Method for improving nitrogen fixation effect of bradyrhizobium japonicum

Abstract

This invention belongs to the field of bioengineering, specifically relating to a method for improving the nitrogen fixation effect of soybean slow-growing rhizobia. This invention improves the nitrogen fixation effect of soybean slow-growing rhizobia DG-688 by knocking out the rsmE gene. Compared to soybeans treated with wild-type DG-688 seeds, soybeans treated with DG-688δE seeds with the rsmE gene knocked out increased the average number of nodules per plant by 31.3%, the root nodule dry weight by 18.8%, and the soybean yield by 11.79%.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a method for improving the nitrogen fixation effect of slow-growing rhizobia in soybeans. 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] Soybeans are an annual herbaceous plant and one of the world's most important legumes. With a protein content of 35%–40%, soybeans are a staple food and oilseed crop, commonly used for various soy products, oil extraction, soy sauce brewing, and protein extraction.

[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. According to field demonstrations, the application of rhizobia to soybeans can increase soybean yield by more than 10%, while simultaneously increasing the protein content by 2% and the crude fat content by 1-2%.

[0005] Currently, soybean cultivation requires a large amount of additional nitrogen fertilizer. Given the soaring prices of chemical fertilizers, promoting soybean rhizobia has significant ecological and environmental value. On the one hand, it directly reduces the input of chemical nitrogen fertilizers, saving planting costs and alleviating the energy pressure from fertilizer production; on the other hand, it can increase soybean yield. Although slow-growing soybean rhizobia that promotes soybean yield has been widely applied, existing soybean rhizobium inoculants suffer from 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 method for improving the nitrogen fixation effect of soybean slow-growing rhizobia. This invention first screened a soybean slow-growing nitrogen-fixing bacterium, DG-688, from the Yellow River Delta in Dongying, Shandong Province. Through genetic modification, an engineered strain was obtained, which enhances the nodulation and nitrogen fixation capabilities of soybean slow-growing rhizobia in symbiosis with soybeans.

[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 method for improving the nitrogen fixation effect of slow-growing rhizobia in soybeans, comprising:

[0009] The rsmE gene of soybean slow-growing rhizobium DG-688 is knocked out to obtain the product.

[0010] The preservation number of the soybean slow-growing rhizobium DG-688 is CCTCC NO: M 20231152, and the nucleotide sequence of the rsmE gene is shown in SEQ ID NO:1.

[0011] Preferably, the specific steps for knocking out the rsmE gene of soybean slow-growing rhizobium DG-688 include:

[0012] The upstream and downstream fragments of the rsmE gene were retrieved and ligated using PCR to obtain the rsmE-UD fragment;

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

[0014] The recombinant plasmid pk18-rsmE-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] Further preferred, the nucleotide sequence of rsmE-UD is shown in SEQ ID NO:2.

[0016] Further preferred, the nucleotide sequences of the upstream homologous arm primers rsmE-F1 and rsmE-R1 of the rsmE gene are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0017] Further preferred, the nucleotide sequences of the downstream homologous arm primers rsmE-F2 and rsmE-R2 of the rsmE gene are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0018] Secondly, the present invention provides a soybean slow-growing rhizobium DG-688δE with improved nitrogen fixation effect, which is obtained by knocking out the rsmE gene of soybean slow-growing rhizobium DG-688.

[0019] The preservation number of the soybean slow-growing rhizobium DG-688 is CCTCC NO: M20231152, and the nucleotide sequence of the rsmE gene is shown in SEQ ID NO:1.

[0020] Thirdly, the present invention provides a microbial agent comprising the soybean slow-growing rhizobium DG-688δE as described in the second aspect.

[0021] Preferably, the microbial agent also includes necessary excipients.

[0022] Preferably, the dosage form of the microbial agent is a liquid, powder, granule or tablet.

[0023] Fourthly, a method for increasing soybean yield includes the following steps:

[0024] Soybean seeds are treated with the slow-growing rhizobium DG-688δE as described in the second aspect or the inoculant as described in the third aspect, and then the soybeans are planted.

[0025] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0026] After measuring soybeans treated with two types of rhizobia, it was found that compared with soybeans treated with wild-type DG-688, soybeans treated with DG-688δE had an average increase of 31.3% in the number of nodules, an increase of 18.8% in the dry weight of nodules, and an increase of 11.79% in soybean yield.

[0027] Preservation Information

[0028] The slow-growing rhizobium of soybean, DG-688 (Bradyrhizobium japonicum DG-687), 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. Attached Figure Description

[0029] 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.

[0030] Figure 1 Electrophoresis diagrams were constructed for the mutant plasmid pK18-rsmE-UD, where (A) shows the amplification of the upstream and downstream homologous arms of rsmE: 1 is the amplification of the upstream homologous arm of rsmE, 2 is the amplification of the downstream homologous arm of rsmE, and 3 is the DNA Ladder DL5000; (B) shows the amplification of the upstream and downstream fusion fragment rsmE-UD of the rsmE gene: 1 is the DNA Ladder DL5000, 2 is the amplification of the fusion fragment of the upstream and downstream arms of the rsmE gene, and 3 is the amplification of the fusion fragment of the upstream homologous arm of rsmE.

[0031] Figure 2 For PCR validation of the rsmE knockout strain, the external primers were: 1 for the wild-type genome template amplification fragment, 2 for the blank control, 3 for DNA Ladder DL5000, and 4 for the rsmE knockout strain genome template amplification fragment; the internal primers were: 1 for the wild-type genome template amplification fragment, 2 for DNA Ladder DL5000, 3 for the rsmE knockout strain genome template amplification fragment, and 4 for the blank control.

[0032] Figure 3The number of nodules per soybean plant after seed dressing of wild DG688 and engineered DG688δE;

[0033] Figure 4 The average dry weight of each root nodule of soybean after seed dressing of wild DG688 and engineered DG688δE;

[0034] Figure 5 Soybean yield after seed dressing of wild strain DG688 and engineered strain DG688δE. Detailed Implementation

[0035] 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.

[0036] Example 1

[0037] Soybean slow-growing rhizobium DG-688 was inoculated into YMB(A + The culture medium was used to extract the genome of soybean slow-growing rhizobium DG-688 overnight at 30°C and shaken at 180 rpm. The genome was then extracted using a genome extraction kit and stored at -20°C for later use.

[0038] The rsmE gene and its upstream and downstream sequences were searched in the sequenced genome data of the soybean slow-growing rhizobium strain DG-688. Using the DG-688 genome as a template, primers rsmE-F1 (nucleotide sequence as shown in SEQ ID NO:3) / rsmE-R1 (nucleotide sequence as shown in SEQ ID NO:4) and rsmE-F2 (nucleotide sequence as shown in SEQ ID NO:5) / rsmE-R2 (nucleotide sequence as shown in SEQ ID NO:6) were used to amplify the upstream fragment rsmE-U and the downstream fragment rsmE-D of the rsmE gene, respectively. Using rsmE-U and rsmE-D as templates, and rsmE-F1 / rsmE-R2 as templates, the upstream and downstream fusion fragment rsmE-UD (nucleotide sequence as shown in SEQ ID NO:2) of the rsmE gene was amplified. Figure 1 As shown, the electrophoresis results indicate that rsmE-UD was successfully amplified.

[0039] The fusion fragment rsmE-UD was ligated with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-rsmE-UD. The recombinant plasmid pk18-rsmE-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-rsmE-UD was introduced into *DG-688*. Gene mutant strains were obtained through sucrose plate screening and copy screening. The knockout strain DG-688δE of DG-688rsmE was verified by PCR. Figure 2 As shown, the rsmE gene in DG-688δE was successfully knocked out.

[0040] Soybean variety He Dou-33 was treated with DG-688 and DG-688δE, 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 plant was measured. It was found that the engineered strain DG-688δE increased the number of nodules by 31.3% compared to the wild-type strain DG-688 (e.g., ...). Figure 3 As shown), the dry weight of root nodules increased by 18.8% (as indicated). Figure 4 (As shown).

[0041] Soybeans were harvested 100-110 days after planting, and the yield was measured and converted. It was found that soybeans treated with the engineered strain DG-688δE yielded 11.79% more soybeans than those treated with the wild strain DG-688 (e.g., ...). Figure 5 (As shown).

[0042] 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 improving the nitrogen fixation effect of slow-growing rhizobia in soybeans, characterized in that, include: The rsmE gene of soybean slow-growing rhizobium DG-688 is knocked out to obtain the product. The preservation number of the soybean slow-growing rhizobium DG-688 is CCTCC NO: M20231152, and the nucleotide sequence of the rsmE gene is shown in SEQ ID NO:

1.

2. The method as described in claim 1, characterized in that, The specific steps for knocking out the rsmE gene of soybean slow-growing rhizobium DG-688 include: The upstream and downstream fragments of the rsmE gene were retrieved and ligated using PCR to obtain the rsmE-UD fragment; The rsmE-UD fragment was combined with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-rsmE-UD; The recombinant plasmid pk18-rsmE-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.

3. The method as described in claim 2, characterized in that, The nucleotide sequence of rsmE-UD is shown in SEQ ID NO:

2.

4. The method as described in claim 2, characterized in that, The nucleotide sequences of the upstream homologous arm primers rsmE-F1 and rsmE-R1 of the rsmE gene are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

5. The method as described in claim 2, characterized in that, The nucleotide sequences of the downstream homologous arm primers rsmE-F2 and rsmE-R2 of the rsmE gene are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

6. A slow-growing soybean rhizobium DG-688δE with improved nitrogen-fixing efficiency, characterized in that, It was obtained by knocking out the rsmE gene of soybean slow-growing rhizobium DG-688; The preservation number of the soybean slow-growing rhizobium DG-688 is CCTCC NO: M20231152, and the nucleotide sequence of the rsmE gene is shown in SEQ ID NO:

1.

7. A microbial agent, characterized in that, Including the soybean slow-growing rhizobium DG-688δE 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 seed is treated with the slow-growing rhizobium DG-688δE as described in claim 6 or the inoculant as described in any one of claims 7-9, and then the soybean is planted.

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