CN102041241A - High-efficiency ammonium-excreting combined azotobacter strain - Google Patents
High-efficiency ammonium-excreting combined azotobacter strain Download PDFInfo
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- CN102041241A CN102041241A CN2009102361319A CN200910236131A CN102041241A CN 102041241 A CN102041241 A CN 102041241A CN 2009102361319 A CN2009102361319 A CN 2009102361319A CN 200910236131 A CN200910236131 A CN 200910236131A CN 102041241 A CN102041241 A CN 102041241A
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Abstract
The invention provides a novel high-efficiency ammonium-excreting combined azotobacter strain which is characterized in that amt B gene is deleted from the whole genome of Pseudomonas stutzeri A1501, and meanwhile, nif A gene is transferred in. The ammonium measurement test proves that the combined azotobacter strain can excrete ammonium at high efficiency and is a potential microbial fertilizer. The invention also provides a method for constructing the high-efficiency ammonium-excreting combined azotobacter strain, which comprises the steps of construction of complete set of expression vectors, triparental mating, screening of recombinants, and identification.
Description
Technical field:
The present invention relates to a plant height and imitate the association nitrogen fixation bacterial strain of secreting ammonium, the invention still further relates to the construction process that this efficiently secretes ammonium association nitrogen fixation bacterial strain.
Background technology:
The research of biological nitrogen fixation helps to produce more food and is free from environmental pollution.Combination azotobacter is a monoid important in the diazotroph, but owing to be a kind of loose combining between combination azotobacter and the plant, fail to form stable symbiotic structure, thereby it is very limited for the nitrogen that plant provides.And secrete the ammonium bacterium to born of the same parents' external environment secretion ammonium, for host plant provides a certain amount of nitrogen, therefore become domestic and international investigator's research focus.Secrete the ammonium bacterium at present and be confined under the laboratory environment more, want real be applied to the land for growing field crops environment and also will improve it: 1) improve the rhizosphere colonization ability of secreting the ammonium bacterium from following factor for the research of the growth-promoting functions of plant; 2) combined nitrogen is to suppress the fixedly principal element of atmospheric nitrogen, improves the nitrogen fixing capacity and the anti-ammonium ability of secreting the ammonium bacterium under the high ammonium condition; 3) in the soil microorganisms flora, make and secrete the ammonium bacterium and become dominant microflora; 4) improve the supply of the fixed nitrogen energy and optimize its composition.
Pseudomonas stanieri A1501 (Pseudomonas stutzeri A1501) is a strain combination azotobacter, be located away from China south rice field in 1980, this bacterium has very strong nitrogen fixing capacity under little aerobic condition, and the fixed nitrogen product can directly be absorbed by paddy rice rapidly.In addition, pseudomonas stanieri A1501 is except having tangible nitrogenase activity, and under anaerobic this bacterium can also be carbon source with lactic acid, NH
4 +For only nitrogen source carries out denitrification, NO
3 -Be final electron acceptor(EA), accept the terminal electronics that the anaerobic respiration chain transmits, through NO
2 -Finally be reduced to N Deng oxynitride
2The N that denitrification produces
2Can under little aerobic condition, be retightened by pseudomonas stanieri A1501 and form NH
4 +(Lin M et al.1987).
The full genome of pseudomonas stanieri A1501 has checked order and has finished (Yan Y et al.2008; Be for GenBank number
CP000304).Though this bacterial strain has tangible nitrogen fixing capacity, their fixed nitrogens are mainly used to satisfy the growth needs of self, and it is limited to offer the nitrogen of plant.Particularly have under the situation of ammonium in the external world, institute's fixed nitrogen just still less even stops fixed nitrogen and secretes ammonium.This exploitation that has just limited combination azotobacter reaches in application in agriculture.
Therefore, improve the fixed nitrogen of pseudomonas stanieri A1501 bacterial strain and secrete the ammonium ability, the ammonium bacterium that secretes that obtains high-efficiency nitrogen-fixing has great importance.
Summary of the invention:
The objective of the invention is pseudomonas stanieri A1501 bacterium to be transformed with the means of gene recombination, develop the new combination azotobacter of efficiently secreting ammonium of a strain, and a kind of construction process of efficiently secreting ammonium association nitrogen fixation bacterial strain is provided, so that the microbial-bacterial fertilizer with application potential to be provided.
The inventor finds that in the full genome of pseudomonas stanieri A1501, if wherein the amtB gene is lacked, for secreting ammonium significance is arranged, described amtB gene has the nucleotide sequence shown in the SEQ ID NO:1.
The invention provides the association nitrogen fixation bacterial strain of efficiently secreting ammonium, feature is:
In the full genome of pseudomonas stanieri A1501, lacked and had the nucleotide sequence shown in the SEQ ID NO:1; Perhaps,
In the full genome of pseudomonas stanieri A1501, lacked and had the nucleotide sequence shown in the SEQ ID NO:1, and changed gene over to, existed with the plasmid form with the nucleotide sequence shown in the SEQ ID NO:2.
The present invention also provides a kind of method of efficiently secreting the structure of ammonium association nitrogen fixation bacterial strain, comprises that the structure of a whole set of expression vector, three parents engage, the screening and the authentication method of reorganization word.Use bacterial strain to be pseudomonas stanieri (Pseudomonas stutzeri) A1501.
The strain construction concrete grammar is as follows:
1. gene clone
From the genome of pseudomonas stanieri (Pseudomonas stutzeri) A1501, utilize round pcr that the both sides flanking sequence of amtB gene is increased respectively.The fragment of amplification is inserted carrier, engage by three parents and import recipient bacterium P.stuteri A1501, make plasmid that the homology single cross take place on target gene and change, thereby disappearance amtB gene builds mutant strain.
2. vector construction
Clone according to a conventional method (New York:Cold Spring Harbor Laboratory Press, 1989).Cloned nitrogenase positive regulator gene (nifA), the dna fragmentation that will contain complete nifA is connected to pVK100, has made up nifA constitutive expression plasmid pVA3.
3. three parents engage
Engage and to import in the recipient bacterium by the self-plasmid that shifts by three parents.
4. the screening of recon and evaluation
After choosing three flat boards of the continuous commentaries on classics of the bacterium colony that partly grows, carry out bacterium colony PCR and identify.Afterwards, identify secreting the ammonium situation.
Survey the ammonium test through adopting the indophenol blue colorimetry, and compare with wild type strain (A1501) (seeing embodiment 3), confirmation is secreted ammonium with the combination azotobacter strain capable of high-efficiency that the present invention makes up, and then can be farm crop nitrogenous fertilizer is provided, and is a kind of potential microbial fertilizer.
Description of drawings:
Fig. 1 is the segmental amplification of amtB1 dna homolog, and M is a dna molecular amount standard among the figure, and 1 is the segmental swimming lane of purpose;
Fig. 2 is the physical map of intestinal bacteria recombinant vectors pK18mob;
Fig. 3 is the physical map of intestinal bacteria recombinant vectors pVA3;
Fig. 4 cuts checking carrier pVA3 electrophorogram for enzyme, and M is a dna molecular amount standard among the figure, and 1 for to cut the result for the EcoRI enzyme, and 2 is pVA3 PCR result; 3 cut the result for Hind III enzyme;
Fig. 5 is that engineering bacteria 1561/pVA3 and wild-type are secreted ammonium relatively.X-coordinate is for cultivating fate, and ordinate zou is an ammonium concentration.
The sequence table explanation
1, the gene order of SEQ ID NO:1 amtB;
2, SEQ ID NO:2 is the Nucleotide of positive regulatory factor nifA of the nitrogenase gene of pseudomonas stanieri A1501;
3, SEQ ID NO:3 is the NifA amino acid sequence coded of deriving from SEQ ID NO:2.
Embodiment
Below used experiment material is originated as follows in the experiment:
Bacterial strain and carrier: coli strain E.coli JM109 is available from Novagen company, and pseudomonas stanieri A1501 and carrier are made up and preserved by this laboratory.
Enzyme and test kit: restriction enzyme, ligase enzyme, Taq enzyme are NEB company product.It is a day root biochemical corp product that genome extracts test kit.
Biochemical reagents: the DNA synthetic agent is a Millipore company product.Primer synthesizes the Cyclone of ABI company dna synthesizer.Reagent such as IPTG, X-Gal, SDS are Sigma company product.
Substratum: the intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).The restricted substratum of A15: potassium primary phosphate 0.4g, dipotassium hydrogen phosphate 0.1g, sodium-chlor 0.1g, magnesium sulfate heptahydrate 0.2g, manganese sulfate monohydrate 0.01g, sulfuric acid monohydrate iron 0.01g, Sodium orthomolybdate 0.01g, Sodium.alpha.-hydroxypropionate 6ml, ammonium sulfate 0.4g is settled to 1000ml, transfers pH6.8.Be used for liquid culture, 28 ℃ of shaking tables, 180-200rpm cultivates 16～18h.Remove ammonium sulfate in the restricted substratum of A15 nitrogen-free agar: A15.
The experimental technique of other unreceipted actual conditions among the embodiment, carry out according to ordinary method, method as people such as Sambrook, molecular cloning is by (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in the laboratory manual, or the condition of advising according to manufacturer.
Embodiment 1amtB mutant strain makes up
1) the segmental amplification of amtB1 dna homolog
With the total DNA of A1501 is template, respectively with
5′-ACAGGATCCTCTGTTAAGCCCAGGCTT-3′
5′-GGGAGGCTTATTGAAGTTGGTGACGCC-3
For primer carries out pcr amplification, amplification is used to make up the dna fragmentation of amtB transgenation strain, and product is through agarose gel electrophoresis checking (Fig. 1).
2) structure of amtB1 gene integration mutant
After the homologous fragment enzyme that PCR is obtained is cut, be connected respectively on the plasmid pK18mob multiple clone site that enzyme is cut, obtain recombinant plasmid.Recombinant plasmid engages importing recipient bacterium P.stuteri A1501 by three parents after enzyme is cut evaluation, make plasmid that the homology single cross take place on target gene and change, and is incorporated on the karyomit(e), obtains three close zygotes through suitable antibiotic-screening.
3) three parents engage experiment
With the donor bacterium, help bacterium pRK2013 overnight incubation in LB respectively, recipient bacterium is cultivated 15～20 hours to OD in the A15 substratum
6000.6～1.0.Centrifugal collection 1ml recipient bacterium, wash twice with physiological saline, then, after centrifugal collection 1ml donor bacterium, 0.5ml help bacterium respectively in same EP pipe, wash 1～2 time with physiological saline again, at last with pipettor will be fully the bacterium mud of mixing transfer on the A15 substratum (not containing any microbiotic) that is placed with nitrocellulose filter.After drying, be inverted in 32 ℃ and cultivate 24h.
The thalline that joint is finished is with 400 μ l ddH
2O wash-out from the filter membrane is coated on the consumption of 200 μ l respectively and contains on the suitable antibiotic A15 culture medium flat plate, screens three close zygotes.Cultivate after 3-4 days, zygote is carried out that enzyme is cut or the PCR checking.
It is with the donor bacterium that solid three parents engage, and helps bacterium and recipient bacterium not containing abundant mixing on any antibiotic A15 substratum, is inverted in 30 ℃ and cultivates two days.Three close zygosporic screenings and checking engage identical with liquid three parents.Mutant strain called after 1561.
The structure and the checking of embodiment 2 1561/pVA3 bacterial strains
1, the clone of nifA gene molecule
Extract the genome of pseudomonas stanieri A1501.With the genome that is extracted is template, carries out pcr amplification.Employed primer is synthetic according to 5 ' and 3 ' terminal sequence of nifA gene, and 2 primer sequences are respectively:
5 ' CCGAAGCTTTCAGATCTTGCGCATATGA 3 ' and
5’CCGAAGCTTACGGTGCATATCGATAGC?3’，
Electrophoresis reclaims the about 1124bp fragment of phytase phyc gene.Obtain complete nifA gene by PCR method, and comprise one section nucleotide sequence of the nifA gene coding region about 70bp in upstream, length is the purpose fragment of 1.64kb.Cut and reclaim the purpose fragment with the HindIII enzyme, be connected with the shuttle vectors pVK100 that same enzyme is cut.Tc
sKm
rResistance screening extracts plasmid, and HindIII and EcoRI enzyme are cut evaluation, and recombinant plasmid pVA3 collection of illustrative plates is seen Fig. 3.
2, three of pVA3 parents engage experiment
Respectively with donor bacterium pVA3, recipient bacterium 1561 helps bacterium pRK2013 according to 1: 1: 1 mixed, by three close bonded methods, recombinant plasmid pVA3 is changed in A1501 and 1561, carries out kantlex (Km) then, tsiklomitsin (Tc) resistance screening.Whether 3 three close zygotes of picked at random extract plasmid checking pVA3 and change in A1501 or 1561.
After extracting plasmid, be template, carry out the PCR checking, obtain the fragment of 1.6Kb, to extract 1561 the negative contrast of empty plasmid with the plasmid DNA; Simultaneously plasmid is cut checking by EcoR I and Hind III enzyme, cut out two band (see figure 4)s respectively, thereby obtained the engineering bacteria of reorganization, called after 1561/pVA3.Because the expression of nifA is subjected to the startup of Km resistance box promoter among the pVA3, after pVA3 changes 1561 over to, transcribing of the nifA among the engineering bacteria 1561/pVA3 of reorganization will not be subjected to the inhibition of ammonium, with constitutive expression.
The ammonium of secreting of embodiment 3 engineering bacteria 1561/pVA3 and wild type strain compares
1) bacterial strain is secreted the ammonium measuring method
NH
4 +The mensuration of concentration is according to indophenol blue colorimetry (Bender et al.1977): A solution (100ml): 5g phenol; 0.025g Na-nitroprusside (Na
2Fe[CN]
5NO2H
2O); B solution (100ml): 62.5ml1M NaOH; 3.3ml NaOCl (available chlorine 5.25%).Get 100 μ l microbial culture supernatant liquors, add 100 μ l B solution again after adding 100 μ l A solution, the depth of observing response liquid color can judge whether to have NH behind the 20min
4 +Suitably extended volume can be drawn NH
4 +The typical curve (NH of serial dilution 1M
4 +Be 0.1,0.2,0.4,0.6,0.8,1.0,1.5,2.0mM; After adding A, B solution reaction 20min, at OD
625Colorimetric).According to the typical curve NH in the supernatant liquor as can be known
4 +Concentration.
2) result
Under the fixed nitrogen condition, the nitrogenase of 1561/pVA3 (14.24U/mgprotein) alive is about 1.95 times of wild type strain (A1501).With 1561/pVA3, A1501 is inoculated in the restricted substratum of semi-solid no nitrogen, is being full of N
2, 0.5%O
2, cultivate under 30 ℃ of conditions, cultivated 15 days, survey its nitrogenase and live the ammonium amount of secreting and pH value in the substratum.Cultivate after 15 days, wild-type, A1501/pVA3 and 1561 concentration by ammonium in the indophenols blue laws detection substratum are found all to show blue, promptly do not secrete ammonium; And show blue at the substratum of 1561/pVA3, and secrete the ammonium amount and reach 5.15mM, its substratum be weakly alkaline (pH value=7.42, and the wild-type cultivation does not detect among 15 days and secretes ammonium, amine concentration is 0, the result is as shown in Figure 5.Result of study shows that 1561/pVA3 has stronger nitrogen fixation activity, and can be to the cell exocrine ammonium.
Sequence table
＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences
＜120〉efficiently secrete the association nitrogen fixation bacterial strain of ammonium
<160>3
<170>PatentIn?version?3.1
<210>1
<211>1317
<212>DNA
＜213〉fixed nitrogen pseudomonas stanieri A1501 (Pseudomonas stutzeri)
<400>1
atgactctgc?gaagattcgc?agggctagga?gcccttttgt?ctctgttaag?cccaggcctg 60
gccatggcgc?aggaggcaac?gctggattca?ggcgatacgg?catggatgct?gacggccact 120
gcgctggtgc?tgttcatgac?catccccgga?ctggcgctgt?tctacggcgg?catggtccgc 180
tcgaagaaca?tcctctcggt?gatgatgcag?tgcttcgcca?tcaccggcct?gatgagcatc 240
ctctggatgg?tctatggcta?cagcctcgca?ttcgatacca?ccggtatgga?agcgggcgtc 300
accaacttca?attccttcgt?cggtggtctg?gatcgcgcct?tcctcagcgg?cctgactcct 360
gacagcctgg?tcggcgcctt?cccggaaagc?gtcttcatca?cgttccagat?gaccttcgcc 420
atcatcaccc?cggcgctcat?cgtcggcgcc?ttcgccgagc?gcatgaagtt?ctcggcgatg 480
ctggtgttca?tgggcgtgtg?gttcaccctg?gtctatgcgc?cgatcgcgca?tatggtctgg 540
ggcggtgacg?gtggcctgat?gtgggactgg?ggcgtgctgg?atttcgccgg?tggcaccgtg 600
gtgcacatca?atgccggtat?cgccggtctg?gtggcctgcc?tggtcctggg?caagcgcaag 660
ggcttcccga?ccacgccgat?ggcaccgcac?aacctggggc?tgaccctggt?cggcgcggcg 720
atgctgtgga?ttggctggtt?cggcttcaac?gccggttcgg?ccgtggctgc?caacggcacc 780
gctggcatgg?ccatgctggt?cacccagatc?gccaccgccg?cagctgcgtt?gggctggatg 840
ttcgccgagt?ggatcggcca?cggcaaaccc?agcgcgctgg?gcatcgcctc?gggcgtggtc 900
gccggtctgg?tcgccatcac?cccggctgcc?ggcaccgtcg?ggccgatggg?cgcgctgatc 960
atcggcctgg?tctcgggcgt?ggtctgcttc?ttctgcgcca?ccagcctgaa?acgcaagctc 1020
ggctacgacg?attcgctgga?tgcctttggc?gtgcacggcg?tgggcggcat?cgtcggtgcg 1080
ctgctcaccg?gcatcttcgc?cgcacccatg?ctgggcggtt?tcggtgaggt?ggagaacatc 1140
ggcctgcagc?tgtggattca?gttcaagggc?gtgctcttca?ccgtcgtcta?caccggcatc 1200
gtcacctacc?tgatcctcaa?ggcgatcgac?ctggtgatgg?gcctgcgggt?gaacgaggag 1260
caagagacca?tcggcctcga?cctcagcctg?cacaacgagc?gcggctacaa?cctgtaa 1320
<210>2
<211>1566
<212>DNA
＜213〉fixed nitrogen pseudomonas stanieri A1501 (Pseudomonas stutzeri)
<400>1
atgaacgcca?cattcgccga?acgccccagc?gcgccaaccc?gcaacgaact?gctggatgcc 60
caactgcagg?cgctggcgca?gatcgcccgc?atccttaacc?gcggccggcc?catcgaggaa 120
ctgctggccg?agatcctcgc?cgtgctgcac?gaagacctcg?gcctgctgca?cgggctggtc 180
tccatctgca?acccgaagga?cggcagcctg?caggtgggcg?ccgtgcacag?cgactccgaa 240
accgtggtac?gggcctgcga?aagcacccgc?taccgcatcg?gcgaaggcgt?gttcggcaac 300
atcctcaagc?atggcaacag?cgtggtgctc?gggcgtatcg?acgccgaacc?gcgctttctc 360
gaccgactgg?cgctgtacga?catggacctg?cccttcatcg?ccgtgccgat?caaggccgtc 420
gacggcacca?ccatcggcgt?gctggctgcc?cagcccgacc?gccgcgccga?cgagctgatg 480
cccgaacgca?cccgtttgat?ggaaatcgtc?gcccgcctac?tggcgcagac?cgtgcgcctg 540
gtggtgaacc?tcgaggacgg?ccaggaagtg?gtcgacgagc?gcgacgagct?acgccgcgaa 600
gtccgcgcca?agtacggctt?cgagaacatg?gtggtgggcc?acaccgcctc?catgcgccgg 660
gttttcgacc?aggttcgacg?ggtcgccaag?tggaacagca?ccgtgctgat?cctcggcgaa 720
tccggcaccg?gcaaggagct?gatcgccagc?gccatccact?acaactcacc?gcgcgctcac 780
cagccgctgg?tacgcctgaa?ctgcgccgcg?ctaccggaaa?ccctgctcga?atcggaactg 840
ttcggtcacg?agaaaggcgc?cttcaccggc?gccgtgaagc?agcgcaaggg?acgtttcgaa 900
caggccgacg?gcggcaccct?gttcctcgac?gagatcggcg?agatctcgcc?gatgttccag 960
gccaagctgc?tgcgcgtgct?gcaggaaggc?gagctggagc?gcgtcggcgg?cagccagacg 1020
gtgaaggtca?acgtgcgcat?cgtcgccgcc?accaaccgcg?acctggagca?cgaggtggag 1080
caaggcaagt?tccgcgaaga?cctctactac?cgcctcaacg?tcatggccat?ccgcgtcccg 1140
ccgctgcgcg?agcgcagcgc?cgacatcccg?gaactggccg?aattcctcct?cgacaagatc 1200
gcccgccagc?agggtcgcaa?actcaagctg?accgacagcg?ccctgcgtct?gctgatgagc 1260
caccgctggc?cgggcaacgt?gcgcgaactg?gaaaactgcc?tggaacgctc?ggccatcatg 1320
agcgaggatg?gcaccatcag?ccgcgacgtg?gtctccctca?ccggcctcga?ccacgacgcc 1380
acgccgctgg?cgccggtccc?cgaagtcgac?ctcgccgacg?acagcctcga?cgaccgcgag 1440
cgcgtcatcg?ccgcgctgga?acaggccggc?tgggtccagg?ccaaggccgc?ccgcctgctc 1500
ggcatgacgc?cccggcagat?cgcctaccga?gtgcagacgc?tgaacattca?tatgcgcaag 1560
atctga 1620
<210>3
<211>521
<212>PRT
＜213〉fixed nitrogen pseudomonas stanieri A1501 (Pseudomonas stutzeri)
<400>2
MET?Asn?Ala?Thr?Phe?Ala?Glu?Arg?Pro?Ser?Ala?Pro?Thr?Arg?Asn?Glu
1 5 10 15
Leu?Leu?Asp?Ala?Gln?Leu?Gln?Ala?Leu?Ala?Gln?Ile?Ala?Arg?Ile?Leu
20 25 30
Asn?Arg?Gly?Arg?Pro?Ile?Glu?Glu?Leu?Leu?Ala?Glu?Ile?Leu?Ala?Val
35 40 45
Leu?His?Glu?Asp?Leu?Gly?Leu?Leu?His?Gly?Leu?Val?Ser?Ile?Cys?Asn
50 55 60
Pro?Lys?Asp?Gly?Ser?Leu?Gln?Val?Gly?Ala?Val?His?Ser?Asp?Ser?Glu
65 70 75 80
Thr?Val?Val?Arg?Ala?Cys?Glu?Ser?Thr?Arg?Tyr?Arg?Ile?Gly?Glu?Gly
85 90 95
Val?Phe?Gly?Asn?Ile?Leu?Lys?His?Gly?Asn?Ser?Val?Val?Leu?Gly?Arg
100 105 110
Ile?Asp?Ala?Glu?Pro?Arg?Phe?Leu?Asp?Arg?Leu?Ala?Leu?Tyr?Asp?MET
115 120 125
Asp?Leu?Pro?Phe?Ile?Ala?Val?Pro?Ile?Lys?Ala?Val?Asp?Gly?Thr?Thr
130 135 140
Ile?Gly?Val?Leu?Ala?Ala?Gln?Pro?Asp?Arg?Arg?Ala?Asp?Glu?Leu?MET
145 150 155 160
Pro?Glu?Arg?Thr?Arg?Leu?MET?Glu?Ile?Val?Ala?Arg?Leu?Leu?Ala?Gln
165 170 175
Thr?Val?Arg?Leu?Val?Val?Asn?Leu?Glu?Asp?Gly?Gln?Glu?Val?Val?Asp
180 185 190
Glu?Arg?Asp?Glu?Leu?Arg?Arg?Glu?Val?Arg?Ala?Lys?Tyr?Gly?Phe?Glu
195 200 205
Asn?MET?Val?Val?Gly?His?Thr?Ala?Ser?MET?Arg?Arg?Val?Phe?Asp?Gln
210 215 220
Val?Arg?Arg?Val?Ala?Lys?Trp?Asn?Ser?Thr?Val?Leu?Ile?Leu?Gly?Glu
225 230 235 240
Ser?Gly?Thr?Gly?Lys?Glu?Leu?Ile?Ala?Ser?Ala?Ile?His?Tyr?Asn?Ser
245 250 255
Pro?Arg?Ala?His?Gln?Pro?Leu?Val?Arg?Leu?Asn?Cys?Ala?Ala?Leu?Pro
260 265 270
Glu?Thr?Leu?Leu?Glu?Ser?Glu?Leu?Phe?Gly?His?Glu?Lys?Gly?Ala?Phe
275 280 285
Thr?Gly?Ala?Val?Lys?Gln?Arg?Lys?Gly?Arg?Phe?Glu?Gln?Ala?Asp?Gly
290 295 300
Gly?Thr?Leu?Phe?Leu?Asp?Glu?Ile?Gly?Glu?Ile?Ser?Pro?MET?Phe?Gln
305 310 315 320
Ala?Lys?Leu?Leu?Arg?Val?Leu?Gln?Glu?Gly?Glu?Leu?Glu?Arg?Val?Gly
325 330 335
Gly?Ser?Gln?Thr?Val?Lys?Val?Asn?Val?Arg?Ile?Val?Ala?Ala?Thr?Asn
340 345 350
Arg?Asp?Leu?Glu?His?Glu?Val?Glu?Gln?Gly?Lys?Phe?Arg?Glu?Asp?Leu
355 360 365
Tyr?Tyr?Arg?Leu?Asn?Val?MET?Ala?Ile?Arg?Val?Pro?Pro?Leu?Arg?Glu
370 375 380
Arg?Ser?Ala?Asp?Ile?Pro?Glu?Leu?Ala?Glu?Phe?Leu?Leu?Asp?Lys?Ile
385 390 395 400
Ala?Arg?Gln?Gln?Gly?Arg?Lys?Leu?Lys?Leu?Thr?Asp?Ser?Ala?Leu?Arg
401 405 410 415
Leu?Leu?MET?Ser?His?Arg?Trp?Pro?Gly?Asn?Val?Arg?Glu?Leu?Glu?Asn
420 425 430
Cys?Leu?Glu?Arg?Ser?Ala?Ile?MET?Ser?Glu?Asp?Gly?Thr?Ile?Ser?Arg
435 440 445
Asp?Val?Val?Ser?Leu?Thr?Gly?Leu?Asp?His?Asp?Ala?Thr?Pro?Leu?Ala
450 455 460
Pro?Val?Pro?Glu?Val?Asp?Leu?Ala?Asp?Asp?Ser?Leu?Asp?Asp?Arg?Glu
465 470 475 480
Arg?Val?Ile?Ala?Ala?Leu?Glu?Gln?Ala?Gly?Trp?Val?Gln?Ala?Lys?Ala
485 490 495
Ala?Arg?Leu?Leu?Gly?MET?Thr?Pro?Arg?Gln?Ile?Ala?Tyr?Arg?Val?Gln
500 505 510
Thr?Leu?Asn?Ile?His?MET?Arg?Lys?Ile
515 520
Claims (3)
1. efficiently secrete the association nitrogen fixation bacterial strain of ammonium, feature is to have lacked to have the nucleotide sequence shown in the SEQ ID NO:1 in the full genome of pseudomonas stanieri A1501.
2. efficiently secrete the association nitrogen fixation bacterial strain of ammonium, feature is to have lacked to have the nucleotide sequence shown in the SEQ ID NO:1 in the full genome of pseudomonas stanieri A1501, and changes the gene with the nucleotide sequence shown in the SEQ ID NO:2 over to.
3. make up the method for claim 1 or 2 described bacterial strains, comprise the structure of expression vector, screening and the authentication methods that three parents engage experiment, recon, it is characterized in that the nucleotides sequence shown in the SEQ ID NO:1 is listed in the fixed nitrogen pseudomonas stanieri and use.
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| CN2009102361319A CN102041241A (en) | 2009-10-20 | 2009-10-20 | High-efficiency ammonium-excreting combined azotobacter strain |
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|---|---|---|---|
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|---|---|
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ID=43907790
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| CN103525830A (en) * | 2013-09-30 | 2014-01-22 | 中国农业科学院生物技术研究所 | Gene capable of enhancing ammonium-secreting ability of nitrogen-fixing bacteria |
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| US11479516B2 (en) | 2015-10-05 | 2022-10-25 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored NIF clusters |
| US11739032B2 (en) | 2015-07-13 | 2023-08-29 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11946162B2 (en) | 2012-11-01 | 2024-04-02 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104136599A (en) * | 2011-11-24 | 2014-11-05 | 国家科学和技术研究委员会(Conicet) | Recombinant nitrogen-fixing bacterial strain, inoculum containing the same and application methods |
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| CN104136599B (en) * | 2011-11-24 | 2017-05-24 | 国家科学和技术研究委员会(Conicet) | Restructuring nitrogen-fixing bacteria bacterial strain, the inoculum that contains described bacterial strain and application method |
| US11946162B2 (en) | 2012-11-01 | 2024-04-02 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
| CN103525830A (en) * | 2013-09-30 | 2014-01-22 | 中国农业科学院生物技术研究所 | Gene capable of enhancing ammonium-secreting ability of nitrogen-fixing bacteria |
| US11739032B2 (en) | 2015-07-13 | 2023-08-29 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11479516B2 (en) | 2015-10-05 | 2022-10-25 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored NIF clusters |
| CN110799474A (en) * | 2017-01-12 | 2020-02-14 | 皮沃特生物公司 | Methods and compositions for improving plant traits |
| CN110799474B (en) * | 2017-01-12 | 2022-07-26 | 皮沃特生物公司 | Methods and compositions for improving plant traits |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| CN107119000B (en) * | 2017-04-19 | 2019-04-12 | 山东大学 | The screening technique of mutant strains of pseudomonas fluorescens and its application in biological control |
| CN107119000A (en) * | 2017-04-19 | 2017-09-01 | 山东大学 | The screening technique of mutant strains of pseudomonas fluorescens and its application in biological control |
| WO2019034992A1 (en) * | 2017-08-17 | 2019-02-21 | Miklens Bio Private Limited | Naturally deriving bio-available forms of nitrogen, phosphorous and potassium using microbial fermentation |
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