Hopane synthetic gene combination and application thereof in enhancing environmental stress resistance of broad-host rhizobium

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to hopane synthetic gene combination and application thereof in enhancing environmental stress resistance of broad-host rhizobium, and particularly relates to 1) regulation and control of sucrose osmotic pressure stress tolerance of rhizobium; (2) regulating and controlling the salt stress tolerance of the rhizobium; 3) regulating and controlling the adaptive capacity of the rhizobium to high temperature; and (4) regulating and controlling the alkaline environment adaptability of the rhizobium.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to a combination of hopanoids (hopane) hopane synthesis genes and their application in enhancing the environmental stress resistance of broad-host rhizobia. Background Art

[0002] The symbiotic nitrogen fixation system formed by rhizobia and legumes is crucial to the nitrogen cycle in the ecosystem. Rhizobia convert nitrogen in the atmosphere into ammonia that can be absorbed and utilized by plants, thereby improving nitrogen utilization, reducing nitrogen fertilizer pollution to the environment, and reducing agricultural production costs. Long-term use of rhizobia can increase soil organic matter content and microbial activity, improve soil structure and fertility, and thus contribute to the sustainable development of agriculture. However, rhizobia are very sensitive to changes in soil conditions and physical and chemical properties (such as temperature, osmotic pressure, and pH), which can affect their survival, growth, and reproduction, and thus their ability to establish a beneficial symbiotic relationship with host plants.

[0003] Highly efficient nitrogen-fixing Bradyrhizobium USDA110 ( Bradyrhizobium diazoefficiens USDA110) is a widely studied and used rhizobium strain. It has been developed into a commercial rhizobium agent because it has a certain ability to resist stress and can form efficient nitrogen-fixing nodules with legumes such as soybeans. Hopanes are widely present in bradyrhizobia, which helps to enhance cell membrane stability, ensure bradyrhizobia resist a series of environmental stresses (such as osmotic pressure, pH fluctuations, oxidative stress, etc.), and help bradyrhizobia survive and reproduce under various adverse conditions.

[0004] Bradyrhizobium has a long generation time, and as a commercial bacterial agent, it takes a long time to ferment, has a high production cost, and its competitive nodulation ability in saline-alkali soil is significantly lower than that of Sinorhizobium freundii ( Sinorhizobium fredii ), while the soil in the main planting areas of leguminous crops such as soybeans in my country is mostly alkaline, which also affects the field application effect of bradyrhizobium agents in my country. On the other hand, Sinorhizobium freundii has the characteristics of a wide host range, salt-alkali tolerance and a short generation time, making it an ideal base bacteria for the development of broad-spectrum resistant rhizobia. About 3 / 4 of Sinorhizobium freundii strains can synthesize C30 hopane compounds, but lack the transporter protein encoding genes for the synthesis and transport of C35 hopane substances to the outer membrane; and C35 hopane substances are necessary for bradyrhizobia to adapt to acidic conditions, osmotic pressure, oxidative stress, etc., and hopane substances can bind to different phospholipid molecules on the membrane to regulate the fluidity of the membrane, thereby maintaining the environmental resilience of the cell.

[0005] Artificially designing and modifying the gene circuit of hopane synthesis metabolism to enhance the broad-spectrum stress resistance of the broad-host Sinorhizobium freundii will help further enhance the contribution of the rhizobium-leguminous plant symbiotic nitrogen fixation system to the sustainable development of my country's agriculture and is of great significance to improving the new quality productivity of agriculture. Summary of the invention

[0006] The present invention provides an application of hopanoids synthesis metabolic pathway gene circuit modification to enhance the stress tolerance ability of broad-host Sinorhizobium freundii, which is used to solve the bottleneck of poor environmental resilience of commercial rhizobium agents in the field application in the prior art, and obtains a modified strain with enhanced environmental stress adaptability by modifying rhizobia.

[0007] In order to solve the above technical problems, the present invention provides rhizobia with improved environmental stress resistance and a preparation method and application thereof. The first aspect of the present invention is to provide a rhizobium with enhanced tolerance to environmental stress, characterized in that the rhizobium increases the expression of genes related to C30 hopane basic anabolism through promoter or enhancer elements.

[0008] Furthermore, the promoter is Praph, and preferably, its sequence is shown in SEQ ID NO: 10; Furthermore, the C30 hopane basic anabolism-related genes include fni, fps, hpnX, hpnC , preferably, their sequences are shown in SEQ ID NOs: 1 to 4 respectively; Further, wherein the gene combination induced to overexpress is fni, fps, hpnX or fni, fps, hpnC.

[0009] Furthermore, the rhizobium is Sinorhizobium freundii, preferably Sinorhizobium fredii CCBAU45436 (SF45436); Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0010] The second aspect of the present invention is the use of a promoter in preparing rhizobia resistant to environmental stress, characterized in that the promoter increases the expression of genes related to the basic anabolism of C30 hopane.

[0011] Furthermore, the promoter is Praph, and preferably, its sequence is shown in SEQ ID NO: 10; Furthermore, the C30 hopane basic anabolism-related genes include fni, fps, hpnX, hpnC Preferably, their sequences are shown in SEQ ID NOs: 1 to 4.

[0012] Furthermore, the rhizobium is Sinorhizobium freundii, preferably Sinorhizobium fredii CCBAU45436 (SF45436); Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0013] The third aspect of the present invention provides a rhizobium with enhanced tolerance to environmental stress, characterized in that the rhizobium overexpresses genes related to the synthesis and transport of heterologous C35 hopane anabolism pathway.

[0014] Furthermore, the heterologous gene refers to the synthesis and transport-related genes of the C35 hopane anabolism pathway from the highly efficient nitrogen-fixing Bradyrhizobium BD110; Furthermore, the synthesis and transport related genes of the C35 hopane anabolism pathway include hpnH , hpnG , hpnP , hpnO , hpnN; Preferably, the nucleotide sequences are shown in SEQ ID NOs: 5 to 9 respectively; Furthermore, the synthesis and transport-related genes of the C35 hopane anabolism pathway are induced to express by a high-efficiency promoter, and the high-efficiency promoter includes Praph, PrpoB, PrpoD, and PcdnL; preferably, the sequence of Praph is SEQ ID NO: 10, the sequence of PrpoB is SEQ ID NO: 11, the sequence of PrpoD is SEQ ID NO: 12, and the sequence of PcdnL is SEQ ID NO: 14; preferably, the connection order of the promoter-induced gene expression is Praph- hpnH 、PrpoB- hpnG 、PrpoD- hpnP + hpnO 、PcdnL- hpnN ; Furthermore, the rhizobium is Sinorhizobium freundii, preferably Sinorhizobium fredii CCBAU45436 (SF45436); Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0015] The fourth aspect of the present invention is to provide a C35 hopane anabolism pathway gene circuit for use in improving the ability of rhizobia to resist environmental stress, characterized in that the rhizobia overexpress heterologous C35 hopane anabolism pathway synthesis and transport-related genes; Furthermore, the heterologous gene refers to the synthesis and transport-related genes of the C35 hopane anabolism pathway from the highly efficient nitrogen-fixing Bradyrhizobium BD110; Furthermore, the synthesis and transport related genes of the C35 hopane anabolism pathway include hpnH , hpnG , hpnP , hpnO , hpnN; Preferably, the nucleotide sequences are shown in SEQ ID NOs: 5 to 9 respectively; Furthermore, the synthesis and transport-related genes of the C35 hopane anabolism pathway are induced to express by a high-efficiency promoter, and the high-efficiency promoter includes Praph, PrpoB, PrpoD, and PcdnL; preferably, the sequence of Praph is SEQ ID NO: 10, the sequence of PrpoB is SEQ ID NO: 11, the sequence of PrpoD is SEQ ID NO: 12, and the sequence of PcdnL is SEQ ID NO: 14; preferably, the connection order of the promoter-induced gene expression is Praph- hpnH 、PrpoB- hpnG 、PrpoD- hpnP + hpnO 、PcdnL- hpnN ; Furthermore, the rhizobium is Sinorhizobium freundii, preferably Sinorhizobium fredii CCBAU45436 (SF45436); Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0016] The fifth aspect of the present invention is to provide a method for increasing the production of C35 hopane in rhizobia, wherein the method comprises overexpressing heterologous genes related to the synthesis and transport of the C35 hopane anabolism pathway in rhizobia; Furthermore, the heterologous gene refers to the synthesis and transport-related genes of the C35 hopane anabolism pathway from the highly efficient nitrogen-fixing Bradyrhizobium BD110; Furthermore, the synthesis and transport related genes of the C35 hopane anabolism pathway include hpnH , hpnG , hpnP , hpnO , hpnN; Preferably, the nucleotide sequences are shown in SEQ ID NOs: 5 to 9 respectively; Furthermore, the synthesis and transport-related genes of the C35 hopane anabolism pathway are induced to express by a high-efficiency promoter, and the high-efficiency promoter includes Praph, PrpoB, PrpoD, and PcdnL; preferably, the sequence of Praph is SEQ ID NO: 10, the sequence of PrpoB is SEQ ID NO: 11, the sequence of PrpoD is SEQ ID NO: 12, and the sequence of PcdnL is SEQ ID NO: 14; preferably, the connection order of the promoter-induced gene expression is Praph- hpnH 、PrpoB- hpnG 、PrpoD- hpnP + hpnO 、PcdnL- hpnN ; Furthermore, the rhizobium is Sinorhizobium freundii, preferably Sinorhizobium fredii CCBAU45436 (SF45436).

[0017] The sixth aspect of the present invention is to provide a rhizobium with enhanced tolerance to environmental stress, characterized in that the rhizobium increases the expression of genes related to the basic anabolism of C30 hopanes through a promoter or enhancer element; at the same time, the rhizobium overexpresses genes related to the synthesis and transport of heterologous C35 hopane anabolism pathways; Furthermore, the promoter is Praph, and preferably, its sequence is shown in SEQ ID NO: 10; Furthermore, the C30 hopane basic anabolism-related genes include fni, fps, hpnX, hpnC , preferably, their sequences are shown in SEQ ID NOs: 1-4 respectively; Furthermore, the synthesis and transport related genes of the C35 hopane anabolism pathway include hpnH , hpnG , hpnP , hpnO , hpnN ; Preferably, the nucleotide sequences are shown in SEQ ID NOs: 5 to 9 respectively; Furthermore, the synthesis and transport-related genes of the C35 hopane anabolism pathway are induced to express by a high-efficiency promoter, and the high-efficiency promoter includes Praph, PrpoB, PrpoD, and PcdnL; preferably, the sequence of Praph is SEQ ID NO: 10, the sequence of PrpoB is SEQ ID NO: 11, the sequence of PrpoD is SEQ ID NO: 12, and the sequence of PcdnL is SEQ ID NO: 14; preferably, the connection order of the promoter-induced gene expression is Praph- hpnH 、PrpoB-hpnG 、PrpoD- hpnP + hpnO 、PcdnL- hpnN ; Furthermore, the rhizobium is Sinorhizobium freundii, preferably Sinorhizobium fredii CCBAU45436 (SF45436); Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0018] The seventh aspect of the present invention provides a biomaterial combination, wherein the gene combination can improve the ability of rhizobia to resist environmental stress, characterized in that the biomaterial combination includes one or more of the following 1) fni Gene; 2) fps Gene; 3) hpnX Gene; 4) hpnC Gene; 5) hpnH Gene; 6) hpnG Gene; 7) hpnP Gene; 8) hpnO Gene; 9) hpnN Gene 10) Improve fni expressed nucleic acid molecules; 11) Improve fps expressed nucleic acid molecules; 12) Improve hpnX expressed nucleic acid molecules; 13) Improve hpnC A nucleic acid molecule that expresses a gene; 14) Contains hpnH , hpnG , hpnP , hpnO , hpnN A nucleic acid molecule that expresses a gene; 15) Contains fni Gene and / or 10) a recombinant vector of the nucleic acid molecule; 16) Contains fps Gene and / or 11) a recombinant vector of the nucleic acid molecule; 17) Contains hpnX Gene and / or 12) a recombinant vector of the nucleic acid molecule; 18) ContainshpnC Gene and / or 13) a recombinant vector of the nucleic acid molecule; 19) Contains hpnH , hpnG , hpnP , hpnO , hpnN Gene and / or 14) a recombinant vector of the nucleic acid molecule; 20) Contains fni Gene and / or 10) the nucleic acid molecule, or a recombinant microorganism containing 15) the recombinant vector; 21) Contains fps Gene and / or 11) the nucleic acid molecule or a recombinant microorganism containing 16) the recombinant vector; 22) Contains hpnX Gene and / or 12) the nucleic acid molecule, or a recombinant microorganism containing 17) the recombinant vector; 23) Contains hpnC and / or 13) the nucleic acid molecule, or a recombinant microorganism containing 18) the recombinant vector; contain hpnH , hpnG , hpnP , hpnO , hpnN Gene and / or 14) the nucleic acid molecule, or a recombinant microorganism containing 19) the recombinant vector.

[0019] The eighth aspect of the present invention provides gene sequences related to the synthetic metabolic pathway for synthesizing C35 hopane compounds: 1) hpnH The nucleic acid sequence is the sequence shown in SEQ ID NO.5; 2) hpnG The nucleic acid sequence is the sequence shown in SEQ ID NO.6; 3) hpnP The nucleic acid sequence is the sequence shown in SEQ ID NO.7; 4) hpnO The nucleic acid sequence is the sequence shown in SEQ ID NO.8; 5) hpnN The nucleic acid sequence is the sequence shown in SEQ ID NO.9; The ninth aspect of the present invention provides a variety of promoter sequences for increasing gene expression: 1) The above improvements fni , fps , hpnX , hpnC The expressed sequence is the sequence shown in SEQ ID NO.10; 2) The above expression hpnH The gene promoter sequence is the sequence shown in SEQ ID NO.10; 3) The above expression hpnG The gene promoter sequence is the sequence shown in SEQ ID NO 11; 4) The above expression hpnP The gene promoter sequence is the sequence shown in SEQ ID NO.12; 5) The above expression hpnO The gene promoter sequence is the sequence shown in SEQ ID NO.12; 6) The above expression hpnN The gene promoter sequence is the sequence shown in SEQ ID NO.14.

[0020] The beneficial effects of the present invention are: 1) SF45436( fps ) and SF45436( fni ) The growth of the strain under high salt (150 mM NaCl) and high alkaline (pH9) stress environment was significantly improved compared with the starting strain SF45436; 2) SF45436 obtained by the present invention ( fni + fps )、SF45436( hpnX )、SF45436( fni + fps + hpnX ) showed significantly improved growth in high salt (150 mM NaCl), high alkali (pH9) and high temperature (37°C) stress environments compared with the original strain SF45436; 3) SF45436 obtained by the present invention:: hpnN and SF45436:: hpnNH The growth under high temperature (37°C) stress conditions was significantly higher than that of SF45436; 4) SF45436 obtained by the present invention:: hpnNHGPO, SF45436( fni ):: hpnNHGPO 、SF45436( fps ):: hpnNHGPO 、SF45436( fni + fps ):: hpnNHGPO 、SF45436( hpnX ):: hpnNHGPO The growth of the strain under high temperature (37℃) and hyperosmotic (15% sucrose) stress conditions was significantly improved compared with that of SF45436. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 for Bradyrhizobium diazoefficiens USDA110 (BD110) Hopane synthesis pathway; Figure 2 for Sinorhizobium fredii CCBAU45436 (SF45436) Hopane synthesis pathway; Figure 3 Growth detection of strains with enhanced gene expression in hopane metabolic pathway and SF45436 under high temperature (37°C), sucrose (15%), salt (150 mM NaCl) and alkaline (pH9) stress conditions; Figure A is a heat map of high temperature growth curves of different strains, Figure B is the growth curve of strains under dual stress of high temperature (37°C) and sucrose (15%), and Figure C is the colony area of ​​the modified strain under sucrose (15%), salt (150 mM NaCl, Figure D) and alkaline (pH9, Figure E) stress (normalized by the colony area of ​​SF45436); Figure 4 Growth test of the constructed C35 hopane synthesis gene circuit strain and SF45436 under high temperature (37°C), sucrose (15%), salt (150 mM NaCl) and alkaline (pH9) stress conditions; Figure A is a heat map of high temperature growth curves of different strains, Figure B is the growth curve of strains under dual stress of high temperature (37°C) and sucrose (15%), and Figure C is the colony area of ​​the modified strain under sucrose (15%), salt (150 mM NaCl, Figure D) and alkaline (pH9, Figure E) stress (normalized by the colony area of ​​SF45436); Figure 5 Growth detection of strains containing C35 hopane synthesis gene circuit and enhanced basic anabolic pathway genes and SF45436 under high temperature (37℃), sucrose (15%), salt (150 mM NaCl) and alkaline (pH9) stress conditions; Figure A is a heat map of high temperature growth curves of different strains, Figure B is the growth curve of strains under dual stress of high temperature (37℃) and sucrose (15%), and Figure C is the colony area of ​​the modified strain under sucrose (15%), salt (150 mM NaCl, Figure D) and alkaline (pH9, Figure E) stress (normalized by the colony area of ​​SF45436).

[0022] Figure 6 Figure A Bradyrhizobium diazoefficiens USDA110 (BD110) Hopane metabolic pathway and hopane substances, Figure B is SF45436 and SF45436:: hpnNHGPOMass spectrometry results of the strains under high temperature (37°C) and sucrose (15%) osmotic stress. DETAILED DESCRIPTION

[0023] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] 1. Experimental Materials (1) Strains: Bradyrhizobium diazoefficiens USDA110 (BD110), Sinorhizobium fredii CCBAU45436 (SF45436), SF45436( fni )、SF45436( fps )、SF45436( fni + fps )、SF45436( hpnX )、SF45436( fni + fps + hpnX )、SF45436( fni + fps + hpnC )、SF45436( hpnN )、SF45436 ( hpnNH )、SF45436:: hpnNHGPO 、SF45436( fni ):: hpnNHGPO 、SF45436( fps ):: hpnNHGPO 、SF45436( fni + fps ):: hpnNHGPO 、SF45436( hpnX ):: hpnNHGPO 、SF45436( fni + fps + hpnX ):: hpnNHGPO、SF45436( fni + fps + hpnC ):: hpnNHGPO .

[0027] (2) Culture media and reagents used ① TY (Tryptone-Yeast Extract) medium / L: 3 g yeast extract, 5 g tryptone, 0.6 g CaCl2, pH 7.0. Add 5% sucrose to the sucrose screening medium. Add 15 g agar powder to the solid medium. Sterilize at 121℃ with high pressure and wet heat for 30 min.

[0028] ② LB (Luria Bertani medium) medium / L: 5 g yeast extract, 10 g tryptone, 10 g NaCl, pH 7.2. Add 15 g agar powder to the solid medium. Sterilize at 121℃ with high pressure and wet heat for 30 min.

[0029] ③ 0.85% saline / L: add 8.5g NaCl to deionized water to make up to 1 L, and sterilize at 121℃ with high pressure wet heat for 30 min.

[0030] ④ Nalidixic acid (NA): stored at a concentration of 30 mg / mL, dissolved in ddH2O, promoted by NaOH solution, sterilized by 0.22 µm filtration, and stored at -20°C after aliquoting; ⑤ Trimethoprim (TMP): storage concentration 10 mg / mL, dissolved in ddH2O, dissolve in glacial acetic acid, sterilize by 0.22 µm filtration, and store at -20°C after aliquoting; ⑥ Gentamycin (Gen): stored at 30 mg / mL, dissolved in ddH2O, sterilized by 0.22 µm filtration, and stored at -20°C after aliquoting.

[0031] ⑦ N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO): Add 9.0912 g AMPSO powder to TY medium, adjust the pH to 9 with HCl or KOH, make up to 1 L, and sterilize at 121℃ for 30 min.

[0032] (3) Plasmid vector: ①pJQ200SK plasmid, pJQ200SK is a suicide plasmid used to construct vectors for subsequent gene enhancement and to supplement C35 hopane synthesis. The strain constructed through subsequent three-parent conjugation experiments does not carry any resistance markers, which is a traceless gene editing method; ②pRJPaph-bjGFP is used to clone the promoter Praph sequence; (4) Triparental conjugation experiment: It is a classic genetic manipulation method used to transfer plasmids from donor bacteria to target recipient bacteria, especially widely used in bacteria that are difficult to directly transform (such as some Gram-negative bacteria). Triparental conjugation introduces a third bacterium as a "helper bacterium" to provide the necessary conjugation function.

[0033] 2. Description of the hopane synthesis metabolic pathway in rhizobia The synthesis of hopanes by rhizobia starts from the mevalonate pathway (MEP), and the intermediate product farnesyl pyrophosphate (FPP) is the precursor for the synthesis of squalene and hopanes, such as Figure 1 and Figure 2 As shown in the figure, only C30 hopanes are synthesized in Sinorhizobium freundii SF45436, namely squalene and hopane, a cyclized product of squalene. fni and fps The transcription level of the gene is very low, and the synthesis of squalene hpnCDEF The gene cluster was also transcribed at a low level. hpnH , various modifier genes hpnG , hpnP , hpnO , and those required for transport to the outer membrane hpnN Gene.

[0034] Example 1 Construction and growth identification of a modified strain of Sinorhizobium freundii SF45436 with enhanced hopane basic biosynthesis metabolic pathway.

[0035] 1.1 Construction of promoter-enhanced strains 1) Extract pJQ200SK plasmid, digest it with SmaI restriction endonuclease and recover the linear plasmid to determine its purity and concentration; 2) Extract pRJPaph-bjGFP plasmid, pathway fps , fni , hpnCDEFThe gene cluster expression is very low. In order to enhance the transcription of these three elements, primers were designed according to the gene sequences of the three elements to amplify the promoter Praph (SEQ ID NO: 10). Upstream and downstream primers were designed according to the gene sequences that needed to be enhanced. 20 bp homology arms were designed at the junction of the upstream and downstream fragments, and 20 bp homology arms of pJQ200SK were added to both ends of the upstream and downstream fragments. The genome of Sinorhizobium freundii SF45436 was used as a DNA template to obtain the target fragment by PCR amplification. 3) Recover the amplified fragment and clone it seamlessly with the linearized vector to obtain; 4) After heat shock transformation, colony PCR was performed to test and obtain the vector for enhanced expression of genes related to hopane synthesis, and the vector was preserved; 5) The vector was introduced into Sinorhizobium freundii SF45436 in a three-parent conjugation experiment to obtain a modified strain with enhanced promoter.

[0036] 1.2 Identification of promoter-enhanced strains The stress experiment was then carried out after the transformation-positive strains were identified and selected correctly by PCR.

[0037] 1.2.1 High temperature growth curves of promoter-enhanced strains and wild-type S. frugiperda SF45436 The growth of strains under high temperature (37°C) was tested in TY medium. The operation was as follows: all modified strains and wild-type strains were activated on TY medium containing NA / TMP antibiotics, and single colonies were picked and transferred to TY liquid medium containing corresponding antibiotics. They were cultured at 28°C and 200 rpm to the logarithmic phase, and the cells were collected by centrifugation at 6500 rpm for 5 min. The cells were washed twice with physiological saline and adjusted to OD 600 =0.02; 300 µL of bacterial suspension of each strain was added to a honeycomb plate (Bioscreen C), and placed in the Bioscreen C fully automatic growth curve analyzer of Growth Curves Company of Finland to measure the growth curve.

[0038] The results are as follows Figure 3 A shows: promoter-enhanced strain SF45436 ( fni + fps ) (t-test; ****, P <0.0001), SF45436( hpnX ) (t-test; ****, P < 0.0001), SF45436( fni + fps + hpnC ) (t-test; ****, P < 0.0001), SF45436( fni + fps + hpnX) (t-test; ****, P < 0.0001) grew significantly higher than the wild-type strain under high temperature stress.

[0039] 1.2.2 Growth curves of promoter-enhanced strains and wild-type SF45436 under dual stress of high temperature and sucrose osmotic stress The growth of strains under 15% sucrose stress at high temperature (37°C) was tested in TY medium. The operation was as follows: all modified strains and wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics, and single colonies were picked and transferred to TY liquid medium containing corresponding antibiotics. They were cultured at 28°C and 200 rpm to the logarithmic phase, and the cells were collected by centrifugation at 6500 rpm for 5 min. The cells were washed twice with physiological saline and adjusted to OD 0. 600 =0.02; 300 µL of each bacterial strain was added to a honeycomb plate (Bioscreen C), and placed in the Bioscreen C fully automatic growth curve analyzer of Growth Curves Company, Finland, to measure the growth curve. Figure 3 B shows: promoter-enhanced strain SF45436 ( fni + fps )(t-test; ****, P < 0.0001), SF45436( hpnX ) (t-test; **, P < 0.01), SF45436( fni + fps + hpnC ) (t-test; ****, P < 0.0001), SF45436( fni + fps + hpnX ) (t-test; **, P < 0.01) showed significantly higher growth than that of the wild-type strain under the dual stress of high temperature and sucrose.

[0040] 1.2.3 Promoter-enhanced strains and wild-type S. frugiperda SF45436 adapt to 15% sucrose osmotic stress Similarly, the transformed strains and wild-type strains in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken on TY solid medium containing 15% sucrose and cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawn or colony was calculated using ImageJ and standardized with the area of ​​wild-type strain. The results are shown in Figure 2.Figure 3 C shows: promoter-enhanced strain Sinorhizobium freundii SF45436 ( fps ) (t-test; P =0.0547) grew better than the wild-type strain SF45436 under 15% sucrose stress.

[0041] 1.2.4 Promoter-enhanced strains and wild-type SF45436 adapt to 150 mM NaCl osmotic stress The transformed strains and wild-type strains in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken for each dilution on TY solid medium containing 150 mM NaCl. The samples were cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawns or colonies was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 3 D shows: promoter-enhanced strain SF45436 ( fni ) (t-test; ***, P < 0.001), SF45436 ( fps ) (t-test; **, P < 0.01), SF45436( fni + fps ) (t-test; **, P < 0.01), SF45436 ( hpnX ) (t-test; *, P < 0.05), SF45436( fni + fps + hpnX ) (t-test; **, P <0.01) The growth ability of SF45436 under 150 mM NaCl stress was significantly higher than that of wild type SF45436.

[0042] 1.2.5 Promoter-enhanced strains and wild-type SF45436 adapt to high alkaline (pH 9) stress The transformed strains and wild-type strains in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6The samples were diluted in a gradient manner, and 10 µL was taken on pH 9 TY solid medium and cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawn or colony was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 3 E shows: promoter-enhanced strain SF45436 ( fni ) (t-test; ***, P < 0.001), SF45436( fps ) (t-test; ***, P < 0.001), SF45436( fni + fps ) (t-test; ***, P < 0.001), SF45436( hpnX ) (t-test; ***, P < 0.001), SF45436( fni + fps + hpnX ) (t-test; ***, P < 0.001) The growth ability of the strain SF45436 under pH 9 stress was significantly higher than that of the wild-type strain SF45436.

[0043] Example 2 Construction and growth identification of the hopane synthesis metabolic pathway strain of Sinorhizobium freundii SF45436 C35 2.1 Construction of C35 Hopane Biosynthesis Pathway Strain 1) Extract the genomes of Sinorhizobium freundii SF45436 and Bradyrhizobium BD110, and identify the genes involved in the synthesis and transport of C35 hopane biosynthesis pathway of BD110 hpnH , hpnG , hpnP , hpnO , hpnN According to the insertion site sequence of SF45436, upstream and downstream fragments, promoter and hpnHGPON Primers, PCR amplification of upstream and downstream fragments as well as promoters and gene fragments: Praph- hpnH 、PrpoB- hpnG 、PrpoD- hpnP + hpnO 、PcdnL- hpnN .

[0044] The sequence of Praph is SEQ ID NO: 10, the sequence of PrpoB is SEQ ID NO: 11, the sequence of PrpoD is SEQ ID NO: 12, and the sequence of PcdnL is SEQ ID NO: 14; hpnH The nucleic acid sequence is the sequence shown in SEQ ID NO.5; hpnGThe nucleic acid sequence is the sequence shown in SEQ ID NO.6; hpnP The nucleic acid sequence is the sequence shown in SEQ ID NO.7; hpnO The nucleic acid sequence is the sequence shown in SEQ ID NO.8; hpnN The nucleic acid sequence is the sequence shown in SEQ ID NO.9; 2) Recover the amplified fragment and perform seamless cloning with the linearized vector; 3) After heat shock transformation, colony PCR was performed to test and obtain the heterologous expression vector of Hopani gene, and the vector was preserved; 4) The vector was introduced into Sinorhizobium freundii SF45436 in a three-parent conjugation experiment, thereby obtaining a modified strain that heterologously expresses genes related to C35 hopane synthesis and transport.

[0045] 2.2 Identification of strains expressing heterologous genes related to C35 hopane synthesis and transport 2.2.1 High temperature growth curves of heterologous expression strains of genes related to C35 hopane synthesis and transport and wild-type SF45436 The growth of strains under high temperature (37°C) was tested in TY medium. The operation was as follows: all modified strains and wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics, and single colonies were picked and transferred to TY liquid medium containing corresponding antibiotics. They were cultured at 28°C and 200 rpm until the logarithmic phase, and the cells were collected by centrifugation at 6500 rpm for 5 min. The cells were washed twice with physiological saline and adjusted to OD 600 =0.02; 300 µL of each bacterial strain was added to a honeycomb plate (Bioscreen C), and placed in the Bioscreen C fully automatic growth curve analyzer of Growth Curves Company, Finland, to measure the growth curve. Figure 4 A shows: C35 hopane synthesis and transport related genes heterologous expression strain SF45436:: hpnN (t-test; ****, P < 0.0001), SF45436:: hpnNH (t-test; ****, P < 0.0001), SF45436:: hpnNHGPO (t-test; ****, P < 0.0001) The growth under high temperature stress was significantly higher than that of the wild-type strain SF45436.

[0046] 2.2.2 Growth curves of the strain expressing C35 hopane biosynthesis and transport-related genes and wild-type SF45436 under dual stress of high temperature and sucrose osmotic pressure The growth of strains under 15% sucrose stress at high temperature (37°C) was tested in TY medium. The operation was as follows: all modified strains and wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics, and single colonies were picked and transferred to TY liquid medium containing corresponding antibiotics. They were cultured at 28°C and 200 rpm to the logarithmic phase, and the cells were collected by centrifugation at 6500 rpm for 5 min. The cells were washed twice with physiological saline and 15% sucrose TY was adjusted to OD 600 =0.02; 300 µL of each bacterial strain was added to a honeycomb plate (Bioscreen C), and placed in the Bioscreen C fully automatic growth curve analyzer of Growth Curves Company, Finland, to measure the growth curve. Figure 4 B shows: C35 hopane synthesis and transport related genes heterologous expression strain SF45436:: hpnN (t-test; **, P < 0.01 (72h) ) The growth rate under high temperature and sucrose stress in the early logarithmic growth phase was significantly higher than that of the wild-type strain SF45436; SF45436:: hpnNH (t-test; ***, P < 0.001), SF45436:: hpnNHGPO (t-test; **, P < 0.01) The growth rate under the dual stress of high temperature and sucrose was significantly higher than that of the wild-type strain SF45436.

[0047] 2.2.3 Ability of heterologous expression strains of C35 hopane biosynthesis and transport-related genes and wild-type SF45436 to adapt to 15% sucrose osmotic stress The transformed strains and wild-type strain SF45436 in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken on TY solid medium containing 15% sucrose and cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawn or colony was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 4 C shows: C35 hopane synthesis and transport related genes heterologous expression strain SF45436:: hpnN (t-test; P = 0.0520), SF45436:: hpnNHGPO (t-test; P = 0.0365) grew better than wild-type SF45436 under 15% sucrose stress.

[0048] 2.2.4 Ability of heterologous expression strains of C35 hopane biosynthesis and transport-related genes and wild-type SF45436 to adapt to 150 mM NaCl osmotic stress The transformed strains and wild-type strain SF45436 in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken for each dilution on TY solid medium containing 150 mM NaCl. The samples were cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawns or colonies was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 4 As shown in D: The growth ability of each modified strain under 150 mM NaCl stress had no significant difference with the wild type SF45436.

[0049] 2.2.5 Ability of heterologous expression strains of C35 hopane biosynthesis and transport-related genes and wild-type SF45436 to adapt to high alkaline (pH 9) stress The transformed strains and wild-type strain SF45436 in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken on pH 9 TY solid medium and cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawn or colony was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 4 E: SF45436:: hpnN (t-test; *, P < 0.05) The growth ability under pH 9 stress was significantly higher than that of wild type SF45436.

[0050] Example 3 Identification of strains expressing enhanced expression of hopane basic anabolism genes and heterologous expression of C35 hopane synthesis-related genes in Sinorhizobium freundii CCBAU45436 (SF45436) 3.1 Enhanced expression of the basic hopane anabolism gene circuit and identification of heterologous expression strains of the C35 hopane anabolism pathway The five genes described in Example 2 were sequentially transformed on the basis of the strain obtained in Example 1 to obtain the strain of this example, and stress resistance test was performed after PCR verification was correct.

[0051] 3.1.1 High temperature growth curves of enhanced expression of hopane basic anabolism gene circuits and heterologous expression strains of C35 hopane anabolism pathway and wild-type SF45436 The growth of strains under high temperature (37°C) was tested in TY medium. The operation was as follows: all modified strains and wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics, and single colonies were picked and transferred to TY liquid medium containing corresponding antibiotics. They were cultured at 28°C and 200 rpm until the logarithmic phase, and the cells were collected by centrifugation at 6500 rpm for 5 min. The cells were washed twice with physiological saline and adjusted to OD 600 =0.02; 300 µL of each bacterial strain was added to a honeycomb plate (Bioscreen C), and placed in the Bioscreen C fully automatic growth curve analyzer of Growth Curves Company, Finland, to measure the growth curve. Figure 5 As shown in A: Transformed strain SF45436 ( fni ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( fps ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( fni + fps ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( hpnX ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( fni + fps + hpnC ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( fni + fps + hpnX ):: hpnNHGPO (t-test; ****, P < 0.0001) The growth under high temperature stress was significantly higher than that of the wild-type strain SF45436.

[0052] 3.1.2 Growth curves of enhanced expression of hopane basic anabolism gene circuits and heterologous expression strains of C35 hopane anabolism pathway and wild-type SF45436 under dual stress of high temperature and sucrose osmotic stress The growth of strains under 15% sucrose stress at high temperature (37°C) was tested in TY medium. The operation was as follows: all modified strains and wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics, and single colonies were picked and transferred to TY liquid medium containing corresponding antibiotics. They were cultured at 28°C and 200 rpm to the logarithmic phase, and the cells were collected by centrifugation at 6500 rpm for 5 min. The cells were washed twice with physiological saline and adjusted to OD 0. 600 =0.02; 300 µL of each bacterial strain was added to a honeycomb plate (Bioscreen C), and placed in the Bioscreen C fully automatic growth curve analyzer of Growth Curves Company, Finland, to measure the growth curve. Figure 5 B shows: Transformed strain SF45436 ( fni ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( fps ):: hpnNHGPO (t-test; **, P < 0.01), SF45436( fni + fps ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( hpnX ):: hpnNHGPO (t-test; *, P < 0.05), SF45436( fni + fps + hpnC ):: hpnNHGPO (t-test; ****, P < 0.0001), SF45436( fni + fps + hpnX ):: hpnNHGPO (t-test; *, P < 0.05) The growth rate under the dual stress of high temperature and sucrose was significantly higher than that of the wild-type strain SF45436.

[0053] 3.1.3 Enhanced expression of hopane basic anabolism gene circuits and the ability of heterologous expression strains of C35 hopane anabolism pathway and wild-type SF45436 to adapt to 15% sucrose osmotic stress Similarly, the transformed strains and wild-type strains in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6The samples were diluted in a gradient manner, and 10 µL was taken on TY solid medium containing 15% sucrose and cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawn or colony was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 5 C shows: Transformed strain SF45436 ( fni ):: hpnNHGPO (t-test; P = 0.0452), SF45436 ( fps ):: hpn NHGPO (t-test; P = 0.0299), SF45436 ( fni + fps ):: hpnNHGPO (t-test; P = 0.0384), SF45436( hpnX ):: hpnNHGPO (t-test; P = 0.0281) grew better than wild type SF45436 under 15% sucrose stress.

[0054] 3.1.4 Enhanced expression of hopane basic anabolism gene circuits and the ability of heterologous expression strains of C35 hopane anabolism pathway and wild-type SF45436 to adapt to 150 mM NaCl osmotic stress The transformed strains and wild-type strain SF45436 in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 - 5, 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken for each dilution on TY solid medium containing 150 mM NaCl. The samples were cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawns or colonies was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 5 D shows: Transformed strain SF45436 ( fni + fps ):: hpnNHGPO (t-test; *, P < 0.05) The growth ability under 150 mM NaCl stress was significantly higher than that of wild-type SF45436.

[0055] 3.1.5 Enhanced expression of hopane basic anabolism gene circuits and the ability of heterologous expression strains of C35 hopane anabolism pathway and wild-type SF45436 to adapt to high alkaline (pH 9) stress The transformed strains and wild-type strain SF45436 in the logarithmic phase were adjusted to OD 600 =0.2, and then press 10 -1, 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The samples were diluted in a gradient manner, and 10 µL was taken on pH 9 TY solid medium and cultured in a 28°C incubator for 7 days. The area of ​​bacterial lawn or colony was calculated using ImageJ and standardized with the area of ​​wild-type strain SF45436. The results are shown in Figure 2. Figure 5 As shown in E: There is no significant difference in the ability of each modified strain to adapt to high alkaline (pH 9) stress compared with the wild type SF45436.

[0056] Example 4 Identification of Hopane Substances Produced by C35 Hopane Synthesis and Transport Related Genes Heterologous Expression Strains SF45436:: hpnNHGPO The wild-type strain SF45436 was activated on TY medium containing NA / TMP antibiotics, and a single colony was picked and transferred to TY liquid medium containing the corresponding antibiotics. The culture was cultured at 28°C and 200 rpm until the logarithmic phase, and the bacteria were collected by centrifugation at 6500 rpm for 5 min; the bacteria were washed twice with physiological saline, and the bacterial liquid was inoculated into 200 mL TY+15% sucrose medium to a final concentration of OD 600 = 0.05, 37℃ expansion culture, culture the bacterial liquid to OD 600 = 0.8, centrifuged at 4°C and 5000 rpm for 15 min to collect the cells, extracted the total lipids with dichloromethane, dried under vacuum, and dissolved with dichloromethane / methanol (volume ratio 9:1) to a final concentration of 1 mg / mL. Diluted 100 times with dichloromethane for mass spectrometry analysis. The results are shown in Figure 6 AB shows: SF45436:: hpnNHGPO It can produce the methylated hopane substance 2-methyl bacteriohopanetetrol (2-methyl BHT).

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene combination, characterized in that: The gene combination includes the promoter Praph and the C30 hopane basic anabolism gene fni, fps, hpnX, hpnC, The sequence of Praph is shown in SEQ ID NO: 10 ， Gene fni, fps, hpnX, hpnC The sequences are shown in SEQ ID NOs: 1 to 4 respectively.

2. A biomaterial combination, characterized in that: The biological material can improve the ability of rhizobia to resist environmental stress. The biological material combination encodes the gene combination of claim 1, wherein the biological material comprises: 1) an expression cassette encoding the gene; 2) a vector containing the expression cassette described in 1); 3) A recombinant microorganism containing 1) the expression cassette or 2) the vector.

3. Use of the gene combination according to claim 1 or the biological material combination according to claim 2 in improving the ability of rhizobia to resist environmental stress.

4. A rhizobium for improving environmental stress tolerance, characterized in that: The rhizobium induces the C30 hopane basic synthesis metabolism gene of claim 1 through the promoter of claim 1 fni, fps, hpnX, hpnC Overexpression, wherein the gene combination that induces overexpression is fni, fps, hpnX or fni, fps, hpnC .

5. A gene combination, characterized in that: The gene combination includes a promoter and genes related to synthesis and transport of the C35 hopane anabolism pathway, wherein the promoter includes Praph, PrpoB, PrpoD, and PcdnL; the sequence of Praph is SEQ ID NO: 10, the sequence of PrpoB is SEQ ID NO: 11, the sequence of PrpoD is SEQ ID NO: 12, and the sequence of PcdnL is SEQ ID NO: 14; the genes related to synthesis and transport of the C35 hopane anabolism pathway include HkDJ , HkDJ , hp , oeLh , hpnN, The sequences thereof are shown in SEQ ID NOs: 5 to 9, respectively.

6. A biomaterial combination, characterized in that: The biological material can improve the ability of rhizobia to resist environmental stress. The biological material combination encodes the gene combination of claim 5, wherein the biological material comprises: 1) an expression cassette encoding the gene; 2) a vector containing the expression cassette described in 1); 3) A recombinant microorganism containing 1) the expression cassette or 2) the vector.

7. Use of the gene combination according to claim 5 or the biological material combination according to claim 6 for improving the ability of rhizobia to resist environmental stress.

8. A rhizobium for improving environmental stress tolerance, characterized in that: The rhizobium induces the C35 hopane synthesis metabolism and transport related genes in claim 5 through the promoter in claim 5 HkDJ , HkDJ , hp , oeLh , n Overexpression.

9. The rhizobium according to claim 8, characterized in that The connection sequence of the promoter inducing the gene expression is Praph- HkDJ 、PrpoB- HkDJ 、PrpoD- hp + oeLh 、PcdnL- n .

10. A rhizobium for improving environmental stress tolerance, characterized in that: The rhizobium is based on the rhizobium described in claim 4, and the promoter described in claim 5 is used to induce overexpression of the C35 hopane synthesis metabolism and transport related genes described in claim 5. HkDJ , HkDJ , hp , oeLh , n get.

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