A method for promoting symbiotic nodulation of soybean by bradyrhizobium japonicum by gene insertion

Abstract

The application belongs to the field of bioengineering and particularly relates to a method for promoting symbiotic nodule formation of soybean rhizobium and soybean by gene insertion. The method comprises the following steps: inserting the nod gene of Bradyrhizobium sp. (Sinorhizobium) yuanmingense FG-486 into Bradyrhizobium sp. (Sinorhizobium) DG-688 to obtain the improved strain. The improved strain is obtained by inserting the nod gene of Bradyrhizobium sp. (Sinorhizobium) yuanmingense FG-486 into the genome of Bradyrhizobium sp. (Sinorhizobium) DG-688, and the nodule formation ability and nitrogen fixation ability of the improved strain after symbiosis with soybean are improved. Compared with the soybean inoculated with the wild strain DG-688, the average nodule number of the soybean inoculated with the improved strain DG-688nod:FGnod increases by 27.15%, the nodule dry weight is basically unchanged, and the soybean yield increases by 6.55%.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a method for promoting symbiotic nodulation between soybean rhizobium and soybean through gene insertion. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Proper water and fertilizer management plays a crucial role in increasing soybean yield. Therefore, chemical fertilizers, including nitrogen fertilizer, are consumed annually. The excessive use of chemical fertilizers not only increases soybean planting costs and reduces farmers' enthusiasm for planting, but also causes soil compaction and decreased fertility. The rational use of chemical fertilizers and the enhancement of fertilizer efficiency through biological methods are receiving increasing attention. The use of soybean rhizobium inoculants is a good example of this approach.

[0004] Soybean rhizobia is a live microbial preparation. The rhizobia within the root nodules live in a mutually beneficial symbiotic relationship with legumes: some of the organic matter produced by the legumes through photosynthesis is supplied to the rhizobia; conversely, the ammonia produced by the rhizobia through biological nitrogen fixation is supplied to the legumes. While slow-growing soybean rhizobia that promotes soybean yield has been widely applied, soybean rhizobia inoculants still suffer from problems such as low nodulation rate and low nitrogen fixation efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for promoting nodulation in soybean rhizobia through gene insertion. This invention involves inserting the nod gene from *Rhizobium freundii* FG-486 into the genome of *Syntropha curcas* DG-688, obtaining an engineered strain that enhances the nodulation and nitrogen-fixing abilities of *Syntropha curcas* in symbiosis with soybean.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides a method for promoting symbiotic nodulation between soybean rhizobia and soybean through gene insertion, comprising:

[0008] The nod gene of *Rhizobium freundii* FG-486 is inserted into *DG-688*, a slow-growing rhizobium of soybean, to obtain the desired result.

[0009] The accession number of the slow-growing soybean rhizobium DG-688 is CCTCC NO: M20231152, the accession number of the *Rhizobium freundii* FG-486 is CCTCC NO: M20231153, and the nucleotide sequence of the nod gene of the *Rhizobium freundii* is shown in SEQ ID NO.1.

[0010] Preferably, the specific steps for inserting the nod gene of *Rhizobium freundii* FG-486 into *Stachyzoa stolonifera* DG-688 include:

[0011] Amplify the nod sequence fragment of *Sinocyclophosphamide* root nod, the upstream nod-U fragment of the nod gene of *Sinocyclophosphamide*, and the downstream nod-D fragment of the nod gene;

[0012] The nod fusion fragment nod-UD was amplified using nod fragments, nod-U fragments, and nod-D fragments as templates;

[0013] The nod-UD fragment was combined with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-nod-UD;

[0014] The recombinant plasmid pk18-nod-UD was introduced into Escherichia coli S17-1(λ) and subjected to parental hybridization culture with soybean slow-growing rhizobium DG-688 to screen for positive clones.

[0015] More preferably, the nucleotide sequence of the nod fusion fragment nod-UD is shown in SEQ ID NO.2.

[0016] Further preferred, the nucleotide sequences of primers F1 and R1 for amplifying the nod-U fragment are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0017] Further preferred, the nucleotide sequences of primers F2 and R2 for amplifying the nod fragment are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0018] Further preferred, the nucleotide sequences of primers F3 and R3 for amplifying the nod-D fragment are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0019] More preferably, when amplifying the nod fusion fragment nod-UD, the ratio of the nod fragment, nod-U fragment and nod-D fragment is 1:1:1, and the nucleotide sequences of amplification primers F1 and R3 are shown in SEQ ID NO:3 and SEQ ID NO:8, respectively.

[0020] Secondly, the present invention provides a gene-inserted soybean rhizobium DG-688nod:FGnod, which is obtained by inserting the nod gene of Rhizobium freundii FG-486 into soybean slow-growing rhizobium DG-688.

[0021] The accession number of the slow-growing soybean rhizobium DG-688 is CCTCC NO: M20231152, the accession number of the *Rhizobium freundii* FG-486 is CCTCC NO: M20231153, and the nucleotide sequence of the nod gene is shown in SEQ ID NO.1.

[0022] Thirdly, the present invention provides a microbial agent comprising soybean rhizobium DG-688nod:FGnod as described in the second aspect.

[0023] Preferably, the microbial agent also includes necessary excipients.

[0024] Preferably, the dosage form of the microbial agent is a liquid, powder, granule or tablet.

[0025] Fourthly, the present invention provides a method for increasing soybean yield, comprising the following steps:

[0026] Soybean seeds are treated with soybean rhizobium DG-688nod:FGnod as described in the second aspect or with the inoculant as described in the third aspect, and the soybeans are then planted.

[0027] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0028] After measuring soybeans treated with the two rhizobia, it was found that compared with soybeans treated with wild-type DG-688, soybeans treated with DG-688nod:FGnod had an average increase of 27.15% in the number of nodules, while the root nodule dry weight remained basically unchanged, and the soybean yield increased by 6.55%.

[0029] Preservation Information

[0030] The slow-growing rhizobium of soybean, DG-688 (Bradyrhizobium japonicum DG-688), was deposited at the China Center for Type Culture Collection (CCTCC) on June 30, 2023, with accession number CCTCC NO: M 20231152, at Wuhan University, Wuhan, China.

[0031] Sinorhizobium fredii FG-486 was deposited at the China Center for Type Culture Collection (CCTCC) on June 30, 2023, with accession number CCTCC NO: M 20231153, at Wuhan University, Wuhan, China. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 Electrophoresis diagrams were constructed for the mutant plasmid pK18-nod-UD, where (A) shows the amplification of the upstream and downstream homologous arms of nod and the nod gene: 1 is the amplification of the upstream homologous arm nod-U, 2 is the amplification of the downstream homologous arm nod-D, 3 is DNALadder DL5000, and 4 is the amplification of the nod gene; (B) shows the amplification of the nod-UD fusion fragment: 1 is the nod-UD fusion fragment, 2 is the nod-UD fusion fragment, and 3 is DNALadder DL5000.

[0034] Figure 2 PCR validation of the nod gene insert was performed. External primers were used for detection: 1 was a blank control, 2 was the wild-type genome as a template for amplification, 3 was DNALadder DL5000, and 4 was the nod gene insert as a template for amplification. Internal primers were used for detection: 1 was the nod insert DG-688 nod:FG nod as a template for amplification, 2 was the wild-type DG-688 genome as a template for amplification, 3 was DNA Ladder DL5000, and 4 was a blank control.

[0035] Figure 3 The number of nodules per soybean plant after seed dressing of wild-type DG688 and engineered DG-688nod:FGnod;

[0036] Figure 4 The average dry weight of each root nod in soybeans after seed dressing with wild-type DG688 and engineered DG-688nod:FGnod;

[0037] Figure 5 Soybean yield after seed dressing of wild strain DG688 and engineered strain DG-688nod:FGnod. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0039] Example 1

[0040] Soybean slow-growing rhizobium DG-688 and *Rhizobium freundii* FG-486 were inoculated into YMB(A) + The culture medium was used to extract the genome of soybean slow-growing rhizobium DG-688 and Sinorhizobium freundii FG-486 using a genome extraction kit. The genomes were stored at -20℃ for later use.

[0041] The nod gene (nucleotide sequence as shown in SEQ ID NO.1) and its upstream and downstream sequences were searched in the sequenced genome data of Rhizobium freundii FG-486. Using the genome of Rhizobium freundii FG-486 as a template, the nod sequence fragment was amplified using F2 (nucleotide sequence as shown in SEQ ID NO.5) / R2 (nucleotide sequence as shown in SEQ ID NO.6) as primers.

[0042] Using F1 (nucleotide sequence as shown in SEQ ID NO.3) / R1 (nucleotide sequence as shown in SEQ ID NO.4) and F3 (nucleotide sequence as shown in SEQ ID NO.7) / R3 (nucleotide sequence as shown in SEQ ID NO.8) as primers, the upstream homologous arm nod-U and the downstream homologous arm nod-D fragment of nod were amplified using the genome of soybean slow-growing rhizobium DG-688 as a template.

[0043] The amplified nod, nod-U, and nod-D fragments were placed in a PCR system at a 1:1:1 ratio as a template. The nod fusion fragment nod-UD (nucleotide sequence shown in SEQ ID NO.2) was amplified using F1 / R3 primers. The fusion fragment nod-UD was then ligated with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-nod-UD. Figure 1 As shown, the nod fusion fragment nod-UD was successfully amplified, and the recombinant plasmid pk18-nod-UD was successfully constructed.

[0044] The recombinant plasmid pk18-nod-UD was introduced into *E. coli* S17-1(λ) via heat shock transformation. *E. coli* S17-1(λ) was then crossbred with *DG-688*, a slow-growing rhizobium of soybean, and the recombinant plasmid pk18-nod-UD was introduced into *DG-688*. Mutant strains were obtained through sucrose plate selection and photocopy selection. The DG-688nod gene insert strain, DG-688nod:FGnod, was verified by PCR. Figure 2 As shown, the nod gene was successfully inserted into DG-688nod:FGnod.

[0045] Soybean variety He Dou 33 was treated with DG-688 and DG-688nod:FGnod, and the soybeans were then planted. During the soybean growing period, appropriate water and fertilizer management was implemented. Soybean roots were randomly collected 70-80 days after planting, and the number of nodules on each soybean plant was measured. It was found that the engineered strain DG-688nod:FGnod increased the number of nodules by 27.15% compared to the wild-type strain DG-688 (e.g., ...). Figure 3 As shown), the dry weight of the root nodule remains basically unchanged (as shown). Figure 4(As shown). Soybeans were harvested 100-110 days after planting, and the yield was measured and converted. It was found that the soybean yield treated with the engineered strain DG-688nod:FGnod was 6.55% higher than that treated with the wild strain DG-688 (e.g., ...). Figure 5 (As shown).

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for promoting symbiotic nodulation between soybean rhizobia and soybean through gene insertion, characterized in that, include: Rhizobium freundii FG-486 nod The gene is inserted into soybean slow-growing rhizobium DG-688 to obtain the product; The soybean slow-growing rhizobium ( Bradyrhizobium japonicum The accession number for DG-688 is CCTCC NO: M20231152, and the *Rhizobium freundii* described therein is... Sinorhizobium fredii The accession number for FG-486 is CCTCC NO: M20231153. nod The nucleotide sequence of the gene is shown in SEQ ID NO.1; Rhizobium freundii FG-486 nod The specific steps for inserting the gene into soybean slow-growing rhizobium DG-688 include: Amplification of Rhizobium freundii nod Sequence fragments, soybean slow-growing rhizobia nod upstream sequence of gene nod-U fragment and nod The downstream sequence of the gene, the nod-D fragment; by nod Fragments, nod-U fragments, and nod-D fragments were used as templates for amplification. nod Merge fragment nod-UD; The nod-UD fragment was combined with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-nod-UD; The recombinant plasmid pk18-nod-UD was introduced into Escherichia coli S17-1(λ) and subjected to parent hybridization culture with soybean slow-growing rhizobium DG-688 to screen for positive clones. The nod The nucleotide sequence of the fusion fragment nod-UD is shown in SEQ ID NO.

2.

2. The method as described in claim 1, characterized in that, The nucleotide sequences of primers F1 and R1 for amplifying the nod-U fragment are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

3. The method as described in claim 1, characterized in that, Amplification nod The nucleotide sequences of primers F2 and R2 for the fragment are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

4. The method as described in claim 1, characterized in that, The nucleotide sequences of primers F3 and R3 for amplifying the nod-D fragment are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

5. The method as described in claim 1, characterized in that, Amplification nod When merging fragments nod-UD, nod The ratio of the fragment, nod-U fragment, and nod-D fragment is 1:1:1, and the nucleotide sequences of amplification primers F1 and R3 are shown in SEQ ID NO:3 and SEQ ID NO:8, respectively.

6. A soybean rhizobium DG-688nod:FGnod with inserted gene, characterized in that, By using Rhizobium freundii FG-486 nod The gene was obtained by inserting it into soybean slow-growing rhizobium DG-688; The preservation number of the slow-growing soybean rhizobium DG-688 is CCTCC NO: M 20231152, and the preservation number of the *Rhizobium freundii* FG-486 is CCTCC NO: M 20231153. nod The nucleotide sequence of the gene is shown in SEQ ID NO.1; Rhizobium freundii FG-486 nod The specific steps for inserting the gene into soybean slow-growing rhizobium DG-688 include: Amplification of Rhizobium freundii nod Sequence fragments, soybean slow-growing rhizobia nod upstream sequence of gene nod-U fragment and nod The downstream sequence of the gene, the nod-D fragment; by nod Fragments, nod-U fragments, and nod-D fragments were used as templates for amplification. nod Merge fragment nod-UD; The nod-UD fragment was combined with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-nod-UD; The recombinant plasmid pk18-nod-UD was introduced into Escherichia coli S17-1(λ) and subjected to parent hybridization culture with soybean slow-growing rhizobium DG-688 to screen for positive clones. The nod The nucleotide sequence of the fusion fragment nod-UD is shown in SEQ ID NO.

2.

7. A microbial agent, characterized in that, Including the soybean rhizobium DG-688nod:FGnod as described in claim 6.

8. The microbial agent as described in claim 7, characterized in that, The microbial agent also includes necessary excipients.

9. The microbial agent as described in claim 7, characterized in that, The dosage form of the microbial agent is liquid, powder, granules or tablets.

10. A method for increasing soybean yield, characterized in that, Includes the following steps: The soybean rhizobium DG-688nod:FGnod as described in claim 6 or the inoculant as described in claim 7 is used to treat soybean seeds, and then the soybeans are planted.

Images