CN1746304A - The structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene - Google Patents
The structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene Download PDFInfo
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Abstract
The present invention relates to the structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene.The present invention screening and cloned the fixed nitrogen negative regulator gene, and with this gene constructed recombinant plasmid that contains negative regulator gene through transforming, be converted into recipient bacterium after, what obtained that negative regulator gene suddenlys change secretes the ammonium engineering bacteria.The nitrogenase of secreting the ammonium engineering bacteria of the present invention is lived and not suppressed by high ammonium, can high-efficiency nitrogen-fixing, can be used for preparing the bacterial manure of efficient association nitrogen fixation.
Description
Technical field:
The present invention relates to the structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene, the clone and the structure that are specifically related to the fixed nitrogen negative regulator gene are secreted the ammonium engineering strain.The invention still further relates to the purposes of this project bacterial strain.
Background technology:
Combination azotobacter is distributed widely in the rhizosphere of food crop such as paddy rice, wheat, corn and various fruit and vegetable plants, has all more important biological characteristicses such as fixed nitrogen and secretion plant hormone as a kind of biological source of manure.But what set up between combination azotobacter and the host crop is a kind of looser syntaxial system, combination azotobacter mainly is incorporated into the root table, can not resemble and be immersed in inside plants the symbiotic nitrogen-fixing bacteria and form root nodule,, can not satisfy the needs of plant-growth far away so nitrogen-fixing efficiency is lower.This loose syntaxial system be subjected to environmental factor particularly in the soil each factor affecting bigger, wherein ammonium, oxygen are the main limiting factor that influences association nitrogen fixation efficient.
Existing people has found the fixed nitrogen negative regulator gene in some vinelandii.(Azotobactervinelandii, A.v), (Klebsiella pneumoniae K.p) He in the fixed nitrogen vibrios (Azoarcus sp.BH72) determines to exist fixed nitrogen negative regulator gene (nifL) to klebsiella pneumoniae as azotobacter vinelandii.K.p is the pattern bacterium of research azotobacter fixed nitrogen regulation mechanism.Its main characteristic is that combined nitrogen (NH is especially arranged
4 +Or NO
3 -) and can grow fast during aerobic, but fixed nitrogen not; Only just fixed nitrogen growth under the condition of no combined nitrogen and anaerobic.The nif of K.p bunch, total length 23Kb, (nif gene) forms by 20 nif, belongs to 8 transcription units (Merrick M.J., 1988).In K.p and A.v, nifLA forms a manipulator.NifA is the positive regulator gene of nitrogenase, and nifL is the negative regulator gene of nitrogenase.NifL is impression of an external environment ammonium and oxygen level, if one of them is excessive or not enough for ammonium and oxygen, then NifL makes the NifA inactivation.Be commonly considered as forming certain mixture, cause the inactivation of NifA, thereby close the expression of nif by the interaction between NifL negative regulator and the NifA positive regulator.
In azotobacter vinelandii, the nifL mutant strain can cause the ammonium of bacterium secretion up to 30mM, and the work of the nitrogenase of this mutant strain is not subjected to high ammonium to suppress (Bail et al., 1992).The nifL mutant strain of klebsiella pneumoniae can be secreted a spot of ammonium.
Pseudomonas stanieri A1501 (Pseudomonas stutzeri) is a kind of combination azotobacter, separates from the paddy rice rhizosphere, belongs to the gamma subtribe of bacteroid, Gram-negative.Have mobility, can utilize maltose, glucose, lactic acid salt etc. to be the sole carbon source growth.Show fixed nitrogen, urge good characteristics such as living and salt tolerant.It also has the main characteristic of combination azotobacter, i.e. fixed nitrogen under the condition of no combined nitrogen and anaerobic is only having combined nitrogen (NH especially
4 +Or NO
3 -) and fixed nitrogen not during aerobic.
If can determine the fixed nitrogen negative regulator gene that exists in the combination azotobacter, manage to break the restraining effect that ammonium and oxygen are expressed nif under the field fertilization condition, make the combination azotobacter also can high-efficiency nitrogen-fixing when ammonium and oxygen concn are higher and can secrete ammonium.But still fail head it off in the prior art.
Summary of the invention:
The objective of the invention is to clone the fixed nitrogen negative regulator gene in the vinelandii, and make up negative regulator gene (nifL) sudden change secrete the ammonium engineering bacteria, be used to prepare can efficient association nitrogen fixation bacterial manure.
Technical scheme of the present invention is:
1. clone negative regulator gene (nifL)
Make up the clay genomic library of pseudomonas stanieri A1501; method is CTAB method (Wilson; K.Inf.M.etal 1987); promptly extract total DNA of pseudomonas stanieri A1501; the partially digested total DNA of restriction enzyme Sau3AI; the suitable fragments that enzyme is cut is connected on the carrier pLA2917, adopts the package kit packing to connect product, transfection Escherichia coli XLblue.Containing on the antibiotic LB flat board bacterial strain that screening contains recombinant plasmid.
Partial sequence with the negative regulator gene of azotobacter vinelandii is a probe, screening and clone's negative regulator gene from the gene library of A1501, and this gene size is 1.614Kb, sequence is shown in SEQ ID NO:1;
The method of clone's negative regulator gene (nifL) is, in the genomic library that has built, obtain containing the bacterium colony of negative regulator gene recombinant plasmid by bacterium colony in-situ hybridization method (DNA-DNA hybridization), with the recombinant plasmid order-checking that obtains, analyze correct reading frame, and then obtain required gene.
2. make up the recombinant plasmid that contains negative regulator gene, be about to negative regulator gene and be inserted in the tetracycline resistance gene (EcoRI) of suicide plasmid, again kantlex (Km) resistant gene is inserted into the ApaI site of negative regulator gene through transforming.
3. what made up the negative regulator gene sudden change secretes the ammonium engineering bacteria, is about to the above-mentioned recombinant plasmid transformed that contains the negative regulator gene through transforming to recipient bacterium, and screening has penbritin (Ap) resistance but do not have the mutant strain of tetracyclin resistance.
Described suicide plasmid comprises plasmid pSUP202, pPHU281 and PSZ21 etc.;
Described recipient bacterium refers to exist the bacterium of fixed nitrogen negative regulator gene, Ru Sishi pseudomonas A1501, azotobacter vinelandii (Azotobacter vinelandii, A.v), and klebsiella pneumoniae (Klebsiella pneumoniae, K.p) and fixed nitrogen vibrios (Azoarcus sp.BH72) etc.
The nitrogenase activity of secreting the ammonium engineering bacteria of negative regulator gene (nifL) sudden change that the present invention makes up is not suppressed by high ammonium, and when with nitrogen being the only nitrogen source growth, ammonium can be secreted in the substratum when exponential phase finishes.This is secreted the ammonium engineering bacteria and can be used for preparing the efficiently bacterial manure of association nitrogen fixation.
Embodiment:
The plasmid of being lifted in following examples, bacterial strain etc. just are used for for example the present invention being described in further detail, and flesh and blood of the present invention are not limited.In fact, with gene and the method that the present invention finds, what those skilled in the art can make up other transgenation secretes the ammonium engineering bacteria.
The structure of embodiment 1 pseudomonas stanieri A1501 genomic library
Extract total DNA of pseudomonas stanieri A1501 with the CTAB method, with the Sau3AI restriction enzyme with total DNA partially digested be that scope is after the fragment between 15～23kb, be connected on the BglII site that is arranged in the last kalamycin resistance gene of carrier pLA2917, for determining that karyomit(e) inserts the suitableeest condition of contact of fragment, by carrier with insert segmental different ratios (1: 1,1: 2 and 1: 3) and establish three processing, establish simultaneously and do not add the control reaction of inserting DNA.To connect mixture 4 ℃ spend the night connect after, adopt the stratagene package kit GigapackIII XL-4 of company packing to connect product, transfection Escherichia coli XLblue.The transfection bacterium is coated on the LB solid medium with tetracyclin resistance, the bacterium colony that grows put respectively in tetracyclin resistance and tsiklomitsin add on the LB solid medium of kalamycin resistance, the transformant of growing on two kinds of resistance substratum is that cosmid vector is from connecting conversion, the subtracting background colony number calculates titre.
The mensuration of clay packing reaction titre:
Titre should be greater than 10
6
The concrete operations step is as follows:
1, the CTAB method is extracted bacteria total DNA
1) gets single colony inoculation in the corresponding substratum of 5ml, under suitable temperature, cultivate.
2) get 1.0ml bacterium liquid in the 1.5ml centrifuge tube, 13, the centrifugal 2min of 000rpm abandons supernatant.
3) precipitation is resuspended among the 1.0ml 0.85%NaCl.
4) room temperature 13, and the centrifugal 2min of 000rpm abandons supernatant.
5) precipitation is resuspended among 550 μ l, 1 * TE.
6) add 17 μ l N,O-Diacetylmuramidases (35mg/ml), 37 ℃ of incubation 30min.
7) add 3 μ l Proteinase Ks (20mg/ml), 37 ℃ of incubation 30min.
8) add 30 μ l 10%SDS, 37 ℃ of incubation 30min.
9) add the abundant mixing of 100 μ l 5M NaCl.
10) add 80 μ l CTAB/NaCl solution, mixing, 65 ℃ of water-bath 10min.
11) (0.7～0.8ml) chloroform/primary isoamyl alcohol (24: 1), mixing gently vibrates to add equal-volume.Room temperature, 13, the centrifugal 10min of 000rpm.
12) supernatant liquor is transferred in the new 1.5ml centrifuge tube, added equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1) mixing that vibrates gently.
13) repeat the 12nd) step.
14) supernatant liquor is transferred in the new 1.5ml centrifuge tube, added equal-volume chloroform/primary isoamyl alcohol (24: 1) mixing that vibrates gently.
15) repeat the 12nd) step.
16) supernatant liquor is transferred in the new 1.5ml centrifuge tube, added 0.6 volume Virahol, room temperature leaves standstill 60min behind the mixing.
17) 20 ℃ 13, the centrifugal 20min of 000rpm.
18) abandon supernatant, add 500 μ l, 70% ethanol, put upside down (desalinization of soil by flooding or leaching) for several times gently.
19) 4 ℃ 15, the centrifugal 20min of 000rpm.
20) repeat twice of the desalinization of soil by flooding or leaching.
21) be inverted centrifuge tube, dry DNA precipitation 10～15min.
The DNA precipitation is dissolved in 100 μ l, 1 * TE damping fluid, and-20 ℃ of preservations are standby.
2, enzyme is cut dna segment and plasmid pLA2917 ligation
10×buffer 2μl
pLA2917 100ng
Enzyme is cut dna segment 350ng
T4 ligase enzyme (3U/ μ l) 2 μ l
Add water to 20 μ l, 4 ℃ of connections of spending the night.
3, connect the packing and the transfection of product
Adopt the transfection of the stratagene package kit GigapackIII XL-4 of company packing.
1) phage albumen packing connects product
2) from 80 ℃ of refrigerators, take out packaging extract, place on ice.
3) in hand, melt packaging extract rapidly.
4) add connection DNAl-4ul (0.1-1.0ug) immediately.
5) gentle piping and druming mixing does not produce bubble.
6) centrifugal as required 3-5 second is to guarantee that all solution are at the pipe end.
7) room temperature (22 ℃) incubation is 2 hours.
8) add 500ul SM buffer.
9) add 20ul chloroform, gentle mixing.
10) of short duration centrifugation fragment.
11) get supernatant liquor and measure titre, can preserve one month in 4 ℃.
4, wrapping body transfection Escherichia coli
1) guarantees to deposit the bacterial classification from glycerine and draw single bacterium colony, 37 ℃ of incubated overnight
2) picking list bacterium colony from the solid medium is inoculated in the LB liquid nutrient medium that contains 10mM MgSO4 and 0.2% maltose.
3) 200rpm, 37 ℃ shaking culture 4-6 hour, or 30 ℃ of incubated overnight (OD is less than 1.0).
4) 500g is centrifugal 10 minutes, collects thalline.
5) with the gentle resuspended thalline of the aseptic 10mM MgSO4 of half volume.
6) with 10mM MgSO4 thalline being diluted to OD600 is 0.5.
7) pack reaction solution (1: 10,1: 50) with SM buffer dilution
8) 25ul being packed reaction solution and 25ul, to be diluted to the OD600 value be 0.5 thalline mixing, room temperature incubation 30 minutes.
9) add 200ul LB substratum, 37 ℃ of incubations one hour, every 15 minutes gentle mixings once.
10) centrifugal one minute, thalline was resuspended in 50ul LB substratum.
The clone of embodiment 2 fixed nitrogen negative regulator genes (nifL)
Compare the genes involved nucleotide sequence, conserved sequence with the azotobacter vinelandii negative regulator gene is an Auele Specific Primer, be respectively nifL1 (5 '-CGCCATTTCCATCACCGA-3 ') and nifL2 (5 '-GACCACCGCCTCGATGATCAT-3 '), obtain the 0.45kb product through PCR.Purified pcr product through the boiling water bath sex change, is used the DIG-High-Primer mark, obtains screening the required probe of positive reorganization thalline.The bacterium colony that is grown on the substratum is forwarded on the nylon membrane to alkaline process cracking thalline, 80 ℃ of baking fixed dnas.Carry out bacterium colony in situ hybridization with aforementioned PCR product probe.The probe of non-specific combination on the wash-out Hybond membrane adds antibody-solutions and carries out immunology detection subsequently.Show results of hybridization with the exposure of X-exographX at last.
Hybridization obtains 15 positive reorganization bacterium that contain goal gene, by recombinant plasmid is extracted, cuts identification and analysis through PCR and enzyme, therefrom chooses to contain the recombinant plasmid that is inserted with goal gene and carry out sequencing analysis, obtains inserting the genetic construction of gene.
The concrete operations step is as follows:
1, PCR reaction system:
10×PCR buffer 2μl
2.5mM dNTP 2μl
10×BSA 2μl
50pM primer 12 μ 1
50pM primer 22 μ l
Template DNA 5ng
5U/ μ lTaq enzyme 0.25 μ l
Add sterilized water to 20 μ l
2, PCR reaction conditions:
1) 94 ℃ of pre-sex change 20s
2)94℃ 0s
54℃ 0s
72℃ 40s
(25 circulations)
3)72℃ 5min
3, DIG-DNA probe mark
1) adds 1 μ g template DNA (linearity or superhelix), sterilization ddH
2The O final volume is 16 μ l.
2) denatured DNA 10min in the heating boiling water places ice bath to cool off rapidly.
3) mix the DIG primer, get 4 μ l DIG primers and join in the denatured DNA, mixing, and do centrifugal slightly.
4) cultivated 20 hours for 37 ℃.
5) add 2 μ l 0.2M EDTA (pH8.0) or 65 ℃ of heating 10min termination reactions.
4, bacterium colony in situ hybridization:
The cultivation of bacterium colony on the nitrocellulose filter:
1) nitrocellulose filter is laid on contains on the selective antibiotic flat board.
2) bacterium colony is transferred to one by one accordingly on the filter membrane of brassboard with aseptic toothpick and contains on the antibiotic main flat board.
3) each standardized clone who contains non-recombinant plasmid on the brassboard and on the main flat board.
4) being inverted dull and stereotyped 37 ℃ cultivated 16 hours.
5) at three or more asymmetric position mark filter membranes, also do same mark in the position that mainboard is close.
6) with sealing film mainboard is sealed, put upside down and be placed on 4 ℃ of preservations, until the result who draws hybrid experiment.
The cracking of bacterium colony and DNA combine with filter membrane:
1) filter paper is cut into size and suitable three of shape, makes it to be suitable for the bottom of three glass pallets, three filter paper are immersed in respectively in 10%SDS, sex change liquid, three kinds of liquid of neutralizer.
2) excess liquid of inclining with a glass stick roll extrusion filter paper, is removed the bubble between filter paper and tray bottom.
3) take nitrocellulose filter on the agar plate off with blunt-ended forceps, bacterium colony towards on place on the SDS wetted filter paper 3min.
4) film is forwarded to on second filter paper of sex change liquid wetted, soak 5min.
5) filter membrane is forwarded on the 3rd filter paper that in neutralizer, soaks, soak 5min.
6) in pallet, place 100ml 2 * SSC, make filter membrane float over fluid surface number minute.After this, in rocking the process of container, allow filter membrane sink to below the liquid level and stay in the liquid 5min.
7) dry filter membrane: filter membrane is placed on the dried filter paper, and bacterium colony is towards last, in room temperature 30min at least.
8) fixed dna:, in baking box, dried 1-2 hour for 80 ℃ with double team filter membrane about two filter paper.
Hybridization:
1) DIG Easy Hyb (10ml/100cm
2Filter membrane) suitable volume is preheated to hybridization temperature 37-42 ℃.
2) in proper container, shake gently and carry out prehybridization 30min.
3) boiling water bath 5min makes the sex change of DIG label probe, places 0 ℃ rapidly.
4) with the DIG Easy Hyb (3.5ml/100cm of sex change DIG label probe and thermal pretreatment
2Film) mixing.
5) prehybridization solution that inclines adds probe/hybridization mixed solution, shakes gently and reacts 4hr.
6) a large amount of 2 * SSC/0.1%SDS wash film, and 15-25 ℃, 5min shakes liquid frequently; Repeat once.
7) wash film with the 0.5 * SSC/0.1%SDS that is preheated to 65-68 ℃ in a large number, 5min shakes liquid frequently; Repeat once.
Immunoassay:
1) after film is washed in hybridization and strictness, with lavation buffer solution rinsing film 1-5min.
2) react 30min in the 100ml stop buffer.
3) the 20ml antibody liquid is cultivated 30min.
4) 100ml lavation buffer solution rinsing 15min washes 2 times.
5) 20ml measures balance 2-5min in the damping fluid.
6) film (DNA face marking) is placed hybridization bag, add 1mlCSPD ready-to-use (kingpin), cultivate 5min for 15-25 ℃.
7) extrude excessive solution, seal.
8) moistening film is cultivated 10min in 37 ℃, react with enhanced chemiluminescence.
9) under 15-25 ℃ of condition, X-ray sensitization 15-25min.
Embodiment 3 makes up the recombinant plasmid that contains the negative regulator gene through transforming
By analyzing, finding has unique EcoRI restriction enzyme site in the tetracycline resistance gene of suicide plasmid pSUP202, after enzyme is cut, is that the complete negative regulator gene of 1.614kb is inserted into this site with size.Owing to contain unique ApaI restriction enzyme site in the nifL gene, after kantlex (Km) the box ApaI enzyme of pUC4KAPA is cut, be inserted into the ApaI site of negative regulator gene (nifL), obtain recombinant plasmid pSUL1.Specific implementation method is as follows:
1, alkaline lysis method of extracting pSUP202, pUC4KAPA and contain recombinant plasmid (NifLT) DNA (seeing " molecular cloning experiment guide " second edition) of negative regulator gene.
2, plasmid pSUP202 is through restriction enzyme EcoRI digestion, and plasmid pUC4KAPA and NifLT are respectively through restriction enzyme A palI digestion (seeing " molecular cloning experiment guide " second edition).
3, the recovery of dna fragmentation and purifying
Use Promega Wiard PCR Preps DNA Purification System test kit.
1) under long-wave ultra violet lamp, downcuts target stripe, get the Eppendorf pipe that 300mg puts into 1.5ml.
2) add the 1ml resin, temperature bath 5min melts fully up to glue in about 65 ℃ of water-baths.
3) draw resin/DNA mixture in syringe tube, slowly add syringe piston, softly push away homogenate in Minicolumn.
4) Virahol of drawing 2ml 80% is washed pillar in syringe.
5) Minicolumn is forwarded in the 1.5ml Eppendorf tube to the centrifugal Minicolum 2min of 10,000 * g dry resin.
6) shift in Minicolumn to the new Eppendorf tube, add the water of 50ul or TE buffer to Minicolumn, static then 1min.The centrifugal Minicolumn 20sec of 10,000 * g, target DNA fragment under the wash-out.
4, carrier and target fragment is connected
Carrier 100ng
Segment 100ng
Buffer 1μl
T4DNAligase 1μl(3U/μl)
H
2O is to final volume 10 μ l
To connect mixture 16 ℃ of connections of spending the night.
5, the preparation of competent cell
Institute all operates under condition of ice bath in steps:
1) the fresh single bacterium colony on the picking LB solid medium is inoculated in the 20ml LB liquid nutrient medium, 37 ℃ of 220rpm shaking culture 2 hours, and bacterium liquid OD value reaches 0.5-0.6, cooled on ice culture to 0 ℃.
2) change bacterium liquid over to the sterilization centrifuge tube, 4 ℃ of 2700g collect thalline, remove supernatant, are inverted empty doing on thieving paper.
3) precooling 10% glycerine of adding 20ml places on ice, puts upside down centrifuge tube frequently gently, thalline is steeped molten.
4) 4 ℃ of 2700g collect thalline, remove supernatant, are inverted empty doing on thieving paper.
5) precooling 10% glycerine of adding 10ml, the jog centrifuge tube steeps thalline molten.
6) repeating step (4)
7) precooling 10% glycerine of adding 10ml, the jog centrifuge tube steeps thalline molten.
8) repeating step (4)
9) precooling 10% glycerine of adding 200 μ l, the dissolving thalline is distributed into 50 μ l/1.5ml centrifuge tubes.
6, connect product transformed competence colibacillus cell
1) electric conversion instrument (BIO-RAD Gene PulserII System) is transferred to 2.4kV, pulse manipulator is transferred to 400 Ω.
2) 1 μ l plasmid DNA (water-soluble) is joined fill in the frozen competent cell tubule 50 μ l prepared fresh or that melt mixing.
3) transformation mixture is transferred in the electric conversion pool of precooling, blotted the outside surface in pond, put into sample cell then.
4) carry out pulsed electrical and transform, take out electric conversion pool then, add 1ml LB nutrient solution, at once in 37 ℃ of middling speed shaking culture 60min.
5) getting 200 μ l coats and contains on the corresponding antibiotic LB flat board.
The bacterium colony that grows put respectively in tetracyclin resistance and kantlex add on the LB solid medium of penbritin resistance, screening contains the bacterium colony of recombinant plasmid.Alkaline process extracts plasmid and carries out enzyme and cut the evaluation and screening transformant.
Embodiment 4 pseudomonas stanieris are secreted the structure of ammonium engineering bacteria A1601
After recombinant plasmid pSUL1 transformed pseudomonas stanieri A1501, screening had kalamycin resistance but does not have the mutant strain of amicillin resistance, and what made up the negative regulator gene sudden change secretes the ammonium engineering bacteria.The nitrogenase work that pseudomonas stanieri of the present invention is secreted the ammonium engineering bacteria is not suppressed by high ammonium, and when growing with the awkward nitrogenous source of nitrogen, when exponential phase finishes ammonium 10mM is secreted in the substratum.
The concrete operations step is as follows:
1, the conjugal transfer of plasmid
1) donor bacterium DH5 α (pSUL1), recipient bacterium A1501 and the bacterial strain that contains helper plasmid pRK2013 are respectively cultivated in LB (Km 50) and A15 substratum incubated overnight on a small quantity.
2) transfer with 2% ratio and be cultured to OD600=0.4～0.6 in the 5ml LB substratum next day, it is centrifugal to take out 1.5ml bacterium liquid respectively, abandons supernatant, with the physiological saline precipitation and centrifugal that suspends, repeats once, and the thalline after centrifugal is suspended in the 100 μ l physiological saline.
3) drip 15 μ l donor bacterium on the LB flat board with pipettor, drip 15 μ l recipient bacteriums again after air-dry, recipient bacterium will all be covered the donor bacterium, drips the bacterium liquid that 15 μ l contain pRK2013 again.Drip donor bacterium, recipient bacterium and bacterium liquid (pRK2013) after air-dry respectively again one time.
4) cultivate after 6 hours for 30 ℃, scrape mycoderm with inoculating needle and put into the 1ml sterilized water, suspend and shake up, doubling dilution is to 10-5.
5) be extension rate 10 respectively
-1, 10
-3, 10
-5Bacterium liquid 100 μ l be applied on the A15 flat board that contains Km, cultivated 36 hours for 30 ℃.
6) zygote that screening is obtained is transferred purifying once again, and does Km and the evaluation of Tc resistance.
Embodiment 5 pseudomonas stanieris are secreted ammonium engineering bacteria A1601 nitrogenase activity and are measured
1. experiment purpose: the difference of relatively secreting ammonium engineering bacteria A1601 and the nitrogenase activity of wild type strain A1501 under different ammonium concentrations (0mM, 0.1mM and 10mM) condition
2. laboratory apparatus and method: adopt SP-2305 type gas chromatograph, detected the nitrogenase activity of two kinds of bacterium under different ammonium concentrations with acetylene reduction method (ARA), specific implementation method is as follows:
1) picking list bacterium colony places to contain corresponding antibiotic LB liquid nutrient medium incubated overnight.
2) it is centrifugal to get an amount of bacterium liquid, and twice back of aseptic washing makes bacterium liquid OD600 value be about 0.6 with the sterilized water thalline that suspends again.
3) get 10 μ l bacterium liquid and be inoculated in the 7ml penicillin bottle that 3ml does not have nitrogen A15 semisolid medium is housed, add plug.
4) 30 ℃ leave standstill to cultivate after 24 hours and inject acetylene gas, volume be in the bottle gas 10%, continue cultivation.
5) be that 24hr and 48hr survey acetyiene reduction activity respectively at incubation time.Measurement result is as shown in table 1.
Calculate the thalline acetyiene reduction activity with following formula:
Unit: nmol ethene/hour/bottle; C: inject acetylene volume (ml); T: the reaction times (hour)
Table 1 pseudomonas stanieri is secreted the acetyiene reduction activity of ammonium engineering bacteria
| Bacterial strain | The acetyiene reduction activity of different N H4 concentration * | ||
| 0mM | 0.1mM | 10mM | |
| A1501(CK) A1601 | 50.92 85.92 | 61.01 80.62 | 3.95 45.86 |
*Incubation time is 24hr.
Unit: nmol ethene/hour/bottle
3. experiment conclusion: show that by the result nitrogenase activity and the anti-ammonium ability of secreting ammonium engineering bacteria A1601 all are higher than wild type strain A1501.
Embodiment 6 pseudomonas stanieris are secreted the mensuration that ammonium engineering bacteria A1601 secretes the ammonium ability
1. experiment purpose: relatively engineering bacteria A1601 and wild type strain A1501 secrete the difference of ammonium ability
2. experimental technique: engineering bacteria and the ammonium concentration of wild type strain in different cultivation stage substratum when being the only nitrogen source growth by being determined at nitrogen, detect it and secrete the ammonium ability.(Bergersen F.J.1980) detects the two ammonium concentration (table 3) under different time (0hr, 15hr and 30hr) to adopt indigo-blue blue colorimetry.
3. experimental result: when with nitrogen being the only nitrogen source growth, when exponential phase finishes ammonium is secreted in the substratum, ammonium concentration can reach 10mM in the substratum.Concrete data see Table 2:
Table 2 pseudomonas stanieri is secreted the ammonium concentration (mM) of ammonium engineering bacteria
| Bacterial strain | The ammonium concentration of different time | ||
| 0hr | 15hr | 30hr | |
| A1501(CK) A1601 | 0 0 | 0 0.51 | 0 10 |
4. experiment conclusion: the result shows that pseudomonas stanieri of the present invention is secreted the ammonium of ammonium engineering bacteria A1601 fixed nitrogen justacrine higher concentration.
Sequence table
＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences
＜120〉structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene
<130>04-02
<160>1
<170>PatentIn version 3.1
<210>1
<211>1614
<212>DNA
＜213〉pseudomonas stanieri A1501 (Pseudomonas stutzeri)
<400>
1 ATGGCTTTGC AACGGATACC GGCGCACAGA CGTCGGCTTC GCCGCATCGA TGAGGACATG
61 CACATGACCC AGGCCACCCC CGAGCGCGAC GCGGGGCAAC CCGCTGCGTC CGATGGATTG
121 GCGCCCGAAG TGTTCCAGCA AGCCGTGGAG CACGCGCCGA TCGCCATTTC CATCACCGAT
181 CTGAAGGCCA ACATCCTCTA TGCCAACCGC GCCTTCAGCG CCATCACCGG CTACGACAGC
241 AGCGAGGTGA TCGGCAAGAA CGAATCGGTG CTGTCCAACG GCACCACCCC GCGGCTGGTC
301 TACCAGGCGC TCTGGAGCCG CCTGGCGCAG AAAAAGGCCT GGTCCGGCAT GCTGGTCAAC
361 CGGCGCAAGG ACGACAGCTG CTACCTCGCC GAGCTGACCG TGGCGCCGGT GCTCGACGAG
421 CACGAGCGGA CCATCCATTA CCTGGGCATG CACCGCGACA GCAGCGACCA GCACAAGCTC
481 GAACAGCGCG TCAGCAACCA GCGGCTGATC ATCGAGGCGG TGGTCGACAG CGCACCGGCG
541 GCCATCGTGG TGCTCGATCA CGCCCTGCGC ATCCGCCTGT CCAACCCCAG CTTCAACCGC
601 CTGGCCGCGG AACTCGGCGA CCAGGCCACG CCCGCGCAAC TGGTGAGCCT GCTGCAGGAC
661 AACCTCGGCG GCGCCATCGA GGCGCTGAAG GCGCATGGCC AGGCCTTCAC CGGCAAGGAA
721 GTCACCTTCG ACCTCGGCGG CCACACACCG CGCTGGCTGT CCTGCCATGG CCGCGCCATC
781 CTCATCGAAG GCGAACGCGC CGACGACTTC TTCGACCCGG GCGAGGAAAA CTACCTGCTG
841 CTGACGGTCA ACGACATCAC CGGGCTGCGC CAGCAACAGC AGGCCTCCCA GCTCAACGCC
901 CTGAAGGTGC TGATGGCCGA GGAAGAGCTG CTCGACGGCA TGCGCGAAAC CTTCAACGGC
961 GCCATCCACC GCCTGCAGGG CCCGGTCAAC CTGATCAGTG CGGCGCTGCG CATGCTCGAA
1021 CGACGCCTGG GCGACAGCGC CGAGGGCGAT CCGGTGCTCA GCGCCATGCG CGAAGCCAGC
1081 CAGGCTGGCA TGGATGCGCT GGAAAGCCTC AGCGGCTCGA TTCCGCAGCG CCGGGCCGGC
1141 GGCTGCGTGC CGGTCAACAT CAACCAGCTG ATCCGCGAAG TGGTCAGCCT GATGACCGAC
1201 CAGTTGCTCG CCCAGGGCAT CGTCGTCGAC TGGCAGCCGG CGCTGCGCCT ACCCTGGGTG
1261 ATGGGCGCCG AAGGCCGGCT GCGCAGCATG ATCAAGCAGC TGCTGGAAAA CGCCATCGAG
1321 GCCATGAGCC AGAACCAGGA CAACCCGCGC ACGCTGTCGA TCGTCACCCG TGTCCAGGGC
1381 CAGCGGGTGG TGCGCCTGGA GATCGCCGAC AGCGGCCCGG GCATCGCGCC GGAGCTGGCG
1441 CTCAAGGTAT TCGAACCCTT CTTCAGCACC AAGCCGCCGC ACAAGACCGG TCGCGGCATG
1501 GGCCTGGCCA TGGTGCAGGA AACCGTCACC GAGCATGCCG GCACGGTGCA TATCGATAGC
1561 GGCTACGACC AGGGCTGCAG GATCGTCGTC GAACTGCCCT TCTCGGCCAG CTGA
Claims (10)
1.SEQ the dna sequence dna shown in the ID NO:1.
2. recombinant plasmid, feature is to contain the gene shown in the SEQ ID NO:1.
3. the described recombinant plasmid of claim 2 is that described gene is inserted in the tetracycline resistance gene of suicide plasmid, and the ApaI site that again kalamycin resistance gene is inserted into described gene obtains.
4. the preparation method of claim 2 or 3 described recombinant plasmids is that the gene shown in the SEQ ID NO:1 is inserted in the tetracycline resistance gene of suicide plasmid, kalamycin resistance gene is inserted into the ApaI site of this gene again.
5. the preparation method of the described recombinant plasmid of claim 4, described suicide plasmid comprises plasmid pSUP202, pPHU281 and pSZ21.
6. the purposes of claim 2 or 3 described recombinant plasmids is to be used for preparation to secrete the ammonium engineering bacteria.
7. secrete the ammonium engineering bacteria for one kind, feature is to contain claim 3 or 4 described recombinant plasmids.
8. the described ammonium engineering bacteria of secreting of claim 7 is that pseudomonas stanieri is secreted the ammonium engineering bacteria.
9. the described preparation method who secretes the ammonium engineering bacteria of claim 8, be with claim 3 or 4 described recombinant plasmid transformed to recipient bacterium, screening has amicillin resistance but does not have the mutant strain of tetracyclin resistance.
10. the described purposes of secreting the ammonium engineering bacteria of claim 7 is to be used to prepare bacterial manure.
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| CN 200410009540 CN1746304A (en) | 2004-09-10 | 2004-09-10 | The structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene |
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| CN 200410009540 CN1746304A (en) | 2004-09-10 | 2004-09-10 | The structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102041241A (en) * | 2009-10-20 | 2011-05-04 | 中国农业科学院生物技术研究所 | High-efficiency ammonium-excreting combined azotobacter strain |
| CN105063166A (en) * | 2015-09-16 | 2015-11-18 | 东莞市保得生物工程有限公司 | Nitrogen-free solid medium and method for judging ammonia secretion of free living nitrogen fixing bacteria by utilizing solid medium |
| CN108602729A (en) * | 2015-07-13 | 2018-09-28 | 皮沃特生物公司 | Improve the method and composition of plant trait |
| CN112481169A (en) * | 2020-12-08 | 2021-03-12 | 中国科学院微生物研究所 | Saline-alkali tolerant ammonium secreting type self-generating nitrogen fixing strain and application thereof |
| CN113248582A (en) * | 2021-05-11 | 2021-08-13 | 西安交通大学 | Transformation and regulation method of nitrogen-fixing microorganisms |
| CN113817732A (en) * | 2021-08-18 | 2021-12-21 | 中国农业科学院生物技术研究所 | Artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof |
| US11479516B2 (en) | 2015-10-05 | 2022-10-25 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored NIF clusters |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | 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 |
| US11993778B2 (en) | 2017-10-25 | 2024-05-28 | Pivot Bio, Inc. | Methods and compositions for improving engineered microbes that fix nitrogen |
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- 2004-09-10 CN CN 200410009540 patent/CN1746304A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102041241A (en) * | 2009-10-20 | 2011-05-04 | 中国农业科学院生物技术研究所 | High-efficiency ammonium-excreting combined azotobacter strain |
| US11946162B2 (en) | 2012-11-01 | 2024-04-02 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
| US11739032B2 (en) | 2015-07-13 | 2023-08-29 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| CN108602729A (en) * | 2015-07-13 | 2018-09-28 | 皮沃特生物公司 | Improve the method and composition of plant trait |
| CN108602729B (en) * | 2015-07-13 | 2022-07-05 | 皮沃特生物公司 | Methods and compositions for improving plant traits |
| CN105063166A (en) * | 2015-09-16 | 2015-11-18 | 东莞市保得生物工程有限公司 | Nitrogen-free solid medium and method for judging ammonia secretion of free living nitrogen fixing bacteria by utilizing solid medium |
| US11479516B2 (en) | 2015-10-05 | 2022-10-25 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored NIF clusters |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11993778B2 (en) | 2017-10-25 | 2024-05-28 | Pivot Bio, Inc. | Methods and compositions for improving engineered microbes that fix nitrogen |
| CN112481169A (en) * | 2020-12-08 | 2021-03-12 | 中国科学院微生物研究所 | Saline-alkali tolerant ammonium secreting type self-generating nitrogen fixing strain and application thereof |
| CN113248582A (en) * | 2021-05-11 | 2021-08-13 | 西安交通大学 | Transformation and regulation method of nitrogen-fixing microorganisms |
| WO2023019693A1 (en) * | 2021-08-18 | 2023-02-23 | 中国农业科学院生物技术研究所 | Artificial non-coding rna module capable of turning off nifl gene expression |
| CN113817732B (en) * | 2021-08-18 | 2023-12-01 | 中国农业科学院生物技术研究所 | Artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof |
| CN113817732A (en) * | 2021-08-18 | 2021-12-21 | 中国农业科学院生物技术研究所 | Artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof |
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