USE OF SOYBEAN BROAD-SPECTRUM DISEASE RESISTANCE RELATED GENE

Abstract

The GmLMM1 gene, which is involved in the regulation of PTI immune responses, Phytophthora resistance, bacterial blight of soybean disease, soybean halo disease, etc., is cloned in soybeans. The PTI immune response and pathogen resistance of plants can be negatively regulated via the GmLMM1 gene. By reducing the expression of GmLMM1, the PTI immune response of plants can be effectively enhanced, and the pathogen resistance of plants can be increased. Cloning and functional discovery of the GmLMM1 gene provide important foundations and theoretical support for research on the related mechanisms of soybean disease resistance and provide valuable genetic resources for advancing the research and application of plant defense systems. Additionally, cloning and functional delivery of the GmLMM1 gene allows for breeding new soybean varieties with high disease resistance.

Claims

1. (canceled)

2. A method for enhancing the PTI immune response of plants, the method comprising: reducing the expression of a GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene.

3. (canceled)

4. A method for plant genetic breeding or transgenic plant preparation, the method comprising: regulating the expression of a soybean GmLMM1 gene or a mutant soybean GmLMM1 gene or a protein encoded by the soybean GmLMM1 gene or the mutant soybean GmLMM1 gene.

5. The method according to claim 2, wherein the protein encoded by the soybean GmLMM1 gene comprises any one of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO. 1; (2) an amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence represented by SEQ ID NO. 1, and having the same function as the amino acid sequence represented by SEQ ID NO. 1; or (3) an amino acid sequence comprising at least 80% homology to the amino acid sequence represented by SEQ ID NO. 1; the protein encoded by the mutant gene of the soybean GmLMM1 gene has any one of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO. 3; (2) an amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence represented by SEQ ID NO. 3, and having the same function as the amino acid sequence represented by SEQ ID NO. 3; and (3) an amino acid sequence having at least 80% homology to the amino acid sequence represented by SEQ ID NO. 3.

6. The method according to claim 2, further comprising increasing a soybean pathogen resistance by reducing the expression of the GmLMM1 gene via targeted knockout of the GmLMM1 gene using CRISPR/Cas9 technology.

7. The method according to claim 6, wherein the gRNA comprises a nucleotide sequence represented by SEQ ID NO. 5.

8. A method for regulating a pathogen resistance of plants, the method comprising: regulating the expression of GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the soybean GmLMM1 gene or a mutant GmLMM1 gene in plants, wherein the protein encoded by the soybean GmLMM1 gene comprises any one of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO. 1; (2) an amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence represented by SEQ ID NO. 1, and having the same function as the amino acid sequence represented by SEQ ID NO. 1; or (3) an amino acid sequence comprising at least 80% homology to the amino acid sequence represented by SEQ ID NO. 1; and wherein the protein encoded by the mutant GmLMM1 gene of the soybean GmLMM1 gene comprises any one of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO. 3; (2) an amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence represented by SEQ ID NO. 3, and having the same function as the amino acid sequence represented by SEQ ID NO. 3; or (3) an amino acid sequence comprising at least 80% homology to the amino acid sequence represented by SEQ ID NO. 3.

9. The method according to claim 8, wherein the pathogen resistance of plants is increased by reducing the expression of the GmLMM1 gene or the protein encoded by the GmLMM1 gene in the plants.

10. The method according to claim 8, wherein the pathogen resistance is increased by reducing the expression of the GmLMM1 gene via targeted knockout of the soybean GmLMM1 gene, wherein targeted knockout comprises using CRISPR/Cas9 technology.

11. The method according to claim 2, wherein reducing the expression of the expression of a GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene comprises reducing the expression of a protein encoded by the GmLMM1 gene, wherein the protein encoded by the GmLMM1 gene comprises any one of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO. 1; (2) an amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence represented by SEQ ID NO. 1, and having the same function as the amino acid sequence represented by SEQ ID NO. 1; or (3) an amino acid sequence comprising at least 80% homology to the amino acid sequence represented by SEQ ID NO. 1.

12. The method according to claim 11, wherein reducing the expression of a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene comprises reducing the expression of a protein encoded by the GmLMM1 gene, wherein the protein encoded by the GmLMM1 gene comprises an amino acid sequence comprising at least 90% homology to the amino acid sequence represented by SEQ ID NO. 1.

13. The method according to claim 12, wherein reducing the expression of a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene comprises reducing the expression of a protein encoded by the GmLMM1 gene, wherein the protein encoded by the GmLMM1 gene comprises an amino acid sequence comprising 95% homology to the amino acid sequence represented by SEQ ID NO. 1.

14. The method according to claim 2, wherein reducing the expression of the expression of a GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene comprises reducing the expression of a protein encoded by the mutant GmLMM1 gene, wherein the protein encoded by the mutant GmLMM1 gene comprises any one of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO. 3; (2) an amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence represented by SEQ ID NO. 3, and having the same function as the amino acid sequence represented by SEQ ID NO. 3; or (3) an amino acid sequence comprising at least 80% homology to the amino acid sequence represented by SEQ ID NO. 3.

15. The method according to claim 14, wherein reducing the expression of the expression of a GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene comprises reducing the expression of a protein encoded by the mutant GmLMM1 gene, wherein the protein encoded by the mutant GmLMM1 gene comprises an amino acid sequence comprising at least 90% homology to the amino acid sequence represented by SEQ ID NO. 3.

16. The method according to claim 15, wherein reducing the expression of the expression of a GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the GmLMM1 gene or the mutant GmLMM1 gene comprises reducing the expression of a protein encoded by the mutant GmLMM1 gene, wherein the protein encoded by the mutant GmLMM1 gene comprises an amino acid sequence comprising 95% homology to the amino acid sequence represented by SEQ ID NO. 3.

17. The method according to claim 5, wherein the protein encoded by the soybean GmLMM1 gene comprises an amino acid sequence comprising at least 90% homology to the amino acid sequence represented by SEQ ID NO. 1.

18. The method according to claim 5, the protein encoded by the mutant gene of the soybean GmLMM1 gene comprises an amino acid sequence having at least 90% homology to the amino acid sequence represented by SEQ ID NO. 3.

19. The method according to claim 8, wherein regulating the expression of GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the soybean GmLMM1 gene or a mutant GmLMM1 gene in plants comprises regulating the expression of the protein encoded by the soybean GmLMM1 gene, wherein the protein encoded by the soybean GmLMM1 gene comprises an amino acid sequence comprising at least 90% homology to the amino acid sequence represented by SEQ ID NO. 1.

20. The method according to claim 8, wherein regulating the expression of GmLMM1 gene or a mutant GmLMM1 gene or a protein encoded by the soybean GmLMM1 gene or a mutant GmLMM1 gene in plants comprises regulating the expression of the protein encoded by the mutant gene of the soybean GmLMM1 gene, wherein the protein encoded by the mutant GmLMM1 gene of the soybean GmLMM1 gene comprises an amino acid sequence comprising at least 90% homology to the amino acid sequence represented by SEQ ID NO. 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 shows the observation of pathogen infection under ultraviolet light 48 hours after wild-type Williams 82 leaves and GmLMM1 mutant leaves were inoculated with Phytophthora sojae (P. sojae) P7076 hyphae blocks in Example 1 of the present disclosure. Scale, 1 cm. Inside the dashed circle are the lesions infected by pathogen.

[0051] FIG. 2 shows the location of GmLMM1 gene on soybean chromosome 13 in Example 1 of the present disclosure.

[0052] FIG. 3 shows the observation of infection and the statistics of the lesion area 60 hours after of the leaves of the transgenic plant with GmLMM1 gene knocked out were inoculated with Phytophthora sojae (P. sojae) P7076 hyphae blocks in Example 2 of the present disclosure. Scale, 1 cm. *** represents significant difference at a level of p<0.001.

[0053] FIG. 4 shows the statistics of the bacterial colony number 4 days after the leaves of the transgenic plant with GmLMM1 gene knocked out were inoculated with pathogen Pseudomonas syringae pv. glycinea (Psg) of bacterial blight of soybeans in Example 2 of the present disclosure. ** represents significant difference at a level of p<0.01.

[0054] FIG. 5 shows the statistics of the bacterial colony number 4 days after the leaves of transgenic plant with GmLMM1 gene knocked out were inoculated with pathogen Pseudomonas syringae pv. phaseolicola (Psp) of halo blight of the common bean, Phaseolus vulgaris in Example 2 of the present disclosure. *** represents significant difference at a level of p<0.001.

[0055] FIG. 6 shows that GmLMM1 inhibits the active oxygen burst in the PTI immune response induced by flg22 in Example 3 of the present disclosure. The active oxygen burst of tobacco leaves treated with 1 μM flg22 or water-treated and transiently expressing GFP or GmLMM1 gene was shown, wherein GFP represents the control group, GmLMM1-HA represents the experimental group, H.sub.2O represents the blank control group treated with H.sub.2O, flg22 represents treatment with flg22 small peptide, and the expression of GmLMM1 was detected by Western blot with Rubisco as the internal reference.

DETAILED DESCRIPTION

[0056] The preferred embodiments of the present disclosure will be described in detail below in conjunction with Examples. It should be understood that the following Examples are given for illustrative purposes only, and are not intended to limit the scope of the present disclosure. A person skilled in the art can make various modifications and substitutions to the present disclosure without departing from the purpose and spirit of the present disclosure.

[0057] The experimental methods used in the following Examples are conventional methods unless otherwise specified.

[0058] The materials and reagents used in the following Examples can be obtained from commercial sources unless otherwise specified.

Example 1: Identification of Phytophthora Resistance Phenotype of GmLMM1 Mutant to Phytophthora

[0059] The mutant GmLMM1 was obtained by screening the mutant library obtained by performing EMS mutagenesis on Williams 82. Phytophthora sojae (P. sojae) P7076 hyphae blocks were used to inoculate leaves of wild-type “Williams 82” and leaves of GmLMM1 mutant. After 48 hours of infection, pathogen infected leaves were observed under ultraviolet light (FIG. 1). The results show that the pathogen infected area of the mutant GmLMM1 is significantly smaller than that of the wild-type Williams 82, indicating that the mutant GmLMM1 significantly improves resistance to soybean Phytophthora compared with the wild-type Williams 82. The above experimental results indicate that the GmLMM1 mutant has the characteristics of being resistant to soybean Phytophthora. Therefore, it is speculated that the GmLMM1 gene may be involved in the regulation of plant immune response. By using the F2 segregated population obtained by the hybridization of GmLMM1 and “Hedou 12” for gene mapping, it was found that the GmLMM1 gene was located in the 131 kb interval between 15.08 Mb and 15.21 Mb on chromosome 13 (FIG. 2), and in combination with the whole genome resequencing result of GmLMM1, it is finally determined that the GmLMM1 gene is Glyma.13G054400 (the amino acid sequence of the protein encoded thereby is represented by SEQ ID NO. 1, and the CDS sequence is represented by SEQ ID NO. 2), and there is a single-base mutation (C to T) in the second exon region of Glyma.13G054400 (GmLMM1) gene in GmLMM1 (the amino acid sequence of the protein encoded by the mutant gene is represented by SEQ ID NO. 3, and the CDS sequence of the mutant gene is represented by SEQ ID NO. 4) (FIG. 2).

Example 2: Significantly Enhanced Pathogen Resistance in the Transgenic Plants with GmLMM1 Gene Knocked Out

[0060] A CRISPR/Cas9 recombinant plasmid with gRNA (sequence as represented by SEQ ID NO. 5) driven by GmU6 promoter and Cas9 protein driven by GmUbi3 promoter was constructed, and the recombinant plasmid was introduced into the wild-type soybean variety Dongnong 50 (DN50) with an Agrobacterium-mediated genetic transformation system. After genetic transformation, Ti generation transgenic plants were finally obtained. Phytophthora sojae (P. sojae) P7076 hyphae blocks were used to inoculate leaves of wild-type DN50 and leaves of transgenic plant CRISPR with GmLMM1 gene knocked out. After 60 hours of infection, trypan blue staining was used to observe the difference in infection, and the statistics of the area of lesion formation was performed (FIG. 3). The results show that the lesion area of the leaves of the transgenic plant CRISPR is significantly smaller than the lesion area of the wild-type DN50, indicating that the resistance to soybean Phytophthora is significantly improved after the GmLMM1 gene is knocked out. The leaves of wild-type DN50 and the leaves of the transgenic plant CRISPR with GmLMM1 gene knocked out were inoculated with the pathogen Pseudomonas syringae pv. glycinea (Psg) of bacterial blight of soybeans (FIG. 4) and the pathogen Pseudomonas syringae pv. phaseolicola (Psp) of halo blight of the common bean, Phaseolus vulgaris (FIG. 5) by injection, and after 4 days, statistical analysis of the number of pathogen colonies formed was performed. Statistical analysis results show that the number of pathogen colonies in the leaves of the transgenic plant CRISPR is significantly less than the number of colonies in the leaves of the wild-type DN50, indicating that the resistance to bacterial blight of soybeans and halo blight of the common bean, Phaseolus vulgaris is significantly improved after the GmLMM1 gene is knocked out. The results of genetic transformation experiment further illustrate the important role of GmLMM1 gene in immune response. The discovery of new functions of GmLMM1 gene provides valuable genetic resources for plant genetic breeding.

Example 3: Inhibition of PTI Immune Response of Plants by GmLMM1 Gene

[0061] Agrobacterium was injected into tobaccos to transiently express GFP (control group) and GmLMM1 (experimental group), and after 24 hours, water and flg22 (a small peptide containing 22 amino acids from conserved N-terminus of flagellin and commonly used in plant immunity research) were used for treatment, respectively. The active oxygen burst was detected after treatment, and it was found that transient overexpression of GmLMM1 can inhibit the active oxygen burst induced by flg22 (FIG. 6). The above experiments show that the GmLMM1 gene can inhibit the active oxygen burst in the PTI immune response, and negatively regulate the PTI immune response of plants.

[0062] Although the present disclosure has been described in detail with general descriptions, specific embodiments and tests above, it is obvious to a person skilled in the art that some modifications or improvements can be made on the basis of the present disclosure. Therefore, all these modifications or improvements made without departing from the spirit of the present disclosure belong to the scope of the present disclosure.

INDUSTRIAL APPLICABILITY

[0063] The present disclosure provides the use of a soybean broad-spectrum disease resistance related gene GmLMM1. In the present disclosure, the GmLMM1 gene, which is involved in the regulation of PTI immune response, Phytophthora resistance, bacterial blight of soybeans disease, halo blight of the common bean, Phaseolus vulgaris, etc., is cloned in soybeans. The PTI immune response and pathogen resistance of plants can be negatively regulated by the GmLMM1 gene; and by reducing the expression of the GmLMM1 gene, the PTI immune response of plants can be effectively enhanced, and the pathogen resistance of plants can be increased. The cloning and functional discovery of the GmLMM1 gene provide important gene foundations and theoretical support for research on the related mechanisms of soybean disease resistance, and provide valuable gene resources for advancing the research and application of plant defense systems and for breeding new soybean varieties with high disease resistance, and thus have an important application value and prospects in the genetic engineering-based breeding of soybean disease resistance.