CN102660651A - Degenerate primer for detecting streptomyces two-domain laccase gene and detection method of degenerate primer - Google Patents
Degenerate primer for detecting streptomyces two-domain laccase gene and detection method of degenerate primer Download PDFInfo
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Abstract
The invention discloses a degenerate primer for detecting a streptomyces two-domain laccase gene and the detection method of the degrnerate primer. The degenerate primer comprises the following upstream and downstream primers: Cu2SF:5'-TACTGGYACTACCACGACCAY-3'and Cu4SR:5'-RTGVSWCTGSACRTGRCAGTG-3'. The detection method comprises the following steps of: carrying out PCR (polymerase chain reaction) amplification by utilizing the degenerate primer based on DNA (deoxyribonucleic acid) in a sample to be detected as a template; after the reaction, carrying out agarose gel electrophoresis detection and preliminary screening according to the fact that whether an electrophoretogram contains a 380-430bp single strip; cloning and sequencing DNA segments which are recovered after the preliminary screening; and putting the DNA sequence in a nucleic acid database for sequence alignment to determine whether the sample to be detected contains the streptomyces two-domain laccase gene. By adopting the detection method, the streptomyces two-domain laccase gene can be rapidly, accurately and specifically detected.
Description
Technical field
The invention belongs to the environmental field, relate in particular to a kind of primer and detection method thereof that detects streptomycete two-domain laccase gene.
Background technology
Laccase is that (p-diphenol oxidase EC.1.10.3.2), belongs to blue many copper oxydase family to one type of copper bearing polyphenoloxidase.Laccase is very active research focuses of fields such as biotechnology, chemical engineering, enzyme engineering, Materials science and environmental science always; Laccase effect substrate is quite extensive; Can demonstrate stronger catalyzed degradation ability to multiple phenolic comp ' ds pollution, so laccase demonstrates good prospects for application in the processing of phenolic comp ' ds pollution and the aspects such as structure of biosensor.
Laccase has the ability of oxidative lignin, can participate in the degraded or the polymerization of xylogen, is that the three big enzymes of participating in lignin degradation or depolymerization one of are.The structures shape of laccase the characteristic of laccase, thereby also just determined the ability of laccase oxidative degradation xylogen.Difference according to the laccase source can be divided into lacquer tree laccase, fungal laccase and bacterial laccase three major types with it, and the ability of the laccase oxidative degradation xylogen of different sources differs greatly.Compare with fungal laccase, bacterial laccase has more advantage, and is wide etc. like the optimum pH scope that do not need glycosylation, Heat stability is good and enzymic activity.
The bacterial species of lignin degrading is a lot, and actinomycetes are one type of stronger filamentous bacteriums of degradation capability of generally acknowledging, comprise streptomycete (
Streptomyces), Arthrobacter (
Arthrobacter), small single-cell bacteria (
Micromonospora) and Nocardia bacteria (
Nocardia) etc.Because actinomycetes can penetrate insoluble matrix such as lignocellulose, therefore in neutrality, slight alkalinity soil or compost, actinomycetes are participated in organic initial degraded and humify.The foreign scholar studies the streptomycete in the actinomycetes, finds that this bacterium mycelia branch is many and output is big, and the bacterium colony quality is tight on Gauss's substratum, combines with substratum firmly to be difficult for provoking.In the elementary metabolism stage, this bacterium has the ability of lignin degrading, and it mainly is through demethylation, aromatic ring fracture and three kinds of approach lignin degradings of oxide side chain.Have research once to show, streptomycete occupies very big proportion in compost and soil actinomycete, is the dominant population in the compost actinomycetes group; Especially in the compost pliotherm period; The quantity of mesophilic bacterias such as fungi reduces because the high temperature resistant and weakly alkaline environment of streptomycete, the compost pliotherm period streptomycete breed growth in a large number; Become the dominant population of compost between the pliotherm period; It produces microbiotic at hot stage and suppresses pathogenic bacterium, produces lignocellulose extracellular enzyme decomposing lignocellulose simultaneously, and keeps the decomposition semicellulose in the long period.Therefore, streptomycete has been brought into play important effect for difficult degradation organic matters such as lignocellulose degradations in the agricultural solid waste compost that the wood fibre cellulose content enriches.
Present research mainly focuses on the structure and the mechanism of action of conventional t hree-domain laccase gene, and is fewer for two-domain laccase gene and the research of lignin degradation bonding mechanism thereof, still belongs to a relative barren survey region now.The Two-domain laccase is one type of laccase than traditional three-domain laccase amino-acid residue lacks, molecular weight is little.The Two-domain laccase is extensively thought to be present in one type of novel laccase enzyme in the streptomycete,
Streptomyces griseus,
Streptomyces ipomoeae,
Streptomyces coelicolor,
Streptomyces scabieiIn the laccase-like laccase amino acid of all having found to have two-domain in the streptomycete.Advantage such as streptomycete two-domain laccase has optimum pH higher than general laccase thermostability, enzyme is wider.Therefore, the research of streptomyces two-domain laccase gene has crucial meaning for the expansion and the molecular biology research thereof of streptomyces two-domain laccase environment of applications condition and Application Areas.Understand according to us in addition, all mainly focus on conventional t hree-domain basidiomycete, ascomycetes and bacterial laccase gene to the multifarious research of laccase gene in recent years.For the Auele Specific Primer of a certain type of laccase gene of check, mainly contain basidiomycete laccase primer Cu1F/Cu2R (many copper combine a district and many copper to combine the design of two districts), ascomycetes laccase gene special primer LAC2F/LAC3R (many copper combine two districts and many copper to combine the design of three districts) and bacterial laccase specific C u1AF/Cu2R (many copper combine a district and many copper to combine the design of two districts).The Auele Specific Primer of above laccase gene is aimed at all that traditional three-domain laccase gene designed, and for the Auele Specific Primer of check streptomycete two-domain laccase gene, does not see that relevant report is arranged in the existing document.Streptomycete two-domain laccase is compared with the laccase of traditional three-domain, lacks second and combines the territory, and amino-acid residue is few, and molecular weight is little, but still contains the binding site of four copper.Streptomycete two-domain laccase is compared with traditional laccase at the conservative region amino acid of many copper binding sites has very big difference; Therefore; For how designing the degenerated primer that can effectively detect streptomycete two-domain laccase gene; And have no precedent to go by, but the exploitation of this degenerated primer has crucial meaning again in environment, detecting and obtain unknown streptomycete two-domain laccase gene and then grasping the biological action mechanism of streptomycete.
Summary of the invention
The object of the present invention is to provide a kind of degenerated primer that is used to detect streptomycete two-domain laccase gene, and corresponding a kind of method that can detect streptomycete two-domain laccase gene fast, accurately, specifically is provided.
The technical scheme that the present invention proposes is a kind of degenerated primer that is used to detect streptomycete two-domain laccase gene, and said degenerated primer comprises the upstream primer and the downstream primer of following nucleotide sequence:
Upstream primer Cu2SF:5 '-TAC TGG YAC TAC CAC GAC CAY-3 ';
Downstream primer Cu4SR:5 '-RTG VSW CTG SAC RTG RCA GTG-3 ';
Wherein, Y=C/T; R=A/G; V=A/G/C; W=A/T; S=G/C.
The above-mentioned degenerated primer that the present invention proposes is the specificity degenerated primer of universal chain mould two-domain laccase gene, and this degenerated primer can check whether there is streptomycete two-domain laccase gene in pure culture state and the environmental sample specifically.
Above-mentioned degenerated primer of the present invention can be obtained by following method design:
(1) is chosen in the laccase amino acid of the streptomycete two-domain that delivers on the Genbank and the cDNA sequence that correspondence is encoded;
(2) streptomycete two-domain laccase gene aminoacid sequence of having reported according to GenBank and common three-domain aminoacid sequence (comprising bacterium, actinomycetes, basidiomycetes and ascomycetes); Utilize the DNAman biological software that amino acid is carried out homology analysis, manually the result of comparison is as shown in Figure 1; Combine in two districts and four districts at the many copper of streptomycete two-domain laccase amino acid; These two regional aminoacid sequences are more conservative; And bigger with conventional t hree-domain laccase amino acid difference, so the amino acid of degenerated primer designing institute of the present invention reference is YWHYHDH and HCHVQSH (referring to Fig. 1);
(3) in many copper of streptomycete two-domain laccase gene conservative region land (copper binding sites) with three-domain laccase amino acid between the different conservative region; Observe through contriver's TE, test and detection, degenerated primer reference of the present invention be that many copper combine two districts and the conserved amino acid in four districts and the cDNA sequence of corresponding codes; According to aminoacid sequence and the cDNA sequence among the state-run biomolecule information database NCBI of the U.S. of place; The cDNA sequence of the amino acid whose correspondence of degenerated primer designing institute reference of the present invention (different in the corresponding cDNA sequence is that runic indicates) as shown in table 1 below is designed degenerated primer of the present invention according to the preference type of codon at last.
Table 1: the conserved amino acid of the degenerated primer institute reference of the present invention's design and the cDNA sequence of pairing coding
| The primer title | Forward（Cu2SF） | Reverse（Cu4SR） |
| The conserved amino acid of design primer reference | Y W H Y H D H | H? C H V Q ? S ? H |
| The degenerated primer that is designed | 5’-TAC TGG YAC TAC CAC GAC CAY-3’ | 5’-RTG VSW CTG SAC RTG RCA GTG-3’ |
| Streptomyces coelicolor | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG AGC CAC |
| Streptomyces ipomoeae | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CA T GT G CAG AGC CAC |
| Streptomyces ghanaensis | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG AGC CAC |
| Streptomycessp. ACTE | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CA T GT G CAG AGC CAC |
| Streptomyces griseus subsp. | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CA T GT G CAG AGC CAC |
| Streptomyces scabiei | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG AG T CAC |
| Streptomyces virido | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GT G CAG AGC CAC |
| Streptomyces roseosporus | TAC TGG CAC TAC CAC GAC CA T | CAC TGC CA T GTC CAG AGC CAC |
| Streptomyces griseoflavus | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG AGC CAC |
| Streptomyces sviceus | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CA T GT G CAG AGC CAC |
| Streptomyces coelicoflavus | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GT G CAG AGC CAC |
| Streptomyces griseus | TAC TGG CAC TAC CAC GAC CAC | CAC TG T CAC GT GCAG AGC CAC |
| Streptomycessp. W007 | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CA T GTC CAG AGC CA T |
| Streptomyces albus | TAC TGG AAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG AGC CAC |
| Streptomyces sp. SA3 | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG TCG CAC |
| Streptomyces sp. S4 | TAC TGG AAC TAC CAC GAC CAC | CAC TGC CAC GTC CAG AGC CAC |
| Streptomyces violaceusniger | TAC TGG CAC TAC CAC GAC CAC | CAC TGC CA T GTC CAG AGC CAC |
| Streptomyces pristinaespiralis | TAC TGG CAC TAC CAC GAC CA T | CAC TGC CAC GTC CAG AGC CAC |
Annotate: go up writing a Chinese character in simplified form in the table 1: Y=C/T; R=A/G; V=A/G/C; W=A/T; S=G/C.
According to the number (referring to Fig. 1) of two-domain laccase amino acid at many copper combination two districts and amino acid whose quantity in four districts and base pair, we judge that the product sheet segment length that obtains when adopting degenerated primer of the present invention to carry out pcr amplification is the single band of 380bp～430bp.
As a total technical conceive; The present invention also provides the above-mentioned degenerated primer of a kind of usefulness to detect the method that whether contains streptomycete two-domain laccase gene in the testing sample; May further comprise the steps: at first; With the DNA in the testing sample is template (this template can be template according to the genomic dna with pure strain or environmental sample in the ncbi database); Utilize said degenerated primer to carry out pcr amplification; Pcr amplification reaction utilizes sepharose that reacted pcr amplification product is carried out electrophoresis detection after accomplishing, and carries out primary dcreening operation according to the single band that whether contains 380bp～430bp in the electrophoretogram after detecting to whether containing streptomycete two-domain laccase gene in the testing sample; If contain the single band of 380bp～430bp; Then pcr amplification product is reclaimed; Dna fragmentation cloning and sequencing after reclaiming is obtained the dna sequence dna of certain fragment length; Again this dna sequence dna is inserted in the nucleic acid database and compare, and then whether contain streptomycete two-domain laccase gene in definite testing sample.Said testing sample can be sample under the pure culture state or environmental sample.
In the pcr amplification of aforesaid method; The composition of 50 μ L pcr amplification reaction systems preferably includes: 25 μ L, 2 * Taq PCR MasterMix (purchase of sky, Beijing root biotech firm); Concentration is each 1～2.5 μ L of solution of solution and downstream primer of the said upstream primer of 25 μ M; DNA 100n as said template adds no enzyme deionized water and complements to 50 μ L.
In the above-mentioned detection method, the reaction conditions of said pcr amplification reaction is preferably: 94 ℃ of sex change 3～5 min; 94 ℃ of sex change 45～60 s then, 58 ℃～60 ℃ annealing 30～45 s, 72 ℃ are extended 40～60 s, circulate altogether 35 times; Last 72 ℃ are extended 7～10 min, end at 4 ℃.
In the above-mentioned detection method, the concentration of said sepharose is preferably 2.0%.
In the above-mentioned detection method, said cloning and sequencing is meant that preferably the order-checking of picking clone obtains said dna sequence dna with cloning in the dna fragmentation insertion intestinal bacteria T carrier after reclaiming.
Compared with prior art; The invention has the advantages that: degenerated primer of the present invention and corresponding detecting method can judge whether contain this streptomycete two-domain laccase gene in pure strain and the environmental sample fast, accurately, specifically; And can obtain new streptomycete two-domain laccase fragment laccase gene, use for the molecular biology of streptomycete two-domain laccase gene and lay a good foundation.
Description of drawings
Fig. 1 is the relevant aminoacid sequence comparison diagram of design specific amplified streptomycete two-domain laccase gene institute reference among the present invention.In this Fig. 1, the position of amino acid in this aminoacid sequence of the digitized representation numeral back before each cbr, the Site II refers to the design site of forward primer, and the Site IV is the design site of reverse primer, cbr:copper binding sites.
Fig. 2 is the sepharose figure behind the purify DNA in the embodiment of the invention.Wherein, Marker is dna molecular amount mark λ DNA/
HindIII (sky, Beijing root), 1,2,3 represent three parallel compost samples among the embodiment respectively, and 4,5,6 represent three parallel pedotheques among the embodiment respectively, and 7～11 is pure strain (model animals), is respectively
Streptomyces coelicolor(ATCC 23899)
, Streptomyces griseus(ATCC 13273),
Streptomyces ipomoeae(ATCC 25462),
Coriolus versicolor(ATCC 51171) reach
Escherichia coli(ATCC 8739).The size of numeral DNA, unit is bp.
Fig. 3 is the agarose gel electrophoresis photo of the streptomycete two-domain laccase gene product that obtains through pcr amplification in the embodiment of the invention.Wherein, Marker is dna molecular amount mark Marker I (purchase of sky, Beijing root biotech firm); 1,2,3 represent three parallel compost samples among the embodiment respectively, 4,5,6 represent three parallel pedotheques among the embodiment, and 7～11 is pure strain (model animals).The size of numeral DNA, unit is bp.
The comparison figure that relevant laccase cDNA sequence is carried out among the sequence of the base pair that obtains after the PCR product cloning order-checking of Fig. 4 for the pedotheque of degenerated primer amplification in the embodiment of the invention and compost sample and the NCBI and the phylogenetic tree of structure.Wherein, AWC1～AWC10: the laccase gene that compost sample is measured; TR1～TR10: the laccase gene that pedotheque is measured.Gi ︱ 24418971 ︱
Streptomyces coliecolorA3 (2) and gi ︱ 3450007964 ︱
Streptomyces violaceusnigerRefer to corresponding streptomycete two-domain laccase gene in the ncbi database.
Embodiment
Below in conjunction with Figure of description and specific embodiment the present invention is further described.
Embodiment:
A kind of degenerated primer (hereinafter to be referred as " degenerated primer Cu2SF/Cu2SR ") that is used to detect streptomycete two-domain laccase gene of the present invention, this degenerated primer comprises the upstream primer and the downstream primer of following nucleotide sequence:
Upstream primer Cu2SF:5 '-TAC TGG YAC TAC CAC GAC CAY-3 ';
Downstream primer Cu4SR:5 '-RTG VSW CTG SAC RTG RCA GTG-3 ';
Wherein, Y=C/T; R=A/G; V=A/G/C; W=A/T; S=G/C.
Utilize the degenerated primer Cu2SF/Cu2SR of present embodiment to detect the method that whether contains streptomycete two-domain laccase gene in the testing sample, particular embodiment is in following implementation process.
At first carry out the sampling of compost and pedotheque.Compost is taken from the composting device of the experimental size of a 20L; Composting material mainly comprises straw 1.074 kg, dish leaf 0.9 kg, leaf 0.216 kg, wheat bran 0.24 kg and soil 0.721 kg, and sampling point is 5 cm below the compost surface, gets three samples (being numbered 1,2,3 respectively) altogether; Each sample 1g; Be at compost treatment 4d, 10d, 15d sample time.Pedotheque is taken from the forest topsoil on Changsha Yue Lu mountain, is numbered 4,5,6.Available from China Committee for Culture Collection of Microorganisms common micro-organisms center, bacterial classification is respectively the pure culture bacterial classification respectively
Streptomyces coelicolor(ATCC 23899),
Streptomyces griseus(ATCC 13273),
Streptomyces ipomoeae(ATCC 25462),
Coriolus versicolor(ATCC 51171) reach
Escherichia coli(ATCC 8739), numbering is respectively 7～11.
1. the extraction of the total DNA of environmental sample: rotten damping fluid is removed in the consumption interpolation with 8～10 ml/g in above (DNA's to be extracted) compost sample of purchasing and pedotheque, with centrifugal 4～6 min of 5000～6000 * g, removes supernatant again; Repeat the supernatant color of this step after centrifugal and the color no significant difference that removes rotten damping fluid.The above-mentioned sample that takes off after the corruption is added DNA extraction damping fluid and 10 μ L Proteinase Ks (20 μ g/mL) with the consumption of 1.0～1.5 ml/g, and under 37 ℃ of temperature with 150～200 rpm vibration, 30～45 min; Consumption adding sodium dodecyl sulfate solution with 250～400 μ L/g compost gets mixed solution, 65 ℃ of water-bath 1.5～2.0 h then; With centrifugal 4～6 min of mixing solutions 5000～6000 * g after the water-bath, the supernatant mixing solutions is drawn to clean centrifuge tube; Add and the isopyknic chloroform of said mixed solution-primary isoamyl alcohol reagent again, shake up the back, reclaim water with centrifugal 6～8 min of 8000～12000 * g; The Virahol that adds 0.6～0.8 times of water volume toward the aqueous phase that reclaims precipitates 1h in ice-water bath, with centrifugal 10～15 min of 12000～13000 * g speed; Deposition after centrifugal adds ice-cold in advance 70% ethanolic soln with the consumption of 1.5～2.0 ml/g compost and washs; With the centrifugal 1min of 10000～13000 * g speed; Sample after will washing with 200～400 μ l/g consumptions then is dissolved in the TE damping fluid, obtains the dna solution of each environmental sample after slightly carrying.
2. the extraction of pure culture bacterial classification DNA: with the spore lyophilize of pure culture bacterial classification liquid culture gained; After adopting liquid nitrogen grinding that spore is ground to white powder then; Consumption with 1.0～1.5 ml/g adds DNA extraction damping fluid and 10 μ L Proteinase Ks (20 μ g/mL) again; Remaining step is identical with above-mentioned environmental sample total DNA extraction, obtains the dna solution of each pure culture bacterial classification sample after slightly carrying at last.
3. the purifying of DNA: the above-mentioned dna solution of slightly carrying is carried out purifying with common DNA product purification test kit (sky, Beijing root); In purified product, adding final concentration is the ribonuclease A of 0.25～0.5 μ g/mL; Digest 20～25 min in 37 ℃ of water-baths, obtain total dna solution of each sample behind the purifying.
4. with DNA electrophoresis in the sepharose of 1% concentration of each environmental sample behind the above-mentioned purifying and pure culture bacterial classification, dna molecular marker is the λ DNA/ that root Bioisystech Co., Ltd buys from sky, Beijing
HindIII; Used electrophoretic buffer is 1 * TAE electrophoretic buffer, and the volts lost of 200V/cm is electrophoresis 15 min on the horizontal strip electrophoresis groove, and electrophoresis finishes the back gel with 1 * Dured dyeing 10min; The detection of on Gel Doc2000 gel imaging system (Bio-Rad), taking pictures then, the result is as shown in Figure 2.Can be seen that by Fig. 2 the DNA band behind the purifying is single, does not have conditions of streaking, dna fragmentation length is about 23kb.
5. with the dna profiling in each sample solution of said extracted, adopt the degenerated primer Cu2SF/Cu2SR of the present invention's design to carry out pcr amplification.The composition of 50 μ L pcr amplification reaction systems comprises: 25 μ L, 2 * Taq PCR MasterMix (purchase of sky, Beijing root biotech firm); Each 1～2.5 μ L (concentration 25 μ M) of the solution of upstream primer and the solution of downstream primer, template DNA 100ng.The reaction conditions of pcr amplification reaction is: 94 ℃ of sex change 3～5 min; 94 ℃ of sex change 45～60 s then, 58 ℃～60 ℃ annealing 30～45 s, 72 ℃ are extended 40～60 s, circulate altogether 35 times; Last 72 ℃ are extended 7～10 min, end at 4 ℃.
6. the reaction product behind the above-mentioned pcr amplification reaction is detected through 2.0% agarose gel electrophoresis.Dna molecular marker is the Marker I that the biochemical ltd of root buys from sky, Beijing; Used electrophoretic buffer is 1 * TAE electrophoretic buffer; Volts lost electrophoresis 20 min on the horizontal strip electrophoresis groove with 130V/cm; Electrophoresis finishes the back gel with 1 * Dured dyeing 10min, the detection of on Gel Doc2000 gel imaging system (Bio-Rad), taking pictures then, and the result is as shown in Figure 3.Can find out by Fig. 3; The degenerated primer Cu2SF/Cu2SR that the present invention designed from 1～No. 9 sample the DNA band that increases out single; No non-specific amplification; The DNA cloning fragment length is 380～430bp, can tentatively judge thus to contain streptomycete two-domain laccase gene in streptomycete type strain and the environmental sample.In addition, No. 10 basidiomycetes
Coriolus versicolorWith No. 11 bacteriums
Escherichia coliThe all known conventional t hree-domain laccase that contains, but after adopting the degenerated primer Cu2SF/Cu2SR of the present invention's design to carry out pcr amplification, do not have band, can judge the two-domain laccase gene that does not contain the streptomyces class in these two kinds of bacterial strains in view of the above.This shows that the degenerated primer of the present invention's design has specificity and specificity to the detection of the two-domain laccase gene of streptomyces class.
7. dna fragmentation is recombined into plasmid vector: whether what obtain for further checking pcr amplification is the two-domain laccase gene of correct streptomyces class; Amplification is obtained multi-functional recovery test kit specification sheets that correct segmental PCR product buys with reference to the biochemical ltd of sky, Beijing root to be carried out dna fragmentation and reclaims purifying; Product after reclaiming is connected with pGMET (available from Promega) carrier; Transform competent cell behind 25 ℃ of reaction 0.5 h, through blue hickie screening positive clone in intestinal bacteria DH-5 α.Positive colony is resuspended in the 10 μ L sterilized waters processes bacteria suspension.The order-checking of bacterium liquid entrusts Mei Ji biological ltd in Shanghai to accomplish; BlastN among the sequence employing NCBI that records and the sequence in the DB are compared; Whether the streptomycete two-domain laccase gene sequence through in checking and the DB has higher similarity, and whether check contains streptomycete two-domain laccase gene from environmental sample and pure culture sample.Also can the sequence that obtain be translated into amino acid in addition, judge the peculiar amino acid such as many copper land that whether contain laccase in the amino acid judge whether contain streptomycete two-domain laccase gene in the environmental sample.The higher sequence of homology adopts the Neighbor-Joining method to use MEGA 5.0 constructing systems to grow tree among sequence that experiment is measured and the NCBI.The result is as shown in Figure 4, and numerical value is 1000 on the branch.Can know that by Fig. 4 the known streptomycete two-domain laccase gene from compost sample and pedotheque in resulting gene order and the Genbank DB can construct good phylogenetic tree, and the genetic evolution distance is less.
It is following to carry out the agarose gel electrophoresis agents useful for same in the present embodiment:
1 * TAE electrophoretic buffer: 40 mM Tris alkali, 20 mM acetate, 2 mM EDTA;
1.0% (W/V) agarose: 1.0 g agaroses add 100ml 1 * TAE electrophoretic buffer heating for dissolving, cooling then;
2.0% (W/V) agarose: 2.0 g agaroses add 100ml 1 * TAE electrophoretic buffer heating for dissolving, cooling then;
10000 times of 1 * Dured staining fluid: 10000 * Dured dilutions.
< 110>Hunan University
< 120>be used to detect the degenerated primer and the detection method thereof of streptomycete two-domain laccase gene
<160> 2
<210> 1
<211> 21bp
<212> DNA
< 213>artificial sequence
<400>
tactggyact?accacgacca?y 21
<210> 2
<211> 21bp
<212> DNA
< 213>artificial sequence
<400>
rtgvswctgs?acrtgrcagt?g 21
Claims (6)
1. a degenerated primer that is used to detect streptomycete two-domain laccase gene is characterized in that, said degenerated primer comprises the upstream primer and the downstream primer of following nucleotide sequence:
Upstream primer Cu2SF:5 '-TAC TGG YAC TAC CAC GAC CAY-3 ';
Downstream primer Cu4SR:5 '-RTG VSW CTG SAC RTG RCA GTG-3 ';
Wherein, Y=C/T; R=A/G; V=A/G/C; W=A/T; S=G/C.
2. one kind is detected the method that whether contains streptomycete two-domain laccase gene in the testing sample with the described degenerated primer of claim 1; May further comprise the steps: at first; With the DNA in the testing sample is template; Utilize said degenerated primer to carry out pcr amplification; Pcr amplification reaction utilizes sepharose that reacted pcr amplification product is carried out electrophoresis detection after accomplishing, and carries out primary dcreening operation according to the single band that whether contains 380bp～430bp in the electrophoretogram after detecting to whether containing streptomycete two-domain laccase gene in the testing sample; If contain the single band of 380bp～430bp; Then pcr amplification product is reclaimed; Dna fragmentation cloning and sequencing after reclaiming is obtained the dna sequence dna of certain fragment length; Again this dna sequence dna is inserted in the nucleic acid database and compare, and then whether contain streptomycete two-domain laccase gene in definite testing sample.
3. method according to claim 2; It is characterized in that; In the said pcr amplification; The composition of 50 μ L pcr amplification reaction systems comprises: 25 μ L, 2 * Taq PCR MasterMix, concentration is each 1～2.5 μ L of solution of solution and downstream primer of the said upstream primer of 25 μ M, as the DNA 100ng of said template.
4. according to claim 2 or 3 described methods, it is characterized in that the reaction conditions of said pcr amplification reaction is: 94 ℃ of sex change 3～5 min; 94 ℃ of sex change 45～60 s then, 58 ℃～60 ℃ annealing 30～45 s, 72 ℃ are extended 40～60 s, circulate altogether 35 times; Last 72 ℃ are extended 7～10 min, end at 4 ℃.
5. according to claim 2 or 3 described methods, it is characterized in that the concentration of said sepharose is 2.0%.
6. according to claim 2 or 3 described methods, it is characterized in that said cloning and sequencing is meant that the order-checking of picking clone obtains said dna sequence dna with cloning in the dna fragmentation insertion intestinal bacteria T carrier after reclaiming.
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| CN112553354A (en) * | 2020-12-24 | 2021-03-26 | 福安药业集团烟台只楚药业有限公司 | Molecular biological method for rapidly detecting gentamicin high-yield strain |
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| CN103060448A (en) * | 2012-12-28 | 2013-04-24 | 中华人民共和国湖北出入境检验检疫局 | Method for high sensitivity detection of Phytophthora hibernalis Carne |
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| CN112553354A (en) * | 2020-12-24 | 2021-03-26 | 福安药业集团烟台只楚药业有限公司 | Molecular biological method for rapidly detecting gentamicin high-yield strain |
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