Preparation method and application of double-auxotroph engineering strain

Abstract

The invention discloses a preparation method and application of a double-auxotroph engineering strain, and belongs to the technical field of gene engineering. The invention discloses a preparation method of a double-auxotroph engineering strain, which comprises the following steps: knocking out a leuA gene in a host cell, and constructing an auxotroph engineering strain EHA105leuA-; the method comprises the following steps: further knocking out a hisD gene from an EHA105uA-strain, and constructing a double-auxotroph type engineering strain EHA105hisD-uA-; the gene ID of the leuA gene is WP010972185.1, and the gene ID of the hisD gene is WP004432479.1. The gene ID of the leuA gene is WP010972185.1. Results of the embodiment of the invention show that survival cells of the double-auxotroph engineering strain EHA105hisD-leuA-in soil and plants are greatly reduced, the probability that agrobacterium escapes into the natural environment is reduced, and the biological safety in the plant genetic transformation process is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and more specifically, relates to a preparation method and application of a double auxotrophic engineering strain. Background Art

[0002] Agrobacterium tumefaciens is a Gram-negative soil bacterium that can infect plants under natural conditions and cause crown galls. Agrobacterium tumefaciens contains Ti plasmids. The T-DNA in the Ti plasmids is transferred into plant cells and integrated into chromosomes during the infection process. T-DNA contains auxin and cytokinin synthesis genes. T-DNA integrated into plant chromosomes can interfere with plant hormone metabolism and lead to the formation of crown galls. Artificially modified Agrobacterium tumefaciens and Ti plasmids are widely used in the genetic transformation of crops and play a key role in creating new varieties of transgenic crops with high economic value.

[0003] Plant genetic transformation mediated by Agrobacterium tumefaciens usually involves two stages: co-cultivation and screening culture. The co-cultivation stage requires full contact between Agrobacterium and explants to ensure that Agrobacterium successfully transfers T-DNA to plant cells, while the screening culture stage requires the removal of Agrobacterium to ensure that the explants can dedifferentiate and redifferentiate smoothly. In the screening culture stage, antibiotics such as timentin, cephalosporin, carbenicillin and meropenem are usually added to inhibit the growth of Agrobacterium. The addition of antibiotics undoubtedly increases the cost of the experiment, and various antibiotics inhibit the dedifferentiation and redifferentiation of explants to varying degrees. In addition, due to drug-resistant mutations of Agrobacterium, or Agrobacterium is located in an area far away from the culture medium, excessive growth of Agrobacterium may occur during the screening culture stage, resulting in browning and death of the explants.

[0004] Agrobacterium tumefaciens used for plant genetic transformation usually carries antibiotic or herbicide resistance genes, so Agrobacterium tumefaciens escaping from the laboratory to the natural environment may pose a potential biosafety risk. The growth of auxotrophic Agrobacterium tumefaciens depends on the addition of corresponding nutrients, which can reduce its adaptability in the natural environment and provide better bioprotection for plant genetic transformation. However, no research has reported the application of auxotrophic Agrobacterium tumefaciens in improving bioprotection.

[0005] Nutritionally deficient Agrobacterium tumefaciens grows slowly or cannot grow in a culture medium lacking corresponding nutrients. Using these strains for plant genetic transformation can reduce the use of antibacterial antibiotics. At present, there are many papers and patents related to the preparation of nutritionally deficient Agrobacterium tumefaciens, but most of them are single nutritional deficiencies that rely on a single nutrient, including methionine, cysteine, leucine, adenine, threonine, tryptophan and thymine. Single nutritional deficiency strains are prone to recovery mutations, loss of nutritional deficiency traits, or easy to obtain corresponding nutrients in explants. Obviously, compared with single nutritional deficiency strains, double nutritional deficiency can further increase the dependence of Agrobacterium tumefaciens on supplementary nutrients, reduce the possibility of recovery mutations, and reduce the excessive growth of Agrobacterium on explants. However, there has been no research report on the creation of double nutritional deficiency Agrobacterium tumefaciens by gene deletion. In addition, there has been no research report on the deletion of leuA or hisD genes to create nutritional deficiency types of Agrobacterium tumefaciens. Summary of the invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a double auxotrophic engineered strain, and the adaptability of the constructed Agrobacterium tumefaciens auxotrophic EHA105hisD-leuA- in the natural environment is significantly reduced, providing better biological protection for plant genetic transformation experiments. Another technical problem to be solved by the present invention is to provide an application of the above double auxotrophic engineered strain in plant genetic transformation. Another purpose of the present invention is to provide an auxotrophic EHA105leuA- and its application.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a double auxotrophic engineered strain, comprising the following steps:

[0009] (1) Knocking out the leuA gene in the Agrobacterium tumefaciens strain EHA105 to construct an auxotrophic engineered strain EHA105leuA-;

[0010] (2) The hisD gene was further knocked out in the EHA105leuA- strain to construct the double auxotrophic engineered strain EHA105hisD-leuA-;

[0011] The gene ID of the leuA gene is WP_010972185.1; the gene ID of the hisD gene is WP_004432479.1; and the double auxotrophic engineered strain is a leucine and histidine double auxotrophic strain.

[0012] In step (1), the process of constructing the auxotrophic engineered strain EHA105leuA- is specifically as follows:

[0013] 1) Design primers and construct leuA gene knockout vector;

[0014] 2) The leuA gene knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were obtained by screening;

[0015] 3) The positive colonies were co-incubated with Agrobacterium tumefaciens EHA105 for diparental conjugation, and the leucine auxotrophic strain EHA105leuA- was obtained by screening and identification.

[0016] The construction method of the leuA gene knockout vector is:

[0017] Primers leuA-up-F and leuA-up-R were used to amplify a 1000bp fragment upstream of the leuA gene, and primers leuA-down-F and leuA-down-R were used to amplify a 1000bp fragment downstream of the leuA gene. The upstream and downstream fragments were cloned into the SalI and EcoRI sites of pK18mobSacB using Gibson recombination technology to obtain the leuA knockout vector. The primer sequences are as follows:

[0018] leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3',

[0019] leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3';

[0020] leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3',

[0021] leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3';

[0022] The gene ID of the leuA gene is WP_010972185.1.

[0023] In step (2), the process of constructing the double auxotrophic engineered strain EHA105hisD-leuA- is specifically as follows:

[0024] 1) Design primers and construct hisD gene knockout vector;

[0025] 2) The hisD knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened;

[0026] 3) Escherichia coli S17-1 carrying the hisD knockout vector was co-incubated with the leuA gene knockout engineering strain for 12 hours, and the leucine and histidine double auxotrophic strain EHA105hisD-leuA- was screened and identified.

[0027] The construction method of the hisD gene knockout vector is:

[0028] Primers hisD-up-F and hisD-up-R were used to amplify a 1000 bp fragment upstream of the hisD gene, and primers hisD-down-F and hisD-down-R were used to amplify a 1000 bp fragment downstream of the hisD gene. The upstream and downstream fragments were cloned into the SalI and EcoRI sites of pK18mobSacB using Gibson recombination technology to obtain a hisD knockout vector. The primer sequences are as follows:

[0029] hisD-up-F: 5'-CAGTACCGATTTTCCCGATGTAA-3',

[0030] hisD-up-R: 5'-GCGATGGCTTCAGGCGAT-3';

[0031] hisD-down-F: 5'-GCCATTACCCTTTCATTGTCCC-3',

[0032] hisD-down-R: 5'-GCGTTCGCAGGGCGTCTC-3';

[0033] The gene ID of the hisD gene is WP_004432479.1.

[0034] The preparation method of the double auxotrophic engineered strain comprises the following specific steps:

[0035] 1) Use primers leuA-up-F and leuA-up-R to amplify the 1000 bp fragment upstream of the leuA gene, and use primers leuA-down-F and leuA-down-R to amplify the 1000 bp fragment downstream of the leuA gene; use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB to obtain the leuA knockout vector;

[0036] 2) The leuA knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing kanamycin; the Escherichia coli S17-1 carrying the leuA knockout vector was co-incubated with Agrobacterium tumefaciens, and the positive colonies that were successfully transformed and integrated into the chromosome of Agrobacterium tumefaciens were screened using LB medium containing rifampicin and kanamycin;

[0037] The positive colonies were plated on LB medium containing sucrose, and the leuA gene between the vector and the upstream and downstream sequences was removed by secondary recombination; the positive colonies were amplified using primers leuA-up-F and leuA-down-R, and the auxotrophic engineered strain EHA105leuA- with successful knockout of the leuA gene was obtained by screening;

[0038] 3) Use primers hisD-up-F and hisD-up-R to amplify the 1000 bp fragment upstream of the hisD gene, and use primers hisD-down-F and hisD-down-R to amplify the 1000 bp fragment downstream of the hisD gene; use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB to obtain the hisD knockout vector;

[0039] 4) The hisD knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing kanamycin; the Escherichia coli S17-1 carrying the hisD knockout vector was co-incubated with the EHA105leuA- strain, and screened using LB medium containing rifampicin and kanamycin to obtain positive colonies that were successfully transformed and integrated into the chromosome;

[0040] The positive colonies were plated on LB medium containing sucrose, and the hisD gene between the vector and the upstream and downstream sequences was removed by secondary recombination; the positive colonies were amplified using primers hisD-up-F and hisD-down-R to obtain the double auxotrophic engineered strain EHA105hisD-leuA- that successfully knocked out the leuA gene and the hisD gene;

[0041] The primer sequences are as follows:

[0042] leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3',

[0043] leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3';

[0044] leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3',

[0045] leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3';

[0046] hisD-up-F: 5'-CAGTACCGATTTTCCCGATGTAA-3',

[0047] hisD-up-R: 5'-GCGATGGCTTCAGGCGAT-3';

[0048] hisD-down-F: 5'-GCCATTACCCTTTCATTGTCCC-3',

[0049] hisD-down-R: 5'-GCGTTCGCAGGGCGTCTC-3';

[0050] The gene ID of the leuA gene is WP_010972185.1;

[0051] The gene ID of the hisD gene is WP_004432479.1.

[0052] The method for preparing a double auxotrophic engineered strain obtains a double auxotrophic engineered strain EHA105hisD-leuA-.

[0053] Application of the double auxotrophic engineered strain EHA105hisD-leuA- in plant genetic transformation.

[0054] A leucine auxotrophic strain is prepared by the following method:

[0055] 1) Use primers leuA-up-F and leuA-up-R to amplify the 1000 bp fragment upstream of the leuA gene, and use primers leuA-down-F and leuA-down-R to amplify the 1000 bp fragment downstream of the leuA gene; use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB to obtain the leuA knockout vector;

[0056] 2) The leuA knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing kanamycin; the Escherichia coli S17-1 carrying the leuA knockout vector was co-incubated with Agrobacterium tumefaciens, and the positive colonies that were successfully transformed and integrated into the chromosome of Agrobacterium tumefaciens were screened using LB medium containing rifampicin and kanamycin;

[0057] The positive colonies were plated on LB medium containing sucrose, and the leuA gene between the vector and the upstream and downstream sequences was removed by secondary recombination; the positive colonies were amplified using primers leuA-up-F and leuA-down-R, and the auxotrophic engineered strain EHA105leuA- with successful knockout of the leuA gene was obtained by screening;

[0058] The primer sequences are as follows:

[0059] leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3',

[0060] leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3';

[0061] leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3',

[0062] leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3';

[0063] The gene ID of the leuA gene is WP_010972185.1.

[0064] Application of leucine auxotrophic strains in plant genetic transformation.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] 1) The present application knocks out the leuA gene in the host cell to construct an auxotrophic engineered strain EHA105leuA-; the hisD gene is further knocked out in the EHA105leuA- strain to construct a double auxotrophic engineered strain EHA105hisD-leuA-; the results of the examples of the present application show that EHA105hisD-leuA- cannot grow in an AB liquid culture medium without any amino acids, nor can it grow in an AB liquid culture medium with 100 mg / L of leucine or histidine added alone, but grows normally in an AB liquid culture medium with 100 mg / L of leucine and histidine added at the same time. When 700 mg / L leucine and 200 mg / L histidine were added to the co-cultivation medium, the EHA105hisD-leuA- strain had the same genetic transformation ability as the wild-type EHA105 strain; without the addition of antibacterial antibiotics, no excessive growth of Agrobacterium was observed on the leaf discs after culturing on the screening medium for 10 days, and resistant calli expressing exogenous genes appeared after culturing on the screening medium for 40 days.

[0067] 2) In the examples of the present application, suspensions of wild-type EHA105, EHA105leuA- and EHA105hisD-leuA- were vacuum infiltrated into leaves of four plants (Nicotiana benthamiana, Populus tomentosa, Chinese rose, and Crabapple) and incubated for 7 days. The results of the surviving cell evaluation showed that compared with the wild-type Agrobacterium EHA105, the surviving cells of EHA105leuA- and EHA105hisD-leuA- decreased to 0.1%-0.3%, a decrease of 1000 to 333 times. In addition, the suspensions of wild-type EHA105, EHA105leuA- and EHA105hisD-leuA- were stirred and mixed with sterilized nutrient soil and incubated for 7 days; the results of the survival cell evaluation showed that compared with the wild-type Agrobacterium EHA105, the survival cells of EHA105leuA- and EHA105hisD-leuA- decreased to 0.61% and 0.23%, respectively, a decrease of 164 to 435 times.

[0068] 3) It can be seen that the adaptability of the two Agrobacterium tumefaciens auxotrophic types EHA105leuA- and EHA105hisD-leuA- constructed in this application in the natural environment is significantly reduced, providing better biological protection for plant genetic transformation experiments. In particular, the auxotrophic type EHA105hisD-leuA- involves mutations in two genes, and the possibility of restoring mutations is extremely low, which can provide the best protection for plant genetic transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Flow chart for knocking out target genes in Agrobacterium tumefaciens (red arrows indicate detection primers);

[0070] Figure 2 PCR detection diagram of leuA (A, M is DL5000 Marker of Novazonics) and ilvE (B, M is MarkerIII of Tiangen; lane 1 is wild-type EHA105 colony; lanes 2-5 are sucrose-resistant colonies) gene knockout engineering strains;

[0071] Figure 3 The nutritional deficiency phenotype diagram of the leuA and ilvE gene knockout engineering strains in AB medium (+ means 100 mg / L leucine was added to the medium; - means no amino acid was added);

[0072] Figure 4 This is the PCR detection diagram of the double auxotrophic gene knockout engineering strain EHA105hisD-leuA- (M is the DL5000 Marker of Novazonics; lane 1 is the wild-type EHA105 colony; lanes 2-9 are sucrose-resistant colonies);

[0073] Figure 5This is the nutritional deficiency phenotype diagram of the double nutritional deficiency gene knockout engineering strain EHA105hisD-leuA- in AB medium (+ means 100 mg / L leucine is added to the medium; - means no amino acid is added);

[0074] Figure 6 This is a transient expression diagram of the double auxotrophic gene knockout engineering strain EHA105hisD-leuA- in Nicotiana benthamiana;

[0075] Figure 7 This is a growth diagram of the co-cultivation of the double auxotrophic gene knockout engineered strain EHA105hisD-leuA-transformed into Nicotiana benthamiana;

[0076] Figure 8 This is the growth diagram of the double auxotrophic gene knockout engineering strain EHA105hisD-leuA- in MS medium;

[0077] Fig. 9 The growth diagram of Nicotiana benthamiana leaf discs transformed with the double auxotrophic gene knockout engineering strain EHA105hisD-leuA- (+ means 200 mg / L histidine and 700 mg / L leucine were added during co-cultivation; - means no amino acids were added during co-cultivation);

[0078] Fig.10 The survival of the double auxotrophic gene knockout strain EHA105hisD-leuA- in four plants and one soil (A is Nicotiana benthamiana; B is Populus tomentosa; C is Rose; D is Malus; E is nutrient soil; Y axis, the number of surviving cells is log 10 (logarithmic scale)). DETAILED DESCRIPTION

[0079] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0080] The method flow of this application for knocking out the target gene in Agrobacterium tumefaciens is as follows Figure 1 shown.

[0081] Example 1

[0082] 1. Construction of leuA and ilvE gene knockout vectors

[0083] The leuA gene (gene ID WP_010972185.1) and ilvE gene (gene ID WP_003509326.1) of the leucine biosynthesis pathway were selected, relevant primers were designed, and knockout vectors were constructed as follows:

[0084] 1) Use primers leuA-up-F and leuA-up-R to amplify the 1000bp fragment upstream of the leuA gene, and use primers leuA-down-F and leuA-down-R to amplify the 1000bp fragment downstream of the leuA gene. Use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB (suicide vector containing the SacB gene) to obtain the leuA knockout vector. The primer sequences are as follows:

[0085] leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3',

[0086] leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3';

[0087] leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3',

[0088] leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3'.

[0089] 2) Use primers ilvE-up-F and ilvE-up-R to amplify the 995 bp fragment upstream of the ilvE gene, and use primers ilvE-down-F and ilvE-down-R to amplify the 1000 bp fragment downstream of the ilvE gene. Use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB to generate the ilvE knockout vector. The primer sequences are as follows:

[0090] ilvE-up-F: 5'-CTTCGCGGTTTTCCTCGC-3',

[0091] ilvE-up-R: 5'-AACGGTCTCCTGTGTCAAACATT-3';

[0092] ilvE-down-F: 5'-AATCCGCATACATTGCGTTAAA-3',

[0093] ilvE-down-R: 5'-TGTTGAGGTCTTCGATGAGCTTG-3'.

[0094] 2. Construction of leuA and ilvE gene knockout strains

[0095] The knockout vectors were transformed into E. coli S17-1 strains, and positive colonies were screened using LB medium containing 50 mg / L kanamycin. 100 μL of E. coli S17-1 carrying the knockout vector (OD 600 =1) and 100 μL Agrobacterium tumefaciens EHA105 (OD 600 =1) and incubated on an LB plate without antibiotics for 12 hours to achieve diphilic conjugation of the two bacteria. The bacteria on the plate were then collected and resuspended in 1 mL of liquid LB medium, from which 50 μL was coated on an LB plate (rifampicin 50 mg / L and kanamycin 50 mg / L) to screen for positive colonies that were successfully transformed and integrated into the chromosome of Agrobacterium tumefaciens.

[0096] The above positive colonies were plated on LB medium containing 10% sucrose, and the leuA or ilvE gene between the vector and the upstream and downstream sequences was removed by secondary recombination. The positive colonies were amplified using primers leuA-up-F and leuA-down-R. The strain with successful knockout of the leuA gene (EHA105leuA-) should produce a 2000bp product, while the wild type produced a 3710bp product. The positive colonies were amplified using primers ilvE-up-F and ilvE-down-R. The strain with successful knockout of the ilvE gene (EHA105ilvE-) should produce a 2000bp product, while the wild type produced a 2872bp product ( Figure 2 ).

[0097] 3. Verification of leucine auxotrophic strains

[0098] The wild-type EHA105 strain, as well as single colonies of the EHA105leuA- and EHA105ilvE- strains were picked and added to 5 mL of liquid AB medium and cultured at 28°C with shaking at 200 rpm for two days.

[0099] The results are as follows Figure 3 As shown, the EHA105leuA- strain with the leuA gene completely knocked out could not grow in AB liquid medium, but after the medium was supplemented with 100 mg / L leucine, EHA105leuA- resumed growth. The wild-type EHA105 strain and the EHA105ilvE- strain with the ilvE gene completely knocked out could grow normally in AB liquid medium. The results show that the leuA gene is the preferred gene for knocking out the leucine synthesis pathway.

[0100] Example 2

[0101] 1. Construction of hisD gene knockout vector

[0102] The hisD gene (gene ID WP_004432479.1) in the leucine biosynthesis pathway was selected, relevant primers were designed, and a knockout vector was constructed as follows:

[0103] Primers hisD-up-F and hisD-up-R were used to amplify the 1000bp fragment upstream of the leuA gene, and primers hisD-down-F and hisD-down-R were used to amplify the 1000bp fragment downstream of the leuA gene. The upstream and downstream fragments were cloned into the SalI and EcoRI sites of pK18mobSacB using Gibson recombination technology to obtain the hisD knockout vector. The primer sequences are as follows:

[0104] hisD-up-F: 5'-CAGTACCGATTTTCCCGATGTAA-3',

[0105] hisD-up-R: 5'-GCGATGGCTTCAGGCGAT-3';

[0106] hisD-down-F: 5'-GCCATTACCCTTTCATTGTCCC-3',

[0107] hisD-down-R: 5'-GCGTTCGCAGGGCGTCTC-3'.

[0108] 2. Further knockout of hisD gene in the leuA knockout engineered strain (EHA105leuA-)

[0109] The above hisD knockout vector was transformed into Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing 50 mg / L kanamycin.

[0110] 100 μL of E. coli S17-1 (OD 600 =1) and 100 μL of leuA knockout strain (OD 600 =1) and incubated on an LB plate without antibiotics for 12 hours to achieve diphilic conjugation of the two bacteria. The bacteria on the plate were then collected and resuspended in 1 mL of liquid LB medium, from which 50 μL was coated on an LB plate (rifampicin 50 mg / L and kanamycin 50 mg / L) to screen for positive colonies that were successfully transformed and integrated into the EHA105leuA- chromosome.

[0111] The positive colonies were plated on LB medium containing 10% sucrose, and the hisD gene between the vector and the upstream and downstream sequences was removed by secondary recombination. The positive colonies were amplified using primers hisD-up-F and hisD-down-R. The strain with successful knockout of the hisD gene (EHA105hisD-leuA-) should produce a 2000bp product, while the wild-type EHA105 produced a 3299bp product ( Figure 4 ).

[0112] 3. Verification of strains with double auxotrophy of leucine and histidine

[0113] A single colony of the above-mentioned EHA105hisD-leuA- strain was cultured in AB liquid culture medium, AB liquid culture medium supplemented with 100 mg / L leucine alone, AB liquid culture medium supplemented with 100 mg / L histidine alone, and AB liquid culture medium supplemented with both 100 mg / L leucine and 100 mg / L histidine at 28°C and 200 rpm for two days.

[0114] The results are as follows Figure 5 As shown, the wild-type EHA105 strain can grow normally in AB liquid medium, while the EHA105hisD-leuA- strain cannot grow in AB liquid medium; the growth of the EHA105hisD-leuA- strain still cannot be restored after supplementing with 100 mg / L leucine or 100 mg / L histidine alone; however, in AB liquid medium supplemented with 100 mg / L leucine and 100 mg / L histidine at the same time, EHA105hisD-leuA- resumed growth. The results showed that EHA105hisD-leuA- had a double auxotrophic phenotype.

[0115] Example 3

[0116] 1. Transform the binary vector into EHA105hisD-leuA-

[0117] The binary vector PH35GG carrying enhanced green fluorescent protein (eGFP) was transformed into EHA105hisD-leuA- and wild-type EHA105 strains by electroporation, and positive colonies were screened on LB medium containing 50 mg / L rifampicin and 100 mg / L spectinomycin.

[0118] 2. Instantaneous transformation of tobacco

[0119] After the above positive strains were expanded and cultured, they were resuspended in infiltration buffer (1 / 4MS liquid culture medium, with different concentrations of leucine and histidine, and 100 μM acetosyringone), shaken for 3 hours, and the concentration was adjusted to OD 600 =0.5, injected into tobacco leaves, and observed fluorescence after 48 hours.

[0120] The results are as follows Figure 6 As shown, the fluorescence intensity is comparable to that when the wild-type EHA105 is used, indicating that when 700 mg / L leucine and 200 mg / L histidine are added to the infiltration buffer, the EHA105hisD-leuA- strain has the same genetic transformation ability as the wild-type EHA105 strain.

[0121] 3. Genetic transformation of Nicotiana benthamiana

[0122] After the EHA105 carrying PH35GG (whose T-DNA has the 35S::eGFP sequence) and EHA105hisD-leuA- were expanded and cultured, they were resuspended in the infiltration buffer and the concentration was adjusted to OD 600 = 0.6, incubated with Nicotiana benthamiana leaf discs for 10 minutes, blotted on sterile filter paper, transferred to co-cultivation medium (MS solid medium, 6-BA 1 mg / L and NAA 0.1 mg / L, supplemented with 700 mg / L leucine and 200 mg / L histidine), 23°C, dark culture, 48 hours. Then the leaf discs were transferred to regeneration medium (MS solid medium, 6-BA 1 mg / L and NAA 0.1 mg / L) and cultured for 8 days.

[0123] The results are as follows Figure 7 As shown, when wild-type EHA105 was used for co-cultivation without adding antibacterial antibiotics, a large number of Agrobacterium colonies appeared around and at the bottom of the leaf disc; while when EHA105hisD-leuA- strain was used for co-cultivation, no Agrobacterium colonies were observed.

[0124] The wild-type EHA105 strain and EHA105hisD-leuA- were directly spread on MS medium plates and incubated at 23°C in the dark for 48 hours.

[0125] The results are as follows Figure 8 As shown, EHA105hisD-leuA- was completely unable to grow in MS medium, but supplementing the medium with 100 mg / L of histidine and leucine restored bacterial growth.

[0126] Therefore, using the nutrient-deficient Agrobacterium EHA105hisD-leuA- for plant genetic transformation can reduce the addition of large amounts of antibacterial antibiotics during the subsequent regeneration process and avoid excessive growth of Agrobacterium.

[0127] 4. Screening of positive transgenic tobacco

[0128] The co-cultivated leaf discs were washed 2-3 times with liquid MS, and the liquid was dried on sterile filter paper. The leaves were transferred to screening medium (MS solid medium, 6-BA 1 mg / L and NAA 0.1 mg / L, kanamycin 50 mg / L) for 40 days. The calli that were successfully transformed and expressed eGFP appeared green under ultraviolet light.

[0129] The results are as follows Fig. 9 As shown, when 700 mg / L leucine and 200 mg / L histidine were added during the co-cultivation stage, the conversion efficiency of EHA105hisD-leuA- was comparable to that of wild-type EHA105, while when no additional amino groups were added during the co-cultivation stage, the conversion efficiency of EHA105hisD-leuA- was much lower than that of wild-type EHA105.

[0130] Example 4

[0131] 1) The suspensions of wild-type EHA105, EHA105leuA- and EHA105hisD-leuA- (OD 600 = 0.1) were vacuum infiltrated into the leaves of four plants (Nicotiana benthamiana, Populus tomentosa, Chinese rose, and Malus chinensis), incubated at 23°C, 12 h light / 12 h dark for 7 days; then cut 1 cm 2 The leaf disks were ground by adding 1 mL of 10 mM MgCl2 solution, and the resulting suspension was diluted 10 to 100 times and then plated on LB plates (rifampicin 100 mg / L), and the surviving cells were evaluated by counting the number of colonies.

[0132] The results are as follows Fig.10 As shown in AD, compared with the wild-type Agrobacterium EHA105, the survival cells of EHA105leuA- and EHA105hisD-leuA- decreased to 0.1%-0.3%, which was decreased by 1000 to 333 times.

[0133] 2) 1 mL of suspension (OD 600 =0.1) was mixed with 50 g sterilized nutrient soil and incubated at 23°C in the dark for 7 days; then 1 g of soil was added with 1 mL of 10 mM MgCl2 solution and mixed. After the soil particles were naturally precipitated, the supernatant was diluted 10 to 100 times and spread on LB plates (rifampicin 100 mg / L), and the surviving cells were evaluated by counting the number of colonies.

[0134] The results are as follows Fig.10As shown in E, compared with the wild-type Agrobacterium EHA105, the survival cells of EHA105leuA- and EHA105hisD-leuA- decreased to 0.61% and 0.23%, respectively, which were decreased by 164 to 435 times.

[0135] During the genetic transformation of plants, Agrobacterium carries various antibiotic or herbicide resistance genes, as well as exogenous genes from various organisms. These genetically modified Agrobacterium escape into the natural environment, which may lead to certain biosafety risks. In summary, the adaptability of the two Agrobacterium nutrient-deficient types EHA105leuA- and EHA105hisD-leuA- constructed in this application in the natural environment is significantly reduced, providing better biological protection for plant genetic transformation experiments. In particular, the nutrient-deficient type EHA105hisD-leuA- involves mutations in two different metabolic pathways, so the possibility of recovery mutations is extremely low, which can provide the best protection for plant genetic transformation.

[0136] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a double auxotrophic engineered strain, characterized in that: (1) Knocking out the leuA gene in the Agrobacterium tumefaciens strain EHA105 to construct an auxotrophic engineered strain EHA105leuA-; (2) The hisD gene was further knocked out in the EHA105leuA- strain to construct the double auxotrophic engineered strain EHA105hisD-leuA-; The gene ID of the leuA gene is WP_010972185.1; the gene ID of the hisD gene is WP_004432479.1; and the double auxotrophic engineered strain is a leucine and histidine double auxotrophic strain.

2. The method for preparing a dual auxotrophic engineered strain according to claim 1, characterized in that: In step (1), the process of constructing the auxotrophic engineered strain EHA105leuA- is specifically as follows: 1) Design primers and construct leuA gene knockout vector; 2) The leuA gene knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were obtained by screening; 3) The positive colonies were co-incubated with Agrobacterium tumefaciens EHA105 for diparental conjugation, and the leucine auxotrophic strain EHA105leuA- was obtained by screening and identification.

3. The method for preparing a dual auxotrophic engineered strain according to claim 2, characterized in that: The construction method of the leuA gene knockout vector is: Primers leuA-up-F and leuA-up-R were used to amplify a 1000 bp fragment upstream of the leuA gene, and primers leuA-down-F and leuA-down-R were used to amplify a 1000 bp fragment downstream of the leuA gene; The upstream and downstream fragments were cloned into the SalI and EcoRI sites of pK18mobSacB using Gibson recombination technology to obtain the leuA knockout vector; the primer sequences are as follows: leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3', leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3'; leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3'; leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3'; The gene ID of the leuA gene is WP_010972185.

1.

4. The method for preparing a dual auxotrophic engineered strain according to claim 1, characterized in that: In step (2), the process of constructing the double auxotrophic engineered strain EHA105hisD-leuA- is specifically as follows: 1) Design primers and construct hisD gene knockout vector; 2) The hisD knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened; 3) Escherichia coli S17-1 carrying the hisD knockout vector was co-incubated with the leuA gene knockout engineering strain for 12 hours, and the leucine and histidine double auxotrophic strain EHA105hisD-leuA- was screened and identified.

5. The method for preparing a dual auxotrophic engineered strain according to claim 4, characterized in that: The construction method of the hisD gene knockout vector is: Primers hisD-up-F and hisD-up-R were used to amplify a 1000 bp fragment upstream of the hisD gene, and primers hisD-down-F and hisD-down-R were used to amplify a 1000 bp fragment downstream of the hisD gene. The upstream and downstream fragments were cloned into the SalI and EcoRI sites of pK18mobSacB using Gibson recombination technology to obtain a hisD knockout vector. The primer sequences are as follows: hisD-up-F: 5'-CAGTACCGATTTTCCCGATGTAA-3', hisD-up-R: 5'-GCGATGGCTTCAGGCGAT-3'; hisD-down-F: 5'-GCCATTACCCTTTCATTGTCCC-3', hisD-down-R: 5'-GCGTTCGCAGGGCGTCTC-3'; The gene ID of the hisD gene is WP_004432479.

1.

6. The method for preparing a double auxotrophic engineered strain according to any one of claims 1 to 5, characterized in that: The specific steps include: 1) Use primers leuA-up-F and leuA-up-R to amplify the 1000 bp fragment upstream of the leuA gene, and use primers leuA-down-F and leuA-down-R to amplify the 1000 bp fragment downstream of the leuA gene; use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB to obtain the leuA knockout vector; 2) The leuA knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing kanamycin; the Escherichia coli S17-1 carrying the leuA knockout vector was co-incubated with Agrobacterium tumefaciens, and the positive colonies that were successfully transformed and integrated into the chromosome of Agrobacterium tumefaciens were screened using LB medium containing rifampicin and kanamycin; The positive colonies were plated on LB medium containing sucrose, and the leuA gene between the vector and the upstream and downstream sequences was removed by secondary recombination; the positive colonies were amplified using primers leuA-up-F and leuA-down-R, and the auxotrophic engineered strain EHA105leuA- with successful knockout of the leuA gene was obtained by screening; 3) Use primers hisD-up-F and hisD-up-R to amplify the 1000 bp fragment upstream of the hisD gene, and use primers hisD-down-F and hisD-down-R to amplify the 1000 bp fragment downstream of the hisD gene; use Gibson recombination technology to clone the upstream and downstream fragments into the SalI and EcoRI sites of pK18mobSacB to obtain the hisD knockout vector; 4) The hisD knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing kanamycin; the Escherichia coli S17-1 carrying the hisD knockout vector was co-incubated with the EHA105leuA- strain, and screened using LB medium containing rifampicin and kanamycin to obtain positive colonies that were successfully transformed and integrated into the chromosome; The positive colonies were plated on LB medium containing sucrose, and the hisD gene between the vector and the upstream and downstream sequences was removed by secondary recombination; the positive colonies were amplified using primers hisD-up-F and hisD-down-R to obtain the double auxotrophic engineered strain EHA105hisD-leuA- that successfully knocked out the leuA gene and the hisD gene; The primer sequences are as follows: leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3', leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3'; leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3', leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3'; hisD-up-F: 5'-CAGTACCGATTTTCCCGATGTAA-3', hisD-up-R: 5'-GCGATGGCTTCAGGCGAT-3'; hisD-down-F: 5'-GCCATTACCCTTTCATTGTCCC-3', hisD-down-R: 5'-GCGTTCGCAGGGCGTCTC-3'; The gene ID of the leuA gene is WP_010972185.1; The gene ID of the hisD gene is WP_004432479.

1.

7. The double auxotrophic engineered strain EHA105hisD-leuA- obtained according to the preparation method of the double auxotrophic engineered strain according to any one of claims 1 to 6.

8. Use of the double auxotrophic engineered strain EHA105hisD-leuA- according to claim 7 in plant genetic transformation.

9. A leucine auxotrophic strain, characterized in that Prepared by the following method: 1) Primers leuA-up-F and leuA-up-R were used to amplify a 1000 bp fragment upstream of the leuA gene, and primers leuA-down-F and leuA-down-R were used to amplify a 1000 bp fragment downstream of the leuA gene; The upstream and downstream fragments were cloned into the SalI and EcoRI sites of pK18mobSacB using Gibson recombination technology to obtain the leuA knockout vector; 2) The leuA knockout vector was transformed into the Escherichia coli S17-1 strain, and positive colonies were screened using LB medium containing kanamycin; the Escherichia coli S17-1 carrying the leuA knockout vector was co-incubated with Agrobacterium tumefaciens, and the positive colonies that were successfully transformed and integrated into the chromosome of Agrobacterium tumefaciens were screened using LB medium containing rifampicin and kanamycin; The positive colonies were plated on LB medium containing sucrose, and the leuA gene between the vector and the upstream and downstream sequences was removed by secondary recombination; the positive colonies were amplified using primers leuA-up-F and leuA-down-R, and the auxotrophic engineered strain EHA105leuA- with successful knockout of the leuA gene was obtained by screening; The primer sequences are as follows: leuA-up-F: 5'-TCGAGACCAAATCACCGGAT-3', leuA-up-R: 5'-GAAGCCTCAAGGGTGGGGT-3'; leuA-down-F: 5'-GTCGCGTCCTCCTTGCGC-3', leuA-down-R: 5'-CTTCGGTGTCGATATTCCCG-3'; The gene ID of the leuA gene is WP_010972185.

1.

10. Use of the leucine auxotrophic strain according to claim 9 in plant genetic transformation.

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