CN114507620B - Rhodococcus Fan Qingsheng strain and acquisition method and application thereof - Google Patents
Rhodococcus Fan Qingsheng strain and acquisition method and application thereof Download PDFInfo
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Abstract
The invention discloses a rhodococcus Fan Qingsheng strainRhodococcus qingshenggii) Srq-21 and an acquisition method and application thereof, belonging to the technical field of microorganism application. Fan Qingsheng rhodococcus furiosus @Rhodococcu sqingshenggii) Srq-21 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23566, and the Fan Qingsheng rhodococcus garvieae is preservedRhodococcus qingshenggii) Srq-21 belongs to the eubacteria kingdom, the phylum actinomycota, the order Corynebacterium, the family Nocardia, and the genus Rhodococcus. The obtaining method comprises the procedures of separation, screening and purification, and is applied to the rhodococcus Fan Qingsheng #Rhodococcus qingshenggii) The application of Srq-21 in preparing single azotobacter seed bacterial agent for promoting plant growth.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and further belongs to the technical field of microorganism separation culture application, and in particular relates to a Fan Qingsheng rhodococcus garvieae strain, an acquisition method and application thereof.
Background
Apples are the second fruit of the world yield rank, next to bananas (statistically cut-off to 2016). The cultivation area of Chinese apples and the total yield are the first of the world apple major countries, and are the first apple producing major countries. With intensive planting of apples in large quantities, the problems of improving the utilization rate of fertilizer and reducing the environmental load of soil are increasingly important. This applies firstly to nitrogen fertilizers, which are the most limiting nutrient substances for crop yield, which combine the properties of high energy intensity and high pollution to the environment. High doses of nitrogen fertilizer application can negatively impact soil properties and fruit quality.
The nitrogen fixed by biological nitrogen fixation can be combined with organic matters and is not easy to run off, and can be completely utilized by organisms without polluting the environment. Therefore, the research of biological nitrogen fixation has important significance in improving ecological balance and promoting agricultural sustainable development. Endophytes are located inside plants and live in plant tissues without causing plant disorders or injury. They can form complex co-association with plants, promote the growth and nutrient absorption of host plants in natural environment, and improve the stress resistance and resistance to pathogenic bacteria of plants. The endophyte is an endophyte which establishes a combined nitrogen fixation system in plant tissues so as to form a close reciprocal symbiotic relationship with plants. In the summary report of the fifth international non-leguminous plant nitrogen fixation meeting called in 1993, the evaluation of the endophytic nitrogen fixation bacteria opens up a new way for expanding biological nitrogen fixation to non-nodulation plants.
Disclosure of Invention
The invention belongs to the technical field of microorganisms, and further belongs to the technical field of microorganism separation culture application, and in particular relates to a Fan Qingsheng rhodococcus garvieae strain, an acquisition method and application thereof.
A first object of the present invention is to provide a strain of rhodococcus Fan Qingsheng; a second object is to provide the method for obtaining the Fan Qingsheng rhodococcus; a third object is to provide the use of said rhodococcus Fan Qingsheng.
The first object of the present invention is achieved by a strain of rhodococcus Fan Qingsheng #Rhodococcusqingshengii) Named Srq-21 and identified as Fan Qingsheng rhodococcus erythropolis @Rhodococcusqingshengii) Is separated from the bark of Fuji apples, and is preserved in China general microbiological culture collection center (CGMCC, address is: the collection number of the rhodococcus Fan Qingsheng is CGMCC No.23566, the rhodococcus Fan Qingsheng belongs to the eubacteria kingdom, actinomycota, corynebacterium, nocardia, rhodococcus, and the ability of combined nitrogen fixation in apple tree bark.
The endophyte nitrogen-fixing bacteria is obtained by separating in sugarcane in the earliest 1986. Later, related researches on the separation and application of endophytic azotobacter of gramineous crops are more. As shown by researches, the sugarcane endophyte can provide up to 70% of nitrogen requirement for plants; under certain conditions, the endophyte in the rice can provide 36-42% nitrogen for plants; at present, the study on the endophytic azotobacter of woody plants is relatively few, but the study is gradually in progress. As has been used 15 The N marking method proves that the poplar endophyte has the function of fixing nitrogen. The invention has verified that abundant endophytic nitrogen-fixing bacteria exist in each tissue part of apples by a high-throughput sequencing method. Meanwhile, a large number of endophytic nitrogen-fixing strains are obtained by a traditional separation method. The application of the new microorganism resource of the apple endophyte is expected to reduce the use of chemical fertilizers in the apple cultivation process.
The second object of the present invention is achieved by: the method for obtaining the Fan Qingsheng rhodococcus garvieae comprises the steps of separation, purification, molecular verification and strain identification. The method specifically comprises the following steps:
A. separation
1) Washing fresh red Fuji branches with running water, transferring to an ultra-clean workbench, cutting into small segments of about 5 cm, sterilizing with 75% ethanol solution for 30 s, sterilizing with 0.1% mercuric chloride solution for 1min, and cleaning with sterile water for 3 times.
2) Tearing off the bark of the sterilized sample, weighing 3 g, shearing into tissue blocks of about 0.5 cm, adding 27 mL deionized water into a sterilized mortar, grinding, standing for 10min, and gradient diluting the supernatant to 10 -1 、10 -2 And 10 -3 100 μl of each gradient was plated on Jensen nitrogen-free medium, and 3 replicates of each gradient.
3) After coating, the petri dish is sealed by a sealing film, and is inverted to be cultured in a constant temperature incubator at 28 ℃. After daily observation of colonies, about 5. 5d, a distinct single colony was visible. To examine the surface disinfection effect, 100 μl of sterile water from the last wash sample was spread on NA medium. If the colony grows, the surface is thoroughly disinfected.
The culture conditions are as follows: the 28 ℃ time is as follows: 5d
Jensen nitrogen-free medium composition in g/L: 15.0 to 25.0 portions of sucrose, 0.5 to 1.5 portions of dipotassium hydrogen phosphate (K2 HPO 4), 0.5 to 1.5 portions of magnesium sulfate heptahydrate (MgSO4.7H2O), 0.4 to 0.6 portions of NaCl, 10.0 to 20.0 portions of agar, 0.05 to 0.15 portions of ferrous sulfate (FeSO 4), and sodium molybdate (Na 2 MoO 4 ) 0.004-0.006, 1.5-2.5 of calcium carbonate (CaCO 3) and 6.5-7.5 of pH.
B. Purification
The screened strain is streaked and purified by a purification culture medium plate to obtain vigorous single colony, namely Fan Qingsheng rhodococcus acidilacticiRhodococcus qingshengii）Srq-21；
The culture conditions are as follows: 28 ℃ for 4-5 d;
the components of the purification culture medium are as follows in g/L: 15.0 to 25.0% of sucrose, and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 0.5 to 1.5, magnesium sulfate heptahydrate (MgSO) 4 •7H 2 O) 0.5-1.5, naCl 0.4-0.6, agar 10.0-20.0, ferrous sulfate (FeSO) 4 ) 0.05 to 0.15, sodium molybdate (Na) 2 MoO 4 ) 0.004-0.006, calcium carbonate (CaCO) 3 ）1.5~2.5、pH6.5~7.5。
C. Molecular validation
By amplifying nitrogen-fixing genesnifHMolecular biology verification of strain Fan Qingsheng rhodococcusRhodococcus qingshengii) Srq-21 is an endophytic azotobacter of apples. The verification process comprises the steps of extracting total DNA of the strain,nifHAnd (3) gene PCR amplification and electrophoresis detection of gene fragments. The method specifically comprises the following steps:
a. total DNA extraction from strains
Total DNA of the purified bacterial strain was extracted with Ezup column bacterial genomic DNA extraction kit. The kit was purchased from Shanghai Bioengineering Co., ltd.
The extraction steps are as follows:
1) 1mL of the bacterial liquid cultured overnight was added to a 2mL centrifuge tube, centrifuged at 10000rpm at room temperature for 5min, and the supernatant was discarded to collect the bacterial cells. 100. Mu. L Buffer Digestion and 80. Mu.L of lysozyme solution were added. Resuspension of the bacteria solution, water bath at 37 ℃ for 30min (corresponding lysozyme is added into Enzymatic lysis buffer before use to prepare 20mg/ml lysozyme solution);
2) 20 mu LProteinase K solution is added and mixed by shaking. Water bath at 56 ℃ for 40min until the cells are completely lysed;
3) Adding 200 mu LBuffer BD, fully reversing and uniformly mixing, and carrying out water bath at 70 ℃ for 10min;
4) Adding 200 mu L of absolute ethyl alcohol, fully reversing and uniformly mixing;
5) Placing the adsorption column into a collecting pipe, adding the solution and semitransparent fibrous suspension into the adsorption column by using a liquid transfer device, standing for 2min, centrifuging at 12000rmp at room temperature for 1min, and pouring out waste liquid in the collecting pipe;
6) The adsorption column was put back into the collection tube, 500. Mu.L PW Solution was added, and the filtrate was poured off by centrifugation at 10000rmp for 1 min.
7) The column was returned to the collection tube, 500. Mu.L Wash Solution was added, and the filtrate was decanted by centrifugation at 10000rmp for 1 min.
8) The column was replaced in the collection tube and centrifuged at 12000rmp for 3min at room temperature to leave a residual Wash Solution.
9) The column was removed, placed in a new 1.5. 1.5mL centrifuge tube, added with 30. Mu.L of CE Buffer, left to stand for 5min, centrifuged at 12000rmp for 2min at room temperature, and the DNA solution was collected. The DNA solution was stored to-20 ℃.
10 2 μl of the DNA solution was measured with nanodrop photometer to have a DNA concentration of 1000 ng/μl, a260/280=1.91
b、nifHGene PCR amplification:
nifHthe gene verification adopts a nested PCR method.
The first round amplification primer pairs were: FGPHl9:5'-TACGGCAARGGTGGNATHG-3' and POLR:5'-ATSGCCATCATYTCRCCGGA-3'.
The reaction system: (20. Mu.L of the total system) wherein 2. Mu.L of the template, 1. Mu.L of each of the primers FGPHl9 and POLR, 10. Mu.L of 2 XPCR mix, and 6. Mu.L of water were added.
Reaction conditions: 95℃5min,95℃1min,55℃1min,72℃2min,30 cycles, 72℃10min.
The second round of PCR amplification takes the first round of PCR product as a template.
The primer pair is as follows: AQER:5'-GACGATGTAGATYTCCTG-3' and POLF:5'-TGCGAYCCSAARGCBGACTC-3'.
The reaction system: (20. Mu.L of the total system) wherein 2. Mu.L of the template, 1. Mu.L of each of the primers AQER and POLF, 10. Mu.L of 2 XPCR mix, and 6. Mu.L of water were supplemented.
Reaction conditions: 95℃for 5min,95℃for 1min, 50℃for 1min,72℃for 2min,30 cycles, 72℃for 10min.
c、nifHAnd (3) gene fragment electrophoresis detection:
the agarose gel electrophoresis is used for detection, clear bands can be observed at about 330bp, and the strain is proved to havenifHGenes (FIG. 1).
D. Identification of strains
The bacterial colony is observed for morphology, size, concave-convex degree, edge, gram staining and physiological and biochemical characteristic test, and the specific method is referred to the common bacteria System identification Manual. The 16S rDNA sequence amplification primer of the strain adopts F27/R1492, and the amplified product is entrusted to the Shanghai biological engineering Co.
1) Colony morphology observation: the colony is white, round, neat in edge, smooth in surface protrusion and opaque on the Jensen nitrogen-free culture medium, and the diameter of the colony is 1.0-2.0 mm.
2) Microscopic morphology observation: the strain was observed under a microscope as a short rod. Gram staining was positive.
3) Physiological and biochemical test: the methyl red reaction is negative; catalase, VP test, indole production test, nitrate reduction test, ammonia production test were positive.
4) 16S rDNA sequence analysis: the strain was subjected to 16S rDNA sequencing to obtain a 1447bp sequence. The similarity of the Srq-21 strain 16S rDNA sequence and the strain of Fan Qingsheng rhodococcus reaches 99.72%, and the strain is identified to belong to Fan Qingsheng rhodococcus rhodochrous by combining the strain morphology, colony characteristics and physiological and biochemical characteristicsRhodococcus qingshengii）。
The 16S rDNA sequence of the Srq-21 strain is shown as SEQ ID NO: 1.
The third object of the present invention is achieved by the method of Fan Qingsheng rhodococcus acidizing fluid @Rhodococcus qingshenggii) The application of Srq-21 in preparing single azotobacter seed bacterial agent for promoting plant growth.
Drawings
FIG. 1 shows Srq-21 strainnifHSchematic representation of gene amplification;
fig. 2 is a schematic drawing of an ethylene standard curve.
Detailed Description
The invention is further described below without limiting the invention in any way, and any alterations or modifications based on the teachings of the invention fall within the scope of the invention.
Fan Qingsheng rhodococcus furiosus strainRhodococcus qingshenggii) Srq-21 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23566, and the Fan Qingsheng rhodococcus garvieae is preservedRhodococcus qingshenggii) Srq-21 belongs to the eubacteria kingdom, the phylum actinomycota, the order Corynebacterium, the family Nocardia, and the genus Rhodococcus.
The Fan Qingsheng rhodococcus acidizing fluid isRhodococcus qingshenggii) Acquisition of Srq-21The method comprises the steps of separation and purification, and specifically comprises the following steps:
A. separating:
1) Cleaning fresh red Fuji branches, cutting the fresh red Fuji branches into sections, and sterilizing and cleaning the sections to obtain a branch material a;
2) Taking paper strip bark from a branch material a, shearing into tissue blocks with the length of 0.4-0.6 cm, adding deionized water for grinding, standing for precipitation for 8-12 min, and taking supernatant for gradient dilution to 10 -1 、10 -2 And 10 -3 100 μl of each gradient was plated on Jensen nitrogen-free medium, 3 replicates of each gradient;
the Jensen nitrogen-free culture medium comprises the following components in g/L: sucrose 15.0-25.0, K 2 HPO 4 0.5 to 1.5, magnesium sulfate heptahydrate (MgSO) 4 •7H 2 O) 0.5-1.5, naCl 0.4-0.6, agar 10.0-20.0, ferrous sulfate 0.05-0.15, sodium molybdate 0.004-0.006, calcium carbonate 1.5-2.5, pH 6.5-7.5;
3) Sealing the coated culture dish, and culturing for 4-6 d at the temperature of 26-30 ℃ to obtain obvious single colonies;
B. purifying:
the obvious single colony is taken and subjected to streak purification culture by a purification culture medium flat plate to obtain the single colony with vigorous growth vigor, namely the target strain Fan Qingsheng rhodococcus erythropolis @Rhodococcus qingshengii）Srq-21；
The components of the purification culture medium are as follows in g/L: sucrose 15.0-25.0, K 2 HPO 4 0.5 to 1.5, magnesium sulfate heptahydrate (MgSO) 4 •7H 2 O) 0.5-1.5, naCl 0.4-0.6, agar 10.0-20.0, ferrous sulfate 0.05-0.15, sodium molybdate 0.004-0.006, calcium carbonate 1.5-2.5, and pH 6.5-7.5.
And A, sterilizing in the step 1) by using a 75% ethanol solution for 20-40 s and a 0.1% mercuric chloride solution for 0.5-1.5 min.
The cleaning in the step A1) is carried out by using sterile water for 2-4 times.
A step 1) also comprises a step of checking the surface disinfection effect after cleaning, wherein the surface disinfection effect is that 100 mu L of sterile water of a last cleaning sample is coated on an NA culture medium, and if the sterile colony grows, the surface disinfection is thorough.
The components of the Jensen nitrogen-free culture medium in the step A are as follows in g/L: sucrose 20.0, K 2 HPO 4 1.0 magnesium sulfate heptahydrate (MgSO) 4 •7H 2 O) 1.0, naCl 0.5, agar 15.0, ferrous sulfate 0.10, sodium molybdate 0.005, calcium carbonate 2.0, ph7.0.
Step A3) is to seal the coated dish and culture 5d at 28 ℃ to obtain obvious single colony.
The components of the purification culture medium in the step B are expressed as g/L: sucrose 20.0, K 2 HPO 4 1.0 magnesium sulfate heptahydrate (MgSO) 4 •7H 2 O) 1.0, naCl 0.5, agar 15.0, ferrous sulfate 0.10, sodium molybdate 0.005, calcium carbonate 2.0, ph7.0.
And B, purifying and culturing at 28 ℃ for 4-5 d.
The application of the Fan Qingsheng rhodococcus rhodochrous (rhodococcus qingshengggii) Srq-21 is the application of the Fan Qingsheng rhodococcus rhodochrous (rhodococcus qingshengggii) Srq-21 in preparing a single azotobacter seed bacterial agent for promoting plant growth.
The following is described by way of example:
example 1
Acquisition and identification of rhodococcus rhodochrous (rhodococcus qingshengii) Srq-21-Fan Qingsheng
(1) Acquisition of rhodococcus Fan Qingsheng (Rhodococcus qingshengggii) Srq-21
A. Separation
1) Washing fresh red Fuji branches with running water, transferring to an ultra-clean workbench, cutting into small segments of about 5 cm, sterilizing with 75% ethanol solution for 30 s, sterilizing with 0.1% mercuric chloride solution for 1min, and cleaning with sterile water for 3 times.
2) Tearing off the bark of the sterilized sample, weighing 3 g, shearing into tissue blocks of about 0.5 cm, adding 27 mL deionized water into a sterilized mortar, grinding, standing for 10min, and gradient diluting the supernatant to 10 -1 、10 -2 And 10 -3 100 μl of each gradient was applied to JeOn nsen nitrogen-free medium, 3 replicates per gradient.
3) After coating, the petri dish is sealed by a sealing film, and is inverted to be cultured in a constant temperature incubator at 28 ℃. After daily observation of colonies, about 5. 5d, a distinct single colony was visible. To examine the surface disinfection effect, 100 μl of sterile water from the last wash sample was spread on NA medium. If the colony grows, the surface is thoroughly disinfected.
The culture conditions are as follows: the 28 ℃ time is as follows: 5d
Jensen nitrogen-free medium composition in g/L: sucrose 20.0, K 2 HPO 4 1.0, sulfuric acid (MgSO) 4 •7H 2 O) 1.0, naCl 0.5, agar 15.0, ferrous sulfate 0.1, sodium molybdate 0.005, calcium carbonate 2.0, ph7.0.
B. Purification
The screened strain is streaked and purified by a purification culture medium plate to obtain vigorous single colony, namely Fan Qingsheng rhodococcus acidilacticiRhodococcusqingshengii）Srq-21；
The culture conditions are as follows: 28 ℃ for 4-5 d;
the components of the purification culture medium are as follows in g/L: sucrose 20.0, K 2 HPO 4 1.0, sulfuric acid heptahydrate (MgSO) 4 •7H 2 O) 1.0, naCl 0.5, agar 15.0, ferrous sulfate 0.1, sodium molybdate 0.005, calcium carbonate 2.0, ph7.0.
C. Molecular validation
By amplifying nitrogen-fixing genesnifHMolecular biology verification of strain Fan Qingsheng rhodococcusRhodococcus qingshengii) Srq-21 is an endophytic azotobacter of apples. The verification process comprises the steps of extracting total DNA of the strain,nifHAnd (3) gene PCR amplification and electrophoresis detection of gene fragments. The method specifically comprises the following steps:
a. total DNA extraction from strains
Total DNA of the purified bacterial strain was extracted with Ezup column bacterial genomic DNA extraction kit. The kit was purchased from Shanghai Bioengineering Co., ltd.
The extraction steps are as follows:
1) 1mL of the bacterial liquid cultured overnight was added to a 2mL centrifuge tube, centrifuged at 10000rpm at room temperature for 5 minutes, and the supernatant was discarded to collect the bacterial cells. 100. Mu. L Buffer Digestion and 80. Mu.L of lysozyme solution were added. Resuspension of the bacteria solution, water bath at 37 ℃ for 30min (corresponding lysozyme is added into Enzymatic lysis buffer before use to prepare a 20mg/ml lysozyme solution);
2) 20 mu LProteinase K solution is added and mixed by shaking. Water bath at 56 ℃ for 40min until the cells are completely lysed;
3) Adding 200 mu LBuffer BD, fully reversing and uniformly mixing, and carrying out water bath at 70 ℃ for 10min;
4) Adding 200 mu L of absolute ethyl alcohol, fully reversing and uniformly mixing;
5) Placing the adsorption column into a collecting pipe, adding the solution and semitransparent fibrous suspension into the adsorption column by using a liquid transfer device, standing for 2min, centrifuging at 12000rmp at room temperature for 1min, and pouring out waste liquid in the collecting pipe;
6) The adsorption column was put back into the collection tube, 500. Mu.L PW Solution was added, and the filtrate was poured off by centrifugation at 10000rmp for 1 min.
7) The column was returned to the collection tube, 500. Mu.L Wash Solution was added, and the filtrate was decanted by centrifugation at 10000rmp for 1 min.
8) The column was replaced in the collection tube and centrifuged at 12000rmp for 3min at room temperature to leave a residual Wash Solution.
9) The column was removed, placed in a new 1.5mL centrifuge tube, added with 30. Mu.L of CE Buffer, left to stand for 5min, centrifuged at 12000rmp for 2min at room temperature, and the DNA solution was collected. The DNA solution was stored to-20 ℃.
10 2 μl of the DNA solution was measured with nanodrop photometer to have a DNA concentration of 1000 ng/μl, a260/280=1.91
b、nifHGene PCR amplification:
nifHthe gene verification adopts a nested PCR method.
The first round amplification primer pairs were: FGPHl9:5'-TACGGCAARGGTGGNATHG-3' and POLR:5'-ATSGCCATCATYTCRCCGGA-3'.
The reaction system: (20. Mu.L of the total system) wherein 2. Mu.L of the template, 1. Mu.L of each of the primers FGPHl9 and POLR, 10. Mu.L of 2 XPCR mix, and 6. Mu.L of water were added.
Reaction conditions: 95℃5min,95℃1min,55℃1min,72℃2min,30 cycles, 72℃10min.
The second round of PCR amplification takes the first round of PCR product as a template. The primer pair is as follows: AQER:5'-GACGATGTAGATYTCCTG-3' and POLF:5'-TGCGAYCCSAARGCBGACTC-3'.
The reaction system: (20. Mu.L of the total system) wherein 2. Mu.L of the template, 1. Mu.L of each of the primers AQER and POLF, 10. Mu.L of 2 XPCR mix, and 6. Mu.L of water were supplemented.
Reaction conditions: 95℃for 5min,95℃for 1min, 50℃for 1min,72℃for 2min,30 cycles, 72℃for 10min.
c、nifHAnd (3) gene fragment electrophoresis detection:
the agarose gel electrophoresis is used for detection, clear bands can be observed at about 330bp, and the strain is proved to havenifHGenes (FIG. 1).
D. Identification of strains
The bacterial colony is observed for morphology, size, concave-convex degree, edge, gram staining and physiological and biochemical characteristic test, and the specific method is referred to the common bacteria System identification Manual. The 16S rDNA sequence amplification primer of the strain adopts F27/R1492, and the amplified product is entrusted to the Shanghai biological engineering Co.
1) Colony morphology observation: the colony is white, round, neat in edge, smooth in surface protrusion and opaque on the Jensen nitrogen-free culture medium, and the diameter of the colony is 1.0-2.0 mm.
2) Microscopic morphology observation: the strain was observed under a microscope as a short rod. Gram staining was positive.
3) Physiological and biochemical test: the methyl red reaction is negative; catalase, VP test, indole production test, nitrate reduction test, ammonia production test were positive.
4) 16S rDNA sequence analysis: the strain was subjected to 16S rDNA sequencing to obtain a 1447bp sequence. The similarity of the Srq-21 strain 16S rDNA sequence and the strain of Fan Qingsheng rhodococcus reaches 99.72%, and the strain is identified to belong to Fan Qingsheng rhodococcus rhodochrous by combining the strain morphology, colony characteristics and physiological and biochemical characteristicsRhodococcu sqingshengii）。
The 16S rDNA sequence of the Srq-21 strain is shown as SEQ ID NO:1, specifically as follows:
TGCTGACCATGAGAGTCGAGCGGTAAGGCCTTTCGGGGTACACGAGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTCCTATCGCATGGTGGGTGGTGGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGAAAGCTGCAGAGATGTGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCACGTTATGGTGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAGACCGTGAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGTTAACCCCTTGTGGGAGGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAGCACCAAAGGGGGGGGCCACAAATT。
the activity of the immobilized enzyme was measured by acetylene reduction. That is, under certain conditions, the amount of acetylene reduced to ethylene by the nitrogen fixation enzyme is positively correlated with the activity of the nitrogen fixation enzyme. Ethylene content was determined using a Furicgc 9790 type II gas chromatograph (Kromat Corporation). Ethylene (80%), acetylene (99.99%) gas was purchased from Yunnan copper Inc., and the ethylene balance gas was nitrogen. Gas transfer was performed using a 0.5L Tesla sampling bag (available from Shanghai Pond instruments Co.).
1. Standard curve formulation
Into 7 25mL penicillin bottles, 0, 100, 200, 300, 400, 500, 600, 700 μl of ethylene gas was injected, and 200 μl of the corresponding peak value was measured in a weather chromatograph using a microsyringe needle (about syringe needle injector experimental equipment from melancholy), and the final drawn standard curve is shown in fig. 2.
2. Bacterial strain nitrogen fixation enzyme activity determination method
1) The strain to be tested is taken out from a refrigerator at the temperature of minus 80 ℃ to be melted on ice, 200 mu L of the strain to be tested is absorbed and inoculated into 2mL of NA culture solution, and the strain to be tested is placed at the temperature of 28 ℃ for 180r/min to shake culture for 48 hours.
2) The strain was cultured in NA culture medium, and 5 μl of the strain was measured with Nanodrop photometer until the concentration of the strain became od600=0.5 (logarithmic growth phase).
3) 200. Mu.L of the bacterial liquid is inoculated into a 25mL penicillin bottle containing 5mL of a nitrogen-free culture medium Jensen, and the culture is carried out at 28 ℃ for 180r/min for 48 hours.
4) 10% of the gas (2 mL) was evacuated, the same volume of acetylene gas was charged, and after further culturing for 24. 24 h, 200. Mu.L of the gas was taken and the amount of ethylene produced was measured by a gas chromatograph (GC 9790 type II chromatograph). The temperature of the sample injector is 200 ℃, the temperature of the detector is 230 ℃ and the temperature of the column temperature box is 80 ℃.
5) In C 2 H 4 Mu mol/(h.mL) represents the activity of the enzyme
C 2 H 4 Concentration (μmol/(h·ml))=sample C 2 H 4 Peak area x standard C 2 H 4 Concentration x penicillin bottle volume/standard C 2 H 4 Peak area×c 2 H 4 Reaction time X24.9
Since the nitrogen-fixing bacteria were cultured on different media to have an effect on the nitrogen fixing ability, the difference in nitrogen fixing ability of the strain on Jensen, ash, jnfb media was tested in this study, and the results are shown in the following table.
TABLE 1 Nitrogen fixation Capacity of Srq-21 strains in different culture media
| Culture medium | Nitrogen fixation enzyme Activity (mu mol/(h mL)) |
| Jensen | 260.75±4.098a |
| Jnfb | 243.38±4.911b |
| Ash | 182.34±1.730c |
From Table 1, it can be seen that the strain has a significant difference in nitrogen fixation capacity in Jensen, ash, jnfb nitrogen-free media. Wherein the nitrogen fixation capacity is strongest in the culture medium Jensen, and reaches 260.75 mu mol/(h.mL).
Sequence listing
<110> university of southwest forestry
<120> rhodococcus Fan Qingsheng strain, and acquisition method and application thereof
<130> 2022
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1447
<212> DNA
<213> Strain (Srq-2116S rDNA sequence)
<400> 1
tgctgaccat gagagtcgag cggtaaggcc tttcggggta cacgagcggc gaacgggtga 60
gtaacacgtg ggtgatctgc cctgcacttc gggataagcc tgggaaactg ggtctaatac 120
cggatatgac ctcctatcgc atggtgggtg gtggaaagat ttatcggtgc aggatgggcc 180
cgcggcctat cagcttgttg gtggggtaat ggcctaccaa ggcgacgacg ggtagccgac 240
ctgagagggt gaccggccac actgggactg agacacggcc cagactccta cgggaggcag 300
cagtggggaa tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg tgagggatga 360
cggccttcgg gttgtaaacc tctttcagca gggacgaagc gcaagtgacg gtacctgcag 420
aagaagcacc ggctaactac gtgccagcag ccgcggtaat acgtagggtg caagcgttgt 480
ccggaattac tgggcgtaaa gagttcgtag gcggtttgtc gcgtcgtttg tgaaaaccag 540
cagctcaact gctggcttgc aggcgatacg ggcagacttg agtactgcag gggagactgg 600
aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg 660
gtctctgggc agtaactgac gctgaggaac gaaagcgtgg gtagcgaaca ggattagata 720
ccctggtagt ccacgccgta aacggtgggc gctaggtgtg ggttccttcc acggaatccg 780
tgccgtagct aacgcattaa gcgccccgcc tggggagtac ggccgcaagg ctaaaactca 840
aaggaattga cgggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg 900
aagaacctta cctgggtttg acatataccg gaaagctgca gagatgtggc cccccttgtg 960
gtcggtatac aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1020
cccgcaacga gcgcaacccc tatcttatgt tgccagcacg ttatggtggg gactcgtaag 1080
agactgccgg ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat 1140
gtccagggct tcacacatgc tacaatggcc agtacagagg gctgcgagac cgtgaggtgg 1200
agcgaatccc ttaaagctgg tctcagttcg ggatcggggt ctgcaactcg accccgtgaa 1260
gtcggagtcg ctagtaatcg cagatcagca acgctgcggt gaatacgttc ccgggccttg 1320
tacacaccgc ccgtcacgtc atgaaagtcg gtaacacccg aagccggtgg ttaacccctt 1380
gtgggaggga gccgtcgaag gtgggatcgg cgattgggac gagcaccaaa ggggggggcc 1440
acaaatt 1447
<210> 2
<211> 19
<212> DNA
<213> FGPHl9
<220>
<221> misc_feature
<222> (15)..(15)
<223> n is a, c, g, or t
<400> 2
tacggcaarg gtggnathg 19
<210> 3
<211> 20
<212> DNA
<213> POLR
<400> 3
atsgccatca tytcrccgga 20
Claims (2)
1. Fan Qingsheng rhodococcus furiosus strainRhodococcus qingshengii) Srq-21 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23566, and the Fan Qingsheng rhodococcus garvieae is preservedRhodococcus qingshengii) Srq-21 belongs to the eubacteria kingdom, the phylum actinomycota, the order Corynebacterium, the family Nocardia, and the genus Rhodococcus.
2. A rhodococcus Fan Qingsheng strain as defined in claim 1Rhodococcu sqingshengii) The application of Srq-21 is characterized in that the rhodococcus Fan Qingsheng strainRhodococcus qingshengii) The application of Srq-21 in preparing single azotobacter seed bacterial agent for promoting plant growth.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589665A (en) * | 2013-10-23 | 2014-02-19 | 浙江工业大学 | Rhodococcus qingshengii and application thereof in preparation of ethyl (S)-4-chloro-3-hydroxy butyrate |
| CN113337426A (en) * | 2021-05-31 | 2021-09-03 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Rhodococcus celebrati RYCS-1 and culture method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589665A (en) * | 2013-10-23 | 2014-02-19 | 浙江工业大学 | Rhodococcus qingshengii and application thereof in preparation of ethyl (S)-4-chloro-3-hydroxy butyrate |
| CN113337426A (en) * | 2021-05-31 | 2021-09-03 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Rhodococcus celebrati RYCS-1 and culture method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Differential protein profiling of soil diazotroph Rhodococcus qingshengii S10107 towards low-temperature and nitrogen deficiency.;Deep Chandra Suyal 等;Sci Rep.;第9卷(第1期);文章号：20378 * |
| Genome-Based Characterization of Plant-Associated Rhodococcus qingshengii RL1 Reveals Stress Tolerance and Plant-Microbe Interaction Traits.;Theresa Kuhl 等;Front Microbiol.;第12卷;文章号：708605 * |
| Impacts of Bioinoculants Pseudomonas jesenii MP1 and Rhodococcus qingshengii S10107 on Chickpea (Cicer arietinum L.) Yield and Soil Nitrogen Status.;JOSHI Divya 等;Pedosphere.;第29卷(第3期);388-399 * |
| 新造地马铃薯根际固氮解磷微生物的 分离与鉴定;王惟帅 等;西北农林科技大学学报(自然科学版);第47卷(第8期);127-133、143 * |
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