Gene editing vector of sinorhizobium meliloti and gene editing method therefor, and edited strain of sinorhizobium meliloti

Abstract

Provided are a gene editing vector of Sinorhizobium meliloti and a gene editing method therefor, and an edited strain of Sinorhizobium meliloti. The gene editing vector of Sinorhizobium meliloti comprises the following genetic elements: a plasmid backbone, an inducible promoter Plac, an spCas9 gene, a gRNA, and an upstream homology arm and a downstream homology arm of a target gene. The gene editing vector of Sinorhizobium meliloti comprises the spCas9 gene, and the inducible promoter Plac is used for driving the expression of the spCas9 gene. However, the Plac exhibits a certain degree of leaky expression in Sinorhizobium meliloti. Therefore, there is no need to add an IPTG inducer to induce the expression of an spCas9 protein, and the leaky expression of the Plac promoter results in that the expression of the spCas9 protein is sufficient to cause a CRISPR system to edit the genome of Sinorhizobium meliloti, so that a mutant strain of Sinorhizobium meliloti can be obtained.

Description

Gene editing vector of sinorhizobium meliloti, gene editing method thereof and sinorhizobium meliloti editing strain TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a gene editing vector of sinorhizobium meliloti, a gene editing method thereof and a sinorhizobium meliloti editing strain. BACKGROUND

[0002] Sinorhizobium meliloti belongs to gram-negative alpha-proteobacteria and is an intracellular nitrogen-fixing symbiont of leguminous plants. A series of genes involved in vitamin B12 synthesis exist in the body of sinorhizobium meliloti. Through genetic manipulation of sinorhizobium meliloti, high-efficiency production of vitamin B12 can be achieved. However, traditional gene editing techniques for sinorhizobium meliloti have the disadvantages of low efficiency, complex operation steps and inability to obtain mutant strains without screening markers, which cause technical obstacles for the research and application of sinorhizobium meliloti.

[0003] In 2016, Johannes et al. used Cre / loxP site-specific recombination to edit the genome of sinorhizobium meliloti. This method can achieve deletion and insertion of large fragments of the genome of sinorhizobium meliloti. In 2020, Carlos Eduardo Flores-Tinoco et al. used transposon mutation to mutate the genome of sinorhizobium meliloti, and identified some nitrogen fixation-related genes through this method. In 2021, Longxiang Wang et al. used CRISPR / Cas9 system and various deaminases to achieve single-base editing of the genome of sinorhizobium meliloti. Under the guidance of gRNA, this system successfully achieved the conversion of adenine to guanine, cytosine to thymine and cytosine to guanine without forming double-strand breaks. Although Cre / loxP site-specific recombination can achieve deletion and insertion of large fragments of the genome of sinorhizobium meliloti, this method introduces exogenous DNA fragments into sinorhizobium meliloti, and the method is time-consuming and laborious. Transposon mutation randomly mutates any gene in the genome using a transposon system. The disadvantage of this technology is that it cannot achieve targeted editing of specific genes, and the screening is time-consuming and laborious. CRISPR / Cas9 system and deaminase-mediated single-base editing can only edit a certain base in the genome of sinorhizobium meliloti, and cannot achieve knockout and knockin of the entire gene.

[0004] SUMMARY

[0005] Therefore, the present application provides a gene editing vector of sinorhizobium meliloti, which can achieve single-gene knockout of sinorhizobium meliloti with high gene editing efficiency.

[0006] A Sinorhizobium meliloti gene editing vector, comprising the following genetic elements: a plasmid backbone, an inducible promoter Plac, a spCas9 gene, a gRNA, an upstream homologous arm of a target gene, and a downstream homologous arm of the target gene.

[0007] The Sinorhizobium meliloti gene editing vector contains a spCas9 gene, and the expression of the spCas9 gene is driven by an inducible promoter Plac. However, Plac has a certain degree of leakage in Sinorhizobium meliloti, so it is not necessary to induce the expression of the spCas9 protein with an IPTG inducer. Through the leakage of the Plac promoter, the expression of the spCas9 protein is sufficient to cause the editing of the Sinorhizobium meliloti genome by the CRISPR system, so that a Sinorhizobium meliloti editing strain can be obtained. The Sinorhizobium meliloti gene editing vector based on the CRISPR / spCas9 system can efficiently and conveniently realize single gene knockout of Sinorhizobium meliloti.

[0008] In some embodiments, the target gene comprises a tdk gene.

[0009] In some embodiments, the gRNA comprises at least one of the sequences shown in SEQ ID NO. 20, SEQ ID NO. 23, and SEQ ID NO. 26.

[0010] In some embodiments, among the plasmid backbone, the inducible promoter Plac, the spCas9 gene, the gRNA, the upstream homologous arm of the target gene, and the downstream homologous arm of the target gene, a repeat sequence is connected between two adjacent genetic elements.

[0011] In some embodiments, the length of the repeat sequence is 30 bp.

[0012] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone, and the nucleotide sequence of the pSJ-backbone plasmid backbone is shown in SEQ ID NO. 1.

[0013] The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO. 4.

[0014] The nucleotide sequence of the spCas9 gene is shown in SEQ ID NO. 5.

[0015] A method for editing the gene of Sinorhizobium meliloti, comprising the following steps:

[0016] Constructing the Sinorhizobium meliloti gene editing vector described above.

[0017] transforming the Sinorhizobium meliloto gene editing vector into E. coli competent cells, culturing to obtain E. coli containing the Sinorhizobium meliloto gene editing vector;

[0018] mixing the Sinorhizobium meliloto and the E. coli containing the Sinorhizobium meliloto gene editing vector, transferring to an antibiotic-free LB solid plate for conjugation, and then screening using an LB solid plate containing a screening marker to obtain a Sinorhizobium meliloto editing strain.

[0019] In some embodiments, the culture medium containing a screening marker is an antibiotic-resistant and inducer-free culture medium.

[0020] In some embodiments, the step of constructing the Sinorhizobium meliloto gene editing vector comprises assembling each of the genetic elements by Gibson technology to obtain the Sinorhizobium meliloto gene editing vector.

[0021] In some embodiments, the Sinorhizobium meliloto is a wild-type Sinorhizobium meliloto, and the mixing volume ratio of the Sinorhizobium meliloto and the E. coli containing the Sinorhizobium meliloto gene editing vector is 0.5:1 to 2:1.

[0022] In some embodiments, the E. coli competent cells are WM6026 E. coli.

[0023] In some embodiments, the step of screening using an LB solid plate containing a screening marker is followed by the steps of transferring the screened Sinorhizobium meliloto editing strain to the antibiotic-free LB liquid medium for culturing, and then performing PCR identification to obtain the Sinorhizobium meliloto editing strain.

[0024] A Sinorhizobium meliloto editing strain prepared by the above gene editing method. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a pSJ-HRs-Plac-spCas9-P23119-gRNA plasmid map;

[0026] Figure 2 is a tdk gene knockout PCR detection result;

[0027] Figure 3 is a column chart of knockout efficiency of three different gRNAs of the tdk gene;

[0028] Figure 4 is a phenotype identification diagram of the tdk gene knockout strain. DETAILED DESCRIPTION

[0029] In order to make the above objectives, characteristics and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the specific embodiments and drawings. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0030] An embodiment of the present application provides a Sinorhizobium meliloti gene editing vector, and the gene editing vector can realize single gene knockout of Sinorhizobium meliloti and has high gene editing efficiency.

[0031] In some embodiments, the Sinorhizobium meliloti gene editing vector comprises the following gene elements: a plasmid backbone, an inducible promoter Plac, a spCas9 gene, a gRNA, an upstream homologous arm and a downstream homologous arm of a target gene.

[0032] The CRISPR / spCas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. The principle of the CRISPR / spCas9 system is as follows: ① recognition component: the CRISPR sequence is transcribed to form sgRNA with a targeted recognition function; ② cutting component: the spCas9 protein is a kind of endonuclease, which forms a combination with sgRNA, and when sgRNA specifically recognizes and combines with a specific DNA sequence, the spCas9 protein cuts the targeted DNA strand, causes a gap in the gene, and realizes the breaking of the target gene; then, the gene repair is performed in the cell through a repair system, such as homologous recombination or non-homologous end joining, to achieve the purpose of gene editing. However, these designs are not enough to make the CRISPR / spCas9 system work efficiently in Sinorhizobium meliloti. We further optimized the promoter driving the spCas9 gene, the concentration of the promoter inducer and the length of the homologous arm, which are described in detail as follows: ① two kinds of inducible promoters, Plac and Pvan, were tested in the present study, and the results showed that the Plac promoter has a relatively high gene editing efficiency when inducing the expression of the spCas9 gene; ② a lower concentration of inducer is conducive to the occurrence of gene editing when inducing the expression of the spCas9 gene; ③ due to the limitation of plasmid size, the homologous arm should not be longer than 2K.

[0033] The system has the following advantages: ① In the CRISPR / spCas9 system, the spCas9 gene is driven by the inducible promoter Plac, which strictly controls the expression amount of the spCas9 protein, and is beneficial to maximize the editing efficiency of the CRISPR / spCas9 system; ② By screening different lengths of homologous arms, the most suitable length of homologous arm is assembled into the CRISPR / spCas9 system, and the obtained gene editing efficiency is relatively high.

[0034] The above-mentioned Sinorhizobium meliloti gene editing vector contains a spCas9 gene, and the expression of the spCas9 gene is driven by an inducible promoter Plac, but Plac has a certain degree of leakage in Sinorhizobium meliloti. Therefore, it is not necessary to induce the expression of the spCas9 protein by adding an IPTG inducer. Through the leakage of the Plac promoter, the expression of the spCas9 protein is sufficient to cause the editing of the Sinorhizobium meliloti genome by the CRISPR system, so that a Sinorhizobium meliloti editing strain can be obtained. The above-mentioned Sinorhizobium meliloti gene editing vector based on the CRISPR / spCas9 system can efficiently and conveniently realize single gene knockout of Sinorhizobium meliloti.

[0035] In some embodiments, the target gene includes, for example, a tdk gene. Further, the gRNA includes at least one of the sequences shown in SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26. It should be noted that the target gene is not limited to the tdk gene, and other genes in Sinorhizobium meliloti can also be genetically edited by designing corresponding gRNAs. It should be noted that the tool used for gRNA design is the CHOPCHOP online tool, and its specific website is: http: / / chopchop.cbu.uib.no / .

[0036] In some embodiments, a repeat sequence is connected between two adjacent gene elements in the plasmid backbone, the inducible promoter Plac, the spCas9 gene, the gRNA, the upstream homologous arm of the target gene and the downstream homologous arm of the target gene. Further, the length of the repeat sequence is 30 bp.

[0037] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone.

[0038] Specifically, the nucleotide sequence of the pSJ-backbone plasmid backbone is shown in SEQ ID NO. 1. The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO. 4. The nucleotide sequence of the spCas9 gene is shown in SEQ ID NO. 5.

[0039] The above-mentioned Sinorhizobium meliloti gene editing vector can realize single gene knockout in Sinorhizobium meliloti, and can realize streamlined gene editing. The Sinorhizobium meliloti gene editing vector can be used for gene editing to obtain a Sinorhizobium meliloti editing strain without a screening marker.

[0040] The application also provides a gene editing method of Sinorhizobium meliloti, including the following steps S110-S130:

[0041] S110, constructing the above-mentioned Sinorhizobium meliloti gene editing vector;

[0042] S120, transforming the Sinorhizobium meliloti gene editing vector into an E. coli competent cell, and culturing to obtain an E. coli containing the Sinorhizobium meliloti gene editing vector;

[0043] S130, mixing the Sinorhizobium meliloti and the E. coli containing the Sinorhizobium meliloti gene editing vector, transferring to an LB solid plate without antibiotics for conjugation, and then screening on an LB solid plate containing a screening marker to obtain a Sinorhizobium meliloti editing strain (i.e. a Sinorhizobium meliloti after gene editing).

[0044] The above-mentioned gene editing method of Sinorhizobium meliloti can realize streamlined gene editing of Sinorhizobium meliloti, and can obtain a Sinorhizobium meliloti editing strain without adding an inducer, and the operation is more simple and convenient.

[0045] The specific description of the Sinorhizobium meliloti gene editing vector is shown above, and will not be repeated here.

[0046] In some embodiments, the step of constructing the Sinorhizobium meliloti gene editing vector includes: assembling each gene element by Gibson technology to obtain the Sinorhizobium meliloti gene editing vector.

[0047] In some embodiments, the E. coli competent cell is an E. coli WM6026. Specifically, S120 includes: taking 5 microliters of the assembled Sinorhizobium meliloti gene editing vector, adding 100 microliters of the E. coli WM6026 competent cell, placing on ice for 30 minutes, heating at 42°C for 90 seconds, placing on ice for 5 minutes, adding 1000 microliters of LB liquid medium, then placing in a 37°C incubator, 200 rpm, 1 hour, finally culturing on an LB solid plate containing DAP and gentamicin resistance, to obtain the E. coli containing the Sinorhizobium meliloti gene editing vector.

[0048] In some embodiments, the Sinorhizobium meliloti is wild type, and the volume ratio of the Sinorhizobium meliloti and the E. coli containing the gene editing vector of the Sinorhizobium meliloti is 0.5:1 to 2:1. Further, the volume ratio of the Sinorhizobium meliloti and the E. coli containing the gene editing vector of the Sinorhizobium meliloti is 1:1.

[0049] In some embodiments, the culture medium containing the screening marker is an antibiotic-resistant LB solid plate without an inducer.

[0050] Specifically, S130 comprises: culturing the WM6026 E. coli containing the plasmid and the wild type Sinorhizobium meliloti to the logarithmic phase, respectively, so that the OD600 of the two strains is between 0.4 and 0.6, mixing them in a volume ratio of 1:1, centrifuging the mixture at a speed of 5000 rpm for 5 minutes, discarding the supernatant, using 100 microliters of LB liquid to suspend the bacterial mixture and the precipitate, and transferring the mixture to an antibiotic-free LB solid plate containing DAP and culturing it at 28°C for 6 hours. Using a swab, the bacterial lawn is scraped and resuspended in 1 milliliter of liquid LB medium, and cultured at 28°C and 220 rpm for 2 hours. Then, 150 microliters of the culture is taken and spread on a gentamicin-resistant LB plate, and cultured at 28°C for 3 days.

[0051] In some embodiments, after the screening step using the culture medium containing the screening marker, the method further comprises the following steps: transferring the screened Sinorhizobium meliloti editing strain to an antibiotic-free liquid LB medium for culture, and then performing PCR identification to obtain a Sinorhizobium meliloti editing strain without the screening marker and the exogenous plasmid. The specific steps comprise: picking the Sinorhizobium meliloti editing strain into an antibiotic-free LB liquid medium, culturing it at 28°C and 220 rpm for 3 days, diluting and plating it on an antibiotic-free LB solid plate on the fourth day, and identifying it by plasmid-specific primers to obtain a Sinorhizobium meliloti editing strain without the screening marker and the exogenous plasmid.

[0052] The above-mentioned gene editing method of Sinorhizobium meliloti can realize streamlined gene editing and obtain a Sinorhizobium meliloti editing strain without an exogenous plasmid.

[0053] In the above-mentioned gene editing method, the expression of the spCas9 gene is driven by an inducible promoter Plac. Plac has a certain degree of leakage in Sinorhizobium meliloti, so the method can induce the expression of the spCas9 protein without IPTG inducer. Through the leakage of the Plac promoter, the expression of the spCas9 protein is sufficient to cause the editing of the Sinorhizobium meliloti genome by the CRISPR system.

[0054] An embodiment of the present application also provides a Sinorhizobium meliloti editing strain prepared by the above-mentioned gene editing method.

[0055] The following are specific examples.

[0056] The drug reagents and instruments used in the examples are all selected according to the conventional selection in the art, unless otherwise specified. The experimental methods not specified in the examples are usually carried out according to the conventional conditions, for example, the conditions described in the literature, books or the methods recommended by the kit manufacturers.

[0057] The sequences of the genes and primers involved in the following Example 1 are shown in Table 1. In Table 1, pJ23119 is the promoter of each sgRNA in the examples, which drives the expression of each sgRNA in the examples; sgRNA scaffold is the sequence of sgRNA together with gRNA sequence, which plays a role in targeting DNA in gene editing, and sgRNA scaffold sequence plays a role in binding spCas protein; in Table 1, the capital letter part is the corresponding designed homologous arm.

[0058] Table 1 Sequences of genes and primers involved in Example 1

[0059] Knockout of wild-type Sinorhizobium meliloti tdk gene in Example 1

[0060] (1) Design of gRNA: CHOPCHOP online tool is used for design, and its specific website is: http: / / chopchop.cbu.uib.no / .

[0061] Specifically, the targeted genes of the present embodiment include tdk gene, and the specific sequences of the designed gRNA are shown in SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26 of Table 1.

[0062] (2) Design of plasmid:

[0063] The vector map designed in the present embodiment is shown in Figure 1, which mainly contains 6 parts, which are pSJ-backbone plasmid backbone, IPTG inducible promoter Plac, spCas9 gene, gRNA and homologous arm. Among them, gRNA and repair template (homologous arm) can be replaced according to the difference of target gene. The sequences of each element are shown in Table 1 gRNA: SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; repair template: SEQ ID NO. 10 and SEQ ID NO. 13.

[0064] The main method for assembling each element into a plasmid is Gibson assembly method, and the specific operation steps are as follows:

[0065] (a) Design primers to linearize the pSJ-backbone plasmid backbone and Plac. After linearization, there will be 30 bp of repeated sequences between the two elements (this needs to be considered before designing the primers, and the primers used for linearization are shown in Table 1). Then, follow the operation instructions of the Invitrogen Seamless Cloning Kit to assemble. The reaction system is 20 microliters, including 0.1 pmol of pSJ-backbone plasmid backbone, 0.2 pmol of Plac inducible system element, 2 microliters of Exnase II, 4 microliters of 5X CE II Buffer, and water to make up to 20 microliters. Then, place the PCR instrument at 37°C for 30 min. Then, transform the assembly product into DH5α competent cells, and screen on LB gentamicin-resistant plates to obtain the plasmid pSJ-Plac.

[0066] (b) Design primers to linearize the plasmid pSJ-Plac and spCas9 gene. Similarly, there are 30 bp of repeated sequences between the two fragments (the primers used are shown in Table 1). Follow the method of step (a) above to assemble, and the molar ratio of the two elements is 1:1. Other methods are the same as step (a) above, and the pSJ-Plac-spCas9 plasmid can be obtained.

[0067] (c) Design primers to assemble the plasmid pSJ-Plac-spCas9, P23119-sgRNA scaffold, the upstream homologous arm and the downstream homologous arm of the target gene (the primers used are shown in Table 1). After linearization of the plasmid pSJ-Plac-spCas9, P23119-sgRNA scaffold and the upstream and downstream homologous arms, there are 30 bp of repeated sequences between each element. Use the method of step (a) above to assemble, and the molar ratio of each element is 1:1:1:1. The total molar number is not more than 0.2 pmol. Other methods are the same as the method of step (a) above, and the pSJ-Plac-spCas9-P23119-sgRNA scaffold-HRs plasmid can be obtained.

[0068] (d) Design gRNA sequences on primers, and perform loop PCR on the plasmid pSJ-Plac-spCas9-P23119-sgRNA scaffold-HRs to obtain a linear fragment. Then, transform the linear fragment into DH5α competent cells, and screen on LB gentamicin-resistant plates to realize the connection of gRNA between P23119 and sgRNA scaffold, and obtain the gene editing vector pSJ-Plac-spCas9-P23119-sgRNA-HRs.

[0069] (3) Genome editing of S. sinorice

[0070] The gene editing vector was transformed into WM6026 E. coli competent cells to obtain WM6026 E. coli containing the gene editing vector, and the wild-type S. sinorice (the wild-type S. sinorice was derived from the literature: Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing, Applied and Environmental Microbiology, SR Khan, J Gaines, RM Roop, SK Farrand, et al.) were cultured to the logarithmic phase, and the OD 600 of the two strains was between 0.4 and 0.6. They were mixed at a volume ratio of 1:1, centrifuged at a speed of 5000 rpm for 5 minutes, and the supernatant was discarded. The bacterial mixture was suspended in 100 microliters of LB liquid medium, and the suspension was transferred to an antibiotic-free LB solid plate containing DAP and cultured at 28°C for 6 hours. The bacterial lawn was scraped using a inoculation loop, eluted in 1 milliliter of liquid LB liquid medium, and cultured at 28°C and 220 rpm for 2 hours. 150 microliters were taken and plated on a gentamicin-resistant LB solid plate, and cultured at 28°C for 3 days until colonies appeared on the plate. The colonies on the LB plate were directly identified by PCR to obtain the S. sinorice editing strain. The principle of designing PCR primers is that both the upstream and downstream primers are designed in the genomic part outside the homologous arm. The specific primers are shown in Table 1.

[0071] (4) Discarding the gene editing vector in the S. sinorice editing strain:

[0072] The S. sinorice editing strain was inoculated into antibiotic-free LB liquid medium and cultured at 28°C and 220 rpm for 3 days. On the fourth day, the bacterial solution was gradiently diluted (100-fold, 1000-fold, and 10000-fold) and plated on antibiotic-free LB solid plates, and cultured in a 28°C incubator. The specific primers on the gene editing vector (see Table 1 for specific primers) were amplified to determine whether the gene editing vector was lost. If the gene editing vector is lost, no band will be produced after PCR amplification of the specific primers; if the gene editing vector exists, a band will be produced after PCR amplification of the specific primers. The S. sinorice editing strain without exogenous plasmid can be obtained by identifying the specific primers of the gene editing vector.

[0073] (5) Result detection

[0074] PCR combined with gel electrophoresis was used for tdk gene knockout PCR identification, and the detection results are shown in Figure 2; PCR was used to detect the knockout efficiency of three different gRNAs of the tdk gene, and the results are shown in Figure 3; and the phenotype of the tdk gene knockout mutant of Sinorhizobium meliloti was identified by nutrient metabolism deficiency method, as shown in Figure 4. In Figure 4, the first culture dish is the result of phenotype identification using PYE solid medium without 5-fluorodeoxyuridine, and the second culture dish is the result of phenotype identification using PYE solid medium containing 5-fluorodeoxyuridine.

[0075] As can be seen from Figure 2, after knocking out the tdk gene, the PCR amplification band in the mutant strain is obviously smaller than that in the wild type strain. As can be seen from Figure 3, the knockout efficiency of the tdk gene is affected by the position of the gRNA, and the knockout efficiency at different gRNA positions is different, but the overall knockout efficiency is very high. As can be seen from Figure 4, the wild type Sinorhizobium meliloti cannot grow on PYE solid medium containing 5-fluorodeoxyuridine (5 FudR), while the tdk gene knockout mutant strain can grow on PYE solid medium containing 5-fluorodeoxyuridine. It can be seen that the tdk gene knockout in Sinorhizobium meliloti is achieved in this embodiment, and the phenotype of the mutant is identified.

[0076] The Sinorhizobium meliloti gene editing vector and editing method of the present application realize single gene knockout of Sinorhizobium meliloti, and construct an efficient, traceless gene editing system for streamlined gene editing, and can obtain a mutant strain without a screening marker.

[0077] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered as within the scope of the present disclosure.

[0078] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

Claims

1. A Sinorhizobium meliloti gene editing vector, characterized in that, The Sinorhizobium meliloti gene editing vector comprises the following gene elements: a plasmid backbone, an inducible promoter Plac, a spCas9 gene, a gRNA, an upstream homologous arm of a target gene, and a downstream homologous arm of the target gene.

2. The Mesorhizobium huanghuaiensis gene editing vector of claim 1, characterized in that, The target gene comprises, for example, a tdk gene. Further, the gRNA comprises at least one of the sequences shown in SEQ ID NO. 20, SEQ ID NO. 23, and SEQ ID NO.

26.

3. The Mesorhizobium huanghuaiensis gene editing vector of claim 1, characterized in that, Among the plasmid backbone, the inducible promoter Plac, the spCas9 gene fragment, the gRNA, the upstream homologous arm of the target gene, and the downstream homologous arm of the target gene, a repeat sequence is connected between two adjacent gene elements. Further, the repeat sequence has a length of 30 bp.

4. The Mesorhizobium huanghuaiensis gene editing vector of any one of claims 1-3, wherein, The plasmid backbone is a pSJ-backbone plasmid backbone, and the nucleotide sequence of the pSJ-backbone plasmid backbone is shown in SEQ ID NO.

1. The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO.

4. The nucleotide sequence of the spCas9 gene fragment is shown in SEQ ID NO.

5.

5. A method of gene editing of Sinorhizobium meliloti, comprising, The method comprises the following steps: constructing the Sinorhizobium meliloti gene editing vector according to any one of claims 1-4; transforming the Sinorhizobium meliloti gene editing vector into an E. coli competent cell, culturing, to obtain an E. coli containing the Sinorhizobium meliloti gene editing vector; mixing the Sinorhizobium meliloti and the E. coli containing the Sinorhizobium meliloti gene editing vector, transferring to an LB solid plate without antibiotics for conjugation, and then screening using an LB solid plate containing a screening marker to obtain a Sinorhizobium meliloti editing strain.

6. The method of gene editing of claim 5, wherein, The culture medium containing a screening marker is an LB solid plate resistant to antibiotics and free of an inducer.

7. The method of gene editing of claim 5, wherein, The step of constructing the Sinorhizobium meliloti gene editing vector comprises assembling each of the gene elements by Gibson technology to obtain the Sinorhizobium meliloti gene editing vector.

8. The method of gene editing of claim 5, wherein, The Sinorhizobium meliloti is a wild-type Sinorhizobium meliloti, and the mixing volume ratio of the Sinorhizobium meliloti to the E. coli containing the Sinorhizobium meliloti gene editing vector is 0.5:1-2:

1. Further, the E. coli competent cell is an E. coli WM6026.

9. The method of gene editing according to any one of claims 5-8, wherein, After the step of screening using the LB solid plate containing a screening marker, the method further comprises the following steps: transferring the Sinorhizobium meliloti editing strain obtained by screening to an LB liquid culture medium without antibiotics for culturing, and then performing PCR identification to obtain the Sinorhizobium meliloti editing strain.

10. A Sinorhizobium meliloti editing strain, characterized in that, The Sinorhizobium meliloti editing strain is prepared by the gene editing method according to any one of claims 4-9.

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