Use of glyma.07g090400 gene in regulating soybean yield and quality

Abstract

The application discloses Glyma.07G090400 application of a gene in regulating soybean yield and quality belongs to the technical field of genetic engineering. Glyma.07G090400 The CDS sequence of the gene is shown as SEQ ID NO. 1. Glyma.07G090400 The application develops a gene for simultaneously improving soybean yield and protein content Glyma.07G090400 Compared with a wild type strain, the transgenic soybean seed with overexpressed gene has obvious advantages, and has higher long diameter, short diameter, hundred-grain weight and protein content. The application provides new gene resources and technical support for soybean trait improvement, high-yield soybean and high-quality soybean breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to... Glyma.07G090400 Application of genes in regulating soybean yield and quality. Background Technology

[0002] Soybeans are an important cereal crop, with seeds typically containing about 40% protein and 20% oil, making them a sustainable source of plant protein and oil. Improving the protein content, oil content, or yield of soybean seeds has significant economic and practical value. Existing genes related to soybean protein include... Glyma.20G085100 ( POWR1 ), Glyma.20G005900 ( GmSop20 ), Glyma.08G35760 ( HSSP1 These genes, such as [list of genes], play a role in soybean protein regulation, but they all have significant limitations. They cannot achieve a synergistic increase in soybean seed protein content and yield; some genes even negatively impact soybean yield while affecting protein content. Although genes affecting soybean seed protein content have been identified, no gene has been found that can simultaneously increase both. Therefore, further research and application of genes regulating soybean seed protein content and yield are of significant theoretical and practical value for breeding high-yielding, high-protein soybean varieties, ensuring soybean quality and yield, and promoting the sustainable development of the soybean industry. Summary of the Invention

[0003] The purpose of this invention is to provide Glyma.07G090400 This invention addresses the problems of existing technologies by applying genes to regulate soybean yield and quality. Specifically, it develops a gene that simultaneously increases soybean yield and protein content. Glyma.07G090400 It has been confirmed that, compared to wild-type strains, overexpression... Glyma.07G090400 The genetically modified soybean seeds exhibit significant advantages, with higher long diameter, short diameter, 100-seed weight, and protein content. This invention provides new genetic resources and technical support for soybean trait improvement, high-yield soybean breeding, and high-quality soybean breeding.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides Glyma.07G090400 The application of genes in regulating soybean yield and quality, the aforementioned Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation of soybean yield and quality refers to the process of overexpressing the aforementioned... Glyma.07G090400 Genes to increase soybean yield and soybean seed protein content.

[0005] The present invention also provides the application of Glyma.07G090400 protein in regulating soybean yield and quality, wherein the amino acid sequence of Glyma.07G090400 protein is shown in SEQ ID NO.2; The regulation of soybean yield and quality refers to increasing soybean yield and soybean seed protein content by increasing the expression level of the Glyma.07G090400 protein in soybeans.

[0006] This invention also provides an overexpression Glyma.07G090400 The application of recombinant gene vectors in regulating soybean yield and quality, the aforementioned Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation of soybean yield and quality refers to the process of overexpressing the aforementioned... Glyma.07G090400 Genes to increase soybean yield and soybean seed protein content.

[0007] This invention also provides an overexpression Glyma.07G090400 The application of recombinant microorganisms in regulating soybean yield and quality, the Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation of soybean yield and quality refers to the process of overexpressing the aforementioned... Glyma.07G090400 Genes to increase soybean yield and soybean seed protein content.

[0008] This invention also provides Glyma.07G090400 The application of genes in breeding high-yield or high-quality soybean varieties, the aforementioned Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

[0009] The present invention also provides the application of Glyma.07G090400 protein in the breeding of high-yield soybean varieties or high-quality soybean varieties, wherein the amino acid sequence of Glyma.07G090400 protein is shown in SEQ ID NO.2; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

[0010] This invention also provides an overexpression Glyma.07G090400 The application of gene recombinant vectors in breeding high-yield or high-quality soybean varieties, the aforementioned Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

[0011] This invention also provides an overexpression Glyma.07G090400 The application of recombinant microorganisms in breeding high-yield or high-quality soybean varieties, the aforementioned Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

[0012] This invention also provides a method for breeding high-yielding soybean varieties, including using genetic transformation technology to... Glyma.07G090400 The steps involved in transferring genes into soybeans and stably overexpressing them to obtain high-yielding soybean varieties. The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0013] This invention also provides a method for breeding high-quality soybean varieties, including using genetic transformation technology to... Glyma.07G090400 The steps involved in transferring genes into soybeans and stably overexpressing them to obtain high-quality soybean varieties. The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

[0014] The present invention discloses the following technical effects: This invention achieves this by overexpressing in soybeans Glyma.07G090400 Genes, constructing overexpression Glyma.07G090400 Transgenic lines, by examining the seed traits produced, were found to have overexpression of [a specific gene / form] compared to wild-type lines. Glyma.07G090400 The genetically modified seeds exhibit significant advantages, with higher long diameter, short diameter, 100-seed weight, and protein content, indicating that... Glyma.07G090400 This is a gene that has the potential to increase seed protein content and yield, and can be used for crop yield and protein variety improvement. This invention develops a gene that simultaneously increases soybean yield and protein content. Glyma.07G090400 This provides new genetic resources and technical support for improving soybean traits and breeding high-yield and high-quality soybeans. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 For wild-type "Zhonghuang 13" and overexpression Glyma.07G090400Results of detection of target gene expression levels in genetically modified soybean seeds; Figure 2 For wild-type "Zhonghuang 13" and overexpression Glyma.07G090400 Results of protein content analysis in genetically modified soybean seeds; Figure 3 For wild-type "Zhonghuang 13" and overexpression Glyma.07G090400 Results of the detection of the length and diameter of genetically modified soybean seeds; Figure 4 For wild-type "Zhonghuang 13" and overexpression Glyma.07G090400 Results of testing for short diameter of genetically modified soybean seeds; Figure 5 For wild-type "Zhonghuang 13" and overexpression Glyma.07G090400 Results of testing 100 seeds of genetically modified soybeans. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Glyma.07G090400 The CDS sequence of the gene is as follows: ATGGCAAGTTATCAGAAGCACTACGATGATCAGGGTCGCAAGGTCGACGAGTATGGCAATGTTGAGAGGCAAACTGACGAATATGGCAACCCGGTTCATGCCACTAGTGTCACTTATGTAGCCACCAAAAGTGTTGGTGGCTACAATGATGACGCTAATAAGCAACATGATATTACTGGTGTCTATCCCGAAAAAGACACCGGTAGACATCATTTTGGTCGTGGTTACGACGGTGACACTAACAAGCAACATGATGCTACTGGTGTTTATCCCGGAATAGACATTGGTAGAGATCATGGGACTACCGGTGTTTATGGCCTAAACACCGACAGACATCATGGAAGTACTGGTGTCAATCCCGGGATAGACACCCATAACCAACAACATGGGACTACCGGTGGTTATGCTGGTGACACTGGAAGGCAGCATGGGAATACAGGTGGCCTTTACTATGGAACCGACACCGCGGACACAGGAGCCGGTCCTAGAAGTGGAAACACCGGTGGCACCGGTTATGGAGGCACTGGTGGTACTGATTATGGAACAGCTGGTGGCACTGGTTATGGAAGTGGAACTGGCTATGGAATCAACACTGGGGGTGCGCACACTGAAGCAGGGTATGGGAAGGAGCATCGTCAGCATGAGCAATCTCATGGTGGTCAGCACGAGAAGAAAGGGATACTGGACAAGATTAAGGAGAAGCTTCCTGGAGGACACAGTGACAAGTAG, SEQ ID NO.1; Glyma.07G090400 The amino acid sequence of the protein encoded by the gene is as follows: MASYQKHYDDQGRKVDEYGNVERQTDEYGNPVHATSVTYVATKSVGGYNDDANKQHDITGVYPEKDTGRHHFGRGYDGDTNKQHDATGVYPGIDIGRDHGTTGVYGLNTDRHHGSTGVNPGID SEQ ID NO.2.

[0023] The overexpression vector pSM101-3HA used in this invention is disclosed in the literature "Zhu Qun, Guo Xiaoli, Zhang Lei". GmMYB15 Positive regulation of soybean resistance to soybean cyst nematode [J]. Plant Protection, 2024, 50(06):42-50. DOI:10.16688 / j.zwbh.2024186.”

[0024] Example 1 1. Glyma.07G090400 Acquisition of gene coding regions (1) Take 100 mg of “Zhonghuang 13” soybean seeds and put them into a 2 mL grinding tube. Add 1 mL of Trizol and homogenize thoroughly. Let stand at room temperature for 10 min. (2) Add 0.2 mL of chloroform, shake well for 15 s, and let stand for 2 min; (3) Centrifuge at 12000 rpm for 15 min at 4℃, and transfer 0.5 mL of the supernatant to a 1.5 mL centrifuge tube; (4) Add 0.5 mL of pre-cooled isopropanol to the tube, mix by inverting the tube, and let stand at room temperature for 10 min. (5) Centrifuge at 4℃ and 12000 rpm for 15 min, then discard the supernatant; (6) Add 1 mL of 75% ethanol, wash the precipitate, centrifuge at 12000 rpm for 2 min at 4℃, and discard the supernatant; (7) Dry the RNA precipitate and add an appropriate amount of enzyme-free water to dissolve the RNA precipitate. (8) cDNA was obtained by reverse transcription using the Novizan Reverse Transcription Kit R333; (9) Using cDNA as a template, amplify according to the reaction system shown in Table 1 and the reaction procedure below. Glyma.07G090400 The coding region sequence of the gene. The primer sequences used are as follows: Forward primer: ATGGCAAGTTATCAGAAGCACTACG, SEQ ID NO.3; Reverse primer: CTACTTGTCACTGTGTCCTCCAGGA, SEQ ID NO.4; Table 1 PCR reaction system The reaction program was as follows: 98℃ for 5 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 10 s, 35 cycles; 72℃ for 5 min. (10) Sequencing the amplification products yielded... Glyma.07G090400 The coding region sequence of the gene is shown in SEQ ID NO.1.

[0025] 2. Glyma.07G090400 Construction of gene overexpression vectors (1) Using the amplification product obtained in step 1 as a template, amplify the homologous arms of the vector according to the reaction system shown in Table 2 and the following reaction procedure. Glyma.07G090400 The coding region sequence of the gene, and the primer sequences used are as follows: Forward primer: atgttgactcgacagtcgacATGGCAAGTTATCAGAAGCACTACG, SEQ ID NO.5; Reverse primer: cggggaaattcgagcggatccCTACTTGTCACTGTGTCCTCCAGGA, SEQ ID NO.6; Table 2 PCR reaction system The reaction program was as follows: 98℃ for 5 min; 98℃ for 10 s, 60℃ for 5 s, 72℃ for 10 s, 35 cycles; 72℃ for 5 min. (2) Following the reaction system shown in Table 3 and the reaction procedure below, the pSM101-3HA vector was linearized by double enzyme digestion to obtain the linearized vector; the enzyme digestion conditions were 37℃ for 3 h. Table 3 Enzyme digestion system (3) Recombinant vectors were constructed using the CloneExpress® II One Step Cloning Kit from Novizan. The recombinant vectors were obtained according to the reaction system shown in Table 4 and the following reaction procedure; 37°C, 30 min; immediately cooled to 4°C; Table 4 Recombinant Vector Construction System (4) Transform the product obtained in step (3) into Tiangen's DH5α competent cells, and extract plasmids according to the instructions to obtain... Glyma.07G090400 Gene overexpression recombinant vector.

[0026] 3. Overexpression Glyma.07G090400 Stable plant creation and positive detection of genes 3.1 Glyma.07G090400 Gene overexpression recombinant vector transformed into EHA105 Agrobacterium (1) Take 1 µL of plasmid and add it to 50 µL of EHA105 Agrobacterium competent cells. After mixing thoroughly, transfer it to an electroporation cup. (2) After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate in a shaker at 30°C and 180 rpm for 30 min. (3) Take 50 µL of the activated Agrobacterium tumefaciens culture and inoculate it onto LB solid medium. Incubate in the dark at 30°C for 48 h.

[0027] 3.2 Soybean genetic transformation (1) Select plump, disease-free mature “Zhonghuang 13” soybean seeds and sterilize them for 14-16 hours using chlorine disinfection (add 5 mL of concentrated hydrochloric acid to 100 mL of sodium hypochlorite with an effective concentration of 10%). This experiment was conducted in a fume hood. (2) Inoculate the sterilized seeds with the hilum facing down onto the germination medium and place them in a 25°C incubator for dark incubation for 1 day; (3) Carrying Glyma.07G090400 Agrobacterium overexpression vectors were initially activated by spreading them on culture medium. After 48 hours of incubation, the cells were collected and activated again on fresh culture medium. After 24 hours of incubation, the cells were collected in the infection solution, vortexed to mix, and the OD of the bacterial culture was adjusted. 600 =0.5 available; (4) After the germinated soybeans are scratched, they are placed in the prepared bacterial solution for 2 hours and then the solution is discarded. (5) Then place the explants on a culture medium lined with filter paper and culture them together at 25°C in the dark for 3-5 days; (6) Select explants that are free from contamination after co-culture, cut off the end of the hypocotyl, insert them into the recovery solid culture medium, and recover culture for 7-10 days; (7) The explants that have grown clustered buds after recovery culture were inoculated onto the selection medium and cultured for 16h / 8h (light / dark) for 21 days; (8) When the young shoots grow to about 5 cm, they are transferred to the rooting medium for further screening. They are cultured for 16 h / 8 h (light / dark) for 21 days. (9) Take half a leaf and test it using transgenic PAT / bar colloidal gold test strips; (10) Remove the positive seedlings detected by Bar test strips from the culture medium, wash off the culture medium attached to the seedling roots, and transplant them into seedling trays filled with nutrient soil. Cultivate at 27℃ for 16h / 8h (light / dark) and harden off for 21-28 days. (11) Transfer the seedlings to a greenhouse to produce seeds, and harvest seeds from each individual plant.

[0028] 4. Positive identification of genetically modified soybean seeds (1) RNA was extracted from transgenic soybean seeds and reverse transcribed into cDNA; (2) Using qRT-PCR technology to detect the presence of transgenic soybean seeds Glyma.07G090400 Gene expression was detected; the reaction system is shown in Table 5, and the reaction procedure is shown in Table 6. The primer sequences used are as follows: Forward primer: TCGCAAGGTCGACGAGTATG, SEQ ID NO.7; Reverse primer: CACCATCGTAACCACGACCA, SEQ ID NO.8.

[0029] The results are as follows Figure 1 As shown. After transformation with the expression vector, the soybean seeds... Glyma.07G090400 Gene expression levels were significantly higher than those of wild-type “Zhonghuang 13” seeds.

[0030] Table 5 Reaction System Table 6 Reaction Procedure 5. Overexpression Glyma.07G090400 Determination of protein content and yield indicators in genetically modified soybean seeds 5.1 Determination of Soybean Seed Protein Content The protein content of soybean seeds was determined using the method described in NY / T 3-1982 standard.

[0031] 5.2 Determination of soybean seed yield indicators (1) Randomly select 10 seeds and use vernier calipers to measure the long diameter and short diameter; (2) Randomly select 100 seeds and use a 1 / 1000 balance to measure the weight of 100 seeds.

[0032] The results are as follows Figure 2-Figure 5 As shown, it can be seen that overexpression Glyma.07G090400 The protein content, seed diameter, and 100-seed weight of the genetically modified soybean were significantly higher than those of wild-type soybean seeds. This result indicates that... Glyma.07G090400The gene is a potential target for increasing seed protein content and yield, and can be used to improve crop yield and protein content, as well as to develop new high-quality and high-yield soybean varieties.

[0033] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Glyma.07G090400 The application of genes in regulating soybean yield and quality is characterized by, The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation of soybean yield and quality refers to the process of overexpressing the aforementioned... Glyma.07G090400 Genes to increase soybean yield and soybean seed protein content.

2. The application of Glyma.07G090400 protein in regulating soybean yield and quality, characterized by, The amino acid sequence of the Glyma.07G090400 protein is shown in SEQ ID NO.2; The regulation of soybean yield and quality refers to increasing soybean yield and soybean seed protein content by increasing the expression level of the Glyma.07G090400 protein in soybeans.

3. An overexpression Glyma.07G090400 The application of recombinant gene vectors in regulating soybean yield and quality is characterized by, The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation of soybean yield and quality refers to the process of overexpressing the aforementioned... Glyma.07G090400 Genes to increase soybean yield and soybean seed protein content.

4. An overexpression Glyma.07G090400 The application of recombinant microorganisms in regulating soybean yield and quality is characterized by, The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation of soybean yield and quality refers to the process of overexpressing the aforementioned... Glyma.07G090400 Genes to increase soybean yield and soybean seed protein content.

5. Glyma.07G090400 The application of genes in breeding high-yield or high-quality soybean varieties is characterized by, The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

6. The application of Glyma.07G090400 protein in the breeding of high-yield or high-quality soybean varieties, characterized in that... The amino acid sequence of the Glyma.07G090400 protein is shown in SEQ ID NO.2; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

7. An overexpression Glyma.07G090400 The application of gene recombinant vectors in breeding high-yield or high-quality soybean varieties is characterized by, The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

8. An overexpression Glyma.07G090400 The application of recombinant microorganisms in breeding high-yield or high-quality soybean varieties is characterized by, The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

9. A method for cultivating a high-yielding soybean variety, characterized in that, This includes using genetic transformation technology to... Glyma.07G090400 The steps involved in transferring genes into soybeans and stably overexpressing them to obtain high-yielding soybean varieties. The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.

1.

10. A method for cultivating a high-quality soybean variety, characterized in that, This includes using genetic transformation technology to... Glyma.07G090400 The steps involved in transferring genes into soybeans and stably overexpressing them to obtain high-quality soybean varieties. The Glyma.07G090400 The CDS sequence of the gene is shown in SEQ ID NO.1; The high-quality soybean varieties refer to soybean varieties with high seed protein content.

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