CN103627805A - Method for rapidly detecting industrial saccharomyces pastorianus higher alcohol metabolizing genes - Google Patents
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Abstract
The invention relates to biotechnologies and particularly relates to a method for rapidly detecting industrial saccharomyces pastorianus higher alcohol metabolizing genes. The method comprises the following steps: extracting RNA, preparing a cDNA template through reverse transcription reaction, performing multiple PCR, performing electrophoresis on blood capillaries, analyzing product segments, and metabolizing related genes BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc and BAT2-Sb for the industrial saccharomyces pastorianus higher alcohol. According the invention, the GeXP multiple-gene expression quantitative analysis technology is adopted, compared with the conventional gene expression quantitative analysis technology (fluorescent quantitative PCR), the GeXP multiple-gene expression quantitative analysis technology has the characteristics of relatively strong specificity and sensitivity, and high degree of automation, so that the reliability and repeatability of results are ensured, and the experimental period is shortened greatly. The metabolism and content of higher alcohol are controlled during the process of fermentation under such condition that beneficial to different productions, and the typical tastiness and uniformity of beer are improved; product features are highlighted and new products are developed by rapidly regulating tastiness of the higher alcohol in the beer.
Description
Technical field
The present invention relates to biotechnology, particularly a kind of method of rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene.
Background technology
Beer is a kind of low ethanol content being loved by the people and nutritious beverage.It is the beer of Lager type that current global Beer surpasses 90%, and therefore the Saccharomyces uvarum (being commonly called as " Lager yeast " or " saccharomyces pastorianus ") for brew Lager beer is widely used in brewing industry most.Lager yeast is also different from the yeast saccharomyces cerevisiae (S.cerevisiae) that carries out genome sequencing for 1996 as model animals, but a kind ofly there is highly coloured body ploidy, chromosome structure forms complicated allopolyploid bacterial classification, except carrying a part of genes of brewing yeast, also carry the genetic information of Bei Shi yeast (S.bayanus), be named as saccharomyces pastorianus (Saccharomyces pastorianus).Therefore for the most gene in saccharomyces pastorianus, all there is Sc-(S.cerevisiae) and Sb-(S.bayanus) two homologous genes.Yet, compare with the yeast saccharomyces cerevisiae entirely checking order, for gene and the expression of described gene in Process of Beer Brewing of the specific participation brewing character of bottom yeast, very limited at present.
In the polycomponent local flavor system of beer, higher alcohols is the essential substance that forms the whole special favor of beer, and the formation of the quality of beer and local flavor typicalness is played to critical effect.Higher alcohols can promote wine body to have plentiful fragrance and taste, and increases the Harmony of wine.Higher alcohols is by cereuisiae fermentum metabolism, to be produced in beer fermentation process.Yeast cell is affected and is participated in the relevant synthetic yeast gene expression of flavour substances by transcriptional control, finally causes the variation of higher alcohols content.Therefore, the research of expressing participating in the synthetic key gene of higher alcohols in industrial saccharomyces pastorianus fermenting process, is the key of Reasonable Regulation And Control higher alcohols content.
Synthesizing of higher alcohols is directly related with amino acid whose metabolic process, has two pathways metabolisms: catabolism approach and metabolic pathway of synthesizing.Catabolism approach also claims Ehrlich degradation pathway, and amino acid forms alpha-ketoacid under the effect of transaminase; Ketone acid decarboxylation becomes aldehyde; Aldehyde is further reduced to higher alcohols under the effect of desaturase.By Ehrlich approach, specific amino acid can form specific higher alcohols.Anabolism is to provide biosynthesizing amino acid whose carbon skeleton by carbohydrate, in its synthetic mesophase stage, forms alpha-ketoacid intermediate, and decarboxylation and reduction thus, forms corresponding higher alcohols.
At present, identified and participated in Ehrlich approach and the synthetic portion gene of higher alcohols.Branched-chain amino acid in yeast, comprises leucine, Isoleucine and α-amino-isovaleric acid, is all that the movement system that the branched-chain amino acid permease by BAP2 genes encoding mediates is transported in yeast cell.The disappearance of BAP2 gene will cause yeast to reduce 20-50% to the absorbed dose of leucine, Isoleucine and α-amino-isovaleric acid.Because amino acid whose accumulation in cell is relevant with Ehrlich approach, so just there is dependency in the expression of BAP2 gene and the formation of higher alcohols.The primary rate-limiting step of branched-amino acid metabolic is exactly by the transamination that is positioned at plastosome and the catalysis of intracellular branched-chain amino acid transaminase (BCAT) institute.This enzyme is formed by BAT1 and BAT2 coding, and catalysis branched-chain amino acid is to the transamination of alpha-ketoacid, and wherein alpha-ketoacid is the synthetic precursor of higher alcohols.Research shows, in yeast, the overexpression of BAT1 gene and BAT2 gene all can cause the increase of higher alcohols output.The deletion mutantion strain of BAT2 gene causes the content of primary isoamyl alcohol and isopropylcarbinol to compare and reduced respectively 40% and 72% with wild strain.Therefore, BAP2, BAT1, BAT2 gene is considered to the key gene in higher alcohols metabolic process, detects BAP2, BAT1, the level of BAT2 genetic expression can be for studying industrial saccharomyces pastorianus higher alcohols metabolic regulation mechanism.
At present, fluorescence quantifying PCR method is the method for present the most conventional quantification of mrna.But due to the not high limitation of the method flux, therefore need a kind of technology fast and accurately to detect a plurality of higher alcohols Metabolism-Related Genes Expressions of saccharomyces pastorianus simultaneously.GenomeLab GeXP Genetic Analysis System(U.S. Beckman Coulter company) be the technology platform that is mainly used in studying multi-gene expression quantitative analysis, adopt the method for capillary electrophoresis and fluorescent mark amplified production, can detect the nearly gene expression abundance of 30 genes simultaneously.Multi-gene expression regulation and control, medicine virulence Mechanism Study, early diagnosis of cancer research etc. have been widely used at present.
Summary of the invention
The object of the invention is to disclose a kind of employing GeXP multiple gene expression quantitative analysis tech and detect industrial saccharomyces pastorianus higher alcohols metabolism related gene (BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb) method expressed, and then be conducive under different working conditions, the Higher Alcohols metabolism of controlled fermentation process and content, local flavor typicalness and the consistence of raising beer; And can pass through rapid adjustment High Alcohols local flavor, and save R&D costs, accelerate the exploitation of product innovation.
Technical scheme of the present invention is:
A kind of method of rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene, comprise the extraction of total RNA, reverse transcription reaction is prepared cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, for industrial saccharomyces pastorianus higher alcohols metabolism related gene BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb, it is SEQ ID NO.2 that reverse transcription reaction is prepared cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, in multi-PRC reaction, apply 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.
On the basis of above scheme, also comprise industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, ACT1-Sb as internal reference gene, it is SEQ ID NO.14, SEQ ID NO.16 that reverse transcription reaction is prepared cDNA template application primer; In multi-PRC reaction, apply primer SEQ ID NO.13, SEQ ID NO.15.
Higher alcohols in beer of the present invention, be commonly called as potato spirit, refer to 3 monovalence alcohols more than carbon atom, mainly contain n-propyl alcohol, propyl carbinol, isopropylcarbinol, amylalcohol, primary isoamyl alcohol, phenylethyl alcohol, tryptophol, alcohol, sugar alcohol etc., wherein primary isoamyl alcohol accounts for the more than 50% of total amount.
Concrete detecting step is as follows:
(1) use following multi-primers to carry out the detection of industrial saccharomyces pastorianus higher alcohols metabolism key gene:
Design meets GeXP multi-PRC reaction feature, detects the specificity upstream and downstream primer of industrial saccharomyces pastorianus higher alcohols 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 higher alcohols metabolism related gene and internal reference gene
Wherein every primer is mixed with respectively the storage liquid of 100 μ M, and upstream primer working fluid is 200nM;
(2) total RNA extraction and purification: adopt general RNA extracting method and purification process, extract the total RNA of yeast that obtains purifying from yeast cell;
(3) reverse transcription reaction is prepared cDNA template: the total RNA of the yeast of take is synthetic cDNA the first chain of template, and the downstream primer of above-mentioned multi-primers in is (1) Auele Specific Primer, and reaction system is 20 μ L; Wherein NTC and RT
-negative contrast.
Table 2 reverse transcription reaction is prepared the reaction conditions of cDNA template
The reverse primer concentration of each gene of table 3
Reverse transcription reaction is prepared cDNA template reaction parameter and is arranged as follows:
48℃1minutes；42℃60minutes；95℃5minutes。
(4) multiplex PCR: above-mentioned synthetic cDNA the first chain of take is template, the upstream primer of above-mentioned multi-primers in is (1) Auele Specific Primer, carries out multiplex PCR amplified reaction, and each sample is established 3 parallel pipes, and archaeal dna polymerase is purchased from Beckman Coulter;
The reaction conditions of table 4 multiplex PCR
| Composition | Volume (μ L) |
| 25mMMgCl 2 | 4.0 |
| 5 * PCR damping fluid | 4.0μL |
| Archaeal dna polymerase | 0.7μL |
| Forward primer (200nM) | 2μL |
| CDNA the first chain | 9.3μL |
Pcr amplification parameter arranges as follows:
95 ℃ of denaturation 10minutes; 94 ℃ of sex change 30seconds; 56 ℃ of annealing temperatures, 30seconds; 71 ℃ are extended 1minute, circulate 35 times.
(5) multiple PCR products capillary electrophoresis: get the multiple product of 1 μ lPCR and be added in the hole of minute 95% deionized formamide (SLS) of 39 μ l and the upper model of 400bp Marker mixed solution being housed, mix rear covering one dropstone wax oil with liquid-transfering gun.In the every hole of damping fluid plate, add in addition the dissociating buffer of 250 μ l.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 test kit of the present invention and detection method thereof, can detect BAP2-Sc sensitive, special, exactly, BAP2-Sb, BAT1-Sc, BAT1-Sb, the level of BAT2-Sc and BAT2-Sb genetic expression.The variation of higher alcohols metabolism key gene expression amount, has reacted the generation situation of yeast higher alcohols, so can be for studying industrial saccharomyces pastorianus higher alcohols metabolic regulation mechanism.
(2) the GeXP multiple gene expression quantitative analysis tech that the present invention adopts compare with conventional Quantitative analysis of gene expression technology (quantitative fluorescent PCR) there is stronger specificity and susceptibility, level of automation high, guaranteed reliability and the repeatability of result.In addition, because GeXP multiple gene expression quantitative analysis tech can detect the nearly gene expression abundance of 30 genes simultaneously, greatly shortened experimental period.
(3) the present invention is conducive under different working conditions, the Higher Alcohols metabolism of controlled fermentation process and content, local flavor typicalness and the consistence of raising beer; And can pass through rapid adjustment High Alcohols local flavor, give prominence to selling point and carry out the exploitation of product innovation.
Accompanying drawing explanation
Accompanying drawing 1 is the amplified production of ACT1-Sb gene and KANr gene;
Accompanying drawing 2 is the amplified production of BAT2-Sb gene and KANr gene;
Accompanying drawing 3 is the amplified production of ACT1-Sc gene and KANr gene;
Accompanying drawing 4 is the amplified production of BAP2-Sb gene and KANr gene;
Accompanying drawing 5 is the amplified production of BAT1-Sc gene and KANr gene;
Accompanying drawing 6 is the amplified production of BAP2-Sc gene and KANr gene;
Accompanying drawing 7 is the amplified production of BAT1-Sb gene and KANr gene;
Accompanying drawing 8 is the amplified production of BAT2-Sc gene and KANr gene;
Accompanying drawing 9 is the multiplex amplification product of higher alcohols genes involved and KANr gene;
Accompanying drawing 10 is the variation of expression amount during the fermentation of higher alcohols genes involved;
Embodiment
The specific embodiment of the present invention is as follows:
Embodiment 1
A kind of method of rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene, comprise the extraction of total RNA, reverse transcription reaction is prepared cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, for industrial saccharomyces pastorianus higher alcohols metabolism related gene BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb, it is SEQ ID NO.2 that reverse transcription reaction is prepared cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, in multi-PRC reaction, apply 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.Also comprise industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, ACT1-Sb as internal reference gene, it is SEQ ID NO.14, SEQ ID NO.16 that reverse transcription reaction is prepared cDNA template application primer; In multi-PRC reaction, apply primer SEQ ID NO.13, SEQ ID NO.15.
According to BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb gene and reference gene ACT1-Sc, the full length sequence of ACT1-Sb, is used Primer Premier5 software design to be applicable to the Auele Specific Primer that GeXP detects, and primer sequence is as follows:
Design meets GeXP multi-PRC reaction feature, detects the specificity upstream and downstream primer of industrial saccharomyces pastorianus higher alcohols 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 higher alcohols metabolism related gene and internal reference gene
Single primer capillary electrophoresis result shows that the product length after amplification is consistent with expectation product length, and is single band, and result is as shown in Fig. 1-8.The primer that design is described has very strong specificity, is suitable for GeXP and detects.
2000L pilot scale fermentation:
Carry out 13 degree wheat juice fermentation tests of 2000L scale.
Canful yeast number 22.0 * 10
6individual/ml, 8.5 ℃ of canful temperature, 24 hours deslagginves of canful once, are warming up to 9.5 ℃ of fermentations naturally.When pol is down to 5.2 ° of BX, be warming up to 12 ℃, carry out di-acetyl reduction.When pol is down to 3.8 ° of BX, sealed cans pressurize 0.1Mpa, deslagging is once.When di-acetyl is down to 0.05mg/L, be cooled to 5 ℃, after 24 hours, thoroughly drain yeast and bits at the bottom of clean tank, then be cooled to 0 ℃, during storage wine, arrange every three days yeast one time.In different time sections, get yeast cell sample, extract rapidly RNA.
1, the extraction and purification (RiboPure of total RNA
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 LLysis Buffer+48 μ L10%SDS+480 μ LPhenol:Cloroform:IAA), resuspended.Vortex vibration 10-15s;
(3) mixed solution is transferred in ready 750 μ L zirconia bead centrifuge tubes, covered tightly;
(4) centrifuge tube is placed in to vortex vibrator 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 1.9mLBinding Buffer, mix;
(7) add 1.25mL100% ethanol, mix;
(8) get 700 μ L mixed solutions in Filter Cartridge+Collection Tube, the centrifugal 1min of 12000g, abandons collection liquid; Repeat this step, extremely above-mentioned mixed solution all shifts;
(9) on Filter Cartridge, add 700 μ LWash Solution1, the centrifugal 1min of 12000g, abandons collection liquid;
(10) add 500 μ LWash Solution2/3, the centrifugal 1min of 12000g, abandons collection liquid.Repeat this step;
(11) the centrifugal 1min of 12000g, thoroughly removes liquid on film, and Filter Cartridge is transferred in new Collection Tube;
(12) add the Elution Solution25-50 μ L of 95-100 ℃ of preheating to film central authorities, the centrifugal 1min of 12000g.Add 25-50 μ LElution Solution to film central authorities, the centrifugal 1min of 12000g, obtains RNA and collects liquid again;
(13) DNaseI processes
50-100 μ LRNA Sample+1/10thvol10 * DNase I Buffer+4 μ LDNaseI, 37 ℃ of reaction 30min.First vortex before adding 1/10th vol DNase Inactivation Reagent(to use), after vortex concussion, mix, room temperature is placed after 5min, the centrifugal 2-3min of 12000g, make DNase Inactivation Reagent precipitation, RNA supernatant liquor is transferred in new pipe and is preserved, can obtain total RNA of purifying.
2, reverse transcription reaction is prepared cDNA template:
The yeast cell RNA of purifying described above of take is template, uses 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 the total RNA of yeast cell of take is synthetic cDNA the first chain of template, and reaction system is 20 μ L.
GenomeLab
tMgeXP starts test kit (production code member: PNA85017), contain reverse transcription reaction damping fluid, ThermoScript II, PCR reaction buffer, KAN
rrNA, the water without DNA enzyme/RNA enzyme, DNAMarker (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 the first chain synthesis reaction system arranges as follows, the wherein negative contrast of NTC and RT-:
Table 2 reverse transcription reaction is prepared the reaction conditions of cDNA template
| Every pipe composition | NTC | RT - | Standard reaction | |
| Without DNA enzyme/RNA enzyme water | | 4μL | 3μL | |
| 5 * reverse transcription damping fluid | 4μL | 4μL | 4μL | |
| KAN rRNA (1:50 dilution) | 5μL | 5μL | 5μL | |
| | 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 (bp) | Reverse primer concentration (nM) |
| BAT1- |
286 | 500 |
| BAT1- |
233 | 125 |
| BAT2- |
142 | 500 |
| BAT2- |
332 | 500 |
| BAP2- |
225 | 500 |
| BAP2- |
280 | 500 |
| ACT1- |
135 | 0.5 |
| ACT1- |
176 | 7.8125 |
| |
325 | 50 |
CDNA the first chain synthesis reaction parameter arranges as follows:
48℃ 1minutes；
42℃ 60minutes；
95℃ 5minutes。
3、RT-PCR：
RT-PCR adopts archaeal dna polymerase and the GenomeLab of BeckmanCoulter company
tMgeXP starts test kit and carries out.Synthetic cDNA the first chain in above-mentioned 2 of take is template, and the upstream primer of the multi-primers in above-mentioned table 1 is Auele Specific Primer, carries out RT-PCR amplified reaction, and each sample is established 3 parallel pipes.
RT-PCR amplification reaction system arranges as follows:
The reaction conditions of table 4 multiplex PCR
| Composition | Volume (μ L) |
| 25mMMgCl 2 | 4.0 |
| 5 * PCR damping fluid | 4.0μL |
| Archaeal dna polymerase | 0.7μL |
| Forward primer (200nM) | 2μL |
| CDNA the first chain | 9.3μL |
RT-PCR Amplification arranges as follows:
95 ℃ of denaturation 10minutes; 94 ℃ of sex change 30seconds; 56 ℃ of annealing temperatures, 30seconds; 71 ℃ are extended 1minute, circulate 35 times.
4, multiple PCR products capillary electrophoresis: get the multiple product of 1 μ lPCR and be added in the hole of minute 95% deionized formamide (SLS) of 39 μ l and the upper model of 400bpMarker mixed solution being housed, mix rear covering one dropstone wax oil with liquid-transfering gun.In the every hole of damping fluid plate, add in addition the dissociating buffer of 250 μ l.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, see Fig. 9.
Figure 10 is that the expression during the fermentation of industrial saccharomyces pastorianus higher alcohols metabolism key gene changes.The expression amount of BAP2-Sc and BAP2-Sb gene slowly increases with fermentation time as seen from the figure, but significantly increases at the latter two expression amount of 86h, especially BAP2-Sb gene.The expression amount of BAT1-Sc and BAT1-Sb gene continues to increase during the fermentation, but the expression amount of BAT1-Sc is higher than the expression amount of BAT1-Sb.The expression of BAT2-Sc and BAT2-Sb gene continued to increase in early stage, and the latter two expression amount of 110h all starts to reduce.
According to method provided by the invention, can detect fast industrial saccharomyces pastorianus higher alcohols metabolism key gene (BAP2-Sc, BAP2-Sb simultaneously, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb) expression level, for studying industrial saccharomyces pastorianus higher alcohols metabolic regulation mechanism.The present invention is conducive under different working conditions, the Higher Alcohols metabolism of controlled fermentation process and content, local flavor typicalness and the consistence of raising beer; And can pass through rapid adjustment High Alcohols local flavor, give prominence to selling point and carry out the exploitation of product innovation.
Sequence table
<110> Qingdao Beer Co., Ltd.
The method of <120> rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 35
<212> primer
<213> BAP2-Sc
<400> 1
aggtgacact atagaatagc tggtccaaga ctgat 35
<210> 2
<211> 35
<212> primer
<213> BAP2-Sc
<400>2
gtacgactca ctatagggat tgtctgaatc gcagatg 37
<210> 3
<211> 36
<212> primer
<213> BAP2-Sb
<400>3
aggtgacact atagaatagt agtatttttg gcaccg 36
<210> 4
<211> 42
<212> primer
<213> BAP2-Sb
<400>4
gtacgactca ctatagggaa gctttatatc ctaactcatc ta 42
<210> 5
<211> 36
<212> primer
<213> BAT1-Sc
<400>5
aggtgacact atagaatatc agaacactcg ctactg 36
<210> 6
<211> 39
<212> primer
<213> BAT1-Sc
<400>6
gtacgactca ctatagggaa gagaaagatt accgtaagg 39
<210> 7
<211> 37
<212> primer
<213> BAT1-Sb
<400>7
aggtgacact atagaatagc cttatgctaa tttatcg 37
<210> 8
<211> 37
<212> primer
<213> BAT1-Sb
<400>8
gtacgactca ctatagggac cattcccttc aggaact 37
<210> 9
<211> 37
<212> primer
<213> BAT2-Sc
<400>9
aggtgacact atagaatacc gaacagtgag ttagtgt 37
<210> 10
<211> 40
<212> primer
<213> BAT2-Sc
<400>10
gtacgactca ctatagggat tagggtaatc aactcttctg 40
<210> 11
<211> 36
<212> primer
<213> BAT2-Sb
<400>11
aggtgacact atagaataga cgaatttgtc tgccag 36
<210> 12
<211> 37
<212> primer
<213> BAT2-Sb
<400>12
gtacgactca ctatagggaa agagtagccc tttccct 37
<210> 13
<211> 36
<212> primer
<213> ACT1-Sc
<400>13
aggtgacact atagaatata acgttccagc cttcta 36
<210> 14
<211> 39
<212> primer
<213> ACT1-Sc
<400>14
gtacgactca ctatagggag tgaggtagag agaaaccag 39
<210> 15
<211> 37
<212> primer
<213> ACT1-Sb
<400>15
aggtgacact atagaatagt atcttgacat tgcgtta 37
<210> 16
<211> 40
<212> primer
<213> ACT1-Sb
<400>16
gtacgactca ctatagggat ctcaactcgt tgtaaaaggt 40
Claims (4)
1. the method for rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene, the extraction, the reverse transcription reaction that comprise total RNA are prepared cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, it is characterized in that for industrial saccharomyces pastorianus higher alcohols metabolism related gene
bAP2-Sc,
bAP2-Sb,
bAT1-Sc,
bAT1-Sb,
bAT2-Sc,
bAT2-Sb, it is SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 that reverse transcription reaction is prepared cDNA template application primer, applies 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 in multi-PRC reaction.
2. the method for rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene according to claim 1, characterized by further comprising the industrial saccharomyces pastorianus β-actin gene as internal reference gene
aCT1-Sc,
aCT1-Sb, it is SEQ ID NO.14, SEQ ID NO.16 that reverse transcription reaction is prepared cDNA template application primer; In multi-PRC reaction, apply primer SEQ ID NO.13, SEQ ID NO.15.
3. the method for rapid detection according to claim 1 and 2 industry saccharomyces pastorianus higher alcohols metabolic gene, it is characterized in that described reverse transcription reaction prepared in cDNA template reaction system, described application primer SEQ ID NO.2 concentration is that 500nM, SEQ ID NO.4 concentration are that 500nM, SEQ ID NO.6 concentration are that 125nM, SEQ ID NO.8 concentration are that 500nM, SEQ ID NO.10 concentration are that 500nM, SEQ ID NO.12 concentration are that 500nM, SEQ ID NO.14 concentration are that 7.8125nM, SEQ ID NO.16 concentration are 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 concentration are 200nM.
4. the method for rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene according to claim 3, is characterized in that in the reaction system of described multi-PRC reaction, and total reaction system is 20 μ L, wherein, and 25 mM MgCl
24.0 μ L, 5 * PCR damping fluid, 4.0 μ L, application primer 2 μ L, DNA polysaccharase 0.7 μ L; 95 ℃ of denaturation 10 minutes; 94 ℃ of sex change 30 seconds; 56 ℃ of annealing temperatures, 30 seconds; 71 ℃ are extended 1 minute, circulate 35 times.
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| CN104131099A (en) * | 2014-07-28 | 2014-11-05 | 青岛啤酒股份有限公司 | Method for rapidly monitoring mix-cultured yeast proliferation |
| CN107142312A (en) * | 2017-05-26 | 2017-09-08 | 青岛啤酒股份有限公司 | A kind of method of the industrial saccharomyces pastorianus aromatic alcohol metabolic gene of quick detection |
| CN108342458A (en) * | 2018-04-13 | 2018-07-31 | 青岛啤酒股份有限公司 | Malt leaching rate evaluation method based on gene expression monitoring |
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| CN102199556A (en) * | 2011-04-15 | 2011-09-28 | 天津科技大学 | Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof |
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| CN102199556A (en) * | 2011-04-15 | 2011-09-28 | 天津科技大学 | Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131099A (en) * | 2014-07-28 | 2014-11-05 | 青岛啤酒股份有限公司 | Method for rapidly monitoring mix-cultured yeast proliferation |
| CN104131099B (en) * | 2014-07-28 | 2016-03-23 | 青岛啤酒股份有限公司 | The method of Fast Monitoring miscegenation Yeast proliferation |
| CN107142312A (en) * | 2017-05-26 | 2017-09-08 | 青岛啤酒股份有限公司 | A kind of method of the industrial saccharomyces pastorianus aromatic alcohol metabolic gene of quick detection |
| CN108342458A (en) * | 2018-04-13 | 2018-07-31 | 青岛啤酒股份有限公司 | Malt leaching rate evaluation method based on gene expression monitoring |
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