CN108913807B - Multiple primers, kit and screening method for screening yeast strains with low protease A gene expression - Google Patents
Multiple primers, kit and screening method for screening yeast strains with low protease A gene expression Download PDFInfo
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention provides a multiplex primer, a kit and a screening method for screening a yeast strain with low protease A gene expression, belongs to the field of food detection, and can solve the problems of long time consumption, complex detection process and long detection period of the traditional method for screening the yeast strain with low protease A gene expression by determining the activity of protease A. The technical scheme comprises the steps of designing multiple primers, sequentially carrying out reverse transcription to synthesize a cDNA template by taking yeast strain total RNA as a template, carrying out multiple PCR amplification reaction and capillary electrophoresis separation, and analyzing a capillary electrophoresis separation result by utilizing a GeXP system; and calculating the expression quantity of the protease A gene by taking the internal reference gene as a control and combining the peak area of the corresponding peak of the key gene, and judging the yeast strain to be a low-yield protease A strain, a medium-yield protease A strain or a high-yield protease A strain according to the expression quantity of the protease A gene. By the method, the yeast strain with low protease A gene expression level can be accurately and quickly screened out.
Description
Technical Field
The invention relates to the field of food detection, in particular to a multiplex primer, a kit and a screening method for screening a yeast strain with low protease A gene expression.
Background
The pure white, fine and durable foam is one of the important marks of high-quality beer, the durability of the foam is measured by a foam sustainability index, the foam sustainability standard of the high-quality beer is specified in the national standard, the bottled wine is more than or equal to 180 seconds, and the canned wine is more than or equal to 150 seconds. Protein is a skeleton component constituting beer foam, and the deterioration of foam retention is mainly caused by the destruction of protein by protease A. The protease A is derived from beer yeast, and is released when the yeast autolyzes, so that the hydrophobic protein in the fermentation liquor is gradually decomposed or the characteristics of the hydrophobic protein are changed, and the foam stability of the fermentation liquor is influenced. Therefore, screening of yeast strains with low protease A gene expression is of great significance to the field of beer fermentation.
The yeast strain with low protease A gene expression is screened mainly by a method for measuring the activity of the protease A in fermentation liquor, wherein the protease A in the fermentation liquor is derived from beer yeast, and the activity reflects the protease A gene expression amount of the beer yeast, so that the yeast strain with low protease A gene expression can be screened by the method. However, the method for detecting the activity of the protease A is long in time consumption and complicated in detection process. And the yeast strain needs to be subjected to a fermentation test before detection, so that the detection period is long.
Disclosure of Invention
The application provides a multiplex primer, a kit and a screening method for screening a yeast strain with low protease A gene expression aiming at the problems of long time consumption, complex detection process and long detection period of screening the yeast strain with low protease A gene expression by determining the activity of protease A.
In order to achieve the above objects, the present invention provides a multiplex primer for screening yeast strains with low protease A gene expression, comprising a key gene according to yeast protease APEP4Homologous gene of (2)PEP4-ScAndPEP4-Sbrespectively designing specific upstream and downstream primers, which specifically comprises the following steps:
the invention also provides a kit, which comprises the multiple primers for screening the yeast strain with low protease A gene expression.
The invention also provides a method for screening a yeast strain with low protease A gene expression by using the multiple primers in the technical scheme, which comprises the following steps:
carrying out reverse transcription synthesis of a cDNA template, multiple PCR amplification reaction and capillary electrophoresis separation in sequence by taking the total RNA of the yeast strain as a template, and analyzing the capillary electrophoresis separation result by utilizing a GeXP system;
and calculating the expression quantity of the protease A gene by taking the internal reference gene as a control and combining the peak area of the corresponding peak of the key gene, and judging the yeast strain to be a low-yield protease A strain, a medium-yield protease A strain or a high-yield protease A strain according to the expression quantity of the protease A gene.
Preferably, the reference gene comprisesACT1Homologous gene of (2)ACT1-Sc、ACT1-SbDesigning specific upstream and downstream primers according to an internal reference gene, which specifically comprises the following steps:
preferably, the specific calculation and judgment method for calculating the expression level of the protease A gene by using the internal reference gene as a control and combining the peak area of the corresponding peak of the key gene comprises the following steps:
PEP4-Scgene expression level =PEP4-ScPeak area/(/)ACT1-ScPeak area +ACT1-SbPeak area)/2
When the expression amount of the protease A gene is less than 3, judging that the yeast strain is a low-yield protease A strain; when the expression quantity of the 3< protease A gene is less than 10, judging the yeast strain to be a medium-yield protease A strain; when the expression quantity of the protease A gene is more than or equal to 10, the yeast strain is judged to be the high-yield protease A strain.
Preferably, the first strand of cDNA template is synthesized by reverse transcription by using yeast strain total RNA as template and downstream primers shown by sequences SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8 as specific primers.
Preferably, the reaction parameters of the reverse transcription synthetic cDNA template are set as follows: 48 ℃ for 1 min; 60 min at 42 ℃; 95 ℃ for 5 min.
Preferably, the first strand of the synthesized cDNA template is used as a template, and the upstream primers shown by the sequences SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7 are used as specific primers to perform a multiplex PCR amplification reaction.
Preferably, the parameters of the multiplex PCR amplification reaction are set as: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 s; the annealing temperature is 55 ℃ and 30 s; the extension is carried out for 1 min at 70 ℃, and the whole PCR amplification reaction process is circulated for 35 times.
Compared with the prior art, the invention has the advantages and positive effects that:
1. by detecting the expression level of the protease A gene of the yeast, the yeast strain with low expression level of the protease A gene can be accurately screened out, and the operation is simple and quick.
2. By adopting the GeXP multifunctional genetic analysis system, the expression quantity of the key gene of the yeast protease A can be detected quickly, accurately and in high flux, the GeXP multifunctional genetic analysis system has the characteristics of stronger specificity, sensitivity, automation degree and the like, and the reliability and repeatability of results are ensured.
Drawings
FIG. 1 shows the results of electrophoresis of the multiple amplification products of the key gene of yeast protease A and the reference gene in example 1.
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
A method for rapidly detecting industrial Saccharomyces pastorianus proteinase A gene comprises the steps of total RNA extraction, reverse transcription reaction for preparing cDNA template, multiple PCR reaction, capillary electrophoresis and product fragment analysis.
(1) Multiplex primer design
According to the industrial Saccharomyces pastorianus protease A genePEP4-Sc，PEP4-SbAnd reference geneACT1-Sc、ACT1-SbThe full-length sequence of (1) is designed into a specific upstream and downstream multiplex primer suitable for GeXP detection (Table 1).
TABLE 1 specific upstream and downstream primers for the protease A gene of industrial Saccharomyces pastorianus and the reference gene
(2) Total RNA extraction and purification
1) Activated culture of yeast
Taking a loop of the slant strain by using an inoculating needle, inoculating the slant strain into a test tube containing 5 mL of sterile wort, and culturing for 24 h at 25 ℃;
3-5 loops were placed in another tube containing 5 mL of wort and incubated at 25 ℃ for 24 h.
2) Pretreatment of yeast
Centrifuging the bacterial suspension at 12000 rpm for 2 min;
discard the supernatant, every 3X 108Rapidly adding 0.5-1 mL of RNAlater solution into each yeast cell;
storing in a refrigerator at 4 ℃ for 1 h or treating at night;
centrifuging at 12000 rpm for 2 min, discarding supernatant, storing in refrigerator at-80 deg.C, and taking out when extracting RNA.
3) Extraction of Yeast cell RNA
And (3) extracting the yeast RNA by stepwise operation according to the flow of the RNA extraction kit.
200 mL of phosphate buffer was added to the sample, centrifuged at 12000 rpm, and the supernatant was removed. Adding 200 mL of phosphate buffer solution for resuspension, adding 20 mL of muramidase, and treating at 30 ℃ for 45 min;
adding 600 mL of lysis solution, adding 10 mL of 2-mercaptoethanol into 1 mL of lysis solution, centrifuging at 12000 rpm for 2 min at room temperature, and transferring the supernatant to a centrifuge tube of RNase-free;
587 mL of absolute ethanol was added and vortexed thoroughly. Taking 500 mL to a column, and centrifuging at 12000 rpm for 15 s;
700 mL of washing buffer I was added and centrifuged at 12000 rpm for 15 s. Adding 500 mL of washing buffer solution II, and centrifuging at 12000 rpm for 15 s;
adding 30 mL of RNase-Free water with the temperature of 80 ℃, and centrifuging for 2 min at the room temperature of 12000 rpm;
adding 2 mL of DNase I, treating at 37 ℃ for 40 min, adding 2 mL of DNase inactivating agent, standing at room temperature for 5 min, centrifuging at 12000 rpm for 2 min to obtain purified total RNA, immediately detecting the concentration, and storing in a refrigerator at-80 ℃; and (3) RNA purity determination: if the measured OD260/280 is between 1.8-2.1, it can be used in the subsequent experiment, otherwise it is re-extracted.
(3) Preparation of cDNA template by reverse transcription reaction
The first strand of cDNA was synthesized using yeast total RNA as template, and the downstream primer of the multiplex primers in Table 1 was the specific primer.
The reaction parameters for preparing cDNA template by reverse transcription reaction are set as follows:
48℃，1 min；42℃，60 min；95℃，5 min。
(4) multiplex PCR reaction
The first strand of the cDNA synthesized above was used as a template, and the upstream primer of the multiplex primers in Table 1 was used as a specific primer to perform a multiplex PCR amplification reaction.
The PCR amplification parameters were set as follows:
pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 s; the annealing temperature is 55 ℃ and 30 s; the extension is carried out for 1 min at 70 ℃, and the whole PCR amplification reaction process is circulated for 35 times.
(5) Multiple PCR product capillary electrophoresis
mu.L of the PCR multiplex product was added to the wells of the upper plate containing 39. mu.L of a mixture of 95% deionized formamide (SLS) and 400 bp Marker, mixed well with a pipette and covered with a drop of paraffin oil. Additionally 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
Detecting the expression quantity of the protease A gene according to the detection process of the GeXP multifunctional genetic analysis system: firstly, performing capillary electrophoresis separation, and analyzing and recording the result by utilizing GeXP system parameters, wherein the result is shown in figure 1. Each peak represents the corresponding gene, and the area of each peak corresponds to the expression level of the gene, wherein the peak area results of each peak are shown in Table 2. And searching the corresponding peak area according to the size of the protease A gene, the size of the internal reference gene and the size of the system control. The peak image of the system control represents whether the capillary electrophoresis result is normal or not.
TABLE 2 Peak area results corresponding to protease A Key genes and reference genes
| Serial number | Name of Gene | Peak area |
| 1 | PEP4-Sc | 37063 |
| 2 | PEP4-Sb | 7946 |
| 3 | ACT1-Sc | 3702 |
| 4 | ACT1-Sb | 1293 |
(7) And calculating the expression level of the protease A gene by taking the internal reference gene as a control.
PEP4-ScGene expression level =PEP4-ScPeak area/[ (/), (ACT1-ScPeak area +ACT1-SbPeak area)/2]
PEP4-SbGene expression level =PEP4-SbPeak area/[ (/), (ACT1-ScPeak area +ACT1-SbPeak area)/2]
Is calculated to obtainPEP4-ScThe gene expression level was 14.84,PEP4-Sbthe gene expression level is 3.18, and the Sc-derived protease A gene expression level is far higher than that of the Sb-derived protease A gene expression level, and the Sc-derived protease A gene expression level has larger fluctuation under different conditions and is greatly influenced by external environment, so that the Sc-derived protease A gene plays a leading role in the yeast strain, and therefore, the yeast strain is screened according to the Sc-derived protease A gene expression level and judged to be the X-producing protease A strain.
Example 2
A, B, C three different yeast strains were selected, and the expression level of the protease A gene was measured in the same manner as in example 1.
The three yeast strains are respectively expanded and cultured step by step and are used for brewing experiments of 100L beer. Except for the strain, the production process parameters are kept consistent.
(1) Yeast expanding culture process
Selecting a ring from the strain on the preservation slant by using an inoculating needle, inoculating into a test tube containing 5 mL of sterile wheat juice, shaking uniformly, and performing activation culture at 25 ℃ for 24 hours; sucking 0.5 mL of the bacterial suspension, inoculating the bacterial suspension into another test tube containing 5 mL of sterile wort, shaking up, and performing activation culture at 25 ℃ for 24 hours; sucking 1 mL of bacterial suspension, inoculating into a test tube containing 10 mL of sterile wort, shaking up, and performing activated culture at 25 ℃ for 24 hours; inoculating 10 mL of the bacterial suspension into a triangular flask containing 200 mL of sterile wort, shaking up, and performing activated culture at 22 ℃ for 24 hours; inoculating 200 mL of the bacterial suspension into a triangular flask containing 2L of sterile wort, shaking up, and performing activated culture at 19 ℃ for 24 hours; inoculating 2L of the bacterial suspension into a Kaschin jar containing 20L of sterile wort, shaking up, and performing activated culture at 16 ℃ for 24 h; 20L of the bacterial suspension was inoculated into a fermentor containing 100L of wort.
(2) Fermentation process conditions
The temperature of the full tank is 10 ℃, the yeast number of the full tank of 13-DEG P wort is 22.0 MM/mL, the temperature of the main fermentation is 10 ℃, the apparent sugar degree is reduced to 5 DEG P, the temperature is raised to 12 ℃, the yeast is recovered, the tank is sealed when the apparent sugar degree is reduced to 3.0 ℃, diacetyl reduction is carried out, the diacetyl is cooled quickly when the diacetyl is reduced to 10 mg/L, the temperature of wine storage is 0 to-1.5 ℃, the wine storage time is 7 days, and the soaking property of the cold-stored wine is measured after filtering.
(3) Analysis of results
TABLE 3 results of different strains
| Yeast strains | Amount of protease A Gene expressed | Protease A Activity | Capacity of cold storage wine (second) |
| A | 1.6 | 15 | 225 |
| B | 8.3 | 39 | 203 |
| C | 15.5 | 71 | 167 |
From the results in Table 3, it was found that the protease A gene expression level of yeast A was low and the cold storage wine-retention property was high; the expression level of protease A gene of yeast C is high, and the cold storage wine soaking property is low. The expression quantity of the protease A gene directly reflects the activity of the protease A, so that the protease A gene expression yeast strain can be rapidly screened by using the protease A gene expression yeast strain. Compared with a yeast strain with low protease A gene expression screened by measuring the activity of the protease A, the yeast strain with low protease A gene expression screened by detecting the protease A gene expression has the advantages of short time consumption and accurate result, and meanwhile, because the GeXP multifunctional genetic analysis system is adopted in the process, the expression quantity of the key genes of the yeast protease A can be rapidly, accurately and high-flux detected, and the method has the characteristics of stronger specificity, sensitivity, automation degree and the like, and ensures the reliability and repeatability of the result.
Sequence listing
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Claims (5)
1. A method for screening a yeast strain with low protease A gene expression by using multiple primers is characterized by comprising the following steps:
carrying out reverse transcription synthesis of a cDNA template, multiple PCR amplification reaction and capillary electrophoresis separation in sequence by taking the total RNA of the yeast strain as a template, and analyzing the capillary electrophoresis separation result by utilizing a GeXP system;
calculating the expression quantity of the protease A gene by taking the internal reference gene as a control and combining the peak area of the peak corresponding to the key gene, and judging the yeast strain to be a low-yield protease A strain, a medium-yield protease A strain or a high-yield protease A strain according to the expression quantity of the protease A gene;
the multiple primers comprise key genes according to the yeast protease APEP4Homologous gene of (2)PEP4-ScAndPEP4-Sbrespectively designing specific upstream and downstream primers, which comprises the following steps:
the reference gene comprisesACT1Homologous gene of (2)ACT1-Sc、ACT1-SbDesigning specific upstream and downstream primers according to an internal reference gene, comprising:
the calculation and judgment method for calculating the expression quantity of the protease A gene by taking the internal reference gene as a control and combining the peak area of the corresponding peak of the key gene comprises the following steps:
PEP4-Scgene expression level =PEP4-ScPeak area/[ (/), (ACT1-ScPeak area +ACT1-SbPeak area)/2]
When the expression amount of the protease A gene is less than 3, judging that the yeast strain is a low-yield protease A strain; when the expression quantity of the 3< protease A gene is less than 10, judging the yeast strain to be a medium-yield protease A strain; when the expression quantity of the protease A gene is more than or equal to 10, the yeast strain is judged to be the high-yield protease A strain.
2. The method of claim 1, wherein the first strand of the cDNA template is synthesized by reverse transcription using yeast strain total RNA as a template and downstream primers of the sequences SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, and SEQ ID No.8 as specific primers.
3. The method of claim 2, wherein the reaction parameters for reverse transcription of the synthetic cDNA template are set as: 48 ℃ for 1 min; 60 min at 42 ℃; 95 ℃ for 5 min.
4. The method of claim 1, wherein the multiplex PCR amplification reaction is performed using the first strand of the synthesized cDNA template as a template and the forward primers of the sequences SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7 as specific primers.
5. The method of claim 4, wherein the parameters of the multiplex PCR amplification reaction are set to: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 s; the annealing temperature is 55 ℃ and 30 s; the extension is carried out for 1 min at 70 ℃, and the whole PCR amplification reaction process is circulated for 35 times.
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| CN104403957A (en) * | 2014-12-16 | 2015-03-11 | 天津科技大学 | Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain |
| CN105385615A (en) * | 2015-12-28 | 2016-03-09 | 天津科技大学 | Saccharomyces cerevisiae strain with high yield of ester and low yield of higher alcohol as well as building and application of saccharomyces cerevisiae strain |
| CN108866027A (en) * | 2014-12-02 | 2018-11-23 | 天津科技大学 | Application of the VPS10 gene in the low secretory protein A of Wine brewing yeast strain |
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| CN108866027A (en) * | 2014-12-02 | 2018-11-23 | 天津科技大学 | Application of the VPS10 gene in the low secretory protein A of Wine brewing yeast strain |
| CN104403957A (en) * | 2014-12-16 | 2015-03-11 | 天津科技大学 | Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain |
| CN105385615A (en) * | 2015-12-28 | 2016-03-09 | 天津科技大学 | Saccharomyces cerevisiae strain with high yield of ester and low yield of higher alcohol as well as building and application of saccharomyces cerevisiae strain |
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