CN117778425A - Use and method of methyltransferase in enhancing nitrogen fixation capacity of nitrogen fixation microorganism - Google Patents
Use and method of methyltransferase in enhancing nitrogen fixation capacity of nitrogen fixation microorganism Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The present disclosure relates to a use and a method of methyltransferase in enhancing nitrogen fixation capacity of nitrogen fixation microorganisms, and a use of methyltransferase in enhancing nitrogen fixation capacity of nitrogen fixation microorganisms, wherein an amino acid sequence of methyltransferase is shown as SEQ ID NO. 1. The present disclosure also provides a method of enhancing the nitrogen fixation capacity of a nitrogen-fixing microorganism, the method comprising the steps of: the gene encoding methyltransferase is introduced into a nitrogen-fixing microorganism and overexpressed. The gene for encoding the methyltransferase can improve the expression level of the active nitrogen fixation gene of the nitrogen fixation enzyme in the nitrogen fixation microorganism, thereby improving the nitrogen fixation capacity of the nitrogen fixation microorganism, in particular the nitrogen fixation capacity of pseudomonas stutzeri.
Description
Technical Field
The present disclosure relates to the technical field of microorganisms and genetic engineering, and in particular, to the use and method of methyltransferases in enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms.
Background
DNA methylation refers to a chemical modification process in which a specific base on a DNA sequence is subjected to covalent bonding under the catalysis of DNA methyltransferase, and S-adenosylmethionine (S-adenosyl methionine, SAM) is used as a methyl donor to obtain a methyl group. Eukaryotes such as plants activate self-defense reactions in the presence of abiotic stresses such as oxygen, drought, high temperature and the like through epigenetic modifications such as DNA methylation, and microorganisms regulate and control gene expression mainly through the DNA methylation mode in order to adapt to complex and changeable external environments.
Biological nitrogen fixation in nitrogen fixation bacteria is a complex regulation and control system, is sensitive to oxygen and depends on energy very much in the nitrogen fixation process. Therefore, the nitrogen fixation regulating element and the functional module are transformed from the perspective of epigenetic modification by means of synthetic biology, and the artificial efficient nitrogen fixation system for the rhizosphere of crops is constructed. In the current research, the DNA methyltransferase encoding gene is not applied to azotobacter and further applied to the aspect of regulating and controlling the activity of azotase.
Disclosure of Invention
The purpose of the present disclosure is to provide an application and a method of methyltransferase in enhancing nitrogen fixation capacity of nitrogen fixation microorganisms, wherein a gene encoding methyltransferase can increase expression level of nitrogen fixation genes of nitrogen fixation microorganisms, increase activity of the methyltransferase in the nitrogen fixation microorganisms, and further increase nitrogen fixation capacity of the nitrogen fixation microorganisms.
To achieve the above object, the present disclosure provides a use of a methyltransferase having an amino acid sequence as shown in SEQ ID NO.1 for enhancing nitrogen fixation ability of a nitrogen-fixing microorganism in a first aspect.
Alternatively, the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO. 2.
Optionally, the nitrogen-fixing microorganism is pseudomonas stutzeri.
Optionally, enhancing the nitrogen fixation capacity of the nitrogen fixation microorganism comprises increasing the activity of the nitrogen fixation enzyme and the expression level of the nitrogen fixation gene in the nitrogen fixation microorganism.
Optionally, the nitrogen fixation gene isnifH、nifDAndnifK。
a second aspect of the present disclosure provides a method of enhancing nitrogen fixation capacity of a nitrogen-fixing microorganism, the method comprising the steps of: introducing a gene encoding a methyltransferase into a nitrogen-fixing microorganism and overexpressing it; the amino acid sequence of the methyltransferase is shown as SEQ ID NO. 1; the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO. 2.
Optionally, the recombinant expression vector contains a constitutive promoter; the nucleotide sequence of the constitutive promoter is shown in the 1 st position to the 176 th position in SEQ ID NO. 3.
Optionally, the nitrogen-fixing microorganism is pseudomonas stutzeri.
Optionally, enhancing the nitrogen fixation capacity of the nitrogen fixation microorganism includes increasing the activity of the nitrogen fixation enzyme and the expression level of the nitrogen fixation gene in the nitrogen fixation microorganism.
Optionally, the nitrogen fixation gene isnifH、nifDAndnifK。
through the technical scheme, the application and the method of methyltransferase in enhancing the nitrogen fixation capacity of nitrogen fixation microorganisms are provided. The gene for encoding the methyltransferase is introduced into the nitrogen-fixing microorganism and is subjected to over-expression, so that the expression of the nitrogen-fixing gene in the nitrogen-fixing microorganism can be improved, the activity of the nitrogen-fixing enzyme in the nitrogen-fixing microorganism is improved, the nitrogen-fixing capability of the nitrogen-fixing microorganism is further improved, and the nitrogen-fixing capability of pseudomonas stutzeri is especially improved.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a diagram showing analysis of homology among genes encoding methyltransferases.
FIG. 2 is a deletion mutantΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Is a graph of growth ability analysis.
FIG. 3 is a deletion mutantΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Is a bar graph of the activity of the immobilized enzyme.
FIG. 4 is a deletion mutantΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Is a bar graph of the relative expression level of the nitrogen fixation gene.
Sequence list information
SEQ ID NO.1: amino acid sequence of methyltransferase.
SEQ ID NO.2: nucleotide sequence of a gene encoding methyltransferase.
SEQ ID NO.3: the nucleotide sequence of a recombinant expression vector of one embodiment of the present disclosure.
Detailed Description
Specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
In a first aspect, the present disclosure provides the use of a methyltransferase having the amino acid sequence shown in SEQ ID NO.1 for enhancing the nitrogen fixation capacity of a nitrogen fixation microorganism.
In a preferred embodiment of the present disclosure, the nitrogen-fixing microorganism is pseudomonas stutzeri.
The inventors of the present disclosure have found, through extensive studies, that by analyzing the DNA methyltransferase domain, which has a class I SAM-dependent methyltransferase domain, a homology analysis with other proteins in the family is less than 40% similar, possibly a novel DNA methyltransferase. The DNA methyltransferase can participate in the regulation and control of the activity of the nitrogen fixation enzyme in the nitrogen fixation microorganism, and the nitrogen fixation capacity of the nitrogen fixation microorganism is improved. Especially, the expression quantity of the nitrogen fixation gene in the pseudomonas stutzeri can be improved, the activity of the nitrogen fixation enzyme in the pseudomonas stutzeri is improved, and the nitrogen fixation capacity of the pseudomonas stutzeri is further improved.
According to the present disclosure, the nucleotide sequence of the gene encoding the methyltransferase is shown as SEQ ID NO. 2.
According to the present disclosure, enhancing the nitrogen fixation capacity of a nitrogen fixation microorganism includes increasing the activity of a nitrogen fixation enzyme and the expression level of a nitrogen fixation gene in the nitrogen fixation microorganism.
According to the present disclosure, the nitrogen fixation gene isnifH、nifDAndnifK。
a second aspect of the present disclosure provides a method of enhancing nitrogen fixation capacity of a nitrogen-fixing microorganism, the method comprising the steps of: introducing a gene encoding a methyltransferase into a nitrogen-fixing microorganism and overexpressing it;
the amino acid sequence of the methyltransferase is shown as SEQ ID NO. 1;
the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO. 2.
In the present disclosure, the gene encoding methyltransferase may be introduced into the nitrogen-fixing microorganism by methods conventionally employed by those skilled in the art, for example, a method of tri-parental ligation.
According to the present disclosure, a gene encoding the methyltransferase is introduced into a nitrogen-fixing microorganism through a recombinant expression vector; the recombinant expression vector contains a constitutive promoter; the nucleotide sequence of the constitutive promoter is shown in the 1 st position to the 176 th position in SEQ ID NO. 3.
According to the present disclosure, enhancing the nitrogen fixation capacity of a nitrogen fixation microorganism includes increasing the activity of a nitrogen fixation enzyme and the expression level of a nitrogen fixation gene in the nitrogen fixation microorganism. Preferably, the nitrogen fixation gene isnifH、nifDAndnifK。
in the present disclosure, the over-expression engineering strain A1501 constructed by the present disclosurePST2700) The activity of the nitrogen fixation enzyme is improved by 20 percent compared with A1501. The over-expression engineering strain A1501 constructed by the methodPST2700) The expression level of the nitrogen fixation gene is up-regulated by 7-13 times compared with A1501.
The present disclosure is further illustrated by the following examples, but the present disclosure is not limited thereby.
The materials, reagents, instruments and equipment involved in the embodiments of the present disclosure, unless otherwise specified, are all available commercially.
Specific experimental conditions are not noted in the examples of the present disclosure, and are all conventional conditions well known to those skilled in the art, such as molecular cloning by Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Example 1
This example is used to illustrate constructionPST2700Deletion mutants.
(1) The experimental method comprises the following steps:
constructed DNA methyltransferase encoding genePST2700Deletion mutants are mainly based on homologous recombination of DNA.
The specific method is as follows: PCR amplification to obtain GenePST2700The upstream and downstream homologous fragments (above 500bp each) of (a) were ligated to a resistance cassette (spectinomycin resistance cassette, available from Thermo Fisher Co.) to obtain recombinant fragments; suicide vector with restriction endonucleases EcoRI and BamHIpk18mobsacBDouble cleavage was performed and the recombinant fragment was ligated into a suicide vector by means of a seamless cloning kit (available from Vazyme Corp.)pK18mobsacBThe recombinant vector was constructed and transformed into Pseudomonas stutzeri (A1501) by the method of three-parent ligation. At this time, the homologous fragments undergo single exchange, so that the vector is integrated into the whole genome, single exchange mutant strains are selected by the resistance of the vector itself, and suicide vector is used in the second exchangesacBWhen excised from the genome, it carries part of the gene in a single crossover, and two products, namely the deletion mutant and the wild-type strain, are produced in a ratio of about 1:1.
Double-exchanged mutants can be screened by 10% sucrose.
(2) Experimental results:
verified by PCR sequencingPST2700Successful knockout of the Gene, designatedΔPST2700。
Example 2
This example is used to illustrate constructionPST2700Functional anaplerotic strain and gene of deletion mutant strainPST2700And (5) over-expressing the recombinant engineering strain.
The experimental method comprises the following steps:
obtaining the whole by PCR amplificationPST2700Gene, using broad host range plasmidpLAFR3The BamH I and HindIII cleavage sites were selected for double cleavage and the genes were cloned using a seamless cloning kit (available from Vazyme Co.)PST2700With the carrier after enzyme digestionpLAFR3Homologous recombination is carried out, and finally, single colony is obtained by transforming escherichia coli, and the single colony is selected for PCR verification to successfully obtain the DNA methyltransferase recombinant plasmidpLA-PST700。
Respectively subjecting donor bacteria toE.coli(PLAFR 3-PST 2700), helper plasmid PRK2013, recipient bacterium: deltaPST2700Or A1501 is cultivated in LB culture medium for overnight, 1mL of the bacterial liquid cultivated overnight is collected and centrifuged at 6000rpm for 10min, supernatant is poured out and resuspended with equal volume of physiological saline, the supernatant is centrifuged again, bacterial precipitates washed twice are resuspended and mixed with 1mL of physiological saline and centrifuged, 50 mu L of physiological saline is used for resuspension of bacterial bodies and then dripped on a solid plate without resistance, and the plate is placed in a 30 ℃ incubator for static cultivation. After 24 hours, streaking on plates of 1/2 chloramphenicol, spectinomycin and spectinomycin, kanamycin respectively, picking single colony for verification, and obtaining the product after sequencing correctlyPST2700Functional compensation strain of deletion mutant strain and over-expression engineering strain A1501%PST2700）。
Example 3
This example is used to demonstrate DNA methyltransferase sequence homology analysis
(1) The experimental method comprises the following steps:
sequence alignment was performed on the coding DNA methyltransferase genes by CDD domain database (https:// www.ncbi.nlm.nih.gov/CDD), analysis websites ClustalW (https:// www.genome.jp/tools-bin/ClustalW) and ESPript 3.0 (https:// espipt.ibcp. Fr/ESPript/ESPript/index. Php)PST2700Bioinformatic analysis was performed.
(2) Experimental results and conclusions:
genes encoding DNA methyltransferasesPST2700The class I SAM-dependent methyltransferase identified by domain predictive analysis was aligned with the DNA methyltransferase of the isogenic family with a 36% similarity. As shown in FIG. 1, the gene encoding DNA methyltransferasePST2700Novel DNA methyltransferases in the family of class I SAM-dependent methyltransferases were identified.
Example 4
This example is for illustrating an overexpressed engineering strain A1501 #PST2700) Growth capacity analysis.
(1) The experimental method comprises the following steps:
detection of deletion mutants in LB MediumΔPST2700、PST2700Deletion mutant functional compensation strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Analyzing whether the DNA methyltransferase affects the growth of the strain, the specific steps are as follows:
(a) Deletion mutant strainsΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Culturing in LB liquid culture medium, 220rpm, and culturing at 30deg.C overnight;
(b) Centrifuging the thalli for 10min at 5000rpm on the next day;
(d) Resuspension the thalli with physiological saline, and washing the thalli twice;
(e) Suspending thalli by using an LB culture medium, and adjusting the OD600 to 1.0;
(f) Inoculating the thalli into an LB culture medium for culture;
(g) Measurement of deletion mutants Using full-automatic growth Curve apparatusΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) The OD600 was measured every 4 hours at 30 ℃ for the growth curve in LB medium, parameter set.
(2) Experimental results and conclusions:
as shown in FIG. 2, the over-expression engineering strain A1501 is in LB culture mediumPST2700) The growth trend is similar to that of the chassis strain A1501.
Thus, in LB medium, the gene encoding DNA methyltransferasePST2700Does not affect the growth of the strain.
Example 5
This example is for illustrating an overexpressed engineering strain A1501 #PST2700) Is used for the determination of the activity of the nitrogen fixation enzyme.
(1) The experimental method comprises the following steps:
detection of deletion mutants by acetylene reductionΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) The specific steps are as follows:
(a) Picking up freshly activated deletion mutantsΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Is inoculated into 20mL of fresh LB liquid medium and cultured at 220rpm and 30 ℃ overnight.
(b) The following day the bacterial liquid was centrifuged at 5000rpm at 4℃for 10min, the supernatant was removed and the cells were washed 2 times with 20mL of a limiting K nitrogen-free medium and centrifuged at the same conditions for 10min.
(c) Washing the bacterial liquid OD 600 To 1.0, 9mL of the limiting K nitrogen-free medium and 1mL of OD were added to a sterile 60mL saline flask 600 1.0 bacterial liquid, initial OD 600 Adjusted to 0.1, 3 replicates were made per sample.
(d) The sterile black rubber plug is tightly covered, marks are made on the bottle body, and the aluminum cap is tightly pressed on each brine bottle by the capping device so as to ensure the tightness of the bottle.
(e) Argon is filled into each brine bottle for 4min so as to exhaust air in the bottle, and oxygen accounting for 0.5 percent of the space in the bottle and acetylene accounting for 10 percent of the space in the bottle are respectively injected into each bottle filled with the argon.
(f) The gas-filled brine bottle is put into a shaking table for culture at 220rpm and 30 ℃, and the enzyme activity is measured every 2 hours.
(2) Experimental results and conclusions:
as shown in FIG. 3, the over-expressed engineering strain was compared with the chassis strain A1501A1501（PST2700) The activity of the azotase is improved by 20 percent. Indicating a gene encoding a DNA methyltransferasePST2700The nitrogen fixation capacity of the chassis strain A1501 can be remarkably improved.
Example 6
This example is for illustrating an overexpressed engineering strain A1501 #PST2700) Analysis of the expression level of the azotase gene.
(1) Experimental method
Deletion mutant strains by qRT-PCR methodΔPST2700、PST2700Functional compensation strain of deletion mutant strain, chassis strain A1501 and over-expression engineering strain A1501PST2700) Nitrogen fixation genenifH、nifD、nifK) The expression level under nitrogen fixation conditions was analyzed.
(2) Experimental results and conclusions:
as shown in FIG. 4, compared with the azotobacter chassis A1501, the over-expression engineering strain A1501 is shownPST2700) Nitrogen fixation genenifH、nifD、nifKThe expression level is respectively improved by 7.5 times, 13.5 times and 10 times under the nitrogen fixation condition. Indicating that under nitrogen fixation condition, the gene encoding DNA methyltransferasePST2700Can increase the expression level of nitrogen fixation gene after transcription.
The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. The use of methyltransferase in enhancing nitrogen fixation capability of nitrogen fixation microorganism is characterized in that the amino acid sequence of methyltransferase is shown in SEQ ID NO. 1.
2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID No. 2.
3. The use according to claim 1, wherein the nitrogen-fixing microorganism is pseudomonas stutzeri.
4. The use according to any one of claims 1 to 3, wherein enhancing the nitrogen fixation capacity of the nitrogen fixation microorganism comprises increasing the activity of a nitrogen fixation enzyme and the expression level of a nitrogen fixation gene in the nitrogen fixation microorganism.
5. The use according to claim 4, wherein the nitrogen fixation gene isnifH、nifDAndnifK。
6. a method of enhancing the nitrogen fixation capacity of a nitrogen-fixing microorganism, the method comprising the steps of: introducing a gene encoding a methyltransferase into a nitrogen-fixing microorganism and overexpressing it;
the amino acid sequence of the methyltransferase is shown as SEQ ID NO. 1;
the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO. 2.
7. The method according to claim 6, wherein the gene encoding the methyltransferase is introduced into the nitrogen-fixing microorganism by a recombinant expression vector;
the recombinant expression vector contains a constitutive promoter; the nucleotide sequence of the constitutive promoter is shown in the 1 st position to the 176 th position in SEQ ID NO. 3.
8. The method of claim 7, wherein the nitrogen-fixing microorganism is pseudomonas stutzeri.
9. The method of any one of claims 6-8, wherein enhancing the nitrogen fixation capacity of the nitrogen-fixing microorganism comprises increasing the activity of a nitrogen fixation enzyme and the expression level of a nitrogen fixation gene in the nitrogen-fixing microorganism.
10. The method of claim 9, wherein the nitrogen fixation gene isnifH、nifDAndnifK。
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