CN114480444B - Degenerate primers for amplifying microbial alcohol dehydrogenase gene fragments and uses of the degenerate primers - Google Patents
Degenerate primers for amplifying microbial alcohol dehydrogenase gene fragments and uses of the degenerate primers Download PDFInfo
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- 238000012408 PCR amplification Methods 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
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- 238000012257 pre-denaturation Methods 0.000 claims description 3
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- 241000228212 Aspergillus Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 241000043309 Enterobacter hormaechei Species 0.000 description 5
- 241000566145 Otus Species 0.000 description 4
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- 241000589220 Acetobacter Species 0.000 description 2
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- 241000233866 Fungi Species 0.000 description 2
- 241000186359 Mycobacterium Species 0.000 description 2
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- 238000010586 diagram Methods 0.000 description 2
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- 241000221020 Hevea Species 0.000 description 1
- 108010011927 Long-chain-alcohol dehydrogenase Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical group [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
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- 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 provides a degenerate primer for amplifying microbial alcohol dehydrogenase gene fragments, and also provides a detection method for amplifying microbial alcohol dehydrogenase gene fragments by using the degenerate primer. The invention starts from the structural domain of alcohol dehydrogenase amino acid, designs degenerate primers for amplifying microbial alcohol dehydrogenase gene fragments, and applies the degenerate primers to detection to determine alcohol dehydrogenase genes, thereby determining the types and abundance of potential higher alcohol-producing microorganisms in a detection sample, and laying a foundation for further research on the compositions of the potential higher alcohol-producing microorganisms in the sample.
Description
[ Field of technology ]
The invention belongs to the technical field of gene detection, and particularly relates to a degenerate primer and a detection method for amplifying a microbial alcohol dehydrogenase gene fragment, and a method for detecting the abundance of a microorganism which potentially produces higher alcohol in Daqu by using the primer.
[ Background Art ]
Higher alcohols (alcohols having more than 2 carbon atoms) have sweet and fragrant functions, are also important precursors for forming flavor substances such as esters and aldehydes, and play an important role in the composition of traditional fermented food flavor substances. However, when the higher alcohol content is too high, not only abnormal flavor but also health risks are caused.
The method is an effective method for controlling the content of higher alcohols in fermented foods by determining the microorganisms with higher alcohol production capacity in the mixed microorganism fermentation process involving various microorganisms and further selecting suitable seeds (yeast) for fermentation through proportioning. However, current methods of associative analysis based on microbial diversity do not establish a direct link between higher alcohols and microorganisms.
Functional genes in metabolic pathways can be used as marker genes, which not only characterize community metabolic functions, but also further study potential metabolite producers in mixed microorganisms. Alcohol dehydrogenases (Alcohol dehydrogenase, ADHs) are critical in microbial higher alcohol metabolism according to existing microbial higher alcohol metabolism pathways (FIG. 1). The microbial alcohol dehydrogenase gene fragment in the strain (yeast) is amplified by designing degenerate primers, the sequence information is obtained by utilizing a high-throughput sequencing technology, and the strain proportion is determined by further analyzing the potential higher alcohol-producing microorganisms by utilizing a bioinformatics technology, so that the product quality is ensured.
[ Invention ]
The invention aims at solving the blank of the prior art in the aspect of analyzing potential higher alcohol-producing microorganisms, and provides a degenerate primer capable of amplifying a microbial alcohol dehydrogenase gene fragment and realizing the detection of the abundance of the potential higher alcohol-producing microorganisms based on the degenerate primer.
In view of the above, the present invention provides degenerate primers for amplifying a fragment of a microbial alcohol dehydrogenase gene, the primers comprising:
L.ADHs-F：5′ATAGAATACTGCGGCgtntgyca3′
L.ADHs-R：5′TTTGTACGCTGTTACnccngcrca3′
based on the primers, the invention also provides a detection method for amplifying the microbial alcohol dehydrogenase gene fragment by using the degenerate primers, which comprises the following steps:
(1) Extracting the total DNA of the microorganism of the sample to be detected;
(2) Taking the total DNA of the microorganism in the step (1) as a template, and carrying out PCR amplification reaction by using the degenerate primer to obtain an amplification product A1;
(3) Purifying the amplified product A1 to obtain a purified amplified product B1;
(4) And (3) carrying out high-throughput sequencing on the purified amplification product B1, and judging whether the microorganism of the sample to be detected contains alcohol dehydrogenase or not by acquiring an operation classification unit of whether the sequencing result contains alcohol dehydrogenase and GroES-like protein.
In the present invention, the alcohol dehydrogenase is zinc-dependent long-chain alcohol dehydrogenase, which is a key enzyme in the microbial higher alcohol metabolic pathway and is responsible for the conversion between alcohol and aldehyde.
Further, the method for analyzing and judging the result in the step (4) is as follows:
After the high-usage sequencing result is spliced by FLASH, carrying out operation classification unit (OTU) division on the sequencing result by using Usearch according to the similarity of 95% -100%, comparing the OTU representative sequence with a Non-redundant protein (nr) database by Basic Local ALIGNMENT SEARCH Tool (blastx) and annotating protein information of the OTU representative sequence, wherein the OTU containing Alcohol dehydrogenase and GroES-like protein in the annotated information is the alcohol dehydrogenase gene OTU.
Wherein, the primer in the step (2) is as follows:
L.ADHs-F：5′ATAGAATACTGCGGCgtntgyca3′
L.ADHs-R：5′TTTGTACGCTGTTACnccngcrca3′
the PCR amplification reaction conditions of step (2) are:
The PCR amplification procedure was: pre-denaturation at 94-95 ℃ for 3-5min, denaturation at 94-95 ℃ for 30s-60s, annealing at 55-60 ℃ for 30s-60s, extension at 72 ℃ for 30s-60s, cycle number of 30-35, and extension at 72 ℃ for 10min.
As a preferred embodiment, the sample to be tested is Daqu, and the detection method of the present invention can be used for detecting whether or not there is a microorganism containing alcohol dehydrogenase in a mixed microorganism sample such as Daqu.
The invention selects a plurality of alcohol dehydrogenase amino acid sequences from the structural domain of alcohol dehydrogenase amino acid, and designs the degenerate primer for amplifying the microbial alcohol dehydrogenase gene fragment after comparison in Bioedit through ClusterW. The Basic Local ALIGNMENT SEARCH Tool (blastx) and Non-redundant protein (nr) databases are adopted for comparison and annotation of the information of the amplified products, and the alcohol dehydrogenase genes are determined so as to determine the types and abundance of the microorganisms containing potential higher alcohols in the detection sample, thereby laying a foundation for further research on the compositions of the microorganisms containing potential higher alcohols in the sample.
[ Description of the drawings ]
FIG. 1 shows the role of Alcohol Dehydrogenases (ADHs) in the metabolic process of microbial higher alcohols;
FIG. 2 is a diagram of the fragment WebLogo of the amino acid sequence of alcohol dehydrogenase (L.ADHs);
FIG. 3 is a gel electrophoresis diagram of PCR products;
FIG. 4 Daqu alcohol dehydrogenase (L.ADHs) gene fragment-derived microorganism genus composition;
FIG. 5 composition of microorganism species derived from Daqu alcohol dehydrogenase (L.ADHs) gene fragment.
[ Detailed description ] of the invention
The following examples serve to illustrate the technical solution of the invention without limiting it.
Example 1
Daqu samples from 3 different regions, labeled as Hevea (HT-1, HT-2, HT-3), shandong (SD-1, SD-2, SD-3) and Sichuan (SC-1, SC-2, SC-3) samples, respectively.
1. Alcohol dehydrogenase primer design
The amino acid sequence of L.ADHs is searched from the Uniport (https:// www.uniprot.org /) database. Based on the reported results of the study on the diversity of microorganisms in Daqu at medium and high temperatures, 51 L.ADHs amino acid sequences (including bacteria and fungi, table 1) were selected. The resulting 51 l.adhs amino acid sequences were subjected to multiple sequence alignment using ClusterW in Bioedit (8.1.0). Based on these L.ADHs amino acid sequences, degenerate primers were designed to amplify the L.ADH gene fragments using j-CODEHOP (https:// 4virology. Net/virology-ca-tools/j-CODEHOP /). The quality of the primers was checked using Oligo Calc (http:// biotools. Nubic. Northwestern. Edu/Oligocalc. Html) and Primer-Blast.
TABLE 1L amino acid sequence of ADHs
2. Amplification and sequencing of L.ADHs fragments in Daqu
The genome of 9 samples was extracted by indirect extraction: 7.0g of Daqu was weighed, and the cells were collected by washing with 0.1mol/L PBS buffer, and extracted by using Omega EZNA TM soil genome extraction kit as described above.
The resulting degenerate primers were used to amplify microbial L.ADHs gene fragments in the amplification under PCR conditions of 20. Mu.l reaction system: 5X FastPfu Buffer. Mu.L, 2.5mM dNTPs 2. Mu.L, fastPfu Polymerase 0.4.4. Mu.L, 0.8. Mu.L each of the upstream primer (L.ADHs-F) and the downstream primer (L.ADHs-R), TEMPLATE DNA ng, ddH 2 O were supplemented to 20. Mu.L. The PCR amplification procedure was: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 45s, cycle number of 35, and extension at 72℃for 10min. The amplified products were purified and high throughput sequenced, which was done by Shanghai Ling En Biotechnology Co.
After the sequencing data are spliced by FLASH (1.2.11), the sequencing results are divided into operation classification units (Operational Taxonomic Units, OTU) with 100% similarity and 97% similarity by using Usearch (10). The OTU sequences were aligned and annotated using Blastx, the information annotated as l.adhs gene fragments was screened, and the genus and species of alcohol dehydrogenase (l.adhs) derived microorganism were statistically analyzed. The diversity index was calculated using the R language, and the composition of the genus and species from which the microbial alcohol dehydrogenase (L.ADHs) was derived was plotted. The sequence of the L.ADHs gene fragment was analyzed using MEGAX and the evolutionary tree was beautified using iTOL (https:// itol. Emmbl. De /).
3. Degenerate primer validation
The quality of the primers was checked using Oligo Calc (http:// biotools. Nubic. Northwestern. Edu/Oligocalc. Html) and Primer-Blast. Finally, L.ADHs-F and L.ADHs-R were selected as the upstream and downstream primers for amplifying the L.ADHs gene fragment (Table 2), which were able to successfully amplify the desired fragment (426 bp) and fragments of other lengths (FIG. 3). Based on the division of the fragment length, 3 different regions were divided, and 3 different sequencing libraries were constructed for each Daqu for high throughput sequencing analysis (Table 3), L.ADHs of fragments of different lengths were studied, wherein fragments greater than 600bp were verified using TA clone sequencing, and fragments greater than 600bp were found to be non-L.ADHs gene fragments after verification.
TABLE 2 degenerate primer sequences for ADHs Gene fragments
Table 2 Degenerate primer sequence of L.ADHs gene fragment
TABLE 3 sequencing library and fragment Length Range thereof
Table 2 Sequencing library and its fragment length range
4. Potential higher alcohol-producing microbial diversity in different Daqus
After amplification and high throughput sequencing of degenerate primers designed based on the L.ADHs amino acid sequence, a total of 45269 reads were obtained from 3 Daqus. After clustering with 100% similarity, the cells were divided into 8415 OTUs, and 2570 OTUs annotated as L.ADHs were aligned by Blastx (Table 4).
TABLE 4 Daqu L.ADHs Gene fragment sequencing results and diversity index
After diversity analysis of the OTU annotated as L.ADHs, it was found that in sequencing library 2 (200-400 bp), the Shannon index and Chao index of L.ADHs were the greatest in the SC-2 sample (6.416 and 1346), the Shannon index of L.ADHs was the least in the SD-2 sample (4.812), and the Chao index of L.ADHs was the least in the HT-2 sample (401). This result shows that the L.ADHs gene diversity with fragment length of 200bp-400bp is highest in the SC Daqu and lowest in the SD Daqu. The number of species of L.ADHs gene with a length of 200bp-400bp was minimal in HT Daqu.
In sequencing library 3 (400-600 bp), shannon and Chao indices of L.ADHs in the SC-3 sample were the largest (6.128 and 1087), and Shannon and Chao indices of L.ADHs in the HT-3 sample were the smallest (5.152 and 481). This result shows that the L.ADHs gene diversity and species number with fragment length of 400bp-600bp is highest in SC Daqu and lowest in HT Daqu.
SC Daqu was the largest in both Shannon index and Chao index in sequencing library 2 and sequencing library 3. Thus, the SC Daqu is the most abundant Daqu of 3 Daqus containing potential higher alcohol-producing microorganisms. HT Daqu has the smallest Chao index in sequencing library 2 and sequencing library 3, and therefore HT Daqu is the Daqu with the smallest species of potential higher alcohol-producing microorganisms.
In sequencing library 3 (400-600 bp), the coverage index of L.ADHs in 3 Daqu samples was greater than 0.90 and greater than that in the other 2 sequencing libraries. The target fragment length of the L.ADHs gene degenerate primer designed by the invention is 400bp-600bp, and the coverage rate of the sequencing library 3 is larger than that of other 2 sequencing libraries, so that the designed L.ADHs gene degenerate primer can effectively amplify the target fragment and achieve a certain coverage rate in high-throughput sequencing.
5. Microorganism composition for producing higher alcohols in different Daqu
Species annotation was performed using the Blastx method with the Non-redundant protein (nr) database, and OTUs containing the L.ADHs gene fragment were divided into 30 genera. As a result of analysis of the microorganism composition containing the L.ADHs gene fragment, it was revealed (FIG. 4) that the L.ADHs gene fragment was mainly derived from Bacillus (Bacillus), enterobacter (Enterobacter), aspergillus (Aspergillus) and Mycobacterium (Mycobacterium), and that a small amount of the L.ADHs gene fragment was detected in the remaining bacteria and fungi.
The result of diversity analysis on the L.ADHs gene fragments of 3 Daqus shows that the microorganism composition of the L.ADHs gene fragments of 3 Daqus is different. The L.ADHs gene fragment was detected in only SD-1 Daqu in sequencing library 1 and was mainly from Bacillus (FIG. 4A). In sequencing library 2, the L.ADHs gene fragments in HT-2 and SD-2 Daqus were mainly from Aspergillus (Aspergillus). The L.ADHs gene fragment in SC-2 Daqu is mainly from Bacillus and has relative abundance of more than 50%. The L.ADHs gene fragment derived from Acetobacter (Acetobacter) was present only in SC-2 Daqu and the relative abundance was more than 10% (FIG. 4B). In sequencing library 3, the L.ADHs gene fragment in HT-3 Daqu was mainly from Aspergillus (Aspergillus) and the relative abundance was greater than 60%. The L.ADHs gene fragment in SC-3 Daqu is mainly from Bacillus and has the relative abundance of more than 70%. The L.ADHs gene fragment in SD-3 Daqu is mainly from Enterobacter (Enterobacter) and the relative abundance is more than 40%. The L.ADHs gene fragment from Serratia (Serratia) was found only in SD-3 Daqu and the relative abundance was greater than 10% (FIG. 4C). The dominant genus containing the L.ADHs gene remained almost identical at different fragment lengths of the same Daqu, but the sources of the L.ADHs gene fragments varied among the 3 Daqus. No L.ADHs gene fragments from Bacillus and Enterobacter (Enterobacter) were detected in the HT Daqu, but Bacillus and Enterobacter (Enterobacter) were detected in the SC and SD Daqu, and were dominant microorganisms of L.ADHs origin in the SC and SD Daqu, respectively.
By further analysis, 81.31% of microorganisms containing the L.ADHs gene fragment were found to be able to annotate on a species level, for a total of 50 species. The SD-1 Daqu had a lower number of OTUs and coverage (Table 3). Overall, the abundance information of the l.adhs gene fragments was similar at the seed level as it was at the genus level. There are 15 species of microorganisms that are more than 1% relatively abundant at the species level. The main dominant microorganisms are Aspergillus fumigatus (Aspergillus fumigatus), bacillus amyloliquefaciens (Bacillus thermoamylovorans), enterobacter cloacae (Enterobacter cloacae) and Enterobacter cholerae (Enterobacter hormaechei).
The difference of the composition of the microorganism species with the potential higher alcohol production in 3 Daqus is more obvious, wherein the proportion of the Aspergillus fumigatus (Aspergillus fumigatus) in the SC-2, the SC-3 and the SD-3 Daqus is smaller, and the proportion of the Aspergillus fumigatus (Aspergillus fumigatus) in the HT-2, the HT-3 and the SD-2 Daqus is larger (the relative abundance is more than 50%). Bacillus amyloliquefaciens (Bacillus thermoamylovorans) was detected only in SC-2 and SC-3 Daqu, and the relative abundance was greater than 50%. Enterobacter cloacae (Enterobacter cloacae) and Enterobacter cholerae (Enterobacter hormaechei) were not detected in HT-2 and HT-3 Daqus, but were detected in other Daqus. The abundance of enterobacter cholerae (Enterobacter hormaechei) was higher than that of enterobacter cloacae (Enterobacter cloacae), enterobacter cholerae (Enterobacter hormaechei) was the most abundant at SD-3 daqu and the relative abundance was greater than 40%, enterobacter cloacae (Enterobacter cloacae) was the least abundant at SD-2 daqu (fig. 5). The sum of the relative abundances of the 4 dominant microorganisms on the species level of the L.ADHs gene fragment information exceeds 60% in each Daqu and up to 95% in SC-3 Daqu. Thus, aspergillus fumigatus (Aspergillus fumigatus), bacillus amyloliquefaciens (Bacillus thermoamylovorans), enterobacter cloacae (Enterobacter cloacae) and Enterobacter cholerae (Enterobacter hormaechei) are 3 dominant microorganisms of the Daqu L.ADHs gene fragment.
Based on the results, the designed degenerate primers can amplify the L.ADHs gene fragments, and dominant microorganisms containing the L.ADHs gene fragments in Daqu in different regions are different. Indicating that the species of the microorganism which is capable of producing higher alcohols in Daqu in different regions are different. Thus, potential higher alcohol producing microorganisms can be studied by studying the compositions of Daqu L.ADHs gene fragments in different regions.
Claims (4)
1. A degenerate primer for amplifying a microbial alcohol dehydrogenase gene fragment, said degenerate primer having a nucleotide sequence as set forth in seq id no:
L.ADHs-F：5´ATAGAATACTGCGGCgtntgyca3´
L.ADHs-R：5´TTTGTACGCTGTTACnccngcrca3´。
2. A method for detecting microbial alcohol dehydrogenase gene fragments in a yeast using the degenerate primer of claim 1, said method comprising the steps of:
(1) Extracting the total DNA of the microorganism of the sample to be detected;
(2) Performing PCR amplification reaction by using the total DNA of the microorganism in the step (1) as a template and using the degenerate primer of claim 1 to obtain an amplified product A1;
(3) Purifying the amplification product A1 to obtain a purified amplification product B1;
(4) Carrying out high-throughput sequencing on the purified amplification product B1, and judging whether the microorganism of the sample to be detected contains alcohol dehydrogenase or not by acquiring an operation classification unit of whether the sequencing result contains alcohol dehydrogenase and GroES-like protein, wherein the method comprises the following steps: after the high-throughput sequencing result is spliced by FLASH, carrying out operation classification unit OTU division on the sequencing result by using Usearch with a similarity of 95% -100%, carrying out Basic Local ALIGNMENT SEARCH Tool comparison on the OTU representative sequence and a Non-redundant protein database, and annotating protein information of the OTU representative sequence, wherein the OTU containing alcohol dehydrogenase and GroES-like protein in the annotated information is the alcohol dehydrogenase gene OTU.
3. The method according to claim 2, wherein the conditions for the PCR amplification reaction in step (2) are:
pre-denaturation at 94-95 ℃ for 3-5min, denaturation at 94-95 ℃ for 30s-60s, annealing at 55-60 ℃ for 30s-60s, extension at 72 ℃ for 30s-60s, cycle number of 30-35, and extension at 72 ℃ for 10min.
4. Use of the degenerate primer of claim 1 for detecting whether a microorganism in Daqu contains an alcohol dehydrogenase gene fragment.
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| Cloning, Expression, and Characterization of a Novel (S)-Specific Alcohol Dehydrogenase from Lactobacillus kefir;Qilei Chen 等;《Applied Biochemistry and Biotechnology》;20081210;第160卷;摘要，第19页第1段至第2页最后1段 * |
| 菌B49乙醛乙醇脱氢酶基因克隆中的引物设计;林海龙 等;《哈尔滨工业大学学报》;20081231(第6期);摘要 * |
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