CN105925667A - Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene - Google Patents

Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene Download PDF

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CN105925667A
CN105925667A CN201610199719.1A CN201610199719A CN105925667A CN 105925667 A CN105925667 A CN 105925667A CN 201610199719 A CN201610199719 A CN 201610199719A CN 105925667 A CN105925667 A CN 105925667A
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primer
mrna expression
acidithiobacillus thiooxidans
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quantitative pcr
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尹华群
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Central South University
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Abstract

The invention provides specific primers for detection of mRNA expression level of an Acidithiobacillus thiooxidans sor gene. The nucleotide sequences are as follows: an upstream primer sequence of 5'-AAGCCCGTGCCTAAAGTG-3', and a downstream primer sequence of 5'-CTGCCATAGTTGGTGTTGT-3'. The specific primers can detect the transcription level of Acidithiobacillus thiooxidans sor gene, and hace the advantages of high sensitivity and good specificity in detecting mRNA gene expression level. Through the real-time monitoring of mRNA expression level of sor gene in sulfur oxidation process, the primers are used to explain the sulfur oxidation mechanism of Acidithiobacillus thiooxidans.

Description

A kind of detection Acidithiobacillus thiooxidanssorThe specificity of mrna expression Primer
Technical field
The invention belongs to biology field, be specifically related to a kind of detection Acidithiobacillus thiooxidanssorGene mRNA expression The specific primer of level.
Background technology
As a kind of inorganic chemosynthetic autotroph, Gram-negative, shaft-like extreme microorganism, Acidithiobacillus thiooxidans with The Bioleaching of sulphide ore system (colliery, Chalkopyrite, pyrite, vitreous copper etc.) is closely related, and it has in industrial application Prospect widely.Acidithiobacillus thiooxidans can be with elementary sulfur and reduced form inorganic sulfide compound (Reduced Inorganic Sulfur Compounds, is called for short RISCs) as energy substance.About between the research the most never of Acidithiobacillus thiooxidans Disconnected, but its Biochemical processes are then needed further to be supplemented and perfect.Sulfur oxidase in Acidithiobacillus thiooxidans Under the effect of system, elementary sulfur or reduced form inorganic sulfide compound finally produce sulphuric acid through a series of enzyme reaction or non-enzyme reaction Salt and be discharged into outside body, sulfate is most important for the pH in maintenance system.
As the initial enzyme in aerobic sulfur oxidizing process, sulfur oxidoreductase (SOR) be present in some hyperthermophilic archaeon strain and In acidophilic bacteria, its addicted to acid hyperthermophilic archaeon strain such as Tengchong addicted to sour both sexes bacterium (Acidianustengchongensis) and Bu Shi addicted to acid Both sexes bacterium (Acidianus ambivalens) and thermophilic thiobacillus (Acidithiobacilluscaldus) all there is report Road.SOR relies on oxygen molecule catalysis elemental sulfur generation dismutation reaction, it is possible to produce sulfide, sulphite and thiosulfuric acid simultaneously Salt.This enzymatic reaction possesses following characteristics: the enzyme 1. reacting relevant is solubility and is positioned at Cytoplasm；2. react Optimum pH and optimum temperature be respectively 7.0 ± 0.5 (Sulfolobusbrierleyi) and 65-85 ° of C (AcidianustengchongensisAndAcidianusambivalens) ；3. this reaction is not required to by external source cofactor And electron donor.Sulfur oxidoreductase has the ability of catalytic elements sulfur generation dismutation reaction, its resulting sulfides, thiosulfuric acid Salt and sulphite all serve as important mesostate in sulfur oxidative pathway, and therefore this enzyme is at elementary sulfur and reduced form The oxidizing process of inorganic sulfide compound is most important.
Fluorescent quantitative PCR technique is on the basis of regular-PCR (Polymerase Chain Reaction, PCR), knot Close real-time fluorescence detection technique and computer analytical technology.It is by fluorescent dye or fluorescently-labeled specific probe, PCR primer is marked tracking, real time and on line monitoring course of reaction, product can be analyzed in conjunction with corresponding software, Calculate the initial concentration of testing sample template.Its appearance, greatly simplifies the process of detection by quantitative, and is truly realized Absolute quantitation.
Summary of the invention
The invention provides a kind of spy detecting Acidithiobacillus thiooxidans sulfur oxide-reductase gene mrna expression level Specific primer, and use fluorescent quantitative PCR technique, by repeatedly testing and optimizing, we obtain one group can effectively detect addicted to Acid oxidase sulfur ThiobacillussorThe primer of gene mRNA expression amount, and sum up the parameter that a set of optimal PCR reaction uses.
The present invention provides a kind of Acidithiobacillus thiooxidanssorThe fluorescence quantitative PCR detection of mrna expression Specific primer, the nucleotides sequence of described primer is classified as:
Forward primer: 5 '-AAGCCCGTGCCTAAAGTG-3 ',
Downstream primer: 5 '-CTGCCATAGTTGGTGTTGT-3 '.
Accordingly, the present invention provides a kind of Acidithiobacillus thiooxidanssorThe quantitative fluorescent PCR of mrna expression Detection method, comprises the following steps:
(1) extraction of Acidithiobacillus thiooxidans total serum IgE and purification；
(2) RNA extracting step (1) carries out reverse transcription generation cDNA, utilizes the specific primer described in claim 1 to enter Row quantitative fluorescent PCR；
(3) mrna expression level is calculated according to the result of quantitative fluorescent PCR.
Wherein the response procedures of the amplification of the PCR in step (3) is: 95 ° of C degeneration 30 sec；Under 56 ° of C of annealing temperature again Property 15s；72 ° of C extend 25 s, 40 circulations.
Preferably, wherein use glyceraldehyde-3-phosphate dehydrogenase gene as transcribing reference gene；Use Δ Δ Ct side Method calculates the relative expression quantity of gene.
The present invention also provides for a kind of detection kit for above-mentioned fluorescent quantitative PCR detection method, this test kit bag Include specific primer as above.
Specifically, described method more specifically step is as follows:
(1) use centrifugal method to reclaim thalline, then use Trizol reagent to extract RNA, be then used by MicroElute RNA Clean-Up Kit (OMEGA), the total serum IgE of extraction is purified according to explanation, then uses RNase-free DNase I (OMEGA) removes the genomic DNA contained in extract；
(2) concentration of NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) detection RNA is used And purity, parameter is set to OD260 and OD280, uses OMEGA MicroElute RNA Clean-Up Kit (50) pure Change RNA；
(3) utilize First Strand cDNA Synthesis Kit (TOYOBO) that 2 μ g total serum IgE are carried out reverse transcription；
(4) using 1 μ l reverse transcription product cDNA as template, QuantiFast is usedTM SYBR® Green PCR Kit (QIAGEN) configure the system of 25 μ l, then use MyIQ Single Color Real-Time PCR Detection System (BIO-RAD) carries out real-time quantitative PCR.
The present invention uses above-mentioned technical scheme, by monitoring different times Acidithiobacillus thiooxidanssorGene mRNA Expression and be used for explaining sulfur oxidation mechanism, and then as this bacterium, element sulphur or reduced form inorganic sulfide compound can be aoxidized The foundation of speed.
The present invention is with in Acidithiobacillus thiooxidanssorGene is template, designs a series of primer, and by optimizing Condition and repeatedly testing, picks out the primer of a pair high specific.This primer is monitored in mRNA level in-sitesorGene different times Differential expression has more specific aim, it is possible to the expression of gene in reflection sulfur oxidizing process effectively such that it is able to as examining Look into an important reference index of sulfur oxidation rate.
Accompanying drawing explanation
Fig. 1 is the sample amplification curve in embodiment 2；
Fig. 2 is the standard curve in embodiment 2；
Fig. 3 is the sample solubility curve in embodiment 2.
Detailed description of the invention
Embodiment 1, the screening of specific primer pair and acquisition
Sulfur oxidoreductase bysorGene code, this enzyme has the ability of catalytic elements sulfur generation dismutation reaction, at elementary sulfur and The oxidizing process of reduced form inorganic sulfide compound is most important.Sulfur oxidoreductase is present in some hyperthermophilic archaeon strain and acidophilic bacteria In, it is equal addicted to acid both sexes bacterium and acidophilic bacteria such as thermophilic thiobacillus addicted to acid both sexes bacterium and Bu Shi addicted to acid hyperthermophilic archaeon strain such as Tengchong Have been reported that.
By genome sequencing, we obtainAcidithiobacillus thiooxidansThe full-length genome of A01 Sequence information, and in this kind, report the existence of sulfur oxidoreduction enzyme coding gene first.According toA. thiooxidans A01'ssorGene (NCBI accession number is KJ483962), is designed by Primer 5 and organizes primer more, meet design of primers as far as possible Rule, avoid primer dimer, mispairing and hairpin structure the most as far as possible.5 pairs of primers are respectively as follows:
Primer 1 upstream sequence: 5 '-ACCAACTATGGCAGTCAGG-3 '
Primer 1 downstream sequence: 5 '-ATCATGGGTCCAAAGAGC-3 '
Primer 2 upstream sequence: 5 '-AAGCCCGTGCCTAAAGTG-3 '
Primer 2 downstream sequence: 5 '-CTGCCATAGTTGGTGTTGTAC-3 '
Primer 3 upstream sequence: 5 '-AAGCCCGTGCCTAAAGTG-3 '
Primer 3 downstream sequence: 5 '-CTGCCATAGTTGGTGTTGT-3 '
Primer 4 upstream sequence: 5 '-CAACACCAACTATGGCAGTC-3 '
Primer 4 downstream sequence: 5 '-ATCATGGGTCCAAAGAGC-3 '
Primer 5 upstream sequence: 5 '-ACCAACTATGGCAGTCAGG-3 '
Primer 5 downstream sequence: 5 '-CATGGGTCCAAAGAGCAT-3 '
By arranging thermograde (50 ° of C ~ 60 ° C；It is incremented by with 2 for step-length), there is many places light and shade not etc. miscellaneous in primer 4 and 5 Band, can determine whether the poor specificity of this primer pair；And the electrophoretogram of primer 1 and 2 shows, occur dark near the place of purpose band Miscellaneous band, preliminary analysis may be primer dimer.And primer 3 has preferable specificity, and according to the reality under different temperatures Test result, finally determine the annealing temperature (56 ° of C) that primer is optimal.
Embodiment 2, Acidithiobacillus thiooxidans be sulfur oxide-reductase gene mrna expression level under different energy sources
One, culture medium and condition of culture
Bacterial strainAcidithiobacillus thiooxidansA01 selects 9K culture medium and Germany's biomaterial resource respectively Center culture medium 71 (DSMZ medium 71) carries out liquid culture.Wherein, the 9K culture medium containing 10.0 g/L elementary sulfurs is used The H2SO4 of 5 mol/L regulates culture fluid initial pH to 2.0, and contains the DSMZ medium 71 of 5.0 g/L sodium thiosulfate Cultivate keynote pH to 4.4.Antibacterial is cultivated in the conical flask (specification is 250 mL) equipped with 100 mL culture fluid, and its antibacterial is initial Concentration is 2.5 × 106Individual/milliliter, cultivation temperature is 30 DEG C, and shaking table frequency of oscillation is 170 rpm.
Two, the extraction of RNA
Centrifugal method is used to reclaim thalline and in order to extract at mid-log phase (54 h) (three are repeated experiment and average) RNA, then uses RNase-free ddH2O cleans sample twice.Specifically comprise the following steps that
(1) cell cracking.12000 rpm are centrifuged 20 min and collect thalline, add the Trizol reagent of 1 ml, and repeatedly Piping and druming liquid-transfering gun, makes cell fully crack.
(2) for making nucleic acid-protein complex be kept completely separate, by homogenised sample at 15-30 ° of C incubation 5 min.
Under (3) 4 ° of C, 10000 rpm are centrifuged 2 min, collect supernatant.
(4) add the ratio interpolation chloroform of 0.2 ml in every 1 ml Trizol, build centrifuge tube, inhale and make a call to 15 s, room temperature temperature Educate 3 min.
Under (5) 4 ° of C, after 10000 rpm are centrifuged 10-15 min, sample is divided into three layers: bottom is the organic facies of yellow, The colourless aqueous phase in intermediate layer and upper strata, and RNA is mainly in aqueous phase, uses liquid-transfering gun carefully aqueous phase (about 600 μ l) to be shifted In new centrifuge tube.
(6) in the new centrifuge tube added with aqueous phase solution, the isopropanol of equal-volume (600 μ l) is added, through fully mixing After, room temperature places 20-30 min.
(7) 4 ° of C, 10000 rpm are centrifuged 10 min, abandon supernatant.
Three, the purification of RNA
(1) RNA sample adds DEPC-Water to cumulative volume is 100 μ l.
(2) 350 μ l QVL Lysis buffer (every 1ml adds 20 μ l beta-mercaptoethanols) are added, another addition 2 μ l Linear Acrylamiole, vortex mixes.
(3) 250 μ l dehydrated alcohol are added in sample mix liquid, mix homogeneously.
(4) shift 700 μ l mixed liquors in the RNeasy purification column overlapping 2 ml collecting pipes, 12,000 rpm from The heart 1 min, abandons collection liquid.
(5) 300 μ l RWB Buffer (diluting) are added in pillar, 10,000 rpm, centrifugal 1 min, abandon collection Liquid.
(6) take another new centrifuge tube, add 73.5 μ l DNase I Digestion Buffer and 1.5 μ l RNase-free DNase I, fully mixes.
(7) the 75 μ l DNase I digestion mixed liquors being made into are transferred to RNeasy purification column pellosil middle, room Temperature (25-30 ° of C) places 15 min.
(8) adding 400 μ l RWB Wash buffer (diluting) in RNeasy purification column, room temperature places 5 min, and 10, 000 rpm is centrifuged 1 min, abandons collection liquid.
(9) adding 500 μ l RWB Buffer (diluting) in RNeasy purification column, 10,000 rpm are centrifuged 1 min, Abandon collection liquid.
(10) step 9 is repeated.
(11) void column 13,000 rpm is centrifuged 2 min skies and dries dry column matrix.
(12) by posts transfer to new 1.5ml centrifuge tube, add 30 μ l DEPC-treated Water and arrive In RNeasy purification column substrate, standing 2 min, 10,000rpm are centrifuged 1 min.
Four, the synthesis of reverse transcription cDNA
(1) configuration reaction system: 11 μ l RNA samples and 1 μ l random primer.
(2) after mixing reaction system gently, it is handled as follows: 65 DEG C of heating 5 min, is placed in 2-3 min on ice, fully After cooling, of short duration low-speed centrifugal.
(3) in reaction system, add 12 μ l degeneration RNA, 4 μ l 5 × RT buffer, 2 μ l dNTP more successively Mixture, 1 μ l RNase Inhibitor and 1 μ l ReverTra Ace.
(4) suction is beaten uniformly the most up and down.
(5) sample processes the most under the following conditions: 30 ° of C, 10 min；42 ° of C, 20 min；99 ° of C, 5 min；4° C, 5 min.
(6) use NanoDrop micro-spectrophotometer detection cDNA mass, then diluted sample is become 200 ng/ μ L, re-uses Real-time PCR and carries out detecting in case stand-by.
Five, real-time quantitative RT-PCR
The parameter of PCR is set to: 95 ° of C, 30 s；56 ° of C, 15s；72 ° of C, 25 s, period is 40.Additionally, glyceraldehyde-3-phosphorus Dehydrogenase gene (gapdh) as transcribing reference gene, and use Δ Δ Ct method to calculate the relative expression quantity of gene, its Multiple change employing 2-ΔΔCtRepresent.
Experimental result refers to Fig. 1 to Fig. 3, Fig. 1 and shows sample amplification curve, and Fig. 2 shows that standard curve, Fig. 3 show sample Product solubility curve.Wherein, sample amplification curve (Fig. 1) shows, the Ct value of sample amplification is 26, and amplification reaches after 38 circulations To plateau.Real-time PCR standard curve (Fig. 2) shows, the kinetics equation correlation coefficient of amplification is 0.999, and PCR expands Increasing Efficiency is 82.1%.Usually, the scope of amplification efficiency is 80%～120%, so the normal equation of this standard curve is preferable 's.This sample solubility curve (Fig. 3) is single absworption peak, generates without nonspecific products and primer dimer etc., thus shows The specificity of this primer is good, it is possible to for subsequent experimental, and Real-time PCR result can to accurately reflect sample the denseest Degree.

Claims (5)

1. an Acidithiobacillus thiooxidanssorThe specific primer of the fluorescence quantitative PCR detection of mrna expression, institute The nucleotides sequence stating primer is classified as:
Forward primer: 5 '-AAGCCCGTGCCTAAAGTG-3 ',
Downstream primer: 5 '-CTGCCATAGTTGGTGTTGT-3 '.
2. an Acidithiobacillus thiooxidanssorThe fluorescent quantitative PCR detection method of mrna expression, including following step Rapid:
The extraction of Acidithiobacillus thiooxidans total serum IgE and purification；
The RNA extracting step (1) carries out reverse transcription and generates cDNA, utilizes the specific primer described in claim 1 to carry out glimmering Fluorescent Quantitative PCR；
Result according to quantitative fluorescent PCR calculates mrna expression level.
Fluorescent quantitative PCR detection method the most according to claim 2, the wherein response procedures of the amplification of the PCR in step (3) For: 95 ° of C degeneration 30 sec；Renaturation 15s under 56 ° of C of annealing temperature；72 ° of C extend 25 s, 40 circulations.
4., according to the fluorescent quantitative PCR detection method described in Claims 2 or 3, wherein use glyceraldehyde-3-phosphate dehydrogenase base Because of as transcribing reference gene；Δ Δ Ct method is used to calculate the relative expression quantity of gene.
5., for a detection kit for the fluorescent quantitative PCR detection method as described in any one of claim 2 to 4, it is special Levy and be to include specific primer as claimed in claim 1.
CN201610199719.1A 2016-04-01 2016-04-01 Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene Pending CN105925667A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108728557A (en) * 2018-05-28 2018-11-02 深圳大学 Primer pair and design method for detecting thiobacillus ferrooxidans and detection method

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033490A (en) * 2007-04-17 2007-09-12 中南大学 Genome chip for analyzing microbial community structure in acidic environment

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN101033490A (en) * 2007-04-17 2007-09-12 中南大学 Genome chip for analyzing microbial community structure in acidic environment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HUA QUN YIN等: "《Whole-genome sequencing reveals novel insights into sulfur oxidation in the extremophile Acidithiobacillus thiooxidans》", 《BMC MICROBIOLOGY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108728557A (en) * 2018-05-28 2018-11-02 深圳大学 Primer pair and design method for detecting thiobacillus ferrooxidans and detection method
CN108728557B (en) * 2018-05-28 2022-06-07 深圳大学 Primer pair for detecting thiobacillus acidocaldarius, design method and detection method

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Application publication date: 20160907