Soybean GmMET1 single-gene and double-gene mutants created based on CRISPR-Cas9 and application of soybean GmMET1 single-gene and double-gene mutants

Abstract

The invention relates to the technical field of gene engineering, and discloses soybean GmMET1 single-gene and double-gene mutants created based on CRISPR-Cas9 and application of the soybean GmMET1 single-gene and double-gene mutants, and the single-gene mutants are single-gene homozygous mutants obtained by editing GmMET1-1 or GmMET1-2 genes through a CRISPR-Cas9 carrier system. The double-gene mutant is obtained by hybridizing two different types of single-gene homozygous mutants and further performing selfing, the GmMET1 gene mutant shows higher survival rate and lower damage degree compared with a wild type under a drought condition, and the mutant also shows higher resistance under a cold damage condition, so that the GmMET1 gene mutant has a better application prospect. The adaptability and the yield stability of crops can be improved by utilizing a gene editing technology, and the method has the characteristics of high practicability and high adaptability.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically to soybean GmMET1 single and double gene mutants created based on CRISPR-Cas9 and their applications. Background Art

[0002] Soybean (Glycine max), as an important cash crop, is not only a major source of protein and vegetable oil but also occupies a key position in global food security. However, with the intensification of global climate change, soybeans are facing more and more abiotic stress challenges, such as drought, extreme temperatures, excessive salinity, etc., which seriously affect its yield and geographical distribution.

[0003] The effects of abiotic stress on soybeans, insufficient water: Under drought conditions, soybean plants will experience problems such as a decrease in cell turgor and leaf wilting, which can lead to death in severe cases. To adapt to this environmental stress, some soybean varieties may adjust their root architecture or change phenotypic characteristics to more effectively obtain water in deep soil; low temperature (cold stress): Under low temperature conditions, the photosynthesis efficiency, respiration rate, and other metabolic activities of soybeans will be affected, which may cause damage to reproductive organs and reduce the seed setting rate; in addition, cold stress may also trigger DNA demethylation in specific regions, change gene expression patterns, and affect overall growth and development.

[0004] Epigenetics, especially DNA methylation, can regulate gene expression without changing the gene sequence and help soybeans adapt to environmental stress. Under drought or cold stress, the DNA methylation pattern of soybeans will change, enhancing its stress resistance; abiotic stress not only causes an immediate response but can also be transmitted to offspring through changes in the DNA methylation state, conferring stronger adaptability.

[0005] With the development of modern biotechnology, especially the emergence of gene editing tools such as the CRISPR / Cas9 system, scientists can now more precisely modify the soybean genome to create ideal varieties that are more resistant to adverse external conditions. Through the research on epigenetic modification of soybeans, not only can the secrets of plant adaptation to the environment be revealed, but it also provides valuable guiding directions for future agricultural practices. By selecting excellent strains that can maintain a high level of DNA methylation under adversity and combining advanced genetic engineering techniques, it is expected to develop new soybean varieties with stronger tolerance and adaptability, which has inestimable significance for improving agricultural production efficiency and ensuring the safety and sustainability of future food supplies. Therefore, it is necessary to design soybean GmMET1 single and double gene mutants created based on CRISPR-Cas9 with strong practicality and high adaptability and their applications. Summary of the Invention

[0006] The object of the present invention is to provide a soybean GmMET1 single-gene and double-gene mutant created based on CRISPR-Cas9 and its application, so as to solve the problems raised in the above-mentioned background art.

[0007] To solve the above technical problems, the present invention provides the following technical solution: A soybean GmMET1 single-gene mutant, the single-gene mutant is a single-gene homozygous mutant obtained by editing the GmMET1-1 or GmMET1-2 gene through a CRISPR-Cas9 vector system. For the GmMET1-1 gene, the editing events include, but are not limited to, inserting one base within the DNMT-RDF domain in the second exon, and its nucleotide sequence is as shown in SEQ ID NO:1, or deleting 171 bp, designated as GmMET1-A, as one of the mutant materials for stress treatment, and its nucleotide sequence is as shown in SEQ ID NO:2. For the GmMET1-2 gene, the editing events include, but are not limited to, deleting 2 bases in the DNMT-RDF domain at the second exon, designated as GmMET1-B, as one of the mutant materials for stress treatment, and its nucleotide sequence is as shown in SEQ ID NO:3, or deleting 6 bases in the cytosine-specific DNA methyltransferase replication focus domain, and its nucleotide sequence is as shown in SEQ ID NO:4. The GmMET1-1 genotype is set as GmMET1-1 - / - GmMET1-2 + / + , and the GmMET1-2 genotype is set as GmMET1-1 + / + GmMET1-2 - / - .

[0008] According to the above technical solution, the double-gene mutant is obtained by hybridizing two different types of single-gene homozygous mutants and further self-crossing. The single-gene homozygous mutants include GmMET1-1 and GmMET1-2.

[0009] According to the above technical solution, the F1 generation double-gene heterozygous mutant GmMET1-1 + / - GmMET1-2 + / - has no significant difference from the wild type in growth and development.

[0010] According to the above technical solution, no GmMET1-1 - / - GmMET1-2 - / - double-gene homozygous mutant can be isolated in the F2 generation, and the GmMET1-1 + / - GmMET1-2 - / - double-gene mutant shows an obvious abnormal phenotype in growth and development. The GmMET1-1 + / + GmMET1-2 + / +, GmMET1-1 + / + GmMET1-2 - / - , GmMET1-1 - / - GmMET1-2 + / + The number of genotypes such as etc. is significantly higher than the theoretical ratio. GmMET1-1 + / - GmMET1-2 + / - , GmMET1-1 + / - GmMET1-1 - / - The number is lower than the theoretical ratio. GmMET1-1 - / - GmMET1-2 + / - The genotype is set as GmMET1-AB and used as one of the mutant materials for stress treatment.

[0011] A method for producing a mutant of soybean GmMET1 gene, comprising the following steps:

[0012] S1. Construct a CRISPR-Cas9 vector containing a gRNA sequence, and this gRNA can target the GmMET1-1 or GmMET1-2 gene;

[0013] S2. Introduce the constructed vector into recipient cells to make the Cas9 protein bind to the gRNA complex and cleave DNA at the target site;

[0014] S3. Screen the plant materials after editing treatment and confirm the expected editing effect.

[0015] A method for producing a double mutant of soybean GmMET1 gene, comprising the following steps:

[0016] S1. Use single-gene homozygous mutants for hybridization to generate F1 generation double-gene heterozygous mutants;

[0017] S2. Self-cross the F1 generation to produce the F2 generation;

[0018] S3. Analyze the genotype distribution of the F2 generation, especially pay attention to GmMET1-1 - / - GmMET1-2 - / - The presence and phenotypic characteristics of double-gene homozygous mutants.

[0019] According to the above technical solution, the application of the mutant of soybean GmMET1 gene in agricultural planting, especially its use in studying the DNA methylation mechanism and crop improvement.

[0020] According to the above technical solution, the application of the double mutant of soybean GmMET1 gene in revealing the gene interaction network and evaluating the effect of loss of function of multiple genes.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0022] (1) A new approach for breeding stress-resistant crops: Reducing the CG methylation level can enhance the tolerance of soybean seedlings to drought and chilling stress, opening up a new path for cultivating crop varieties with stronger stress resistance;

[0023] (2) Improving crop stress resistance: Under drought conditions, mutants (gmmet1-A, gmmet1-B, gmmET1-AB) showed higher survival rates and lower damage levels compared to the wild type (WT); under chilling stress conditions, the mutants also showed stronger resistance, contributing to the use of gene editing technology to improve crop adaptability and yield stability;

[0024] (3) Studying gene regulation: Through different editing events of the GmMET1 gene, the role of DNA methylation in plant growth and development and its impact on environmental stress responses can be studied in depth;

[0025] (4) Exploring gene interaction networks: The application of double mutants helps to reveal the interaction relationships between different genes and the effects of loss of function of multiple genes, providing a new perspective for understanding complex genetic regulatory networks;

[0026] (5) Accelerating the breeding process: Using the CRISPR-Cas9 system to efficiently obtain specific genotypes of soybean mutants can significantly shorten the traditional breeding cycle and accelerate the development of excellent varieties;

[0027] (6) Cultivating new stress-tolerant varieties: Based on the technical solution of the present invention, new soybean lines with stronger tolerance can be developed to cope with the challenges of abiotic stress brought about by climate change and ensure food security;

[0028] (7) Improving yield stability: The improved soybean varieties can maintain a high yield level under adverse environments, thus improving the stability and economic benefits of agricultural production;

[0029] (8) Supporting sustainable development: Enhancing crop stress resistance reduces the dependence on external resources (such as irrigation water), which is conducive to achieving the goals of sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the drawings:

[0031] Figure 1It is a schematic diagram of the sequencing results of the GmMET1 single-gene homozygous mutant of the present invention. (a) Sequencing results of the single-gene mutation of GmMET1-1 with an insertion of 1 bp; (b) Sequencing results of the single-gene mutation of GmMET1-1 with a deletion of 171 bp (gmmet1-A); (c) Sequencing results of the single-gene mutation of GmMET1-2 with a deletion of 2 bp (gmmet1-B); (d) Sequencing results of the single-gene mutation of GmMET1-2 with a deletion of 6 bp; abcd are different editing events.

[0032] Figure 2 It is a schematic diagram of the creation of the GmMET1 double-gene mutant of the present invention;

[0033] Figure 3 It is a schematic diagram of the phenotypes of the representative GmMET1 gene mutations of the present invention;

[0034] Figure 4 It is a schematic diagram of the phenotypes of the GmMET1 single-gene mutants of the present invention;

[0035] Figure 5 It is a schematic diagram of the phenotypes of the GmMET1 F1 double-gene mutants of the present invention;

[0036] Figure 6 It is a schematic diagram of the information of the plant mutant materials used for stress in the present invention;

[0037] Figure 7 It is a schematic diagram of the leaf phenotypes and drought stress survival rates of the drought stress treatment group and the control group of the present invention; (a) Representative leaf phenotypes of different genotypes under drought stress for 10 days and control conditions; (b) Representative leaf phenotypes of different genotypes under drought stress for 12 days and control conditions; (c) Survival rates after drought stress of different genotypes (lowercase letters are the results of multiple comparisons by one-way ANOVA, and different lowercase letters indicate significant differences between groups, p < 0.05, the same below).

[0038] Figure 8 It is a schematic diagram of the fresh weights of the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0039] Figure 9 It is a schematic diagram of the dry weights of the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0040] Figure 10 It is a schematic diagram of the malondialdehyde content and relative conductivity of the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0041] Figure 11 It is a schematic diagram of the soluble sugar content of the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0042] Figure 12It is a schematic diagram of the proline content in the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0043] Figure 13 It is a schematic diagram of the relative chlorophyll content of the drought stress treatment group and the control group of the present invention;

[0044] Figure 14 It is a schematic diagram of the SOD activity in the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0045] Figure 15 It is a schematic diagram of the POD activity in the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0046] Figure 16 It is a schematic diagram of the CAT activity in the leaves and roots of the drought stress treatment group and the control group of the present invention;

[0047] Figure 17 It is a schematic diagram of the statistical results of the change rate of growth indexes after drought stress treatment of the present invention;

[0048] Figure 18 It is a schematic diagram of the leaf phenotypes and cold stress survival rates of the cold stress treatment group and the control group of the present invention;

[0049] Figure 19 It is a schematic diagram of the MDA content in the leaves and roots of the cold stress treatment group and the control group of the present invention;

[0050] Figure 20 It is a schematic diagram of the proline content and soluble sugar content in the leaves and roots of the cold stress treatment group and the control group of the present invention;

[0051] Figure 21 It is a schematic diagram of the SOD activity in the leaves and roots of the cold stress treatment group and the control group of the present invention;

[0052] Figure 22 It is a schematic diagram of the statistical results of the change rate after cold stress treatment of the present invention; Detailed implementation manners

[0053] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0054] Please refer to Figures 1-22, the present invention provides a technical solution: a soybean GmMET1 single gene mutant, which is a single gene homozygous mutant obtained by editing the GmMET1-1 or GmMET1-2 gene through a CRISPR-Cas9 vector system. For the GmMET1-1 gene, the editing events include, but are not limited to, inserting a base within the DNMT-RDF domain in the second exon, and its nucleotide sequence is as shown in SEQ ID NO:1, or deleting 171bp, designated as GmMET1-A, which is one of the mutant materials for stress treatment, and its nucleotide sequence is as shown in SEQ ID NO:2. For the GmMET1-2 gene, the editing events include, but are not limited to, deleting 2 bases in the DNMT-RDF domain at the second exon, designated as GmMET1-B, which is one of the mutant materials for stress treatment, and its nucleotide sequence is as shown in SEQ ID NO:3, or deleting 6 bases in the cytosine-specific DNA methyltransferase replication focus domain, and its nucleotide sequence is as shown in SEQ ID NO:4. The GmMET1-1 genotype is designated as GmMET1-1 - / - GmMET1-2 + / + , the GmMET1-2 genotype is designated as GmMET1-1 + / + GmMET1-2 - / - ;

[0055] In this application, the optimized CRISPR-Cas9 vector system is used to precisely edit the GmMET1-1 and GmMET1-2 genes, and two single gene homozygous mutants with different mutation characteristics are obtained for each gene;

[0056] For the GmMET1-1 gene, two single gene homozygous mutants (genotypes are - / -, + / +) with different editing events are obtained, including: inserting a base within the DNMT-RDF domain in the second exon of the GmMET1-1 gene, resulting in premature termination of translation at the 223rd amino acid of the GmMET1-1 gene (mutation form is a, see Figure 1 a); deleting 171bp in the second exon of the GmMET1-1 gene, resulting in the loss of 57 amino acids in the methylation domain (mutation form is b, see Figure 1 b). The structure and function of a protein are closely related to the integrity of its amino acid sequence. Therefore, such a large fragment deletion not only destroys the integrity of the domain but may also lead to the loss of gene function;

[0057] For the GmMET1-2 gene, single-gene homozygous mutants with 2 different editing events of the GmMET1-2 gene (genotypes: + / +, - / -) were obtained, including: a deletion of 2 bases in the DNMT-RDF domain at the second exon of the GmMET1-2 gene, resulting in premature termination of translation (mutation form: c, see Figure 1 c); a deletion of 6 bases in the cytosine-specific DNA methyltransferase replication focus domain at the second exon of the GmMET1-2 gene, resulting in the deletion of two amino acids in this conserved domain (mutation form: d, see Figure 1 d);

[0058] The phenotypic analysis results of single-gene mutants of GmMET1-1 and GmMET1-2 in generations T1 - T3 showed that the growth and development of single-gene mutants of soybean GmMET1-1 or GmMET1-2 were normal, with no obvious phenotypic differences compared to the wild type (Wild Type, WT) (see Figure 4 );

[0059] Specifically, the double-gene mutants were obtained by crossing two different types of single-gene homozygous mutants and further self-crossing. The single-gene homozygous mutants include GmMET1-1 and GmMET1-2;

[0060] Specifically, the F1 generation double-gene heterozygous mutant GmMET1-1 + / - GmMET1-2 + / - showed no significant differences in growth and development compared to the wild type;

[0061] Specifically, no GmMET1-1 - / - GmMET1-2 - / - double-gene homozygous mutants could be isolated in the F2 generation, and the GmMET1-1 + / - GmMET1-2 - / - double-gene mutants showed obvious abnormal growth and development phenotypes. The numbers of genotypes such as GmMET1-1 + / + GmMET1-2 + / + , GmMET1-1 + / + GmMET1-2 - / - , GmMET1-1 - / - GmMET1-2 + / + were significantly higher than the theoretical ratio, while the numbers of GmMET1-1 + / - GmMET1-2 + / - , GmMET1-1 + / - GmMET1-1 - / - were lower than the theoretical ratio, and GmMET1-1 - / - GmMET1-2+ / - The genotype was set as GmMET1-AB, which was one of the mutant materials for stress treatment;

[0062] where - / -, + / + are the abbreviations of GmMET1-1 - / - GmMET1-2 + / + ; + / +, - / - are the abbreviations of GmMET1-1 + / + GmMET1-2 - / - ; + / -, + / - are the abbreviations of GmMET1-1 + / - GmMET1-2 + / - ; - / -, + / - are the abbreviations of GmMET1-1 - / - GmMET1-2 + / - ; + / -, - / - are the abbreviations of GmMET1-1 + / - GmMET1-2 - / - ; the rest is the same; abc are different editing events. The double-gene heterozygous mutant F1 generation with the genotype of + / -, + / - was obtained by crossing the single-gene homozygous mutants + / +, - / - and - / -, + / + (see Figure 2 ), and there was no significant difference in growth and development between the + / -, + / - double-gene heterozygous mutant and the wild type (see Figure 5 ). By self-crossing the + / -, + / - (ac / bc) double-gene heterozygous mutant, F2 generation double-gene mutants with different genotypes of different editing events were further obtained: + / -, + / - (ac), + / -, + / - (bc), - / -, + / - (ac), - / -, + / - (bc), + / -, - / - (ac), + / -, - / - (bc);

[0063] Among the 480 F2 generation seeds obtained by self-crossing the F1 generation + / -, + / - double-gene heterozygous mutant, no - / -, - / - double-gene homozygous mutant in any mutant form was isolated. Among the offspring of the remaining genotypes, + / -, + / - and + / -, - / - were significantly lower than the theoretical segregation ratio. At the same time, the numbers of + / +, - / -, - / -, + / + and + / +, + / + in the offspring were significantly higher than the theoretical ratio;

[0064] By analyzing the growth and development and phenotypes of the offspring, it was found that the + / -, - / - double-gene mutant showed obvious abnormal phenotypes such as significantly shorter plants, single branches, curled and wrinkled leaves, lighter leaf color, earlier flowering, and severely reduced seed setting rate, while other genotypes had no obvious differences from the wild type (see Figure 3 )

[0065] A method for producing a mutant of soybean GmMET1 gene, comprising the following steps:

[0066] S1. Construct a CRISPR-Cas9 vector containing the gRNA sequence, which can target the GmMET1-1 or GmMET1-2 gene;

[0067] S2. Introduce the constructed vector into recipient cells to bind the Cas9 protein to the gRNA complex and cleave DNA at the target site;

[0068] S3. Screen the plant materials after editing treatment and confirm the expected editing effect;

[0069] A method for producing a double mutant of soybean GmMET1, comprising the following steps:

[0070] S1. Hybridize using single-gene homozygous mutants to generate F1 double-gene heterozygous mutants;

[0071] S2. Self-cross the F1 generation to produce the F2 generation;

[0072] S3. Analyze the genotype distribution of the F2 generation, especially focusing on the presence and phenotypic characteristics of the GmMET1-1 - / - GmMET1-2 - / - double-gene homozygous mutants;

[0073] Specifically, the application of the soybean GmMET1 gene mutant in agricultural cultivation, especially its use in studying the DNA methylation mechanism and crop improvement;

[0074] Specifically, the application of the double mutant of the soybean GmMET1 gene in revealing the gene interaction network and evaluating the effect of loss of function of multiple genes.

[0075] Effects of drought stress on the phenotypic and survival rates of soybean seedlings with different genotypes

[0076] Plant the four experimental materials of gmmet1-A, gmmet1-B, gmmet1-AB and WT in an artificial climate chamber environment, and apply 20% PEG-6000 solution (artificial simulation of drought stress) and equal amount of water treatment to the treatment group and the control group for 10 days and 12 days respectively. Under control conditions, there are no obvious phenotypic differences among the four soybean genotypes ( Figure 7 a and Figure 7 b). Under the condition of 10-day drought stress treatment, WT shows obvious stress damage phenotypes, specifically manifested as leaf curling and wrinkling, and slightly yellowing of the color. The leaf edges of the mutants (gmmet1-A, gmmet1-B, gmmet1-AB) are slightly yellowed, and the damage degree is lower than that of WT. Figure 7 a and Figure 7b). By the 12th day of drought stress treatment, the leaf damage of all four genotypes further intensified. Most leaves of the WT showed obvious yellowing, and some plants wilted. The degree of leaf shrinkage of the three mutants increased, and a small number of plants wilted and their leaves drooped ( Figure 7 a, Figure 7 b, Figure 4 and Figure 5 ), and there were no obvious differences in the leaf phenotypes of the three mutants with different degrees of methylation reduction at 10 days and 12 days of stress treatment ( Figure 7 a, Figure 7 b, Figure 4 and Figure 5 ).

[0077] After continuously simulating drought stress treatment on the WT and the three mutants for 20 days until the WT died completely, the survival rates of the three mutants were counted. The results showed that the survival rates of gmmet1-A, gmmet1-B, and gmmet1-AB were 17%, 13%, and 11% respectively, and there were significant differences in the survival rates among different mutant genotypes.

[0078] Effects of drought stress on the growth indexes of three mutants and wild-type soybean seedlings

[0079] Fresh leaf weight: Compared with the control group, at 10 days and 12 days of treatment, the fresh leaf weights of the three mutants and the wild type were all significantly reduced ( Figure 8 a and Figure 8 b). Compared within the treatment group, at 10 days and 12 days of treatment, the fresh leaf weights of the three mutants were all significantly higher than those of the wild type ( Figure 8 a and Figure 8 b).

[0080] Fresh root weight: Compared with the control group, except that there was no significant difference in the fresh root weight of the wild type at 12 days of treatment, the fresh root weights of the three mutants at 10 days and 12 days of treatment and the wild type at 10 days of treatment were all significantly reduced ( Figure 8 c and Figure 8 d). Compared within the treatment group, at 10 days and 12 days of treatment, there were no significant differences in the fresh root weights of the three mutants and the wild type ( Figure 8 c and Figure 8 d).

[0081] Dry leaf weight: Compared with the control group, at 10 days and 12 days of treatment, there were no significant differences in the dry leaf weights of all genotypes ( Figure 9 a and Figure 9 b). Compared within the treatment group, at 10 days and 12 days of treatment, there were no significant differences among the four groups of materials ( Figure 9 a and Figure 9 b).

[0082] Root dry weight: Compared with the control group, the root dry weight of the wild type was significantly reduced at 10 and 12 days of treatment, and there was no significant difference in the root dry weight of the three mutants ( Figure 9 c and Figure 9 d). Compared within the treatment group, at 10 and 12 days of treatment, the root dry weight of the three mutants was significantly higher than that of the wild type; there was no significant difference in the root dry weight among the three mutants ( Figure 9 c and Figure 9 d).

[0083] The above results indicate that drought stress treatment inhibits the growth of the three mutants and the wild type, damages soybean seedlings, and reduces dry matter accumulation. The comparison results among different genotypes within the treatment group show that most growth indicators of the three mutants are significantly higher than those of the wild type, and there is no significant difference in most growth indicators among the three mutants.

[0084] Effects of drought stress on the cell membrane system of soybean seedlings of three mutants and the wild type

[0085] Malondialdehyde (MDA) content in leaves: Compared with the control group, at 10 and 12 days of treatment, except for no significant difference in the MDA content in the leaves of gmmet1-AB, the MDA content in the leaves of the other three groups of materials increased significantly ( Figure 10 a and Figure 10 b). Compared within the treatment group, at 10 days of treatment, the MDA content in the leaves of gmmet1-AB was significantly lower than that of the WT, and the MDA content in the leaves of gmmet1-A and gmmet1-B was significantly higher than that of the WT; at 12 days of treatment, the MDA content in the leaves of the three mutants was significantly lower than that of the wild type ( Figure 10 a and Figure 10 b).

[0086] MDA content in roots: Compared with the control group, at 10 days of treatment, the MDA content in the roots of the four groups of materials increased significantly; at 12 days of treatment, the MDA content in the roots of the wild type increased significantly, and there was no significant difference among the three mutants ( Figure 10 c and Figure 10 d). Compared within the treatment group, at 10 days of treatment, the MDA content in the roots of gmmet1-A was significantly higher than that of other genotypes, and there was no significant difference among other genotypes; at 12 days of treatment, the MDA content in the roots of the three mutants was significantly lower than that of the wild type ( Figure 10 c and Figure 10 d).

[0087] Relative conductivity: Compared with the control group, at 10 and 12 days of treatment, the relative conductivity of the three mutants and the wild type increased significantly ( Figure 10 e and Figure 10f). Compared within the treatment groups, the relative conductivity of gmmet1-B was significantly lower than that of WT at 10 days of treatment; at 12 days of treatment, the relative conductivity of gmmet1-B was significantly lower than that of WT, and there was no significant difference among the three mutants ( Figure 10 e and Figure 10 f).

[0088] The above results indicate that the biological membrane systems of soybean seedlings of each genotype were damaged to varying degrees under drought stress treatment. After drought stress, the MDA content in the roots and leaves of the three CG methylation-reduced mutants first increased and then decreased, while the MDA content in WT increased with the increase of drought stress treatment time. It can be seen that the CG methylation-reduced mutants showed a certain degree of adaptability, while the damage degree of WT deepened.

[0089] Effects of drought stress on osmotic adjustment substances of three mutants and wild-type soybean seedlings

[0090] Leaf soluble sugar content: Compared with the control group, at 10 days of treatment, the leaf soluble sugar contents of gmmet1-A and gmmet1-AB were significantly increased, and there was no significant difference in other genotypes; at 12 days of treatment, the leaf soluble sugar content of gmmet1-B did not change significantly, while the leaf soluble sugar contents of other genotypes were all significantly increased ( Figure 11 a and Figure 11 b). Compared within the treatment groups, at 10 days and 12 days of treatment, the leaf soluble sugar content of gmmet1-A was significantly higher than that of WT, and there was no significant difference among other genotypes ( Figure 11 a and Figure 11 b).

[0091] Root soluble sugar content: Compared with the control group, at 10 days of treatment, the root soluble sugar content of gmmet1-B was significantly increased, and there was no significant difference in other genotypes; at 12 days of treatment, except for gmmet1-B, the root soluble sugar contents of gmmet1-A, gmmet1-AB and WT were all significantly increased ( Figure 11 c and Figure 11 d). Compared within the treatment groups, at 10 days of treatment, the root soluble sugar content of gmmet1-B was significantly higher than that of WT, gmmet1-A and gmmet1-AB; at 12 days of treatment, there was no significant difference among the four groups of materials ( Figure 11 c and Figure 11 d).

[0092] Leaf proline content: Compared with the control group, at 10 days of treatment, the leaf proline contents of the three mutants were significantly increased, while the leaf proline content of the wild type was significantly decreased; at 12 days of treatment, the leaf proline contents of gmmet1-B and gmmet1-AB were significantly increased, and the leaf proline content of WT was significantly decreasedFigure 12 a and Figure 12 b). When compared within the treatment groups, at 10 and 12 days of treatment, the proline content in the leaves of all three mutants was significantly higher than that of the wild type ( Figure 12 a and Figure 12 b).

[0093] Root proline content: Compared with the control group, the root proline content of all three mutants at 10 and 12 days of treatment and the wild type at 10 days of treatment increased. At 12 days of treatment, there was no significant difference in the root proline content of the wild type ( Figure 12 a and Figure 12 b). When compared within the treatment groups, at 10 days of treatment, the root proline content of all three mutants was significantly higher than that of the wild type; at 12 days of treatment, the root proline content of gmmet1-AB was significantly higher than that of the WT, and there was no significant difference in the root proline content of the other two groups of materials and the wild type ( Figure 12 a and Figure 12 b).

[0094] The above results indicate that drought stress increases the soluble sugar content and proline content in the roots and leaves of soybean seedlings, enhances cell membrane permeability, thereby reducing the damage caused by stress. Among them, the proline content in the leaves of the wild type decreased compared with the control group, probably due to insufficient drought resistance of the wild type.

[0095] Effect of drought stress on the relative chlorophyll content of three mutants and wild type soybean seedlings

[0096] Relative chlorophyll content: Compared with the control group, at 10 and 12 days of treatment, the relative chlorophyll content of all four groups of materials increased significantly ( Figure 13 a and Figure 13 b). When compared within the treatment groups, at 10 days of treatment, there was no significant difference in the relative chlorophyll content between gmmet1-B and the WT, and the relative chlorophyll content of the other two mutant groups was significantly lower than that of the WT; at 12 days of treatment, the relative chlorophyll content of gmmet1-B was significantly higher than that of the WT, and there was no significant difference in the relative chlorophyll content of the other two mutant groups and the WT ( Figure 13 a and Figure 13 b).

[0097] The amount of plant chlorophyll content can be represented by the relative chlorophyll content. The plant chlorophyll content can reflect the plant growth status and growth rate to a certain extent. Appropriate drought can increase the plant chlorophyll content and promote plant growth. Drought stress increased the relative chlorophyll content of all four groups of materials, and gmmet1-B had the highest relative chlorophyll content. It is speculated that gmmet1-B has a strong chlorophyll synthesis ability to resist adverse environments and promote its own growth and development.

[0098] Effect of Drought Stress on Antioxidant Enzyme Activities in Seedlings of Three Mutants and Wild-Type Soybeans

[0099] Leaf SOD Activity: Compared with the control group, the leaf SOD activities of the three mutants and wild-type were significantly increased at 10 days of treatment; at 12 days of treatment, the leaf SOD activities of the three mutants showed no significant change, while the leaf SOD activity of WT decreased significantly ( Figure 14 a and Figure 14 b). Compared within the treatment groups, at 10 days of treatment, the leaf SOD activity of gmmet1-A was higher than that of WT, and the leaf SOD activities of the other two mutant groups had no significant difference from that of WT; at 12 days of treatment, the leaf SOD activities of gmmet1-A and gmmet1-AB were significantly higher than that of WT, and the leaf SOD activity of gmmet1-B had no significant difference from that of WT ( Figure 14 a and Figure 14 b).

[0100] Root SOD Activity: Compared with the control group, the root SOD activities of the three mutants and wild-type were significantly increased at 10 days and 12 days of treatment ( Figure 14 c and Figure 14 d). Compared within the treatment groups, at 10 days and 12 days of treatment, the root SOD activities of gmmet1-B and gmmet1-AB were significantly higher than that of WT, and the root SOD activity of gmmet1-A had no significant difference from that of WT.( Figure 14 c and Figure 14 d).

[0101] Leaf POD Activity: Compared with the control group, the leaf POD activities of the four groups of materials increased at 10 days of treatment; at 12 days of treatment, the leaf POD activities of gmmet1-A and gmmet1-AB increased, and the leaf POD activities of the other two groups showed no significant difference.( Figure 15 a, Figure 15 b). Compared within the treatment groups, at 12 days of treatment, the leaf POD activity of gmmet1-B was significantly higher than that of WT( Figure 15 a, Figure 15 b).

[0102] Root POD Activity: Compared with the control group, at 10 days of treatment, the root POD activities of gmmet1-A and gmmet1-B increased significantly, and the root POD activities of the other two groups of materials showed no significant change; at 12 days of treatment, the root POD activity of gmmet1-B increased significantly, and the root POD activities of the other two groups of materials showed no significant change( Figure 15 c, Figure 15d). Compared within the treatment groups, at 10 days of treatment, the root POD activities of all three mutants were significantly higher than that of the wild type; at 12 days of treatment, the root POD activities of gmmet1-A and gmmet1-B were significantly higher than that of the WT, and there was no significant difference in the root POD activity between gmmet1-AB and the WT( Figure 15 c, Figure 15 d).

[0103] Leaf CAT activity: Compared with the control group, at 10 days and 12 days of treatment, the leaf CAT activity of gmmet1-AB increased significantly, and there was no significant change in the leaf CAT activities of the other three groups of materials( Figure 16 a, Figure 16 b). Compared within the treatment groups, at 12 days of treatment, the leaf CAT activities of gmmet1-A and gmmet1-AB were significantly higher than that of the WT( Figure 16 a, Figure 16 b).

[0104] Root CAT activity: Compared with the control group, at 10 days of treatment, except for no significant change in the root CAT activity of gmmet1-A, the root CAT activities of the other three groups of materials increased significantly; at 12 days of treatment, the root CAT activities of all three mutants increased significantly, and there was no significant change in the root CAT activity of the wild type( Figure 16 c, Figure 16 d). Compared within the treatment groups, at 12 days of treatment, the root CAT activities of gmmet1-A and gmmet1-AB were significantly higher than that of the WT( Figure 16 c, Figure 16 d).

[0105] The above results show that except for the decrease in the SOD activity of wild-type leaves under the condition of 12 days of drought stress treatment, the antioxidant enzyme activities in the other groups were enhanced or unchanged. It shows that the four groups of soybean seedling materials can accumulate antioxidant enzymes to resist the damage caused by stress.

[0106] Statistical analysis of the change rates of growth indexes and physiological indexes of soybean seedlings after drought stress

[0107] Drought stress causes different degrees of increase or decrease in plant biomass. In order to analyze the change degrees of growth indexes and physiological indexes between methylation-reduced mutants of different genotypes and the wild type, the change rates of their measured values were statistically analyzed, and the results are as follows:

[0108] As Figure 17As shown, the calculation method is change rate = (treatment group - control group) / control group. Blue indicates negative values and red indicates positive values. After 10 days of drought stress treatment, the dry and fresh weights of soybean seedlings decreased. The degree of decrease in the dry weight of WT roots was significantly higher than that of gmmet1-A, gmmet1-B, and gmmet1-AB. Under the condition of 10 days of drought stress treatment, the change rate of relative electrical conductivity of gmmet1-B was significantly lower than that of other group materials. The soluble sugar content in the leaves of gmmet1-A after 10 days of treatment, the proline content in the leaves of gmmet1-AB after 12 days of treatment, the proline content in the roots of gmmet1-AB after 10 days of treatment, the SOD activity in the leaves of gmmet1-AB after 12 days of treatment, the SOD activity in the roots of gmmet1-B after 10 days of treatment, the POD activity in the roots of gmmet1-B after 10 days of treatment, the CAT activity in the leaves of gmmet1-AB after 12 days of treatment, and the CAT activity in the roots of gmmet1-AB increased significantly more than those of other groups.

[0109] The above results indicate that drought stress causes varying degrees of changes in the biomass of mutants and wild types. The change rates of osmoregulatory substances and antioxidant enzyme activities in different mutants are generally higher than those of the wild type, and the change rate of membrane lipid peroxidation is generally lower than that of the wild type. The reduction of CG methylation may enhance the drought resistance of mutants by affecting the levels of regulatory substances of drought resistance in gmmet1-A, gmmet1-B, and gmmet1-AB. Effects of cold stress on the phenotypes and survival rates of different genotypes of soybean seedlings

[0110] The growth conditions of the leaves of three MET1 gene mutants and wild type after 24 h of cold stress treatment in a 4°C incubator are as Figure 18 shown in a. Before the cold stress treatment, there were no obvious phenotypic differences among the three mutants and the wild type. When treated at 4°C for 24 h, it was found that the leaves of wild type plants showed damaged phenotypes, mainly manifested as leaf water loss, wilting and wrinkling, local color change to brown, and the appearance of white spots. There were no obvious changes in the leaves of the three mutants. After the cold stress treatment, the leaf colors of the four groups of materials became darker.

[0111] After 25 days of cold stress treatment, the survival rates of different genotypes were counted. The results showed that all WT died, and the survival rates of gmmet1-A, gmmet1-B, and gmmet1-AB were 15%, 8%, and 11%. The survival rates of gmmet1-A, gmmet1-B, and gmmet1-AB were significantly higher than that of WT ( Figure 18 b).

[0112] Effects of cold stress on the malondialdehyde content of three mutants and wild type soybean seedlings

[0113] Malondialdehyde (MDA) content in leaves: Compared with the control group, the MDA content in the leaves of gmmet1-A and gmmet1-B was significantly reduced, while there was no significant change in the MDA content in the leaves of the other two groups of materials. Compared within the treatment groups, the MDA content in the leaves of gmmet1-A was significantly lower than that of the other three groups ( Figure 19 a).

[0114] MDA content in roots: Compared with the control group, the MAD content in the roots of gmmet1-A and gmmet1-AB increased significantly, while there was no significant change in the MDA content in the roots of the other two materials. Compared within the treatment groups, the MAD content in the roots of gmmet1-A and gmmet1-AB was significantly higher than that of the other two groups ( Figure 19 b). Effects of cold stress on osmotic adjustment substances in three mutants and wild-type soybean seedlings

[0115] Proline content in leaves: Compared with the control group, the proline content in the leaves of gmmet1-B increased significantly, while there was no significant change in the proline content in the leaves of the other three groups of materials. Compared within the treatment groups, the proline content in the leaves of gmmet1-A and gmmet1-B was significantly higher than that of the other two groups of materials ( Figure 20 a).

[0116] Proline content in roots: Compared with the control group, the proline content in the roots of all three mutants increased significantly, while there was no significant change in the proline content in the roots of WT. Compared within the treatment groups, the proline content in the roots of all three mutants was significantly higher than that of the wild-type ( Figure 20 b).

[0117] Soluble sugar content in leaves: Compared with the control group, the soluble sugar content in the leaves of gmmet1-AB increased significantly, while the soluble sugar content in the leaves of gmmet1-B decreased significantly, and there was no significant change in the soluble sugar content in the leaves of the other two materials. Compared within the treatment groups, the soluble sugar content in the leaves of gmmet1-AB was significantly higher than that of the other three groups of materials ( Figure 20 c).

[0118] Soluble sugar content in roots: Compared with the control group, the soluble sugar content in the roots of gmmet1-B increased significantly, while there was no significant change in the soluble sugar content in the roots of the other three materials. Compared within the treatment groups, the soluble sugar content in the roots of gmmet1-A was significantly lower than that of WT, and there was no significant difference in the soluble sugar content in the roots of the other two mutants and WT ( Figure 20 d).

[0119] Effects of cold stress on superoxide dismutase (SOD) activity in three mutants and wild-type soybean seedlings

[0120] Leaf SOD activity: Compared with the control group, except for the leaf SOD activity of gmmet1-A showing no significant change, the leaf SOD activities of the other three groups of materials were all significantly increased. Within the treatment groups, the leaf SOD activity of WT was significantly higher than that of the three mutants ( Figure 21 )

[0121] Root SOD activity: Compared with the control group, except for the leaf SOD activity of gmmet1-A showing no significant change, the leaf SOD activities of the other three groups of materials were all significantly increased. The leaf SOD activity of gmmet1-B was significantly higher than that of the wild type, and there was no significant difference in the root SOD activities of the other two materials and that of WT ( Figure 21 )

[0122] The above results indicate that under cold stress, mutants with different degrees of CG methylation reduction and wild type generally showed varying degrees of changes in physiological and biochemical indexes to cope with the adverse environment. This shows that soybean seedlings can all regulate their own metabolism and physiological and biochemical reactions to resist stress, but the resistance abilities of different types of soybean seedlings are different.

[0123] Statistics and analysis of the change rates of physiological indexes of soybean seedlings after cold stress

[0124] Cold stress causes changes in plant biomass. To compare the change degrees of physiological indexes of mutants with reduced methylation of different genotypes and wild type, the change rates of their measured values were statistically analyzed ( Figure 22 ). The results show that under the condition of 24 h treatment, the change rates of SOD activities in the leaves and roots of gmmet1-AB were significantly higher than those of WT, the soluble sugar content in the leaves of gmmet1-AB was significantly higher than that of the other three materials, the soluble sugar content in the roots of WT was significantly lower than that of the other three materials, and there was no significant difference in the MDA contents in the leaves and roots.

[0125] Under the condition of 24 h cold stress treatment, soybean seedlings with reduced CG methylation improved their cold resistance by significantly increasing the activities of antioxidant enzymes and the contents of osmotic adjustment substances in vivo. The damage degree of wild type soybean seedlings was significantly higher than that of soybean treatment groups with different degrees of reduced CG methylation. It shows that CG methylation can participate in regulating the metabolism of soybean physiological related substances and improve the cold resistance of soybean seedlings, but there is no significant difference in the cold resistance of the three types of soybeans with different degrees of reduced methylation in this experiment.

[0126] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0127] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A soybean GmMET1 single gene mutant, characterized in that: The single gene mutants are single gene homozygous mutants obtained by editing the GmMET1-1 or GmMET1-2 gene by the CRISPR-Cas9 vector system. For the GmMET1-1 gene, the editing events include but are not limited to the insertion of a base in the DNMT-RDF domain in the second exon, whose nucleotide sequence is shown in SEQ ID NO:1, or a deletion of 171 bp, which is set as GmMET1-A, as one of the mutant materials for stress treatment, whose nucleotide sequence is shown in SEQ ID NO:

2. For the GmMET1-2 gene, the editing events include but are not limited to the deletion of 2 bases in the DNMT-RDF domain at the second exon, which is set as GmMET1-B, as one of the mutant materials for stress treatment, whose nucleotide sequence is shown in SEQ ID NO:3, or a deletion of 6 bases in the replication focus domain of cytosine-specific DNA methyltransferase, whose nucleotide sequence is shown in SEQ ID NO:

4. The genotype of GmMET1-1 is set as GmMET1-1. - / - GmMET1-2 + / + , the genotype of GmMET1-2 was set to GmMET1-1 + / + GmMET1-2 - / - .

2. A soybean GmMET1 double gene mutant, characterized in that: The double gene mutants were obtained by crossing two different types of single gene homozygous mutants and further selfing. The single gene homozygous mutants include GmMET1-1 and GmMET1-2.

3. The soybean GmMET1 single gene mutant according to claim 2, characterized in that: The F1 generation double gene heterozygous mutant GmMET1-1 + / - GmMET1-2 + / - There was no significant difference in growth and development compared with the wild type.

4. The soybean GmMET1 single gene mutant according to claim 2, characterized in that: No GmMET1-1 was isolated in the F2 generation. - / - GmMET1-2 - / - Double gene homozygous mutant, and GmMET1-1 + / - GmMET1-2 - / - The double gene mutant showed obvious growth and development abnormalities, GmMET1-1 + / + GmMET1-2 + / + 、GmMET1-1 + / + GmMET1-2 - / - 、GmMET1-1 - / - GmMET1-2 + / + The number of isogenic genotypes is significantly higher than the theoretical ratio, GmMET1-1 + / - GmMET1-2 + / - 、GmMET1-1 + / - GmMET1-1 - / - The amount of GmMET1-1 is lower than the theoretical ratio. - / - GmMET1-2 + / - The genotype was set as GmMET1-AB as one of the mutant materials for stress treatment.

5. A method for producing the soybean GmMET1 gene mutant according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1, construct a CRISPR-Cas9 vector containing a gRNA sequence that can target the GmMET1-1 or GmMET1-2 gene; S2, the constructed vector is introduced into the recipient cell, allowing the Cas9 protein to bind to the gRNA complex and cut the DNA at the target site; S3, screening the plant materials after editing and confirming the expected editing effect.

6. A method for producing the soybean GmMET1 double gene mutant according to claim 2, characterized in that: The following steps are involved: S1, using single-gene homozygous mutants to crossbreed and generate F1 generation double-gene heterozygous mutants; S2, self-pollinate the F1 generation to produce the F2 generation; S3, Analyze the genotype distribution of the F2 generation, with a particular focus on GmMET1-1 - / - GmMET1-2 - / - The existence and phenotypic characteristics of double gene homozygous mutants.

7. Use of the soybean GmMET1 gene mutant according to any one of claims 1 to 2 in agricultural planting, especially in studying DNA methylation mechanisms and crop improvement.

8. Use of the soybean GmMET1 gene double mutant as claimed in claim 2 in revealing gene interaction networks and evaluating the effects of multi-gene loss of function.

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