CN113025545B - Blue algae engineering bacterium capable of efficiently fixing nitrogen and preparation method and application thereof - Google Patents
Blue algae engineering bacterium capable of efficiently fixing nitrogen and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a blue algae engineering bacterium for efficiently fixing nitrogen, a preparation method and application thereof, and anabaena delta FTN with the abnormal cell volume about 2 times of the normal abnormal cell volume is obtained by knocking out the gene of a cell division related factor FTN of wild anabaena PCC 7120. The azathionase activity of the anabaena engineering bacterium delta FTN is 2.3 times that of a wild type, the growth speed is high, the generation time is about 20 hours, the anabaena engineering bacterium delta FTN has a prospect of popularization and application in agriculture, and a new choice is provided for application of nitrogen-fixing blue algae in agricultural production.
Description
Technical Field
The invention belongs to the field of industrial microorganisms, and particularly relates to a blue algae engineering bacterium with higher nitrogen-fixing enzyme activity, a preparation method and application thereof, which provides a new choice for the application of nitrogen-fixing blue algae in agriculture.
Background
With the development of nitrogen-fixing blue algae in the aspect of agricultural application basic research, the application potential of the nitrogen-fixing blue algae in agricultural production is more comprehensively and deeply known. In 1939, the nitrogen-fixing blue algae is used for fertilizing the field for the first time reported in India, and a plurality of researches prove that the yield of the nitrogen-fixing blue algae inoculated in the rice field can be increased. The nitrogen-fixing blue algae bred in the rice field can provide nitrogen and organic matters for crops, improve the solubility of phosphate, reduce water and soil pollution, supply oxygen to the roots of the crops, secrete plant hormones, inhibit weed growth and the like, and play roles in maintaining ecological balance and promoting agricultural sustainable development. Especially in the global warming and serious agricultural non-point source pollution, the value of the blue algae capable of fixing nitrogen and carbon in agricultural production and ecological environment protection is increasingly prominent.
However, the application of nitrogen-fixing cyanobacteria in agriculture still has not been realized at present, and the reason for this is related to the large-scale use of fertilizers in the world, and also related to the relative complexity, high cost and low reliability of the self technology. An important technical factor for restricting the application of nitrogen-fixing blue algae in agriculture is the nitrogen-fixing efficiency. Compared with the fertilizer increasing amount of the fertilizer, the nitrogen fixing efficiency of the nitrogen-fixing blue algae is lower. However, under the background that the current country requires agricultural 'reduction' and 'low-carbon' development, nitrogen-fixing blue algae as ecological and environment-friendly biological fertilizer may meet new development opportunities.
Disclosure of Invention
In order to construct engineering bacteria of blue algae with higher nitrogen-fixing enzyme activity, the invention aims to carry out gene modification on wild anabaena, construct engineering bacteria of anabaena with improved nitrogen-fixing enzyme activity and higher growth speed, have popularization and application prospects in agriculture and provide a new choice for application of nitrogen-fixing blue algae in agricultural production.
Anabaena is a class of blue algae that can differentiate heteromorphic cells to fix nitrogen. The heteromorphic cell is formed by metamorphosis of vegetative cells, and nitrogen fixation enzyme is contained in the heteromorphic cell. The nitrogen fixation efficiency of anabaena is directly related to heteromorphic cells, and the number, the size and the enzyme activity of the heteromorphic cells containing azotase are direct factors determining the nitrogen fixation efficiency of anabaena. A plurality of continuous heteromorphic cells and mutant delta PatS or delta HetN with the heteromorphic cell frequency about 2.5 times of that of a wild type are reported in the prior literature, but the nitrogenase activity of the continuous heteromorphic cells is lower than that of normal heteromorphic cells, so that the nitrogen fixation efficiency of the mutant is lower than that of the wild type on the whole; and these continuous heteromorphic mutants grow at a significantly slower rate than the wild type. At present, no literature reports that mutant strains with the abnormal cell volume being remarkably larger than that of wild type strains and the nitrogenase activity being improved.
The invention constructs the Anabaena engineering bacteria with the abnormal cell volume about 2 times of the normal abnormal cell volume by a gene knockout method, finds that the azotase activity of the Anabaena engineering bacteria is 2.3 times of wild Anabaena (Anabaena sp.) PCC7120 by detection, has higher growth speed and about 20 hours of generation time, has the prospect of popularization and application in agriculture, and provides a new choice for the application of azotobacter in agriculture.
Specifically, the gene of a cell division related factor FTN of Anabaena sp.PCC 7120 is knocked out through genetic engineering, so that the Anabaena engineering bacterium delta FTN with the abnormal cell volume about 2 times of the normal abnormal cell volume is obtained.
The invention also provides a preparation method of the anabaena engineering bacterium delta FTN, which comprises the following steps:
(1) constructing a knockout vector, wherein the vector contains upstream and downstream homologous fragments FlankA and FlankB of an FTN gene, a selective marker gene is arranged between the upstream and downstream homologous fragments FlankA and FlankB, and the vector can knock out the gene of a cell division related factor FTN in a genome through homologous recombination;
(2) transforming wild anabaena PCC7120 by using the knockout vector, screening positive strains on a selective plate, and selecting positive homozygous mutant strains delta FTN by gene detection;
(3) and (3) culturing the positive homozygous mutant strain delta FTN selected in the step (2), and measuring the azotobacter activity and the growth rate of the positive homozygous mutant strain delta FTN to obtain the anabaena engineering bacteria delta FTN with high azotobacter activity and good growth.
In the step (1), the lengths of the upstream and downstream homologous fragments FlanKA and FlanKB are about 3Kb, and the selectable marker gene is usually a resistance marker gene, such as erythromycin resistance gene (Em), kanamycin resistance gene (Kan), neomycin resistance gene (Neo), streptomycin resistance gene (Sm), and the like. In one embodiment of the invention, in the step (1), upstream and downstream homologous fragments FlanKA and FlanKB of FTN gene and selectable marker gene are ligated into pRL277 vector by means of PCR amplification and endonuclease cleavage ligation to construct knockout vector pRL277FTN, wherein the sequences of upstream homologous fragment FlanKA and downstream homologous fragment FlanKB are respectively shown in SEQ ID nos: 9 and 10, the selectable marker gene is Em (erythromycin resistance gene).
In the step (2), the positive strains are subcultured and screened on the selective plate by continuous streaking, and whether the positive strains screened by the plate are homozygous mutant strains is verified by PCR.
In the step (3), the abnormal cell shape of the mutant strain delta FTN is observed and counted, and the abnormal cell volume is about 2 times of that of the wild strain, and the azotase activity is more than 2 times of that of the wild strain.
The invention provides a blue algae engineering bacterium for efficiently fixing nitrogen, which is characterized in that a gene of a cell division related factor FTN of wild anabaena PCC7120 is knocked out to obtain the anabaena engineering bacterium delta FTN with the abnormal cell volume about 2 times as large as the normal abnormal cell volume. The nitrogen-fixing enzyme activity of the anabaena engineering bacterium delta FTN is 2.3 times of that of wild anabaena PCC7120, the growth speed is high, and a new choice is provided for the application of nitrogen-fixing blue algae in agricultural production (such as inoculation to a rice field).
Drawings
FIG. 1 is a schematic diagram of FTN knockout mutant obtained by homologous double exchange of knockout vector PRL277FTN and wild-type Anabaena PCC 7120.
FIG. 2 is a gel electrophoresis result diagram of PCR identification of homozygous positive FTN knockout mutants.
FIG. 3 phenotypic graphs of differentiated heteromorphic cells of wild type strain (WT) and positive homozygous mutant strain (. DELTA.FTN) (A) and corresponding statistical size graphs (B1 and B2)
FIG. 4 is a graph showing the enzymatic activities of azotase of a wild-type strain (WT) and a positive homozygous mutant strain (. DELTA.FTN).
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following examples,% is by mass unless otherwise specified.
The pRL277 vector is owned by the laboratory and was given in 1998 by professor Donald A.Bryant, university of State university, Pa., original literature sources T.A.Black, Y.P.Cai and C.P.Wolk, Spatial Expression and Autoreglationof Hetr, a Gene analyzed in the Control of heterologous Development in Anabaena (Vol 9, Pg 77,1993), Mol Microbiol 10(5),1153 and 1153. Coli HB101 was purchased from takara, catalog No.: D9051. the cyanobacterium Anabaena sp.pcc 7120 (derived from freshwater algae seed bank (FACHB) of chinese academy of sciences) and mutants derived therefrom are grown in BG11 liquid or solid medium. BG11 solid medium: agar was added to BG11 liquid medium to a final concentration of 1.2% (mass percent, g/mL). During liquid culture, a fluorescent lamp light source is illuminated, and the light intensity is 25uE/m2S, temperature 30 ℃ and introduction of 1% CO2(v/v) air. Erythromycin Em for selection of growth+The antibiotic concentrations were: 10. mu.g/ml.
The experimental E.coli strain DH5 a was grown in conventional LB medium, with the antibiotic concentrations selected for growth: 75 mu g/ml Sm+，150μg/ml Em+。
BG11 medium: per liter of culture medium
Wherein the Trace metal mix comprises the following components:
BG11 medium without N source: that is, no NaNO was added to the BG11 medium3。
(1) Construction of knockout vector PRL277FTN
Structure of knock-out vector PRL277FTNAs shown in FIG. 1, FlankA and FlankB represent homologous fragments on the genome of wild-type Anabaena PCC7120, respectively (see B in FIG. 1). Using the total DNA of wild Anabaena sp.PCC 7120 genome as a template, and amplifying Flanka and FlankB fragments by using primer pairs P1/P2 and P3/P4; a commercial plasmid pMG36e commonly found on the market is taken as a template, and an erythromycin Em gene fragment is amplified by utilizing a primer pair P5/P6; then using the one-step seamless cloning kit (of the whole gold company) ((
Uni Seamless Cloning and Assembly Kit) three fragments FlanKA, Em, FlanKB were ligated simultaneously into pRL277 plasmid to obtain knock-out vector pRL277 FTN.
(2) Screening and PCR verification of positive mutant strain delta FTN as homozygous strain
The knockout vector PRL277FTN is transferred into wild Anabaena sp.PCC 7120 through triparental mating, the schematic diagram of homologous double exchange between the knockout vector PRL277FTN and a genome is shown as C and D in figure 1, and Em replaces an FTN gene through the homologous double exchange. The method of triparental mating is as follows: 1. the combined plasmid pRL443 and the auxiliary plasmid pRL528 are transformed into Escherichia coli HB101 to obtain recombinant bacterium A containing resistance genes Amp (ampicillin), Sm (streptomycin), Cm (chloramphenicol) and Tc (tetracycline); 2. transferring the knockout carrier PRL277FTN into the recombinant bacterium A to obtain a recombinant bacterium B, and washing for 3 times by using an LB culture medium; 3. wild-type Anabaena sp.pcc 7120 was washed 3 times with Bg11 liquid medium; 4. centrifuging the recombinant strain B in the step 2 and the wild-type Anabaena sp.PCC 7120 treated in the step 3, placing the centrifuged recombinant strain B in a suspension culture medium (a culture medium obtained by mixing an LB culture medium and a Bg11 culture medium in equal volumes), fully mixing the centrifuged recombinant strain B and the culture medium, and standing the mixture for 4 hours under the condition of room-temperature low-light culture; 5. and (3) covering a sterile nitrocellulose membrane on Bg11 solid medium, then coating the thallus mixture on the sterile nitrocellulose membrane, and culturing by illumination until new cyanobacterial colonies grow out.
Positive strain Δ FTN was screened on plates containing BG11 solid with erythromycin sucrose, and mutants were made homozygous by serial streak passage. The positive strains are subjected to PCR identification by using primer pairs P7 and P8 (the identification result is shown in figure 2), and the identification result shows that the positive mutant strains can not see the specific bands of wild bacteria completely, namely the positive mutant strains delta FTN are homozygous.
(3) Delta FTN heteromorphic cell size statistics and azotase activity determination
When a positive homozygous mutant strain Δ FTN was cultured in BG11 liquid medium without N and observed by a microscope, as shown in a in fig. 3, it was found that the size of Δ FTN differentiated heteromorphic cells was 60% larger than that of WT differentiated heteromorphic cells. By counting the length and width of heteromorphic cells (see B1 and B2 in FIG. 3), it can be seen that the average length of Δ FTN differentiated heteromorphic cells is about 2 times that of WT heteromorphic cells. By counting the growth curves, Δ FTN was measured to grow for 19.91 hours and the wild type for 20.12 hours, indicating that the mutant grew slightly faster than the wild type.
The azotase activity of delta FTN is about 4.8nmol-1.h-1And WT azotase activity is 2.1nmol-1.h-1Δ FTN has a nitrogenase activity about 2.3 times that of WT (see FIG. 4).
TABLE 1 primer sequences used in the present invention
SEQUENCE LISTING
<110> Beijing university
<120> blue algae engineering bacteria for efficiently fixing nitrogen and preparation method and application thereof
<130> WX2021-03-057
<160> 10
<170> PatentIn version 3.5
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atacctgggc ggacaaatct tcgccatgtc tggcggtagt aaagaacaca atcttatatc 180
agggaatatt tactcaagac tacgttccca tctgcggggt acttcctgta gcgtcttcat 240
ggctgacata aaaatcagac taaaactcac caatgaagcc aaaaacttat tttactatcc 300
agatgttaca gtcacctgcg actcacaaga caaagaccgc ttttacttaa attatccctg 360
cttaattatc gaagttctat caccaagcac agaactcaca gacagaagag aaaaacttgt 420
taattaccgt actctagaaa gcttacaaga atatatatta atttcccaag atgaaatcaa 480
aatcgaagtt taccgcaaag ataaccaaga caattggtca ctagaaatct taagccaggg 540
agatgaatta aaattgaact ctataggatt aaccctcacg atgccagaaa tttacgaaga 600
tgtcatcact atataaggtc aaataaatca tgcagctagc cctacatatt acaacccgaa 660
ttattcccga tacaaactta tcatcagcat aatatgcggg aatgattaaa taacagtttg 720
ccaagattat cccaacccat tcccattgtg atttgcagcg aagccctgat ataagtagtg 780
ttgttttatg atggacgtaa tttcaacaaa ctgttgctca atcattaatg gtgaacaata 840
cgcccattac cacagtctat gtcaacccag caacggggaa tgacgctaac acgggttcgc 900
ggcagagtcc gtttaaaacc cttacccgcg ctttaaaagc cactccacca aagattgtcc 960
aactgagtgc tgggacttat aacactgcaa ataaagaagt ctttcccttg gttattcctg 1020
agggggtaac agtggtgggg aacgaagcca gtaaaggcgc aggaattttg atttctggag 1080
ggggagagta ccaaagccct agctttagtg tgcagaatgt caccttagtt ttactcaatc 1140
gagcgagtct ccggggtgtg actgtcacca atccgatagc taagggtact ggtgtgtgga 1200
ttgaatcaac tgcacctaac attgctaata atacttttac taattgtggt cgagaaggcg 1260
tatttactag cggtacagcc aaaccagcga ttttagataa tgtgtttttg aaaaatacag 1320
ctagtggttt atttatggca cgcaacagta aaggggaagt gttgcggaat gtgttccaaa 1380
agaatccttt aggtatcgct atcagtgact ttgctgcacc tttactcgtt aataacaaac 1440
tatcagaaaa tcggacagcg atcgctcttt ctcgcaatgc ccgtcctgta ctacgtcata 1500
atttactcgt caaaaatacc caaggcggat tattaatcaa tgaaaacgcc ctccctgact 1560
tgggtagcag ccaagatacg gctggcaata tttttagtaa taatggtgaa tttgatatac 1620
aaaatactac aacccaaaaa ttagtttctg taggcaatca gttaaatccc acccaagtca 1680
agggattggt ggaatttccc gccgccatca tcgatattcc catacagtcg gctaacattc 1740
gttttcttga cctagctgga cattgggcaa ccgcttttgt ggaagcattg gtaaacaagg 1800
gactaatgag tggctttccc gatggcacat ttgcccccga tgcaccaatc acccgcgccc 1860
agtatgcggc ggtgattgcc aaaacctttc aactccccgc caacaatcag ccgcccaaat 1920
ttaccgatat caaatcagat ttctgggcag catcagccat caccaaagcc gcccagatgg 1980
gatttattag cggttttcct gatggaacct tccgccccat gcagaactta agcaaagtgc 2040
aggcaatcgt ttccattgtt aatggtctga aattaactgg aggtagcccc aatgtgttaa 2100
ctgtataccg cgatcgcgcc caaattccca gttacgccac aaatcccctc gccgtcgcca 2160
cccaaaacag cttggttgtc aactatcccc aaaccgatag actagaacca ttgcgagata 2220
ttacccgcgc cgagataggg gctttaattt atcaggcatt agtagctaat gggcaagaaa 2280
aagcgatcgc ttcaccatat atagtcagcc ctgaagttga tactccctcc ttcaccgacc 2340
tgacaggaca ctgggctgaa ccattcattc gcggtttagc cagcatgggg atcaccagtg 2400
gttttgctga tggtagctat caacccaaca aacccatgac ccgcgcccaa tatgcggcga 2460
tggtagcagt agcctttaac ccaccagcta gaagaccagc tacagatttt gctgacgtac 2520
ccaagaattt ttgggcatac aaagccttac aaatcgccgc tagtggcggc ttcgtcagtg 2580
gttttagcga tcgcaccttc cgccccgacc aaaacgtcca gaggctacag gtcatcgtgt 2640
ctctagtcaa cggattagcc ctatcgacag ttgatggcaa taacttactc atctacactg 2700
acaataataa tattcctgaa tatgctcgca aagccgtggt tacagcaacg caacaaaaaa 2760
ttactgtcaa ttatccagac ccgaaacagc tagcaccaac cagagaagcc accagggcag 2820
aagtagccgc aatggtctat caagccttag tcgccataca gagagcgcca agtattaatt 2880
caccctatat tatttcgaca gttggcagtt gaacaagtaa aataaaaaac actgcttttt 2940
tgtcggaaaa ctcttggtaa taaatcctac agccaaaaac acgctaaaca cgctaggcta 3000
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ccaatcttaa aaaagaatgt gacagatatt acagccccaa atacttgtcc tgtcttgatt 180
cttaattttg aattgatatt agtacaacca ctaataaagt tagtcaatct ccgcgcccct 240
ctgcgttaaa aacataactt caatactttg agcagccttc cctggtaggg aaggtgctag 300
ggttaagttc cgtcaaactc cccgtaatcc attaactaat tctgacagtc ttaggtgtat 360
ttctgcgtct cttgttgata ctttcctcat caatatctac ttcttgagaa gagagaacac 420
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ttgtatagct agaagttaat ccagaaagcg tcgtattact actacctgtg tttgacaaag 600
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gcgcttgagc atccgtgaga ttattcctcg cactcagcat cagcgccacc acacccgcaa 720
cgtgaggagc tgccattgat gttcctggca taagaccata tttagcacct ggaagcgtcg 780
aatatatacc tatgtctaag ttgctgtatg caccaggggc ggtgacgtat gccattggag 840
ttgttccagc acgattggaa aaattagtta gggttctatt ataattaact gctcccacag 900
caattccaaa ctggtctgca tagcgagctg ggtagccagg tgctgactca ctttcattcc 960
cagatgcgga gacaacaatt gctcctttac tgatggcata ttgaatagct gagagagtat 1020
caggtgaagg attagatcct cctaagctga gattaatcac acgcgcccca ttatctgcgg 1080
cataacgaat actattgcct acggaattac ctgaatttgc tccttgagaa ccacccaacg 1140
ctttcaatgc cataattttg gcattataag caataccagt cactccaaag gtatttctca 1200
cagcagcaat agtaccagcg acatgagtac catgaccgcc ttcatctagt ggagtattgt 1260
tgttgtcgaa aaagttccaa ccatagatat catcgatgaa accattgcca tcatcatcta 1320
tgccattacc cgcaatttct ctgctattcc tccacatatt ggcactcaag tctgggtgtg 1380
tatagtcaac tccactatcc acaaccgcaa cgataacacc ttgaccagtg tatcccttcg 1440
cccagacttc aggtgcttta atcgcatccg ctccccaatc attaccaccc aagtcaggga 1500
catcagcaaa ggtgctttga ttaagggctt tggctacggc ggcggcggcg ttgactaagc 1560
cataacctgt agtggagtta aatccaccac cattagcagg gtttaaagta atgggtgtgt 1620
ctacgtaaaa accattgtta ctttcattgc tctcagcaac agatcctgct ccatcggctt 1680
tcaatatgac gtagtatttg ccaggattca gagttgaatt aaacgtaata gacgtggagc 1740
gagaaataac gctaccaggt gtcagactag cattagcctc agtttcaata cctaagtaga 1800
tatcatcatc accgaatgcc gtatctcgtg aaagataaaa acttgtctta ctgcctccag 1860
cagttccatt accttggttt ttcagttgat agttaacttg cagtgtagtc ccgatggcag 1920
cactactagc agatgtagca ttttggatca ctaaatctgg tgcagtaatc gtgatcgctc 1980
tcgcagcgac gttattattt tcattgcttt ctcttattgt tccatcacca tcagctctat 2040
acagcagata ataattacca ggaacagtgt tgcttctaat tgttaaagat gtagagcgtg 2100
aagctacggc agagggtgca agactagaga tggaatcaaa gcctaagtag acatcatcat 2160
tactgagagt ttggtcttgg gaaagataaa aattagtgaa atgaaaacca gcattgccat 2220
taccttggtt tcttaattga tagctaagtg aaattgttgt cccaatgttt gcgctggtag 2280
gagccgaagg attctggaca attaaatctg gtgccgcaat tgtaatcgct ttggcggtga 2340
cattgttggt ttcgttgctt tcactgattt cactattgcc atcagcttgc aatagcagat 2400
ggtagttacc aaaattgata tccctactta tagaccaaag gtaagattcc gaactataag 2460
tgccagcatt tagtacagga taaaagtagt tcgggtcaga ggctagcaat atatcatcgt 2520
tactaagagt tgtgtcccta gacaaataaa tcttagttct atgattacca gcactagcat 2580
tacctaaatt cgctacttgg tagttgagtg taaatatgtt accagggtca acaatactag 2640
gagcggaaat attttggatg atcaggtcgg gtcttgggcc attaatcgta atagctctag 2700
aaaccacatt attattttca ttactttctg ccactgtgct atcagcatca gttctgaaca 2760
gcaaatgata gttacctccg gcgatactat tagctataac aatcgaggct gtttcggaac 2820
tgacagcacc tgcggcaatg ctattgactg catctgaacc taacaacaca tcatctgtac 2880
tgaatgttgt gtctctagaa aggtagaatc tggtggtact cgcaacggca tttccagcac 2940
cttggtttct tacctgataa ctcaatgcga tcgcactacc tactgatgct gtcgttgctg 3000
ctgtggcgtt ttgaataatt aaatcagctg tgttaattg 3039
Claims (7)
1. A preparation method of high-efficiency nitrogen-fixing blue algae engineering bacteria, wherein the high-efficiency nitrogen-fixing blue algae engineering bacteria are wild anabaena hyalopecuroidesAnabaena spA mutant of PCC7120 with a knockout of the gene for cell-division associated factor FTN, prepared by a method comprising the steps of:
1) constructing a knockout vector, wherein the knockout vector contains upstream and downstream homologous fragments FlankA and FlankB of a cell division related factor FTN gene, and a selective marker gene is arranged between the upstream and downstream homologous fragments FlankA and FlankB, wherein the sequences of the upstream and downstream homologous fragments FlankA and FlankB are respectively shown as SEQ ID No: 9 and 10;
2) transforming wild anabaena PCC7120 by using the knockout vector, screening positive strains on a selective plate, and selecting positive homozygous mutant strains delta FTN by gene detection;
3) culturing the positive homozygous mutant strain delta FTN selected in the step 2), and measuring the nitrogenase activity and the growth rate of the positive homozygous mutant strain delta FTN to obtain the high-efficiency nitrogen-fixing blue algae engineering bacteria with high nitrogenase activity and good growth.
2. The method according to claim 1, wherein the upstream and downstream homologous fragments FlankA and FlankB in step 1) have a length of 3 Kb.
3. The method according to claim 1, wherein the selectable marker gene in step 1) is a resistance marker gene.
4. The method of claim 3, wherein the resistance marker gene is selected from the group consisting of: erythromycin resistance gene Em, kanamycin resistance gene Kan, neomycin resistance gene Neo, streptomycin resistance gene Sm.
5. The method of claim 1, wherein step 1) comprises constructing a knock-out vector pRL277FTN by ligating FlanKA and FlanKB, which are upstream and downstream homologous fragments of FTN gene, and a selectable marker gene into the pRL277 vector by PCR amplification and seamless cloning, wherein the selectable marker gene is erythromycin resistance gene Em.
6. The method according to claim 1, wherein in step 2), the positive strain is selected by continuous streak subculture on a selective plate, and the positive strain selected by the plate is verified by PCR as to whether the plate is a homozygous mutant.
7. The preparation method of claim 1, wherein the nitrogenase activity of the high-efficiency nitrogen-fixing cyanobacteria engineering bacteria obtained in the step 3) is more than 2 times of that of the wild type cyanobacteria engineering bacteria.
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Non-Patent Citations (2)
| Title |
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| A Novel Gene That Bears a DnaJ Motif Influences Cyanobacterial Cell Division;Olga A. Koksharova等;《JOURNAL OF BACTERIOLOGY》;20021031;第184卷(第19期);第5526页右栏第2段，第5527页左栏第1段 * |
| An amidase is required for proper intercellular communication in the filamentous cyanobacterium Anabaena sp. PCC 7120;Zheng Zhenggao等;《PNAS》;20170203;E1405-E1412 * |
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