Methods for improving soybean traits

Abstract

The present application relates to a method for improving soybean traits. It is used to improve at least one of the characteristics including soybean yield, number of soybean main stem nodes, number of pods per plant, number of grains per plant and plant height, and the method includes reducing or inhibiting the content and / or activity of the following proteins in the recipient soybean to obtain soybean mutants; the proteins include one or more of GmCKX3, GmCKX7 and GmCKX14. The present application edited soybean GmCKX3 and its homologous genes GmCKX7 and / or GmCKX14 for the first time, and created gene-edited soybean homozygous mutants with amino acid changes of GmCKX3, GmCKX7 and / or GmCKX17, respectively, to improve the number of soybean main stem nodes, number of pods per plant, number of grains per plant, yield per plant and plant height, and soybean yield, providing a good seed resource reserve.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and genetic engineering, and in particular to a method for improving soybean traits. Background Art

[0002] Soybean is an important grain and oil crop in the world. It is rich in protein, 2-5 times that of other grain crops. It can provide 30%~40% of the protein content required by the human body, and is the main source of high-quality protein for humans (Han Lide et al., 2003). Soybean yield is composed of the number of effective plants per unit area and the yield per plant. The yield per plant is affected by plant height, number of main stem nodes, number of branches, number of pods per plant, number of grains per pod, number of grains per plant, and 100-grain weight. The ideal yield composition is that several yield components grow at the same time, but these yield components restrict each other. In the same variety, it is difficult to combine the advantages of more pods per plant, more grains per pod, and larger grains. Therefore, it is of great significance to improve soybean yield and breed new soybean varieties with ideal plant types and high yields to improve as many traits as possible.

[0003] Cytokinins regulate plant growth and development by inducing cell division and differentiation. The homeostasis of cytokinins is regulated by multiple processes, among which cytokinin oxidases / dehydrogenases (CKX) catalyze the degradation of CKs. CKX is the only enzyme known to specifically catalyze the irreversible degradation of natural isopentenyl CKs and their nucleosides (Zhang Fen et al., 2008). Summary of the invention

[0004] In order to solve the above technical problems, one embodiment of the present application provides a method for increasing soybean yield, the number of soybean main stem nodes, the number of pods per plant, the number of grains per plant and / or plant height by reducing or inhibiting the content and activity of GmCKX3, GmCKX7 and / or GmCKX14 proteins in recipient soybeans.

[0005] The technical solutions include the following:

[0006] In the first aspect, the present application provides a method for improving soybean traits, which is used to improve at least one of soybean yield, number of soybean main stem nodes, number of pods per plant, plant height and number of grains per plant. The method includes reducing or inhibiting the content and / or activity of the following proteins in the recipient soybean to obtain a soybean mutant; the proteins include one or more of GmCKX3, GmCKX7 and GmCKX14.

[0007] In one embodiment, the protein is selected from any one of the following groups:

[0008] (1) GmCKX3 and GmCKX7 proteins;

[0009] (2) GmCKX3 and GmCKX14 proteins;

[0010] (3) GmCKX3, GmCKX7 and GmCKX14 proteins.

[0011] In one embodiment, the purpose of reducing or inhibiting the content and / or activity of the protein is achieved by mutating or reducing the expression of the gene corresponding to the protein.

[0012] In one embodiment, the method of mutating, reducing expression or knocking out the gene comprises one or more of CRISPR technology, mutagenesis technology, chromosome engineering and homologous recombination technology.

[0013] In one embodiment, the CRISPR technology uses a gene editing system including CRISPR / Cas, CRISPR / Cpf1, CRISPR / Cas12i / j, TALEN or ZFN to mutate the gene.

[0014] In one embodiment, the CRISPR / Cas gene editing system includes one or more gRNAs and Cas proteins targeting the gene.

[0015] In one embodiment, the gRNA targeting the GmCKX3 gene includes the gRNA shown in SEQ ID No.13 and / or SEQ ID No.14; and / or,

[0016] In one embodiment, the gRNA targeting the GmCKX7 gene includes the gRNA shown in SEQ ID No.15 and / or SEQ ID No.16; and / or,

[0017] In one embodiment, the gRNA targeting the GmCKX14 gene includes the gRNA shown in SEQ ID No.17 and / or SEQ ID No.18.

[0018] In a second aspect, the present application provides biological materials, including one or more of nucleic acid molecules, expression vectors and hosts.

[0019] In one embodiment, the nucleic acid molecule comprises one or more gRNAs from the following group:

[0020] (1) The gRNA targeting GmCKX3 includes the gRNA shown in SEQ ID No. 13 and / or SEQ ID No. 14;

[0021] (2) The gRNA targeting GmCKX7 includes the gRNA shown in SEQ ID No. 15 and / or SEQ ID No. 16;

[0022] (3) The gRNA targeting the GmCKX14 gene includes the gRNA shown in SEQ ID No. 17 and / or SEQ ID No. 18;

[0023] In one embodiment, an expression vector comprises the nucleic acid molecule;

[0024] In one embodiment, the host comprises the expression vector, and the host comprises a plant cell, a plant tissue or a plant organ.

[0025] In a third aspect, the present application provides a reagent, comprising a reagent capable of mutating, reducing expression or knocking out at least one of the following genes in a recipient soybean: GmCKX3, GmCKX7 and GmCKX14.

[0026] In one embodiment, the reagent includes the biological material.

[0027] In a fourth aspect, the present application provides the use of the biological material or the reagent in soybean breeding or improving the following soybean traits, wherein the traits include: one or more of soybean yield, number of soybean main stem nodes, number of pods per plant, plant height and number of grains per plant.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] This application edited soybean GmCKX3 (Glyma.17G054500) and its homologous genes GmCKX7 (Glyma.09G063900) and / or GmCKX14 (Glyma.15G170300) for the first time, and created gene-edited soybean homozygous mutants with amino acid changes in GmCKX3, GmCKX7 and / or GmCKX17, respectively, thereby increasing the number of soybean main stem nodes, the number of pods per plant, the number of grains per plant, the yield per plant, the plant height and the soybean yield, and providing a good seed resource reserve. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Design of CRISPR / cas9 gene editing target sites and mutation sites of Gmckx3, Gmckx3 / 7, Gmckx3 / 14, and Gmckx3 / 7 / 14;

[0031] Figure 2 The statistical results of the related shapes of the homozygous mutants of Gmckx3, Gmckx3 / 7, Gmckx3 / 14, and Gmckx3 / 7 / 14, including A. plant height, B. number of main stem nodes, C. number of effective branches, D. number of effective pods, E. number of pods per main stem node, F. number of grains per plant, G. grain weight per plant, and H. 100-grain weight;

[0032] Figure 3 The phenotypes of individual plants of wild-type wm82 and various genotype mutants are shown, scale bar = 5 cm;

[0033] Figure 4 These are the effective pods of individual plants of wild-type wm82 and various genotype mutants. Scale bar = 5 cm. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0036] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0037] In this application, "multiple", "multiple", "multiple times", "multiple groups", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two. "More than" includes the number itself, for example, "more than two" includes two, three or more.

[0038] In the present application, “preferred” is only used to describe an implementation method or example with better effects, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0039] In the present application, the terms "first", "second", "third" and "fourth" in "the first aspect", "the second aspect", "the third aspect" and "the fourth aspect" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first" and "second" only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0040] An embodiment of the present application provides a method for improving soybean traits, which is used to improve at least one of soybean yield, number of soybean main stem nodes, number of pods per plant, number of grains per plant and plant height; the method includes reducing or inhibiting the content and / or activity of the following proteins in the recipient soybean to obtain a soybean mutant; wherein the proteins include one or more of GmCKX3, GmCKX7 and GmCKX14 proteins.

[0041] In a specific example, the amino acid sequence of the recipient soybean GmCKX3 protein is shown as SEQ ID No.2, and optionally, the nucleotide sequence of the gene encoding the GmCKX3 protein is shown as SEQ ID No.1.

[0042] In a specific example, the amino acid sequence of the recipient soybean GmCKX7 protein is shown as SEQ ID No.7, and optionally, the nucleotide sequence of the gene encoding the GmCKX7 protein is shown as SEQ ID No.6.

[0043] In a specific example, the amino acid sequence of the recipient soybean GmCKX14 protein is shown as SEQ ID No.10, and optionally, the nucleotide sequence of the gene encoding the GmCKX14 protein is shown as SEQ ID No.9.

[0044] In a specific example, the protein is selected from any of the following groups:

[0045] (1) GmCKX3 and GmCKX7 proteins;

[0046] (2) GmCKX3 and GmCKX14 proteins;

[0047] (3) GmCKX3, GmCKX7 and GmCKX14 proteins.

[0048] In a preferred embodiment, the proteins are GmCKX3, GmCKX7 and GmCKX14 proteins.

[0049] The present application found that GmCKX3, GmCKX7 and GmCKX14 proteins are all involved in regulating soybean yield-related traits, and inhibiting or reducing their protein content or activity leads to higher yield indicators such as the number of effective pods and the number of pods per main stem node of the plant.

[0050] In a specific example, the purpose of reducing or inhibiting the content and / or activity of the protein is achieved by mutating, reducing expression or knocking out the gene corresponding to the protein.

[0051] In a specific example, a soybean mutant is obtained by mutating, reducing expression or knocking out the gene corresponding to the protein, wherein the gene includes one or more of GmCKX3, GmCKX7 and GmCKX14.

[0052] In a specific example, the method of mutating, reducing expression or knocking out the gene includes one or more of CRISPR technology, mutagenesis technology, chromosome engineering and homologous recombination technology.

[0053] In a specific example, the CRISPR technology uses a gene editing system including CRISPR / Cas, CRISPR / Cpf1, CRISPR / Cas12i / j, TALEN or ZFN to mutate the gene.

[0054] In one specific example, a gene is mutated using a CRISPR / Cas system, which includes one or more gRNAs and Cas proteins targeting the gene.

[0055] In a specific example, the Cas protein includes a Cas9 protein.

[0056] A person skilled in the art may design other gRNAs according to the GmCKX3, GmCKX7 and / or GmCKX14 gene sequences according to conventional techniques to knock out the GmCKX3, GmCKX7 and / or GmCKX14 genes, thereby reducing or inhibiting the content or activity of the GmCKX3, GmCKX7 and / or GmCKX14 proteins, which are all within the scope of protection of the present application.

[0057] In a specific example, the gRNA targeting the GmCKX3 gene includes the gRNA shown in SEQ ID No.13 and / or SEQ ID No.14.

[0058] In a specific example, the gRNA targeting the GmCKX7 gene includes the gRNA shown in SEQ ID No.15 and / or SEQ ID No.16.

[0059] In a specific example, the gRNA targeting the GmCKX14 gene includes the gRNA shown in SEQ ID No.17 and / or SEQ ID No.18.

[0060] In a specific example, the CRISPR / Cas system is used to perform gene editing in recipient soybeans to obtain gene-edited soybean plants.

[0061] Specifically, the CRISPR / Cas system is introduced into microorganisms, and the microorganisms are used to transfect soybean tissue materials to obtain gene-edited soybean plants.

[0062] In one specific example, the microorganism includes Agrobacterium.

[0063] In a specific example, the soybean tissue material includes soybean cells, soybean seeds, soybean tissues or parts thereof.

[0064] In a specific example, homologous recombination technology was used to knock out the GmCKX3, GmCKX7 and / or GmCKX14 genes, which resulted in higher yield indicators such as the number of effective pods and / or the number of pods per main stem node of the plant. The results were consistent with the improved plant traits caused by knockout using other technologies (such as the CRISPR / Cas system), such as the Gmckx3 single mutant and the Gmckx3 / 7 / 14 triple mutant, which had better phenotypes.

[0065] Specifically, the knockout steps of homologous recombination technology include the following:

[0066] The upstream gene fragments and downstream gene fragments of the GmCKX3, GmCKX7 and / or GmCKX14 genes are connected to a suicide vector to obtain a recombinant suicide plasmid vector; the recombinant suicide plasmid vector is introduced into a strain to screen for gene knockout strains. The strain is further used to transfect soybean tissue materials to obtain gene-edited soybean plants.

[0067] One embodiment of the present application also provides a reagent, which includes a reagent capable of mutating, reducing expression or knocking out at least one of the following genes in a recipient soybean: GmCKX3, GmCKX7 and GmCKX14 genes.

[0068] In a specific example, the reagent includes a CRISPR / Cas system, which includes one or more gRNAs targeting the gene.

[0069] In a specific example, the gRNA targeting the GmCKX3 gene includes the gRNA shown in SEQ ID No.13 and / or SEQ ID No.14.

[0070] In a specific example, the gRNA targeting the GmCKX7 gene includes the gRNA shown in SEQ ID No.15 and / or SEQ ID No.16.

[0071] In a specific example, the gRNA targeting the GmCKX14 gene includes the gRNA shown in SEQ ID No.17 and / or SEQ ID No.18.

[0072] In a specific example, the CRISPR / Cas system includes a Cas protein, and optionally, the Cas protein includes a Cas9 protein.

[0073] One embodiment of the present application also provides the use of the above reagent in soybean breeding.

[0074] In one specific example, soybean breeding includes growing soybeans that are one or more of:

[0075] breeding soybeans with increased yields;

[0076] Cultivate soybeans with increased number of main stem nodes;

[0077] Breeding soybeans with increased number of pods per plant;

[0078] Breeding soybeans with increased number of grains per plant.

[0079] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0080] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0081] Example 1 Knockout of soybean genes GmCKX3 / GmCKX7 / GmCKX14 using CRISPR / Cas9 mutation

[0082] The soybean GmCKX3 (Glyma.17G054500) gene sequence (SEQ ID No.1), protein amino acid sequence (SEQ ID No.2), GmCKX7 (Glyma.09G063900) gene sequence (SEQ ID No.6), protein amino acid sequence (SEQ ID No.7), GmCKX14 (Glyma.15G170300) gene sequence (SEQ ID No.9), protein amino acid sequence (SEQ ID No.10) were obtained from the Phytozome database. Figure 1 As shown in Figure 1, two target sites (gRNA1, gRNA2) were designed on the first exon of soybean GmCKX3, GmCKX7 and GmCKX14, respectively. The specific gRNAs are shown in Table 1. They were edited using CRISPR / cas9 gene editing technology, and the vector was stably transformed into the soybean variety William82. The above-mentioned related experimental steps such as CRISPR / Cas9 vector construction, Agrobacterium-mediated soybean genetic transformation, and sequence detection of gene-edited plants were all performed by Weimi Biotechnology (Jiangsu) Co., Ltd.

[0083] Table 1

[0084]

[0085] The base sequence characteristics of the Gmckx3 homozygous mutant, the Gmckx3 / 7 homozygous mutant, the Gmckx3 / 14 homozygous mutant and the Gmckx3 / 7 / 14 homozygous mutant were obtained as follows: Figure 1 As shown in Table 2.

[0086] Table 2

[0087]

[0088] Wild-type GmCKX3 gene sequence (SEQ ID No. 1):

[0089] >G.max Wm82.a4.v1|Glyma.17G054500|Gm17:4146032..4150593 forward CKX3

[0090]

[0091] Wild-type GmCKX3 amino acid sequence (SEQ ID No. 2):

[0092] *

[0093] CKX3 amino acid sequence in the Gmckx3 mutant (SEQ ID No. 3):

[0094] MALNYPFLTYFILLLVTITRSLWEKLSNGKLRFYQSLTLITYPTNSMMTLKPFKWLQGTMAI*

[0095] CKX3 amino acid sequence in Gmckx3 / 7 and Gmckx3 / 14 mutants (SEQ ID No.4):

[0096] MALNYPFLTYFILLLVTITRFDIHCGKN*

[0097] CKX3 amino acid sequence in the Gmckx3 / 7 / 14 mutant (SEQ ID No. 5):

[0098] MIFTVGKTEQWKAPILPELDIDNISHKLHDDPETIQMASRDYGHLTHEFPLAVFRPSSIDDIVTLIKSSYNSFAPFDIAARGQGHSTHGQAMARDGIVVDMASLRKQRNGVAISVSKDPLMGHYADVGGEQLWIDVLHATLEYGLAPVSWTDYLYLTVGGTLSNAGISGQSFRYGPQISNVHEMDVITGKGEFVTCSSQKNLELFHAVLGGLGQFGVIARARIALEPAPKRVKWVRLLYSDFSAFTKDQERLISINGRKQKNALDFLEGMLLMNQGPINNWRSSFFPLSDHPRIASLITEHSILYCLEVAKYYDEQTELNVDKEIEVLLQGLAYIPGFNYEKNVSYVEFLNRVRSGELKLQSQGLWEVPHPWLNLFIPKSQILDFNSGVFKDIVLKRNISSGPVLVYPMNRNKWDDRMSASIPDEDVFYTVGFLHSSGFDTWKAYDAQNREILEFCRDAGIMVKQYLPNHSTQEDWTNHFGAKWMKFLERKHQFDPRMILSPGQKIFHKKLQPVF*

[0099] Wild-type GmCKX7 gene sequence (SEQ ID No.6):

[0100] >G.max Wm82.a4.v1|Glyma.09G063900|Gm09:6122518..6127221 forward genomic CKX7

[0101]

[0102] Wild-type GmCKX7 amino acid sequence (SEQ ID No.7):

[0103] MVAGKYPSPTYFILLLITITRLISTVGKTSQWMKALTPPPELASVSLDDTIFSKLRNDPEALQGRASRDYGNLVREVPSAVFHPTSSSDIARLIKLSYNGSVPFKIAARGQGHSTRGQAMVRDGVVVDMAGFRERGNGEGIRVVMSVVVDPNNKNGYGYYYADVGGEQLWIDVLNATLEHGLAPMSWTDYLYLTVGGTLSNAGISGQTFRYGPQITTVRQMDVITGKGEFVTCSQQTNSELFHAVLGGLGQFGIITRARIALAPAPKRVKWVRLLYNDFSAFTKDQEQLISITRRKQNIALDYLEGLLLMHQGPINNWRSSFFPLADHARIISLVTKHSVLYCLEVAKYYDGQNENNVDKELKVLLQGLSYIPGFYYEKDVSYVEFLNRVRSGELKLQSQGLWDVPHPWLNLFIPKSQIMEFDSGVFKNIILKRNITTGPVLVYPMNRNKWDNRMSASIPDEDIFYTVGFLHSSGFDNWKAYDAQNKEILQFCNDSGIKVKQYLPHYRTQEDWTNHFGPKWRTFVERKHQFDPKMILSPGQRIFNN*

[0104] CKX7 amino acid sequence in Gmckx3 / 7 and Gmckx3 / 7 / 14 mutants (SEQ ID No.8):

[0105] MVAGKYPSPTYFILLLITITRLISTVGKTSQWMKALTPPPELASVSLDDTIFSKLRNDPEALQGRASRDYGNLVARFPRRSSTRPRRATSRG*

[0106] Wild-type GmCKX14 gene sequence (SEQ ID No.9):

[0107]

[0108] Wild-type GmCKX14 amino acid sequence (SEQ ID No.10):

[0109] MQIKVLFHSIYSPYLLHLLSPLQHTNTNNQINPSTLETTKHSLFSHSIIFSHPLILSKPTKKKMVAENYPSPTYFILLFITITRLISTVGKTSQWTKALSLTPELASVSLDDTIFCKLRDDPEALQGRASRDYGNLVREVPLAVFHPASASDIARLIKLSYNGSVPFKIAARGQGHSTRGQAMAREGVVVDMAGFRERGNGVGIRVVSSVDPNNKNGYYYYADVGGEQLWIDVLHATLEHGLAPMSWTDYLYLTLGGTLSNAGISGQTFRYGPQITTVREMDVITGKGEFVTCSQQTNSELFHAVLGGLGQFGIITRARIALAPAPKRVKWVRLLYNDFSAFTKDQEQLISVTGRKQNVSLDYLEGLLLMHQGPINNWRSSFFPLADHARIISLVTKHSVLYCLEVAKYYDGQNENNVDKELQVLLQGLSYIPGFYYEKDVSYFEFLNRVRSGELKLQSQGLWDVPHPWLNLFIPKSQIMEFDSGVFKNIILKRNITTGPVLVYPMNRNKWDNRMSASIPDEDIFYTVGFLHSSGFDNWKAYDAQNKEILQFCNVAGIKVKQYLPHYRTQEDWANHFGPKWRTFVERKHQFDPRMILSPGQRIFNN*

[0110] CKX14 amino acid sequence in the Gmckx3 / 14 mutant (SEQ ID No.11):

[0111] MQIKVLFHSIYSPYLLHLLSPLQHTNTNNQINPSTLETTKHSLFSHSIIFSHPLILSKPTKKKMVAENYPSPTYFILLFITITRLISTVGKTSQWTKALSLTPELASVSLDDTIFCKLRDDPEALQGRGSLGSLPPSLSERHREADQAVVQRLCPLQDCGEGARALDKGPGDGT*

[0112] CKX14 amino acid sequence in the Gmckx3 / 7 / 14 mutant (SEQ ID No. 12):

[0113] MQIKVLFHSIYSPYLLHLLSPLQHTNTNNQINPSTLETTKHSLFSHSIIFSHPLILSKPTKKKMVAENYPSPTYFILLFITITRLISTVGKTSQWTKALSLTPELASVSLDDTIFCKLRDDPEALQGRASRDYGNLVARFPWQSSTQPQRATSRG*

[0114] Example 2 Field test

[0115] Wild-type and mutant soybeans of different genotypes were directly planted in the fields of Beijing (116°39′N, 40°8´W) in early May and fully matured in early October. The whole cycle was about 150 days. After maturity, the whole plant was cut from the ground surface for photography, measurement and statistics. The results are shown in Figure 2-Figure 4 .

[0116] Specific statistical methods and statistical results:

[0117] 1Statistical indicators include:

[0118] ① Plant height: the height from the cotyledon node to the top of the plant (excluding the terminal inflorescence), expressed in cm; the results are as follows Figure 2 As shown in A.

[0119] ②The number of main stem nodes: the number of nodes from the first true leaf node to the top node of the main stem, excluding the cotyledon node and the top inflorescence; the results are as follows Figure 2 As shown in B.

[0120] ③The number of effective branches: refers to the number of branches on the main stem that bear pods. The effective branches have at least 2 nodes, excluding secondary branches. Figure 2 As shown in C.

[0121] ④The number of effective pods per plant: refers to the number of pods containing more than one full seed; the results are as follows: Figure 2 As shown in D.

[0122] ⑤ The number of pods per main stem node: the average number of valid pods in each node from the first true leaf node to the top node of the main stem; the results are as follows Figure 2 As shown in E.

[0123] ⑥ The number of seeds per plant: the number of all immature seeds, insect-eaten seeds, and diseased seeds, excluding unformed seeds. Figure 2 As shown in F.

[0124] ⑦ Seed weight per plant: sieve out impurities from the beans of a single plant, including immature, insect-eaten and diseased seeds, and weigh them (g); the results are as follows: Figure 2 As shown in G in .

[0125] ⑧ 100-seed weight: Randomly select two portions of complete mature beans, 100 seeds each, and weigh them (g). If the weight difference between the two portions of 100 seeds exceeds 0.5 g, re-sample and weigh. The results are as follows: Figure 2 As shown in H.

[0126] 2. According to statistical results

[0127] according to Figure 2 It can be seen that GmCKX3, GmCKX7 and GmCKX14 are all involved in regulating soybean yield-related traits. After mutation, there are significant differences in yield indicators such as the number of effective pods and the number of pods per main stem node. On the other hand, due to the redundancy of gene function, soybean yield indicators have different differences. For example, there is no significant difference in the number of grains per plant and the weight of grains per plant in the double mutation, while the single mutation of Gmckx3 and the triple mutation of Gmckx3 / 7 / 14 have excellent phenotypes.

[0128] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0129] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for improving soybean traits, characterized in that: Used to improve soybean yield traits, the soybean yield traits include the number of soybean main stem nodes, the number of grains per plant, the number of effective pods and the number of pods per main stem node, the method includes reducing or inhibiting the content and / or activity of the following proteins in the recipient soybean to obtain soybean mutants; The proteins are GmCKX3, GmCKX7 and GmCKX14 proteins; In the soybean mutant, the amino acid sequence of the GmCKX3 protein is shown as SEQ ID No.5, the amino acid sequence of the GmCKX7 protein is shown as SEQ ID No.8, and the amino acid sequence of the GmCKX14 protein is shown as SEQ ID No.

12.

2. The method for improving soybean traits according to claim 1, characterized in that: By mutating the gene corresponding to the protein, the content and / or activity of the protein is reduced or inhibited, thereby obtaining a soybean mutant.

3. The method for improving soybean traits according to claim 2, characterized in that: Methods for mutating the gene include one or more of CRISPR technology, mutagenesis technology, chromosome engineering and homologous recombination technology.

4. The method for improving soybean traits according to claim 3, characterized in that: The CRISPR technology uses a gene editing system including CRISPR / Cas, CRISPR / Cpf1, CRISPR / Cas12i / j, TALEN or ZFN to mutate the gene, wherein the CRISPR / Cas gene editing system includes one or more gRNAs and Cas proteins targeting the gene.

5. The method for improving soybean traits according to claim 4, characterized in that: Targeting the GmCKX3 The gRNA of the gene includes the gRNA shown in SEQ ID No. 13 and SEQ ID No. 14; Targeting the GX7 The gRNA of the gene includes the gRNA shown in SEQ ID No. 15; Targeting the GmCKX14 The gRNA of the gene includes the gRNA shown in SEQ ID No.

17.

6. Biomaterial, characterized in that It includes one or more of a nucleic acid molecule and an expression vector; wherein, The nucleic acid molecule includes a gRNA in the following group: (1) Targeting GmCKX3 The gRNA includes the gRNA shown in SEQ ID No.13 and SEQ ID No.14; (2) Targeting GX7 The gRNA includes the gRNA shown in SEQ ID No.15; (3) Targeting GmCKX14 The gRNA of the gene includes the gRNA shown in SEQ ID No. 17; The expression vector comprises the nucleic acid molecule.

7. A reagent, characterized in that The reagents include reagents capable of mutating the following genes in the recipient soybean: GmCKX3, GX7 and GmCKX14 ; The reagent comprises the biological material according to claim 6.

8. Use of the biological material according to claim 6 or the reagent according to claim 7 in soybean breeding or improving the following soybean traits, wherein the traits include soybean yield traits, and the soybean yield traits include the number of soybean main stem nodes, the number of grains per plant, the number of effective pods, and the number of pods per main stem node.

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