Soybean Gmpeamt1 and Gmplmt genes and the encoded proteins in regulating soybean seed quality

Abstract

The application relates to the technical field of genetic engineering, in particular to a soybean GmPEAMT1 and GmPLMT gene and an application of the coded protein in regulating soybean seed quality. GmPEAMT1 and GmPLMT The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO. 1-2, GmPEAMT1 and GmPLMT The amino acid sequence of the protein coded by the gene is shown in the sequence table SEQ ID NO. 3-4. Regulating soybean seed quality includes reducing the water content of soybean seeds, and increasing the total protein content and amino acid content of soybean seeds. Advantages are that, by using the CRISPR-Cas9 technology to knockout the GmPEAMT1 and GmPLMT gene at the same time, a GmPEAMT1 and GmPLMT double-gene-edited soybean is constructed, materials are provided for soybean quality improvement, and the situation that the oil content and the protein and amino acid content in soybean seeds are negatively correlated in traditional molecular breeding is broken.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to a soybean. GmPEAMT1 and GmPLMT Application of genes and their encoded proteins in regulating soybean seed quality. Background Technology

[0002] Soybeans, as an important global oilseed crop and protein source, have their quality formation and stress resistance influenced by the phospholipid metabolism pathway. In this pathway, phosphatidylcholine (PC), a core component of the soybean cell membrane, directly determines the structural stability and permeability of the cell membrane through its content and molecular structure. The cell membrane, as a crucial barrier for the exchange of substances and signal transduction between the cell and its external environment, is essential for the maintenance of normal cell function, which is a fundamental prerequisite for protein synthesis and transport.

[0003] Meanwhile, phosphatidylcholine (PC) and seed oil share similar precursors. Changes in PC content can directly affect the accumulation efficiency and fatty acid ratio of soybean oil. PC synthesis depends on the synergistic action of phosphatidylethanolamine-N-methyltransferase (PEAMT) and phosphatidylcholine methyltransferase (PLMT). Changes in the protein content of these two enzymes directly regulate the efficiency of the PC synthesis pathway. When the protein expression of PEAMT and PLMT is imbalanced, it may lead to a response in oil metabolism through changes in PC. Directly altering genes that regulate oil typically results in a downregulation of amino acid content. By regulating PC expression, it is possible to avoid the competition for energy and substances between amino acid metabolism and oil metabolism, instead using intermediate products of PC and oil as a bridge to reduce the impact on amino acid metabolism. This is an effective means of balancing the oil and protein content in soybean seeds during the breeding of high-quality, high-oil varieties.

[0004] However, the molecular mechanisms by which PEAMT and PLMT synergistically regulate PC synthesis remain unclear. The regulatory network through which they precisely affect cell membrane stability, lipid accumulation efficiency, and amino acid metabolic balance is also poorly understood, and functional validation and application research of related genes are scarce. This technological gap makes it difficult to achieve synergistic optimization of soybean oil and protein content by targeting phospholipid metabolism pathways, thus limiting the breeding process of high-quality, high-oil soybean varieties. Therefore, there is an urgent need to develop molecular breeding methods based on dual-gene synergistic editing, construct efficient and targeted gene editing vectors, and improve soybean seed quality by precisely regulating key nodes in the phospholipid metabolism pathway. Summary of the Invention

[0005] To solve the above problems, the present invention provides a soybean GmPEAMT1 and GmPLMT Application of genes and their encoded proteins in regulating soybean seed quality.

[0006] The primary objective of this invention is to provide a soybean GmPEAMT1 and GmPLMT Application of genes and their encoded proteins in regulating soybean seed quality GmPEAMT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing. GmPEAMT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3 of the sequence listing; GmPLMT The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing. GmPLMT The amino acid sequence of the protein encoded by the gene is shown in the sequence listing SEQ ID NO.4.

[0007] Preferably, regulating soybean seed quality includes reducing soybean seed moisture content and increasing soybean seed total protein content and amino acid content.

[0008] The second objective of this invention is to provide a recombinant vector pYL-GmPEAMT1 / GmPLMT, designed based on GmPEAMT1 Genes and GmPLMT The specific sgRNA target sequence of the conserved region of the gene exon was cloned into the BsaI restriction site of the pYLCRISPR / Cas9 vector to obtain the recombinant vector pYL-GmPEAMT1 / GmPLMT. GmPEAMT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing. GmPLMT The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing.

[0009] The third objective of this invention is to provide a method for regulating soybean seed quality, comprising: constructing the recombinant vector pYL-GmPEAMT1 / GmPLMT and introducing it into Agrobacterium; then performing Agrobacterium-mediated genetic transformation using soybean cotyledonary nodes as explants, and obtaining regenerated plants through co-culture, shoot induction, and rooting culture; obtaining a homozygous double-gene mutant through resistance screening and molecular identification; wherein the soybean seeds of the homozygous double-gene mutant have reduced water content and increased total protein and amino acid content.

[0010] Preferably, the recombinant vector pYL-GmPEAMT1 / GmPLMT is introduced into Agrobacterium tumefaciens EHA105 by electroporation, and then cultured in a mannitol medium containing kanamycin and rifampicin yeast extract to obtain an infection solution; the infection solution is used to infect soybean cotyledon nodes, and after infection, the explants are placed on a co-culture medium lined with sterile filter paper for co-culture.

[0011] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0012] This invention utilizes CRISPR-Cas9 technology to study soybeans.GmPEAMT1 and GmPLMT Genes are knocked out simultaneously to construct GmPEAMT1 and GmPLMT Double-gene editing in soybeans provides materials for soybean quality improvement, breaking the traditional molecular breeding paradigm where there is a negative correlation between oil content and protein and amino acid content in soybean seeds. This invention reveals the regulation of soybean seed quality by double gene deletion, aiming to fill the gap in improving the balance of oil, protein, and amino acid content by inhibiting soybean phospholipid metabolism. It provides potential genetic resources and technical support for breeding high-quality new soybean varieties, meeting the agricultural demand for nutritionally balanced and superior soybean varieties, and promoting the industrial application of soybean molecular breeding technology. Attached Figure Description

[0013] Figure 1 The results of Agrobacterium-mediated genetic transformation of soybean seeds according to embodiments of the present invention are: (A) co-culture of soybean seeds; (B) induction of callus shoot formation; (C) induction of shoot elongation; and (D) induction of root development.

[0014] Figure 2 This is the soybean cultivar "Dongnong 50" based on CRISPR / Cas9 technology provided according to an embodiment of the present invention. GmPEAMT1 and GmPLMT Molecular characterization of dual editing; (A) GmPEAMT1 and GmPLMT (a) Location of the editing target gene sequence; (b) Protein model diagram of GmPEAMT1 and GmPLMT.

[0015] Figure 3 It is provided according to the embodiments of the present invention. GmPEAMT1 and GmPLMT The relative levels of water content, total protein, lipids, and amino acids in double-gene-edited soybean seeds were determined; (A) represents the water, total protein, and lipid content; (B) represents the relative levels of amino acids; significant differences between the control group and the double-gene-edited group were based on P ≤ 0.05 ( ). P≤0.01 ) and P≤0.001 The 18 amino acids detected included phenylalanine (Phe), methionine (Met), lysine (Lys), leucine (Leu), isoleucine (Ile), threonine (Thr), valine (Val), tryptophan (Try), aspartic acid (Asp), alanine (Ala), proline (Pro), glycine (Gly), glutamic acid (Glu), cysteine ​​(Cys), arginine (Arg), serine (Ser), tyrosine (Tyr), and histidine (His). Detailed Implementation

[0016] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0018] This invention provides GmPEAMT1 and GmPLMT The gene and its nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2 of the sequence listing; GmPEAMT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3 of the sequence listing; GmPLMT The amino acid sequence of the protein encoded by the gene is shown in the sequence listing SEQ ID NO.4.

[0019] Specific sgRNAs targeting the aforementioned genes are also provided: these sgRNAs are designed based on the conserved exon regions of the GmPEAMT1 and GmPLMT genes and can specifically guide the Cas9 enzyme to cleave the target genes; a dual-gene editing recombinant vector pYL-GmPEAMT1 / GmPLMT containing this sgRNA is also provided. This vector uses pYLCRISPR / Cas9 as its backbone and clones the sgRNA fragments of the two genes into the vector through the BsaI restriction site. Sequencing has verified that the construction was successful.

[0020] It also provides a method for obtaining [something] using the recombinant vector pYL-GmPEAMT1 / GmPLMT. GmPEAMT1 and GmPLMT A method for double-gene editing of soybean plants: A vector was introduced into Agrobacterium tumefaciens EHA105 (purchased from Beijing Coollab Technology Co., Ltd., catalog number CC403-10) via electroporation. Then, Agrobacterium-mediated genetic transformation was performed using cotyledonary nodes of soybean 'Dongnong 50' as explants. Regenerated plants were obtained through co-culture, shoot induction, and rooting culture. Subsequently, homozygous mutants with both genes were obtained through resistance screening and molecular identification. The method also provides applications for improving seed quality in double-gene-edited soybean plants, including regulating water content and increasing seed protein and amino acid content. The mutant soybean seeds showed significantly increased protein and amino acid (especially essential amino acids) content while maintaining stable oil content, making them suitable for breeding and developing high-protein, high-nutrient soybean varieties.

[0021] Example 1

[0022] This embodiment provides the design and vector construction of single-target gene guide RNA, as detailed below:

[0023] Experimental material preparation: Fresh leaves of soybean variety "Dongnong 50" were selected as experimental materials; specifically, seeds of soybean variety "Dongnong 50" were provided by Wuhan Boyuan Biotechnology Co., Ltd. (Wuhan, China). Total RNA was extracted using the TRIzol method, and RNA integrity was verified by 1% agarose gel electrophoresis. The A260 / A280 ratio (1.8~2.0) was measured by ultraviolet spectrophotometer to ensure that the RNA purity met the experimental requirements.

[0024] GmPEAMT1 (Glyma_05G246500v4) and GmPLMT The exon sequences of the (Glyma_08G218900v4) gene were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Specific sgRNAs were designed using CRISPR-P v2.0 (http: / / cbi.hzau.edu.cn / CRISPR / ) to avoid off-target effects.

[0025] The sgRNA target sequences of the two genes are as follows:

[0026] GmPEAMT1: 5'-ACGGACTTGCCTTCGAATGGAGG-3' and 5'-GGCAAGTCCGTTATTGAGCTTGG-3';

[0027] GmPLMT: 5'-ATGGGGATTGCGGCGGCTATTGG--3' and CACAAGTCCACCCACCACTGTGG-3';

[0028] The dual-gene editing recombinant vector pYL-GmPEAMT1 / GmPLMT was constructed by cloning into the BsaI site of the pYLCRISPR / Cas9 vector using a CRISPR / Cas9 vector construction kit (BGK041, Hangzhou Baige Gene Co., Ltd., Hangzhou, China). Successful vector construction was verified by sequencing after construction.

[0029] The specific method for constructing the carrier is as follows:

[0030] (1) Enzyme digestion reaction: Take 1 μg of pYLCRISPR / Cas9 vector, add 2 μL of BsaI endonuclease, 5 μL of 10×CutSmartBuffer, and add ddH2O to 50 μL. Digest at 37℃ for 3 h, and recover the vector backbone (about 14 kb) by 1% agarose gel electrophoresis.

[0031] (2) Ligation reaction: Mix the vector backbone and double-stranded sgRNA fragments at a molar ratio of 1:3, add 1 μL of T4 DNA Ligase and 2 μL of 10×T4 DNA Ligase Buffer, add ddH2O to make up to 20 μL, and ligate overnight at 16℃;

[0032] (3) Transformation and screening: The ligation product was transformed into Escherichia coli DH5α competent cells (TIANGEN, CB101), plated on LB solid medium containing 50 μg / mL kanamycin, and incubated upside down at 37℃ for 12 h. Single colonies were picked and inoculated into LB liquid medium, and incubated at 200 rpm and 37℃ for 8 h before plasmid extraction.

[0033] (4) Sequencing verification: Sanger sequencing was performed using universal primers for the vector (pYL-F: 5'-GAGCGGATAACAATTTCACACAGG-3'; pYL-R: 5'-GCGTCTTCTGCTTGGTGTAGC-3') to verify that the sgRNA fragment was correctly inserted into the BsaI site of the pYLCRISPR / Cas9 vector and that there were no base mutations in the vector backbone. Finally, the double gene editing recombinant vector was obtained and named pYL-GmPEAMT1 / GmPLMT.

[0034] Example 2

[0035] Agrobacterium-mediated genetic transformation and regeneration culture of soybean cotyledonary nodes, as follows: The recombinant plasmid pYL- was transformed using an electroporation method. GmPEAMT1 / GmPLMT Introducing Agrobacterium tumefaciens ( Agrobacterium tumefaciensIn EHA 105, the soybeans were inoculated onto yeast extract mannitol medium (YMB) plates containing 50 mg / L kanamycin and 50 mg / L rifampin, and cultured at 28 ℃ for 48 h to obtain the infection solution. Dongnong 50 soybean seeds were taken, the seed coat was removed, half of the cotyledons were cut off transversely, leaving a 3-5 mm hypocotyl, and the two cotyledons were separated longitudinally. After removing the apical bud and primary bud, the cotyledon nodes were lightly cut with a blade to obtain cotyledon node explants for transformation. These explants were placed in the infection solution, and the solution was changed after 2 hours, continuing the infection for 3 hours. After infection, the soybeans were removed, the seed coat was removed, and they were placed in a large petri dish lined with sterile filter paper, with excess infection solution absorbed as much as possible. Then, the soybeans were placed flat-side up on a solid co-culture medium lined with sterile filter paper and co-cultured at 26 ℃ in the dark for 5 days. After 5 days of co-culturing, the explants were washed three times with sterile water and then transferred to SI-I medium for recovery. The cotyledons were inserted into the medium with the embryo axis facing downwards and the cotyledon plane facing downwards, leaving half of the cotyledon exposed. 20 explants were placed per dish, and cultured under light for 7 days. Cotyledons from SI-I that had produced clustered buds were selected, large buds were cut, and the explants were vertically inserted into the medium, ensuring the entire bud was submerged. 10-14 explants were placed per dish, and cultured under light for 2 weeks. Undying buds from SI-II were selected, large buds were cut cleanly, and browned parts of the explant surface were removed. The explants were then transferred to SE medium, ensuring each bud was in contact with the medium. The SE medium was changed every 2 weeks, with a maximum of two subcultures. Buds (3-5 cm) emerging from the SE medium were selected, and the cut ends were soaked in IBA solution for 60 seconds before being transferred to RM rooting medium. After roots grew to 3-4 cm, transplanting began. The batch, carrier, and time were labeled, excess medium was washed away with water, and the seedlings were transplanted into pots (soil:vermiculite = 2:1) for hardening off. Results are as follows. Figure 1 As shown. Figure 1 Image A shows the state of soybean cotyledonary explants after co-culturing with Agrobacterium carrying the recombinant vector, with the explants remaining active on the culture medium; Image B shows that the co-cultured explants formed callus tissue and differentiated into shoot clusters in the induction medium, indicating that the transformation by Agrobacterium had been successfully initiated, and soybean cells began to proliferate and differentiate into shoot tissue; Image C shows that the induced shoot clusters further elongated and grew, with their morphology gradually approaching that of normal shoots, indicating that the differentiation capacity of the transformed cells was normal; Image D shows that the elongated shoots formed complete regenerated plants, proving that this transformation system can achieve the regeneration process from explants to complete transgenic plants.

[0036] Example 3

[0037] Soybean plants obtained through regeneration culture, as potential dual-gene-editing materials, need further screening for resistance and molecular identification to clarify their gene-editing status. The specific screening and identification methods are as follows:

[0038] Soybean plants were sprayed with 6-glufosinate. Three days later, surviving plants were collected and tested using PAT / bar EPSPS LFD Strips (LS) (Hangzhou Baige Gene Co., Ltd., Hangzhou, China) to screen for soybean plants with the Bar resistance gene. Genomic DNA was extracted from the surviving soybean plants and targeted to the gene. GmPEAMT1 and GmPLMT Specific primer pairs were designed based on the genome sequence, and the above-mentioned plants were detected by PCR amplification to screen for positive transformants; the specific primer pairs were also used for GmPEAMT1 and GmPLMT The genome sequence was analyzed, and the specific primer sequences are as follows:

[0039] GmPEAMT1 -F:5'-TTTCAGCTATGGATGAGCGT-3'

[0040] GmPEAMT1 -R: 5'-CAAAAGCGTAAGTTGAGCAG-3;

[0041] GmPLMT -F:5'-TCTTCAAACCAAACAAGCCA-3'

[0042] GmPLMT-R (5'-AGAAGTAGAAAGGAGGAGGC-3;

[0043] The amplified products were sequenced using the primers described above to verify the presence of insertion or deletion mutations. Molecular modeling analysis was used to analyze the changes in protein structure after gene editing, and the results are as follows: Figure 2 As shown. Figure 2 The soybean cultivar "Dongnong 50" based on CRISPR / Cas9 technology was showcased. GmPEAMT1 and GmPLMT Molecular characterization of dual editing, where B shows protein model diagrams of GmPEAMT1 and GmPLMT: the original domains of the protein, the mutated amino acids in the domains due to gene editing, and the regions with a predicted local distance difference test (pLDDT) score below 90 are represented by different colors, which can clearly show the impact of gene editing on protein structural stability.

[0044] T0 generation positive soybean plants were self-pollinated to obtain T1 generation, and T1 generation seeds were harvested and planted to obtain T2 generation; samples of T2 generation plants were taken and sequenced and screened at Sangon Biotech (Changchun) Co., Ltd. (Changchun, China) to obtain double gene mutants without wild-type (WT) sequences for subsequent experiments.

[0045] Example 4

[0046] To clarify GmPEAMT1 andGmPLMT The effects of dual gene editing on soybean quality were investigated. Nutritional components of the selected dual-gene mutant seeds were determined. Specific methods and results are as follows:

[0047] Collect a total of 10 g of mature soybean seeds (grown for 146 days) and weigh them. Allow them to air dry naturally in a cool, well-ventilated place for 3-5 days. The mass of the dried seeds was measured using a multi-functional near-infrared spectroscopy analyzer (NIRS™ DS 3F, FORS (Beijing) Technology & Trade Co., Ltd., Beijing, China) to determine moisture content, protein content, and the content of various amino acids in the mature soybean seeds.

[0048] Measurement results ( Figure 3 Clearly revealed GmPEAMT1 and GmPLMT The regulatory effect of dual gene editing on the composition of soybean seeds: Seeds showed decreased water content and increased total protein and amino acid content, while lipid content showed no significant difference. Of the 18 amino acids tested in dual-gene-edited soybean seeds, 12 showed significant increases. These amino acids can serve as direct precursors for protein synthesis in soybean seeds, providing sufficient raw materials for seed protein storage and contributing to an increase in overall soybean protein content. Regarding essential amino acids: threonine (Thr), isoleucine (Ile), leucine (Leu), and valine (Val) are key nutrients that the human body cannot synthesize independently and must obtain from food. Their significant increase has direct and important nutritional value, providing core raw material support for the development of high-nutrient-density soybean processed products. From the perspective of non-essential amino acids: the increased content of aspartic acid (Asp), alanine (Ala), proline (Pro), glycine (Gly), glutamic acid (Glu), cysteine ​​(Cys), arginine (Arg), and serine (Ser) plays an important positive role in the storage tolerance and metabolism of soybean seeds. For example, proline (Pro) can enhance seed tolerance, and cysteine ​​(Cys) participates in the synthesis of acetyl-CoA. As a key hub connecting lipid and protein metabolism, acetyl-CoA promotes fatty acid synthesis and provides precursors for the carbon skeleton of amino acids, which directly affects the synergistic optimization of soybean seed oil content and protein quality.

[0049] The above results prove that GmPEAMT1 and GmPLMT The deletion of genes balances the inverse relationship between lipid content and amino acid metabolism, further illustrating that gene editing shifts carbon sources towards protein synthesis pathways. This optimizes nutritional quality by increasing essential amino acids while simultaneously increasing total protein levels through the reserve of non-essential amino acids, creating a dual regulatory effect that provides both nutritional value and physiological metabolic support. Both of these factors together confirm that... GmPEAMT1 and GmPLMTThe regulatory role of genes in lipid balance and amino acid metabolism further demonstrates that dual gene editing can maintain lipid stability and promote the shift of carbon sources towards protein synthesis pathways, which is a dual regulation that combines the increase in total amount and the optimization of quality.

[0050] In summary, GmPEAMT1 and GmPLMT These are genes that play a key regulatory role in plant nutrient metabolism pathways. Their loss of function (gene editing) can significantly alter the material accumulation pattern of soybean seeds. After gene editing, the oil content of soybean seeds remains stable while the water allocation mechanism is changed, thereby promoting the shift of carbon sources towards the protein synthesis pathway, ultimately increasing the protein content in the seeds. This provides an important gene target for breeding high-protein soybeans and reveals the core role of these two genes in soybean seed quality.

[0051] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0052] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A type of knockout soybean GmPEAMT1 and GmPLMT The application of genes in regulating soybean seed quality is characterized by: GmPEAMT1 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO. 1, GmPEAMT1 The amino acid sequence of the protein encoded by the gene is shown in the sequence listing SEQ ID NO. 3; GmPLMT The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO. 2, GmPLMT The amino acid sequence of the protein encoded by the gene is shown in the sequence listing SEQ ID NO. 4; The regulation of soybean seed quality includes reducing the moisture content of soybean seeds and increasing the total protein content and amino acid content of soybean seeds.

2. A method for regulating soybean seed quality, characterized in that: include: The recombinant vector pYL-GmPEAMT1 / GmPLMT was constructed and introduced into Agrobacterium tumefaciens; Agrobacterium-mediated genetic transformation was then performed using soybean cotyledonary nodes as explants, and regenerated plants were obtained through co-culture, shoot induction, and rooting culture; a double-gene homozygous mutant was obtained through resistance screening and molecular identification; the soybean seeds of the double-gene homozygous mutant had reduced water content and increased total protein and amino acid content; The construction method of the recombinant vector pYL-GmPEAMT1 / GmPLMT includes: designing based on GmPEAMT1 Genes and GmPLMT The specific sgRNA target sequence of the conserved region of the gene exon was cloned into the BsaI restriction site of the pYLCRISPR / Cas9 vector to obtain the recombinant vector pYL-GmPEAMT1 / GmPLMT. GmPEAMT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing. GmPLMT The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing.

3. The method for regulating soybean seed quality according to claim 2, characterized in that: The recombinant vector pYL-GmPEAMT1 / GmPLMT was introduced into Agrobacterium tumefaciens EHA105 via electroporation. The explants were cultured in mannitol medium containing kanamycin and rifampicin yeast extract to obtain an infection solution. The infection solution was used to infect soybean cotyledon nodes. After infection, the explants were placed on a co-culture medium lined with sterile filter paper for co-culture.

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