CN103451130A - Nitrogen fixing gene cluster and application thereof - Google Patents
Nitrogen fixing gene cluster and application thereof Download PDFInfo
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Abstract
The invention provides a nitrogen fixing gene cluster which comprises 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, moeB and nifV9) and is a currently-discovered relatively small gene cluster with a nitrogen fixing function. The invention also provides an expression vector containing the genes of the nitrogen fixing gene cluster as well as gene engineering bacteria containing the vector. The nitrogen fixing gene cluster provided by the invention can finish a nitrogen fixing process in escherichia coli; and the obtained gene engineering bacteria have relatively high nitrogen fixing ability and can eliminate influence of ammonium oxygen on the nitrogen fixation. The nitrogen fixing gene cluster provided by the invention can be used for preparing genetically modified crops to improve the biological nitrogen fixing ability of the crops.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of nif bunch nifBHDKENXV moeB and application thereof with nitrogen fixing capacity.
Background technology
Nitrogen is one of necessary important element of biological existence.Nitrogen in organic sphere animal, plant can not directly utilize atmosphere, can only utilize combined nitrogen.But certain micro-organisms can directly utilize the nitrogen that accounts for atmosphere nearly 80%, having the ability that nitrogen is transformed to ammonification is biological nitrogen fixation.They can, by the activity that in body, a kind of protein with special catalytic capability is nitrogenase, be airborne nitrogen transformation the nitrogenous compound that plant can absorb.Plant draws these nutriment in order to synthetic protein from soil, and animal is the vital movement of become the assign to continuity oneself essential with other from plant there panning protein again.As can be seen here, various nitrogenous compounds comprise protein in the world, and its initial source is all airborne nitrogen, and be all fixed nitrogen movable handle by microorganism it change effective nitrogen into.So biological nitrogen fixation has formed the important step of the whole Nitrogen Cycling of nature, and be directly connected to plant, animal so that the mankind's existence.
Biological nitrogen fixation is a very complicated physiological and biochemical procedure, and a series of biochemistry of biosynthesizing, assembling, enzyme catalysis, regulation and control etc. that relates to nitrogenase is crossed into.The nitrogenase system is structurally all very complex with the catalytic mechanism aspect, form an activated nitrogenase complex of tool, needs many genes to encode.21 nif of klebsiella pneumoniae (Klebesiela pneumoniae) form with gene cluster on karyomit(e) exists, the about 24kb of total length.21 genes form 8 transcription unit: nifJC, nifHDKTY, nifENX, nifUSVW, nifZM, nifF, nifLA and nifBQ.Having 56 on pseudomonas denitrificas (Pseudomonas stutzeri) A1501 karyomit(e) exists with the form of fixed nitrogen genes involved with gene cluster, the nif gene of azotobacter vinelandii (Azotobacter vinelandii) is positioned at two non-conterminous gene clusters, nifHDKTYENXUSVWZMF and nifLABQ.Brasil diazotrophic spirillum (Azospirillum brasilense) fixed nitrogen genes involved mainly is distributed in two gene clusters: a main nif bunch contains nifHDK, nifY, nifE, nifUS, nifW and fixABCX, and another one nif gene cluster is by nifA and nifB genomic constitution.The nif promotor of these G-vinelandii all belongs to σ
54the type promotor, it transcribes strictly the regulation and control that are subject to transcription activating protein NifA according to ammonium and oxygen concn.Helicobacter (Heliobacterium) has 1 10kb, containing the nif bunch of 11 genes: nifI1 nifI2 nifH nifD nifK nifE nifN nifX fdx nifB nifV, clostridium acetobutylicum (C.acetobutylicum) nif bunch is by 9 genomic constitutions: nifH nifI1 nifI2 nifD nifK nifE nifN-B nifVw nifVa; Clostridium beijerinckii nif bunch has 14 genes: nifH nifI1 nifI2 nifD nifK nifE nifN-B fdxA nirJ1 nirJ2 nirD nirH nifVw nifVa; Pasteur's gemma clostridium (C.pasteurianum) nif bunch has 10 genes: nifH2 nifH1 nifD nifK nifE nifN-B modA modB nifVw nifVa.
Fixed nitrogen series bacillus (Paenibacillus) report at present has 17 fixed nitrogen bacterial classifications, its resistance against disadvantage is strong, storage tolerance, a lot of bacterial strains also have growth-promoting except nitrogen fixing capacity is arranged, the multi abilities such as antibiosis, it has unique gene type, different fixed nitrogen Regulation Mechanism and potential using value, therefore studying its nif bunch is conducive to study the fixed nitrogen regulation mechanism, to obtaining better, more transgenic line, has important Research Significance.
Summary of the invention
The object of the present invention is to provide a strain to there is the genus bacillus of nif bunch.
Another object of the present invention is to provide the expression vector that contains above-mentioned nif bunch.
A further object of the present invention is to provide the application of above-mentioned nif bunch.
A strain series bacillus provided by the invention (Paenibacillus sp.) 18WLY, it contains nif bunch nifBHDKENXV moeB.This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 19th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is Paenibacillus sp., and preserving number is CGMCC No.7724.
Nif bunch provided by the invention, it is nifBHDKENXV moeB, nucleotides sequence is classified as:
(1) shown in SEQ ID NO.10, comprise nifB, nifH, nifD, nifK, nifE, nifN, nifX, moeB, nifV9 gene, its nucleotide sequence is respectively as shown in SEQ ID NO.1-9; Or nucleotide sequence shown in (2) SEQ ID No.10 is substituted, lacks and/or increases one or several Nucleotide; Or the nucleotide sequence of (3) DNA sequence dna hybridization of limiting with (1) under stringent condition.
Described stringent condition is is NaCl0.015M at 0.1 * SSC(composition, Trisodium Citrate 0.0015M, pH7.0), in the solution of 0.1%SDS, hybridization under 65 ℃, and wash film with this solution.
In sequence table, SEQ ID NO.1 is comprised of 1501 base pairs, and coded product participates in the synthetic of FeMoco cofactor.SEQ ID NO.2 is comprised of 867 base pairs, the Fe albumen of coding nitrogenase.SEQ ID NO.3 is comprised of 1449 base pairs, a subunit of the Mo-Fe protein of coding nitrogenase.SEQ ID NO.4 is comprised of 1449 base pairs, the β subunit of coding Nitrogenase MoFe Protein.SEQ ID NO.5 is comprised of 1362 base pairs, and coded product participates in the synthetic of FeMoco cofactor.SEQ ID NO.6 is comprised of 1212 base pairs, and coded product participates in the synthetic of FeMoco cofactor.SEQ ID NO.7 is comprised of 390 base pairs, and coded product participates in the synthetic of FeMoco cofactor.SEQ ID NO.8 is comprised of 1147 base pairs, and coded product participates in the synthetic of homocitric acid iron.SEQ ID NO.9 is comprised of 894 base pairs, and coded product participates in the synthetic of FeMoco cofactor.SEQ ID NO.10 is the sequence of gene cluster in whole genome, the nucleotide pair of 11039bp, consists of.
In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be applicable to the codon that specific species are expressed.Thereby nif cluster gene gene of the present invention also comprises by nucleotide sequence shown in SEQ ID No.1-9 and be substituted, lack and/or increase one or several Nucleotide, the relevant nucleotide sequence of fixed nitrogen obtains encoding.
Gene cluster of the present invention can be operably connected with expression vector, obtain expressing the recombinant expression vector of albumen of the present invention, further this recombinant expression vector be imported in appropriate host cell, obtain and express the genetic engineering bacterium that the present invention has nitrogen fixing capacity.
The invention provides the expression vector that contains above-mentioned nif bunch nifBHDKENXV moeB.
In embodiments of the present invention, respectively nif bunch is inserted into to expression vector pUC18, the upper structure of pHY300PLK and pET28b obtains recombinant expression vector pUC18-1, pHY300PLK-2, pET28b-3, and its plasmid map is as shown in Fig. 7 a, Fig. 7 b, Fig. 7 c.Further, these expression vectors are imported to E.coli(JM109) the middle engineering strain with nitrogen fixing capacity that obtains.Therefore the present invention also comprises host cell or the Host Strains that contains above-mentioned expression vector.
Above-mentioned recombinant expression vector pUC18-1, pHY300PLK-2, pET28b-3 build and obtain by the following method respectively:
1) take respectively S3 and S4, S1 and S2 or S5 and S6 as primer, bacillus azotobacter Paenibacillus sp.18WLY genome is template, and pcr amplification nif bunch nifBHDKENXVmoeB is in the purpose fragment;
2) respectively the purpose fragment of expression vector pUC18, pHY300PLK or pET28b and step 1) is used respectively to XbaI and HindIII, XbaI and BamHI, BamHI and HindIII double digestion respectively, reclaimed enzyme and cut product;
3) expression vector pUC18, pHY300PLK or pET28b1 μ l and the 7.5 μ l purpose fragments (mol ratio is about 1:6) after adding respectively enzyme to cut in 10 μ l reaction systems, 10 * ligase buffer1 μ l, T4DNA ligase0.5 μ l, mix; 16 ℃ of water-baths connect 16h, transformed competence colibacillus cell;
4) screening positive clone, obtain the plasmid pUC18-1, pHY300PLK-2, the pET28b-3 that contain nif bunch nifBHDKENXVmoeB.
In one embodiment of the invention, utilize carrier pHY300PLK-2 to build and obtain Recombinant organism strain 78-7 by engineered method, it builds and obtains by the following method:
1) take S1 and S2 as primer, bacillus azotobacter Paenibacillus sp.18WLY genome is template, pcr amplification with the nif bunch nifBHDKENXV moeB of self promotor in the purpose fragment; The sequence of described S1 is: GCTCTAGAGCGGAGACTATTTCCCAAAAT; The S2 sequence is TGCGGATCCGCGTGTAATGGTTATATGAAT;
2) the purpose fragment of expression vector pHY300PLK and step 1) is used respectively to XbaI and BamHI double digestion respectively, reclaimed enzyme and cut product;
3) expression vector pHY300PLK1 μ l and the 7.5 μ l purpose fragments (mol ratio is about 1:6) after adding respectively enzyme to cut in 10 μ l reaction systems, 10 * ligase buffer1 μ l, T4DNA ligase0.5 μ l, mix; 16 ℃ of water-baths connect 16h, transformed competence colibacillus cell;
4) screening positive clone, obtain the plasmid pHY300PLK-2 that contains nif bunch nifBHDKENXVmoeB;
The plasmid pHY300PLK-2 electricity that 5) will contain nif bunch nifBHDKENXVmoeB forwards in E.coli JM109, obtains engineering strain 78-7.
The invention provides the application of Recombinant organism strain 78-7 in preparation biological nitrogen fixation microbiobacterial agent or biological organic fertilizer.
The invention provides the nif bunch purposes of nifBHDKENXV moeB in biological nitrogen fixation.And a nif bunch nifBHDKENXV moeB has the application in the nitrogen fixing capacity transgenic crop in preparation.
The present invention also provides the microbial inoculum that contains Recombinant organism strain 78-7, and the application of this microbial inoculum on Promoting plant growth.The invention provides the application of microbial inoculum in preparation biological nitrogen fixation microbiobacterial agent or biological organic fertilizer that contains Recombinant organism strain 78-7.The microbial inoculum that contains series bacillus 18WLY also belongs to protection scope of the present invention.The invention provides the purposes of series bacillus 18WLY in biological nitrogen fixation.
Beneficial effect of the present invention is mainly reflected in: provide a strain to have the series bacillus 18WLY of nitrogenase activity, it contains nif bunch nifBHDKENXVmoeB gene, through experimental verification by this nif bunch at expression in escherichia coli, can make non-azotobacter strain obtain nitrogen fixing capacity, and the regulation and control of ammonium radical ion to the biological nitrogen fixation function have been removed, it is to improving nitrogen-fixing efficiency, building the high-efficiency nitrogen-fixing engineering strain is worth significantly, the intestinal bacteria 78-2(pUC18 that 3 strains that obtain have nitrogen fixing capacity), 78-7(pHY300PLK), 78-32(pET28b), it is 149.78 that the Acetylene Reduction method measures that its nitrogenase work is respectively, 298.76, 57.69nmolC
2h
4/ (mg protein h).
The accompanying drawing explanation
The structure iron that Fig. 1 is fixed nitrogen series bacillus nif bunch.
Fig. 2 is the pcr amplification figure of nif bunch nifBHDKENXVmoeB; Wherein 1 is primer S1, the gene cluster electrophoretic band that the S2 amplification obtains, and 2 is S3, the gene cluster electrophoretic band that the S4 amplification obtains, 3 is S3, the gene cluster electrophoretic band that the S4 amplification obtains, M is 1kb ladder marker.
Fig. 3 is expression vector pUC18-1, pHY300PLK-2, and pET28b-3 builds enzyme and cuts the agarose gel electrophoresis proof diagram; Wherein Fig. 3 a is pUC18-1 restriction enzyme digestion and electrophoresis figure, and 1 is pUC18-78/XbaI+HindIII, and 2 is nif cluser PCR product/XbaI+HindIII, and 3 is pUC18/XbaI+HindIII; Fig. 3 b is pHY300PLK-2 restriction enzyme digestion and electrophoresis figure, 1 is nif cluser PCR product/XbaI+BamHI, 2 is pHY300PLK-78/XbaI+BamHI, 3 is pHY300PLK/XbaI+BamHI, Fig. 3 c is pET28b-3 restriction enzyme digestion and electrophoresis figure, 1 is nif cluser PCRproduct/BamHI+HindIII, and 2 is pET28b-78/BamHI+HindIII, and 3 is pET28b/BamHI+HindIII.Fig. 4 is engineering strain 78-2(pUC18), 78-7pHY300PLK) and 78-32(pET28b) nitrogenase activity figure, wherein Fig. 4 a is for utilizing the Acetylene Reduction method to measure, Fig. 4 b is
15the N2 labelling method is measured.
Fig. 5 is that nitrogenase activity affects figure; Fig. 5 a and Fig. 5 b are respectively that different oxygen concentrations and different ammonium concentration are on engineering strain 78-7(pHY300PLK) nitrogenase activity impact figure.
Fig. 6 is that the ammonium oxygen that detects of high-fidelity real-time fluorescence quantitative PCR is on engineering strain 78-7(pHY300PLK) the nif transcriptional level affect figure.
Fig. 7 is the recombinant vectors collection of illustrative plates, and wherein Fig. 7 a is pUC18-1, and Fig. 7 b is pHY300PLK-2, the collection of illustrative plates that Fig. 7 c is pET28b-3;
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, in embodiment, chemical reagent used is conventional commercial reagent, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
(1) fixed nitrogen series bacillus complete genome sequencing
The present invention has completed the sequential analysis of a few strain fixed nitrogen series bacillus genome frame diagram, and these fixed nitrogen series bacillus strains have the common more complete nif of ratio bunch nifBHDKENXVmoeB, as shown in Figure 1.It may be to form a transcriptional units that information science is analyzed these 9 genes, and promotor is σ
70, this is the less Natural Nitrogen Fixation gene cluster of nif number of finding at present, comprises 6 basic nif nifBHDKEN and other 3 gene nifXVmoeB.
(2) amplification of nif bunch nifBHDKENXVmoeB
Series bacillus 18WLY of the present invention separates the hundred prestige Garcinia Bark root systems from Beijing, and the method that adopts morphologic observation and 16S rRNA sequencing to combine, be series bacillus by this dientification of bacteria.This bacterial strain 18WLY is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 19th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.7724, classification series bacillus (Paenibacillus sp.) by name.
The fixed nitrogen series bacillus nif bunch nifBHDKENXVmoeB of the bacterial strain Paenibacillus sp.18WLY that the present invention obtains separation clones, according to its genome Frame sequence analytical results, design three couples of primer S1 and S2, S3 and S4, S5 and S6(are in Table 1), the bacterial strain Paenibacillus sp.18WLY genome of take increases to its nif bunch nifBHDKENXVmoeB and shared promotor thereof as template, obtain the fragment that size is about 11kb, sequence is as shown in SEQ ID NO.10.Three fragments that the three pairs of primer amplifications go out are the same, are all the nif bunch of 9 genomic constitutions, and the beginning of fragment that different is is different with the restriction enzyme site at end.And this fragment has been carried out to glue and reclaimed purifying, the amplification purification band is as shown in Figure 2.
Table 1 is for cloning nif bunch primer used
After the purpose band of 3 pairs of primer amplifications is reclaimed to purifying, send genome company's order-checking, sequencing result shows, 3 goal gene sequences of clone are respectively as shown in sequence table (sequence 1 and nifB) (sequence 1 and nifB) (sequence 1 and nifB), illustrate that clone's goal gene is nif bunch nifBHDKENXVmoeB.
The structure of the different expression vectors of embodiment 2 nif bunch nifBHDKENXVmoeB
1.pUC18-1 the structure of expression vector
A. the preparation of intestinal bacteria Electroporation-competent cells
E.coli DH10B is inoculated in the LB substratum to 37 ℃, and the 160r/min overnight incubation prepares seed liquor, and seed liquor is transferred in fresh LB substratum by 1% inoculum size, at 37 ℃, shakes 4 hours under the condition of 200r/min to OD
600=0.6; By this culture ice bath 30min, 4 ℃, the centrifugal collection thalline of 6000rpm; Ice-cold aseptic ddH
2o cleans thalline 3 times, and precooling 10% glycerine is washed thalline 1 time; Then with 10% aseptic glycerine, suspend, cell suspension is by 40 μ l aliquot packing-20 ℃ preservations.
B. connect and transform expression vector pUC18 and take XbaI and HindIII double digestion respectively for the nif bunch nifBHDKENXVmoeB large fragment with himself promotor that S3 and S4 obtain as the primer amplification purifying; Enzyme is cut product and is reclaimed test kit recovery purifying DNA fragment with sky with glue respectively; Add respectively carrier 1 μ l and 7.5 μ l external source fragments (mol ratio is about 1:6) in 10 μ l reaction systems, 10 * ligase buffer1 μ l, T4DNA ligase0.5 μ l, mix; 16 ℃ of water-baths connect 16-24hr.Getting 1 μ l connects product and is added in 40 μ l competent cells and mixes; The electricity that mixture is sucked to the 0.1cm of precooling swashs in cup, and under the condition of 1.6kV, electric shock transforms; Converted product is placed in to fresh LB substratum, 37 ℃ of renewal cultivation 40min; The renewal cultivation thing is applied on the LB culture medium flat plate that contains 100ug/ml Amp and (X-gal and IPTG is arranged), 37 ℃ of incubated overnight, the picking hickie is preserved.
C. the screening of positive colony
Screen doubtful positive colony by the method for bacterial plasmid rapid detection (Cracking); Extract the plasmid of doubtful positive colony with sky root plasmid extraction kit; Restriction enzyme XbaI and HindIII cut respectively transformant; Method validation positive colony with agarose gel electrophoresis and order-checking.
PUC18-1 expression vector restriction enzyme digestion and electrophoresis detects as Fig. 3 a.Obtain the plasmid pUC18-1 that contains nif bunch nifBHDKENXVmoeB.
2.pHY300PLK-2 the structure of expression vector
A. the preparation method of intestinal bacteria Electroporation-competent cells is the same.
B. connect and transform expression vector pHY300PLK and take XbaI and BamHI double digestion respectively for the nif bunch nifBHDKENXVmoeB large fragment with himself promotor that S1 and S2 obtain as the primer amplification purifying; Enzyme is cut product and is reclaimed test kit recovery purifying DNA fragment with sky with glue respectively; Add respectively carrier 1 μ l and 7.5 μ l external source fragments (mol ratio is about 1:6) in 10 μ l reaction systems, 10 * ligase buffer1 μ l, T4DNA ligase0.5 μ l, mix; 16 ℃ of water-baths connect 16-24hr.
Getting 1 μ l connects product and is added in 40 μ l competent cells and mixes; The electricity that mixture is sucked to the 0.1cm of precooling swashs in cup, and under the condition of 1.6kV, electric shock transforms; Converted product is placed in to fresh LB substratum, 37 ℃ of renewal cultivation 40min; The renewal cultivation thing is applied on the LB culture medium flat plate that contains 100ug/ml Amp and 20ug/mlTc, 37 ℃ of incubated overnight, the picking transformant is preserved.
C. the screening of positive colony
Screen doubtful positive colony by the method for bacterial plasmid rapid detection (Cracking); Extract the plasmid of doubtful positive colony with sky root plasmid extraction kit; Restriction enzyme XbaI and BamHI cut respectively transformant; Method validation positive colony with agarose gel electrophoresis and order-checking.Obtain the plasmid pHY300PLK-2 that contains nif bunch nifBHDKENXVmoeB.PHY300PLK-2 expression vector restriction enzyme digestion and electrophoresis detects as Fig. 3 b.
3.pET28b-3 the structure of expression vector
A. the preparation method of intestinal bacteria Electroporation-competent cells is the same.
B. connect and transform expression vector pET28b and take BamHI and HindIII double digestion respectively for the nif bunch nifBHDKENXVmoeB large fragment with himself promotor that S5 and S6 obtain as the primer amplification purifying; Enzyme is cut product and is reclaimed test kit recovery purifying DNA fragment with sky with glue respectively; Add respectively carrier 1 μ l and 7.5 μ l external source fragments (mol ratio is about 1:6) in 10 μ l reaction systems, 10 * ligase buffer1 μ l, T4DNA ligase0.5 μ l, mix; 16 ℃ of water-baths connect 16-24hr.Getting 1 μ l connects product and is added in 40 μ l competent cells and mixes; The electricity that mixture is sucked to the 0.1cm of precooling swashs in cup, and under the condition of 1.6kV, electric shock transforms; Converted product is placed in to fresh LB substratum, 37 ℃ of renewal cultivation 40min; The renewal cultivation thing is applied on the LB culture medium flat plate that contains 50ug/ml Km, 37 ℃ of incubated overnight, the picking transformant is preserved.
C. the screening of positive colony
Screen doubtful positive colony by the method for bacterial plasmid rapid detection (Cracking); Extract the plasmid of doubtful positive colony with sky root plasmid extraction kit; Restriction enzyme BamHI and HindIII cut respectively transformant; Method validation positive colony with agarose gel electrophoresis and order-checking, obtain the plasmid pET28b-3 that contains nif bunch nifBHDKENXVmoeB.Expression vector pET28b-3 restriction enzyme digestion and electrophoresis detects as Fig. 3 c.
The expression of embodiment 3 nif bunch in intestinal bacteria and the acquisition of engineering bacteria
1. the preparation of competent escherichia coli cell
(1) the intestinal bacteria colony inoculation that picking is fresh in 20ml LB substratum, 37 ℃, the 160rpm incubated overnight; (2) by 1% inoculum size, be inoculated in the salt-free LB substratum of 1L, 37 ℃, 160rpm shakes to OD600 and is about 0.5-0.8; (3) by culture ice bath 1 hour, 5000rpm, 4 ℃ of centrifugal 10min, collect thalline; (4) with 500ml precooling sterilized water suspension thalline, 5000rpm, 4 ℃ of centrifugal 10min, collect thalline; (5) repeating step (4); (6) use 10% glycerine of the sterilizing of 1ml precooling to suspend; (7) by 50 μ l aliquot packing cell suspensions, it is standby that liquid nitrogen flash freezer is placed on-80 ℃ of Refrigerator stores.
2. electric shock transforms
(1) get 1 μ l DNA and connect product and join in colibacillary competent cell prepared by 50 μ l, mix; (2) magnitude of voltage being set is that the 1.6kV(0.1cm electric shock transforms the voltage that cup is used); (3) above mixture is joined in the electric shock cup of precooling, solution is thrown to bottom, the electric shock cup is put into to the slide block of precooling, press immediately the beginning key; (4) take out rapidly the electric shock product, add in the LB substratum of 800 μ l, 37 ℃, 80rpm shakes 0.5-1hr.
The plasmid pUC18-1 that will contain nif bunch nifBHDKENXVmoeB, pHY300PLK-2, the voltage that pET28b-3 is used at 1.6kV(0.1cm electric shock conversion cup) during under condition, electricity forwards E.coli JM109 to, obtain respectively engineering strain 78-2(pUC18), 78-7(pHY300PLK) and 78-32(pET28b).
With the negative contrast of E.coli JM109, utilize the Acetylene Reduction method and
15the N labelling method has been measured nitrogenase activity, sees Fig. 4.Result shows that the nif bunch nifBHDKENXVmoeB of fixed nitrogen series bacillus can complete the fixed nitrogen process in intestinal bacteria, the engineering strain 78-7(pHY300PLK that the shuttle plasmid of intestinal bacteria and genus bacillus of wherein take is vector construction) nitrogen fixing capacity is the strongest, Acetylene Reduction method (ARA) shows above-mentioned 3 strain engineering bacteria 78-2(pUC18), 78-7(pHY300PLK) and 78-32(pET28b) all can utilize N
2as nitrogenous source, and have higher biological nitrogen fixation activity, under the pure culture condition, its nitrogenase work is respectively 149.78,298.76,57.69nmolC
2h
4/ (mg protein h).The present invention utilizes nif to realize the process of biological nitrogen fixation first in non-vinelandii, if this nif bunch is proceeded to farm crop, realizes biological nitrogen fixation in farm crop, and this has great significance to Biological Nitrogen Fixation Researches.
Embodiment 4 ammoniums are on engineering strain 78-7(pHY300PLK) nitrogenase impact alive
(1) engineering strain 78-7(pHY300PLK embodiment 3 obtained) be inoculated in the LB substratum of 5mL, 37 ℃ of overnight incubation, inoculum size by 1% is transferred in the fresh LB substratum of 20mL, 37 ℃, 160rpm cultivates 7h, then collect thalline, the substratum of living with appropriate survey enzyme suspends concrete, and adjusts OD
600to 0.3.
(2) add the NH of the 5M of different volumes in the cell suspension of step (1) gained
4the Cl mother liquor, obtain respectively NH
4the Cl final concentration is 0mM, 1mM, 5mM, 20mM, 50mM, 100mM, the cell suspension of 200mM, get respectively the 1ml cell suspension in the anaerobism pipe of 25ml, 30 ℃ coerce and cultivate 12-16h after, inject 10% acetylene gas and continue to cultivate 2h, get 100 μ L gas gas Chromatographic Determination ethylene content.
LB substratum: peptone 10g, yeast extract paste 5g, NaCl5g, H
2o1L, 121 ℃ of sterilizings.
Surveying enzyme substratum alive is: 26.3g Na
2hPO
412H
2o; 3.4g KH
2pO
4; 10 μ g vitamin Hs; 26mg CaCl
22H
2o; 30mg MgSO
4; 0.33mg MnSO
4h
2o; The 36mg ironic citrate; 7.6mg Na
2moO
42H
2o; 10 μ g para-amino benzoic acid; 4g glucose.After 115 ℃ of sterilizing 30min of glucose, add separately, other reagent can prepare rear 121 ℃ of sterilizing 30min.
(3) centrifugal collection thalline, the NaOH that adds 200 μ L0.5M boils 5min, then the HCl that adds 200 μ L0.5M mixes rear centrifugal absorption supernatant liquor 40 μ L, joins in 5mL Bradford working fluid, mixes, and places 3～5min, measures OD
595value carries it in the typical curve equation, calculates protein concentration.
(4) calculate enzyme activity according to following nitrogenase activity calculation formula, and analyze more obstructed ammonium concentration to engineering strain 78-7(pHY300PLK) impact of nitrogenase activity.
Ammonium ion is on engineering strain 78-7(pHY300PLK) impact of nitrogenase activity is as shown in Figure 5 a, result shows, the ammonium radical ion of different concns, on not impact of nitrogenase activity, illustrates the engineering strain 78-7(pHY300PLK that the present invention obtains) removed the impact of ammonium radical ion on biological nitrogen fixation.
(1) engineering strain 78-7(pHY300PLK embodiment 3 obtained) be inoculated in the LB substratum of 5mL, 37 ℃ of overnight incubation, inoculum size by 1% is transferred in the fresh LB substratum of 20mL, 37 ℃, 160rpm cultivates 6h, then collect thalline, the substratum of living with appropriate survey enzyme suspends concrete, and adjusts OD
600to 0.4.
(2) get the 1ml cell suspension in the anaerobism pipe of 25ml, the oxygen that adds certain volume with the minimum gas sampler, obtaining respectively the oxygen final concentration is 0%, 1%, 5%, 10%, 21% different oxygen stress conditions, after 30 ℃ of cultivation 12-16h, inject 10% acetylene gas continuation cultivation 2h, get 100 μ L gas gas Chromatographic Determination ethylene content.
LB substratum: peptone 10g, yeast extract paste 5g, NaCl5g, H
2o1L, 121 ℃ of sterilizings.
Surveying enzyme substratum alive is: 26.3g Na
2hPO
412H
2o; 3.4g KH
2pO
4; 10 μ g vitamin Hs; 26mg CaCl
22H
2o; 30mg MgSO
4; 0.33mg MnSO
4h
2o; The 36mg ironic citrate; 7.6mg Na
2moO
42H
2o; 10 μ g para-amino benzoic acid; 4g glucose.After 115 ℃ of sterilizing 30min of glucose, add separately, other reagent can prepare rear 121 ℃ of sterilizing 30min.
(3) centrifugal collection thalline, the NaOH that adds 200 μ L0.5M boils 5min, then the HCl that adds 200 μ L0.5M mixes rear centrifugal absorption supernatant liquor 40 μ L, joins in 5mL Bradford working fluid, mixes, and places 3～5min, measures OD
595value carries it in the typical curve equation, calculates protein concentration.
(4) calculate enzyme activity according to following nitrogenase activity calculation formula, and analyze more obstructed oxygen concentration to engineering strain 78-7(pHY300PLK) impact of nitrogenase activity.
Oxygen concentration is on engineering strain 78-7(pHY300PLK) impact of nitrogenase activity is as shown in Figure 5 b, result shows, different oxygen concentrations have a certain impact to nitrogenase activity, when oxygen concentration is 5%, nitrogenase activity has been subjected to very large impact, and this is that this could show its nitrogenase activity in the environment of strictly anaerobic due to nitrogenase.
(2) according to preceding method respectively at the fixed nitrogen (NH of 0% oxygen and 0mM
4cl) and the non-fixed nitrogen (NH of 21% oxygen and 100mM
4cl) coerce 2h under condition, collect the thalline liquid nitrogen and preserve.
(3) extract test kit SV Total RNA I solation System (Promega) with RNA and extract RNA ,-80 ℃ of preservations.
(4) use test kit
rT reagent Kit with gDNA Eraser removes genomic dna, and be turned into cDNA, the cDNA of reverse transcription of take carries out Quantitative real-time RT-PCR(primer as table 2 as template), adopt SYBR with reference to the requirement of Applied Biosystem
@green PCR Master Mix is reacted on 96 orifice plates, three repetitions of every sample, and the Real-time pcr amplification instrument model of use is ABI applied biosystem vii A7.
(5) calculate according to the Ct value relative expression quantity that detects gene, the non-fixed nitrogen condition of take is reference, calculates engineering strain 78-7(pHY300PLK) relative expression quantity of each gene under the fixed nitrogen condition, with excel analytical data mapping.
Ammonium oxygen is on engineering strain 78-7(pHY300PLK) impact of nif transcriptional level the results are shown in Figure 6, result shows the not impact on nif bunch transcriptional level of ammonium concentration and oxygen, be engineering strain 78-7(pHY300PLK) removed the impact on its fixed nitrogen of ammonium in the environment and oxygen, can study on its basis the necessary gene of fixed nitrogen, fixed nitrogen process and mechanism are carried out to deep probing into, and this has important directive significance to the oxygen regulation and control of research nif ammonium and the strain of structure high expression level nitrogen-fixing engineering strain.Secondly, the engineering strain of removing ammonium and oxygen regulation and control (has ammonium and oxygen) and can not be subject to the impact of ammonium and oxygen to carry out fixed nitrogen in edatope, for soil provides nitrogen, reduces using of amount of nitrogenous fertilizer.
Table 2 Quantitative real-time RT-PCR the primer
| Primer | Sequence 5-3 | Position |
| nifB-F | GGATAAGCCTGAAGCGAGC | nifB，470-489bp |
| nifB-R | CAATCGGCTTTCCCGTGAC | nifB，637-656bp |
| nifH-F | AACAGCCGGAATACGGACC | nifH，450-469bp |
| nifH-R | ACCTGCCAGCTCTTCATACTC | nifH，614-634bp |
| nifD-F | TCATTCCTGTACGCTGTGAGG | nifD，534-555bp |
| nifD-R | CACCGCCGATATTGTAGTCTC | nifD，670-6９１bp |
| nifK-F | GCGGAGATGATTGCGGTATG | nifK，463-485bp |
| nifK-R | GGCGTCATAGCCTGTAATATGTG | nifK,610-633bp |
| nifE-F | TCCCGCTGTGTTTGTCTATACC | nifE，338-360bp |
| nifE-R | GTGCTGCCGATAATATGCTGG | nifE，509-530bp |
| nifN-F | AGCTACTAATGGATGCCGTACTC | nifN,365-388bp |
| nifN-R | CCGGACAAGGAAGTGGAAATATC | nifN,522-545bp |
| nifX-F | CTGGGGAGCGATGAGAATGAG | nifX，126-147bp |
| nifX-R | TCCTCAATGGTGCTACCGAAG | nifX，269-290bp |
| moeB-F | GGATGGAGACGGCATTACAG | moeB，261-282bp |
| moeBR | GTTCAGCGCATATCTTTCCGG | moeB,401-422bp |
| nifV-F | GCCATAGCTGCCCGTATAGA | nifV,519-539bp |
| nifV-R | CTCCAGCGCGGTATTACCTG | nifV,685-708bp |
| 16s-F | TTTGTCGTCAGCTCGTGTTCGTG | 16s?RNA,901-924bp |
| 16s-R | ATCCCCACCTTCCTCCGGTTTG | 16s?RNA,1021-1043bp |
Claims (10)
1. a strain series bacillus (Paenibacillus sp.) 18WLY, its deposit number is CGMCC No.7724.
2. series bacillus as claimed in claim 1 (Paenibacillus sp.) 18WLY, is characterized in that, contain a kind of nif bunch, it is nifBHDKENXV moeB.
3. a nif bunch, it is nifBHDKENXV moeB, its nucleotides sequence is classified as:
(1) shown in SEQ ID NO.10, comprise nifB, nifH, nifD, nifK, nifE, nifN, nifX, moeB, nifV9 gene, its nucleotide sequence is respectively as shown in SEQ ID NO.1-9; Or
(2) nucleotide sequence shown in SEQ ID No.10 is substituted, lacks and/or increases one or several Nucleotide; Or
(3) nucleotide sequence of the DNA sequence dna hybridization limited with (1) under stringent condition.
4. the expression vector that contains the described nif of claim 3 bunch.
5. expression vector as claimed in claim 4, is characterized in that, it is pHY300PLK-2.
6. the host cell or the Host Strains that contain the arbitrary described expression vector of claim 4-5.
7. Host Strains as claimed in claim 6, it is Recombinant organism strain 78-7, it builds and obtains by the following method:
(1) take S1 and S2 as primer, bacillus azotobacter Paenibacillus sp.18WLY genome is template, pcr amplification with the nif bunch nifBHDKENXV moeB of self promotor in the purpose fragment; The sequence of described S1 is: GCTCTAGAGCGGAGACTATTTCCCAAAAT; The S2 sequence is TGCGGATCCGCGTGTAATGGTTATATGAAT;
(2) the purpose fragment of expression vector pHY300PLK and step (1) is used respectively to XbaI and BamHI double digestion respectively, reclaimed enzyme and cut product;
(3) expression vector pHY300PLK1 μ l and the 7.5 μ l purpose fragments after adding respectively enzyme to cut in 10 μ l reaction systems, mol ratio is about 1:6,10 * ligase buffer1 μ l, T4DNA ligase0.5 μ l, mix; 16 ℃ of water-baths connect 16h, transformed competence colibacillus cell;
(4) screening positive clone, obtain the plasmid pHY300PLK-2 that contains nif bunch nifBHDKENXVmoeB;
(5) the plasmid pHY300PLK-2 electricity that will contain nif bunch nifBHDKENXVmoeB forwards in E.coli JM109, obtains engineering strain 78-7.
8. the application of Recombinant organism strain 78-7 in preparation biological nitrogen fixation microbiobacterial agent or biological organic fertilizer.
9. the microbial inoculum that contains series bacillus 18WLY claimed in claim 1.
10. series bacillus 18WLY claimed in claim 1 or nif claimed in claim 3 purposes bunch in biological nitrogen fixation.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5059533A (en) * | 1987-02-26 | 1991-10-22 | Lubrizol Genetics, Inc. | Rhizobial ferredoxin genes |
| CN1552846A (en) * | 2003-06-02 | 2004-12-08 | 中国农业大学 | Engineering strain of Brasil diazotrophic spirillum DraT* containing multiple copied nifA gene |
| CN102352332A (en) * | 2011-10-21 | 2012-02-15 | 中国农业大学 | Nitrogen fixing Paenibacillus 1-43 and application thereof |
| CN102352331A (en) * | 2011-10-21 | 2012-02-15 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-33 and application thereof |
| CN102352333A (en) * | 2011-10-21 | 2012-02-15 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof |
-
2013
- 2013-07-25 CN CN201310317366.7A patent/CN103451130B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5059533A (en) * | 1987-02-26 | 1991-10-22 | Lubrizol Genetics, Inc. | Rhizobial ferredoxin genes |
| CN1552846A (en) * | 2003-06-02 | 2004-12-08 | 中国农业大学 | Engineering strain of Brasil diazotrophic spirillum DraT* containing multiple copied nifA gene |
| CN102352332A (en) * | 2011-10-21 | 2012-02-15 | 中国农业大学 | Nitrogen fixing Paenibacillus 1-43 and application thereof |
| CN102352331A (en) * | 2011-10-21 | 2012-02-15 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-33 and application thereof |
| CN102352333A (en) * | 2011-10-21 | 2012-02-15 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| QUOK-CHEONG CHOO ET AL.: "Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| SHIN SH.ET AL.: "EMBL-EBI：AET59368.1", 《EMBL-EBI》 * |
| 张海予等: "粪产碱菌nif H,nif D和部分nif K的克隆、定位及序列分析", 《中国科学C辑》 * |
| 曾王勇等: "弗兰克氏菌nifH及nifD基因的定位", 《西北农林科技大学学报(自然科学版)》 * |
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