CN103627815B - Method for rapidly detecting industrial saccharomyces pastorianus ester metabolism genes - Google Patents
Method for rapidly detecting industrial saccharomyces pastorianus ester metabolism genes Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
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- 230000003321 amplification Effects 0.000 description 4
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- 102100023033 Cyclic AMP-dependent transcription factor ATF-2 Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
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- 101150040087 EEB1 gene Proteins 0.000 description 2
- 101150105495 EHT1 gene Proteins 0.000 description 2
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- 241000235072 Saccharomyces bayanus Species 0.000 description 2
- 101100236714 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) EEB1 gene Proteins 0.000 description 2
- 101100236715 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) EHT1 gene Proteins 0.000 description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 2
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- 239000011324 bead Substances 0.000 description 2
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- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 2
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 description 2
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- 229940117955 isoamyl acetate Drugs 0.000 description 2
- 235000015095 lager Nutrition 0.000 description 2
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- 108010080691 Alcohol O-acetyltransferase Proteins 0.000 description 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 101000974934 Homo sapiens Cyclic AMP-dependent transcription factor ATF-2 Proteins 0.000 description 1
- 101000997829 Homo sapiens Glial cell line-derived neurotrophic factor Proteins 0.000 description 1
- 101001019605 Homo sapiens Isoamyl acetate-hydrolyzing esterase 1 homolog Proteins 0.000 description 1
- 101150087107 IAH1 gene Proteins 0.000 description 1
- 102100035008 Isoamyl acetate-hydrolyzing esterase 1 homolog Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 241000582914 Saccharomyces uvarum Species 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 235000013361 beverage Nutrition 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
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- 210000004027 cell Anatomy 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
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Abstract
The invention relates to biotechnology, and particularly relates to a method for rapidly detecting industrial saccharomyces pastorianus ester metabolism genes. The method comprises the following steps: extracting total RNA (Ribose Nucleic Acid), preparing a cDNA (complementary DNA) template by virtue of reverse transcription reaction, performing multiplex-PCR ((Polymerase Chain Reaction), performing capillary electrophoresis, and analyzing product segments, wherein the cDNA template is prepared by virtue of reverse transcription reaction by aiming at industrial saccharomyces pastorianus ester type metabolism-related genes ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2-Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc and IAH1-Sb. Compared with the conventional gene expressing quantitative analysis technology (fluorogenic quantitative PCR), the GeXP (multiplex-Gene Expression) quantitative analysis technology adopted in the method has the characteristics of stronger specificity, stronger sensitiveness, high automatic degree and the like, the reliability and repeatability of results are ensured, and the experiment time is shortened greatly. The method is in favor of controlling the metabolism and content of esters during fermentation under different production conditions and enhancing the typicality and consistency of beer flavor; besides, product characteristics are enabled to be remarkable and new products can be developed by means of rapidly adjusting the flavor of esters in beer.
Description
Technical field
The present invention relates to biotechnology, particularly a kind of method of rapid detection industry saccharomyces pastorianus ester class metabolic gene.
Background technology
Beer is a kind of low ethanol content of being loved by the people and nutritious beverage.Current global Beer is the beer of Lager type more than 90%, and the Saccharomyces uvarum (being commonly called as " Lager yeast " or " saccharomyces pastorianus ") therefore for brew Lager beer is widely used in brewing industry most.Lager yeast is also different from 1996 carry out genome sequencing yeast saccharomyces cerevisiae (S.cerevisiae) as model animals, but a kind of allopolyploid bacterial classification with highly coloured body ploidy, chromosome structure composition complexity, except carrying a part of genes of brewing yeast, also carry the genetic information of bayesian yeast (S.bayanus), be named as saccharomyces pastorianus (Saccharomyces pastorianus).Therefore all Sc-(S.cerevisiae is existed for the most gene in saccharomyces pastorianus) and Sb-(S.bayanus) two homologous genes.But, compared with the yeast saccharomyces cerevisiae entirely checked order, for gene and the expression of described gene in Process of Beer Brewing of bottom yeast specific participation brewing character, very limited at present.
In the polycomponent flavor systems of beer, ester class is the essential substance forming the overall special favor of beer, plays critical effect to the quality of beer and the formation of local flavor typicalness.Appropriate ester class can make beer flavor coordinate, mellow in taste.Ester class about containing more than 100 kinds in beer, the main Ester wherein affecting beer flavor has ethyl acetate, Isoamyl Acetate FCC, ethyl hexanoate etc.Ester class too high levels gives the uncomfortable bitter taste of beer and fruity, too low, makes beer boring, unfavorable to the Harmony of beer flavor.Ester class trace is present in beer, the existence of different ester class can produce synergy, why this explains that ester class content is when lower than the local flavor that still can affect beer when threshold level, and small change in concentration then can produce important impact to beer flavor.Ester class is produced by cereuisiae fermentum metabolism in beer fermentation process.Yeast cell participates in the yeast gene expression of relevant flavour substances synthesis by transcriptional control impact, finally causes the change of ester class content.Therefore, to the research that the key gene participating in the synthesis of ester class in industrial saccharomyces pastorianus fermenting process is expressed, be the key of Reasonable Regulation And Control ester class content.
The biosynthesizing of ester class is by enzymatic polyreaction between acyl-CoA and higher alcohols.Ester class in beer mainly contains two kinds, acetic ester and fatty-acid ethyl ester (FAEEs).Ester class in beer is based on acetate esters, and wherein ethyl acetate and Isoamyl Acetate FCC sum account for more than 80% of beer total ester content.The biosynthesizing of acetic ester is produced by ethanol acetyltransferase (AATase) catalysis, and AATases is the key enzyme in ester class building-up process.Gas chromatographic analysis result shows, the output of acetic ester is obviously subject to the impact of ATF1 and ATF2 gene expression amount in yeast.In fermenting process, FAEEs synthesizes under the katalysis of the acyl-CoA/ethanol O-acyltransferase (AEATases) of EHT1 and EEB1 coding.Condensation reaction between this enzyme catalysis acyl-CoA and ethanol, thus form Medium chain fatty acetoacetic ester.The change of EHT1 and EEB1 gene expression amount provides more information for the regulation and control of Medium chain fatty acetoacetic ester.In addition, research shows to there is a balance between esterase in ester class synthetic enzyme Sum decomposition ester class is as Iah1p, and the net velocity that this balance is accumulated for ester class is also most important.Therefore, ATF1, ATF2, EEB1, EHT1 and IAH1 gene is considered to the key gene in ester class metabolic process, and the level detecting ATF1, ATF2, EEB1, EHT1 and IAH1 genetic expression may be used for studying industrial saccharomyces pastorianus ester class metabolic regulation mechanism.
At present, fluorescence quantifying PCR method is the method for now conventional quantification of mrna.But due to the limitation that the method flux is not high, therefore need one fast and accurately technology saccharomyces pastorianus multiple ester class Metabolism-Related Genes Expression is detected simultaneously.Beckman Coulter company of the GenomeLab GeXP Genetic Analysis System(U.S.) be the technology platform being mainly used in studying multi-gene expression quantitative analysis, adopt the method for capillary electrophoresis and fluorescent mark amplified production, the gene expression abundance of nearly 30 genes can be detected simultaneously.Multi-gene expression regulation and control, medicine virulence Mechanism Study, early diagnosis of cancer research etc. are widely used at present.
Summary of the invention
The object of the invention is the method that open one adopts GeXP multiple gene expression quantitative analysis tech to detect industrial saccharomyces pastorianus ester class metabolism related gene (ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2-Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb) to express, and then be conducive under different working condition, control the metabolism of ester class and content in fermenting process, improve local flavor typicalness and the consistence of beer; And by ester class local flavor in rapid adjustment beer, R&D costs can be saved, accelerate the exploitation of product innovation.
Technical scheme of the present invention is:
A kind of method of rapid detection industry saccharomyces pastorianus ester class metabolic gene, comprise the extraction of total serum IgE, reverse transcription reaction prepares cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, for industrial saccharomyces pastorianus ester class metabolism related gene ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2-Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb, it is SEQ ID NO.2 that reverse transcription reaction prepares cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, primer SEQ ID NO.1 is applied in multi-PRC reaction, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19.
On the basis of above scheme, also comprise industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, the ACT1-Sb as internal reference gene, it is SEQ ID NO.22, SEQIDNO.24 that reverse transcription reaction prepares cDNA template application primer; Primer SEQ ID NO.21, SEQ ID NO.23 is applied in multi-PRC reaction.
Concrete detecting step is as follows:
(1) use following multi-primers to carry out the detection of industrial saccharomyces pastorianus ester class metabolism key gene:
Design meets GeXP multi-PRC reaction feature, detects the specificity upstream and downstream primer of industrial saccharomyces pastorianus ester class Metabolism-Related Genes Expression: and as the specificity upstream and downstream primer (seeing the following form) of internal reference gene:
The specificity upstream and downstream primer of table 1 industrial saccharomyces pastorianus ester class metabolism related gene and internal reference gene
Wherein every bar primer is mixed with the storage liquid of 100 μMs respectively, and upstream primer working fluid is 200nM;
(2) Total RNAs extraction and purifying: adopt general RNA extraction method and purification process, extracts the yeast total serum IgE obtaining purifying from yeast cell;
(3) reverse transcription reaction prepares cDNA template: with yeast total serum IgE for templated synthesis cDNA first chain, above-mentioned (1) in the downstream primer of multi-primers be Auele Specific Primer, reaction system is 20 μ L; Wherein NTC and RT
-for negative control.
Table 2 reverse transcription reaction prepares the reaction conditions of cDNA template
The reverse primer concentration of each gene of table 3
It is as follows that reverse transcription reaction prepares cDNA template reaction optimum configurations:
48℃ 1minutes；42℃60minutes；95℃ 5minutes。
(4) multiplex PCR: with cDNA first chain of above-mentioned synthesis for template, above-mentioned (1) in the upstream primer of multi-primers be Auele Specific Primer, carry out multiplexed PCR amplification reaction, 3 parallel pipes established by each sample, and archaeal dna polymerase is purchased from Beckman Coulter;
The reaction conditions of table 4 multiplex PCR
| Composition | Volume (μ L) |
| 25mM MgCl 2 | 4.0μL |
| 5 × PCR damping fluid | 4.0μL |
| Archaeal dna polymerase | 0.7μL |
| Forward primer (200nM) | 2μL |
| CDNA first chain | 9.3μL |
Pcr amplification optimum configurations is as follows:
95 DEG C of denaturation 10minutes; 94 DEG C of sex change 30seconds; Annealing temperature 56 DEG C, 30seconds; 71 DEG C extend 1minute, circulate 35 times.
(5) multiple PCR products capillary electrophoresis: get 1 μ lPCR multiple products and be added to and point be equipped with in the hole of 95% deionized formamide (SLS) of 39 μ l and the upper model of 400bp Marker mixed solution, cover a dropstone wax oil with after liquid-transfering gun mixing.The dissociating buffer of 250 μ l is added in addition in the every hole of damping fluid plate.After all finishing, upper machine carries out capillary electrophoresis.Separation gel, dissociating buffer are purchased from Beckman Coulter;
(6) product sheet piecewise analysis: utilize GeXP system parameter to analyze capillary electrophoresis result, record result.
The invention has the beneficial effects as follows:
(1) adopt detection method, the level of ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2--Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb genetic expression can be detected sensitive, special, exactly.The change of ester class metabolism key gene expression amount, has reacted the generation situation of yeast ester class, therefore may be used for studying industrial saccharomyces pastorianus ester class metabolic regulation mechanism.
(2) the GeXP multiple gene expression quantitative analysis tech that the present invention adopts has stronger specificity and susceptibility, level of automation high compared with conventional Quantitative analysis of gene expression technology (quantitative fluorescent PCR), ensure that reliability and the repeatability of result, substantially reduce experimental period simultaneously.
(3) the present invention is conducive under different working condition, controls the metabolism of ester class and content in fermenting process, improves typical local flavor and the consistence of beer; And can by ester class local flavor in rapid adjustment beer, outstanding selling point and carry out the exploitation of product innovation.
Accompanying drawing explanation
Accompanying drawing 1 is ATF1-Sc gene and KAN
rthe amplified production of gene;
Accompanying drawing 2 is ATF1-Sb gene and KAN
rthe amplified production of gene;
Accompanying drawing 3 is ATF2-Sc gene and KAN
rthe amplified production of gene;
Accompanying drawing 4 is ATF2-Sb gene and KAN
rthe amplified production of gene;
Accompanying drawing 5 is EEB1-Sc gene and KAN
rthe amplified production of gene;
Accompanying drawing 6 is EEB1-Sb gene and KAN
rthe amplified production of gene;
Accompanying drawing 7 is EHT1-Sc gene and KAN
rthe amplified production of gene;
Accompanying drawing 8 is EHT1-Sb gene and KAN
rthe amplified production of gene;
Accompanying drawing 9 is IAH1-Sc gene and KAN
rthe amplified production of gene;
Accompanying drawing 10 is IAH1-Sb gene and KAN
rthe amplified production of gene;
Accompanying drawing 11 is ACT1-Sc gene and KAN
rthe amplified production of gene;
Accompanying drawing 12 is ACT1-Sb gene and KAN
rthe amplified production of gene;
Accompanying drawing 13 is ester class genes involved and KAN
rthe multiplex amplification product of gene;
Accompanying drawing 14 is the change of ester class genes involved expression amount during the fermentation;
Embodiment
The specific embodiment of the present invention is as follows:
Embodiment 1
A kind of method of rapid detection industry saccharomyces pastorianus ester class metabolic gene, comprise the extraction of total serum IgE, reverse transcription reaction prepares cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, for industrial saccharomyces pastorianus ester class metabolism related gene ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2--Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb, it is SEQ ID NO.2 that reverse transcription reaction prepares cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, primer SEQ ID NO.1 is applied in multi-PRC reaction, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19.Also comprise industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, the ACT1-Sb as internal reference gene, it is SEQ ID NO.22, SEQ ID NO.24 that reverse transcription reaction prepares cDNA template application primer; Primer SEQ ID NO.21, SEQ ID NO.23 is applied in multi-PRC reaction.
According to ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2--Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb gene and reference gene ACT1-Sc, the full length sequence of ACT1-Sb, use Primer Premier5 software design to be applicable to the Auele Specific Primer of GeXP detection, primer sequence is as follows:
Design meets GeXP multi-PRC reaction feature, detects the specificity upstream and downstream primer of industrial saccharomyces pastorianus ester class Metabolism-Related Genes Expression: and as the specificity upstream and downstream primer (seeing the following form 1) of internal reference gene:
The specificity upstream and downstream primer of table 1 industrial saccharomyces pastorianus ester class metabolism related gene and internal reference gene
Product length after single primer capillary electrophoresis result display amplification is consistent with expected product length, and is single band, and result as shown in figs. 1-12.Illustrate that the primer of design has very strong specificity, be suitable for GeXP and detect.
2000L pilot scale fermentation:
Carry out 13 degree of wort fermentation tests of 2000L scale.
Canful yeast number 22.0 × 10
6individual/ml, canful temperature 8.5 DEG C, canful 24 hours deslagginves once, are warming up to 9.5 DEG C of fermentations naturally.When pol is down to 5.2 ° of BX, be warming up to 12 DEG C, carry out di-acetyl reduction.When pol is down to 3.8 ° of BX, sealed cans pressurize 0.1Mpa, deslagging once.When di-acetyl is down to 0.05mg/L, be cooled to 5 DEG C, after 24 hours, thoroughly drain the yeast at the bottom of clean tank and bits, then be cooled to 0 DEG C, during storage wine, arrange a yeast every three days.Get yeast cell sample in different time sections, extract RNA rapidly.
1, the extraction and purification (RiboPure of total serum IgE
tM-YeastKit, Ambion, production code member AM1926):
(1) get 750 μ L zirconium oxide beads in 1.5mL centrifuge tube (band screw-cap), about 2.5cm eminence;
(2) in yeast collection tube, be sequentially added into lysate (480 μ LLysisBuffer+48 μ L10%SDS+480 μ L Phenol:Cloroform:IAA), resuspended.Vortex oscillation 10-15s;
(3) mixed solution is transferred in ready 750 μ L zirconia bead centrifuge tubes, cover tightly;
(4) centrifuge tube is placed in turbula shaker concussion 10min(maximum speed of revolution), lysing cell;
(5) the centrifugal 5min of 16000g room temperature, by upper water phase transition in 10mL centrifuge tube;
(6) add 1.9mL Binding Buffer, mixing;
(7) add 1.25mL100% ethanol, mixing;
(8) get 700 μ L mixed solutions in Filter Cartridge+Collection Tube, the centrifugal 1min of 12000g, abandons collection liquid; Repeat this step, all shift to above-mentioned mixed solution;
(9) on Filter Cartridge, add 700 μ LWash Solution1, the centrifugal 1min of 12000g, abandons collection liquid;
(10) add 500 μ L Wash Solution2/3, the centrifugal 1min of 12000g, abandons collection liquid.Repeat this step;
(11) the centrifugal 1min of 12000g, thoroughly removes liquid on film, is transferred to by Filter Cartridge in new Collection Tube;
(12) add the Elution Solution25-50 μ L of 95-100 DEG C of preheating to film central authorities, the centrifugal 1min of 12000g.Add 25-50 μ L Elution Solution again to film central authorities, the centrifugal 1min of 12000g, namely obtain RNA and collect liquid;
(13) DNase I process
50-100 μ L RNA Sample+1/10th vol 10 × DNase I Buffer+4 μ L DNase I, 37 DEG C of reaction 30min.Add 1/10th vol DNase Inactivation Reagent(and use front first vortex), mixing after vortex concussion, after room temperature places 5min, the centrifugal 2-3min of 12000g, DNase Inactivation Reagent is precipitated, RNA supernatant liquor is transferred in new pipe and preserves, the total serum IgE of purifying can be obtained.
2, reverse transcription reaction prepares cDNA template:
With the yeast cell RNA of purifying described above for template, use the GenomeLab of Beckman Coulter company
tMgeXP starts test kit, and the downstream primer of the multi-primers in above-mentioned table 1 is Auele Specific Primer, and with the total serum IgE of yeast cell for templated synthesis cDNA first chain, reaction system is 20 μ L.
GenomeLab
tMgeXP starts test kit (production code member: PNA85017), containing reverse transcription reaction damping fluid, ThermoScript II, PCR reaction buffer, KAN
rrNA, the water without DNA enzymatic/RNA enzyme, DNA Marker (DSS-400), mineral oil, GeXP sample-loading buffer; Archaeal dna polymerase (production code member: PNA85022); Separation gel (production code member: PNA608010); Dissociating buffer (production code member: PNA608012), all purchased from Beckman Coulter.
CDNA first chain synthesis reaction system arranges as follows, wherein NTC and RT
-for negative control:
Table 2 reverse transcription reaction prepares the reaction conditions of cDNA template
| Often pipe composition | NTC | RT - | Standard reaction |
| Without DNA enzymatic/RNA enzyme water | 8μL | 4μL | 3μL |
| 5 × reverse transcription buffer | 4μL | 4μL | 4μL |
| KAN rRNA (1:50 dilution) | 5μL | 5μL | 5μL |
| ThermoScript II | 1μL | 0 | 1μL |
| Downstream primer (according to concentration in table 2) | 2μL | 2μL | 2μL |
| RNA template (5-20ng/ μ L) | 0 | 5μL | 5μL |
The reverse primer concentration of each gene of table 3
| Gene | Product size | Reverse primer concentration (nM) |
| ATF1-Sc | 363 | 500 |
| ATF1-Sb | 259 | 500 |
| ATF2-Sc | 353 | 1500 |
| ATF2-Sb | 249 | 500 |
| EEB1-Sc | 273 | 62.5 |
| EEB1-Sb | 203 | 7.81 |
| EHT1-Sc | 383 | 1500 |
| EHT1-Sb | 194 | 500 |
| IAH1-Sc | 212 | 500 |
| IAH1-Sb | 159 | 125 |
| ACT1-sc | 176 | 7.8125 |
| ACT1-sb | 135 | 0.5 |
| KAN r | 325 | 50 |
CDNA first chain synthesis reaction optimum configurations is as follows:
48℃ 1minutes；
42℃ 60minutes；
95℃ 5minutes。
3、RT-PCR：
RT-PCR adopts archaeal dna polymerase and the GenomeLab of Beckman Coulter company
tMgeXP starts test kit and carries out.With cDNA first chain of synthesis in above-mentioned 2 for template, the upstream primer of the multi-primers in above-mentioned table 1 is Auele Specific Primer, carries out RT-PCR amplified reaction, and 3 parallel pipes established by each sample.
RT-PCR amplification reaction system arranges as follows:
The reaction conditions of table 4 multiplex PCR
| Composition | Volume (μ L) |
| 25mM MgCl 2 | 4.0μL |
| 5 × PCR damping fluid | 4.0μL |
| Archaeal dna polymerase | 0.7μL |
| Forward primer (200nM) | 2μL |
| CDNA first chain | 9.3μL |
RT-PCR Amplification arranges as follows:
95 DEG C of denaturation 10minutes; 94 DEG C of sex change 30seconds; Annealing temperature 56 DEG C, 30seconds; 71 DEG C extend 1minute, circulate 35 times.
4, multiple PCR products capillary electrophoresis: get 1 μ lPCR multiple products and be added to and point be equipped with in the hole of 95% deionized formamide (SLS) of 39 μ l and the upper model of 400bp Marker mixed solution, cover a dropstone wax oil with after liquid-transfering gun mixing.The dissociating buffer of 250 μ l is added in addition in the every hole of damping fluid plate.After all finishing, upper machine carries out capillary electrophoresis.Separation gel, dissociating buffer are purchased from Beckman Coulter.
5, product sheet piecewise analysis: utilize GeXP system parameter to analyze capillary electrophoresis result, record result, is shown in Figure 13.
Figure 14 is the expression change during the fermentation of industrial saccharomyces pastorianus ester class metabolism key gene.ATF1-Sc does not express all the time in whole fermenting process as seen from the figure, and IAH1-Sc is only in fermentation initial stage trace expression, and ATF2-Sc had the expression of trace before fermentation initial stage and fermentation ends.The expression amount of ATF2-Sb and EEB1-Sc gene is during the fermentation in increase trend, but the expression amount of EEB1-Sc is higher than the expression amount of ATF2-Sb.The expression of ATF1-Sb, EEB1-Sb, EHT1-Sc and EHT1-Sb gene is in rising trend in early stage, and after 110h, expression amount all starts to reduce.The expression amount of IAH1-Sb gene increases gradually with fermentation, starts to reduce after 86h.
According to method provided by the invention, industrial saccharomyces pastorianus ester class metabolism key gene ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2--Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb expression level can be detected fast, for studying industrial saccharomyces pastorianus ester class metabolic regulation mechanism simultaneously.The present invention is conducive under different working condition, controls the metabolism of ester class and content in fermenting process, improves typical local flavor and the consistence of beer; And can by ester class local flavor in rapid adjustment beer, outstanding selling point and carry out the exploitation of product innovation.
Sequence table
The method of <120> rapid detection industry saccharomyces pastorianus ester class metabolic gene
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 36
<212> primer
<213> ATF1-sc
<400>1
aggtgacact atagaataat tattatcgca gttccg 36
<210> 2
<211> 36
<212> primer
<213> ATF1-sc
<400>2
gtacgactca ctatagggag tggatcgaag accgac 36
<210> 3
<211> 36
<212> primer
<213> ATF1-sb
<400> 3
aggtgacact atagaatatg aagttcgagt ggctca 36
<210> 4
<211> 37
<212> primer
<213> ATF1-sb
<400>4
gtacgactca ctatagggat cttgtctttt atcgccc 37
<210> 5
<211> 36
<212> primer
<213> ATF2-sc
<400>5
aggtgacact atagaataac gaaccacata tcacgc 36
<210>6
<211> 39
<212> primer
<213> ATF2-sc
<400>6
gtacgactca ctatagggaa attcaagatg agaaatcac 39
<210> 7
<211> 35
<212> primer
<213> ATF2-sb
<400>7
aggtgacact atagaatatc gctgtttatg gggaa 35
<210> 8
<211> 39
<212> primer
<213> ATF2-sb
<400>8
gtacgactca ctatagggac tccagttcta ggtgaggta 39
<210> 9
<211> 36
<212> primer
<213> EEB1-sc
<400>9
aggtgacact atagaatact gggagagaaa ggaacc 36
<210> 10
<211> 36
<212> primer
<213> EEB1-sc
<400>10
gtacgactca ctatagggac agacccctcc atcaga 36
<210> 11
<211> 35
<212> primer
<213> EEB1-sb
<400>11
aggtgacact atagaataga gaagggcact aaggc 35
<210> 12
<211> 40
<212> primer
<213> EEB1-sb
<400>12
gtacgactca ctatagggag atctccgctc aataagtata 40
<210> 13
<211> 38
<212> primer
<213> EHT1-sc
<400>13
aggtgacact atagaatagc taacaaatat gtcccaac 38
<210> 14
<211> 37
<212> primer
<213> EHT1-sc
<400>14
gtacgactca ctatagggac ctcccgtagt tcttcca 37
<210> 15
<211> 38
<212> primer
<213> EHT1-sb
<400>15
aggtgacact atagaatacg ttatcttcag aacactcg 38
<210> 16
<211> 38
<212> primer
<213> EHT1-sb
<400>16
gtacgactca ctatagggat cttcaaactc aacttcgc 38
<210> 17
<211> 40
<212> primer
<213> IAH1-sc
<400>17
aggtgacact atagaatata tctttgatga agtcttacca 40
<210> 18
<211> 37
<212> primer
<213> IAH1-sc
<400>18
gtacgactca ctatagggaa cattttcctc attggct 37
<210> 19
<211> 38
<212> primer
<213> IAH1-sb
<400> 19
aggtgacact atagaatatt acagtctatt ctgatgcg 38
<210> 20
<211> 37
<212> primer
<213> IAH1-sb
<400>20
gtacgactca ctatagggat ccatccgtta gtaggct 37
<210> 22
<211> 36
<212> primer
<213> ACT1-Sc
<400>22
aggtgacact atagaatata acgttccagc cttcta 36
<210>23
<211> 39
<212> primer
<213> ACT1-Sc
<400>23
gtacgactca ctatagggag tgaggtagag agaaaccag 39
<210> 23
<211> 37
<212> primer
<213> ACT1-Sb
<400>23
aggtgacact atagaatagt atcttgacat tgcgtta 37
<210> 24
<211> 40
<212> primer
<213> ACT1-Sb
<400>24
gtacgactca ctatagggat ctcaactcgt tgtaaaaggt 40
Claims (4)
1. the method for a rapid detection industry saccharomyces pastorianus ester class metabolic gene, comprise the extraction of total serum IgE, reverse transcription reaction prepares cDNA template, multi-PRC reaction, capillary electrophoresis, product sheet piecewise analysis, it is characterized in that for industrial saccharomyces pastorianus ester class metabolism related gene ATF1-Sc, ATF1-Sb, ATF2-Sc, ATF2-Sb, EEB1-Sc, EEB1-Sb, EHT1-Sc, EHT1-Sb, IAH1-Sc, IAH1-Sb, it is 500nM SEQ ID NO.2 that reverse transcription reaction prepares cDNA template application primer, 500nM SEQ ID NO.4, 1.5 μMs of SEQ ID NO.6, 500nM SEQ ID NO.8, 62.5nM SEQ ID NO.10, 7.81nM SEQ ID NO.12, 1.5 μMs of SEQ ID NO.14, 500nM SEQ ID NO.16, 500nM SEQ ID NO.18 and 125nM SEQ ID NO.20 synthesizes cDNA first chain, in multi-PRC reaction with described cDNA first chain for template, application primer be SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17 and SEQ ID NO.19.
2. the method for rapid detection industry saccharomyces pastorianus ester class metabolic gene according to claim 1, characterized by further comprising industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, the ACT1-Sb as internal reference gene, it is that SEQ ID NO.22 and SEQ ID NO.24 synthesize cDNA first chain that reverse transcription reaction prepares cDNA template application primer; In multi-PRC reaction with described cDNA first chain for template, application primer be SEQ ID NO.21 and SEQ ID NO.23.
3. the method for rapid detection industry saccharomyces pastorianus ester class metabolic gene according to claim 2, it is characterized in that described reverse transcription reaction is prepared in cDNA template reaction system, described application primer SEQ ID NO.22 concentration is 7.81nM, SEQ ID NO.24 concentration is 0.5nM; In the reaction system of described multi-PRC reaction, described application primer SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23 concentration are 200nM.
4. the method for rapid detection industry saccharomyces pastorianus ester class metabolic gene according to claim 3, it is characterized in that in the reaction system of described multi-PRC reaction, total reaction system is 20 μ L, wherein, 25mM MgCl
24.0 μ L, 5 × PCR damping fluid 4.0 μ L, application primer 2 μ L, archaeal dna polymerase 0.7 μ L, cDNA the first chain 9.3 μ L; 95 DEG C of denaturation 10minutes; 94 DEG C of sex change 30seconds; Annealing temperature 56 DEG C, 30seconds; 71 DEG C extend 1minute, circulate 35 times.
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| Title |
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| ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究;刘玉兰等;《中国酿造》;20121231;第31卷(第1期);第151-153页 * |
| 付俊淑.重组酿酒酵母酯代谢酶EHT1、EEB1、TGL3的表达及其活性研究.《中国优秀硕士学位论文全文数据库(电子期刊)》.2012,第B024-328页. * |
| 马金鸣.酵母乙酸异戊酯水解酶和V型ATP酶F亚基的结构生物学研究.《中国博士学位论文全文数据库(电子期刊)》.2011,第A006-14页. * |
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