clean bench, peel off the seed coat under a microscope, separate the two cotyledons along the long axis, keep the cotyledon with the complete hypocotyl, and scratch the connection between the hypocotyl and the cotyledon. Generally, one cotyledon is scratched 3-5 times, and then infect in a 28℃ incubator for 2 hours;

[0076] 3) Spread the infected cotyledons with the inner surface (smooth surface) facing up on the co-cultivation medium covered with sterile filter paper and culture in the dark at 22°C for 5 days;

[0077] 4) After 5 days of co-culture, the hypocotyl of the explants elongated to 1-2 cm, and part of the hypocotyl was cut off, leaving 0.5 cm, and then the treated explants were placed in recovery medium and cultured at 28°C with 16 h light / 8 h dark conditions for 7 days;

[0078] 5) Remove the explants from the recovery medium, remove the new buds, cut off part of the hypocotyl, retain 0.5 cm of the hypocotyl, and then transfer the trimmed explants to the screening medium and culture them at 28°C with 16 h light / 8 h dark for 21 days;

[0079] 6) After 21 days of screening and induction, the explants produce a large number of adventitious buds. The cotyledons and brown leaves are removed and the remaining parts are transferred to elongation medium for culture at 28°C with 16 h of light and 8 h of darkness. When the clustered buds grow out 5-8 cm young stems, they are cut off from the base of the adventitious buds. The base of the stem is soaked in 1 mg / LIBA solution for 1 min, and then transferred to rooting medium for culture at 28°C with 16 h of light and 8 h of darkness for one week. After a large number of roots are produced at the base of the stem, they are transplanted into pots. The grown plants are T0 generation transformed soybean plants.

[0080] (4) Identification of transgenic plants

[0081] Cut the leaves of the transformed soybean plants, put them into a 1.5mL centrifuge tube containing 200uL of sterile water, and crush the leaves into a homogenate. Put the PAT test strip (the screening gene on the vector used for transformation is the bar gene) into the tube, and record the number of bands on the test strip within 15 minutes. If two bands appear, it means positive transformation of soybeans, and only one band appears for negative transformation of soybeans.

[0082] (5) Phenotypic identification of transgenic plants

[0083] The T2 transgenic plants were planted in pots under greenhouse conditions, and the height of soybean plants was counted. The results showed that the height of wild-type control plants was 70.14 cm, while the heights of overexpression plants 1 and 2 were 124.14 cm and 122.29 cm, respectively. The height of overexpression plants increased significantly (see Figure 5 ).

[0084] The nucleotide sequence of the soybean GmZG3 gene of the present invention is as follows:

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of soybean GmZG3 gene in regulating soybean plant growth, characterized in that: The nucleotide sequence of the GmZG3 gene is shown in SEQ ID NO.

1.

2. The use of the soybean GmZG3 gene in regulating soybean plant type according to claim 1, characterized in that: The method for regulating soybean plant type is to reduce soybean plant height by mutating soybean GmZG3 gene.

3. The use of the soybean GmZG3 gene in regulating soybean plant type according to claim 1, characterized in that: The soybean plant type is regulated by overexpressing the soybean GmZG3 gene, which can increase the soybean plant height.

4. The use of the soybean GmZG3 gene in regulating soybean plant type according to claim 2, characterized in that: The soybean GmZG3 gene mutation occurs by inserting or deleting bases at any position of the GmZG3 gene, thereby causing the GmZG3 gene to lose its function.

5. The use of the soybean GmZG3 gene in regulating soybean plant type according to claim 4, characterized in that: The soybean GmZG3 gene mutation specifically includes: adding a base A between the 764th and 765th positions of the soybean GmZG3 gene nucleotide sequence; or adding a base T between the 764th and 765th positions of the soybean GmZG3 gene nucleotide sequence; or deleting the 758th to 764th bases of the soybean GmZG3 gene nucleotide sequence.

6. A preparation capable of regulating soybean plant type, characterized in that: Comprising the soybean GmZG3 gene according to claim 1 or a protein encoded by the soybean GmZG3 gene.

7. A preparation capable of regulating soybean plant type, characterized in that: The preparation comprises an agent capable of causing mutation of the soybean GmZG3 gene according to claim 1.

8. A preparation capable of regulating soybean plant type, characterized in that: The preparation comprises an agent capable of overexpressing the soybean GmZG3 gene according to claim 1.

9. A method for preparing a dwarf soybean variety, characterized in that: Dwarf soybean varieties were prepared by mutating the soybean GmZG3 gene.

10. A method for preparing a tall soybean variety, characterized in that: Tall soybean varieties were prepared by overexpressing the soybean GmZG3 gene.

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