Sinorhizobium fredii with improved nodulation ability and a method for constructing the same
By genetically engineering *Rhizobium freundii* FG-486 and knocking out the pip gene, a variant of *Rhizobium freundii* FG-486δP with enhanced nodulation ability was constructed. This solved the problems of low nodulation rate and nitrogen fixation efficiency, and significantly increased the number of soybean nodules, root nodule dry weight, and yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QILU UNIVERSITY OF TECHNOLOGY (SHANDONG ACADEMY OF SCIENCES)
- Filing Date
- 2023-10-30
- Publication Date
- 2026-07-07
Smart Images
Figure CN117467591B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering, specifically relating to a *Rhizobium freundii* strain with enhanced nodulation ability and its construction method. 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] Nitrogen-fixing bacteria are a general term for various prokaryotes that can perform biological nitrogen fixation in nature. They can be divided into several categories, including free-living nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria, and associative nitrogen-fixing bacteria. Because nitrogen-fixing bacteria can convert free gaseous molecules in the air into nitrogen fertilizer that plants can use, the application of nitrogen-fixing bacteria can increase soil fertility and promote plant growth.
[0004] Legumes, such as soybeans, often form mutually beneficial symbiotic relationships with rhizobia, such as *Rhizobium freundii*. Legumes supply some of the organic matter they produce through photosynthesis to the rhizobia; conversely, the ammonia produced by the rhizobia through biological nitrogen fixation is supplied to the legumes. Field demonstrations have shown that applying rhizobia to soybeans can increase yield by over 10%, while simultaneously increasing protein content by 2% and crude fat content by 1-2%. Therefore, applying appropriate rhizobia when planting soybeans and other crops can not only reduce fertilizer use and lower planting costs but also increase soybean yield and economic benefits.
[0005] Although Rhizobium freundii, which promotes soybean yield, has been widely applied, there are still problems such as low nodulation rate and low nitrogen fixation efficiency. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a *Rhizobium freundii* strain with enhanced nodulation ability and its construction method. This invention first screened and obtained a *Rhizobium freundii* strain FG-486, and then improved its nodulation efficiency through genetic engineering, providing a new approach to improving soybean cultivation efficiency.
[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:
[0008] In a first aspect, the present invention provides a *Rhizobium freundii* FG-486δP with enhanced nodulation ability, obtained by knocking out the pip gene of *Rhizobium freundii* FG-486.
[0009] The preservation number of the *Rhizobium freundii* FG-486 is CCTCC NO: M20231153, and the nucleotide sequence of the pip gene is shown in SEQ ID NO.1.
[0010] In a second aspect, the present invention provides a method for constructing *Rhizobium freundii* FG-486δP with enhanced nodulation ability as described in the first aspect, comprising:
[0011] The result is obtained by knocking out the pip gene in Rhizobium freundii FG-486.
[0012] Preferably, the specific steps for knocking out the pip gene in *Rhizobium freundii* FG-486 include:
[0013] The upstream and downstream fragments of the pip gene were retrieved and ligated using PCR to obtain the pip-UD fragment;
[0014] The pip-UD fragment was combined with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-pip-UD;
[0015] The recombinant plasmid pk18-pip-UD was introduced into Escherichia coli S17-1(λ) and subjected to parental hybridization culture with Rhizobium freundii FG-486 to screen for positive clones.
[0016] More preferably, the nucleotide sequence of the pip-UD fragment is shown in SEQ ID NO.2.
[0017] Further preferred, the nucleotide sequences of the upstream homologous arm primers pip-F1 and pip-R1 of the pip gene are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
[0018] Further preferred, the nucleotide sequences of the downstream homologous arm primers pip-F2 and pip-R2 of the pip gene are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.
[0019] Thirdly, the present invention provides a microbial agent comprising *Rhizobium freundii* FG-486δP as described in the first aspect.
[0020] Preferably, the microbial agent also includes necessary excipients.
[0021] Preferably, the dosage form of the microbial agent is a liquid, powder, granule or tablet.
[0022] Fourthly, the present invention provides a method for increasing soybean yield, comprising the following steps:
[0023] Soybean seeds are treated with either *Rhizobium freundii* FG-486δP as described in the first aspect or the inoculant as described in the third aspect, and then the soybeans are planted.
[0024] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:
[0025] After measuring soybeans treated with two types of rhizobia, it was found that compared with the wild strain FG-486, soybeans treated with FG-486δP had an average increase of 31.15% in nodule count, 10.26% in nodule dry weight, and 7.85% in yield.
[0026] Preservation Information
[0027] 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
[0028] 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.
[0029] Figure 1 Electrophoresis diagrams were constructed for the mutant plasmid pK18-pip-ud, where (A) shows the amplification of the upstream and downstream homologous arms of pip: 1 is DNA Ladder DL5000, 2 is the amplification of the upstream homologous arm of pip, and 3 is the amplification of the downstream homologous arm of pip; (B) shows the amplification of the upstream and downstream fusion fragment pip-UD of the pip gene: 1 is DNA Ladder DL2000, 2 is the amplification of the fusion fragment of the upstream and downstream arms of pip, and 3 is the amplification of the fusion fragment of the upstream and downstream homologous arms of pip.
[0030] Figure 2 For PCR validation of the PIP knockout strain, the external primers used were: 1 for blank control, 2 for the PIP knockout strain genome as template amplification fragment, 3 for the wild-type strain genome as template amplification fragment, and 4 for DNALadder DL5000; the internal primers used were: 1 for the wild-type strain genome as template amplification fragment, 2 for the PIP knockout strain genome as template amplification fragment, 3 for DNALadder DL5000, and 4 for blank control.
[0031] Figure 3 The number of nodules per soybean plant after seed dressing with wild FG-486 and engineered FG-486δP;
[0032] Figure 4 The average dry weight of each root nodule of soybean after seed dressing with wild FG-486 and engineered FG-486δP;
[0033] Figure 5 Soybean yield after seed dressing of wild strain FG-486δP and engineered strain FG-486. Detailed Implementation
[0034] 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.
[0035] Example 1
[0036] Rhizobium freundii FG-486 was inoculated into YMB(A) + The culture medium was used to extract the genome of Rhizobium freundii FG-486 from the culture medium at 30℃ and shaken at 180 rpm for 2-3 days. The genome of Rhizobium freundii FG-486 was extracted using a genome extraction kit and stored at -20℃ for later use.
[0037] The *Pip* 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* strain FG-486. Using the FG-486 genome as a template, primers F1 (nucleotide sequence as shown in SEQ ID NO. 3) / R1 (nucleotide sequence as shown in SEQ ID NO. 4) and F2 (nucleotide sequence as shown in SEQ ID NO. 5) / R2 (nucleotide sequence as shown in SEQ ID NO. 6) were used to amplify the upstream fragment *pip-U* and the downstream fragment *pip-D*, respectively. Using *pip-U* and *pip-D* as templates, and F1 / R2 as templates, the upstream and downstream fusion fragment *pip-UD* (nucleotide sequence as shown in SEQ ID NO. 2) of the *Pip* gene was amplified. Figure 1 As shown, the electrophoresis results indicate that pip-UD was successfully amplified.
[0038] The fusion fragment pip-UD was ligated with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-pip-UD. The recombinant plasmid pk18-pip-UD was then introduced into *E. coli* S17-1(λ) via heat shock transformation. *E. coli* S17-1(λ) was then hybridized with *Rhizobium freundii* FG-486, and the recombinant plasmid pk18-pip-UD was introduced into *Rhizobium freundii* FG-486. Mutant strains were obtained through sucrose plate screening and photocopy screening. The knockout strain FG-486δP of the FG-486pip fragment was verified by PCR. Figure 2 As shown, the pip gene was successfully knocked out in FG-486δP.
[0039] FG-486δP and FG-486 were used as seed dressings for the soybean variety He Dou 12, 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 root nodules per soybean plant was measured. It was found that the engineered strain FG-486δP had a 31.15% higher nodule count compared to the wild-type strain FG-486 (e.g., ...). Figure 3 As shown), the dry weight of root nodules increased by 10.26% (as indicated). 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 of the engineered strain FG-486δP was 7.85% higher than that of the wild strain FG-486 (e.g., ...). Figure 5 (As shown).
[0040] 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 *Rhizobium freundii* strain with enhanced nodulation ability ( Sinorhizobium fredii FG-486δP, characterized in that, By knocking out Rhizobium freundii FG-486 pip Gene acquisition; The preservation number of *Rhizobium freundii* FG-486 is CCTCC NO: M 20231153. pip The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. A method for constructing *Rhizobium freundii* FG-486δP with enhanced nodulation ability as described in claim 1, characterized in that, include: Knockout of Rhizobium freundii FG-486 pip Genes, that is.
3. The construction method as described in claim 2, characterized in that, The knockout of Rhizobium freundii FG-486 pip The specific steps involved in gene generation include: Fishing and ligation using PCR pip The upstream and downstream segments of the gene are used to obtain the pip-UD fragment; The pip-UD fragment was combined with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-pip-UD; The recombinant plasmid pk18-pip-UD was introduced into Escherichia coli S17-1(λ) and subjected to parental hybridization culture with Rhizobium freundii FG-486 to screen for positive clones.
4. The construction method as described in claim 3, characterized in that, The nucleotide sequence of the pip-UD fragment is shown in SEQ ID NO.
2.
5. The construction method as described in claim 3, characterized in that, pip The nucleotide sequences of the upstream homologous arm primers pip-F1 and pip-R1 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
6. The method as described in claim 3, characterized in that, pip The nucleotide sequences of the downstream homologous arm primers pip-F2 and pip-R2 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.
7. A microbial agent, characterized in that, Including *Rhizobium freundii* FG-486δP as described in claim 1.
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 *Rhizobium freundii* FG-486δP as described in claim 1 or the inoculant as described in any one of claims 7-9 is used to treat soybean seeds, and the soybeans are then planted.