CN114959097A - Primer, kit and method for judging flocculation strength of yeast - Google Patents
Primer, kit and method for judging flocculation strength of yeast Download PDFInfo
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- 238000005189 flocculation Methods 0.000 title claims abstract description 81
- 230000016615 flocculation Effects 0.000 title claims abstract description 81
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- 241001123227 Saccharomyces pastorianus Species 0.000 claims abstract description 36
- 238000012163 sequencing technique Methods 0.000 claims abstract description 19
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 230000003311 flocculating effect Effects 0.000 claims abstract description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 73
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 239000012224 working solution Substances 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 3
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- 238000009395 breeding Methods 0.000 abstract description 6
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- 239000012634 fragment Substances 0.000 description 4
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- 241000894006 Bacteria Species 0.000 description 3
- 101100066910 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FLO1 gene Proteins 0.000 description 3
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- 210000005253 yeast cell Anatomy 0.000 description 3
- 101100083069 Candida albicans (strain SC5314 / ATCC MYA-2876) PGA62 gene Proteins 0.000 description 2
- 101100106993 Candida albicans (strain SC5314 / ATCC MYA-2876) YWP1 gene Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 101150054379 FLO1 gene Proteins 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
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- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 101100120289 Drosophila melanogaster Flo1 gene Proteins 0.000 description 1
- 101100013145 Drosophila melanogaster Flo2 gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- 101150006710 FLO8 gene Proteins 0.000 description 1
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 description 1
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- 238000012408 PCR amplification Methods 0.000 description 1
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- 101100066906 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FLO10 gene Proteins 0.000 description 1
- 101100066911 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FLO5 gene Proteins 0.000 description 1
- 101100446801 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FLO9 gene Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
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- 238000005842 biochemical reaction Methods 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
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- 235000015095 lager Nutrition 0.000 description 1
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- 108010056929 lyticase Proteins 0.000 description 1
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- 235000008935 nutritious Nutrition 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/6869—Methods for sequencing
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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Abstract
The invention discloses a primer, a kit and a method for judging flocculation strength of yeast, and belongs to the field of yeast detection. The technical scheme comprises a sequencing comparison step, wherein the sequencing comparison step comprises comparison of sequencing results, and the yeast strain is a strong flocculation yeast strain with an amplification product of FLO11-Sc gene in the yeast strain having an insert of 171bp shown as SEQID NO. 3; the yeast strain in which the amplification product of the FLO11-Sc gene lacks the 171bp insert is a weakly flocculating yeast strain. The method is applied to the aspect of flocculation strength of the Lager yeast, solves the problem that flocculation difference judgment cannot be carried out on different yeast strains due to diversity of the Lager yeast when the flocculation strength of the Lager yeast is evaluated by a gene means in the prior art, can evaluate the flocculation capacity of the yeast in advance in a breeding stage, improves the breeding efficiency, and saves time and cost.
Description
Technical Field
The invention belongs to the field of yeast detection, and particularly relates to a primer, a kit and a method for judging the flocculation strength of yeast.
Background
Beer is a low-alcohol and nutritious beverage well liked by people. Over 90% of the global beer market today is Lager-type beer, and therefore the bottom brewer's yeast (commonly known as "Lager yeast" or "pasteur yeast") used to brew Lager beer has been most widely used in the beer industry. Yeast flocculation is the yeast cells from the medium from the suspension state aggregation agglomeration phenomenon. The Lager yeast can quickly settle to the bottom of the fermentation tank after the main fermentation is finished. Among various properties of yeast, flocculation is an extremely important performance index throughout the beer fermentation process, and the yeast cell number should be kept in a relatively stable range in each fermentation stage to ensure the normal operation of beer production and further ensure the consistency of fermentation and the stability of taste quality of products, so that the flocculation of the yeast is closely related to the fermentation performance of the yeast, the generation and reduction of metabolites and other biochemical reactions, and the abnormal fluctuation of the flocculation of the yeast can seriously affect the taste and production efficiency of the products in the beer brewing process. Thus, flocculation is an important property of brewers yeast. For beer production, flocculation capability of beer yeast cells is a key index for evaluating the performance of strains.
Different yeast strains have different physiological characteristics and different flocculation strengths. The flocculation of saccharomyces cerevisiae (s. cerevisiae) is determined by its genetic background. There are a plurality of genes (including FLO1, FLO2, FLO4, FLO5, FLO8, FLO9, FLO10, FLO11, etc.) involved in expression of flocculation protein, forming a flocculation protein gene family. It was found that there are differences in the genetic composition of flocculation families of different yeast strains, of which the most extensively studied is FLO1 in Saccharomyces cerevisiae, whose ORF is about 4.6Kb, with a number of repeats in the middle, encoding a Ser/Thr-rich flocculation protein of 1537 amino acids (Flo1 p). Unlike s.cerevisiae, the industrial Lager yeast s.pastoris strain is an allopolyploid yeast strain with high chromosomal ploidy and complex chromosomal structure composition. For Lager yeast s.pastoris, after long-term use and continuous domestication in different industrial environments, the genome of the strain is changed to different degrees, so that the particularity and complexity of the industrial Lager yeast s.pastoris strain are derived, which is also the essential reason for the diversification of flocculation, fermentation performance and flavor characteristics of different industrial Lager yeast s.pastoris strains.
The determination of the flocculation strength of the yeast strain is very important through the flocculation gene sequence of the Lager yeast, and the flocculation characteristics of the strain can be rapidly known. But as the flocculation property in the yeast is determined by a multigene family, the number of genes is large, and the genes are longer (-4 Kb); sequence similarity among different genes is high, and a large number of repeated fragments exist in the genes, which increases difficulty for primer design work of gene detection; especially for Lager yeast, which contains gene copies (-Sc/-Sb) from different sources and has higher sequence similarity, and the gene from the same source may have multiple copies, so the difficulty of gene detection work is increased.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the technical problem that the flocculation difference judgment of different yeast strains cannot be carried out due to the diversity of Lager yeast when the flocculation strength of the Lager yeast is evaluated by a gene means in the prior art, and provides the primers, the kit and the method for judging the flocculation strength of the yeast, which can evaluate the flocculation capability of the yeast in advance in a breeding stage and improve the breeding efficiency so as to save time and cost.
In order to solve the technical problem, the technical scheme adopted by the invention is as follows:
the invention provides a primer for judging the flocculation strength of yeast, wherein an upstream primer and a downstream primer are designed according to a flocculation gene FLO11-sc of Lager yeast, and the sequences of the primers are as follows:
| name of Gene | Primer sequences | |
| FLO11-sc | FW | SEQ ID NO.1 |
| FLO11-sc | RV | SEQ ID NO.2 |
。
Preferably, the design of the primer comprises:
searching a flocculation gene FLO11-sc in a saccharomyces cerevisiae S.cerevisiae, and searching a homologous gene sequence in a S.pastoris TT-21 genome through BLASTn sequence comparison;
the sequenced gene information of the Lager yeast TT-21 is utilized to carry out CLUSTAL W comparison on the same gene from different sources (-Sc/-Sb), searching for non-homologous sections to carry out primer design respectively, and enabling the non-homologous sections to be positioned at the 3' tail end of the primer.
Preferably, the primer is designed to satisfy the following requirements: in order to obtain longer PCR products for comparison among different yeast strains, the upstream and downstream primers are as close to the two ends of the gene sequence as possible, the Tm value is 55-65 ℃, and the secondary structure and mismatching of the primers are avoided.
In another aspect, the present invention provides a kit comprising the primer according to any one of the above technical schemes.
The invention also provides a method for determining the flocculation strength of the yeast by using the primer in any technical scheme.
Preferably, a sequencing alignment step is included, the sequencing alignment step comprises aligning the sequencing results, and
the yeast strain is a strong flocculation yeast strain with the amplification product of the FLO11-Sc gene having the insert of 171bp shown in SEQ ID NO. 3;
the yeast strain in which the amplification product of the FLO11-Sc gene lacks the 171bp insert is a weakly flocculating yeast strain.
Preferably, the sequencing and aligning step further comprises: a primer preparation step, a template DNA dilution step, a PCR reaction step and an agarose electrophoresis detection step.
Preferably, the method further comprises the step of extracting the DNA of the Lager yeast strain.
Preferably, the primer formulation step comprises: the primers were dissolved in 10mM Tris-HCl, pH8.0, and prepared into 100. mu.M primer stock solution, which was further diluted to 10. mu.M as working solution.
Preferably, the template DNA dilution step comprises diluting the DNA sample to 10 ng. mu.L -1 。
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for judging the flocculation strength of Lager yeast based on a gene sequence, which can quickly judge the flocculation strength of a Lager yeast strain without fermentation experiments, and can evaluate the flocculation capacity of the yeast in advance in a breeding stage to improve the breeding efficiency, thereby saving time and cost.
Detailed Description
The technical solutions in the embodiments of the present invention will be fully described in detail below. It is obvious that the described embodiments are only some specific embodiments, not all embodiments, of the general technical solution of the present invention. All other embodiments, which can be derived by a person skilled in the art from the general idea of the invention, fall within the scope of protection of the invention.
The invention provides a primer for judging the flocculation strength of yeast, wherein an upstream primer and a downstream primer are designed according to a flocculation gene FLO11-sc of Lager yeast, and the sequences of the primers are as follows:
| name of Gene | Primer sequences | |
| FLO11-sc | FW | SEQ ID NO.1 |
| FLO11-sc | RV | SEQ ID NO.2 |
。
In a preferred embodiment, the design of the primers comprises:
s1: searching flocculation gene FLO11-sc in model bacteria Saccharomyces cerevisiae S.cerevisiae, and searching homologous gene sequence in S.pastorianus TT-21 genome by BLASTn sequence comparison. Optionally, a flocculation gene FLO11-sc in a model bacterium Saccharomyces cerevisiae S.cerevisiae is searched on Genbank of NCBI. It should be noted that, regarding the determination of FLO11-sc, the technical scheme of the present application finally locks the key difference gene to be FLO11-sc by comparing 10 different copies of 5 genes of the FLO flocculation family, and the technical scheme of the present application limits the use of gene FLO11-sc because only in the case of selecting the gene, the correlation between the genotype difference and the flocculation strength difference of different Lager yeast strains can be detected, and the gene is used for primer design and detection, so that the flocculation strength of different Lager yeasts can be accurately detected, and the determination that the flocculation property of only one yeast strain can be determined by the existing detection means is overcome. Therefore, the prior art can only detect the change (existence or nonexistence) of the flocculation property of the yeast strain before and after fermentation so as to judge whether the strain can be continuously used by passage, and the scheme can judge the difference of the flocculation property of different yeast strains.
S2: the sequenced gene information of the Lager yeast TT-21 is utilized to carry out CLUSTAL W comparison on the same gene from different sources (-Sc/-Sb), searching for non-homologous sections to carry out primer design respectively, and enabling the non-homologous sections to be positioned at the 3' tail end of the primer. The present embodiment specifically defines that "locating the non-homologous segment at the 3' end of the primer" is advantageous to enhance the specificity of the amplification product.
In a preferred embodiment, the primers are designed such that: the length of the PCR product is as long as possible, the Tm value is 55-65 ℃, and the secondary structure and mismatching of the primer are avoided. The technical scheme specifically limits the three conditions required to be met by the design of the primer so as to enhance the specificity of the amplification product.
In another aspect, the present invention provides a kit comprising the primer according to any one of the above technical schemes.
The invention also provides a method for determining the flocculation strength of the yeast by using the primer in any technical scheme. Preferably, a method for determining the flocculation strength of the Lager yeast by using the primer in any one technical scheme is provided. The primer is used for judging the flocculation degree of the yeast, so that the problem that flocculation difference judgment cannot be carried out on different yeast strains due to the diversification of the Lager yeast can be solved, the flocculation degree of different types of Lager yeast can be accurately detected, and the problem that only one type of yeast can be judged by the existing detection means, and the variation probability in the yeast group is judged is solved.
In a preferred embodiment, a sequencing alignment step is included, the sequencing alignment step comprises aligning the sequencing results, and
the yeast strain is a strong flocculation yeast strain with the amplification product of the FLO11-Sc gene having the insert of 171bp shown in SEQ ID NO. 3;
the yeast strain in which the amplification product of the FLO11-Sc gene lacks the 171bp insert is a weakly flocculating yeast strain. The technical scheme of the application takes the 171bp insert as the judgment index of the flocculation strength of the yeast strain, and the reason is the correlation between the flocculation genotype and the flocculation phenotype of the yeast.
In a preferred embodiment, the sequencing alignment step further comprises: a primer preparation step, a template DNA dilution step, a PCR reaction step and an agarose electrophoresis detection step. Wherein the reaction system of the PCR reaction step is as follows:
| reaction system | Addition amount (μ L) |
| H 2 O | 10.1 |
| 25mM MgCl 2 | 0.1 |
| DNTP | 2 |
| PCR Buffer 5× | 4.0 |
| Phusion DNAPolymerase | 0.2 |
| Primer(Forward&Reverse) | 0.8/Each |
| Template DNA | 2 |
。
The reaction program of the PCR reaction step is as follows:
| step (ii) of | Temperature of | Time |
| 1 | 98℃ | 30s |
| 2 | 98℃ | 10s |
| 3 | Tm℃ | 15s |
| 4 | 72℃ | 1min |
| 5 | N/A | Repeating the steps 2-4 for 29 cycles (total 30 cycles) |
| 6 | 72℃ | 1min |
| 7 | 4℃ | Preservation of |
In a preferred embodiment, the method further comprises a step of extracting the DNA of the Lager yeast strain, and specifically comprises the following steps: culturing yeast in YPD for 48 hr, centrifuging 800 μ L bacterial liquid at 4500rpm for 5 min; discarding the supernatant, adding 500. mu.L PBS buffer solution, and resuspending the thallus; centrifuging at 4500rpm for 5min, removing supernatant, adding 160 μ L PBS buffer solution and 20 μ L Lyticase, and resuspending thallus; carrying out DNA extraction of yeast on the full-automatic nucleic acid extraction; after extraction, DNA concentration and quality are detected by a micro ultraviolet visual instrument.
In a preferred embodiment, the primer formulation step comprises: the primers were dissolved in 10mM Tris-HCl, pH8.0, and prepared into 100. mu.M primer stock solution, which was further diluted to 10. mu.M as working solution.
In a preferred embodiment, the template DNA dilution step comprises diluting the DNA sample to 10 ng. mu.L -1 。
In order to more clearly and specifically describe the primers, the kit and the method for determining the flocculation strength of the yeast provided in the embodiments of the present invention, the following description will be given with reference to specific embodiments.
Example 1
1. Extraction of DNA from Lager yeast strains
1) Culturing yeast in YPD for 48 hr, centrifuging 800 μ L bacterial liquid at 4500rpm for 5 min;
2) discarding the supernatant, adding 500. mu.L PBS buffer solution, and resuspending the thallus;
3) centrifuging at 4500rpm for 5min, discarding supernatant, adding 160 μ L PBS buffer solution and 20 μ L LLyticase, and resuspending thallus;
4) carrying out DNA extraction of yeast on the full-automatic nucleic acid extraction;
5) after extraction, DNA concentration and quality are detected by a micro ultraviolet visual instrument.
2. Designing and synthesizing a flocculation gene FLO11-sc amplification primer of Lager yeast;
flocculation gene FLO11-sc in lager yeast: the flocculation gene FLO11-sc in the model bacterium Saccharomyces cerevisiae S.cerevisiae is searched on the Genebank of NCBI, and the homologous gene sequence in the S.pastoris TT-21 genome is searched through BLASTn sequence comparison.
Primer design of flocculation gene FLO11-sc of Lager yeast:
the sequenced gene information of the Lager yeast TT-21 is utilized to carry out CLUSTAL W comparison on the same gene from different sources (-Sc/-Sb), search non-homologous sections to carry out primer design respectively, and the non-homologous sections are positioned at the 3' tail end of the primer as far as possible. Designing flocculation family gene upstream and downstream primers by using Primer Primier 5.0 software, wherein the design principle is as follows: the PCR product length was as long as possible (differences were observed with more probability), the Tm was between 55-65 ℃ and secondary structure and mismatches of the primers were avoided as much as possible. All primers were synthesized by the manufacturer. The designed upstream and downstream primers are shown in Table 1.
TABLE 1 primer sequences
| FLO11-sc-F-FW | GACAACACATACCAAATCA |
| FLO11-sc-F-RV | GCAGCAGAACATTCAGTA |
。
3. PCR-based method for detecting flocculation gene FLO11-sc of Lager yeast
1) Preparing a primer: each primer (forward & reverse) was dissolved in 10mM Tris-HCl, pH8.0, and prepared as 100. mu.M primer stock; further diluted to 10. mu.M as a working solution.
2) Template DNA: the DNA samples were diluted to 10 ng. mu.L-1 (DNase/RNase Free H) 2 O-Nuclear-Free Water dilution).
3) And (3) PCR reaction: PCR amplification reactions were performed with 2 parallel channels per sample, and the PCR reaction system is shown in Table 2.
TABLE 2 PCR reaction System
The PCR reaction procedure is shown in Table 3.
TABLE 3 PCR reaction procedure
| Step (ii) of | Temperature of | Time |
| 1 | 98℃ | 30s |
| 2 | 98℃ | 10s |
| 3 | Tm℃ | 15s |
| 4 | 72℃ | 1min |
| 5 | N/A | Repeating the steps 2-4 for 29 cycles (30 cycles in total) |
| 6 | 72℃ | 1min |
| 7 | 4℃ | Preservation of |
4) Agarose electrophoresis detection
5) Sequencing all sequencing was done by the worker.
4. And (3) comparing sequencing results, namely comparing and analyzing the sequencing results by utilizing the MegAlign function in the Lasergene software.
Amplification products of FLO11-Sc gene in strongly flocculating yeast strains (TT01& TT 04): an insert of 171bp shown as SEQ ID NO.3 exists in FLO11-Sc gene
Amplification product of FLO11-Sc gene in weakly flocculating yeast strain (TT02& TT03& TT05& TT 06): the 171 bp-deficient fragment
5. Result verification
And (3) optimizing an ASBC absorbance method and detecting the flocculation capacity of the yeast strain. The optimized method comprises the following steps:
(1) mixing the fermented liquids, and respectively taking out the same bacterial concentration (OD) 600 Value is 6.5-7) putting the fermentation liquid for 72h in a 15mL centrifuge tube, and marking as A, B;
(2) the following operations were performed for tube a: centrifuging to collect precipitate, transferring 9mL distilled water and 1mL 250mmol/L EDTA (pH 4.5), suspending the bacterial sludge, shaking, mixing, diluting 1mL suspension 10 times, and measuring OD 600 And the value was recorded as OD A ；
(3) The following operations were performed for tube B: centrifuging to obtain precipitate, adding 10mL of 0.051% CaSO 4 And (3) suspending the bacterial sludge in the solution, centrifuging to obtain a precipitate, adding a calcium sulfate buffer solution for washing, oscillating on a vortex oscillator for 15s, and vertically placing a centrifugal tube for 6 min. 1mL of the top liquid was diluted 3-fold and the OD was measured 600 And the value was recorded as OD B 。
(4) Flocculation number F ═ 100X ((OD) A -OD B )/OD A )
In the formula: f, flocculation ability of yeast strain,%; OD A OD measured after treatment of tube A 600 A value; OD B OD measured after treatment of tube B 600 The results are shown in Table 4.
TABLE 4 flocculation number F
| Yeast strains | Flocculation number |
| TT01 | 77.6±4.3 |
| TT02 | 66.3±2.9 |
| TT03 | 68.1±3.7 |
| TT04 | 73.1±3.7 |
| TT05 | 64.4±2.8 |
| TT06 | 60.9±2.9 |
As can be seen from the flocculation value F, the flocculation properties of strains TT01 and TT04 are significantly higher than those of strains TT02, TT03, TT05 and TT 06. This result is consistent with the judgment from the gene amplification result: the strongly flocculating yeast strain FLO11-Sc gene has an insert of 171bp, while the weakly flocculating yeast strain lacks the 171bp fragment. The flocculation strength of the reaction strain can be accurately reflected by the detection method of the technical scheme of the application, and the method has the characteristics of simplicity, convenience and quickness.
<110> Qingdao beer Ltd
<120> primer, kit and method for judging flocculation strength of yeast
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> upstream primer
<400> 1
GACAACACAT ACCAAATCA
<210> 2
<211> 18
<212> DNA
<213> downstream primer
<400> 2
GCAGCAGAAC ATTCAGTA
<210>3
<211> 171
<212> DNA
<213> Gene fragment
<400> 3
TGCTCCATCT TCAACTCCAT TCAGCTCTAG CACTGAAAGC TCTTCTGTTC CAGTACCAAC CCCATCAAGC TCTACTACTG AAAGCTCTTC TGCTCCAGTA TCCAGCTCCA CCACTGAAAG CTCTGTAGCA CCAGTACCAA CCCCATCTTC CTCTAGCAAC ATCACTTCCT C
Claims (10)
1. A primer for judging the flocculation strength of yeast is characterized in that an upstream primer and a downstream primer are designed according to a flocculation gene FLO11-sc of Lager yeast, and the sequences of the primers are as follows:
。
2. The primer for determining flocculation strength of yeast according to claim 1, wherein the design of the primer comprises:
searching a flocculation gene FLO11-sc in a saccharomyces cerevisiae S.cerevisiae, and searching a homologous gene sequence in a S.pastoris TT-21 genome through BLASTn sequence comparison;
the sequenced gene information of Lager yeast TT-21 is utilized to carry out CLUSTAL W comparison on the same gene from different sources (-Sc/-Sb), searching non-homologous sections to carry out primer design respectively, and enabling the non-homologous sections to be positioned at the 3' tail end of a primer.
3. The primer for determining flocculation strength of yeast according to claim 1, wherein the primer is designed to satisfy the following requirements: in order to obtain longer PCR products for comparison among different yeast strains, the upstream and downstream primers are as close to the two ends of the gene sequence as possible, the Tm value is 55-65 ℃, and the secondary structure and mismatching of the primers are avoided.
4. A kit comprising the primer for determining flocculation strength of yeast according to any one of claims 1 to 3.
5. A method for determining flocculation strength of yeast by using the primer of any one of claims 1 to 3.
6. The method of claim 5, comprising a sequencing alignment step, wherein the sequencing alignment step comprises aligning sequencing results, and wherein
The yeast strain is a strong flocculation yeast strain with the amplification product of the FLO11-Sc gene having the insert of 171bp shown in SEQ ID NO. 3;
the yeast strain in which the amplification product of the FLO11-Sc gene lacks the 171bp insert is a weakly flocculating yeast strain.
7. The method of claim 6, further comprising, prior to the sequencing alignment step: a primer preparation step, a template DNA dilution step, a PCR reaction step and an agarose electrophoresis detection step.
8. The method according to claim 6, further comprising a step of extracting DNA from the Lager yeast strain.
9. The method of claim 6, wherein the primer formulation step comprises: the primers were dissolved in 10mM Tris-HCl, pH8.0, and prepared into 100. mu.M primer stock solution, which was further diluted to 10. mu.M as working solution.
10. The method of claim 6, wherein the template DNA dilution step comprises diluting the DNA sample to 10 ng- μ L -1 。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115895926A (en) * | 2022-09-30 | 2023-04-04 | 青岛啤酒股份有限公司 | FLO1 gene deletion mutant strain based on CRISPR-Cas9 system, construction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090011080A1 (en) * | 2006-02-28 | 2009-01-08 | Suntory Limited | Gene encoding protein responsible for flocculation property of yeast and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090011080A1 (en) * | 2006-02-28 | 2009-01-08 | Suntory Limited | Gene encoding protein responsible for flocculation property of yeast and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| MANUEL FIDALGO等: "Coding repeat instability in the FLO11 gene of Saccharomyces yeasts", YEAST, vol. 25, no. 12, 31 December 2008 (2008-12-31), pages 1 - 3 * |
| SEBASTIAAN E等: "Flocculation gene variability in industrial brewer’s yeast strains", APPL MICROBIOL BIOTECHNOL, vol. 88, 31 August 2010 (2010-08-31), pages 1321, XP019862128, DOI: 10.1007/s00253-010-2843-5 * |
| 郭立芸，等: "应用絮凝基因表达量分析技术评价酵母菌株的絮凝性", 食品与发酵工业, vol. 44, no. 5, 31 January 2018 (2018-01-31), pages 70 - 75 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115895926A (en) * | 2022-09-30 | 2023-04-04 | 青岛啤酒股份有限公司 | FLO1 gene deletion mutant strain based on CRISPR-Cas9 system, construction method and application thereof |
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