CN106086042A - Nitrogen-fixing bacteria ammonium vector gene, mutant, nitrogen-fixing bacteria ammonium carrier mutant and application - Google Patents

Abstract

The present invention relates to nitrogen-fixing bacteria ammonium vector gene, mutant, nitrogen-fixing bacteria ammonium carrier mutant and application, nitrogen-fixing bacteria Kosakonia radicincitans GXGL 4A, being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 2nd, 2016, preservation registration number is CGMCC No.12588；Nitrogen-fixing bacteria ammonium vector gene nrgA, the ammoniacal nitrogen that can be secreted into outside born of the same parents by nitrogen-fixing bacteria is transported intracellular back, is maintained the balance in nitrogen-fixing bacteria nitrogen storehouse, have nucleotide sequence as shown in SEQ ID No.1.Also include nitrogen-fixing bacteria ammonium vector gene mutant, mutant.Described mutant, at nitrogen stress or without in nitrogen environment, can better profit from the nitrogen in air and carry out microorganism fixed nitrogen.Simultaneously, mutant maintains the good speed of growth of wild strain and secretes ammonium ability, barren nitrogen stress soil is used reach volume increase or for preparing microbial-bacterial fertilizer, reduce amount of application of nitrogen fertilizer, to prevent owing to there is good application prospect in the fields such as excess the applied nitrogen soil acidification, greenhouse gas emission and the groundwater azotate pollution that cause.

Description

Nitrogen-fixing bacteria ammonium vector gene, mutant, nitrogen-fixing bacteria ammonium carrier mutant and application

Technical field

The invention belongs to technical field of bioengineering, especially relate to a kind of nitrogen-fixing bacteria ammonium vector gene, mutant, fixed nitrogen Bacterium ammonium carrier mutant and application.

Background technology

Ammonium carrier protein is the hydrophobic protein that a class is present on cell membrane, and in the environment of low ammonium, ammonium carrier leads to The mode crossing Active transport transports the ammonium ion outside born of the same parents back cell, it is ensured that intracellular ammonium storehouse is stable and cell nitrogen metabolism normal Run.Ammonium carrier the earliest is to be isolatable from saccharomycetic mep gene, thereafter, from prokaryote bacillus subtilis and large intestine bar Having cloned ammonium carrier nrgA and amtB gene in bacterium respectively, these 2 gene order similaritys are about 42%.In recent years, ammonium is separated Carrier is also studied it and is secreted ammonium and transporting mechanism and become the study hotspot in biological nitrogen fixation field.

In most antibacterials, ammonium carrier amtB gene and glnK gene belong to a manipulator, at glnK gene In the upstream of amt gene, both are closely coupled, share a promoter, and what it started transcribes by Nitrogen Regulation system (ntr) Regulation and control, promoter is NtrC dependent form.GlnK albumen is soluble protein, and it forms complex with AmtB albumen, plays negative tune Control ammonium carrier AmtB transports NH₃/NH₄ ⁺The effect of activity.On the combination interface of AmtB-GlnK, GlnK protein protomer the 47th Amino acids i.e. arginine (Arg₄₇) with ammonium carrier protein AmtB subunit on the 313rd amino acids i.e. aspartic acid (Asp₃₁₃) Form salt bridge.

The connection complex crystal structure of ammonium carrier protein AmtB and GlnK albumen must compare thoroughly the most after deliberation, up till now Till identified on ammonium carrier protein with NH₃/NH₄ ⁺In conjunction with 6 sites, i.e. Am1-Am6.These researchs are for from molecular water Flat transformation ammonium carrier is effectively secreted ammonium nitrogen-fixing bacteria with structure and is provided theoretical foundation.Utilize molecular biology method, the most closely The CRPSPR-Cas9 gene editing technology in the past few years occurred is oriented sudden change to ammonium vector gene so that it is the ability of delivery ammonium Losing or significantly reduce, that can improve nitrogen-fixing bacteria in theory secretes ammonium ability, and crop can directly absorb nitrogen-fixing bacteria secretion Ammoniacal nitrogen outside born of the same parents, reaches Shaoshi chemical nitrogen fertilizer or purpose that applied nitrogen just can not increase production, thus was prevented effectively from Body eutrophication that amount applied nitrogen causes, the problem such as the soil salinization, it is achieved the sustainable development of agricultural, have well Scientific meaning and actual application value.By genetically engineered and screening, there is under nitrogen stress environment higher nitrogenase activity, The double non-leguminous plant nitrogen-fixing bacteria that can efficiently secrete ammonium become the study hotspot in current microorganism fixed nitrogen field, with ammonium vector gene Editor transform breach as and realizes the effective way of these targets beyond doubt.

Summary of the invention

The purpose of the present invention, it is simply that in order to by Rhizosphere of Crops combination azotobacter Kosakonia radicincitans The ammonium vector gene of GXGL-4A suddenlys change, and by CRPSPR-Cas9 gene editing technology, by this gene silencing, is allowed to lose Or significantly reduce the ability delivering ammonium ion, reach that both there is good biological nitrogen fixation ability, can effectively secrete again ammonium.

The purpose of the present invention can be achieved through the following technical solutions:

One of technical scheme that the present invention takes is: provide a kind of nitrogen-fixing bacteria, nitrogen-fixing bacteria Kosakonia Radicincitans GXGL-4A, is deposited in China Committee for Culture Collection of Microorganisms on June 2nd, 2016 common Microorganism center, preservation registration number is CGMCC No.12588.

The two of the technical scheme that the present invention takes are: a kind of nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Ammonium vector gene, for having nucleotide sequence shown in SEQ ID No.1, this sequential coding ammonium carrier protein, by this carrier Ammonium ion outside fixed nitrogen mycetocyte can be transported back intracellular thus ensure that cell nitrogen storehouse is stable and being normally carried out of nitrogen metabolism by albumen.

The acquisition of described ammonium vector gene sequence can expand nitrogen-fixing bacteria Kosakonia radicincitans by PCR method GXGL-4A genomic DNA realizes.

The three of the technical scheme that the present invention takes are: the sudden change of a kind of ammonium vector gene, for ammonium vector gene DNA sequence 5 ' end 2 sgRNA of design, as target spot, build corresponding Cas9-guide rna expression carrier, convert nitrogen-fixing bacteria GXGL-4A, screens positive transformant.

The four of the technical scheme that the present invention takes are: a strain effective ammonium carrier mutant, and it is by measuring transformant Nitrogenase activity and the outer ammonia nitrogen content Integrated Selection mutant out of born of the same parents, and to containing nrgA-glnK gene land The DNA sequence in territory has carried out sequence verification.

The five of the technical scheme that the present invention takes are: ammonium carrier mutant carries out answering under nitrogen-free or nitrogen restrictive condition With.By using acetylene reduction method to measure the nitrogenase activity of mutant, compared with wild strain GXGL-4A, targeted mutagenesis strain exists Cultivating in Ashby nitrogen-free agar and have higher nitrogenase activity, it is cultivated in the LB culture medium of rich nitrogen, nitrogenase activity It is effectively suppressed, demonstrates more preferable resistance to low nitrogen ability and the high degree of adaptability to extracellular environment.

The ammonium carrier nrgA gene source of the present invention in nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, its Mutant is to dash forward through CRISPR-Cas9 gene editing technology having as shown in SEQ ID No.1 in sequence table nucleotide sequence Become, including the mutant with more preferable nitrogenase activity function of the Base substitution mutations acquisition in multiple sites.In the present invention NrgA mutant Kosakonia radicincitans GXGL-4A (g5-1) azotase in nitrogen-free Ashby culture medium Activity is 346.32 ± 4.16nmol C₂H₄/ (mL h), and compare the fixed nitrogen of wild strain K.radicincitans GXGL-4A Enzymatic activity is 205.14 ± 6.21nmol C₂H₄/ (mL h), both differences reach pole significant level.Meanwhile, nrgA mutant is protected Hold the good speed of growth of wild strain and secreted ammonium ability, having used to reach volume increase or micro-for preparing in barren nitrogen stress soil Bio-bacterial manure, reduces amount of application of nitrogen fertilizer, to prevent soil acidification, greenhouse gas emission and the ground owing to excess applied nitrogen causes There is good application prospect in the fields such as lower water azotate pollution.

Accompanying drawing explanation

Fig. 1 is GXGL-4A nitrogen-fixing bacteria electromicroscopic photograph (Tecnai G2spirit Biotwin, 120kV Bio-TEM)；

Fig. 2 is that the PCR of Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria ammonium carrier nrgA gene expands electrophoresis Collection of illustrative plates；

Fig. 3 is the Cas9 expression vector pET28a-cas9 collection of illustrative plates built；

Fig. 4 is the cas9 gene test PCR amplification of Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria transformant Electrophoresis pattern；

In Fig. 4, M:marker is DL2000 (TaKaRa)；Swimming lane 1-4 is mutant；

Fig. 5 is ammonium carrier mutant nitrogenase activity the selection result in LB culture medium；

In Fig. 5,4A:GXGL-4A (CK), wild strain；5-1,5-2,5-3,5-4 and 5-11 are ammonium carrier mutant；

Fig. 6 is ammonium carrier mutant nitrogenase activity the selection result in Ashby nitrogen-free agar；

Fig. 7 is ammonium carrier mutant growing state on Ashby nitrogen-free agar；

Fig. 8 is the growth curve of ammonium vector gene mutant；

Fig. 9 is ammonium carrier mutant nrgA gene mutation region sequence measurement result.

Detailed description of the invention

The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.

Strain, reagent and material source in the following example:

Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, is isolatable from the milpa root of Guilin, for Combination azotobacter, Gram-negative.This strain is deposited in Chinese microorganism strain preservation management on June 2nd, 2016 Committee's common micro-organisms center, is called for short CGMCC.Address: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, Chinese science Institute of Micro-biology of institute；Preservation registration number is CGMCC No.12588.

This Kosakonia radicincitans GXGL-4A Electronic Speculum figure as it is shown in figure 1, in Fig. 1 A be the bacterium of positive binary fission Group；B is the single thalline in binary fission.

Cas9 expression vector pET28a-cas9 is built voluntarily by this laboratory.

Bacterial genomes extraction purification test kit is purchased from Puluomaige Biological Products Co., Ltd., Shanghai of the U.S.；PCR primer Reclaim test kit and be purchased from Kai Jie biotechnology (Shanghai) Co., Ltd. of Germany；PCR reaction agents useful for same is purchased from Beijing Kang Wei reagent Biotech company；Calcium carbonate, sodium chloride, trisodium citrate, two water sodium nitroprussides, phenol, NaOH, (NH₄)₂SO₄Deng Conventional medication is all domestic analytical pure.Ethylene gas is purchased from Mao Tu gas apparatus (Shanghai) Co., Ltd..

E. coli strain bl21 is purchased from Beijing Quanshijin Biotechnology Co., Ltd；Experiment purification bacterial strain GXGL-4A, matter Grain pET-28a (+) it is that laboratory preserves；GRNA plasmid VK001-03 is given by only Shang Lide bio tech ltd, Beijing； Competent escherichia coli cell, low molecular weight protein Marker, cloning vehicle pMD19 T, restriction endonuclease (notI, speI and NheI), T4DNA ligase is purchased from precious biological (Dalian) company limited (TaKaRa)；Kanamycin is purchased from the raw work biology work in Shanghai Journey company limited (Sangon)；Plasmid DNA Mini Kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd..

Embodiment 1: ammonium vector gene nrgA PCR expands

Using bacterial genomes extraction purification test kit extraction GXGL-4A strain gene group STb gene as template, PCR is just Being nrgA-F (5 '-CGGGATCCATGAAAAACACAACATTAAAAACAGGTC-3 ') to primer, reverse primer is nrgA-R (5′-CCCAAGCTTTCAGGCGTTGTAGGCGTTTTCGCCG-3′).The primer is closed by Shanghai Sheng Gong bio-engineering corporation Become.PCR reaction system cumulative volume is 25 μ L, and reaction system includes: 2 × Taq Master Mix 12.5 μ L, forward primer (10 μ Mol/L) 1.0 μ L, reverse primer (10 μm ol/L) 1.0 μ L, dd H₂O 9.5 μ L, template DNA 1.0 μ L.PCR reaction condition is: 94 DEG C of denaturations 5min；Then carrying out 30 circulations, each circulation is 94 DEG C of degeneration 1min, 55 DEG C of anneal 1min and 72 DEG C of extensions 2min；Finally at 72 DEG C, finally extend 10min.

Pcr amplification product size is that 1287bp is (as in figure 2 it is shown, a left side 1 is DL2000 molecular weight marker thing in Fig. 2；Swimming lane 2- 3 is the pcr amplification product of nrgA gene), electrophoresis detection on 1.2% agarose gel, under 110V voltage after electrophoresis 40min Under gel imaging system (Gel Doc TM XR+ type gel imaging system, Bio-rad, USA) ultra-vioket radiation, imaging is taken pictures, inspection Looking into or without band and non-specific amplification, DNA Marker used by electrophoresis is DL2000 (sky, Beijing limited public affairs of root biochemical technology Department).Purpose band reclaims and is checked order by Shanghai Sheng Gong bio-engineering corporation after purification.

The structure of embodiment 2:Cas9 expression vector pET28a-cas9

According to nrgA gene DNA sequence, the gRNA of 2 CRISPR/Cas9 systems of design, target sequence is shown in Table 1.By gRNA It is connected to carrier VK001-03, obtains plasmid VK001-03-G.Then, use NotI and SpeI double digestion, reclaim one section and be about The long segment of 6.6kb, with as double digestion reacted plasmid pET-28a (+) product connect, connect product through electroporated enter Bacillus coli DH 5 alpha competent cell, uses PCR and enzyme cutting method to identify positive transformant.Extract transformant plasmid pET28a- Cas9 (Fig. 3), more electroporated K.radicincitans GXGL-4A bacterial strain, Screening and Identification goes out targeted transformation.

The design of table 1 nitrogen-fixing bacteria GXGL-4A genome target gene gRNA target spot

GRNA target spot	GRNA target sequence (underline font styles is PAM sequence)
		nrgA-g1	AAACACAACATTAAAAAC<u>AGG</u>
nrgA-g2	TTCACTGGCATTGCTGCC<u>AGG</u>

Embodiment 3: the PCR detection of nitrogen-fixing bacteria transformant cas9 gene

Use PCR method amplification cas9 Gene Partial sequence, screen positive transformant.Pcr amplification primer thing is Cas9-SqF: 5'-ACAAGGACTTCCTGGACAATG-3', Cas9-SqR:5'-CTCGGGCTTATGCCTTCCCAT-3'.Primer is by Shanghai Sangon company synthesizes.PCR reaction system volume is 25 μ L, including 2 × Taq Master Mix 12.5 μ L, forward primer Cas9-SqF (10 μm ol/L) 1.0 μ L, reverse primer Cas9-SqR (10 μm ol/L) 1.0 μ L, dd H₂O 9.5 μ L, conversion subbase Because of group STb gene 1.0 μ L.Wherein, 2 × Taq Master mix (Beijing Kang Wei reagent biotech company) product consists of: 0.1U Taq polymerase/μL、500μmol/L dNTP each、20mmol/L Tris-HCl(pH 8.3)、100mmol/ L KCl、3mmol/L MgCl₂, other stabilizer and reinforcing agent.

The cas9 gene test result of K.radicincitans GXGL-4A nitrogen-fixing bacteria transformant as shown in Figure 4, in Fig. 4, M:marker is DL2000 (TaKaRa)；Swimming lane 1-4 is mutant.

Embodiment 4: mutant nitrogenase activity determination in LB (rich nitrogen) and Ashby (nitrogen-free) culture medium

This research uses acetylene reduction method (ARA) to detect nitrogen-fixing bacteria GXGL-4A, and its principle is to make according to azotase Acetylene Reduction is ethylene, finally with ethylene volume [the nmol C generated₂H₄/ (mL h)] number represent the big of its nitrogen fixing capacity Little.

By inoculation in the 50mL triangular flask having rubber stopper to seal, 180r/min, cultivate at 28～30 DEG C 24～ 48h.Then from each bottle, extract the gas of 2mL with 10mL asepsis injector out, then be injected separately into 2mL C₂H₂After, it is placed in training Support and case is cultivated 12～48h.From each culture bottle, take gas sample 200 μ L respectively, in injection gas chromatography instrument, detect C₂H₄Generation Situation.Observe C in gas chromatograph display screen₂H₄Peak value determines whether C₂H₄Generation.To inoculate but unimplanted C₂H₂Cultivation Bottle is as comparison 1, not connect bacterium but injection C₂H₂Culture bottle do comparison 2.With nmol C₂H₄/ (mL h) represents that azotase is lived Property (N).By following equation calculating nitrogenase activity:

$N=\frac{hx\×C\×V}{hs\×v\×t}$

In formula: hx sample peak value；

Hs standard C₂H₄Peak value；

C standard C₂H₄Concentration (nmol/mL)；

V test tube volume (mL)；

V standard C₂H₄Volume (mL) when test；

T sample culturing time (h)；

The C that N produces₂H₄Concentration.

(Fig. 5, Fig. 6, Fig. 5, in 6,4A:GXGL-4A (CK), wild strain after measured；5-1、5-2、5-3、5-4、5-5、5-6、 5-7,5-8,5-9,5-10,5-11 are ammonium carrier mutant), from 21 mutants, filter out 1 strain targeted mutagenesis strain g5-1, its Show more more preferable nitrogenase activity than wild strain GXGL-4A in without nitrogen environment, be that a strain (can lack in lean soil Nitrogen) the middle mutant used.

Embodiment 5: ammonium carrier mutant growing state on Ashby solid medium is observed

IPTG induce the mutant bacterium solution after 6h coat on Ashby nitrogen-free agar solid plate, with Ashby nitrogen-free In culture medium, interpolation ammonium sulfate is as comparison, each flat board coating 200uL, cultivates, each bacterium of routine observation in 28 DEG C of calorstats Fall growing state.Result shows, compared with the bacterium GXGL-4A that sets out with comparison, most ammonium carrier mutant growths significantly slow down, Individual other even can not grow, reduce the Utilization ability of carbon source, hydrolysis circle diminishes (Fig. 7).

Embodiment 6: ammonium carrier mutant growth curve measures

Take out the ammonium carrier mutant of preservation respectively from-20 DEG C in 37 DEG C, 180r/min activated overnight, then, connect with 2% Plant amount switching LB culture medium, measure 1 its OD per hour₆₀₀Value, is repeated 3 times, and draws growth curve chart (Fig. 8).

Embodiment 7: ammonium carrier mutant nrgA gene mutation site checks order

For verifying ammonium carrier mutant target-gene sequence catastrophe, use PCR method amplification nrgA gene gRNA site Flanking sequence, upstream and downstream each about 300bp place design PCR primer in this site, expands the band of a segment length 607bp respectively. PCR forward primer g5/g6-Cas9-F:5'-CGTGGATATACTGATAGATGCTGCC-3', reverse primer g5/g6-Cas9-R: 5'-GCTGTACCGCGGTGCGGAATATAGCGTC-3'.PCR amplification system composition and amplification program are with the nrgA in embodiment 1 Gene amplification.Amplified production reclaims through agarose gel electrophoresis and serves the order-checking of sea Sangon company after purification., ammonium carrier mutant NrgA gene mutation district is with wild strain GXGL-4A Multiple Sequence Alignment as shown in Figure 9.

The above-mentioned description to embodiment is to be understood that for ease of those skilled in the art and use invention. These embodiments obviously easily can be made various amendment by person skilled in the art, and described herein typically Principle is applied in other embodiments without through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability Field technique personnel should be the present invention's according to the announcement of the present invention, the improvement made without departing from scope and amendment Within protection domain.

Claims (5)

1. a nitrogen-fixing bacteria ammonium vector gene nrgA, it is characterised in that the ammoniacal nitrogen that can be secreted into outside born of the same parents by nitrogen-fixing bacteria transports born of the same parents back In, maintain the balance in nitrogen-fixing bacteria nitrogen storehouse, there is nucleotide sequence as shown in SEQ ID No.1.

2. a nitrogen-fixing bacteria ammonium carrier, it is characterised in that containing albumen nucleotide sequence coded as shown in SEQ ID No.1 Matter.

3. a nitrogen-fixing bacteria ammonium vector gene mutant, it is characterised in that there is nucleotide sequence warp as shown in SEQ ID No.1 Gained nucleotide sequence after the Base substitution mutations in multiple sites, has more preferable nitrogenase activity function simultaneously.

4. a strain comprises the mutant of nitrogen-fixing bacteria ammonium vector gene mutant as claimed in claim 3.

5. the application of a mutant as claimed in claim 4, it is characterised in that at nitrogen stress or without in nitrogen environment, can utilize Nitrogen in air carries out microorganism fixed nitrogen, has more higher than wild strain Kosakonia radicincitans GXGL-4A Diazotroph activity.