Method for improving biocontrol photosynthetic bacteria through distant hybridization and genome rearrangement technology

Abstract

The invention discloses a method for improving biocontrol photosynthetic bacteria through a far-source hybridization and genome rearrangement technology. The method comprises the following steps: inoculating a rhodopseudomonas palustris GJ-22 seed into a photosynthetic bacteria liquid culture medium for culturing; inoculating the bacillus subtilis KY-2 into an LB liquid culture medium for culturing; the cultured GJ-22 is taken and subjected to treatment and centrifugation, and then a GJ-22 protoplast is obtained; kY-2 is taken to be treated, and KY-2 protoplast is obtained after centrifugation; fusing the obtained GJ-22 protoplast and KY-2 protoplast, diluting a photosynthetic bacterium hypertonic liquid culture medium, and regenerating on a photosynthetic bacterium soft agar culture medium to obtain a fused strain; a fusion strain is screened out through an induced tobacco core leaf anti-TMV test to serve as a starting strain for genome rearrangement. Preparing protoplast from the original strain; dividing the prepared protoplast into two groups, and respectively carrying out ultraviolet inactivation and thermal inactivation; respectively fusing the inactivated protoplasts, and regenerating to obtain fusant strains; finally, a target strain with genetic stability is screened through a fusant strain induced tobacco core leaf anti-TMV test.

Description

Technical Field

[0001] The invention relates to the technical field of bio-control photosynthetic bacteria variety improvement, and in particular to a method for improving bio-control photosynthetic bacteria through distant hybridization and genome rearrangement technology. Background Art

[0002] Distant hybridization and genome rearrangement technology is a new breeding technology used to overcome the incompatibility of distant hybridization and to quickly and efficiently improve microorganisms without knowing the genetic background of the parents, while reducing the separation of parents in hybrid offspring. Protoplast fusion can enable microorganisms to quickly obtain diverse and complex phenotypes, and genome rearrangement technology can further quickly improve and stabilize the excellent traits of the fusion strain, that is, the hybrid offspring. The method of the present invention is to overcome the incompatibility of distant hybridization through protoplast fusion technology to obtain a fusion strain of Rhodopseudomonas palustris GJ-22 and Bacillus subtilis KY-2; then use the gene rearrangement technology to use the fusion strain as the starting strain, inactivate it, fuse and regenerate it, repeat several rounds of fusion and regeneration to obtain a fusion bacterial group, and screen it to obtain a fusion strain with stable inheritance of parental traits. Different from traditional microbial mutagenesis breeding, the technology of the present invention has higher efficiency, can aggregate the phenotypes of positive mutations, reduce parental separation, and multiple rounds of fusion greatly increase the probability of gene exchange. Summary of the invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a method for improving biocontrol photosynthetic bacteria through distant hybridization and genome rearrangement technology, so as to make up for the shortcomings of distant hybridization incompatibility, long improvement cycle and poor genetic stability in the existing biocontrol microbial strain improvement.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A palustris Rhodopseudomonas, the palustris Rhodopseudomonas is named (Rhodopseudomonassp) GJ-22, the strain GJ-22 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit number is CGMCC NO: 17356, and the deposit date is March 19, 2019.

[0006] A Bacillus subtilis, wherein the Bacillus subtilis is classified and named as (Bacillus subtilis) KY-2, and the strain KY-2 is preserved in the General Microbiological Center of China Microorganism Culture Collection Administration, the preservation number is CGMCC NO: 18448, and the preservation date is August 30, 2019.

[0007] A method for preparing a distant hybrid strain, wherein the distant hybrid strain is obtained by protoplast fusion of Rhodopseudomonas palustris GJ-22 and Bacillus subtilis KY-2;

[0008] The method comprises:

[0009] S11), inoculating Rhodopseudomonas palustris GJ-22 seeds into photosynthetic bacteria liquid culture medium for culture; inoculating Bacillus subtilis KY-2 into LB liquid culture medium for culture;

[0010] S12), treating the cultured GJ-22 with cefotaxime sodium and lysozyme, and centrifuging to obtain GJ-22 protoplasts; treating the cultured KY-2 with lysozyme, and centrifuging to obtain KY-2 protoplasts;

[0011] S13), 40% (V / V) PEG6000 was used to induce the fusion of GJ-22 protoplasts and KY-2 protoplasts, and the fusion strains were obtained after gradient dilution with photosynthetic bacteria hypertonic liquid medium and regenerated on photosynthetic bacteria soft agar medium;

[0012] S14) The fusion strain selected from the experiment of inducing heartleaf tobacco to resist TMV was used as the starting strain.

[0013] A method for improving bio-defense photosynthetic bacteria by distant hybridization and genome rearrangement technology, comprising:

[0014] S21), the starting strain is treated with cefotaxime sodium and lysozyme and then incubated and centrifuged to obtain the protoplasts of the fusion strain;

[0015] S22), dividing the obtained fusion strain protoplasts into two groups and performing UV inactivation and heat inactivation respectively;

[0016] S23), taking equal volumes of the two groups of inactivated protoplasts, fusing, centrifuging, washing, and regenerating to obtain fused sub-bacteria;

[0017] S24), select strains with better growth in the fusion bacterial community to induce TMV resistance test of heartleaf tobacco. Further, the seed inoculation amount of Rhodopseudomonas palustris GJ-22 in S11) is 2% inoculated in photosynthetic bacterial liquid culture medium, and cultured at 30°C and anaerobic light for 72 hours to the logarithmic growth phase; the Bacillus subtilis KY-2 is inoculated in LB liquid culture medium at an inoculation amount of 1%, and cultured at 37°C and 180r / min for 16 hours to the logarithmic growth phase.

[0018] Furthermore, in the S12), GJ-22 was cultured with sodium fotaxime at a final concentration of 60 μg / mL at 37° C. for 2 h with shaking, and then treated with lysozyme at a final concentration of 1.2 mg / mL; KY-2 was treated with lysozyme at a final concentration of 1.0 mg / mL and then cultured with shaking at 37° C. for 1 h.

[0019] Furthermore, the S13) comprises taking equal volumes of successfully prepared GJ-22 and KY-2 protoplasts, digesting DNA released by bacterial lysis with DNaseI, incubating at 25°C with 40% (V / V) PEG6000 to induce protoplast fusion; gradiently diluting the fused bacteria with a photosynthetic bacteria hypertonic liquid culture medium and regenerating them on a photosynthetic bacteria soft agar culture medium, and obtaining fusion strains RH-0110, RH-0112, RH-0203, RH-0303, and RH-0308 through an induced heartleaf tobacco anti-TMV test.

[0020] Further, the S21) comprises adjusting the concentration of the obtained fusion strain to 1×10 6 cell / mL, treated with 60 μg / mL ceftriaxone sodium and 1.2 mg / mL lysozyme, and obtained the protoplasts of the fusion strain after centrifugation.

[0021] Furthermore, the S22) includes dividing the fusion strain protoplasts into two groups for inactivation, one group is inactivated by ultraviolet light and the other group is inactivated by heat.

[0022] Further, the S23) includes taking two groups of inactivated protoplasts of equal volume and randomly fusing them with 40% (V / V) PEG6000, washing the cells with SMM buffer, and regenerating and culturing them on a photosynthetic bacteria soft agar medium at 30°C for 7-14 days to obtain regenerated colonies, which are F1 generation fusion sub-bacteria groups; using the F1 generation fusion sub-bacteria groups as the starting strains, inactivating them, fusing them again, and regenerating them, and obtaining the regenerated strains as the second round of fusion bacteria groups, i.e., F2 generation; and repeatedly performing cyclic protoplast fusion and regeneration. Different fusion sub-generations are marked as F1, F2, and F3.

[0023] Compared with the prior art, one or more embodiments of the present invention may have the following advantages:

[0024] The fusion strain screened by the present invention can effectively improve the level of heartleaf tobacco's resistance to TMV infection, and due to the occurrence of gene rearrangement, the separation of parental traits of the fusion strain is greatly reduced. Multiple rounds of fusion greatly increase the probability of gene exchange and enhance the stability of the fusion strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a flow chart of a method for preparing a distant hybrid strain;

[0026] Figure 2 This is a flow chart of the method for improving biodefense photosynthetic bacteria through genome rearrangement technology. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the embodiments and drawings. The method of the present invention is to overcome the incompatibility of distant hybridization by protoplast fusion technology to obtain a fusion strain of Rhodopseudomonas palustris GJ-22 and Bacillus subtilis KY-2; then use the gene rearrangement technology to use the fusion strain as the starting strain, inactivate it, fuse it, regenerate it, repeat several rounds of fusion and regeneration to obtain a fusion bacterial group, and screen it to obtain a fusion substrain with stable inheritance of parental traits. Specific examples are as follows:

[0028] Example 1

[0029] like Figure 1 As shown, it is a method for preparing protoplasts and distant hybrid strains, wherein the protoplasts are obtained by culturing Rhodopseudomonas sp. GJ-22 and Bacillus subtilis KY-2; both strains GJ-22 and KY-2 are deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, wherein the deposit number of strain GJ-22 is CGMCC NO: 17356; the deposit number of strain KY-2 is CGMCC NO: 18448; the method comprises the following steps;

[0030] S11), inoculating Rhodopseudomonas palustris GJ-22 seeds into photosynthetic bacteria liquid culture medium for culture; inoculating Bacillus subtilis KY-2 into LB liquid culture medium for culture;

[0031] The seed inoculation amount of Rhodopseudomonas palustris GJ-22 was 2% and inoculated into photosynthetic bacterial liquid culture medium, and cultured at 30°C and anaerobic light for 72 hours until the logarithmic growth phase. The seed inoculation amount of Bacillus subtilis KY-2 was 1% and inoculated into LB liquid culture medium, and cultured at 37°C and 180r / min for 16 hours until the logarithmic growth phase.

[0032] S12), treating the cultured GJ-22 with cefotaxime sodium and lysozyme, and centrifuging to obtain GJ-22 protoplasts; treating the cultured KY-2 with lysozyme, and centrifuging to obtain KY-2 protoplasts;

[0033] A certain amount of GJ-22 was treated with ceftriaxone sodium at a final concentration of 60 μg / mL and lysozyme at a final concentration of 1.2 mg / mL, incubated at room temperature and then centrifuged to obtain GJ-22 protoplasts; similarly, a certain amount of KY-2 was treated with lysozyme at a final concentration of 1.0 mg / mL, then shaken cultured at 37°C and centrifuged to obtain KY-2 protoplasts.

[0034] S13), 40% (V / V) PEG6000 was used to induce the fusion of GJ-22 protoplasts and KY-2 protoplasts, and the fusion strains were obtained after gradient dilution with photosynthetic bacteria hypertonic liquid medium and regenerated on photosynthetic bacteria soft agar medium;

[0035] Take 0.5mL of successfully prepared GJ-22 and KY-2 protoplasts, and 1U of DNaseI to digest the DNA released by bacterial lysis. Incubate with 0.5mL 40% (V / V) PEG6000 at 25℃ to induce protoplast fusion, and wash with SMM buffer. Resuspend the cells in photosynthetic bacteria hypertonic liquid culture medium, and regenerate on photosynthetic bacteria soft agar medium after gradient dilution.

[0036] S14) The fusion strain was selected as the starting strain through the test of inducing TMV resistance of Nicotiana tabacum.

[0037] The fusion strains with better growth were selected for inducing TMV resistance test in heartleaf tobacco.

[0038] For heart-leaf tobacco, 6-8 weeks after transplanting, select heart-leaf tobacco plants with uniform growth, irrigate the roots of each plant with 5mL of the above-obtained fusion strain for three consecutive days, use the bacterial solution of Rhodopseudomonas palustris GJ-22 as a positive control, and sterile water as a negative control. Three days later, the third and fourth leaves were inoculated with TMV virus particles. Sprinkle emery on the leaves, and inoculate each leaf with 25μL TMV virus particles (concentration of 0.74mg / ml). After TMV inoculation, wash the bottle and rinse. Cultivate under light at 25℃, with 10 plants as one treatment, and repeat three times. Count the number of dead spots after 72h.

[0039] Conclusion: The experimental data showed that 3 days after the plants were inoculated with the bacterial solution of the fusion strains RH-0110, RH-0112, RH-0203, RH-0303, and RH-0308, the number of necrotic spots on the leaves of the heartleaf tobacco was significantly lower than that in the negative control group and the positive control group (Table 1 Statistics of the average number of necrotic spots in the fusion strains of Rhodopseudomonas palustris GJ-22 and Bacillus subtilis KY-2 and the induced heartleaf tobacco anti-TMV test), that is, the forward mutant strains RH-0110, RH-0112, RH-0203, RH-0303, and RH-0308 with significantly enhanced disease resistance were screened out from the fusion strains obtained by distant hybridization of Rhodopseudomonas palustris GJ-22 and Bacillus subtilis KY-2.

[0040] Table 1 Statistics of average nematode number in the biocontrol experiment of distant hybrid strains of Rhodopseudomonas palustris GJ-22 and Bacillus subtilis KY-2 and induced heartleaf tobacco against TMV

[0041]

[0042]

[0043] Note: Different lowercase letters in the same column indicate significant differences among treatments (P<0.05).

[0044] Example 2

[0045] like Figure 2 As shown, this embodiment provides a method for improving bio-defense photosynthetic bacteria by genome rearrangement technology, comprising the following steps:

[0046] S21), taking out the spore-forming strain, treating it with ceftriaxone sodium and lysozyme, and centrifuging it after incubation to obtain the protoplasts of the fusion strain;

[0047] The selected fusion strains RH-0110, RH-0112, RH-0203, RH-0303, and RH-0308 were respectively selected as starting strains and the concentration was adjusted to 1×10 6 cell / mL, treated with 60 μg / mL ceftriaxone sodium and 1.2 mg / mL lysozyme, incubated at room temperature and centrifuged to obtain protoplasts of the fusion strain.

[0048] S22), dividing the obtained fusion strain protoplasts into two groups for inactivation;

[0049] The fusion strains of RH-0110, RH-0112, RH-0203, RH-0303, and RH-0308 were randomly divided into two groups, A and B.

[0050] Group A was inactivated by ultraviolet light: the protoplasts of the fusion strain were transferred into a 6cm sterile plate and irradiated under a 30W ultraviolet lamp for 115s, and the lethality of the protoplasts reached 100%. Group B was heat inactivated: the protoplasts were treated in a 60℃ constant temperature water bath for 5min, and the lethality of the protoplasts reached 100%.

[0051] S23), taking equal volumes of two groups of inactivated protoplasts, fusing, centrifuging, washing, and regenerating to obtain fused sub-bacteria;

[0052] Take the two groups of inactivated protoplasts of equal volume and mix them evenly, incubate at 25℃ for 30min, centrifuge at 4000r / min and 4℃ for 20min, and resuspend in 40% (V / V) PEG 6000 for random fusion. After centrifugation at 4000r / min and 4℃, wash the bacteria with SMM solution. After resuspending in photosynthetic bacteria hypertonic liquid culture medium, regenerate and culture on photosynthetic bacteria soft agar medium at 30℃ for 7-14d, and collect all the regenerated colonies to be the F1 generation fusion sub-colony; then use the F1 generation fusion sub-colony as the starting strain, inactivate it, fuse it, and regenerate it. The regenerated strain is the second round fusion sub-colony, i.e., the F2 generation. Repeat several rounds of protoplast fusion and regeneration. Different fusion progeny are marked as F1, F2, and F3;

[0053] S24) Test on TMV resistance of Nicotiana tabacum induced by fusion strain.

[0054] The strains with better growth in the fusion bacterial colony were selected for the test of inducing TMV resistance in heartleaf tobacco.

[0055] For heart-leaf tobacco, 6-8 weeks after transplanting, select heart-leaf tobacco plants with uniform growth, and irrigate the roots of each plant with 5mL of ultrasonically treated PSB-06 bacterial solution of Rhodopseudomonas palustris for three consecutive days. The bacterial solution of RH-0203, which has the best effect of inducing heart-leaf tobacco resistance to TMV among the fusion strains, is used as a positive control, and sterile water is used as a negative control. Three days later, the third and fourth leaves are inoculated with TMV virus particles. Sprinkle emery on the leaves, and inoculate each leaf with 25μL TMV virus particles (concentration of 0.74mg / m1). After TMV inoculation, wash the bottle and rinse. Cultivate under light at 25℃, with 10 plants as one treatment, and repeat three times. Count the number of dead spots after 72h.

[0056] Conclusion: The experimental data showed that a total of three rounds of fusion were carried out in this experiment, that is, the target fusion strains were not screened in F1 and F2, and two target strains were screened from the fusion colonies selected from F3. In the test of inducing heart-leaf tobacco to resist TMV, two fusion strains, RHZ-F3-07 and RHZ-F3-10, were screened, whose disease resistance was significantly better than (or equivalent to) the positive control (Table 2 Statistics of the average number of dead spots in the test of inducing heart-leaf tobacco to resist TMV by further improving the fusion strains using genome rearrangement technology). Three days after the plants in the treatment groups of fusion RHZ-F3-07 and RHZ-F3-10 were infected with the virus, the number of dead spots on the leaf surface of heart-leaf tobacco was significantly lower than that of the negative control group, that is, the fusion strains screened by this method can effectively improve the level of heart-leaf tobacco's resistance to TMV infection, and due to the occurrence of gene rearrangement, the separation of the parental traits of the fusion strains will be greatly reduced, and the stability of the fusion strains will be enhanced.

[0057] Table 2 Statistics of average necrotic spots in the TMV resistance test of heartleaf tobacco induced by further improved fusion strains using genome rearrangement technology

[0058]

[0059]

[0060] Note: Different lowercase letters in the same column indicate significant differences among treatments (P<0.05).

[0061] Although the embodiments disclosed in the present invention are as above, the above contents are only embodiments adopted for facilitating the understanding of the present invention and are not intended to limit the present invention. Any technician in the technical field to which the present invention belongs can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed in the present invention, but the patent protection scope of the present invention shall still be subject to the scope defined in the attached claims.

Claims

1. A Rhodopseudomonas palustris, characterized in that The Rhodopseudomonas palustris was named as (Rhodopseudomonassp) GJ-22, and the strain GJ-22 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC NO:17356.

2. A Bacillus subtilis, characterized in that The Bacillus subtilis is classified and named as (Bacillus subtilis) KY-2, and the strain KY-2 is preserved in the General Microbiological Center of China Microorganism Culture Collection Administration, and the preservation number is CGMCC NO: 18448.

3. A method for preparing a distant hybrid strain, characterized in that: The distant hybrid strain is obtained by protoplast fusion of the Rhodopseudomonas palustris GJ-22 described in claim 1 and the Bacillus subtilis KY-2 described in claim 2; The preparation method comprises: S11), inoculating Rhodopseudomonas palustris GJ-22 seeds into photosynthetic bacteria liquid culture medium for culture; inoculating Bacillus subtilis KY-2 into LB liquid culture medium for culture; S12), treating the cultured GJ-22 with cefotaxime sodium and lysozyme, and centrifuging to obtain GJ-22 protoplasts; treating the cultured KY-2 with lysozyme, and centrifuging to obtain KY-2 protoplasts; S13), inducing the fusion of GJ-22 protoplasts and KY-2 protoplasts, diluting them with photosynthetic bacteria hypertonic liquid culture medium, and regenerating them on photosynthetic bacteria soft agar culture medium to obtain distant hybrid strains, i.e., fusion strains; S14) The fusion strain was selected as the starting strain for genome rearrangement through the test of inducing TMV resistance of Nicotiana tabacum.

4. The method for improving biocontrol photosynthetic bacteria by distant hybridization and genome rearrangement technology according to claim 3, characterized in that: include: S21), taking out the spore-forming strain, treating it with ceftriaxone sodium and lysozyme, and then centrifuging it to obtain the protoplasts of the fusion strain; S22), dividing the obtained fusion strain protoplasts into two groups and performing UV inactivation and heat inactivation respectively; S23), taking equal volumes of the two groups of inactivated protoplasts, fusing, centrifuging, washing, and culturing to obtain fused sub-bacteria; S24) Select strains with better growth in the fusion bacterial colony to carry out the test of inducing heartleaf tobacco to resist TMV.

5. The method for improving biocontrol photosynthetic bacteria by distant hybridization and genome rearrangement technology according to claim 3, characterized in that: In the S11), the seed inoculation amount of Rhodopseudomonas palustris GJ-22 is 2% and inoculated into a photosynthetic bacterial liquid culture medium, and cultured at 30° C. and anaerobic illumination for 72 hours until the logarithmic growth phase; the Bacillus subtilis KY-2 is inoculated into an LB liquid culture medium at a 1% inoculation amount and cultured at 37° C. and 180 r / min for 16 hours until the logarithmic growth phase.

6. The method for improving biocontrol photosynthetic bacteria by distant hybridization and genome rearrangement technology according to claim 3, characterized in that: In the S12), GJ-22 was cultured with ceftriaxone sodium at a final concentration of 60 μg / mL at 37° C. for 2 h with shaking, and then treated with lysozyme at a final concentration of 1.2 mg / mL; KY-2 was treated with lysozyme at a final concentration of 1.0 mg / mL and then cultured with shaking at 37° C. for 1 h.

7. The method for improving biocontrol photosynthetic bacteria by distant hybridization and genome rearrangement technology according to claim 3, characterized in that: The step S13) comprises taking equal volumes of successfully prepared GJ-22 and KY-2 protoplasts, digesting DNA released by bacterial lysis with DNaseI, and incubating with 40% (V / V) PEG6000 at 25° C. to induce protoplast fusion; The fused bacteria were graded diluted with photosynthetic bacteria hypertonic liquid culture medium and then regenerated on photosynthetic bacteria soft agar medium. The fusion strains RH-0110, RH-0112, RH-0203, RH-0303 and RH-0308 were obtained by screening the induced TMV resistance test of Nicotiana tabacum.

8. The method for improving biocontrol photosynthetic bacteria by distant hybridization and genome rearrangement technology according to claim 7, characterized in that: The S21) comprises adjusting the concentration of the obtained fusion strain to 1×10 6 cell / mL, treated with 60 μg / mL ceftriaxone sodium and 1.2 mg / mL lysozyme, and centrifuged to obtain protoplasts of the fusion strain.

9. The method for improving biocontrol photosynthetic bacteria by distant hybridization and genome rearrangement technology according to claim 7, characterized in that: The step S23) comprises taking two groups of inactivated protoplasts of equal volumes and fusing them with 40% (V / V) PEG6000, washing the cells with SMM buffer, and regenerating and culturing them on a photosynthetic bacteria soft agar medium at 30° C. for 7-14 days to obtain regenerated colonies, namely the F1 generation fusion sub-bacterial colony; using the F1 generation fusion sub-bacterial colony as the starting strain, inactivating it, fusing it again, and regenerating it to obtain a regenerated strain, which is the second round of fusion bacterial colony, namely the F2 generation; The protoplast fusion and regeneration were repeated, and the different fusion progeny were marked as F1, F2, and F3.

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