Method for synergistically improving soybean yield and seed quality

Abstract

The invention belongs to the technical field of plant genetic engineering and molecular breeding, and particularly relates to a method for synergistically improving soybean yield and seed quality. The invention provides a method for synergistically improving soybean yield and seed quality, which comprises the following steps: simultaneously performing targeted editing on soybean cytokinin oxidase / dehydrogenase genes GmCKX3 and GmCKX14 by using a gene editing technology to generate frame-shift mutation and lead to early termination of protein translation. According to the method, two key cytokinin oxidase genes are knocked out at the same time, the negative correlation between the yield and the protein content in traditional soybean breeding is broken, the yield and the protein content of the seeds are increased synchronously, and meanwhile the stability of the oil content is kept.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering and molecular breeding technology, specifically involving a method for synergistically improving soybean yield and seed quality. Background Technology

[0002] Soybeans, as a globally important dual-purpose crop for both food and oil, are one of the main sources of high-quality plant protein and edible oil for humankind. Improving soybean yield and quality (especially protein content) has always been a core objective of breeding efforts. However, there is usually a significant negative correlation between soybean yield and quality traits (such as protein and oil content); that is, high yield is often accompanied by a decrease in protein content or a reduction in oil content. This has become a long-standing challenge in soybean genetic improvement.

[0003] Several existing technologies have disclosed methods to improve specific traits in soybeans by regulating single genes or using molecular marker-assisted selection. For example, CN118291671A discloses the gene GmPGK5, which is related to soybean oil content and 100-grain weight; CN110041416A discloses the application of the GmABCA9 gene in improving soybean protein content and grain weight; and CN106701784A discloses the application of the oil body protein gene GmOLE01 in improving soybean oil content. However, these technologies all focus on the genetic improvement of a single trait and have failed to achieve a synergistic improvement in yield and quality traits.

[0004] Currently, there are no reports of existing technical solutions that can effectively break the negative correlation between soybean yield and quality traits, achieve synergistic improvement in yield and protein content, and maintain stable oil content. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a method for synergistically improving soybean yield and seed quality.

[0006] This application provides a method for synergistically improving soybean yield and seed quality, including:

[0007] Gene editing was performed on the target region of the second exon of the target gene in soybean to construct the soybean GmCKX3 / GmCKX14 mutant;

[0008] The target region includes the target region shown in SEQ ID NO.1 and the target region shown in SEQ ID NO.2.

[0009] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant undergoes deletion and / or insertion mutations in the target region.

[0010] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant inserts a single base into the target region shown in SEQ ID NO.1.

[0011] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has multiple bases missing in the target region shown in SEQ ID NO.2;

[0012] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has 3-5 bases missing in the target region shown in SEQ ID NO.2.

[0013] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has the fragments shown in SEQ ID NO.5 and SEQ ID NO.7.

[0014] In some embodiments, gene editing includes one or more of CRISPR / Cas9, CRISPR / Cpf1, CRISPR / Cas12i / j, TALEN, ZFN, and replacement after knocking out the relevant target gene;

[0015] In some of these embodiments, gene editing employs CRISPR / Cas9 technology.

[0016] In some embodiments, the soybean variety includes Williams 82.

[0017] This application also provides a planting method for synergistically improving soybean yield and seed quality, the method comprising:

[0018] The soybean GmCKX3 / GmCKX14 mutant was constructed using the method described above; and the soybean GmCKX3 / GmCKX14 mutant was then planted.

[0019] This application provides a method for synergistically improving soybean yield and seed quality. The method involves using gene editing technology to edit a target region on the second exon of a soybean target gene. By simultaneously targeting and editing the soybean cytokinin oxidase / dehydrogenase genes GmCKX3 and GmCKX14, a frameshift mutation is generated, leading to premature termination of protein translation. This method, by simultaneously knocking out two key cytokinin oxidase genes, breaks the traditional negative correlation between yield and protein content in soybean breeding, achieving simultaneous increases in yield and seed protein content while maintaining stable oil content.

[0020] Among them, the synergistic knockout of GmCKX3 and GmCKX14 genes can effectively regulate the homeostasis of cytokinins. By affecting cell division and differentiation, it can both promote the increase of yield components and maintain the normal process of seed protein synthesis and metabolism, thereby breaking through the trade-off between "high yield and low protein". Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The CRISPR / Cas9 gene editing target design and Gmckx3 / 14 mutation site provided in one embodiment of this application;

[0023] Figure 2 This serves as a verification of the yield results of single plants with double mutants and single mutants provided in an embodiment of this application;

[0024] Figure 3 This serves as a verification of the yield results of double mutant and single mutant plots provided in an embodiment of this application;

[0025] Figure 4 This serves as a verification of the seed protein content results of the double mutant and single mutant provided in an embodiment of this application;

[0026] Figure 5 This is to verify the results of oil content in double mutants and single mutants provided in an embodiment of this application. Detailed Implementation

[0027] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0030] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0031] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0032] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0033] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0034] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0035] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0036] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0037] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0038] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0039] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0040] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0041] The term "gene editing technology" refers to a technique that allows modification of specific DNA segments within an organism's genome. In this field, gene editing technology specifically identifies and cuts target DNA sequences, utilizing the organism's own DNA repair mechanisms to insert, delete, or replace genes. This technology is characterized by high precision, efficiency, and reproducibility, making it an important tool in modern molecular biology and genetic engineering. Specific sub-concepts include CRISPR / Cas9 technology, TALEN technology, and ZFN technology.

[0042] The term "CRISPR / Cas9 technology," short for Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9, is a gene editing technology based on the bacterial adaptive immune system. This technology uses guide RNA (sgRNA) to guide the Cas9 nuclease to recognize and cleave specific DNA sequences, achieving gene editing through non-homologous end joining (NHEJ) or homologous recombination repair (HDR) pathways. CRISPR / Cas9 technology is characterized by its ease of operation, low cost, and high editing efficiency, making it one of the most widely used gene editing technologies currently available.

[0043] The term "cytokinin oxidase / dehydrogenase (CKX)" refers to a class of key enzymes that catalyze the degradation of cytokinins. Cytokinins are important plant hormones involved in the regulation of various biological processes, including cell division, differentiation, and organogenesis. CKX enzymes degrade cytokinins through oxidation or dehydrogenation reactions, thereby maintaining cytokinin homeostasis within plants. In soybean, the CKX gene family includes several members, among which GmCKX3 and GmCKX14 are key genes regulating yield and quality traits.

[0044] The term "frameshift mutation" refers to a type of gene mutation caused by the insertion or deletion of bases that are not multiples of 3 in the DNA sequence. This mutation alters the reading frame of subsequent codons, often producing premature stop codons, leading to premature termination of protein translation and the production of truncated, non-functional or dysfunctional proteins. Frameshift mutations are a commonly used mutation type in gene function research, effectively disrupting the normal function of target genes.

[0045] The term "exon" refers to the DNA sequence region in a gene that is ultimately transcribed and translated into protein. Unlike introns, exon sequences are preserved during RNA splicing, becoming part of mature mRNA and guiding protein synthesis during translation. Exons typically contain key sequences encoding protein functional domains; therefore, gene editing in exon regions can effectively disrupt gene function.

[0046] The term "sgRNA," short for single-stranded guide RNA, is a small RNA molecule used in the CRISPR / Cas9 system to guide the Cas9 nuclease to recognize and cleave target DNA sequences. sgRNA consists of two parts: a crRNA (CRISPR RNA) that can complementaryly pair with the target DNA sequence, and a tracrRNA (trans-activating CRISPR RNA) with a fixed sequence structure. sgRNA specifically recognizes the target DNA sequence through the base pairing principle, guiding the Cas9 protein to cleave it.

[0047] The term "homozygous mutant" refers to an individual in an organism where both alleles of a gene have undergone the same type of mutation. In diploid organisms, each gene typically has two alleles, one from the father and one from the mother. When both alleles undergo the same mutation, the individual is considered a homozygous mutant. Homozygous mutants stably express the mutant phenotype and are important materials for gene function research and breeding applications.

[0048] The term "double mutant" refers to an organism in which two different genes are mutated simultaneously. In this application, a double mutant specifically refers to an individual in which both the GmCKX3 and GmCKX14 genes are mutated simultaneously. Double mutants can disrupt the function of two genes simultaneously and usually exhibit more significant phenotypic changes than single mutants, making them important materials for studying gene interactions and synergistic effects.

[0049] The term "premature termination of protein translation" refers to the premature appearance of a stop codon (UAA, UAG, or UGA) in the mRNA sequence due to a gene mutation. This causes the ribosome to terminate protein synthesis prematurely during translation, resulting in a truncated protein that is shorter than the normal protein. This truncated protein typically loses its normal function, thus achieving gene knockout. Premature termination of protein translation is a common phenomenon in gene knockout research and is also an important mechanism for achieving the functional disruption of the GmCKX3 and GmCKX14 genes in this application.

[0050] This application provides a method for synergistically improving soybean yield and seed quality, including:

[0051] Gene editing was performed on the target region of the second exon of the target gene in soybean to construct the soybean GmCKX3 / GmCKX14 mutant;

[0052] The target region includes the target region shown in SEQ ID NO.1 and the target region shown in SEQ ID NO.2.

[0053] Specifically, the method includes the following steps: using gene editing technology to simultaneously target and edit the soybean cytokinin oxidase / dehydrogenase genes GmCKX3 and GmCKX14, producing frameshift mutations that lead to premature termination of protein translation. This method, by simultaneously knocking out two key cytokinin oxidase genes, breaks the traditional negative correlation between yield and protein content in soybean breeding, achieving a simultaneous increase in yield and seed protein content while maintaining stable oil content.

[0054] Specifically, the synergistic knockout of the GmCKX3 and GmCKX14 genes can effectively regulate the homeostasis of cytokinins. By affecting cell division and differentiation, it promotes the increase of yield components while maintaining the normal process of seed protein synthesis and metabolism, thus overcoming the trade-off between "high yield and low protein". Specifically, the GmCKX3 gene is Glyma.17G054500, and the GmCKX14 gene is Glyma.15G170300. These two genes have clear sequence information and functional annotations in the soybean genome.

[0055] In some embodiments, the targeted editing is performed on the second exon of the GmCKX3 and GmCKX14 genes. An exon is a region in a gene that encodes a protein, and the second exon is often a critical region for gene function. Editing at this location can effectively disrupt normal gene function, producing frameshift mutations, thereby achieving a complete loss of gene function. Specifically, conserved sequence regions within the second exon can be selected as target sites to ensure maximum editing effectiveness.

[0056] This application successfully broke the negative correlation between yield and protein content by simultaneously knocking out the GmCKX3 and GmCKX14 genes, achieving simultaneous improvement of the two traits while maintaining stable oil content, providing a new technical approach for breeding soybean varieties with excellent comprehensive traits.

[0057] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant undergoes deletion and / or insertion mutations in the target region.

[0058] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant inserts multiple bases into the target region shown in SEQ ID NO.1;

[0059] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has one base inserted into the target region shown in SEQ ID NO.1.

[0060] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has multiple bases missing in the target region shown in SEQ ID NO.2;

[0061] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has 3-5 bases missing in the indicated target region.

[0062] The target sequence for GmCKX3 is 5'-CTTGGAGGCTTAGGACAATTTGG-3' (SEQ ID NO.1).

[0063] The target sequence for GmCKX14 is 5'-CTTGGAGGCTTGGGACAATTTGG-3' (SEQ ID NO.2).

[0064] In some embodiments, the soybean GmCKX3 / GmCKX14 mutant has the fragments shown in SEQ ID NO.5 and SEQ ID NO.7.

[0065] SEQ ID NO.5:>Glyma.17G054500-ckx3 / 14 (CKX3 amino acid sequence in the double mutant)

[0066] MALNYPFLTYFILLLVTITRLIFTVGKTEQWKAPILPELDIDNISHKLHDDPETIQMASRDYGHLTHEFPLAVFRPSSIDDIVTLIKSSYNSFAPFDIAARGQGHSTHGQAMARDGIVVDMAS LRKQRNGVAISVSKDPLMGHYADVGGEQLWIDVLHATLEYGLAPVSWTDYLYLTVGGTLSNAGISGQSFRYGPQISNVHEMDVITGKGEFVTCSSQKNLELFHAVLGELGTIWSYSKGENCS*

[0067] SEQ ID NO.7:>Glyma.15G170300-ckx3 / 14 (CKX14 amino acid sequence in the double mutant)

[0068] MQIKVLFHSIYSPYLLHLLSPLQHTNTNNQINPSTLETTKHSLFSHSIIFSHPLILSKPTKKKMVAENYPSPTYFILLFITITRLISTVGKTSQWTKALSLTPELASVSLDDTIFCKLRDDPEALQGRASRDYGNLVREVPLAVFHPASASDIARLIKLSYNGS VPFKIAARGQGHSTRGQAMAREGVVVDMAGFRERGNGVGIRVVSSVDPNNNKNGYYYYADVGGEQLWIDVLHATLEHGLAPMSWTDYLYLTLGGTLSNAGISGQTFRYGPQITTVREMDVITGKGEFVTCSQQTNSELFHAVLGLRTIWNYNKGKNCSCASSEEG*

[0069] In some embodiments, gene editing includes one or more of CRISPR / Cas9, CRISPR / Cpf1, CRISPR / Cas12i / j, TALEN, ZFN, and replacement after knocking out the relevant target gene;

[0070] In some embodiments, gene editing employs CRISPR / Cas9 technology. CRISPR / Cas9 technology is one of the most widely used and efficient gene editing technologies, offering advantages such as ease of operation, high specificity, and high editing efficiency. This technology uses designed specific sgRNAs to guide the Cas9 nuclease to precisely cut the target gene, thereby achieving targeted gene editing.

[0071] In some embodiments, the soybean variety includes Williams 82. This is a widely cultivated soybean variety, and gene-edited mutants show promising application prospects.

[0072] This application also provides a planting method for synergistically improving soybean yield and seed quality, the method comprising:

[0073] The soybean GmCKX3 / GmCKX14 mutant was constructed using the method described above; and the soybean GmCKX3 / GmCKX14 mutant was then planted.

[0074] This method has important application value in soybean breeding, and can effectively cultivate new soybean varieties with high yield, high protein content and stable oil content, providing technical support for the sustainable development of the soybean industry.

[0075] This application successfully broke the negative correlation between soybean yield and seed protein content by simultaneously knocking out the GmCKX3 and GmCKX14 genes, achieving a synergistic improvement in both traits and solving a long-standing technical problem in soybean breeding. The method described in this application can increase yield and protein content while maintaining stable seed oil content, avoiding the "high yield - low oil" trade-off problem in traditional breeding, and providing a new technical approach for cultivating soybean varieties with excellent comprehensive traits.

[0076] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0077] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0078] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0079] Example 1: This example provides a method for synergistically improving soybean yield and seed quality.

[0080] I. Method for preparing raw materials:

[0081] Soybean variety: William82

[0082] CRISPR / Cas9 vector: pCAMBIA1300-Cas9

[0083] sgRNA design tool: CRISPR-P 2.0

[0084] Agrobacterium strain: EHA105

[0085] II. Preparation of a method to synergistically improve soybean yield and seed quality:

[0086] Target design: Using the CRISPR-P 2.0 online tool, sgRNA target sequences were designed on the second exon of soybean GmCKX3 (Glyma.17G054500) and GmCKX14 (Glyma.15G170300), respectively.

[0087] The target sequence for GmCKX3 is 5'-CTTGGAGGCTTAGGACAATTTGG-3' (SEQ ID NO.1).

[0088] The target sequence for GmCKX14 is 5'-CTTGGAGGCTTGGGACAATTTGG-3' (SEQ ID NO.2).

[0089] III. Carrier Construction:

[0090] The designed sgRNA sequence was cloned into the pCAMBIA1300-Cas9 vector to construct a dual-target editing vector containing both GmCKX3 and GmCKX14 sgRNAs. The vector was constructed using the Golden Gate assembly method and digested and ligated using the BsaI restriction endonuclease.

[0091] IV. Agrobacterium Transformation: The constructed dual-target editing vector was introduced into Agrobacterium EHA105 competent cells via electroporation. The electroporation parameters were: voltage 2.5 kV, capacitance 25 μF, and resistance 200 Ω. Positive clones of the transformed Agrobacterium were screened on YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin.

[0092] V. Soybean Genetic Transformation: An Agrobacterium-mediated transformation method was employed for soybean cotyledonary nodes. William82 soybean seeds were surface-sterilized and germinated, with cotyledonary nodes used as explants. Agrobacterium-mediated transformation using a dual-target editing vector (OD) was then performed. 600 =0.6) Infected for 20 minutes, co-cultured for 3 days, then transferred to selection medium. The selection medium contains 50 mg / L kanamycin and 250 mg / L cephalosporin, and the medium is changed every 2 weeks.

[0093] VI. Obtaining Regenerated Plants: After 6-8 weeks of selection and culture, resistant callus tissue was transferred to differentiation medium to induce shoot differentiation. The differentiation medium contained 2 mg / L 6-BA and 0.2 mg / L NAA. When the shoots grew to 2-3 cm, they were cut off and transferred to rooting medium to induce rooting. The rooting medium contained 0.5 mg / L IBA.

[0094] VII. Mutant Identification: Genomic DNA was extracted from the obtained regenerated plants, and the target regions of GmCKX3 and GmCKX14 were amplified by PCR. The mutation type was identified by sequencing. Homozygous single mutants of Gmckx3 and homozygous double mutants of Gmckx3 / 14 were obtained. The Gmckx3 target site was deleted by 4 bp; the double mutants showed a single A insertion in Gmckx3 sgRNA and a 2 bp deletion in Gmckx14 sgRNA1. All editing resulted in frameshift mutations, leading to premature termination of protein translation.

[0095] SEQ ID NO.3: >G.max Wm82.a4.v1|Glyma.17G054500|CKX3 (CKX3 amino acid sequence in wild type) MALNYPFLTYFILLLVTITRLIFTVGKTEQWKAPILPELDIDNISHKLHDDPETIQMASRDYGHLTHEFPLAVFRPSSIDDIVTLIKSSYNSFAPFDIAARGQGHSTHGQAMARDGIVVDMASLRKQRNGVAISVSKDPLMGHYADVGGEQLWIDVLHATLEYGLAPVSWTDYLYLTVGGTLSNAGISGQSFRYGPQISNVHEMDVITGKGEFVTCSSQKNLELFHAVLGGLGQFGVIARARIALEPAPKRVKWVRLLYSDFSAFTKDQERLISINGRKQKNALDFLEGMLLMNQGPINNWRSSFFPLSDHPRIASLITEHSILYCLEVAKYYDEQTELNVDKEIEVLLQGLAYIPGFNYEKNVSYVEFLNRVRSGELKLQSQGLWEVPHPWLNLFIPKSQILDFNSGVFKDIVLKRNISSGPVLVYPMNRNKWDDRMSASIPDEDVFYTVGFLHSSGFDTWKAYDAQNREILEFCRDAGIMVKQYLPNHSTQEDWTNHFGAKWMKFLERKHQFDPRMILSPGQKIFHKKLQPVF*

[0096] SEQ ID NO.4: >Glyma.17G054500-ckx3 (CKX3 amino acid sequence in single mutant)

[0097] MALNYPFLTYFILLLVTITRLIFTVGKTEQWKAPILPELDIDNISHKLHDDPETIQMASRDYGHLTHEFPLAVFRPSSIDDIVTLIKSSYNSFAPFDIAARGQGHSTHGQAMARDGIVVDMASLRKQRNGVAISVSKDPLMGHYADVGGEQLWIDVLHATLEYGLAPVSWTDYLYLTVGGTLSNAGISGQSFRYGPQISNVHEMDVITGKGEFVTCSSQKNLELFHAVLGGLGLEL*

[0098] SEQ ID NO.5:>Glyma.17G054500-ckx3 / 14 (CKX3 amino acid sequence in the double mutant)

[0099] MALNYPFLTYFILLLVTITRLIFTVGKTEQWKAPILPELDIDNISHKLHDDPETIQMASRDYGHLTHEFPLAVFRPSSIDDIVTLIKSSYNSFAPFDIAARGQGHSTHGQAMARDGIVVDMAS LRKQRNGVAISVSKDPLMGHYADVGGEQLWIDVLHATLEYGLAPVSWTDYLYLTVGGTLSNAGISGQSFRYGPQISNVHEMDVITGKGEFVTCSSQKNLELFHAVLGELGTIWSYSKGENCS*

[0100] SEQ ID NO.6:>G.max Wm82.a4.v1|Glyma.15G170300|GmCKX14 (Cellular amino acid sequence of wild type)

[0101] MQIKVLFHSIYSPYLLHLLSPLQHTNTNNQINPSTLETTKHSLFSHSIIFSHPLILSKPTKKKMVAENYPSPTYFILLFITITRLISTVGKTSQWTKALSLTPELASVSLDDTIFCKLRDDPEALQGRASRDYGNLVREVPLAVFHPASASDIARLIKLSYNGSVPFKIAARGQGHSTRGQAMAREGVVVDMAGFRERGNGVGIRVVSSVDPNNKNGYYYYADVGGEQLWIDVLHATLEHGLAPMSWTDYLYLTLGGTLSNAGISGQTFRYGPQITTVREMDVITGKGEFVTCSQQTNSELFHAVLGGLGQFGIITRARIALAPAPKRVKWVRLLYNDFSAFTKDQEQLISVTGRKQNVSLDYLEGLLLMHQGPINNWRSSFFPLADHARIISLVTKHSVLYCLEVAKYYDGQNENNVDKELQVLLQGLSYIPGFYYEKDVSYFEFLNRVRSGELKLQSQGLWDVPHPWLNLFIPKSQIMEFDSGVFKNIILKRNITTGPVLVYPMNRNKWDNRMSASIPDEDIFYTVGFLHSSGFDNWKAYDAQNKEILQFCNVAGIKVKQYLPHYRTQEDWANHFGPKWRTFVERKHQFDPRMILSPGQRIFNN*

[0102] SEQ ID NO.7:>Glyma.15G170300-ckx3 / 14 (Amino acid sequence of CKX14 in the double mutant)

[0103] MQIKVLFHSIYSPYLLHLLSPLQHTNTNNQINPSTLETTKHSLFSHSIIFSHPLILSKPTKKKMVAENYPSPTYFILLFITITRLISTVGKTSQWTKALSLTPELASVSLDDTIFCKLRDDPEALQGRASRDYGNLVREVPLAVFHPASASDIARLIKLSYNGS VPFKIAARGQGHSTRGQAMAREGVVVDMAGFRERGNGVGIRVVSSVDPNNNKNGYYYYADVGGEQLWIDVLHATLEHGLAPMSWTDYLYLTLGGTLSNAGISGQTFRYGPQITTVREMDVITGKGEFVTCSQQTNSELFHAVLGLRTIWNYNKGKNCSCASSEEG*

[0104] Performance testing methods:

[0105] Single-plant yield measurement: The total weight of all seeds harvested from a single soybean plant. Plot yield measurement: The plot has a row spacing of 50 cm, a row length of 3 m, 3 rows of each material, a plant spacing of 15 cm, and an area of ​​4.5 m². 2 Each step was repeated three times. The total weight of seeds from all plants in each plot was calculated.

[0106] Seed protein content determination: The Kjeldahl method was used to determine the seed protein content.

[0107] Seed oil content determination: The seed oil content was determined by Soxhlet extraction.

[0108] Test results as follows Figures 2-5 As shown.

[0109] The results showed that Gmckx3 / 14 (double gene modification) significantly increased the seed weight per plant and the yield of wheat plots, but had a slight negative effect on protein content, while oil content and seed protein content did not change significantly.

[0110] Gmckx3 (single-gene modification): It has the highest protein content, but its seed weight and yield are not significantly different from the wild type (Wm82), and its oil content is also unchanged.

[0111] Wm82 (wild-type control): All indicators are baseline values. Seed weight and yield are lower than Gmckx3 / 14, protein content is lower than Gmckx3 and Gmckx3 / 14, and oil content is similar to Gmckx3.

[0112] In summary, Gmckx3 / 14 can significantly increase the weight of individual seeds and the yield of wheat plots while maintaining oil content and seed protein content.

[0113] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for synergistically improving soybean yield and seed quality, characterized in that, include: Gene editing was performed on the target region of the second exon of the target gene in soybean to construct the soybean GmCKX3 / GmCKX14 mutant; The target region includes the target region shown in SEQ ID NO.1 and the target region shown in SEQ ID NO.

2.

2. The method for synergistically improving soybean yield and seed quality according to claim 1, characterized in that, The soybean GmCKX3 / GmCKX14 mutant exhibits deletion and / or insertion mutations in the target region.

3. The method for synergistically improving soybean yield and seed quality according to claim 2, characterized in that, The soybean GmCKX3 / GmCKX14 mutant inserts a single base into the target region shown in SEQ ID NO.

1.

4. The method for synergistically improving soybean yield and seed quality according to claim 2, characterized in that, The soybean GmCKX3 / GmCKX14 mutant has multiple bases missing in the target region shown in SEQ ID NO.

2.

5. The method for synergistically improving soybean yield and seed quality according to claim 2, characterized in that, The soybean GmCKX3 / GmCKX14 mutant has a deletion of 3-5 bases in the target region shown in SEQ ID NO.

2.

6. The method for synergistically improving soybean yield and seed quality according to any one of claims 1 to 5, characterized in that, The soybean GmCKX3 / GmCKX14 mutant has the fragments shown in SEQ ID NO.5 and SEQ ID NO.

7.

7. The method for synergistically improving soybean yield and seed quality according to claim 6, characterized in that, Gene editing includes one or more of the following: CRISPR / Cas9, CRISPR / Cpf1, CRISPR / Cas12i / j, TALEN, ZFN, and replacement after knocking out the relevant target gene.

8. The method for synergistically improving soybean yield and seed quality according to claim 7, characterized in that, Gene editing uses CRISPR / Cas9 technology.

9. The method for synergistically improving soybean yield and seed quality according to any one of claims 1 to 5, characterized in that, The soybean varieties mentioned include Williams 82.

10. A planting method for synergistically improving soybean yield and seed quality, characterized in that, The method includes: The soybean GmCKX3 / GmCKX14 mutant was constructed using the method described in any one of claims 1 to 9; and, The soybean GmCKX3 / GmCKX14 mutant was planted.

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