An overexpression of a gene engineering rhizobium which codes purine nucleoside phosphorylase, construction method and application thereof

Abstract

This invention discloses a genetically engineered rhizobium strain overexpressing purine nucleoside phosphorylase, its construction method, and its applications, belonging to the fields of agricultural biotechnology and genetic engineering. This invention uses *Rhizobium fischeri* (Fragariae spp.) as the basis for its development. Sinorhizobium fredii CCBAU 45436 was used as the starting strain. The strain was introduced by introducing a sample containing the strain itself. rpoD Strong promoter sequences of genes, encoding purine nucleoside phosphorylases deoD The recombinant strain CCBAU 45436 deoD OE was obtained by using a recombinant plasmid composed of the gene sequence and the gene encoding a 3×Flag tag sequence. This recombinant strain has the accession number CGMCC No. 38578 at the China General Microbiological Culture Collection Center (CGMCC). This invention experimentally demonstrates that inoculation... deoD deoD deoD deoD deoD deoD deoD deoD Soybeans overexpressing the recombinant strain (CGMCC No. 38578) showed significantly better plant height and root nodule fresh weight than soybeans inoculated with unmodified wild-type rhizobium strains.

Description

Technical Field

[0001] This invention belongs to the fields of agricultural biotechnology and genetic engineering, specifically relating to a genetically engineered rhizobium that overexpresses purine nucleoside phosphorylase, its construction method, and its application. Background Technology

[0002] Soybeans Glycine max (L.) Merr.) is one of the world's most important dual-purpose crops for both food and oil, a core source of dietary protein and vegetable oil for humans, and an irreplaceable raw material for the feed industry. As a typical leguminous symbiotic nitrogen-fixing crop, soybean can fix nitrogen through interaction with *Rhizobium fischeri* (L.) Merr. Sinorhizobium fredii ), Ehrlich. spp. ( Bradyrhizobium elkanii Soybeans establish a symbiotic relationship with rhizobia, forming root nodules on the roots. Using a nitrogenase system within mature rhizobia, they reduce atmospheric molecular nitrogen (N2) to ammonia (NH3), which is then converted into organic nitrogen available to plants. Studies have shown that biological symbiotic nitrogen fixation can meet approximately 60%–70% of the nitrogen requirements of soybeans throughout their entire growth cycle. This is a key technological pathway to reduce the input of chemical nitrogen fertilizers in agricultural production, mitigate agricultural non-point source pollution, and achieve green and sustainable production. Fully exploring the potential of the soybean-rhizobium symbiotic nitrogen fixation system and improving biological nitrogen fixation efficiency is of great strategic significance for ensuring national food security and promoting sustainable agricultural development.

[0003] Purine nucleoside phosphorylase (EC 2.4.2.1; abbreviated as PNP) is a core enzyme in the purine salvage pathway of organisms, composed of... deoD Encoded in genes, this enzyme catalyzes the reversible phosphorylation reaction between purine nucleosides (such as adenosine, inosine, and guanosine) and inorganic phosphates, generating the corresponding purine bases (such as adenine, hypoxanthine, and guanine) and 1-phosphoribose. In the forward reaction, PNP participates in the catabolism of purine nucleosides, and the generated 1-phosphoribose and purine bases can provide reaction substrates for other metabolic pathways within the cell, supporting intracellular glucose metabolism, replenishing the nucleotide pool, and thus influencing changes in cellular metabolic flux and the synthesis of ATP, DNA / RNA, purine-containing compounds, and downstream signaling molecules. Therefore, the purine metabolic pathway involving PNP plays a multi-level regulatory role in cellular material metabolism and energy metabolism.

[0004] In the soybean-rhizobium symbiotic system, purine metabolism plays a particularly important physiological role. On the one hand, the efficient nitrogen fixation of the bacteriophytes differentiated within the root nodules depends on sufficient ATP supply and energy turnover. On the other hand, the main forms of transport of nitrogen fixation products from soybeans and other legumes to the aboveground parts are allantoin and allantoate, both of which are purine derivatives. Their synthesis requires purine metabolic intermediates; therefore, the level of purine metabolic flux directly affects the efficiency of nitrogen transport from the nitrogen fixation product to the aboveground parts. Besides the symbiotic nitrogen fixation stage, purine compounds (such as adenosine and AMP) may also play a signaling mediator role in the early signal recognition process between rhizobia and the host plant.

[0005] Despite PNP / deoD Its function in the basal metabolism of other non-rhizobium bacteria has been studied to some extent, but currently there is no research on its application in the domestic and international markets. deoD No publicly available reports have described the effects of gene overexpression in rhizobia systems on soybean nodulation and plant growth promotion, nor have any related patented technologies been found. This indicates that targeting the rhizobium purine nucleoside phosphorylase gene is effective. deoD Regulating purine metabolic flux in root nodules by adjusting expression levels, thereby enhancing symbiotic nitrogen fixation and plant growth promotion, is an innovative approach that has not yet been fully understood and developed, and has significant technological gaps and high research value.

[0006] Based on the above analysis, it is urgent to conduct research targeting... deoD The study on the overexpression of genes in *Rhizobium fischeri*, which coexists with soybean, aims to construct an engineered strain of rhizobium that combines efficient nodulation ability with growth-promoting effects on soybeans. This provides new ideas and technical routes for the genetic modification of rhizobia, and offers new germplasm materials and technical support for enhancing nitrogen fixation efficiency in soybean production, thereby meeting the urgent need for sustainable green agriculture that reduces the application of chemical nitrogen fertilizers. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a genetically engineered recombinant rhizobium that can nodulate and fix nitrogen in soybeans, and further promote soybean growth. The technical problem to be solved by this invention is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0008] To address the aforementioned problems, this invention provides a method for constructing a genetically engineered recombinant rhizobium. This recombinant rhizobium is prepared through the following steps: introducing biological material into a recipient rhizobium that increases or / and enhances the content of purine nucleoside phosphorylase (PNP) and / or the expression level of the gene encoding said purine nucleoside phosphorylase, thereby obtaining the recombinant rhizobium.

[0009] In some embodiments of the present invention, the recipient rhizobium is a rhizobium containing purine nucleoside phosphorylase or nucleic acid encoding the purine nucleoside phosphorylase.

[0010] In the above-mentioned recombinant rhizobium, the purine nucleoside phosphorylase gene is derived from the rhizobium itself.

[0011] The purine nucleoside phosphorylase is any one of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:2; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1), which has more than 80% identity with the protein shown in A1) and has purine nucleoside phosphorylase activity. A3) A fusion protein with purine nucleoside phosphorylase activity obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0012] The tag proteins include, but are not limited to, 3×Flag tag proteins.

[0013] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using Blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0014] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0015] In the above method, the biomaterial is any one of the following: B1) Nucleic acid molecules that encode the aforementioned proteins; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

[0016] B2) The expression cassette containing nucleic acid molecules refers to DNA capable of expressing the proteins described above in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all the regulatory sequences necessary for the expression of any of the aforementioned proteins. These regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation.

[0017] In some embodiments of the present invention, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0018] The term "vector" refers to any construct that can introduce heterologous DNA or RNA into a host cell.

[0019] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, or bacteriophages. Constructs typically include one or more expression cassettes.

[0020] The expression cassette is a DNA molecule capable of expressing the protein-coding gene in the host cell (e.g., a microbial cell). In some embodiments, the expression cassette further includes a DNA fragment containing all regulatory sequences necessary for expressing the encoded gene. The regulatory sequences, under compatible conditions, direct the expression of the encoded gene in a suitable host cell. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylated sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter and termination signals for transcription and translation. The regulatory sequence can be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to that of the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in a selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in a selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell used can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0021] In some embodiments, the recombinant vector is a recombinant expression vector containing the coding gene of the protein. When preparing the recombinant expression vector, the coding gene may be located within the vector so as to be operatively linked to an appropriate expression regulatory sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of the coding gene. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector may contain any mechanism that ensures self-replication. Alternatively, the vector is one that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome.

[0022] In this application, the term "operable link" refers to an operable connection between segments of a nucleic acid sequence that are functionally related to each other. For example, operably linked promoters, enhancers, open reading frames (OPFs), 5' UTRs and 3' UTRs, and terminator sequences result in the precise production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements lead to transcription of the OPF and ultimately to the production of a polypeptide (i.e., expression of the OPF).

[0023] In one specific embodiment of the present invention, the expression cassette includes a promoter and a nucleic acid molecule encoding the aforementioned protein, wherein the nucleotide sequence of the promoter is nucleotides 1-420 of SEQ ID NO:1.

[0024] In the above method, the nucleic acid molecule is the gene encoding the purine nucleoside phosphorylase.

[0025] In the above method, the encoding gene is any of the following sequences: K1) A DNA molecule with a nucleotide sequence as shown in nucleotides 421-1215 of SEQ ID NO:1; The DNA molecule that has more than 75% identity with the nucleotide sequence defined in K2) and K1) and encodes the purine nucleoside phosphorylase; K3) hybridizes under stringent conditions with any of the defined nucleotide sequences in K1)-K2) and encodes the DNA molecule of the purine nucleoside phosphorylase.

[0026] Those skilled in the art can readily mutate the nucleotide sequence encoding purine nucleoside phosphorylase of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotide sequences possessing 75% or higher identity with the purine nucleoside phosphorylase of the present invention, as long as they encode and function as purine nucleoside phosphorylase, are derived from and equivalent to the nucleotide sequence of the present invention.

[0027] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the purine nucleoside phosphorylase of this invention. Identity can be evaluated using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0028] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0029] The present invention also provides an expression cassette for a DNA molecule whose nucleotide sequence is shown in nucleotides 1-1215 of SEQ ID NO:1.

[0030] The recombinant rhizobium prepared by the aforementioned method also falls within the scope of protection of this invention.

[0031] The recombinant rhizobium is *Rhizobium fischeri* (Sinapis alfa). Sinorhizobium fredii Its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 38578.

[0032] In a specific embodiment of the present invention, the rhizobium species is specifically: Sinorhizobium fredii Its Chinese name is *Rhizobium fischeri*, and its strain number is CCBAU45436.

[0033] The present invention also provides applications, which include any of the following: C1) The application of the aforementioned recombinant rhizobia in increasing the plant height of soybeans; C2) The application of the aforementioned recombinant rhizobium in the preparation of products (inoculants) that increase the plant height of soybeans; C3) The aforementioned recombinant rhizobium in increasing the fresh weight of soybean root nodules; C4) The application of the aforementioned recombinant rhizobium in the preparation of products that increase the fresh weight of soybean root nodules; C5) The aforementioned application of purine nucleoside phosphorylase in increasing the plant height of soybean; C6) The aforementioned application of purine nucleoside phosphorylase in the preparation of products that increase soybean plant height; C7) The aforementioned application of purine nucleoside phosphorylase in increasing the fresh weight of soybean root nodules; C8) The aforementioned application of purine nucleoside phosphorylase in the preparation of products that increase the fresh weight of soybean root nodules; C9) The aforementioned application of purine nucleoside phosphorylase in the construction of recombinant rhizobia; C10) The application of the aforementioned purine nucleoside phosphorylase in the preparation of products containing recombinant rhizobia.

[0034] The present invention also provides biomaterials, said biomaterials comprising any of the following: B1) Nucleic acid molecules that encode the aforementioned proteins; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

[0035] In the above-mentioned biological materials, the nucleic acid molecule mentioned in B1) is the gene encoding the aforementioned protein.

[0036] In one specific embodiment of the present invention, the nucleotide sequence of the encoding gene is nucleotides 421-1215 of SEQ ID NO:1.

[0037] In one specific embodiment of the present invention, the expression box described in B2) is composed of... rpoD The strong promoter sequence (420bp) (positions 1-420 of SEQ ID NO:1, lowercase letters) encodes the purine nucleoside phosphorylase gene. deoD It consists of the sequence (795bp) (positions 421-1215 of SEQ ID NO:1, uppercase letters) and the 3×Flag tag sequence (99bp) (positions 1216-1314 of SEQ ID NO:1, lowercase letters).

[0038] In one specific embodiment of the present invention, the backbone vector of the recombinant vector is pBBR1MCS-2 (sequence can be found at https: / / www.addgene.org / 85168 / sequences / ), which is a vector capable of delivering exogenous DNA or target genes into host cells for amplification and expression.

[0039] In one specific embodiment of the present invention, the recombinant plasmid vector contains an expression cassette with the aforementioned nucleotide sequence SEQ ID NO:1.

[0040] In one specific embodiment of the present invention, the nucleotide sequence of the recombinant plasmid vector may be SEQ ID NO:3.

[0041] The recombinant vector pBBR1MCS-2-deoD is constructed as follows: the pBBR1MCS-2 plasmid vector is used with restriction endonucleases... Sam I cut, Sam The I recognition site (CCC↓GGG) is located at position 3263 of SEQ ID NO:3, and is then inserted into SEQ ID NO:1 in the sequence listing. After SEQ ID NO:1 is expressed in recombinant rhizobium via the recombinant plasmid vector pBBR1MCS-2-deoD, it produces purine nucleoside phosphorylase, the amino acid sequence of which is identical to that shown in SEQ ID NO:2.

[0042] This invention obtained wild-type rhizobium (such as strain CCBAU45436) through PCR amplification. rpoD The strong promoter sequence and purine nucleoside phosphorylase coding region were obtained from *E. coli* containing the pVO155 plasmid. A 3×Flag tag sequence was then amplified by PCR and recombined into the pBBR1MCS-2 plasmid vector, yielding the recombinant plasmid pBBR1MCS-2-deoD with the nucleotide sequence SEQ ID NO:3. This recombinant plasmid was then introduced into a wild-type rhizobium strain via three-parent hybridization to form a recombinant rhizobium, also known as a genetically engineered recombinant rhizobium. This recombinant rhizobium strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38578. This invention verified the results using qPCR. deoD The overexpression of the gene in recombinant rhizobia was verified by Western blot (WB), confirming the successful expression of PNP protein in recombinant rhizobia.

[0043] By inoculating soybeans with the genetically engineered recombinant rhizobium constructed in this invention, soybeans exhibited significantly better growth than legumes inoculated with unmodified wild-type rhizobia—a phenomenon previously unreported. This invention significantly enhances the growth of the rhizobium-soybean symbiotic system by regulating the expression of the purine nucleoside phosphorylase gene, providing a new strategy for the genetic improvement of rhizobia. The obtained genetically engineered rhizobium can serve as core germplasm or chassis strains for developing highly efficient soybean rhizobium agents, pointing to a new technological direction for creating highly competitive and efficient symbiotic rhizobia, and showing potential applications in the rhizobium agent production industry and soybean cultivation.

[0044] Preservation Instructions Strain name: Rhizobium fischeri Latin name:Sinorhizobium fredii Strain number: CCBAU 45436 deoD OE Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: May 8, 2026 Registration number at the Preservation Center: CGMCC No. 38578. Attached Figure Description

[0045] Figure 1 This includes sequencing peak diagrams and sequence descriptions. Figure 1 In the sequence A, the plasmid vector sequence is from SEQ ID NO:1. rpoD The sequence determination results of the first 50 bases of the strong promoter sequence are as follows: the first 10 nucleotides from left to right (5'-TGGATCCCCC-3') correspond to nucleotides 3254-3263 on the recombinant plasmid pBBR1MCS-2-deoD-3×Flag, and the 11th-60th nucleotides (5'-ATTGGTACATATAGGCCCCCGCCGGAAAGCCCGCGGCCCTTGTCTTTTGG-3') correspond to nucleotides 1-50 in SEQ ID NO:1. Figure 1 B represents the ligation sequencing results of SEQ ID NO:1 and the vector. Nucleotides 1-50 from left to right (5'-GTGATTATAAAGATCACGATATCGATTACAAGGATGACGATGACAAGTGA-3') correspond to nucleotides 4528-4577 on the recombinant plasmid pBBR1MCS-2-deoD-3×Flag (also corresponding to nucleotides 1265-1314 of SEQ ID NO:1), and nucleotides 51-60 (5'-GGGCTGCAGG-3') correspond to nucleotides 4578-4587 on the recombinant plasmid pBBR1MCS-2-deoD-3×Flag. Figure 1 C in the middle represents qPCR verification. deoD Overexpression level (OE-) deoD Significantly higher than wild-type (WT) and those without [specific traits]. deoD The expression level of the empty plasmid vector pBBR1MCS-2 (abbreviated as MCS-2) was compared (****, extremely significant difference, p=0.0001). Figure 1In the image, D represents the results of Western Blot (WB) verification of PNP protein expression in recombinant rhizobia. M represents protein molecular weight standards, from top to bottom: 43 kDa, 34 kDa, and 25 kDa; WT represents wild-type *Rhizobium fischeri* CCBAU45436; MCS-2 represents the control group into which the empty plasmid vector pBBR1MCS-2 was introduced into wild-type rhizobia; OE- deoD This is a recombinant genetically engineered rhizobium strain that has been introduced with the recombinant plasmid pBBR1MCS-2-deoD-3×Flag. In the Western blot image, each sample was loaded in triplicate.

[0046] Figure 2 This study compared and statistically analyzed the growth of soybeans after different rhizobium strains were inoculated onto the roots and grown in a greenhouse for 30 days. Figure 2 Figure A shows a comparison of the growth of soybean plants inoculated with different strains. From left to right: Control treatment (no rhizobium inoculation), soybean plants inoculated with wild-type rhizobium (WT), soybean plants inoculated with rhizobium carrying the empty plasmid vector pBBR1MCS-2 (MCS-2), and soybean plants inoculated with the pBBR1MCS-2-deoD recombinant plasmid overexpression. deoD Genetically engineered recombinant rhizobia (OE-) deoD Soybean plants, inoculation and deletion deoD rhizobium of the gene (Δ deoD Soybean plants. Figure 2 B is Figure 2 The statistical graph of soybean plant height inoculated with different strains in the A group shows that the differences were extremely significant (denoted by the significance symbol a). One-way ANOVA results indicate that the differences were extremely significant. Figure 2 Figure C shows the statistical graph of soybean root nodule fresh weight inoculated with different strains in Figure A. The one-way ANOVA results showed significant differences, indicated by the letter 'a'. Each sample was replicated at least 7 times. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] The antibiotics (stock solutions) used in the following examples are as follows: Nalidixic acid (NA): 30 mg / mL; Kanamycin (Km): 50 mg / mL. All antibiotics were prepared according to the product instructions, sterilized by filtration through a 0.22 μm sterile filter, aliquoted, and stored at -20°C protected from light. These antibiotics are used to screen for resistant strains and inhibit non-resistant strains, thus serving a screening function. The stock solutions of the aforementioned antibiotics, diluted 1000-fold, are the antibiotics used in subsequent examples (i.e., working solutions).

[0050] 0.85% physiological saline: Weigh 0.85 g NaCl, dissolve in 100 mL of deionized water, and autoclave at 121 ℃ for 30 min. Used for adjusting the concentration of Rhizobium strains or washing the cells.

[0051] 5×TAE electrophoresis buffer: Dissolve 242.0 g of Tris in approximately 800 mL of deionized water, stirring thoroughly. Add 57.1 mL of glacial acetic acid and 100 mL of 0.5 M EDTA (pH 8.0), and bring the volume to 1 L with deionized water. This buffer is used for nucleic acid electrophoresis detection. Dilute with deionized water to 1× (i.e., dilute 5 times) before use.

[0052] TY (Tryptone-yeast) liquid medium (1L) (nutrient-rich medium for culturing rhizobia): 5 g tryptone, 3 g yeast extract, 0.6 g CaCl2, adjust pH to 6.8-7.2, the remainder is deionized water.

[0053] LB medium (Luria-Bertani medium) (1 L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, balance deionized water, adjust pH to 6.8-7.2. This medium is used for culturing *Escherichia coli* (…). E. coli ).

[0054] If preparing solid culture media for either of the above two media, 1.5% (by weight) of agar powder should be added to the corresponding media. All media should be autoclaved at 121 °C for 30 min.

[0055] In the quantitative experiments described below, the results of three replicate experiments are presented in a bar chart as mean ± standard deviation. One-way ANOVA is used to test the significance of differences between samples and whether those differences are significant. Lowercase letters are used to indicate differences above the bars; no identical symbols are used for significant differences, while identical symbols are used for partial differences.

[0056] The following examples use the rhizobium strain CCBAU45436 (i.e., wild-type rhizobium, abbreviated as WT) containing plasmid pVO155. E. coli and helper bacteria containing helper plasmid pRK2013 E. coli Published in “YuanHui Liu, Yin Shan Jiao, Li Xue Liu, Dan Wang, Chang Fu Tian, ​​En Tao Wang, Lei Wang, Wen Xin Chen, Shang Ying Wu, Bao Lin Guo, Zha Gen Guan, Véréna Poinsot, Wen FengChen. Nonspecific Symbiosis between Sophora flavescens and Different Rhizobia. Molecular Plant-Microbe Interaction. 2018;31(2):224-232. doi: 10.1094 / MPMI-05-17-0117-R”, the rhizobium CCBAU45436 is named *Rhizobium fischeri* in this literature (( Sinorhizobium fredii (CCBAU45436). The biological material is available to the public from the applicant and is intended solely for the purpose of repeating the relevant experiments of this invention and may not be used for any other purpose.

[0057] The pBBR1MCS-2 plasmid vector used in the following examples was a product of Addgene, catalog number Plasmid #85168. It was preserved in *E. coli* in 20% glycerol at -80°C.

[0058] Example 1: Construction of genetically engineered recombinant rhizobia carrying the pBBR1MCS-2-deoD recombinant plasmid 1. Extraction of Rhizobium Genomic DNA Genomic DNA was extracted from Rhizobium strain CCBAU45436 according to the instructions of the DNA extraction kit (Coolaber, catalog number: DE231). After determining the nucleic acid concentration and purity using a Nanodrop nucleic acid analyzer, the DNA was stored at -20 °C.

[0059] 2. Extraction of pBBR1MCS-2 plasmid vector and pVO155 plasmid from Escherichia coli Plasmid DNA was extracted using a plasmid mini-extraction kit (TIANGEN, catalog number: DC301). The procedure was performed according to the kit instructions. Nanodrop was used. ® After the concentration and purity are measured by the nucleic acid detection instrument, store at -20℃.

[0060] 3. DNA fragment recovery PCR products and enzyme digestion products were recovered and purified using a DNA fragment recovery kit (Vazyme, catalog number: DC301). After determining the concentration and purity using a Nanodrop nucleic acid detector, the products were stored at -20°C.

[0061] 4. Vector construction: Seamless cloning and transformation 1) Identify the target gene deoD The insertion site on plasmid vector pBBR1MCS-2 was used. SmaⅠ The enzyme has a recognition site (CCCGGG) and a cleavage site (CCC↓GGG). The cleavage site is located between position C at position 3263 and position G at position 3264 of the vector pBBR1MCS-2.

[0062] 2) Primer design: Based on CCBAU45436 deoD gene sequence, rpod Primers p.-deoD-F, deoD-3.-R, MCS2-rpoD-F, and prpod-d.-R were designed based on the promoter sequence of plasmid pVO155. Primers d.-3×flag-F and 3×flag-mcs2-R were designed based on the 3×Flag tag sequence of plasmid pVO155. Each primer was designed to carry approximately 20 bp of homologous sequences of adjacent fragments after ligation, as shown below: p.-deoD-F: 5'-GACAAGGAAAGCGACGAATAAATGACCGACGCAGCTGAATTC-3' (SEQ ID NO: 4); deoD-3.-R: 5'-CCACCCGAACCGCCACCGGAGGCTTCTGTCGCGATCATG-3' (SEQ ID NO: 5); MCS2-rpoD-F: 5'-ACTAGTGGATCCCCCATTGGTACATATAGG-3' (SEQ ID NO: 6); prpod-d.-R: 5'-ATTCAGCTGCGTCGGTCATTTATTCGTCGCTTTCCTTG-3' (SEQ ID NO: 7); d.-3×Flag-F: 5'-CATGATCGCGACAGAAGCCTCCGGTGGCGGTTCGGGTG-3' (SEQ ID NO: 8); 3×Flag-mcs2-R: 5'-ATATCGAATTCCTGCAGCCCTCACTTGTCATCGTCATCC-3' (SEQ ID NO: 9).

[0063] 3) Using the primer pairs described above, PCR amplification was performed on the genomic DNA of Rhizobium CCBAU45436 and plasmid pVO155 using high-fidelity DNA polymerase (GenStar, catalog number: A066) to obtain... rpoD The promoter sequence (obtained by amplification of MCS2-rpoD-F and prpod-d.-R), deoD The complete and correct sequences of the gene sequence (obtained by amplification of p.-deoD-F and deoD-3.-R) and the 3×Flag tag sequence (obtained by amplification of d.-3×Flag-F and 3×Flag-mcs2-R).

[0064] The 50 μL PCR amplification system is prepared as shown in Table 1 below.

[0065] Table 1. PCR amplification system

[0066] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 20 s, 55℃ annealing for 30 s, 72℃ extension for 44 s, 35 cycles; 70℃ final extension for 5 min. After amplification, 4 μL of the amplification product was analyzed by 1% agarose gel electrophoresis. deoD gene fragments, rpoD The promoter fragment and 3×Flag tag fragment band sizes were approximately 795bp, 420bp, and 99bp, respectively. After confirming that the band sizes were correct, purification and recovery were performed.

[0067] 4) Using restriction endonucleases Sam I (from NEB, catalog number: R0141V) digested the pBBR1MCS-2 plasmid vector with enzymes, and the cleaved linear plasmid fragments were purified and recovered by agarose gel.

[0068] 5) Ligation: Following the instructions in the Seamless Assembly Cloning Kit (China-US Taihe (Beijing) Biotechnology Co., Ltd., Catalog No.: C5891), ligate the above-mentioned target DNA fragment (with the DNA fragment that has undergone...) SamThe linearized plasmid fragment digested with enzyme I was ligated. The reaction system was as follows: 3 µL of linearized vector + target DNA fragment, 5 µL of mixed solution containing enzyme, and ddH2O to a final volume of 10 µL. The reaction was carried out in a metal bath at 50 °C for 15 min. After the reaction, the ligation product was placed on ice for later use or stored at -20 °C.

[0069] 6) Conversion: The bonding product obtained in 5) is thermally converted to... E. coli In China, using Qingke Company E. coli DH5α chemocompetent cells (product code: S101-02) were transformed under aseptic conditions according to the instructions. The transformed cells were then evenly spread onto LB agar plates containing kanamycin at a final concentration of 50 μg / mL and incubated inverted at 37°C overnight. The following day, single colonies were identified by PCR using the forward primer F and reverse primer R (sequences shown in Table 2) from the plasmid vector. Colony PCR was performed using standard Taq enzyme (GENSTAR, product code A012-101) and 2×Taq PCR SatrMix (Kangrun Biotechnology, product code A012-01). The colony PCR system is shown in Table 2.

[0070] Table 2. Colony PCR Reaction System

[0071] In Table 2: The forward primer F sequence is: 5'-CCCAGTCACGACGTTGTAAAACG-3' (SEQ ID NO:10); The reverse primer R sequence is: 5'-AGCGGATAACAATTTCACACAGG-3' (SEQ ID NO:11).

[0072] The colony PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s; 72℃ extension for 59 s; repeat the last three steps (denaturation, annealing, and extension) for 30-35 cycles.

[0073] After amplification, 5 μL of the PCR product was analyzed by 1% agarose gel electrophoresis. The band size was approximately 1577 bp (reverse primer to...). Sam The sequence length at the I cut site is 150bp, and the forward primer is... SamThe sequence length at the cut site is 113bp, and the sequence length of SEQ ID NO:1 inserted at the cut site is 1315bp. Therefore, 2-3 positive clones (420+795+99+150+113=1577) are sent to a sequencing company (such as Qingke Biotechnology) for sequencing.

[0074] Partial chromatograms of the sequencing results are shown below. Figure 1 As shown in Figures A and B. Where... Figure 1 In the sequence A, SEQ ID NO:1 is the plasmid vector sequence. rpoD The sequence determination results of the linkage of bases 1-50 in the strong promoter sequence, in Figure 1 In sequence A, the 1st to 50th bases of SEQ ID NO:1 match the sequence in the vector. SamI The accurate ligation of the remaining CCC after the enzyme recognition site CCCGGG is cleaved, along with the preceding sequences (GTGGATCCCCC (SEQ ID NO:12), etc.), demonstrates... deoD The upstream has been successfully connected to the carrier; Figure 1 B represents the bases at positions 1265-1314 of SEQ ID NO:1 (3×Flag tag sequence) and the bases in the vector sequence. Sam After the enzyme recognition site CCC↓GGG was cleaved, the remaining GGG and subsequent CTGCAGG vector sequences were accurately ligated, indicating that the 3×Flag tag sequence was successfully ligated into the vector. The sequences in the two peak diagrams also correspond to the base positions on the recombinant plasmid.

[0075] This step yielded the recombinant plasmid vector pBBR1MCS-2-deoD containing SEQ ID NO:1. The nucleotide sequence of the recombinant plasmid pBBR1MCS-2-deoD is shown in SEQ ID NO:3 (nucleotides 3264-4577 are SEQ ID NO:1). This recombinant plasmid vector was then transformed into [the target cell / organization] using a heat shock method. E. coli In the process, donor bacteria are formed. E. coli / pBBR1MCS-2-deoD.

[0076] In SEQ ID NO:1, nucleotides 1-420 are rpoD The promoter sequence (420 bp), nucleotides 421-1215 are deoD The gene sequence (795bp, encoding the protein with amino acid SEQ ID NO:2), nucleotides 1216-1245 are the encoding nucleotides of the flexible linker peptide, nucleotides 1246-1311 are the 3×Flag tag sequence (66bp), and nucleotides 1312-1314 are the stop codons.

[0077] 5. Triparental conjugation experiment 1) Activate wild-type recipient rhizobium CCBAU45436 on TY solid medium containing naphthylpyridinol acid antibiotic at a final concentration of 30 μg / mL. Incubate upside down at 28℃ for 3-4 days. After single colonies grow, pick a single colony and inoculate it into 5 mL of TY liquid medium (containing naphthylpyridinol acid at a final concentration of 30 μg / mL). Incubate at 28℃ and 180 rpm until the logarithmic growth phase, which takes about 2 days.

[0078] 2) Activate the donor bacteria overnight in an incubator at 37°C on LB solid medium containing a final concentration of 50 μg / mL kanamycin. E. coli / pBBR1MCS-2-deoD; activate helper bacteria carrying the helper plasmid pRK2013 overnight in an incubator at 37°C on LB solid medium containing kanamycin at a final concentration of 50 μg / mL. E. coli Each single colony was picked and inoculated into 5 mL of LB liquid medium containing the corresponding antibiotic (the donor and auxiliary bacteria were both resistant to kanamycin, and the concentration was 50 μg / mL). The cultures were incubated at 37°C and 200 rpm for 10–12 h.

[0079] 3) Take the recipient rhizobium (wild type CCBAU45436) and the donor bacteria respectively. E. coli / pBBR1MCS-2-deoD and helper bacteria (containing helper plasmid pRK2013) E. coli Collect 1 mL, 300 μL, and 200 μL of the culture, centrifuge at 8000 g for 5 min at room temperature to collect the bacterial cells, wash the cells twice with TY liquid medium, centrifuge again, and discard the supernatant. Add about 30-50 μL of fresh TY liquid medium, gently suspend the precipitate and mix well.

[0080] 4) Spot the bacterial suspension onto antibiotic-free TY solid culture medium plates. After the bacterial suspension is dried by sterile air, transfer it to a 28°C incubator and incubate upside down for 24 hours.

[0081] 5) Use a sterile spatula to scrape the bacterial growth from the TY solid medium and transfer it to a 1.5 mL sterile Eppendorf centrifuge tube. Resuspend the culture in 1 mL of sterile water. Use a pipette tip to repeatedly pipette and agitate the culture to mix thoroughly. Use sterile water in a gradient to obtain the original solution and 10 mL of the culture medium. -1 Dilution, 10 -2 Three dilution gradients of bacterial suspension were prepared. 200 μL of each dilution gradient was spread onto TY solid medium plates used for screening resistance (containing both donor and recipient bacteria, specifically 30 μg / mL nalidixic acid and 50 μg / mL kanamycin). The plates were then incubated at 28°C for 3-5 days.

[0082] 6) After single colonies grow on the solid culture medium plates from step 5), use a sterile toothpick to pick single colonies and screen for clones that have undergone homologous single crossover on a plate containing antibiotics (final concentration of 30 μg / mL naridinolone acid). Use the forward primer F and reverse primer R on the pBBR1MCS-2 plasmid vector (Table 2), ordinary Taq enzyme (GENSTAR, catalog number A012-101), and 2×Taq PCR StarMix (Kangrun Biotechnology, catalog number A012-01) to perform colony PCR amplification. The reaction system is shown in Table 2.

[0083] The amplification procedure is the same as step 4) 6) colony PCR amplification procedure above.

[0084] After amplification, 5 μL of PCR product was taken and detected by 1% agarose gel electrophoresis. Two to three positive clones with band sizes between 1000 and 2000 bp were sent to a sequencing company (such as Qingke Biotechnology Co., Ltd.) for analysis.

[0085] After screening and identification, the genetically engineered recombinant rhizobium (CCBAU45436 pBBR1MCS-2-deoD) carrying the pBBR1MCS-2-deoD recombinant plasmid was cultured in TY liquid medium containing 30 μg / mL naridinolone acid at 28°C and 200 rpm for 2 days. Sterile glycerol with a final concentration of 20% was added to the culture medium, and the strain was stored at -80°C.

[0086] 6. deoD qPCR validation of gene overexpression 1) RNA extraction Collect bacterial cell material: Take activated WT, MCS-2, OE-deoD, and single colonies into 5 mL of TY medium containing NA, incubate at 28℃ and 180 rpm for 24 h, then centrifuge at 4℃ and 12000 rpm for 2 min. Carefully remove all supernatant, and flash-freeze a portion in liquid nitrogen. Store at -80℃ for use as material for RNA extraction. Set up three independent replicates and three technical replicates for each group. RNA extraction was performed according to the Zhuangmeng RNA Extraction Kit, and the procedure was followed according to the instruction manual ZP403.

[0087] 2) RNA reverse transcription and removal of genomic DNA The FastKing cDNA first-strand synthesis kit (catalog number KR116) from TianGen was used.

[0088] The specific steps are as follows: a. Thaw the template RNA on ice; thaw 5×gDNA Buffer, FQ-RT Primer Mix, 10×King RT Buffer, and RNase-Free ddH2O at room temperature, and immediately place on ice after thawing. Vortex each solution to mix before use, and briefly centrifuge to collect any liquid remaining on the tube wall.

[0089] b. Preparation of a genomic DNA removal system: Take 2 μL of 5×gDNA Buffer and 1000 ng of Total RNA, add RNase-Free ddH2O to 10 μL, mix well, centrifuge briefly, then incubate at 42℃ for 3 min, and then place on ice.

[0090] c. Prepare the reverse transcription reaction system: Take 2 μL of 10×King RT Buffer, 1 μL of FastKing RT Enzyme Mix, and 2 μL of FQ-RT PrimerMix, and add RNase-Free ddH2O to 10 μL to obtain the Mix.

[0091] d. Add the Mix from the reverse transcription reaction to the reaction solution from the gDNA removal step and mix thoroughly. Incubate at 42°C for 15 min, then transfer to 95°C and incubate for 3 min. After that, transfer to ice to obtain cDNA, which can be used for subsequent experiments or cryopreservation.

[0092] 3) Real-time quantitative PCR (RT-qPCR) The 2×RealStar Fast SYBR qPCR Mix (Low Rox) from GenStar was used, catalog number A304.

[0093] a. Dilute the reverse transcription product: Dilute the above cDNA product 10-50 times with RNase-free deionized water.

[0094] b. Establish the qPCR reaction system as follows: Table 3. qPCR reaction system

[0095] The primers used for qPCR in the table above are as follows: Table 4. Primers used for qPCR

[0096] Primers 16S F and 16S R were used to amplify the 16S rRNA gene of rhizobia and served as internal controls; primers qPCR-deoD-F and qPCR-deoD-R were used to amplify... deoD Genes were used to detect and compare expression levels in different strains.

[0097] The calculation method is as follows: .

[0098] Where: C t (Cycle threshold): The number of PCR cycles required for the fluorescence signal to reach the threshold.

[0099] ΔC t = C t (Target gene) – C t (Internal reference gene).

[0100] ΔΔC t = ΔC t (Processing group) – ΔC t (Control group)

[0101] c. The qPCR reaction procedure is as follows: Table 5. qPCR reaction procedure

[0102] Each sample group was configured with three independent replicates and four technical replicates.

[0103] Figure 1 C represents the qPCR verification of the OE-deoD strain. deoD The overexpression level was significantly higher than that of wild-type (WT) and the expression level with empty plasmid (MCS-2).

[0104] 7. Western blot validation of PNP protein expression In order to detect deoD The protein expression level of the gene was determined by adding a 3×Flag tag to its C-terminus, followed by Western blotting experiments. The specific steps are as follows: Prepare the following in advance: Running Buffer, Transformation Buffer, Blocking Solution (prepared with TBST using 5% skim milk powder), Primary Antibody Incubation Solution (add 10000× primary antibody to the blocking solution; the primary antibody is the antibody corresponding to the tag), and Secondary Antibody Incubation Solution (prepared with TBST using 2% skim milk powder, add 10000× secondary antibody; the secondary antibody is generally a mouse or rabbit antibody, corresponding to the primary antibody). Store temporarily at 4°C.

[0105] Preparation of adhesive: After setting up the adhesive frame, add deionized water to check for leakage, add the prepared lower layer separating adhesive, seal with anhydrous ethanol, discard the anhydrous ethanol after it solidifies, and add the prepared concentrated adhesive.

[0106] Protein sample preparation: Centrifugation to collect OD 600 1 mL of each of the same bacteria (OD) 600 =1-2), resuspend in 200 μL of physiological saline, add 50 μL of 5× protein loading to make a 250 μL system, boil the sample and then centrifuge briefly.

[0107] SDS-PAGE electrophoresis: Take 10 μL / 20 μL of the supernatant from the centrifuged sample and load 5-6 μL of marker. Stack the gel at 80 V for about 30 min; separate the gel at 120 V for 60-80 min, until the bromophenol blue reaches the bottom of the gel.

[0108] Note: For the same sample, run two gels: one for Coomassie Brilliant Blue staining (to see all protein bands and verify whether the total protein loading is consistent), and one for Western blotting.

[0109] After the gel running is finished, carefully disassemble the sample gel. Place the gel for Coomassie Brilliant Blue staining on a petri dish and incubate overnight on a shaker.

[0110] Transfer: First, place three layers of filter paper in the transfer buffer, pre-cool the transfer buffer on ice, cut a PVDF membrane to an appropriate size, prepare the transfer "sandwich" structure in the transfer buffer, remove air bubbles, place the sandwich in the transfer tank, fill with pre-cooled transfer solution, maintain a constant flow of 200 mA, and perform electrophoresis for 1.2-1.5 h.

[0111] Blocking: Take out the PVDF membrane with the protein side facing up, transfer the membrane to the incubation box, ensuring that the membrane can be shaken inside, wash twice with TBST, add blocking solution, shake slowly, and incubate at room temperature for 30 min to 1 h.

[0112] Incubation of primary antibody: Soak the PVDF membrane in 5% skim milk powder and incubate overnight with shaking.

[0113] Membrane washing: Discard the primary antibody incubation solution, rinse once with TBST, then add TBST to the box, shake and wash the PVDF membrane 3 times, 15 minutes each time. During the washing process, the PVDF membrane needs to be held with tweezers to prevent the membrane from being poured out or contaminated.

[0114] Secondary antibody incubation: The PVDF membrane was immersed in 2% skim milk powder (containing horseradish peroxidase-labeled secondary antibody, purchased from Abcam) and incubated at low temperature with shaking for 1 h.

[0115] Washing the membrane: Discard the secondary antibody incubation solution, rinse once with TBST, and wash the PVDF membrane three times with TBST for 15 minutes each time. During the washing process, the PVDF membrane needs to be held with tweezers to prevent the membrane from being poured out or contaminated.

[0116] Development: Remove the PVDF membrane with tweezers, use disposable absorbent paper to remove excess buffer from the membrane edge, add ECL and detect the signal intensity with a chemiluminescence analyzer.

[0117] Western blot results are as follows Figure 1 The D-value shows that in recombinant rhizobia (OE-) deoD A significantly increased specific band of approximately 34 kDa was detected in the protein, consistent with the theoretical molecular weight of PNP protein plus the total molecular weight of the 3×Flag tag. In other WT and MCS-2 cells, however, the 3×Flag tag... deoD The concentration of the PNP protein band in gene expression was much lower. Figure 1 (C). The band at this position was relatively weaker in wild-type rhizobia (WT) and the empty vector control, indicating that the PNP-3×Flag fusion protein was specifically expressed in the recombinant rhizobia, proving the success of the genetic engineering.

[0118] In summary, recombinant OE rhizobium (OE-) deoD The difference between this recombinant rhizobium strain and the wild-type strain CCBAU45436 is that the former has an additional recombinant plasmid pBBR1MCS-2-deoD. This genetically engineered recombinant rhizobium has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38578.

[0119] Example 2: Determination of Rhizobium-Soybean Symbiotic Phenotype Recombinant rhizobia (OE-) carrying the pBBR1MCS-2-deoD recombinant plasmid were separately... deoD ), wild-type rhizobia (WT, S. fredii Rhizobium strain CCBAU45436, carrying the pBBR1MCS-2 plasmid (MCS-2) deoD Gene deletion mutant strain Δ deoD After washing twice with 0.85% physiological saline, the OD values ​​of each strain were determined using 0.85% physiological saline. 600 Adjust each to 0.2, take 1 mL of each and inoculate it into the soybean roots. After the soybeans have grown in the greenhouse for 30 days, observe the growth of the above-ground parts and the underground root system, and measure the plant height, fresh weight of root nodules, and fresh weight of the above-ground parts.

[0120] Figure 2In the results shown in A and B, compared to soybeans inoculated with wild-type rhizobia and rhizobia carrying the pBBR1MCS-2 plasmid, soybeans inoculated with rhizobia carrying the pBBR1MCS-2-deoD recombinant plasmid (OE-) showed a significantly higher yield. deoD Soybean plants inoculated with wild-type rhizobia were significantly taller and had more developed root systems, while there was no difference in plant height between soybeans inoculated with wild-type rhizobia and those inoculated with rhizobia carrying the pBBR1MCS-2 plasmid. This indicates that recombinant rhizobia promotes soybean plant height growth more effectively than wild-type rhizobia.

[0121] Figure 2 The statistical results shown in C indicate that, compared to soybeans inoculated with wild-type rhizobia and rhizobia carrying the pBBR1MCS-2 plasmid, soybeans inoculated with rhizobia carrying the pBBR1MCS-2-deoD recombinant plasmid (OE-) showed a significantly higher yield. deoD The fresh weight of soybean root nodules inoculated with recombinant rhizobia was significantly higher than that inoculated with wild-type rhizobia, while there was no difference in fresh weight between soybean root nodules inoculated with wild-type rhizobia and those inoculated with rhizobia carrying the pBBR1MCS-2 plasmid. This indicates that recombinant rhizobia can promote the increase of soybean root nodule quality more effectively than wild-type rhizobia.

[0122] The sequence of SEQ ID NO:1 is as follows:

[0123] The sequence of SEQ ID NO: 2 is as follows: MTDAAEFLGDKLGGLSPRYGIVLGSGLGSLVEAVTEPVRISYADIPGFPVSSVSGHAGELVLGRLGNVPVVMLSGRVHYYERGDANAMRVPIETLKRIGVENLILTNSAGSLREDMPPGSVMRIADHIAFSGANPLIGVESDDRFVGMTNAYDAALAVGMEEAAERLGIPLARGVYMWFSGPSFETPAEIRMARILGADAVGMSTVPEVILARFFGLRVAAASVITNFGAGMTGGELSHQETKEMAPFGGQRLAAILKDMIATEA* The sequence of SEQ ID NO: 3 is as follows:

[0124] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing recombinant rhizobia, characterized in that, The method includes the following steps: introducing biological material into recipient rhizobia that increases or / and improves the content of purine nucleoside phosphorylase and / or the expression level of the gene encoding said purine nucleoside phosphorylase, to obtain recombinant rhizobia.

2. The method according to claim 1, characterized in that, The purine nucleoside phosphorylase is any one of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:2; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1), which has more than 80% identity with the protein shown in A1) and has purine nucleoside phosphorylase activity. A3) A fusion protein with purine nucleoside phosphorylase activity obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

3. The method according to claim 2, characterized in that, The biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

4. The method according to claim 3, characterized in that, The expression cassette includes a promoter and a nucleic acid molecule encoding the protein of claim 1.

5. The method according to claim 4, characterized in that, The promoter is any one of the following: 1) The promoter whose nucleotide sequence is nucleotides 1-420 of SEQ ID NO:1; 2) A DNA molecule having at least 98% nucleotide sequence identity with the nucleotide sequence consisting of nucleotides 1-420 of SEQ ID NO:1 and having the promoter activity of 1).

6. The method according to any one of claims 3-5, characterized in that, The nucleic acid molecule is a DNA molecule with a nucleotide sequence as shown in nucleotides 421-1215 of SEQ ID NO:

1.

7. The expression cassette is a DNA molecule whose nucleotide sequence is shown in nucleotides 1-1215 of SEQ ID NO:

1.

8. Recombinant rhizobium, characterized in that, The recombinant rhizobium was prepared by the method according to any one of claims 1-5.

9. The recombinant rhizobium according to claim 8, characterized in that, The recombinant rhizobium is *Rhizobium fischeri* (Sinapis alfa). Sinorhizobium fredii Its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 38578.

10. Application, characterized in that, The application includes any of the following: C1) The application of the recombinant rhizobium as described in claim 6 or 7 in increasing the plant height of soybeans; C2) The use of the recombinant rhizobium as described in claim 6 or 7 in the preparation of products that increase the plant height of soybeans; C3) The application of the recombinant rhizobium as described in claim 6 or 7 in increasing the fresh weight of soybean root nodules; C4) The use of the recombinant rhizobium as described in claim 6 or 7 in the preparation of products that increase the fresh weight of soybean root nodules; C5) The application of the purine nucleoside phosphorylase as described in claim 1 or 2 in increasing the plant height of soybean; C6) The use of the purine nucleoside phosphorylase as described in claim 1 or 2 in the preparation of products that increase the plant height of soybeans; C7) The application of the purine nucleoside phosphorylase as described in claim 1 or 2 in increasing the fresh weight of soybean root nodules; C8) The use of the purine nucleoside phosphorylase as described in claim 1 or 2 in the preparation of products that increase the fresh weight of soybean root nodules; C9) The use of the purine nucleoside phosphorylase as described in claim 1 or 2 in the construction of recombinant rhizobia; C10) The use of the purine nucleoside phosphorylase as described in claim 1 or 2 in the preparation of products containing recombinant rhizobia.

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