CN103275919B - Escherichia coli engineering bacteria and preparation method and applications thereof - Google Patents
Escherichia coli engineering bacteria and preparation method and applications thereof Download PDFInfo
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- CN103275919B CN103275919B CN201310239938.4A CN201310239938A CN103275919B CN 103275919 B CN103275919 B CN 103275919B CN 201310239938 A CN201310239938 A CN 201310239938A CN 103275919 B CN103275919 B CN 103275919B
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Abstract
The invention relates to an escherichia coli expression vector PcPcS1-pET-22b (+) of callery pear phytochelatin synthase genes PcPCS1 and an escherichia coli strain PcPCS1-Pet-22b (+)/Rosetta (DE3), and the preservation No. is CGMCCNo. 7569. The phytochelatin synthase genes PcPCS1 are derived from callery pear. On the basis, the invention relates to a method for quantitatively determining callery pear phytochelatin synthase genes absorbing cadmium, copper and sodium based on escherichia coli heterologous-expression and an atomic absorption spectrometry. The endurance capacity and the accumulation capacity of recombined escherichia coli strain PcPCS1-Pet-22b (+)/Rosetta (DE3) to Cd2+, Cu2+ and Na+ are increased, and the recombined escherichia coli strain PcPCS1-Pet-22b (+)/Rosetta (DE3) can be used for microbial remediation for liquid-phase carriers polluted by Cd2+, Cu2+ or Na+ metal ions.
Description
Technical field
The present invention relates to beans pears Phytochelatin synthase gene
pcPCS1be building up on colon bacillus expression vector pET-22b (+), and recombinant plasmid transformed is entered E bacteria strain Rosetta (DE3) (
escherichia coli) middle acquisition recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3) (
escherichia coli), PcPCS1-pET-22b (+) makes this E bacteria strain to Cd after expressing in Rosetta (DE3)
2+, Cu
2+and Na
+tolerance and accumulation ability improve, above-mentioned recombinant escherichia coli engineering bacteria can be utilized Cd
2+or Cu
2+or Na
+the liquid phase carrier of metal ion pollution carries out biological restoration, belongs to biological technical field.
Background technology
The heavy metal contamination that various industry and rural activity produce causes serious detrimentally affect (Nriagu J O and Pacyna J M. 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. to human health and the ecosystem
nature, 333:134-139.).In numerous heavy metals, the harm of cadmium to plant maximum (Mohammed A S, Kapri A, Goel R. 2011. Heavy metal pollution:source, impact, and remedies.
environment Pollution, 20:1-28.).Atmospheric pollution and using of commercial fertilizer, sewage sludge, ight soil and lime all can accumulate cadmium (L ó pez-Mil á n A F in soil, Sagardoy R, Solanas M, Abad í A, Abad í J. 2009. Cadmium toxicity in tomato (
lycopersicom esculentummill.) plants grown in hydroponics.
environmental and Experimental Botany, 65:376-385.).The set life of plant battalion, can not the disadvantageous habitat of active avoidance.In order to avoid the murder by poisoning of heavy metal ion, plant evolution goes out the mechanism of distribution in a set of effective adjustment ion born of the same parents, synthesize chief component (the Tennstedt P that different metal ion Binding peptides is this mechanism, Peisker D, Bottcher C, Trampczynska A, Clemens S. 2009. Phytochelatin synthesis is essential for the detoxification of excess Zn and contributes significantly to the accumulation of Zn.
plant Physiology, 149:938-948.).Plant complexing element is heavy metal Binding peptide (the Chaurasia N being rich in halfcystine by gsh as substrate through a class of enzyme' s catalysis, Mishra Y, Rai L C. 2008. Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp PCC 7120 offering multiple stress tolerance in
escherichia coli.
biochemical and Biophysical Research Communications, 376:225-230.).In plant materials after synthesis plant complexing element, this substance is combined with heavy metal ion and transports in vacuole by the latter, thus by heavy metal ion compartmentation, be fixed on (Clemens S. 2006. Evolution and function of phytochelatin synthases. in vacuole with the form of mixture
journal of Plant Physiology, 163:319-332.).PCs energy and Cu
2+, Zn
2+etc. vital movement essential metal ionic bond, and be transported to the position such as vacuole or target enzyme (Tennstedt P, Peisker D, Bottcher C, Trampczynska A, Clemens S. 2008. Phytochelatin synthesis is essential for the detoxification of excess Zn and contributes significantly to the accumulation of Zn.
plant Physiology, 149:938-948.), also can chelating Cd
2+, Pb
2+etc. nonessential metal ion, avoid the combination of itself and enzyme or other active substance, thus reduce toxic action (the Alberich A of heavy metal, Diaz-Cruz J M, Arino C, Esteban M. 2008. Combined use of the potential shift correction and the simultaneous treatment of spectroscopic and electrochemical data by multivariate curve resolution:analysis of a Pb (II)-phytochelatin system.
analyst, 133:470-477.).The gene being responsible for synthesis plant complexing element is phytochelatin synthase (Phytochelatin synthases, PCS, EC 2.3.2.15) (Ramos J, Naya L, Gay M, Abian J, Becana M. 2008. Functional characterization of an unusual phytochelatin synthase
ljPCS3, of
lotus japonicus.
plant Physiology, 148:536-545.).Based on the Process of chelating and detoxicification of plant complexing element heavy metal ion, investigator attempts the Phytochelatin synthase gene microbial colon bacillus of different sources, builds the biological restoration that colon bacillus recombinant bacterial strain carries out heavy metal contamination.In the recombinant escherichia coli cell of experimental result display overexpression heterologous plant complexing element synthase gene, cadmium content is higher than non-transgenic bacterial strain (contrast), the recipient bacterium of above-mentioned experiment comprises E bacteria strain JM105, JM109, M15REP4 and BL21 (DE3) (Bae W, Chen W, Mulchandani A, Mehra R K. 2000. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins.
biotechnology and Bioengineering, 70 (5): 518-524; Bae W, Mulchandani A, Chen W. 2002. Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation.
journal of Inorganic Biochemistry, 88 (2): 223-227; Sauge-Merle S, Cuine S, Carrier P, Lecomte-Pradines C, Luu D, Peltier G. 2003. Enhanced toxic metal accumulation in engineered bacterial cells expressing
arabidopsis thalianaphytochelatin synthase.
applied and Environmental Microbiology, 69 (1): 490-494; Matsumoto S, Shiraki K, Tsujib N, Hiratab K, Miyamoto K, Takagi M. 2004. Functional analysis of phytochelatin synthase from Arabidopsis thaliana and its expression in
escherichia coliand
saccharomyces cerevisiae.
science and Technology of Advanced Materials, 5 (3): 377-381; Kang S H, Singh S, Kim J Y, Lee W, Mulchandani A, Chen W. 2007. Bacteria metabolically engineered for enhanced phytochelatin production and cadmium accumulation.
applied and Environmental Microbiology, 73 (19): 6317-6320.).There is no at present and utilize E bacteria strain Rosetta (DE3) as recipient bacterium, carry out Phytochelatin synthase gene conversion, obtaining can the report of recombinant bacterial strain of heavy metal cadmium accumulation.In addition, Phytochelatin synthase gene proceed to the report that the accumulation that can improve recombinant escherichia coli engineering bacteria heavy metal copper is detected in Sauge-Merle etc., this experiment bacterial strain used is M15REP4(Sauge-Merle S, Cuine S, Carrier P, Lecomte-Pradines C, Luu D, Peltier G. 2003. Enhanced toxic metal accumulation in engineered bacterial cells expressing
arabidopsis thalianaphytochelatin synthase.
applied and Environmental Microbiology, 69 (1): 490-494).And utilize Phytochelatin synthase gene to transform colon bacillus, obtain sodium accumulation recombinant bacterial strain and there is no report, also without overexpression Phytochelatin synthase gene E bacteria strain simultaneously tool accumulate cadmium, copper and sodium 3 report of metal ion species ability.
Beans pears (
pyrus calleryanadcne.) China is originated in, for one of stock conventional in pears production, it absorbs, accumulate and the ability of transport heavy metal directly can determine pear tree overground part fruit heavy metal content, and the heavy metal binding ability therefore studying the Phytochelatin synthase gene of beans pears has great importance.We are separated and obtain a Phytochelatin synthase gene from beans pears
pcPCS1, the expression level of this gene be subject to the induction of heavy metal cadmium and copper rise (Li Hui, Cong Yu, Wang Hongwei contain Baolong, Lin Jing, Chang Youhong. 2010. beans pears phytochelatin synthases
pcPCS1gene clone and expression analysis thereof. gardening journal, 37 (6): 880-890.).But,
pcPCS1the function whether gene possesses in conjunction with heavy metal it be unclear that, also without bibliographical information.Therefore, we will
pcPCS1be connected with colon bacillus expression vector pET-22b (+), and by recombinant plasmid PcPCS1-pET-22b (+) transform E. coli bacterial strain Rosetta (DE3), adopt the heavy metal content in aas determination recombined engineering mycetocyte, can quantitatively illustrate
pcPCS1the Metal Ions Absorption ability of gene, thus discussion utilizes above-mentioned recombinant escherichia coli engineering bacteria to Cd
2+or Cu
2+or Na
+the liquid phase carrier of metal ion pollution carries out the possibility of biological restoration.
Summary of the invention
The object of the invention is to, for the blank field of the quantitative assay heavy metal ion lacked in the functional study of current beans pears plant complexing plain gene, design and build a kind of with beans pears plant complexing plain gene
pcPCS1with the recombinant plasmid PcPCS1-pET-22b (+) of colon bacillus expression vector pET-22b (+), and recombinant plasmid is proceeded in E bacteria strain Rosetta (DE3), obtain a strain recombinant escherichia coli engineering strain, and quantitatively implement beans pears plant complexing plain gene
pcPCS1the different Cd in the external world
2+, Cu
2+and Na
+to the mensuration of the absorbed dose of this three metal ion species under concentration conditions.
The object of the present invention is achieved like this: a strain colon bacillus (
escherichia coli) engineering bacteria, be with beans pears Phytochelatin synthase gene
pcPCS1e bacteria strain PcPCS1-pET-22b (+)/Rosetta (DE3) of recombinating of colon bacillus expression vector PcPCS1-pET-22b (+) and E bacteria strain Rosetta (DE3), its preserving number is CGMCC No. 7569.
A kind of preparation method of above-mentioned colon bacillus engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3), is characterized in that:
With beans pears Phytochelatin synthase gene
pcPCS1with colon bacillus expression vector pET-22b (+) construction recombination plasmid PcPCS1-pET-22b (+);
With chemical transformation, recombinant plasmid PcPCS1-pET-22b (+) is proceeded in E bacteria strain Rosetta (DE3), after mixed solution after conversion being added the LB nutrient solution of antibiotic-free expresses 1 hour in advance in 37 DEG C, bacterium liquid is coated on the screening flat board containing 50 μ g/ml kantlex, cultivate 16 hours for 37 DEG C, after colon bacillus grows, through PCR qualification, obtain recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3).
Preparation method described colon bacillus engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3): with beans pears Phytochelatin synthase gene
pcPCS1refer to colon bacillus expression vector pET-22b (+) construction recombination plasmid PcPCS1-pET-22b (+):
A) be KC936270 for number of registration in GenBank, nucleotide sequence is SEQ ID NO. 1, and aminoacid sequence is the beans pears Phytochelatin synthase gene of SEQ ID NO. 2
pcPCS1the upstream primer SEQ ID NO. 3 of design and downstream primer SEQ ID NO. 4, wherein, contains in SEQ ID NO. 3
ecoRi restriction enzyme site
gAATTC, contain in SEQ ID NO. 4
xhoi restriction enzyme site
gCTCGAG;
B) with the cDNA of beans pears for template, utilize round pcr to increase 5' end band
ecoRi restriction enzyme site, 3' end band
xhoi restriction enzyme site
pcPCS1gene order, the cumulative volume of its PCR reaction system is 50 μ L, wherein concrete composition is: 0.25 U high-fidelity DNA polymerase, the cDNA of 2 μ L, the dimethyl sulfoxide (DMSO) of 2.5 μ L, 4 μ L concentration are the dNTPs of 2.5 mmol/L, 2 μ L concentration are the upstream primer SEQ ID NO. 3 of 10 μm of ol/L, 2 μ L concentration are the downstream primer SEQ ID NO. 4 of 10 μm of ol/L, and the reaction buffer corresponding to high-fidelity DNA polymerase, supplies 50 μ L by appropriate distilled water; PCR reaction process is: 95 DEG C of denaturation 3 min; 95 DEG C of 30 sec subsequently, 58 DEG C of 30 sec, 68 DEG C of 90 sec, carry out 28 circulating reactions; 72 DEG C extend 10 min, terminate reaction;
C) by the PCR primer of b step sodium-acetate alcohol settling, with aseptic ultrapure water solubilize after drying precipitate, solute is used
ecoRi and
xhoi double digestion, glue reclaims digestion products; Use the same method and double digestion and glue are carried out to pET-22b (+) plasmid reclaim;
D) the PCR enzyme getting 7.5 μ L respectively cuts back to close pET-22b (+) the plasmid enzyme restriction recovery product of product and 1 μ L, the 10 × T of 1 μ L
4the T of DNA ligase Buffer and 0.5 μ L
4dNA ligase, 16 DEG C of connection instrument connects 12 hours, will connect product conversion colon bacillus DH5 α competent cell, then containing 37 DEG C of overnight incubation on the LB flat board of 100 mg/L acillins, treats that mono-clonal grows;
E) by single bacterium colony of growing in d step in containing the LB nutrient solution of 100 mg/L penbritins 37 DEG C, 220 rpm shaking table overnight incubation, collect thalline, extract plasmid, warp
ecoRi and
xhoi double digestion qualification of checking order, obtain PcPCS1-pET-22b (+) plasmid of restructuring.
In the preparation method of described colon bacillus engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3): the 2.5 U/ μ L PrimeSTAR HS DNA Polymerase 0.1 μ L that the high-fidelity DNA polymerase described in b step provides for TaKaRa company, high-fidelity DNA polymerase reaction buffer is 5 × Mg
2+plus PrimeSTAR Buffer 10 μ L, the addition of distilled water is 27.4 μ L.
Above-mentioned colon bacillus engineering bacteria is used for Cd
2+or Cu
2+or Na
+the liquid phase carrier of metal ion pollution carries out biological restoration.
The invention has the advantages that: colon bacillus engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3) that the present invention relates to (
escherichia coli) be realized by the synthetic technology of maturation, according to technology disclosed in this invention, this area researchist easily obtains this bacterial strain; This bacterial strain is to Cd
2+, Cu
2+and Na
+all there is adsorptive power, go for the microorganism remediation of multiple heavy metal contamination liquid phase carrier.
accompanying drawing explanation
The structure schema of Fig. 1, PcPCS1-pET-22b (+) colon bacillus expression vector.
Fig. 2, beans pears
pcPCS1gene PCR product figure, in figure: M swimming lane is TaKaRa company DL2000 DNA Marker; L1 swimming lane is beans pears
pcPCS1gene PCR product.
Fig. 3, beans pears
pcPCS1gene PCR product and pET-22b (+) carrier
ecoRi and
xhoi double digestion qualification figure, in figure: M swimming lane is TaKaRa company Wide Range DNA Marker (500-15,000); L1 swimming lane is
pcPCS1pCR primer double digestion (
ecoRi and
xhoi) product; L2 swimming lane be pET-22b (+) double digestion (
ecoRi and
xhoi) product;
PcPCS1-pET-22b (+) plasmid of Fig. 4, restructuring
ecoRi and
xhoi double digestion qualification figure, in figure, M swimming lane is TaKaRa company Wide Range DNA Marker(500-15,000); L1 swimming lane be restructuring PcPCS1-pET-22b (+) plasmid double digestion (
ecoRi and
xhoi) product;
Fig. 5, recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3) PCR identify figure, and in figure, M swimming lane is TaKaRa company DL2000 DNA Marker; L1 swimming lane is blank; L2 swimming lane is for turning pET-22b (+) empty carrier Rosetta (DE3) PCR result; L3 swimming lane is recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3) PCR result.
Fig. 6, recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3) SDS-PAGE identify figure, in figure, M swimming lane is Invitrogen company protein molecular weight standard (Protein molecular weight standard); L1 swimming lane is induce without IPTG; L2 swimming lane is that 0.5 mmol/L IPTG induces 1 hour; L3 swimming lane is that 0.5 mmol/L IPTG induces 3 hours; L4 swimming lane is that 0.5 mmol/L IPTG induces 6 hours;
Fig. 7, recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3) are in different cultured on solid medium situation, and wherein: A is blank LB solid medium, B is interpolation 2.0 mmol/L CdCl
22.5H
2the LB solid medium of O, C is interpolation 4.0 mmol/L CuCl
22H
2the LB solid medium of O, D is the LB solid medium of interpolation 4.5% (w/v) NaCl..
Embodiment
embodiment 1 beans pears
pcPCS1
the structure of gene colon bacillus expression vector
Beans pears
pcPCS1the structure flow process of gene colon bacillus expression vector PcPCS1-pET-22b (+) is as Fig. 1.
In GenBank, number of registration is the beans pears Phytochelatin synthase gene of KC936270
pcPCS1, its coding region nucleotide sequence is SEQ ID NO. 1:
ATGGCGATGG CGGGGTTGTA TCGGCGCCTT CTTCCTTCAC CCCCTGCCGT CGATTTCGCT 60
TCCTCTCAGG GCAAGCAACT TTTTCTTGAA GCGGTTCAAA ATGGAACCAT GGAAAGCTTT 120
TACAGGTTGG TTTCATATTT CCAAACGCAA TCAGAGCCTG CATTTTGTGG CCTCGCGAGC 180
TTGTCCATGG TCCTCAATGC TCTTGCCATT GATCCTGGCA GAAAATGGAA AGGGCCTTGG 240
AGATGGTTTG ATGAATCTAT GTTAGACTGT TGCGAGCCTT TGGAGACTGT CAAAGTAAGA 300
GGCATCTCAT TTGGGAAGCT TGTCTGCTTG GCTCACTGTG CTGGAGCAAA AGTTGAAGCC 360
TTTCGCACAA ATCATAGCAC AATTGATGAG TTTCGTAAAT ATGTATTGAG ATGTTCTACT 420
TCTGATGATT GTCATGTGAT CTCATCATAC GATAGATCAG TTCTCAAACA GACGGGTACT 480
GGTCATTTTT CACCAATTGG TGGCTATCAT GCTGAAAGAG ACTTGGCGCT GATTTTGGAT 540
GTTGCACGTT TTAAGTATCC TCCCCATTGG GTACCCCTCA AAATTCTTTG GGATGCCATG 600
AATAATGTTG ATTCATCTAC TGGACAACGC AGAGGGTTCA TGCTTATATC CAGGCCTCAC 660
AGTGAGCCAG GACAACTTTA TACTCTGAGC TGCAAACATG AGAGTTGGGT TGGTGTTGCA 720
AAATACTTAA TGGATGATGT TCCTCTTCTT CTAAAATCGG AGGAGGTGAA AGACATTCAA 780
GAAGTTCTCT CTGTTGTTTT CAAGTCACTT CCATCCTATT TTGGGCAGTT CATCAAGTGG 840
GTTGCAGAAG TTCGGAGACG AGAGGATGGT GGCCAAAGCT TAAGCCCAGA GGAAAAAGCT 900
AGGCTTGCTG TCAAGGAGGA GGTACTGAGA CAAGTCCAGG ACACTGGCCC TTTTAAACAC 960
GTGGCAGAAT TATTATCTTC AGCACATTCA TGCTGCGGAA ACTTATATTC TGGTCATGAA 1020
GAAAACTTGC CCAACATTGC TGCGAGCGTT TGCTGCCAAG GCGCACAAAT TTTGTCGGGG 1080
AACTCTGCCT TCCCGGGGGT TTATTGCTGT CAGGAAACAT GTGTGAAATG CTTCAAGGCA 1140
AATGGCGACA AACCAGTCAC GGTTGTGAGT GGGACAGTGG TAAATGGTGG AACCGAGGAG 1200
AAGATGGACA TGCTTGTTCC TTCATCAAAA ACAAATTCAG GATGTTCTTG TGCAATTGGG 1260
ATTCATCCTG CTAGCAATGA CGTTCTAACA GCACTTCTAC TGGCTTTGCC CCCAGGTACA 1320
TGGTCTGGGA TCAAAGACGA GAAGCTTTCA AAAGGAATAT CTAACCTTGT TTCCACTGTA 1380
AATCTTCCTA CTTTGCTTCA AGAAGAGGTT TTGCACTTGC GACGCCAGCT CCACCTTCTC 1440
AAGAAATGCC AGGAGGATAG GGTAGACGAC GATCTTGGCT CTCCTCTCGC TTAG 1494
Aminoacid sequence is SEQ ID NO. 2:
MAMAGLYRRL LPSPPAVDFA SSQGKQLFLE AVQNGTMESF YRLVSYFQTQ SEPAFCGLAS 60
LSMVLNALAI DPGRKWKGPW RWFDESMLDC CEPLETVKVR GISFGKLVCL AHCAGAKVEA 120
FRTNHSTIDE FRKYVLRCST SDDCHVISSY DRSVLKQTGT GHFSPIGGYH AERDLALILD 180
VARFKYPPHW VPLKILWDAM NNVDSSTGQR RGFMLISRPH SEPGQLYTLS CKHESWVGVA 240
KYLMDDVPLL LKSEEVKDIQ EVLSVVFKSL PSYFGQFIKW VAEVRRREDG GQSLSPEEKA 300
RLAVKEEVLR QVQDTGPFKH VAELLSSAHS CCGNLYSGHE ENLPNIAASV CCQGAQILSG 360
NSAFPGVYCC QETCVKCFKA NGDKPVTVVS GTVVNGGTEE KMDMLVPSSK TNSGCSCAIG 420
IHPASNDVLT ALLLALPPGT WSGIKDEKLS KGISNLVSTV NLPTLLQEEV LHLRRQLHLL 480
KKCQEDRVDD DLGSPLA 497
For above-mentioned beans pears Phytochelatin synthase gene
pcPCS1design primer pair:
Upstream primer: SEQ ID NO. 3:
5'-
gC gAATTCaTGGCGATGGCGGGGTTG-3'(tilted letter is restriction enzyme site protection base, and underlined letter is
ecoRi restriction enzyme site);
Downstream primer: SEQ ID NO. 4:
5'-
cC gCTCGAGctaagcgagaggagagccaagatc-3'(tilted letter is restriction enzyme site protection base, and underlined letter is
xhoi restriction enzyme site).
With beans pears seedling for vegetable material, with RNAiso Plus(TaKaRa company) extract total serum IgE, operating process is in strict accordance with the extraction flow process of specification sheets.After DNA enzymatic I (without RNA enzyme, TIANGEN company) digestion, get the synthesis (Primescript 1st Strand cDNA Synthesis Kit, TaKaRa company) of 2 μ g total serum IgE for cDNA first chain, reverse transcription primer is Oligo (dT)
15, method is carried out according to test kit specification sheets.CDNA first chain obtained with reverse transcription is for template, primer SEQ ID NO. 3 and SEQ ID NO. 4 is utilized to match, with PrimeSTAR HS DNA Polymerase(TaKaRa company) carry out pcr amplification, the cumulative volume of PCR system is 50 μ L, concrete composition is as follows: 0.1 μ L concentration is the PrimeSTAR HS DNA Polymerase of 2.5 U/ μ L, the cDNA of 2 μ L, the dimethyl sulfoxide (DMSO) of 2.5 μ L, 4 μ L concentration are the dNTPs of 2.5 mmol/L, 2 μ L concentration are the upstream primer SEQ ID NO. 3 of 10 μm of ol/L, 2 μ L concentration are the downstream primer SEQ ID NO. 4 of 10 μm of ol/L, 5 × Mg
2+plus PrimeSTAR Buffer 10 μ L, distilled water 27.4 μ L.
Its PCR response procedures is: 95 DEG C of sex change 3 min, carries out 28 circulating reactions (95 DEG C of 30 sec, 58 DEG C of 30 sec, 68 DEG C of 90 sec) subsequently, and 72 DEG C extend 10 min, and the PCR primer obtained as shown in Figure 2.Isopyknic 3 M sodium-acetates (pH 5.2) are added in the PCR primer obtained, and add 2.5 times of volume dehydrated alcohols at-20 DEG C of precipitation 30 min, 12 000 rpm, 4 DEG C centrifugal 10 minutes, primary sedimentation washed by 70% ethanol, 12 000 rpm again, 4 DEG C are removed supernatant in centrifugal 10 minutes, with aseptic ultrapure water dissolution precipitation; With
ecoRi and
xhoi carries out double digestion to the PCR primer after purifying and pET-22b (+) carrier, and it is as follows that enzyme cuts system: 10x Buffer H(TaKaRa company) 5 μ L, PCR primer 5 μ g or pET-22b (+) carrier 5 μ g,
ecoRi and
xhothe each 3 μ L of I, add aseptic ultrapure water constant volume to 50 μ L, and 37 DEG C of Water Under bath enzymes cut 8 h, and reclaim test kit (TIANGEN company) with plain agar sugar gel DNA subsequently and carry out purifying recovery, enzyme cuts back to close product as shown in Figure 3.Get the PCR digestion products of 5.5 μ L and pET-22b (+) the plasmid enzyme restriction product of 3 μ L respectively, 10 × T4 DNA ligase Buffer of 1 μ L and the T4 DNA ligase (TaKaRa company) of 0.5 μ L, 12 hours are connected at 16 DEG C, after connecting product conversion E bacterium competence cell DH 5 α, 37 DEG C of overnight incubation on the LB substratum containing 100 mg/L penbritins, carry out cultivation screening, define the restructuring positive Escherichia coli transforming PcPCS1-pET-22b (+).By the restructuring positive Escherichia coli that obtains in containing the LB nutrient solution of 100 mg/L penbritins 37 DEG C, 220 rpm shaking culture are spent the night, and collect thalline, extract after plasmid
ecoRi and
xhoi double digestion is identified, it is as follows that enzyme cuts system: 10x Buffer H(TaKaRa company) 5 μ L, PcPCS1-pET-22b (+) plasmid 5 μ g,
ecoRi and
xhothe each 3 μ L of I, add aseptic ultrapure water constant volume to 50 μ L, and 37 DEG C of Water Under bath enzymes cut 8 h, and digestion products as shown in Figure 4.Send Beijing Liuhe Huada Genomics Technology Co., Ltd to check order plasmid correct for digestion verification, by plasmid correct for order-checking in-20 DEG C of preservations, be PcPCS1-pET-22b (+) plasmid of restructuring.
embodiment 2 beans pears
pcPCS1
the acquisition of recombinant escherichia coli cell and qualification
The colon bacillus Rosetta (DE3) that preserves in-70 DEG C of Ultralow Temperature Freezers with inoculating needle picking (
escherichia coli) (buy in Merck KGaA company, Corporation web site is http://www.emdmillipore.com), on LB plate culture medium, classification line, is inverted in 37 DEG C of incubated overnight in constant incubator by the plate culture medium after line.Picking list bacterium colony, is inoculated in LB liquid nutrient medium, and 37 DEG C of 150 rpm shaking culture is to OD
600=0.6, bacterial culture fluid is positioned in ice and stops cultivating.Get above-mentioned bacterial culture fluid 1 mL in 1.5 mL centrifuge tubes, place 10 minutes on ice, 10 000 centrifugal 10 minutes of rpm 4 DEG C, abandon supernatant.Add 50 mmol/L CaCl of precooling in 200 μ L ice
2solution is suspension cell gently, places after 15 minutes on ice, 10 000 centrifugal 10 minutes of rpm 4 DEG C.Supernatant discarded, adds the CaCl of 100 μ L precoolings containing 50 mmol/L of 15% glycerine
2solution, gently suspension cell, be colon bacillus Rosetta (DE3) competent cell.
PcPCS1-pET-22b (+) plasmid containing goal gene and empty carrier pET-22b (+) (content is no more than 50 ng, and volume is no more than 10 μ L) are added colon bacillus Rosetta (DE3) competent cell respectively.Shake up gently, place 30 minutes on ice, 42 DEG C of thermal shocks 90 seconds, are placed in rapidly cooled on ice 3 minutes.In centrifuge tube, add 1 mL LB liquid nutrient medium, mix rear 37 DEG C of shaking culture 1 hour, bacterium is restore normal growth state, and the antibiotics resistance gene (Amp of expression plasmid coding
r).Getting 100 μ L after being shaken up by above-mentioned bacterium liquid is uniformly coated on LB plate culture medium (containing 100 mg/L penbritins), 37 DEG C, be inverted cultivation 16 hours, picking colon bacillus list bacterium colony, shaking culture 16 hours (37 DEG C of 150 rpm) in LB nutrient solution (containing 100 mg/L penbritins), get 2 μ L bacterial culture fluids and carry out PCR qualification as template, the operation in PCR primer and PCR parameter reference embodiment 1, PCR qualification result as shown in Figure 5.Single bacterium colony of the qualification PCR positive is recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3), on May 8th, 2013 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is in No. 3, the good institute in BeiChen West Road, Chaoyang District, BeiJing City 1, Institute of Microorganism, Academia Sinica, postcode is 100101, and preserving number is CGMCC No.7569.
Recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3) is being rule containing on the LB plate culture medium of 100 mg/L penbritins, select single bacterium colony after 37 DEG C of cultivation 16 h and access (containing penbritin 100 mg/L) in 3 mL LB liquid nutrient mediums, 37 DEG C, 200 rpm cultivate about 16 h, then the bacterium liquid that spent the night by 100 μ L is inoculated in fresh LB liquid nutrient medium, add isopropyl-beta D-thio galactopyranoside (Isopropyl-β-D-1-Thiogalactopyranoside simultaneously, IPTG) its concentration is in the medium made to be 0.5 mmol/L.37 DEG C of 200 rpm cultivates after 1,3 or 6 hour, 8 000 rpm, centrifugal 5 minutes collecting cells, adopt the total bacterial protein of Sangon Biotech (Shanghai) Co., Ltd. to extract test kit (Bacterial Protein Extraction Kit) and extract albumen.Detect PcPCS1 albumen by 12% (w/v) SDS-PAGE (SDS-PAGE) whether to express in recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3).SDS-PAGE operating process is with reference to method (the Sambrook J of Sambrook etc., Fritsch E F, Maniatis T. 2001. Molecular cloning, a laboratory manual. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press.).SDS-PAGE electrophoresis result as shown in Figure 6, after 0.5 mmol/L IPTG induces 6 hours, express in recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3) about 55.59 kDa albumen (
pcPCS1coded by said gene molecular weight of albumen is 54.77 kDa+6 His labels).Show that PcPCS1 albumen can successful expression in recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3).
embodiment 3 recombinant escherichia coli PcPCS1-pET-22b (+)/Rosetta (DE3) is to Cd
2+
, Cu
2+
and Na
+
the determination of tolerance
Recombinant escherichia coli bacterial strain PcPCS1-pET-22b (+)/Rosetta (DE3) is being cultured to OD containing on the LB liquid nutrient medium of 100 mg/L penbritins
600=0.5, in contrast with E bacteria strain Rosetta (DE3), LB liquid nutrient medium is cultured to OD simultaneously
600=0.5.Two kinds of bacterium liquid get 5 μ L point samples respectively in interpolation different concns CdCl after 10 times of gradient dilutions
22.5H
2o, CuCl
22H
2on the LB solid medium of O or NaCl, cultivate 16 h in 37 DEG C, observe colony growth situation and take a picture.
Fig. 7 A shows: the bacterium liquid (OD of recombinant escherichia coli bacterial strain PcPCS1-pET-22b (+)/Rosetta (DE3) and non-recombinant Rosetta (DE3)
600be 0.5) dilution 1,10,100 and 1000 times after, the growing state on blank LB solid medium is identical.
Fig. 7 B shows: the bacterium liquid (OD of recombinant escherichia coli bacterial strain PcPCS1-pET-22b (+)/Rosetta (DE3)
600=0.5), after diluting 1,10,100 and 1000 times, this bacterial strain still can contain 2.0 mmol/L CdCl
22.5H
2normal growth on the LB solid medium of O, non-recombinant bacterial strain Rosetta (DE3) bacterium liquid (OD
600=0.5), before undiluted, this bacterial strain can contain 2.0 mmol/L CdCl
22.5H
2the LB cultured on solid medium of O, non-recombinant bacterial strain Rosetta (DE3) bacterium liquid (OD
600=0.5), after diluting 10,100 and 1000 times, this bacterial strain can not grow on the above medium.
Fig. 7 C shows: the bacterium liquid (OD of recombinant escherichia coli bacterial strain PcPCS1-pET-22b (+)/Rosetta (DE3)
600=0.5), after diluting 1,10,100 and 1000 times, this bacterial strain still can contain 4.0 mmol/L CuCl
22H
2normal growth on the LB solid medium of O, non-recombinant bacterial strain Rosetta (DE3) bacterium liquid (OD
600=0.5), before undiluted, this bacterial strain can contain 4.0 mmol/L CuCl
22H
2the LB cultured on solid medium of O, after non-recombinant bacterial strain Rosetta (DE3) bacterium liquid dilution 10,100 and 1000 times, this bacterial strain can not grow on the above medium.
Fig. 7 D shows: the bacterium liquid (OD of recombinant escherichia coli bacterial strain PcPCS1-pET-22b (+)/Rosetta (DE3)
600=0.5) after diluting 1 and 10 times, this bacterial strain still can containing normal growth on the LB solid medium of 4.5% (w/v) NaCl, but not recombinant bacterial strain Rosetta (DE3) can not containing the LB cultured on solid medium of 4.5% (w/v) NaCl.
The above results shows, beans pears
pcPCS1proceeding to of gene, can strengthen colon bacillus Rosetta (DE3) bacterial strain to CdCl
22.5H
2o, CuCl
22H
2the tolerance of O and NaCl.
embodiment 4 recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3) is containing different Cd
2+
, Cu
2+
or Na
+
the mensuration of Metal Ions Absorption amount in the liquid nutrient medium of concentration
Recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3) is being rule containing on the LB plate culture medium of 100 mg/L penbritins, select single bacterium colony after 37 DEG C of cultivation 16 h and access (containing penbritin 100 mg/L) in 3 mL LB liquid nutrient mediums, 37 DEG C, 200 rpm cultivate about 16 h, and the bacterium liquid that then spent the night by 100 μ L is inoculated in fresh in 0.5,1.0 and 1.5 mmol/L CdCl
22.5H
2o, 1.5,2.0 and 2.5 mmol/L CuCl
22H
2in the 25 mL LB liquid nutrient mediums of O or 2.5,3.0 and 3.5% (w/v) NaCl, add isopropyl-beta D-thio galactopyranoside (Isopropyl-β-D-1-Thiogalactopyranoside, IPTG) makes its concentration in the medium be 0.5 mmol/L simultaneously.After 37 DEG C of 200 rpm cultivates 6 hours, 8 000 rpm, centrifugal 5 minutes collecting cells, with the 5 mmol/L 4-hydroxyethyl piperazine ethanesulfonic acid washed cells containing 0.8% (w/v) NaCl, after 65 DEG C of process dry cell in 24 hours, add after 500 μ L concentrated nitric acids clear up 2 days and measure Cd on the atomic absorption spectrum spectrograph (model AAnalyst 700) of Perkin Elmer company
2+, Cu
2+or Na
+content.Result is as shown in table 1: work as Cd
2+when concentration is 0.5 ~ 1.5 mmol/L, Cd in PcPCS1-pET-22b (+)/Rosetta (DE3) strain cell
2+content is non-transgenosis Rosetta (DE3) (contrast) 1.89 ~ 2.19 times; Work as Cu
2+when concentration is 1.5 ~ 2.5 mmol/L, Cd in PcPCS1-pET-22b (+)/Rosetta (DE3) strain cell
2+content is non-transgenosis Rosetta (DE3) (contrast) 2.00 ~ 2.04 times; Work as Na
+when concentration is 2.5 ~ 3.5% (w/v), Na in PcPCS1-pET-22b (+)/Rosetta (DE3) strain cell
+content is non-transgenosis Rosetta (DE3) (contrast) 1.60 ~ 1.76 times.The above results shows, beans pears
pcPCS1proceeding to of gene, can strengthen colon bacillus Rosetta (DE3) bacterial strain to Cd
2+, Cu
2+and Na
+receptivity.
Table 1. PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain
At different Cd
2+, Cu
2+or Na
+the mensuration of Metal Ions Absorption amount under concentration conditions
embodiment 5 recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3) is to Cd
2+
, Cu
2+
or Na
+
the liquid phase carrier of metal ion pollution carries out biological restoration
Recombinant escherichia coli cell PcPCS1-pET-22b (+)/Rosetta (DE3) is being rule containing on the LB plate culture medium of 100 mg/L penbritins, is selecting single bacterium colony after 37 DEG C of cultivation 16 h and access 20,40 and 60 mg Cd respectively
2+1000 mL tap water, 20,40 and 60 mg Cu
2+1000 mL tap water and 100,200 and 300 mg Na
2+1000 mL tap water in, 37 DEG C, static gas wave refrigerator 5 days, then 8 000 rpm, remove E mycetocyte in centrifugal 10 minutes, measures supernatant C d in the Atomic Absorption Spectroscopy AAS (model AAnalyst 700) of Perkin Elmer company is upper
2+, Cu
2+or Na
+content.3 repetitions are carried out in experiment, and experimental result mean value record in table 2.
From table 2: as Cd in tap water
2+when content is 20 ~ 60 mg, inoculate PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain after 5 days, Cd in tap water
2+before content drops to and do not inoculate 61.55 ~ 90.87%; As Cu in tap water
2+when content is 20 ~ 60 mg, inoculate PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain after 5 days, Cu in tap water
2+content drops to 40.05 ~ 80.20% when not inoculating; As Na in tap water
+when content is 100 ~ 300 mg, inoculate PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain after 5 days, the Na in tap water
+content drops to 59.73 ~ 79.01% when not inoculating.The above results shows, inoculation PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain can reduce Cd in tap water
2+, Cu
2+and Na
+content, this bacterial strain can be used for the biological restoration of liquid phase carrier metallic pollution.
PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain pair inoculated by table 2.
Containing different concns Cd
2+, Cu
2+or Na
+tap water repair ability
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> mono-strain colon bacillus engineering bacteria and its preparation method and application
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1494
<212> DNA
<213> beans pears (Pyrus calleryana Dcne.)
<400> 1
atggcgatgg cggggttgta tcggcgcctt cttccttcac cccctgccgt cgatttcgct 60
tcctctcagg gcaagcaact ttttcttgaa gcggttcaaa atggaaccat ggaaagcttt 120
tacaggttgg tttcatattt ccaaacgcaa tcagagcctg cattttgtgg cctcgcgagc 180
ttgtccatgg tcctcaatgc tcttgccatt gatcctggca gaaaatggaa agggccttgg 240
agatggtttg atgaatctat gttagactgt tgcgagcctt tggagactgt caaagtaaga 300
ggcatctcat ttgggaagct tgtctgcttg gctcactgtg ctggagcaaa agttgaagcc 360
tttcgcacaa atcatagcac aattgatgag tttcgtaaat atgtattgag atgttctact 420
tctgatgatt gtcatgtgat ctcatcatac gatagatcag ttctcaaaca gacgggtact 480
ggtcattttt caccaattgg tggctatcat gctgaaagag acttggcgct gattttggat 540
gttgcacgtt ttaagtatcc tccccattgg gtacccctca aaattctttg ggatgccatg 600
aataatgttg attcatctac tggacaacgc agagggttca tgcttatatc caggcctcac 660
agtgagccag gacaacttta tactctgagc tgcaaacatg agagttgggt tggtgttgca 720
aaatacttaa tggatgatgt tcctcttctt ctaaaatcgg aggaggtgaa agacattcaa 780
gaagttctct ctgttgtttt caagtcactt ccatcctatt ttgggcagtt catcaagtgg 840
gttgcagaag ttcggagacg agaggatggt ggccaaagct taagcccaga ggaaaaagct 900
aggcttgctg tcaaggagga ggtactgaga caagtccagg acactggccc ttttaaacac 960
gtggcagaat tattatcttc agcacattca tgctgcggaa acttatattc tggtcatgaa 1020
gaaaacttgc ccaacattgc tgcgagcgtt tgctgccaag gcgcacaaat tttgtcgggg 1080
aactctgcct tcccgggggt ttattgctgt caggaaacat gtgtgaaatg cttcaaggca 1140
aatggcgaca aaccagtcac ggttgtgagt gggacagtgg taaatggtgg aaccgaggag 1200
aagatggaca tgcttgttcc ttcatcaaaa acaaattcag gatgttcttg tgcaattggg 1260
attcatcctg ctagcaatga cgttctaaca gcacttctac tggctttgcc cccaggtaca 1320
tggtctggga tcaaagacga gaagctttca aaaggaatat ctaaccttgt ttccactgta 1380
aatcttccta ctttgcttca agaagaggtt ttgcacttgc gacgccagct ccaccttctc 1440
aagaaatgcc aggaggatag ggtagacgac gatcttggct ctcctctcgc ttag 1494
<210> 2
<211> 497
<212> PRT
<213> beans pears (Pyrus calleryana Dcne.)
<400> 2
Met Ala Met Ala Gly Leu Tyr Arg Arg Leu Leu Pro Ser Pro Pro Ala
1 5 10 15
Val Asp Phe Ala Ser Ser Gln Gly Lys Gln Leu Phe Leu Glu Ala Val
20 25 30
Gln Asn Gly Thr Met Glu Ser Phe Tyr Arg Leu Val Ser Tyr Phe Gln
35 40 45
Thr Gln Ser Glu Pro Ala Phe Cys Gly Leu Ala Ser Leu Ser Met Val
50 55 60
Leu Asn Ala Leu Ala Ile Asp Pro Gly Arg Lys Trp Lys Gly Pro Trp
65 70 75 80
Arg Trp Phe Asp Glu Ser Met Leu Asp Cys Cys Glu Pro Leu Glu Thr
85 90 95
Val Lys Val Arg Gly Ile Ser Phe Gly Lys Leu Val Cys Leu Ala His
100 105 110
Cys Ala Gly Ala Lys Val Glu Ala Phe Arg Thr Asn His Ser Thr Ile
115 120 125
Asp Glu Phe Arg Lys Tyr Val Leu Arg Cys Ser Thr Ser Asp Asp Cys
130 135 140
His Val Ile Ser Ser Tyr Asp Arg Ser Val Leu Lys Gln Thr Gly Thr
145 150 155 160
Gly His Phe Ser Pro Ile Gly Gly Tyr His Ala Glu Arg Asp Leu Ala
165 170 175
Leu Ile Leu Asp Val Ala Arg Phe Lys Tyr Pro Pro His Trp Val Pro
180 185 190
Leu Lys Ile Leu Trp Asp Ala Met Asn Asn Val Asp Ser Ser Thr Gly
195 200 205
Gln Arg Arg Gly Phe Met Leu Ile Ser Arg Pro His Ser Glu Pro Gly
210 215 220
Gln Leu Tyr Thr Leu Ser Cys Lys His Glu Ser Trp Val Gly Val Ala
225 230 235 240
Lys Tyr Leu Met Asp Asp Val Pro Leu Leu Leu Lys Ser Glu Glu Val
245 250 255
Lys Asp Ile Gln Glu Val Leu Ser Val Val Phe Lys Ser Leu Pro Ser
260 265 270
Tyr Phe Gly Gln Phe Ile Lys Trp Val Ala Glu Val Arg Arg Arg Glu
275 280 285
Asp Gly Gly Gln Ser Leu Ser Pro Glu Glu Lys Ala Arg Leu Ala Val
290 295 300
Lys Glu Glu Val Leu Arg Gln Val Gln Asp Thr Gly Pro Phe Lys His
305 310 315 320
Val Ala Glu Leu Leu Ser Ser Ala His Ser Cys Cys Gly Asn Leu Tyr
325 330 335
Ser Gly His Glu Glu Asn Leu Pro Asn Ile Ala Ala Ser Val Cys Cys
340 345 350
Gln Gly Ala Gln Ile Leu Ser Gly Asn Ser Ala Phe Pro Gly Val Tyr
355 360 365
Cys Cys Gln Glu Thr Cys Val Lys Cys Phe Lys Ala Asn Gly Asp Lys
370 375 380
Pro Val Thr Val Val Ser Gly Thr Val Val Asn Gly Gly Thr Glu Glu
385 390 395 400
Lys Met Asp Met Leu Val Pro Ser Ser Lys Thr Asn Ser Gly Cys Ser
405 410 415
Cys Ala Ile Gly Ile His Pro Ala Ser Asn Asp Val Leu Thr Ala Leu
420 425 430
Leu Leu Ala Leu Pro Pro Gly Thr Trp Ser Gly Ile Lys Asp Glu Lys
435 440 445
Leu Ser Lys Gly Ile Ser Asn Leu Val Ser Thr Val Asn Leu Pro Thr
450 455 460
Leu Leu Gln Glu Glu Val Leu His Leu Arg Arg Gln Leu His Leu Leu
465 470 475 480
Lys Lys Cys Gln Glu Asp Arg Val Asp Asp Asp Leu Gly Ser Pro Leu
485 490 495
Ala
<210> 3
<211> 26
<212> DNA
<213> artificial sequence
<400> 3
gcgaattcat ggcgatggcg gggttg 26
<210> 4
<211> 33
<212> DNA
<213> artificial sequence
<400> 4
ccgctcgagc taagcgagag gagagccaag atc 33
Claims (5)
1. a strain colon bacillus (
escherichia coli) PcPCS1-pET-22b (+)/Rosetta (DE3) bacterial strain, its preserving number is CGMCC No.7569.
2. the preparation method of colon bacillus as claimed in claim 1, is characterized in that:
Take nucleotide sequence as beans pears Phytochelatin synthase gene PcPCS1 and colon bacillus expression vector pET-22b (+) construction recombination plasmid PcPCS1-pET-22b (+) of SEQ ID NO. 1;
With chemical transformation, recombinant plasmid PcPCS1-pET-22b (+) is proceeded in E bacteria strain Rosetta (DE3), after mixed solution after conversion being added the LB nutrient solution of antibiotic-free expresses 1 hour in advance in 37 DEG C, bacterium liquid is coated on the screening flat board containing 50 μ g/ml kantlex, cultivate 16 hours for 37 DEG C, after colon bacillus grows, through PCR qualification, obtain recombinant escherichia coli engineering bacteria PcPCS1-pET-22b (+)/Rosetta (DE3).
3. according to the preparation method of colon bacillus described in claim 2, it is characterized in that: refer to beans pears Phytochelatin synthase gene PcPCS1 and colon bacillus expression vector pET-22b (+) construction recombination plasmid PcPCS1-pET-22b (+):
A) be SEQ ID NO. 1 for nucleotide sequence, aminoacid sequence is upstream primer SEQ ID NO. 3 and the downstream primer SEQ ID NO. 4 of the beans pears Phytochelatin synthase gene PcPCS1 design of SEQID NO. 2, wherein, containing EcoR I restriction enzyme site GAATTC in SEQ ID NO. 3, containing Xho I restriction enzyme site GCTCGAG in SEQ ID NO. 4;
B) with the cDNA of beans pears for template, utilize PCR technology amplification 5' end band EcoR I restriction enzyme site, the PcPCS1 gene order of 3' end band Xho I restriction enzyme site, the cumulative volume of its PCR reaction system is 50 μ L, wherein concrete composition is: 0.25U high-fidelity DNA polysaccharase, the cDNA of 2 μ L, the dimethyl sulfoxide (DMSO) of 2.5 μ L, 4 μ L concentration are 2.5 mmol/L's
DNTPs, the downstream primer SEQ ID NO. 4 of 2 μ L concentration to be upstream primer SEQ ID NO. 3, the 2 μ L concentration of 10 μm of ol/L be 10 μm of ol/L, the reaction buffer corresponding to high-fidelity DNA polysaccharase, supplies 50 μ L by appropriate distilled water;
PCR reaction process is: 95 DEG C of denaturation 3 min; 95 DEG C of 30 sec subsequently, 58 DEG C of 30 sec, 68 DEG C of 90 sec, carry out 28 circulating reactions; 72 DEG C extend 10 min, terminate reaction;
C) by the PCR product sodium-acetate alcohol settling of b) step, with aseptic ultrapure water solubilize after drying precipitate, solute EcoR I and Xho I double digestion, glue reclaims digestion products; Use the same method and double digestion and glue are carried out to pET-22b (+) plasmid reclaim;
D) the PCR enzyme getting 7.5 μ L respectively cuts back to close pET-22b (+) the plasmid enzyme restriction recovery product of product and 1 μ L, 10 × T4 DNA ligase enzyme Buffer of 1 μ L and the T4 DNA ligase enzyme of 0.5 μ L, 16 DEG C of connection instrument connect 12 hours, product conversion colon bacillus DH5 α competent cell will be connected, then containing 37 DEG C of overnight incubation on the LB flat board of 100 mg/L acillins, treat that mono-clonal grows;
E) by single bacterium colony of growing in d) step in containing the LB nutrient solution of 100 mg/L penbritins 37 DEG C, 220rpm shaking table overnight incubation, collect thalline, extract plasmid, through EcoR I and Xho I double digestion and qualification of checking order, obtain PcPCS1-pET-22b (+) plasmid of restructuring.
4. according to the preparation method of colon bacillus described in claim 3, it is characterized in that: the 2.5 U/ μ L PrimeSTAR HS DNA Polymerase 0.1 μ L that high-fidelity DNA polysaccharase b) described in step provides for TaKaRa company, high-fidelity DNA polymeric enzyme reaction damping fluid is 5 × Mg
2+plus PrimeSTAR Buffer 10 μ L, the addition of distilled water is 27.4 μ L.
5. the colon bacillus described in claim 1 is to Cd
2+or Cu
2+or Na
+the liquid phase carrier of metal ion pollution carries out the application in biological restoration.
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Citations (2)
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|---|---|---|---|---|
| CN1401771A (en) * | 2002-09-26 | 2003-03-12 | 中国科学院植物研究所 | Garlic heavy metal resistance related gene and use thereof |
| US20110126312A1 (en) * | 2007-10-24 | 2011-05-26 | Juan Pedro Navarro Avino | Method for improving salinity tolerance |
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2013
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1401771A (en) * | 2002-09-26 | 2003-03-12 | 中国科学院植物研究所 | Garlic heavy metal resistance related gene and use thereof |
| US20110126312A1 (en) * | 2007-10-24 | 2011-05-26 | Juan Pedro Navarro Avino | Method for improving salinity tolerance |
Non-Patent Citations (4)
| Title |
|---|
| Enhanced tolerance and accumulation of heavy metal ions by engineered Escherichia coli expressing Pyrus calleryana phytochelatin synthase;Hui Li et al;《Journal of Basic Microbiology》;20141231;第54卷;1-8 * |
| Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase;Sandrine Sauge-Merle et al;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20030131;第69卷(第1期);材料与方法部分 * |
| 植物络合素及其合酶在重金属抗性中的功能研究进展;冯保民 等;《应用与环境生物学报》;20031231;第9卷(第6期);657-661 * |
| 豆梨植物络合素合酶PcPCS1基因克隆及其表达分析;李慧 等;《园艺学报》;20101231;第37卷(第6期);摘要、图2 * |
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