Method for improving salt tolerance of peanut rhizobium

Abstract

The invention relates to the technical field of agriculture, in particular to a method for improving salt tolerance of peanut rhizobium, which specifically comprises the following steps: step 1, constructing an otsA overexpression engineering bacterium strain; 2, carrying out salt tolerance analysis on the engineering bacteria; 3, analyzing the nitrogen fixation capability of the peanuts inoculated with the engineering bacteria; 4, analyzing agronomic characters of the peanuts inoculated with the engineering bacteria; according to the method, otsA overexpression engineering bacteria are constructed, so that the salt tolerance of peanut rhizobium is effectively improved, the nitrogen fixation capacity and agronomic traits of peanuts under salt stress are enhanced, and a sustainable solution for reducing nitrogen fertilizer dependence is provided for peanut planting in saline-alkali soil.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, in particular to a method for improving the salt tolerance of peanut rhizobia. Background Art

[0002] Peanuts are an important edible and oil-bearing legume crop, with China being a major producer. However, the peanut industry faces challenges due to climate change, pests and diseases, and soil salinization. Symbiotic nitrogen fixation between rhizobia and peanuts can reduce reliance on chemical nitrogen fertilizers. However, under salt stress, leguminous plants experience insufficient nutrient supply to their roots, hindering the synthesis of leghemoglobin and homocitrate within the nodules, reducing nitrogen fixation capacity. This, in turn, impacts rhizobia abundance and distribution, reducing symbiotic nitrogen fixation efficiency. Soil salinization poses a serious threat to crop growth, causing reduced grain yields, ecological damage, and economic losses, and is poised to expand. While research on peanut salt tolerance has made some progress, including the discovery of several salt-tolerance genes and successful cultivation in saline-alkali soils through strain improvement and agronomic manipulation, the coordinated response mechanisms between rhizobia and their hosts in saline-alkali soils remain underdeveloped.

[0003] Existing methods for improving the salt tolerance of peanut rhizobia have limitations, and it is difficult to fundamentally solve the problems of low nitrogen fixation efficiency of rhizobia and poor salt tolerance of peanuts in peanut cultivation in saline-alkali land. Summary of the Invention

[0004] In view of the limitations of the existing methods for improving the salt tolerance of peanut rhizobia, it is difficult to fundamentally solve the technical problems of low nitrogen fixation efficiency of rhizobia and poor salt tolerance of peanuts in peanut cultivation in saline-alkali land. The present invention provides a method for improving the salt tolerance of peanut rhizobia.

[0005] The technical solution adopted by the present invention is: a method for improving the salt tolerance of peanut rhizobia, which specifically comprises the following steps:

[0006] Step 1: construct an otsA overexpression engineered bacterial strain;

[0007] Step 2: Conduct salt tolerance analysis of the engineered bacteria;

[0008] Step 3: Analyze the nitrogen fixation ability of peanuts inoculated with engineered bacteria;

[0009] Step 4: Analyze the agronomic traits of peanuts inoculated with engineered bacteria.

[0010] In one embodiment, in step 1, the specific method for constructing an otsA overexpressing engineered bacterial strain is as follows:

[0011] Extraction of rhizobium genomic DNA: The SDS / CTAB method was used to extract rhizobium genomic DNA in preparation for the subsequent cloning of the otsA gene;

[0012] Select vector to construct overexpression vector: Select broad-host expression vector pBBRIMCS-5 to construct overexpression vector pBBRIMCS-5-otsA;

[0013] in,

[0014] The nucleotide sequence of the pBBRIMCS-5-otsA gene is shown in SEQ ID NO. 1;

[0015] The nucleotide sequence of the otsA gene is shown in SEQ ID NO. 2;

[0016] Transfer the vector into rhizobium cells: Use the modified heat shock method to transfer the constructed vector into rhizobium cells, then screen with antibiotics and use colony PCR to select the positive strains with successful transfer.

[0017] In one embodiment, in step 2, the specific method for analyzing the salt tolerance of the engineered bacteria is as follows:

[0018] Rhizobium overexpressing the otsA gene (O-otsA) and wild-type rhizobium (CCBAU25338) were cultured on YMA plates with different salt concentrations (50mM, 100mM, 150mM), and their growth at different dilution multiples was compared.

[0019] In one embodiment, in step 3, the specific method for analyzing the nitrogen fixation ability of peanuts inoculated with engineered bacteria is as follows:

[0020] Evaluation of symbiotic nitrogen fixation efficiency: The symbiotic nitrogen fixation efficiency was evaluated by detecting the number of rhizobia under different NaCl levels;

[0021] Determination of nitrogenase activity: The nitrogenase activity of peanut roots inoculated with different strains was measured when treated with 150 mM NaCl;

[0022] Detection of gene expression: Using the peanut "alcohol dehydrogenase" encoding gene LOC112715878 as the internal reference gene, the qRT-PCR technology was used to determine the expression levels of peanut nodulation reporter genes (Castor, CCaMK, SYMRK) and nitrogen fixation reporter genes (AMT1.1, NRT1.1, NRT1.2).

[0023] In one embodiment, in step 4, the specific method for analyzing the agronomic traits of peanuts inoculated with engineered bacteria is as follows:

[0024] During the 40th day of nitrogen fixation, photosynthesis analysis, plant height, and dry and fresh weight of peanuts inoculated with different strains under salt stress were performed.

[0025] The beneficial effects of the present invention are as follows: compared with the existing technology, the present invention effectively improves the salt tolerance of peanut rhizobia by constructing otsA overexpression engineered bacteria, enhances the nitrogen fixation ability and agronomic traits of peanuts under salt stress, and provides a sustainable solution for reducing nitrogen fertilizer dependence for peanut cultivation in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a map of the vector for overexpressing the otsA gene in the present invention;

[0027] Figure 2 is the qRT-PCR gene expression level O-otsA in the present invention;

[0028] Figure 3 This is a graph showing the effects of different strains on peanut nodule number and nitrogenase activity under different salt concentrations in the present invention;

[0029] Figure 4 This is a graph showing the effects of different strains on the expression of genes related to peanut nodulation and nitrogen fixation under different salt concentrations in the present invention. DETAILED DESCRIPTION

[0030] In the description of the present invention, it should be noted that the terms "front", "up", "down", "left", "right", "vertical", "horizontal", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limiting the present invention.

[0031] refer to Figure 1-Figure 4 In order to solve the problems existing in the background technology, the present application proposes the following technical solution: a method for improving the salt tolerance of peanut rhizobia, specifically comprising the following steps:

[0032] Step 1: construct an otsA overexpression engineered bacterial strain;

[0033] In step 1, the specific method for constructing an otsA overexpression engineered bacterial strain is as follows:

[0034] Extraction of rhizobium genomic DNA: The SDS / CTAB method was used to extract rhizobium genomic DNA in preparation for the subsequent cloning of the otsA gene;

[0035] Select a vector to construct an overexpression vector: select the broad-host expression vector pBBRIMCS-5 and construct the overexpression vector pBBRIMCS-5-otsA; this vector can overexpress the otsA gene under the control of its lac promoter.

[0036] in,

[0037] The nucleotide sequence of the pBBRIMCS-5-otsA gene is shown in SEQ ID NO. 1;

[0038] The nucleotide sequence of the otsA gene is shown in SEQ ID NO. 2;

[0039] Transfer the vector into rhizobium cells: Use the modified heat shock method to transfer the constructed vector into rhizobium cells, then screen with antibiotics, and then use colony PCR (primers only need the target gene sequence) to select the positive strains with successful transfer. Finally, send them to the company for sequencing to confirm that the positive strains have been successfully transferred.

[0040] refer to Figure 1 and Figure 2 In order to determine the specific function of otsA in the trehalose metabolism process, the present invention first constructed an overexpression strain in rhizobia. The otsA gene was cloned, and the SDS / CTAB method was used to extract the rhizobium genomic DNA. The wide-host expression vector pBBRIMCS-5 was selected to construct the overexpression vector pBBRIMCS-5-otsA (O-otsA), which can overexpress the target gene under the control of its lac promoter. The constructed vector was transferred into the rhizobium cells through a modified heat shock method. After antibiotic screening, colony PCR (primers only need the target gene sequence) was performed, and the positive strains with successful transfer were selected and sent to the company for sequencing to confirm that the positive strains were successfully transferred.

[0041] Step 2: Conduct salt tolerance analysis of the engineered bacteria;

[0042] In step 2, the specific method for analyzing the salt tolerance of the engineered bacteria is as follows:

[0043] Rhizobia overexpressing the otsA gene (O-otsA) and wild-type rhizobia (CCBAU25338) were cultured on YMA plates at different salt concentrations (50mM, 100mM, and 150mM), and their growth at different dilutions was compared. The results showed that O-otsA exhibited significantly better salt tolerance than the wild-type at all salt concentrations, demonstrating that overexpression of the otsA gene can enhance salt tolerance in rhizobia.

[0044] Table 1 Effect of overexpression of otsA gene on salt tolerance of rhizobia

[0045]

[0046] Table 1 shows the effect of overexpressing the otsA gene on the salt tolerance of rhizobia in the present invention (according to the order from top to bottom in Table 1, they are YMA, YMA+50mMNaCl, YMA+100mMNaCl, and YMA+150mMNaCl; according to the order from left to right in Table 1, they are the cases of dilution 1, 2, 5, 10, and 50 times, respectively;

[0047] In Table 1, + indicates that there is colony growth, but the number is small; ++ indicates that the number of colonies is moderate (between a small amount and a large amount); +++ indicates that there is a large amount of colonies; ++++ indicates that there is excessive colony growth; - indicates that there is no colony growth.

[0048] Referring to Table 1, under salt stress conditions, the salt tolerance of rhizobia overexpressing the otsA gene (O-otsA) was significantly better than that of the wild type CCBAU25338): at a salt concentration of 50 mM, O-otsA could still grow normally after being diluted 10 times (the wild type could only tolerate a dilution of 2 times); at 100 mM, O-otsA could tolerate a dilution of 5 times (the wild type could still tolerate a dilution of 2 times); at 150 mM, O-otsA could still partially survive after being diluted 1-2 times, while the wild type could not grow at all.

[0049] Step 3: Analyze the nitrogen fixation ability of peanuts inoculated with engineered bacteria;

[0050] In step 3, the specific method for analyzing the nitrogen fixation ability of peanuts inoculated with engineered bacteria is as follows:

[0051] First, the symbiotic nitrogen fixation efficiency was evaluated by the number of rhizobia under different NaCl levels;

[0052] Then, under 300 mM NaCl treatment, inoculation of rhizobia (wt and engineered strains) resulted in an increase in the number of rhizobia compared with the control, among which the O-otsA strain increased by 30% compared with the wild type (wt) (p<0.05, n=6).

[0053] refer to Figure 4 To further determine the nitrogen-fixing capacity of the engineered rhizobium strains in their host plants, we assessed nitrogenase activity in peanut roots. Under 150 mM NaCl treatment, nitrogenase activity in peanut roots inoculated with O-otA was significantly higher than that in roots inoculated with other strains (p < 0.05, n = 6). This suggests that overexpression of otsA specifically enhances nitrogen fixation under ionic stress.

[0054] The expression levels of peanut nodulation reporter genes Castor, CCaMK, SYMRK and nitrogen fixation reporter genes AMT1.1, NRT1.1, NRT1.2 were measured by qRT-PCR technology using the peanut "alcohol dehydrogenase" encoding gene LOC112715878 as the internal reference gene.

[0055] Step 4: Analyze the agronomic traits of peanuts inoculated with engineered bacteria.

[0056] In step 4, the specific method for analyzing the agronomic traits of peanuts inoculated with engineered bacteria is as follows:

[0057] First, in the 300 mM NaCl-treated group, the O-otsA strain was inoculated;

[0058] However, the net photosynthetic rate of peanuts inoculated with the O-otsA strain was 27.2% higher than that of WT (p < 0.05, n = 6), and the plant height was 18.7% higher (p < 0.05, n = 6), confirming that the otsA gene promoted symbiotic performance by coordinating osmotic regulation and carbon metabolism.

[0059] Next, the agronomic traits at 40 days of nitrogen fixation period showed obvious phenotypes under salt stress;

[0060] Secondly, in the photosynthesis analysis, in the 300mMNaCl treatment group, the net photosynthetic rate of peanuts inoculated with the O-otsA strain was 27.2% higher than that of WT (p<0.05, n=6), indicating that the photosynthetic recovery ability of peanuts inoculated with the O-otsA strain was enhanced under lethal salt stress.

[0061] In addition, under 300 mM NaCl treatment, the plant height of the inoculated O-otsA increased by 18.7% (p<0.05, n=6) compared with the WT.

[0062] These data thus confirm that otsA is a key gene that promotes symbiotic performance by coordinating osmotic regulation and carbon metabolism under ionic stress.

[0063] In summary, it was confirmed that the otsA gene is a key gene that promotes symbiotic performance by coordinating osmotic regulation and carbon metabolism under ion stress.

[0064] Table 2 Effects of inoculation with different strains on peanut growth at different salt levels

[0065]

[0066] In summary, under 150 mM salt stress, rhizobia overexpressing otsA (O-otsA) still showed some growth (although not completely normal) when grown on a 1-2-fold diluted medium, whereas the wild-type strain (CCBAU25338) was completely unable to grow at this salt concentration. This suggests that the O-otsA strain still possesses some salt tolerance under 150 mM NaCl stress, significantly superior to the wild-type and knockout strains.

[0067] In addition, rhizobia overexpressing the otsA gene (O-otsA) significantly improved the tolerance of peanuts to salt stress. Under 150mMNaCl stress, the nitrogenase activity of peanuts inoculated with O-otsA was significantly higher than that of the wild type (WT, i.e., the original strain CCBAU25338), and the expression levels of nitrogen fixation genes (such as AMT1.1, NRT1.1) and nodulation genes (Castor, CCaMK, etc.) in their nodules increased by 1.8-2.3 times (p<0.05).

[0068] Under lethal salt concentration (300mMNaCl), the number of rhizobia in the O-otsA inoculation group increased by 30% compared with the WT, while the net photosynthetic rate and plant height increased by 27.2% and 18.7%, respectively (p<0.05), indicating that the strain systematically improved the salt tolerance and physiological recovery ability of the host peanut by enhancing the efficiency of symbiotic nitrogen fixation and coordinating carbon metabolism and osmotic regulation.

[0069] The specific methods for constructing the engineered bacterial strain and the otsA sequence are as follows:

[0070] By cloning the otsA gene and selecting the broad-host expression vector pBBRIMCS-5, the overexpression vector pBBRIMCS-5-otsA (O-otsA) was constructed. This vector can overexpress the target gene under the control of its lac promoter. The constructed vector was transferred into rhizobium cells through a modified heat shock method. After antibiotic screening, colony PCR (primers only need the target gene sequence) was performed, and the positive strains with successful transfer were selected and sent to the company for sequencing to confirm that the positive strains were successfully transferred.

[0071] In addition, the otsA gene significantly enhances the salt tolerance of rhizobia by regulating trehalose synthesis. In saline soil, peanuts inoculated with the O-otsA strain show stronger stress resistance. This technology can reduce dependence on nitrogen fertilizers and provide a sustainable solution for the cultivation of legume crops in saline-alkali land. It is especially suitable for the adverse cultivation of economic crops such as peanuts and soybeans.

[0072] In summary, the present invention successfully improved the salt tolerance of rhizobia by constructing otsA-overexpressing engineered bacteria. Under 150mM NaCl stress, wild-type rhizobia were completely unable to grow, while rhizobia overexpressing otsA (O-otsA) still partially survived when diluted 1-2 times, greatly improving the survival of rhizobia in saline-alkali environments.

[0073] In addition, the present invention significantly enhances the tolerance and physiological recovery ability of peanuts under salt stress. This method reduces the dependence of peanut cultivation on nitrogen fertilizer, provides a sustainable solution for the cultivation of legume crops in saline-alkali land, effectively alleviates the constraints of soil salinization on the peanut industry, reduces economic losses, and has important ecological and economic benefits.

[0074] Thus, by constructing an otsA overexpression engineered bacterium in the present invention, the salt tolerance of peanut rhizobia was effectively improved, the nitrogen fixation ability and agronomic traits of peanuts under salt stress were enhanced, and a sustainable solution for reducing nitrogen fertilizer dependence for peanut cultivation in saline-alkali land was provided.

[0075] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the salt tolerance of peanut rhizobia, characterized in that: The specific steps include: Step 1: construct an otsA overexpression engineered bacterial strain; Step 2: Conduct salt tolerance analysis of the engineered bacteria; Step 3: Analyze the nitrogen fixation ability of peanuts inoculated with engineered bacteria; Step 4: Analyze the agronomic traits of peanuts inoculated with engineered bacteria.

2. A method for improving the salt tolerance of peanut rhizobia according to claim 1, characterized in that: In step 1, the specific method for constructing an otsA overexpression engineered bacterial strain is as follows: Extraction of rhizobium genomic DNA: The SDS / CTAB method was used to extract rhizobium genomic DNA in preparation for the subsequent cloning of the otsA gene; Select vector to construct overexpression vector: Select broad-host expression vector pBBRIMCS-5 to construct overexpression vector pBBRIMCS-5-otsA; Transform the vector into rhizobium cells: Use a modified heat shock method to transform the constructed vector into rhizobium cells, then screen with antibiotics and use colony PCR to select the positive strains with successful transformation; in, The nucleotide sequence of the pBBRIMCS-5-otsA gene is shown in SEQ ID NO. 1; The nucleotide sequence of the otsA gene is shown in SEQ ID NO.

2.

3. A method for improving the salt tolerance of peanut rhizobia according to claim 1, characterized in that: In step 2, the specific method for analyzing the salt tolerance of the engineered bacteria is as follows: Rhizobia overexpressing the otsA gene and wild-type rhizobia were cultured on YMA plates with different salt concentrations, and their growth at different dilution multiples was compared.

4. The method for improving the salt tolerance of peanut rhizobia according to claim 1, wherein: In step 3, the specific method for analyzing the nitrogen fixation ability of peanuts inoculated with engineered bacteria is as follows: Evaluation of symbiotic nitrogen fixation efficiency: The symbiotic nitrogen fixation efficiency was evaluated by detecting the number of rhizobia under different NaCl levels; Determination of nitrogenase activity: The nitrogenase activity of peanut roots inoculated with different strains was measured when treated with 150 mM NaCl; Detection of gene expression: Using the peanut "alcohol dehydrogenase" encoding gene LOC112715878 as the internal reference gene, the expression levels of peanut nodulation reporter gene and nitrogen fixation reporter gene were determined by qRT-PCR technology.

5. The method for improving the salt tolerance of peanut rhizobia according to claim 1, wherein: In step 4, the specific method for analyzing the agronomic traits of peanuts inoculated with engineered bacteria is as follows: During the 40th day of nitrogen fixation, photosynthesis analysis, plant height, and dry and fresh weight of peanuts inoculated with different strains under salt stress were performed.

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