Hopane synthesis gene combination and its application in enhancing environmental stress tolerance of broad-host-range rhizobia

Abstract

The present invention relates to the field of agricultural microbiology, and relates to hopane synthesis gene combinations and their application in enhancing the environmental stress tolerance of broad-host rhizobia, specifically including 1) regulating the sucrose osmotic stress tolerance of rhizobia; 2) regulating the salt stress tolerance of rhizobia; 3) regulating the high-temperature adaptability of rhizobia; and 4) regulating the alkaline environment adaptability of rhizobia.

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] Enhancing the broad-spectrum stress resistance of Sinorhizobium fredii with a wide host range by artificially designing and modifying the hopanoid biosynthesis gene circuit is beneficial to further improving the contribution of the rhizobium-legume symbiotic nitrogen fixation system to the sustainable development of agriculture in China, and is of great significance for enhancing the new quality productivity of agriculture. Summary of the Invention

[0006] The present invention provides the application of modifying the hopanoid biosynthesis gene circuit to enhance the stress tolerance of Sinorhizobium fredii with a wide host range, so as to solve the bottleneck problem of poor environmental toughness of commercial rhizobia agents in field applications in the prior art, and obtain modified strains with enhanced environmental stress adaptation ability by modifying rhizobia.

[0007] To solve the above technical problems, the present invention provides rhizobia with enhanced environmental stress resistance, their preparation methods and applications. Specifically

[0008] The first aspect of the present invention is to provide a rhizobium with enhanced tolerance to environmental stress, characterized in that the expression of genes related to the basic biosynthesis of C30 hopanoids in the rhizobium is increased through promoter or enhancer elements.

[0009] Further, the promoter is Praph, preferably, its sequence is shown as SEQ ID NO:10;

[0010] Further, the genes related to the basic biosynthesis of C30 hopanoids include fni, fps, hpnX, hpnC , preferably, their sequences are respectively shown as SEQ ID NOs: 1-4;

[0011] Further, among them, the gene combination for induced overexpression is fni, fps, hpnX or fni, fps, hpnC.

[0012] Further, the rhizobium is Sinorhizobium fredii, preferably Sinorhizobium fredii CCBAU45436 (SF45436);

[0013] Further, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0014] The second aspect of the present invention is the application of a promoter in the preparation of rhizobia resistant to environmental stress, characterized in that the promoter increases the expression of genes related to the basic biosynthesis of C30 hopanoids.

[0015] Further, the promoter is Praph, preferably, its sequence is shown as SEQ ID NO:10;

[0016] Further, the genes related to the basic biosynthesis of C30 hopanoids includefni, fps, hpnX, hpnC , preferably, their sequences are shown as SEQ ID NOs: 1 to 4 respectively.

[0017] Furthermore, the rhizobia are Sinorhizobium fredii, preferably Sinorhizobium fredii CCBAU45436 (SF45436);

[0018] Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0019] The third aspect of the present invention provides a rhizobium with enhanced tolerance to environmental stress pressure, characterized in that the rhizobium overexpresses genes related to the synthesis and transport of the heterologous C35 hopane synthesis metabolic pathway.

[0020] Furthermore, the heterologous refers to that the genes related to the synthesis and transport of the C35 hopane synthesis metabolic pathway come from the efficient nitrogen-fixing Bradyrhizobium sp. BD110;

[0021] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthesis metabolic pathway include hpnH , hpnG , hpnP , hpnO , hpnN; , preferably, their nucleotide sequences are shown as SEQ ID NO: 5 to 9 respectively;

[0022] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthesis metabolic pathway are induced to express by a highly efficient promoter, and the highly efficient promoters include Praph, PrpoB, PrpoD, 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 ;

[0023] Furthermore, the rhizobia are Sinorhizobium fredii, preferably Sinorhizobium fredii CCBAU45436 (SF45436);

[0024] Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0025] The fourth aspect of the present invention is to provide the application of the C35 hopane synthetic metabolic pathway gene circuit in improving the ability of rhizobia to tolerate environmental stress, characterized in that the rhizobia overexpress the genes related to the synthesis and transport of the heterologous C35 hopane synthetic metabolic pathway;

[0026] Furthermore, the heterologous refers to that the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway are from the slow-growing rhizobium Bradyrhizobium diazoefficiens BD110 with high nitrogen fixation efficiency;

[0027] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway include hpnH 、 hpnG 、 hpnP 、 hpnO 、 hpnN; Preferably, their nucleotide sequences are respectively as shown in SEQ ID NO: 5-9;

[0028] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway are induced to express by a highly efficient promoter, and the highly efficient promoters include Praph, PrpoB, PrpoD, 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 inducing gene expression is Praph- hpnH 、PrpoB- hpnG 、PrpoD- hpnP + hpnO 、PcdnL- hpnN ;

[0029] Furthermore, the rhizobia are Sinorhizobium fredii, preferably Sinorhizobium fredii CCBAU45436 (SF45436);

[0030] Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0031] The fifth aspect of the present invention is to provide a method for increasing the C35 hopane production of rhizobia, and the method is to overexpress the genes related to the synthesis and transport of the heterologous C35 hopane synthetic metabolic pathway in rhizobia;

[0032] Furthermore, the heterologous refers to that the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway are from the slow-growing rhizobium Bradyrhizobium diazoefficiens BD110 with high nitrogen fixation efficiency;

[0033] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway includehpnH , hpnG , hpnP , hpnO , hpnN; Preferably, their nucleotide sequences are respectively as shown in SEQ ID NO:5-9;

[0034] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway are induced to express by a highly efficient promoter, and the highly efficient promoter includes Praph, PrpoB, PrpoD, 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 ;

[0035] Furthermore, the rhizobium is Sinorhizobium fredii, preferably Sinorhizobium fredii CCBAU45436 (SF45436).

[0036] The sixth aspect of the present invention is to provide a rhizobium with enhanced tolerance to environmental stress, characterized in that the rhizobium improves the expression of genes related to the basic synthesis metabolism of C30 hopane through a promoter or enhancer element; meanwhile, the rhizobium overexpresses genes related to the synthesis and transport of the heterologous C35 hopane synthetic metabolic pathway;

[0037] Furthermore, the promoter is Praph, preferably, its sequence is as shown in SEQ ID NO:10;

[0038] Furthermore, the genes related to the basic synthesis metabolism of C30 hopane include fni, fps, hpnX, hpnC , preferably, their sequences are respectively as shown in SEQ ID NOs:1-4;

[0039] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthetic metabolic pathway include hpnH , hpnG , hpnP , hpnO , hpnN ; preferably, their nucleotide sequences are respectively as shown in SEQ ID NO:5-9;

[0040] Furthermore, the genes related to the synthesis and transport of the C35 hopane synthesis metabolic pathway are induced to express by highly efficient promoters, and the highly efficient promoters include 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 ;

[0041] Furthermore, the rhizobium is Sinorhizobium fredii, preferably Sinorhizobium fredii CCBAU45436 (SF45436);

[0042] Furthermore, the environmental stress includes but is not limited to high temperature, alkaline environment, high salt, and high sugar osmotic pressure.

[0043] The seventh aspect of the present invention provides a biological material combination. The gene combination can improve the environmental stress tolerance of rhizobia. It is characterized in that the biological material combination includes one or more of the following

[0044] 1) fni gene;

[0045] 2) fps gene;

[0046] 3) hpnX gene;

[0047] 4) hpnC gene;

[0048] 5) hpnH gene;

[0049] 6) hpnG gene;

[0050] 7) hpnP gene;

[0051] 8) hpnO gene;

[0052] 9) hpnN gene

[0053] 10) Nucleic acid molecules that enhance fni expression;

[0054] 11) Nucleic acid molecules that enhancefps Nucleic acid molecules expressed;

[0055] 12) To increase hpnX Nucleic acid molecules expressed;

[0056] 13) To increase hpnC Nucleic acid molecules for gene expression;

[0057] 14) Containing hpnH , hpnG , hpnP , hpnO , hpnN Nucleic acid molecules for gene expression;

[0058] 15) A recombinant vector containing fni a gene and / or the nucleic acid molecule of 10);

[0059] 16) A recombinant vector containing fps a gene and / or the nucleic acid molecule of 11);

[0060] 17) A recombinant vector containing hpnX a gene and / or the nucleic acid molecule of 12);

[0061] 18) A recombinant vector containing hpnC a gene and / or the nucleic acid molecule of 13);

[0062] 19) A recombinant vector containing hpnH , hpnG , hpnP , hpnO , hpnN a gene and / or the nucleic acid molecule of 14);

[0063] 20) A recombinant microorganism containing fni a gene and / or the nucleic acid molecule of 10), or containing the recombinant vector of 15);

[0064] 21) A recombinant microorganism containing fps a gene and / or the nucleic acid molecule of 11), or containing the recombinant vector of 16);

[0065] 22) A recombinant microorganism containing hpnX a gene and / or the nucleic acid molecule of 12), or containing the recombinant vector of 17);

[0066] 23) A recombinant microorganism containing hpnC and / or the nucleic acid molecule of 13), or containing the recombinant vector of 18);

[0067] Containing hpnH , hpnG, hpnP , hpnO , hpnN genes and / or 14) the nucleic acid molecule, or the recombinant microorganism containing 19) the recombinant vector.

[0068] The eighth aspect of the present invention provides gene sequences related to the synthetic metabolic pathway of C35 hopanoids:

[0069] 1) hpnH The nucleic acid sequence is the sequence shown in SEQ ID NO.5;

[0070] 2) hpnG The nucleic acid sequence is the sequence shown in SEQ ID NO.6;

[0071] 3) hpnP The nucleic acid sequence is the sequence shown in SEQ ID NO.7;

[0072] 4) hpnO The nucleic acid sequence is the sequence shown in SEQ ID NO.8;

[0073] 5) hpnN The nucleic acid sequence is the sequence shown in SEQ ID NO.9;

[0074] The ninth aspect of the present invention provides a variety of promoter sequences for enhancing gene expression:

[0075] 1) The above-mentioned sequence for enhancing fni , fps , hpnX , hpnC expression is the sequence shown in SEQ ID NO.10;

[0076] 2) The promoter sequence of the above-mentioned expressed hpnH gene is the sequence shown in SEQ ID NO.10;

[0077] 3) The promoter sequence of the above-mentioned expressed hpnG gene is the sequence shown in SEQ ID NO 11;

[0078] 4) The promoter sequence of the above-mentioned expressed hpnP gene is the sequence shown in SEQ ID NO.12;

[0079] 5) The promoter sequence of the above-mentioned expressed hpnO gene is the sequence shown in SEQ ID NO.12;

[0080] 6) The promoter sequence of the above-mentioned expressed hpnN gene is the sequence shown in SEQ ID NO.14.

[0081] The beneficial effects of the present invention are as follows:

[0082] 1) The growth of SF45436( fps ) and SF45436( fni ) obtained in the present invention is significantly improved compared with the starting strain SF45436 under high-salt (150 mM NaCl) and high-alkali (pH9) stress environments;

[0083] 2) The growth of SF45436( fni + fps ), SF45436( hpnX ), and SF45436( fni + fps + hpnX ) obtained in the present invention is significantly improved compared with the starting strain SF45436 under high-salt (150 mM NaCl), high-alkali (pH9), and high-temperature (37°C) stress environments;

[0084] 3) The growth of SF45436:: hpnN and SF45436:: hpnNH is significantly improved compared with SF45436 under high-temperature (37°C) stress conditions;

[0085] 4) The growth of SF45436:: hpnNHGPO, SF45436( fni ):: hpnNHGPO , SF45436( fps ):: hpnNHGPO , SF45436( fni + fps ):: hpnNHGPO , and SF45436( hpnX ):: hpnNHGPO is significantly improved compared with SF45436 under high-temperature (37°C) and high-osmotic (15% sucrose) stress conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is Bradyrhizobium diazoefficiens the hopane synthesis pathway of USDA110 (BD110);

[0087] Figure 2 is Sinorhizobium fredii the hopane synthesis pathway of CCBAU45436 (SF45436);

[0088] Figure 3Growth detection of the strain with enhanced gene expression in the hopane metabolic pathway and SF45436 under high temperature (37 °C), sucrose (15%), salt (150 mM NaCl), and alkaline (pH 9) stress conditions; Panel A is a heat map of the high-temperature growth curves of different strains, Panel B is the growth curve of the strains under the dual stress of high temperature (37 °C) and sucrose (15%), and Panel C is the colony area of the engineered strain under the stress of sucrose (15%), salt (150 mM NaCl, Panel D), and alkaline (pH 9, Panel E) (normalized by the colony area of SF45436);

[0089] Figure 4 Growth detection of the strain with the constructed C35 hopane synthesis gene circuit and SF45436 under high temperature (37 °C), sucrose (15%), salt (150 mM NaCl), and alkaline (pH 9) stress conditions; Panel A is a heat map of the high-temperature growth curves of different strains, Panel B is the growth curve of the strains under the dual stress of high temperature (37 °C) and sucrose (15%), and Panel C is the colony area of the engineered strain under the stress of sucrose (15%), salt (150 mM NaCl, Panel D), and alkaline (pH 9, Panel E) (normalized by the colony area of SF45436);

[0090] Figure 5 Growth detection of the strain containing the C35 hopane synthesis gene circuit and with enhanced genes in the basic synthetic metabolic pathway and SF45436 under high temperature (37 °C), sucrose (15%), salt (150 mM NaCl), and alkaline (pH 9) stress conditions; Panel A is a heat map of the high-temperature growth curves of different strains, Panel B is the growth curve of the strains under the dual stress of high temperature (37 °C) and sucrose (15%), and Panel C is the colony area of the engineered strain under the stress of sucrose (15%), salt (150 mM NaCl, Panel D), and alkaline (pH 9, Panel E) (normalized by the colony area of SF45436).

[0091] Figure 6 Panel A shows Bradyrhizobium diazoefficiens The hopane metabolic pathway and hopane substances of USDA110 (BD110), and Panel B shows the mass spectrometry results of SF45436 and SF45436:: hpnNHGPO strains under the osmotic stress of high temperature (37 °C) and sucrose (15%). Specific implementation manners

[0092] The following further elaborates on the concept and technical effects of the present invention in conjunction with specific embodiments to fully understand the purpose, features, and effects of the present invention. Unless otherwise specified, the methods are all conventional methods. Unless otherwise specified, the materials can all be obtained from public commercial channels. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0093] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0094] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0095] 1. Experimental Materials

[0096] (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 .

[0097] (2) Culture media and reagents used

[0098] ① TY (Tryptone - Yeast Extract) medium / L: 3 g of yeast extract, 5 g of tryptone, 0.6 g of CaCl 2 0.6 g, pH 7.0. 5% sucrose is added to the sucrose screening medium. 15 g of agar powder is added to the solid medium. Autoclave at 121°C for 30 min.

[0099] ② LB (Luria Bertani medium) medium / L: 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, pH 7.2. 15 g of agar powder is added to the solid medium. Autoclave at 121°C for 30 min.

[0100] ③ 0.85% normal saline / L: 8.5 g of NaCl is made up to 1 L with deionized water and autoclaved at 121°C for 30 min.

[0101] ④ Nalidixic acid (NA): The storage concentration is 30 mg / mL, dissolved in ddH2O, solubilized with NaOH solution, filtered and sterilized through a 0.22 µm filter, and stored at -20°C after aliquoting;

[0102] ⑤ Trimethoprim (TMP): The storage concentration is 10 mg / mL, dissolved in ddH2O, solubilized with glacial acetic acid, filtered and sterilized through a 0.22 µm filter, and stored at -20°C after aliquoting;

[0103] ⑥ Gentamicin (Gen): The storage concentration is 30 mg / mL, dissolved in ddH2O, filtered and sterilized through a 0.22 µm filter, and stored at -20°C after aliquoting.

[0104] ⑦ N-(1,1 - dimethyl - 2 - hydroxyethyl)-3 - amino - 2 - hydroxypropanesulfonic acid (AMPSO): 9.0912 g of AMPSO powder is added to the TY medium, adjusted to pH 9 with HCl or KOH, made up to 1 L, and autoclaved at 121°C for 30 min.

[0105] (3) Plasmid vector:

[0106] ① The plasmid pJQ200SK is a suicide plasmid used to construct vectors for subsequent gene enhancement and complementation of C35 hopane synthesis. The strains constructed through subsequent triparental mating experiments do not carry any resistance markers, representing a markerless gene editing method.

[0107] ② pRJPaph-bjGFP is for cloning the promoter Praph sequence.

[0108] (4) Triparental mating experiment: A classic genetic manipulation method used to transfer plasmids from donor bacteria to target recipient bacteria, especially widely applied in bacteria where direct transformation is difficult (such as some Gram-negative bacteria). Triparental mating introduces a third bacterium as an "auxiliary bacterium" to provide the necessary conjugation functions.

[0109] 2. Description of the hopane synthesis metabolic pathway in rhizobia

[0110] For the hopane synthesis in rhizobia, it starts from the mevalonate pathway (MEP). The intermediate product farnesyl pyrophosphate (FPP) generated is the precursor for synthesizing squalene and hopane substances, as Figure 1 and Figure 2 shown: Sinorhizobium fredii SF45436 only synthesizes C30 hopane substances, namely squalene and hopene after squalene cyclization. In the basic metabolic pathway, fni and fps the transcriptional levels of genes are very low, and the transcriptional level of the gene cluster for synthesizing squalene is also very low. Sinorhizobium fredii SF45436 lacks the hpnCDEF genes for synthesizing C35 hopane, various modification genes hpnH , hpnG , hpnP , hpnO , as well as the hpnN gene required for transport to the outer membrane.

[0111] Example 1 Construction and growth identification of a transformed strain with enhanced hopane basic synthesis metabolic pathway in Sinorhizobium fredii SF45436.

[0112] 1.1 Construction of promoter-enhanced strains

[0113] 1) Extract the pJQ200SK plasmid, digest it with SmaI restriction endonuclease and recover it to measure the purity and concentration of the linear plasmid;

[0114] 2) Extract the pRJPaph-bjGFP plasmid, on the pathway fps , fni , hpnCDEFThe gene cluster expression is very low. To enhance the transcription of these three components, primers were designed according to the gene sequences of the three components to amplify the promoter Praph (SEQ ID NO:10). Upstream and downstream primers were designed according to the gene sequence to be enhanced, with a 20 bp homologous arm designed at the junction of the upstream and downstream fragments, and 20 bp homologous arms of pJQ200SK were added to both ends of the upstream and downstream fragments. Using the genome of Sinorhizobium fredii SF45436 as the DNA template, the target fragment was obtained by PCR amplification;

[0115] 3) The amplified fragment was recovered and seamlessly cloned with the linearized vector to obtain;

[0116] 4) After heat shock transformation, colony PCR was used for inspection and a vector with enhanced expression of the hopane synthesis-related gene was obtained, and the vector was preserved;

[0117] 5) In the triparental mating experiment, the vector was introduced into Sinorhizobium fredii SF45436 to obtain a modified strain with enhanced promoter.

[0118] 1.2 Identification of promoter-enhanced strains

[0119] PCR was used for identification, and after selecting the correct transformed positive strains, stress experiments were carried out.

[0120] 1.2.1 High-temperature growth curves of promoter-enhanced strains and wild-type Sinorhizobium fredii SF45436

[0121] The growth of the strains was detected in TY medium at high temperature (37°C) as follows: All modified strains and wild-type strains were activated on TY medium containing NA / TMP antibiotics. Single colonies were picked and transferred to TY liquid medium containing the corresponding antibiotics, and cultured at 28°C and 200 rpm until the logarithmic phase. The cells were harvested 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 the cell suspension of each strain was added to a honeycomb plate (BioscreenC) and placed in a Bioscreen C fully automatic growth curve analyzer of Growth Curves, Finland to measure the growth curve.

[0122] The results are as Figure 3 shown in 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.

[0123] 1.2.2 Growth curves of promoter - enhanced strains and wild - type SF45436 under dual stresses of high temperature and sucrose osmosis

[0124] The growth of the tested strains under 15% sucrose stress was detected in TY medium under the premise of high temperature (37°C) as follows: All the modified strains and the wild - type strain SF45436 were activated on TY medium containing NA / TMP antibiotics. Single colonies were picked and transferred to TY liquid medium containing the corresponding antibiotics. They were cultured at 28°C and 200 rpm until the logarithmic phase, and the cells were harvested by centrifugation at 6500 rpm for 5 min; The cells were washed twice with physiological saline and adjusted to OD 600 = 0.02 with 15% sucrose TY; 300 µL of the cell 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 Finland to measure the growth curve. The results are as Figure 3 shown in Figure B: The 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) grew significantly higher than the wild - type strain under dual stresses of high temperature and sucrose.

[0125] 1.2.3 Ability of promoter - enhanced strains and wild - type Sinorhizobium fredii SF45436 to adapt to 15% sucrose osmotic stress

[0126] Similarly, the cell suspensions of each modified strain and the wild - type strain in the logarithmic phase were adjusted to OD 600 = 0.2, and then the cell suspensions were diluted by 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10-6 Dilute by gradient, take 10 μL respectively and place them on TY solid medium containing 15% sucrose, and incubate statically in an incubator at 28 °C for 7 days. Calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain. The results are as Figure 3 shown in C: The promoter-enhanced strain Sinorhizobium fredii SF45436( fps ) (t-test; P = 0.0547) grows better than the wild-type strain SF45436 under 15% sucrose stress.

[0127] 1.2.4 Ability of promoter-enhanced strains and wild-type SF45436 to adapt to 150 mM NaCl osmotic stress

[0128] Adjust the logarithmic-phase transformed strains and wild-type strain to OD 600 = 0.2, and then dilute the bacterial solution by gradients of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Take 10 μL respectively and place them on TY solid medium containing 150 mM NaCl, and incubate statically in an incubator at 28 °C for 7 days. Calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain SF45436. The results are as Figure 3 shown in D: The promoter-enhanced strains 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) have significantly higher growth ability than the wild-type SF45436 under 150 mM NaCl stress.

[0129] 1.2.5 Ability of promoter-enhanced strains and wild-type SF45436 to adapt to high-alkali (pH 9) stress

[0130] Adjust the logarithmic-phase transformed strains and wild-type strain to OD 600 = 0.2, and then dilute the bacterial solution by gradients of 10 -1 , 10-2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution was performed. 10 μL was taken respectively and cultured statically in a pH 9 TY solid medium in an incubator at 28 °C for 7 days. The area of the bacterial lawn or colony was calculated using ImageJ and normalized with the area of the wild-type strain SF45436. The results are shown in Figure 3 E: 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) had significantly higher growth ability than the wild-type strain SF45436 under pH 9 stress.

[0131] Example 2 Construction and growth identification of Sinorhizobium fredii SF45436 strain with C35 hopane synthesis and metabolism pathway

[0132] 2.1 Construction of strain with C35 hopane synthesis and metabolism pathway

[0133] 1) Genomes of Sinorhizobium fredii SF45436 and Bradyrhizobium diazoefficiens BD110 were extracted. According to the genes related to the synthesis and transport of the C35 hopane synthesis and metabolism pathway of BD110 hpnH , hpnG , hpnP , hpnO , hpnN , and according to the insertion site sequence of SF45436, upstream and downstream fragments, promoters and hpnHGPON primers were designed. Upstream and downstream fragments, promoters and each gene fragment were amplified by PCR: Praph- hpnH , PrpoB- hpnG , PrpoD- hpnP + hpnO , PcdnL- hpnN .

[0134] Among them, 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; hpnG The 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;

[0135] 2) Recover the product of the amplified fragment and perform seamless cloning with the linearized vector;

[0136] 3) After heat shock transformation, perform colony PCR verification to obtain the heterologous expression hopane gene vector and preserve the vector;

[0137] 4) Through a triparental mating experiment, introduce the vector into Sinorhizobium fredii SF45436 to obtain a modified strain heterologously expressing genes related to C35 hopane synthesis and transport.

[0138] 2.2 Identification of the heterologous expression strain of genes related to C35 hopane synthesis and transport

[0139] 2.2.1 High-temperature growth curves of the heterologous expression strain of genes related to C35 hopane synthesis and transport and the wild-type SF45436

[0140] Detect the growth of the strains in TY medium under high temperature (37 °C) conditions as follows: Activate all modified strains and the wild-type strain SF45436 on TY medium containing NA / TMP antibiotics, pick a single colony and transfer it to TY liquid medium containing the corresponding antibiotics, culture at 28 °C and 200 rpm until the logarithmic phase, and collect the cells by centrifugation at 6500 rpm for 5 min; Wash the cells twice with physiological saline and adjust to OD 600 = 0.02; Take 300 µL of the cell suspension of each strain and add it to a honeycomb plate (Bioscreen C), and place it in the Bioscreen C fully automatic growth curve analyzer of Growth Curves, Finland, to measure the growth curve. The results are as Figure 4 shown in A: The heterologous expression strain of genes related to C35 hopane synthesis and transport SF45436:: hpnN (t-test; ****, P < 0.0001), SF45436:: hpnNH(t - test; ****, P < 0.0001), SF45436:: hpnNHGPO (t - test; ****, P < 0.0001) showed significantly higher growth than the wild - type strain SF45436 under high - temperature stress.

[0141] 2.2.2 Growth curves of heterologous - expression strains related to C35 hopane synthesis and transport and wild - type SF45436 under dual stress of high temperature and sucrose osmosis

[0142] The growth of the tested strains under 15% sucrose stress was detected in TY medium at high temperature (37°C) as follows: All the modified strains and the wild - type strain SF45436 were activated on TY medium containing NA / TMP antibiotics. Single colonies were picked and transferred to TY liquid medium containing the corresponding antibiotics, and cultured at 28°C and 200 rpm until the logarithmic phase. The cells were harvested by centrifugation at 6500 rpm for 5 min; The cells were washed twice with physiological saline, and adjusted to OD 600 = 0.02 with 15% sucrose - containing TY; 300 μL of the cell suspension of each strain was added to a honeycomb plate (Bioscreen C) and placed in the Bioscreen C fully automatic growth curve analyzer of Finland Growth Curves Company to measure the growth curve. The results are as Figure 4 shown in Figure B: The heterologous - expression strains related to C35 hopane synthesis and transport, SF45436:: hpnN (t - test; **, P < 0.01 (72 h)) showed significantly higher growth than the wild - type strain SF45436 under high - temperature and sucrose stress in the early logarithmic growth phase; SF45436:: hpnNH (t - test; ***, P < 0.001), SF45436:: hpnNHGPO (t - test; **, P < 0.01) showed significantly higher growth than the wild - type strain SF45436 under dual stress of high temperature and sucrose.

[0143] 2.2.3 Ability of heterologous - expression strains related to C35 hopane synthesis and transport and wild - type SF45436 to adapt to 15% sucrose osmotic stress

[0144] The cell suspensions of each modified strain and the wild - type strain SF45436 in the logarithmic phase were adjusted to OD 600 = 0.2, and then the cell suspensions were diluted by 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6Dilute by gradient, take 10 µL respectively and place them on the TY solid medium containing 15% sucrose, and statically culture them in an incubator at 28 °C for 7 days. Calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain SF45436. The results are as Figure 4 shown in C: The heterologous expression strains SF45436:: hpnN (t-test; P = 0.0520), SF45436:: hpnNHGPO (t-test; P = 0.0365) grow better than the wild-type SF45436 under 15% sucrose stress.

[0145] 2.2.4 Ability of the heterologous expression strains related to C35 hopane synthesis and transport and the wild-type SF45436 to adapt to 150 mM NaCl osmotic stress

[0146] Adjust the logarithmic-phase modified strains and the wild-type strain SF45436 to OD 600 = 0.2, and then dilute the bacterial liquid by gradients of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Take 10 µL respectively and place them on the TY solid medium containing 150 mM NaCl, and statically culture them in an incubator at 28 °C for 7 days. Calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain SF45436. The results are as Figure 4 shown in D: There is no significant difference in the growth ability of each modified strain under 150 mM NaCl stress compared with the wild-type SF45436.

[0147] 2.2.5 Ability of the heterologous expression strains related to C35 hopane synthesis and transport and the wild-type SF45436 to adapt to high-alkali (pH 9) stress

[0148] Adjust the logarithmic-phase modified strains and the wild-type strain SF45436 to OD 600 = 0.2, and then dilute the bacterial liquid by gradients of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Take 10 µL respectively and place them on the TY solid medium with pH 9, and statically culture them in an incubator at 28 °C for 7 days. Calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain SF45436. The results are asFigure 4 As shown in E: SF45436:: hpnN (t-test; *, P < 0.05) Its growth ability under pH9 stress was significantly higher than that of the wild-type SF45436.

[0149] Example 3 Identification of Strains with Enhanced Expression of Hopane Basic Anabolic Genes and Heterologous Expression of Genes Related to C35 Hopane Synthesis in Sinorhizobium fredii CCBAU45436 (SF45436)

[0150] 3.1 Identification of Strains with Enhanced Expression of Hopane Basic Anabolic Gene Circuit and Heterologous Expression of C35 Hopane Anabolic Pathway

[0151] On the basis of the strains obtained in Example 1, the 5 genes described in Example 2 were sequentially transformed to obtain the strains of this example. After correct PCR verification, stress resistance detection was carried out.

[0152] 3.1.1 High-temperature Growth Curves of Strains with Enhanced Expression of Hopane Basic Anabolic Gene Circuit and Heterologous Expression of C35 Hopane Anabolic Pathway and Wild-type SF45436

[0153] The growth of the strains was detected in TY medium under high temperature (37°C) conditions as follows: All the modified strains and the wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics. Single colonies were picked and transferred to TY liquid medium containing the corresponding antibiotics. They were cultured at 28°C and 200 rpm until the logarithmic phase, and the cells were harvested 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 the cell suspension of each strain was added to a honeycomb plate (Bioscreen C) and placed in a Bioscreen C fully automatic growth curve analyzer of Growth Curves, Finland, to measure the growth curve. The results are as Figure 5 shown in A: The modified 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) showed significantly higher growth than the wild-type strain SF45436 under high temperature stress.

[0154] 3.1.2 Growth curves of strains with enhanced expression of hopane basic synthetic metabolic gene circuits and heterologous expression of the C35 hopane synthetic metabolic pathway under dual stresses of high temperature and sucrose osmosis compared with the wild-type SF45436

[0155] The growth of the tested strains under 15% sucrose stress was detected in TY medium at a high temperature (37°C) as follows: All the engineered strains and the wild-type strain SF45436 were activated on TY medium containing NA / TMP antibiotics. Single colonies were picked and transferred to TY liquid medium containing the corresponding antibiotics, and cultured at 28°C and 200 rpm until the logarithmic phase. The cells were harvested by centrifugation at 6500 rpm for 5 min; the cells were washed twice with physiological saline, and adjusted to OD 600 = 0.02 with 15% sucrose TY; 300 µL of the cell 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 Finland to measure the growth curve. The results are as Figure 5 shown in B: The engineered 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 under the dual stress of high temperature and sucrose was significantly higher than that of the wild-type strain SF45436.

[0156] 3.1.3 Ability of the strains with enhanced expression of the hopane basic synthetic metabolic gene circuit and heterologous expression of the C35 hopane synthetic metabolic pathway and the wild-type SF45436 to adapt to 15% sucrose osmotic stress

[0157] Similarly, the logarithmic-phase modified strains and the wild-type strain were adjusted to OD 600 = 0.2, and then the bacterial liquid was diluted in gradients of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 respectively. 10 μL of each dilution was taken and cultured statically on TY solid medium containing 15% sucrose in an incubator at 28 °C for 7 days. The areas of the bacterial lawns or colonies were calculated using ImageJ and normalized with the area of the wild-type strain SF45436. The results are shown in Figure 5 Figure C: The modified 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 the wild-type SF45436 under 15% sucrose stress.

[0158] 3.1.4 Ability of the strains with enhanced expression of the hopane basic synthetic metabolic gene circuit and heterologous expression of the C35 hopane synthetic metabolic pathway and the wild-type SF45436 to adapt to 150 mM NaCl osmotic stress

[0159] The logarithmic-phase modified strains and the wild-type strain SF45436 were adjusted to OD 600 = 0.2, and then the bacterial liquid was diluted in gradients of 10 -1 、10 -2 、10 -3 、10 -4 、10 - 5、10 -6Dilute by gradient, take 10 µL respectively and place them on TY solid medium containing 150 mM NaCl, statically culture in an incubator at 28 °C for 7 days, calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain SF45436. The results are as Figure 5 shown in fni + fps D: The reconstructed strain SF45436( hpnNHGPO )(t-test; *, P < 0.05) has significantly higher growth ability than the wild-type SF45436 under 150 mM NaCl stress.

[0160] 3.1.5 Ability of strains with enhanced expression of hopane basic synthetic metabolic gene circuits and heterologous expression of C35 hopane synthetic metabolic pathways to adapt to high-alkali (pH9) stress compared with the wild-type SF45436

[0161] Adjust the logarithmic-phase reconstructed strains and the wild-type strain SF45436 to OD 600 = 0.2, then dilute the bacterial liquid by gradients of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Take 10 µL respectively and place them on pH9 TY solid medium, statically culture in an incubator at 28 °C for 7 days, calculate the area of the bacterial lawn or colony using ImageJ, and standardize it with the area of the wild-type strain SF45436. The results are as Figure 5 shown in E: There is no significant difference in the ability of each reconstructed strain to adapt to high-alkali (pH9) stress compared with the wild-type SF45436.

[0162] Example 4 Identification of hopane substances produced by strains with heterologous expression of genes related to C35 hopane synthesis and transport

[0163] SF45436:: hpnNHGPO and the wild-type strain SF45436 are activated on TY medium containing NA / TMP antibiotics. Pick a single colony and transfer it to TY liquid medium containing the corresponding antibiotics. Culture at 28 °C and 200 rpm until the logarithmic phase, and collect the bacterial cells by centrifugation at 6500 rpm for 5 min; wash the bacterial cells twice with physiological saline, and inoculate the bacterial liquid into 200 mL TY + 15% sucrose medium until the final concentration is OD 600 = 0.05, and expand the culture at 37 °C. When the bacterial liquid is cultured to OD 600= around 0.8. The cells were harvested by centrifugation at 4 °C and 5000 rpm for 15 min. The total lipids were extracted with dichloromethane, dried in vacuo and dissolved in dichloromethane / methanol (volume ratio 9:1) to a final concentration of 1 mg / mL. Dilute 100-fold with dichloromethane for mass spectrometry analysis. The results are as Figure 6 shown in A-B: SF45436:: hpnNHGPO can produce the methylated hopane substance 2-methyl bacteriohopanetetrol (2-methyl BHT).

[0164] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rhizobium for improving environmental stress tolerance, characterized in that: The rhizobium expresses the C30 hopane basic anabolism gene in the following manner: Promoter Praph starts gene f , fps , x co-expression of; or, Promoter Praph starts gene f , fps , HkDJ co-expression of Wherein, 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; The rhizobium is Sinorhizobium freundii.

2. A rhizobium for improving environmental stress tolerance, characterized in that: The rhizobium simultaneously expresses five C35 hopane anabolism and transport-related genes in the following manner: Praph-mediated promoter HkDJ The expression Promoter PrpoB mediated HkDJ The expression Promoter PrpoD-mediated hp + oeLh expression, and PcdnL-mediated promoter n Expression; The sequence of the promoter 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 C35 hopane anabolism and transport related genes HkDJ , HkDJ , hp , oeLh , hpnN The sequences are shown in SEQ ID NOs: 5 to 9 respectively; The rhizobium is Sinorhizobium freundii.

3. A rhizobium for improving environmental stress tolerance, characterized in that: The rhizobium is based on the rhizobium described in claim 1, and the five C35 hopane synthesis metabolism and transport related genes described in claim 2 are induced by the method described in claim 2. HkDJ , HkDJ , hp , oeLh , n Overexpression of The rhizobium is Sinorhizobium freundii.

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