CN107142312B - Method for rapidly detecting aromatic alcohol metabolism genes of industrial Saccharomyces pastorianus - Google Patents
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to the technical field of biological engineering, in particular to a method for rapidly detecting industrial barbiennis aromatic alcohol metabolism genes, which comprises the steps of total RNA extraction, reverse transcription reaction for preparing a cDNA template, multiple PCR reaction, capillary electrophoresis and product fragment analysis, and aims at industrial barbiennis aromatic alcohol metabolism related genes ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO 80-Sb. The GeXP multiple gene expression quantitative analysis technology adopted by the invention has the characteristics of stronger specificity and sensitivity, high automation degree and the like, ensures the reliability and repeatability of results, and greatly shortens the experimental time. The invention is beneficial to controlling the metabolism and the content of aromatic alcohol in the fermentation process under different production conditions, and improving the typical flavor and the consistency of beer; and the flavor of aromatic alcohol in the beer can be quickly adjusted, the product characteristics are highlighted, and a new product is developed.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for rapidly detecting aromatic alcohol metabolism genes of industrial Saccharomyces pastorianus.
Background
Beer is a beverage with low alcohol content and rich nutrition which is well liked by people. Over 90% of the global beer market today is Lager-type beer, and therefore the bottom brewer's yeast (commonly referred to as "Lager yeast") used to brew Lager beer has been most widely used in the beer industry. Lager yeast (Saccharomyces pastorianus) is different from Saccharomyces cerevisiae (s.cerevisiae) which has been used as a model organism in 1996 to perform whole genome sequencing, but is an allopolyploid strain with high chromosome ploidy and complex chromosome structure, and carries genetic information of Saccharomyces bayanus (s.bayanus) in addition to a part of Saccharomyces cerevisiae genes. Therefore, two homologous genes, Sc- (s.cerevisiae) and Sb- (s.bayanus), are present for most of the genes in Lager yeast.
Beer contains at least 45 different alcohols, the most important of which are aliphatic and aromatic alcohols. Wherein the content of isoamyl alcohol in the fatty alcohol is the most abundant, and is about 25-120 mg/L; the content of phenethyl alcohol in the aromatic alcohol is also higher, and is about 5-100 mg/L. Beta-phenylethyl alcohol is an aromatic alcohol with a rose fragrance, and brewers yeast has the ability to de novo synthesize and biotransform L-phenylalanine to beta-phenylethyl alcohol (i.e., the alder pathway). In yeast cells, which route β -phenylethyl alcohol synthesis takes depends on the nitrogen source in the medium. Glutamic acid, ammonium salts and the like are generally used as high-quality and readily available nitrogen sources, while amino acids, urea and the like are used only as secondary nitrogen sources. However, the Ehrlich pathway predominates only when L-phenylalanine is the sole nitrogen source. If other more readily available nitrogen sources are present in the environment, L-phenylalanine is partly metabolized by other pathways even at higher concentrations of L-phenylalanine. In addition, the influence of tyrosol and tryptophol, which are both aromatic alcohols, on the flavor of beer is not known to be relatively positive at present. Tryptophol is important in the top fermented beer, but is present in the bottom fermented beer in very low amounts, which is caused by the metabolic differences of yeast towards tryptophan.
At present, the fluorescent quantitative PCR method is the most commonly used method for quantifying mRNA. However, due to the limitation of low throughput of the method, a rapid and accurate technology is needed to simultaneously detect the expression of a plurality of aromatic alcohol metabolism related genes of Lager yeast.
Disclosure of Invention
The invention provides a method for rapidly detecting aromatic alcohol metabolism genes of industrial Saccharomyces pastorianus, in particular a method for detecting aromatic alcohol metabolism related genes (ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO80-Sb) expression of the industrial Saccharomyces pastorianus by adopting a GeXP multiple gene expression quantitative analysis technology, which is favorable for controlling the metabolism and the content of aromatic alcohol in a fermentation process under different production conditions and improving the flavor stability and consistency of beer; and the aromatic alcohol flavor in the beer can be quickly adjusted, so that the research and development cost is saved, and the development of a new product is accelerated.
In order to achieve the above purpose, the invention provides a method for rapidly detecting industrial barbiennis aromatic alcohol, especially beta-phenethyl alcohol metabolism genes, which comprises the steps of total RNA extraction, reverse transcription reaction for preparing cDNA templates, multiple PCR reaction, capillary electrophoresis, product fragment analysis, and specific to industrial barbiennis aromatic alcohol metabolism related genes ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO80-Sb, wherein the primers for preparing cDNA templates by reverse transcription reaction are SEQ ID NO.2, 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.22, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.20, SEQ ID NO., SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO.30, wherein primers SEQID NO.1, SEQID NO.3, SEQID NO.5, SEQID NO.7, SEQID NO.9, SEQID NO.11, SEQID NO.13, SEQID NO.15, SEQID NO.17, SEQID NO.19, SEQID NO.21, SEQID NO.23, SEQID NO.25, SEQID NO.27 and SEQID NO.29 are used in the multiplex PCR reaction.
As a preferred technical scheme, the gene vector also comprises industrial Saccharomyces pastorianus beta-actin genes ACT1-Sc and ACT1-Sb which are used as internal reference genes, and application primers for preparing a cDNA template through reverse transcription reaction are SEQ ID NO.32 and SEQ ID NO. 34; primers SEQID NO.31 and SEQID NO.33 are applied in the multiplex PCR reaction.
The specific detection steps are as follows:
(1) the following multiplex primers were used for the detection of the key genes of aromatic alcohol metabolism in industrial Saccharomyces pastorianus:
designing specific upstream and downstream primers which accord with the GeXP multiplex PCR reaction characteristics and can detect the expression of industrial barbiennis aromatic alcohol metabolism related genes, and the specific upstream and downstream primers serving as internal reference genes (see table 1):
TABLE 1 specific upstream and downstream primers for aromatic alcohol metabolism related genes and internal reference genes of industrial Pasteurella multocida
Wherein each primer is prepared into 100 mu M storage solution, the working solution of the downstream primer is 500nM, and the working solution of the upstream primer is 200 nM;
(2) total RNA extraction and purification: extracting purified yeast total RNA from yeast cells by adopting a general RNA extraction method and a purification method;
(3) preparing a cDNA template by reverse transcription reaction: synthesizing a first cDNA chain by using total yeast RNA as a template, wherein a downstream primer of the multiple primers in the step (1) is a specific primer, and a reaction system is 20 mu L; wherein, NTC and RT-As a negative control, the specific reaction conditions are shown in tables 2 and 3.
TABLE 2 reaction conditions for reverse transcription to prepare cDNA templates
| Composition per tube | NTC | RT- | Standard reaction |
| DNase/RNase-free water | 8μL | 4μL | 3μL |
| 5X reverse transcription buffer solution | 4μL | 4μL | 4μL |
| KANrRNA (1:50 dilution) | 5μL | 5μL | 5μL |
| Reverse transcriptase | 1μL | 0 | 1μL |
| Downstream primer (500nM) | 2μL | 2μL | 2μL |
| RNA template (5-20 ng/. mu.L) | 0 | 5μL | 5μL |
TABLE 3 downstream primer concentrations for each gene
| Gene | Size of product | Concentration of downstream primer (nM) |
| ADH1- |
187 | 62.5 |
| ADH1-Sc | 244 | 125 |
| ADH3-Sb | 165 | 7.8125 |
| ADH3-Sc | 169 | 500 |
| ADH4-Sb | 406 | 7.8125 |
| ADH4-Sc | 142 | 500 |
| ADH5-Sb | 200 | 500 |
| ADH5-Sc | 146 | 125 |
| ARO8-Sc | 251 | 500 |
| ARO9-Sb | 240 | 500 |
| ARO9-Sc | 389 | 62.5 |
| ARO10-Sb | 316 | 500 |
| ARO10-Sc | 219 | 500 |
| ARO80-Sb | 331 | 500 |
| ARO80-Sc | 264 | 1500 |
| ACT1-Sb | 284 | 0.5 |
| ACT1-Sc | 362 | 500 |
The reaction parameters for preparing cDNA template by reverse transcription reaction are set as follows:
1 minute at 48 ℃; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.
(4) Multiplex PCR: performing multiple PCR amplification reaction by using cDNA template prepared by the synthesized reverse transcription reaction as template and upstream primer of the multiple primer in (1) as specific primer, wherein each sample is provided with 3 parallel tubes, and DNA polymerase and genomeLab of Beckman Coulte company are adoptedTMGeXP start kit, the specific reaction conditions are shown in Table 4.
TABLE 4 reaction conditions for multiplex PCR
| Composition (I) | Volume (μ L) |
| 25mM MgCl2 | 4.0μL |
| 5 XPCR buffer | 4.0μL |
| DNA polymerase | 0.7μL |
| Upstream primer (200nM) | 2μL |
| First strand cDNA | 9.3μL |
The RT-PCR amplification parameters were set as follows:
10 minutes at 95 ℃; 30 seconds at 94 ℃; 56 ℃ for 30 seconds; 71 ℃ for 1 minute (35 cycles).
(5) Capillary electrophoresis of multiplex PCR products: mu.L of the PCR multiplex product was added to the wells of the upper plate containing 39. mu.L of the SLS + DSS mixture, mixed well with a pipette and covered with a drop of paraffin oil. In addition, 250. mu.L of separation buffer was added to each well of the buffer plate. After all the preparations are finished, the capillary electrophoresis is carried out on a machine. The separation gel and separation buffer were purchased from Beckman Coulter.
(6) Analysis of product fragments: analyzing the capillary electrophoresis result by utilizing GeXP system parameters, and recording the result.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) the detection method can sensitively, specifically and accurately detect the expression levels of ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO80-Sb genes. The change of the expression quantity of the key genes of aromatic alcohol metabolism reflects the generation condition of the aromatic alcohol of the yeast, so that the method can be used for researching the industrial barbiennis aromatic alcohol metabolism regulation mechanism.
(2) Compared with the conventional gene expression quantitative analysis technology (fluorescent quantitative PCR), the GeXP multiple gene expression quantitative analysis technology adopted by the invention has the characteristics of stronger specificity and sensitivity, high automation degree and the like, and ensures the reliability and repeatability of results. In addition, the GeXP multiple gene expression quantitative analysis technology can simultaneously detect the expression abundance of up to 30 genes, thereby greatly shortening the experimental time.
(3) The invention is beneficial to controlling the metabolism and the content of aromatic alcohol in the fermentation process under different production conditions, and improving the flavor typicality and consistency of beer; and the flavor of aromatic alcohol in the beer can be quickly adjusted, so that the product characteristics are highlighted and new products are developed.
Drawings
FIG. 1 is a schematic diagram of analyzing a result of capillary electrophoresis by using GeXP system parameters according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of A, B expression levels of key genes for aromatic alcohol metabolism in industrial Saccharomyces pastorianus.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) The embodiment provides a method for rapidly detecting industrial barbiennis aromatic alcohol metabolism genes, which comprises the steps of total RNA extraction, reverse transcription reaction for preparing cDNA templates, multiple PCR reactions, capillary electrophoresis and product fragment analysis, and aiming at industrial barbiennis aromatic alcohol metabolism related genes ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO80-Sb, the reverse transcription reaction for preparing cDNA templates 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, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.20, SEQ ID NO.26, PCR reactions, SEQID NO.3, SEQID NO.5, SEQID NO.7, SEQID NO.9, SEQID NO.11, SEQID NO.13, SEQID NO.15, SEQID NO.17, SEQID NO.19, SEQID NO.21, SEQID NO.23, SEQID NO.25, SEQID NO.27 and SEQID NO. 29.
On the basis, the gene vector also comprises industrial Saccharomyces pastorianus beta-actin genes ACT1-Sc and ACT1-Sb which are used as internal reference genes, and application primers for preparing a cDNA template by reverse transcription reaction are SEQ ID NO.32 and SEQ ID NO. 34; primers SEQID NO.31 and SEQID NO.33 are applied in the multiplex PCR reaction.
According to the full-length sequences of ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc, ARO80-Sb genes and internal reference genes ACT1-Sc and ACT1-Sb, Primer Premier 5 software is used for designing specific upstream and downstream primers which meet the characteristics of GeXP multiplex PCR reaction and can detect the expression of industrial Saccharomyces pastorianus aromatic alcohol metabolism related genes, and specific upstream and downstream primers serving as internal reference genes, the Primer sequences are shown in the following Table 5:
TABLE 5 specificity of the genes related to aromatic alcohol metabolism of industrial Pasteurella multocida and the internal reference genes
The result of single primer capillary electrophoresis shows that the length of the amplified product is consistent with the length of the expected product, which indicates that the designed primer has strong specificity and is suitable for GeXP detection.
(2) Taking full-yeast cell samples in A, B two yeast strain fermentors with consistent fermentation conditions, and rapidly extracting RNA
(3) Extraction and purification of Total RNA (Ribopure)TM-Yeast Kit, Ambion, product number AM 1926):
1) taking 750 mu L of zirconia beads in a 1.5mL centrifuge tube (with a spiral cover) and keeping the height at about 2.5 cm;
2) lysis Buffer (480. mu.L Lysis Buffer + 48. mu.L 10% SDS + 480. mu.L Phenol: cloroform: IAA), resuspension. Carrying out vortex oscillation for 10-15 s;
3) transferring the mixed solution into a prepared 750 mu L zirconium oxide bead centrifugal tube, and tightly covering the centrifugal tube;
4) placing the centrifugal tube in a vortex oscillator to shake for 10min (maximum rotation speed), and lysing cells;
5)16000g, centrifuging at room temperature for 5min, and transferring the upper aqueous phase into a 10mL centrifuge tube;
6) adding 1.9mL Binding Buffer, and mixing uniformly;
7) adding 1.25mL of 100% ethanol, and mixing uniformly;
8) taking 700 mu L of mixed solution to a Filter card + Collection Tube, centrifuging for 1min at 12000g, and removing the collected solution; repeating the steps until the mixed solution is completely transferred;
9) adding 700 mu L of Wash Solution 1 on a Filter Cartridge, centrifuging for 1min at 12000g, and discarding the collected liquid;
10) adding 500 μ L of Wash Solution 2/3, centrifuging at 12000g for 1min, and discarding the collected Solution. Repeating the steps;
11) centrifuging at 12000g for 1min to completely remove the liquid on the membrane, and transferring the Filter Cartridge to a new Collection Tube;
12) adding 25-50 μ L of Solution preheated to 95-100 deg.C to the center of the membrane, and centrifuging at 12000g for 1 min. Adding 25-50 μ L of precipitation Solution to the center of the membrane, and centrifuging at 12000g for 1min to obtain RNA collection Solution;
13) DNase I treatment
50-100. mu.L of RNA sample +1/10th vol 10 XDNase I Buffer + 4. mu.L DNase I, reacted at 37 ℃ for 30 min. Adding 1/10th vol DNase Inactivity Reagent (vortex before use), mixing uniformly after vortex oscillation, standing at room temperature for 5min, centrifuging at 12000g for 2-3min to precipitate the DNase Inactivity Reagent, transferring the RNA supernatant to a new tube for storage, and obtaining the purified total RNA.
(4) Preparing a cDNA template by reverse transcription reaction:
using the purified yeast cell RNA as a template, a genome Lab of Beckman Coulter was usedTMIn the GeXP start kit, the downstream primer of the multiple primers in the table 5 is a specific primer, the total RNA of the yeast cells is used as a template to synthesize a first strand of cDNA, and the reaction system is 20 mu L.
GenomeLabTMGeXP start kit (product number: PN A85017) contains reverse transcription reaction buffer solution, reverse transcriptase, PCR reaction buffer solution and KANrRNA, DNase/RNase-free water, DNA Marker (DSS-400), mineral oil and GeXP loading buffer; DNA polymerase(product No. PN A85022); separating gel (product number: PN A608010); separation buffers (product number: PN A608012), both from Beckman Coulter.
The cDNA first strand synthesis reaction system setup is shown in Table 6 and Table 7, in which NTC and RT-Negative control:
TABLE 6 reaction conditions for reverse transcription to prepare cDNA templates
| Composition per tube | NTC | RT- | Standard reaction |
| DNase/RNase-free water | 8μL | 4μL | 3μL |
| 5X reverse transcription buffer solution | 4μL | 4μL | 4μL |
| KANrRNA (1:50 dilution) | 5μL | 5μL | 5μL |
| Reverse transcriptase | 1μL | 0 | 1μL |
| Downstream primer (500nM) | 2μL | 2μL | 2μL |
| RNA template (5-20 ng/. mu.L) | 0 | 5μL | 5μL |
TABLE 7 downstream primer concentrations for each gene
The reaction parameters for preparing cDNA template by reverse transcription reaction are set as follows:
1 minute at 48 ℃;
60 minutes at 42 ℃;
95 ℃ for 5 minutes.
(5)RT-PCR：
DNA polymerase and genome Lab from Beckman CoulterTMGeXP start kit. RT-PCR amplification reaction was performed using cDNA templates prepared by reverse transcription reaction synthesized in the above Table 2 as templates and the upstream primers of the multiplex primers in the above Table 5 as specific primers, each sample was provided with 3 parallel channels, and the specific reaction conditions are shown in Table 8.
TABLE 8 reaction conditions for RT-PCR
| Composition (I) | Volume (μ L) |
| 25mM MgCl2 | 4.0μL |
| 5 XPCR buffer | 4.0μL |
| DNA polymerase | 0.7μL |
| Upstream primer (200nM) | 2μL |
| First strand cDNA | 9.3μL |
The RT-PCR amplification parameters were set as follows:
10 minutes at 95 ℃; 30 seconds at 94 ℃; 56 ℃ for 30 seconds; 71 ℃ for 1 minute (35 cycles).
(6) Capillary electrophoresis of multiplex PCR products: mu.L of the PCR multiplex product was added to the wells of the upper plate containing 39. mu.L of the SLS + DSS mixture, mixed well with a pipette and covered with a drop of paraffin oil. In addition, 250. mu.L of separation buffer was added to each well of the buffer plate. After all the preparations are finished, the capillary electrophoresis is carried out on a machine. The separation gel and separation buffer were purchased from Beckman Coulter.
(7) Analysis of product fragments: analyzing the capillary electrophoresis result by utilizing GeXP system parameters, and recording the result, wherein each peak with different sizes corresponds to a corresponding gene, and the height of each peak corresponds to the expression level of the gene as shown in figure 1.
FIG. 2 shows A, B expression levels of key genes in aromatic alcohol metabolism of two industrial Saccharomyces pastorianus. It can be seen from FIG. 2 that there is a difference in the aromatic alcohol metabolism performance between the two yeast strains. Specifically, the expression level of the key genes of aromatic alcohol metabolism of the strain B is higher than that of the strain A, and the significant difference exists between the expression levels of ARO8-sc and ARO80-sc genes.
According to the method provided by the invention, the expression levels of industrial Saccharomyces pastorianus aromatic alcohol metabolism key genes (ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO80-Sb) can be rapidly and simultaneously detected, and the method is used for researching Lager yeast aromatic alcohol metabolism regulation mechanism. The invention is beneficial to controlling the metabolism and the content of aromatic alcohol in the fermentation process under different production conditions, and improving the flavor stability and consistency of beer; and the flavor of aromatic alcohol in the beer can be quickly adjusted, so that the product characteristics are highlighted and new products are developed.
Sequence listing
<110> Qingdao beer Ltd
<120> method for rapidly detecting aromatic alcohol metabolism genes of industrial Saccharomyces pastorianus
<160> 34
<170> PatentIn version 3.3
<210> 1
<211> 38
<212> primer
<213> ADH1-Sc
<400> 1
AGGTGACACT ATAGAATACT GAATGTGCTC CAGTCTTG 38
<210> 2
<211> 40
<212> primer
<213> ADH1-Sc
<400> 2
GTACGACTCA CTATAGGGAC ACCAATGGAT CTGAATAATT 40
<210> 3
<211> 37
<212> primer
<213> ADH1-Sb
<400> 3
AGGTGACACT ATAGAATATG TTCAATATGC TAAGGCG 37
<210> 4
<211> 38
<212> primer
<213> ADH1-Sb
<400> 4
GTACGACTCA CTATAGGGAT TGGTAGCCTT AACGACTG 38
<210> 5
<211> 36
<212> primer
<213> ADH3-Sc
<400> 5
AGGTGACACT ATAGAATACA ACATTGTTCA CCAGGC 36
<210> 6
<211> 37
<212> primer
<213> ADH3-Sc
<400> 6
GTACGACTCA CTATAGGGAT GTAATGCAGC TTCCCCT 37
<210> 7
<211> 42
<212> primer
<213> ADH3-Sb
<400> 7
AGGTGACACT ATAGAATAAG TCTACTTGCT AGAAATATCC TC 42
<210> 8
<211> 37
<212> primer
<213> ADH3-Sb
<400> 8
GTACGACTCA CTATAGGGAT TTGGTTCTGG GACTGGA 37
<210> 9
<211> 36
<212> primer
<213> ADH4-Sc
<400> 9
AGGTGACACT ATAGAATAGG GATCACGTAT GCCGTT 36
<210> 10
<211> 37
<212> primer
<213> ADH4-Sc
<400> 10
GTACGACTCA CTATAGGGAC TGCCCTTTTT TCCTGTT 37
<210> 11
<211> 35
<212> primer
<213> ADH4-Sb
<400> 11
AGGTGACACT ATAGAATAGC TTACAACGACG GCTCT 35
<210> 12
<211> 38
<212> primer
<213> ADH4-Sb
<400> 12
GTACGACTCA CTATAGGGAG TTCTCAGCCA AGATACCG 38
<210> 13
<211> 36
<212> primer
<213> ADH5-Sc
<400> 13
AGGTGACACT ATAGAATAGC CTTCGCAAGT CATTCC 36
<210> 14
<211> 40
<212> primer
<213> ADH5-Sc
<400> 14
GTACGACTCA CTATAGGGAA ATTTCGTTAG GCTTAGGTTC 40
<210> 15
<211> 35
<212> primer
<213> ADH5-Sb
<400> 15
AGGTGACACT ATAGAATATCT TCTCAACCCA TCCCG 35
<210> 16
<211> 36
<212> primer
<213> ADH5-Sb
<400> 16
GTACGACTCA CTATAGGGAC GTGCCATGCGT GTAAAT 36
<210> 17
<211> 37
<212> primer
<213> ARO8-Sc
<400> 17
AGGTGACACT ATAGAATAGA TGAAACCAAT GCTCGTA 37
<210> 18
<211> 38
<212> primer
<213> ARO8-Sc
<400> 18
GTACGACTCA CTATAGGGAA ATCTTTGTTG ACGGTGTA 38
<210> 19
<211> 37
<212> primer
<213> ARO9-Sc
<400> 19
AGGTGACACT ATAGAATACC CCTGTTGATT ACACTTC 37
<210> 20
<211> 39
<212> primer
<213> ARO9-Sc
<400> 20
GTACGACTCA CTATAGGGAC GATTAATTCT GGACACAAA 39
<210> 21
<211> 34
<212> primer
<213> ARO9-Sb
<400> 21
AGGTGACACT ATAGAATAGC GTCATCGAGC TGGC 34
<210> 22
<211> 37
<212> primer
<213> ARO9-Sb
<400> 22
GTACGACTCA CTATAGGGAC GGTGGCAGAC CTCTAGT 37
<210> 23
<211> 37
<212> primer
<213> ARO10-Sc
<400> 23
AGGTGACACT ATAGAATAAA TCAAGAAGAA CGACACC 37
<210> 24
<211> 36
<212> primer
<213> ARO10-Sc
<400> 24
GTACGACTCA CTATAGGGAA CCCATCTTAG GCCAGC 36
<210> 25
<211> 34
<212> primer
<213> ARO10-Sb
<400> 25
AGGTGACACT ATAGAATAAA TGCTGCCTAT GCCG 34
<210> 26
<211> 40
<212> primer
<213> ARO10-Sb
<400> 26
GTACGACTCA CTATAGGGAG TTATCCTGAA CCATGTCATG 40
<210> 27
<211> 35
<212> primer
<213> ARO80-Sc
<400> 27
AGGTGACACT ATAGAATACT ACGGACGAGG GGTTG 35
<210> 28
<211> 39
<212> primer
<213> ARO80-Sc
<400> 28
GTACGACTCA CTATAGGGAC TCTATGAGTT CAATCGCCT 39
<210> 29
<211> 36
<212> primer
<213> ARO80-Sb
<400> 29
AGGTGACACT ATAGAATATA TCGCCCAGCA TGATTA 36
<210> 30
<211> 42
<212> primer
<213> ARO80-Sb
<400> 30
GTACGACTCA CTATAGGGAA TGAGTGCAAA GAGTTTACAT AT 42
<210> 31
<211> 35
<212> primer
<213> ACT1-Sc
<400> 31
AGGTGACACT ATAGAATAAC GCTCCTCGTG CTGTC 35
<210> 32
<211> 37
<212> primer
<213> ACT1-Sc
<400> 32
GTACGACTCA CTATAGGGAT AGAAGGCTGGA ACGTTAA 37
<210> 33
<211> 35
<212> primer
<213> ACT1-Sb
<400> 33
AGGTGACACT ATAGAATAGG CATCACACCTT TTACA 35
<210> 34
<211> 35
<212> primer
<213> ACT1-Sb
<400> 34
GTACGACTCA CTATAGGGAC CGGCGTAGAT TGGGA 35
Claims (6)
1. A method for rapidly detecting industrial Saccharomyces pastorianus aromatic alcohol metabolism genes comprises the steps of total RNA extraction, cDNA template preparation through reverse transcription reaction, multiple PCR reaction, capillary electrophoresis and product fragment analysis, and is characterized in that for industrial Saccharomyces pastorianus aromatic alcohol metabolism related genes ADH1-Sc, ADH1-Sb, ADH3-Sc, ADH3-Sb, ADH4-Sc, ADH4-Sb, ADH5-Sc, ADH5-Sb, ARO8-Sc, ARO9-Sc, ARO9-Sb, ARO10-Sc, ARO10-Sb, ARO80-Sc and ARO80-Sb, the primers for preparing the cDNA template through reverse transcription reaction are SEQ ID NO.2, 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.22 and SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, primers 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 I DNO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29 are used in a multiplex PCR reaction;
in the reaction system of the multiplex PCR reaction, the total reaction system is 20 μ L, wherein, 25mM MgCl24.0. mu.L, 5 XPCR buffer 4.0. mu. L, DNA polymerase 0.7. mu.L, primer 2. mu.L in multiplex PCR reaction, cDNA template 9.3. mu.L in multiplex PCR reaction; the amplification parameters were: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds; the annealing temperature is 56 ℃ and 30 seconds; extension at 71 ℃ for 1min and 35 cycles.
2. The method of claim 1, further comprising industrial Saccharomyces pastorianus beta-actin genes ACT1-Sc and ACT1-Sb as internal reference genes, wherein the primers for preparing cDNA templates by reverse transcription are SEQ ID NO.32 and SEQ ID NO. 34; primers SEQ ID NO.31 and SEQ ID NO.33 were used in multiplex PCR reactions.
3. The method according to claim 2, wherein the primer used is SEQ ID No.2 at 125nM, SEQ ID No.4 at 62.5nM, SEQ ID No.6 at 500nM, SEQ ID No.8 at 7.8125nM, SEQ ID No.10 at 500nM, SEQ ID No.12 at 7.8125nM, SEQ ID No.14 at 125nM, SEQ ID No.16 at 500nM, SEQ ID No.18 at 500nM, SEQ ID No.20 at 62.5nM, SEQ ID No.22 at 500nM, SEQ ID No.24 at 500nM, SEQ ID No.26 at 500nM, SEQ ID No.28 at 1.5. mu.M, SEQ ID No.30 at 500nM, SEQ ID No.32 at 500nM, and SEQ ID No.34 at 0.5 nM.
4. The method of claim 2, wherein the primers 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, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29, SEQ ID No.31, and SEQ ID No.33 are used at a concentration of 200 nM.
5. The method of any one of claims 2 to 4, wherein the product size of the industrial Saccharomyces pastorianus aromatic alcohol metabolism-related gene ADH1-Sc is 244bp, the product size of ADH1-Sb is 187bp, the product size of ADH3-Sc is 169p, the product size of ADH3-Sb is 165bp, the product size of ADH4-Sc is 142bp, the product size of ADH4-Sb is 406bp, the product size of ADH5-Sc is 146bp, the product size of ADH5-Sb is 200bp, the product size of ARO8-Sc is 251bp, the product size of ARO9-Sc is 389bp, the product size of ARO9-Sb is 240bp, the product size of ARO10-Sc is 219bp, the product size of ARO10-Sb is 316bp, the product size of ARO80-Sc is 264bp, and the product size of ARO80-Sb is 331 bp.
6. The method of claim 5, wherein the size of the product of the industrial Saccharomyces pastorianus beta-actin gene ACT1-Sc is 362bp, and the size of the product of ACT1-Sb is 284 bp.
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