Application of GmCDPKb gene in regulating soybean symbiotic nodule number

Abstract

The application belongs to the technical field of plant genetic engineering and soybean breeding, and discloses a gene GmCDPKb Application of the gene in regulating the number of symbiotic nodule of soybean, GmCDPKb The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The application constructs GmCDPKb The mutant and overexpression soybean stable genetic transformation plants are obtained, through trait investigation, it is found that the nodule number of the mutant plant is significantly lower than that of the wild type, and the nodule number of the overexpression plant is significantly higher than that of the wild type. It is shown that GmCDPKb The gene plays a regulatory role on the nodule number of soybean. The application discloses that GmCDPKb The gene is a key regulatory factor for regulating the number of symbiotic nodule of soybean, the nodule number of soybean can be stably and significantly regulated by regulating the expression amount of the gene, and important gene resources and germplasm materials are provided for analyzing the symbiotic nitrogen fixation molecular mechanism of soybean and cultivating high-efficiency nitrogen fixation soybean new varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and soybean breeding technology, specifically involving GmCDPKb Application of genes in regulating the number of symbiotic nodules in soybeans. Background Technology

[0002] Calcium-dependent protein kinases (CDPKs) are a class of serine / threonine-specific protein kinases widely found in plants and serve as important calcium ion sensors. CDPK genes exhibit diverse functions. Overexpression of some CDPK genes, such as AtCPK1, AtCPK4, and AtCPK6, can enhance plant tolerance to drought and salt stress, manifested by increased proline content, decreased malondialdehyde (MDA) and hydrogen peroxide (H2O2) content, and reduced stomatal aperture, thereby reducing water loss and improving plant survival under adverse conditions. The regulation of plant secondary metabolism by CDPKs has also attracted considerable attention. Studies have found that CDPKs can influence the synthesis of plant secondary metabolites by phosphorylating enzymes involved in secondary metabolism, such as phenylalanine ammonia-lyase (PAL), and transcription factors that regulate the expression of genes related to secondary metabolism. Therefore, CDPKs play a crucial role in plant cells, and in-depth research on their functions is of great significance for breeding superior crop varieties and improving agricultural production efficiency.

[0003] Soybeans, an important crop originating in China and used for grain, oil, and feed, occupy a central position in agricultural production and food supply due to their rich protein content. Nitrogen is a crucial nutrient element indispensable for soybean growth and development, and its acquisition mainly relies on a symbiotic nitrogen-fixing system established with rhizobia. The root nodules formed by soybean roots can convert atmospheric nitrogen into usable ammonium salts. This process not only meets the soybean's own nitrogen needs but also improves soil nitrogen levels, which is of great significance to agricultural ecology.

[0004] However, the symbiotic nodulation of soybeans and rhizobia is a complex process involving multiple stages, including rhizobia recognition and infection, symbiotic signal transduction, and nodule organ formation. It is also highly susceptible to environmental factors, and its molecular regulatory mechanisms remain incompletely understood. This gap in understanding limits the precise regulation of the symbiotic nitrogen fixation system and hinders the development of highly efficient nitrogen-fixing soybean varieties. Therefore, identifying key genes regulating symbiotic nodulation is of significant scientific value and application potential for elucidating the molecular mechanisms of symbiotic nitrogen fixation and promoting sustainable agricultural development. Summary of the Invention

[0005] The purpose of this invention is to provide GmCDPKbThe application of this gene in regulating the number of symbiotic nodules in soybeans fills the gap in existing research on the role of this gene in regulating symbiotic nodules in soybeans, and provides gene resources and germplasm materials for elucidating the molecular mechanism of symbiotic nitrogen fixation in soybeans and for breeding new varieties of soybeans with high efficiency in nitrogen fixation.

[0006] To achieve the above objectives, the present invention provides GmCDPKb The application of genes or their encoded proteins in regulating the number of symbiotic nodules in soybeans, the GmCDPKb The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, knocking out soybeans GmCDPKb Genes that reduce the number of symbiotic nodules in soybeans and overexpress the gene in soybeans GmCDPKb Genes that increase the number of symbiotic nodules in soybeans.

[0008] The present invention also provides GmCDPKb Application of protein in regulating the number of symbiotic nodules in soybeans GmCDPKb The amino acid sequence of the protein is shown in SEQ ID NO.2. GmCDPKb The gene encoding the protein is GmCDPKb Gene, GmCDPKb The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] This invention also provides a method for regulating the number of symbiotic nodules in soybeans, by knocking out nodules in soybeans. GmCDPKb Genes that reduce the number of symbiotic nodules in soybeans and overexpress the gene in soybeans GmCDPKb Genes that increase the number of symbiotic nodules in soybeans; GmCDPKb The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] Furthermore, methods to reduce the number of symbiotic nodules in soybeans include the following steps:

[0011] S1. Designing targets using CRISPR-GE online tools GmCDPKb The sgRNA in the gene coding region is modified with adapter bases according to the sequence information of the vector to ensure efficient binding of the sgRNA to the Cas9 protein;

[0012] S2. Obtain double-stranded oligonucleotides using PCR amplification technology;

[0013] S3. The sgRNA sequence was constructed into the pCBSG015 vector using homologous recombination.

[0014] S4. The successfully constructed sgRNA-pCBSG015 vector was introduced into EHA105 Agrobacterium competent cells;

[0015] S5. Use the Agrobacterium obtained in step S4 to infect soybeans;

[0016] S6. Perform plant tissue culture on the soybean tissue obtained in step S5;

[0017] S7. Extract DNA from the tissue culture seedlings obtained in step S6, perform PCR amplification of sgRNA, and select positive plants by agarose gel electrophoresis and DNA sequencing to obtain... GmCDPKb Soybean plants with genetic mutations.

[0018] Furthermore, in step S1, the target sequences of the sgRNA are GACGGTGATGCCGTTGTGGTGGG, as shown in SEQ ID NO.5 and GCTTGGGGAGTTGGGGTGAAGGG, as shown in SEQ ID NO.6.

[0019] Furthermore, methods to increase the number of symbiotic nodules in soybeans include the following steps:

[0020] S1: Using reverse-transcribed Dongnong 50 cDNA as a template, amplification... GmCDPKb Encoding area;

[0021] S2: Using the enzyme digestion and ligation method, GmCDPKb Constructed onto the Fu28 vector;

[0022] S3: Utilizing Gateway technology, GmCDPKb LR reacts onto the target vector pSOYI;

[0023] S4: Successfully constructed GmCDPKb -pSOYI vector was introduced into EHA105 Agrobacterium competent cells;

[0024] S5: Soybeans infected with Agrobacterium obtained using S4;

[0025] S6: Plant tissue culture was performed on the soybean tissue obtained in S5;

[0026] S7: Positive plants were selected by qRT-PCR and Western blot detection to obtain... GmCDPKb Soybean plants with overexpressed genes.

[0027] Therefore, the present invention provides GmCDPKb The advantages and positive effects of using genes to regulate the number of symbiotic nodules in soybeans are:

[0028] (1) The invention establishes GmCDPKb Gene editing technology and soybean cotyledon node transformation system are highly efficient and stable, providing important materials for elucidating the molecular mechanism of soybean symbiotic nodulation and for breeding new soybean varieties with high efficiency in nitrogen fixation.

[0029] (2) This invention creates a protein kinase that does not contain calcium-dependent protein kinase. GmCDPKb Soybean mutant plants, and GmCDPKb Overexpression of the transgenic plant. CDPK, as an important protein kinase, is closely related to plant growth and development. Knockout of this gene significantly reduced the number of soybean nodules, while overexpression significantly increased the number of nodules, revealing its positive regulatory role in soybean nodulation. This invention provides a basis for analysis... GmCDPKb It provides important genetic material for the establishment of the soybean-rhizobium symbiotic nitrogen fixation relationship.

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 for GmCDPKb The expression pattern analysis diagram, where A is GmCDPKb Gene expression in 50 tissues of Dongnong, B is GmCDPKb Gene expression at different stages after inoculation;

[0033] Figure 2 A map of the sgRNA-pCBSG015 recombinant vector;

[0034] Figure 3 Electrophoresis image of bacterial culture constructed using the sgRNA-pCBSG015 vector;

[0035] Figure 4 for gmcdpkb Mutant construction process;

[0036] Figure 5 for gmcdpkb Mutation type detection diagram, where A is the agarose gel electrophoresis detection of PCR amplification products of sgRNA target region in T2 generation transgenic seedlings, and B is the wild-type (WT) and... gmcdpkb Mutant lines (Line 1, Line 2, Line 3) GmCDPKb Sequence alignment of gene target regions;

[0037] Figure 6 for GmCDPKb Spectrum of the -pSOYI recombinant vector;

[0038] Figure 7 for GmCDPKb PCR electrophoresis detection image of bacterial culture constructed with the -pSOYI vector;

[0039] Figure 8 for GmCDPKb Image showing detection of overexpression-positive plants, where A represents T2 generation plants. GmCDPKb Expression level detection, B represents protein level detection in T2 generation plants;

[0040] Figure 9 for GmCDPKb The effect of gene mutation and overexpression on soybean nodule number is shown in the figure, where A represents wild type (WT). gmcdpkb Mutants (cdpkb-1, cdpkb-2, cdpkb-3) and GmCDPKb Comparison of nodulation phenotypes in overexpressing plants (OE1, OE2). B is a bar chart showing the number of nodules per plant in wild type, each mutant line and overexpressing line. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0044] In this example, the soybean cultivar used was Dongnong 50. Dongnong 50 is a small-grained Electron soybean variety introduced from Canada in 2003. This variety is a typical medium-maturing soybean with a well-developed root system and stable nodulation phenotype, making it suitable for genetic transformation research. Its approval number is Heishendou 2007022.

[0045] pCBSG vector (containing the Cas9 nuclease coding sequence and glufosinate resistance selection marker) is one of the commonly used expression vectors in plant genetic engineering. This vector has passed safety assessment and has stable replication and expression capabilities in Escherichia coli and Agrobacterium.

[0046] pSOYI vector, Fu28 vector, Gateway reaction kit: Gateway® LRClonase® II are available commercially.

[0047] GmCDPKb The gene was independently screened by the inventor and identified through whole-genome characterization (WGS sequencing) and expression level analysis (RNA sequencing) of the soybean CDPK family. GmCDPKb Genes can participate in the symbiotic nodulation process in soybeans.

[0048] In this invention, GmCDPKb The CDS sequence of the gene is shown in SEQ ID NO.1:

[0049]

[0050] GmCDPKb The amino acid sequence encoded by the gene is shown in SEQ ID NO.2:

[0051] MGNCSSGAGAPTTYSDDHPHHNGITVLPPNSNPSPQLPKPPPTSSSSSSLGRVLGRPMEDVRSIYIFGRELGRGQFGVTYLVTHKATKEQFACKSIATRKLVNRDDIDDIRREVQIMHHLTGHRNIVEL KGAYEDRHSVNLVMELCAGGELFDRIITKGHYSERAAANSCRQIVTVVHNCHSMGVMHRDLKPENFLLLNKNDDSPLKATDFGLSVFFKPGDVFRDLVGSAYYVAPEVLRRSYGPEADIWSAGVILYILL SGVPPFWAENEQGIFDAILRGHIDFASDPWPSISSSAKDLVKKMLRADPKERLSAVEVLNHPWMRVDGDAPDKPLDIAVLTRMKQFRAMNKLKVALKVIAENLSEEEIIGLKEMFKSMDTDNSGTITFE ELKAGLPKLGTKLSESEVRQLMEAADVDGNGTIDYIEFITATMHMNRMEREDHLYKAFEYFDNDKSGYITMEELESALKKYNMGDEKTIKEIIAEVDTDNDGRINYDEFVAMMRKGNPDITHITQRRRK.

[0052] Example 1 Soybeans GmCDPKb Gene tissue specificity and expression pattern analysis

[0053] First, real-time quantitative primers qRT-F (sequence shown in SEQ ID NO.3) and qRT-R (sequence shown in SEQ ID NO.4) were designed to... GmCDPKb Gene expression was measured, and the primer sequences are as follows:

[0054] SEQ ID NO.3: CATATTTTGGCTGCAGAGGTAC;

[0055] SEQ ID NO.4:ACTGATGTTCGTATATAGCGCA.

[0056] GmCDPKb Gene expression patterns in 50 tissues of Dongnong Agricultural University, as follows Figure 1 As shown in Figure A. Statistical analysis based on real-time quantitative PCR shows... GmCDPKb The gene exhibited distinct tissue-specific expression characteristics, with significantly higher transcription levels in root tissues compared to other organs. Furthermore, expression levels were also relatively high in root nodules. Fresh roots were collected at different time points after Dongnong 50 plants were inoculated with rhizobium HH103 for analysis. GmCDPKb Expression patterns at different stages of rhizobium infection. For example... Figure 1 As shown in Figure B, the quantitative results indicate that... GmCDPKb Gene expression levels exhibited an alternating trend of upregulation and downregulation. This further illustrates... GmCDPKb It plays a role in the symbiotic nodulation process of soybean-rhizobium.

[0057] Example 2 Soybean mutant gmcdpkb Construction

[0058] First, through the CRISPR-GE website design GmCDPKb sgRNA targets from genomic sequences. Based on rigorous target design principles (including PAM sequence matching, GC content optimization, and off-target effect assessment), two high-priority target sequences were ultimately identified: GACGGTGATGCCGTTGTGGTGGG (SEQ ID NO.5) and GCTTGGGGAGTTGGGGTGAAGGG (SEQ ID NO.6). The targets were amplified by PCR and ligated into the pCBSG015 vector. Figure 2 After transformation into Agrobacterium EHA105 competent cells, detection was performed by colony PCR using a combination of vector-specific primers and target primers for amplification, such as... Figure 3 As shown, the target sequence was finally detected between 1000-2000 bp (the expected target band was 1488 bp). Combined with the company's sequencing results, it was preliminarily confirmed that the sgRNA target was successfully ligated into the pCBSG015 vector.

[0059] Based on an optimized soybean cotyledon node transformation method, through infection and co-culture, screening, shoot induction, elongation, and hardening, a successful system was constructed. GmCDPKb Stable genetic mutant lines ( gmcdpkb The conversion process is as follows: Figure 4 As shown.

[0060] The specific implementation steps are as follows:

[0061] S1. Co-cultivation: Select plump, disease-free Dongnong 50 soybean seeds and sterilize them by chlorine fumigation. Transfer the sterilized seeds to sterile petri dishes and soak them overnight. Use EHA105 Agrobacterium containing the target vector as the infection solution. After the seeds have swelled, remove the seed coat with a sterile scalpel, divide the seeds in half, and remove the true leaves. Make multiple incisions at the cotyledon growing point with a sterile scalpel to create wounds, and place the treated seeds in the infection solution for co-cultivation for 30 min. After infection, insert the cotyledons diagonally into a solid culture medium and cultivate for 2 weeks at 25℃ under 16 h light / 8 h dark conditions.

[0062] S2. Screening and bud induction: Observe the growth of the clustered buds, cut off the clustered buds with a sterile scalpel, and put the clustered buds back into the elongation medium. Continue to culture for 3-5 weeks at 25℃ under 16 h light / 8 h dark conditions.

[0063] S3. Elongation and Hardening-off: Using a sterile scalpel, cut the appropriately elongated seedlings from the base and insert them into rooting medium for 2-3 weeks, observing the rooting progress regularly. Transplant healthy seedlings into sterile peat moss and continue culturing at 25℃ under 16h light / 8h darkness conditions for 1-2 weeks.

[0064] Soybean mutant gmcdpkb The seeds are cultivated using the following methods:

[0065] High-quality soil and vermiculite were mixed in a 3:1 ratio and sterilized at high temperature to serve as the cultivation substrate. Soybean mutant T2 generation seeds were sown in the sterilized substrate and cultured in an incubator with 16 hours of light and a constant temperature of 25℃, during which precise water and fertilizer management was implemented according to the plant growth stages. After the plants were robust and stable, young leaf tissue was collected for DNA extraction. Using the extracted DNA as a template, PCR detection of sgRNA in the T2 generation transgenic seedlings yielded the target band (580 bp). Figure 5 As shown in Figure A, the PCR product of the expected size was sequenced for DNA. Soybean mutant. gmcdpkb mutation information such as Figure 5 As shown in B, 3 out of 16 lines caused GmCDPKb The early termination.

[0066] Example 3 Soybean overexpression plants GmCDPKb Construction

[0067] First, PCR amplification was obtained. GmCDPKb The coding region sequence and primer sequences are as follows: BamH I- GmCDPKb -F: CGCGGATCCATGGGAAACTGCAGCAGC (SEQ ID NO.7); Indian III- GmCDPKb -R: CCAAGCTTTGCGACGTCTTTGGGTT (SEQ ID NO.8); constructed using enzyme digestion and ligation method. GmCDPKb -Fu28 vector, the selected endonuclease is BamH I and Indian III. Using Gateway recombination technology (Gateway Reaction Kit: Gateway® LRClonase® II), GmCDPKb LR reacts onto the pSOYI vector ( Figure 6 ).

[0068] After the successfully sequenced plasmid was transformed into Agrobacterium EHA105 competent cells, detection was performed by bacterial culture PCR, and the target sequence was finally detected in the range of 1000-2000 bp. Figure 7 Following the same genetic transformation process for soybean cotyledonary nodes as in Example 2, a new type of soybean cotyledonary node was successfully created. GmCDPKb Overexpression strains.

[0069] Using the soybean seed cultivation method described in Example 2, T2 generation plants overexpressing the protein were cultivated. After the plants were robust and stable, young leaf tissues were collected for RNA and plant protein extraction. RNA and protein levels were detected, such as... Figure 8 As shown, two overexpression positive lines were screened.

[0070] Example 4 Wild-type soybean (WT) GmCDPKb Identification of nodulation phenotype in knockout and overexpression plants

[0071] Sterilized soybeans were planted in double-layered pots and cultured in an incubator with 16 hours of light and a constant temperature of 25°C. Once the seedlings reached the three-leaf stage, they were inoculated with rhizobium HH103 (OD) using a sterile syringe. 600 =0.6-0.8), the root nodulation phenotype was investigated when the plants entered the peak nodulation stage. Wild type (WT), gmcdpkb mutants and GmCDPKb Overexpression of root phenotypes and number of root nodules, such as Figure 9 As shown. The survey results show... GmCDPKb The number of nodules was significantly reduced in homozygous mutant plants and significantly increased in overexpression plants.

[0072] Therefore, the present invention adopts the above-mentioned GmCDPKb The application of this gene in regulating the number of symbiotic nodules in soybeans fills the gap in existing research on the regulation of symbiotic nodules in soybeans, and provides gene resources and germplasm materials for elucidating the molecular mechanism of symbiotic nitrogen fixation in soybeans and for breeding new varieties of soybeans with high efficiency in nitrogen fixation.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. GmCDPKb The application of genes in regulating the number of symbiotic nodules in soybeans is characterized by: The GmCDPKb The nucleotide sequence of the gene is shown in SEQ ID NO.1; Knock out soybeans GmCDPKb Genes that reduce the number of symbiotic nodules in soybeans and overexpress the gene in soybeans GmCDPKb Genes that increase the number of symbiotic nodules in soybeans.

2. The application of GmCDPKb protein in regulating the number of symbiotic nodules in soybean, characterized by: The amino acid sequence of the GmCDPKb protein is shown in SEQ ID NO.2, and the gene encoding the GmCDPKb protein is... GmCDPKb Gene, GmCDPKb The nucleotide sequence of the gene is shown in SEQ ID NO.1; Knock out soybeans GmCDPKb Genes that reduce the number of symbiotic nodules in soybeans and overexpress the gene in soybeans GmCDPKb Genes that increase the number of symbiotic nodules in soybeans.

3. A method for regulating the number of symbiotic nodules in soybeans, characterized in that, Knock out soybeans GmCDPKb Genes that reduce the number of symbiotic nodules in soybeans and overexpress the gene in soybeans GmCDPKb The gene increases the number of symbiotic nodules in soybeans; the nucleotide sequence of the GmCDPKb gene is shown in SEQ ID NO.1.

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