CN114480444A - Degenerate primer for amplifying microbial alcohol dehydrogenase gene fragment and application of degenerate primer - Google Patents
Degenerate primer for amplifying microbial alcohol dehydrogenase gene fragment and application of degenerate primer Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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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]
Abstract
The invention provides a degenerate primer for amplifying a microbial alcohol dehydrogenase gene segment and a detection method for amplifying a microbial alcohol dehydrogenase gene segment by using the degenerate primer. The invention designs a degenerate primer for amplifying the gene segment of the microbial alcohol dehydrogenase from the structural domain of the amino acid of the alcohol dehydrogenase, and the degenerate primer is applied to detection to determine the alcohol dehydrogenase gene, and further determine the type and abundance of the microorganism which potentially produces higher alcohol in a detected sample, thereby laying a foundation for further research on the composition of the microorganism which potentially produces higher alcohol in the sample.
Description
[ technical field ] A
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 further relates to a method for detecting the abundance of a microorganism potentially producing higher alcohol in a yeast by using the degenerate primer.
[ background of the invention ]
Higher alcohols (alcohols with carbon number more than 2) have the functions of alcohol sweetness and fragrance aid, are also important precursors for forming flavor substances such as esters and aldehydes and the like, and play an important role in the flavor substance composition of traditional fermented foods. However, when the content of the higher alcohol is too high, the flavor abnormality is caused, and even the health risk is caused.
The method is an effective method for controlling the content of the higher alcohol in the fermented food by determining the microorganisms with the higher alcohol production capacity in the mixed microorganism fermentation process in which various microorganisms participate and further proportioning and selecting proper seed (koji) fermentation. However, current correlation analysis methods 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, thereby not only characterizing community metabolic functions, but also further researching potential metabolite producers in mixed microorganisms. Alcohol Dehydrogenases (ADHs) are very critical in the microbial higher Alcohol metabolism process in view of the existing microbial higher Alcohol metabolism pathways (FIG. 1). The method comprises the steps of amplifying microbial alcohol dehydrogenase gene segments in strains (koji) by designing degenerate primers, obtaining sequence information by using a high-throughput sequencing technology, and further determining the strain ratio by using a bioinformatics technology to analyze potential microorganisms producing higher alcohols, thereby ensuring the product quality.
[ summary of the invention ]
The invention aims to provide a degenerate primer capable of amplifying a microbial alcohol dehydrogenase gene fragment aiming at the blank of the prior art in the analysis of potential higher alcohol-producing microorganisms, and realize the detection of the abundance of the potential higher alcohol-producing microorganisms based on the degenerate primer.
In view of the above objects, the present invention provides degenerate primers for amplifying a microbial alcohol dehydrogenase gene fragment, 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 degenerate primers, which comprises the following steps:
(1) extracting total DNA of the microorganism of a sample to be detected;
(2) carrying out PCR amplification reaction by using the total DNA of the microorganism in the step (1) as a template and the degenerate primer to obtain an amplification product A1;
(3) purifying the amplification product A1 to obtain a purified amplification product B1;
(4) and (3) carrying out high-throughput sequencing on the purified amplification product B1, and judging whether the microorganism to be tested contains alcohol dehydrogenase or not by an operation classification unit for obtaining whether the sequencing result contains the alcohol dehydrogenase and GroES-like protein or not.
In the present invention, the alcohol dehydrogenase is a 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) comprises the following steps:
after the high-dose sequencing result is spliced by FLASH, the sequencing result is subjected to operation classification unit (OTU) division by using Usearch with 95% -100% similarity, the OTU representative sequence is compared with Non-redundant protein (nr) database by Basic Local Alignment Search Tool (blastx) and the protein information of the OTU representative sequence is annotated, wherein the OTU containing Alcohol dehydrogenase and GroES-like protein in the annotation information is the Alcohol dehydrogenase gene OTU.
Wherein, the primers in the step (2) are as follows:
L.ADHs-F：5′ATAGAATACTGCGGCgtntgyca3′
L.ADHs-R：5′TTTGTACGCTGTTACnccngcrca3′
the PCR amplification reaction conditions of the step (2) are as follows:
the PCR amplification procedure was: pre-denaturation at 94-95 ℃ for 3-5min, denaturation at 94-95 ℃ for 30-60 s, annealing at 55-60 ℃ for 30-60 s, extension at 72 ℃ for 30-60 s, cycle number of 30-35, and final extension at 72 ℃ for 10 min.
In 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 a microorganism containing alcohol dehydrogenase is present in a mixed microorganism sample such as Daqu.
According to the invention, a plurality of alcohol dehydrogenase amino acid sequences are selected from the structural domain of alcohol dehydrogenase amino acid, and are compared by Cluster W in Bioedit to design the degenerate primer for amplifying the microbial alcohol dehydrogenase gene fragment. Comparing Basic Local Alignment Search Tool (blastx) with Non-redundant protein (nr) database, annotating the information of the amplified product, determining alcohol dehydrogenase gene and further determining the type and abundance of the potential higher alcohol-producing microorganisms in the detected sample, thereby laying a foundation for further research on the composition of the potential higher alcohol-producing microorganisms in the sample.
[ description of the drawings ]
FIG. 1 shows the effect of Alcohol Dehydrogenases (ADHs) on the metabolism of higher alcohols by microorganisms;
FIG. 2 is a Weblogo diagram of an amino acid sequence fragment of alcohol dehydrogenase (L.ADHs);
FIG. 3 is a gel electrophoresis of PCR products;
FIG. 4 shows the composition of microorganisms from which a Daqu alcohol dehydrogenase (L.ADHs) gene fragment is derived;
FIG. 5 shows the composition of a microorganism from which a Daqu alcohol dehydrogenase (L.ADHs) gene fragment is derived.
[ detailed description ] embodiments
The following examples serve to illustrate the technical solution of the present invention without limiting it.
Example 1
Daqu samples taken from 3 different regions were labeled as samples from the Hetao region (HT-1, HT-2, HT-3), Shandong region (SD-1, SD-2, SD-3) and Sichuan region (SC-1, SC-2, SC-3).
1. Alcohol dehydrogenase primer design
The amino acid sequence of L.ADHs was searched from the Uniport (https:// www.uniprot.org /) database. And selecting 51 L.ADHs amino acid sequences (containing bacteria and fungi and attached table 1) according to the reported research result of the microbial diversity of the medium-high temperature Daqu. The obtained 51 l.adhs amino acid sequences were subjected to multiple sequence alignment using ClusterW in Bioedit (8.1.0). Based on these amino acid sequences of L.ADHs, degenerate primers for amplifying the L.ADH gene fragments were designed using j-CODEHOP (https://4virology. net/virology-ca-tools/j-CODEHOP /). The quality of the primers was verified using Oligo Calc (http:// biolols. nubic. northwestern. edu/OligoCalc. html.) and Primer-Blast.
TABLE 1 L. ADHs amino acid sequence
2. Amplification and sequencing of L.ADHs fragment in Daqu
The genome of 9 samples was extracted by indirect extraction: weighing Daqu 7.0g, washing with PBS buffer solution of 0.1mol/L, and collecting thallusTMExtracting with a soil genome extraction kit.
Amplifying a microorganism L.ADHs gene fragment in the yeast by using the obtained degenerate primer, wherein the PCR amplification condition is 20 mul of reaction system: 5 XFastPfu Buffer 4. mu.L, 2.5mM dNTPs 2. mu.L, FastPfu Polymerase 0.4. mu.L, each of the upstream primer (L.ADHs-F) and the downstream primer (L.ADHs-R) 0.8. mu.L, Template DNA 10ng, ddH2O was 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 final extension at 72 ℃ for 10 min. The amplification products were purified and subjected to high throughput sequencing, which was performed by Shanghai Linn Biotech Ltd.
The data obtained by sequencing are spliced by FLASH (1.2.11), and the sequencing result is divided into operation classification Units (OTU) by Usearch (10) with 100% and 97% similarity. OTU sequences were aligned and annotated with information using Blastx, screened for information annotated as l.adhs gene fragments, and statistically analyzed for the genus and species of the alcohol dehydrogenase (l.adhs) -derived microorganisms. And calculating a diversity index by using the R language, and drawing a composition diagram of the source genus and species of the microbial alcohol dehydrogenase (L.ADHs). Evolution analysis was performed on the sequence of the L.ADHs gene fragment using MEGAX, and beautification of the evolutionary tree was performed using iTOL (https:// itol.embl. de /).
3. Degenerate primer validation
The quality of the primers was verified using Oligo Calc (http:// biolols. nubic. northwestern. edu/OligoCalc. html.) and Primer-Blast. Finally, L.ADHs-F and L.ADHs-R are selected as upstream and downstream primers for amplifying L.ADHs gene fragments (Table 2), and the primers can successfully amplify the target fragment (426bp) and fragments with other lengths (FIG. 3). The fragments are divided into 3 different regions according to the length of the fragments, each Daqu constructs 3 different sequencing libraries for high-throughput sequencing analysis (table 3), L.ADHs of the fragments with different lengths are researched, the fragments larger than 600bp are verified by TA clone sequencing, and the fragments larger than 600bp are found to be non-L.ADHs gene fragments after verification.
TABLE 2 L. ADHs Gene fragment degenerate primer sequences
Table 2 Degenerate primer sequence of L.ADHs gene fragment
TABLE 3 sequencing libraries and fragment Length ranges thereof
Table 2 Sequencing library and its fragment length range
4. The diversity of microorganisms potentially producing higher alcohol in different Daqu
After amplification of degenerate primers designed based on the amino acid sequence of l.adhs and high throughput sequencing, a total of 45269 reads were obtained from 3 master batches. Clustering at 100% similarity was performed and then divided into 8415 OTUs, and 2570 OTUs annotated as l.adhs by Blastx alignment (table 4).
TABLE 4 Daqu L.ADHs Gene fragment sequencing results and diversity index
After diversity analysis of OTU annotated with L.ADHs, it was found that the Shannon index and Chao index of L.ADHs in SC-2 samples were maximal (6.416 and 1346), the Shannon index of L.ADHs in SD-2 samples was minimal (4.812), and the Chao index of L.ADHs in HT-2 samples was minimal (401) in sequencing library 2 (200-400 bp). This result indicates that the diversity of the L.ADHs gene with fragment length of 200bp-400bp is highest in SC Daqu and lowest in SD Daqu. The number of species of L.ADHs gene with length of 200bp-400bp is the least in HT Daqu.
In sequencing library 3(400-600bp), the Shannon index and the Chao index of L.ADHs in SC-3 samples were the largest (6.128 and 1087) and the Shannon index and the Chao index of L.ADHs in HT-3 samples were the smallest (5.152 and 481). This result indicates that the diversity and species number of the L.ADHs genes with fragment lengths of 400bp to 600bp are the highest in SC Daqu and the lowest in HT Daqu.
SC master was the largest in both sequencing library 2 and sequencing library 3 for Shannon index and Chao index. Therefore, SC Daqu is the most abundant Daqu containing potential higher alcohol-producing microorganisms in 3 kinds of Daqu. The HT Daqu has the smallest Chao index in the sequencing library 2 and the sequencing library 3, so the HT Daqu is the Daqu with the smallest variety of potential higher alcohol-producing microorganisms.
In sequencing library 3(400-600bp), 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 length of a target fragment of the degenerate primer of the L.ADHs gene designed by the invention is between 400bp and 600bp, and the coverage rate of the sequencing library 3 is greater than that of other 2 sequencing libraries, so that the degenerate primer of the L.ADHs gene can effectively amplify the target fragment and achieve a certain coverage rate in high-throughput sequencing.
5. Composition of microorganisms capable of potentially producing higher alcohol in different Daqu
Species annotation was performed using the Blastx method with the Non-redundant protein (nr) database, and OTUs containing l.adhs gene fragments were divided into 30 genera. Analysis of microbial composition of the l.adhs gene-containing fragment showed (fig. 4) that the l.adhs gene fragment was mainly derived from Bacillus (Bacillus), Enterobacter (Enterobacter), Aspergillus (Aspergillus), and Mycobacterium (Mycobacterium), and a small amount of the l.adhs gene fragment was detected in the remaining bacteria and fungi.
The diversity analysis result of the L.ADHs gene segments of the 3 types of Daqu shows that the microbial compositions of the L.ADHs gene segments contained in the 3 types of Daqu are different. The l.adhs gene fragment was detected in only SD-1 large curve in sequencing library 1 and was mainly from Bacillus (Bacillus) (fig. 4A). In sequencing library 2, the L.ADHs gene fragment in HT-2 and SD-2 Daqu was mainly from Aspergillus (Aspergillus). The L.ADHs gene segment in the SC-2 Daqu is mainly from Bacillus (Bacillus) and has relative abundance of more than 50%. The l.adhs gene fragment derived from Acetobacter (Acetobacter) is present only in SC-2 daqu, and the relative abundance is greater than 10% (fig. 4B). In sequencing library 3, the L.ADHs gene fragment in HT-3 Daqu was mainly from Aspergillus (Aspergillus) and was present in relative abundance of greater than 60%. The L.ADHs gene segment in the SC-3 Daqu is mainly from Bacillus (Bacillus) and has the relative abundance of more than 70 percent. The L.ADHs gene fragment in the SD-3 Daqu is mainly from Enterobacter (Enterobacter) and has the relative abundance of more than 40 percent. The l.adhs gene fragment derived from Serratia (Serratia) was present only in SD-3 koji with a relative abundance of more than 10% (fig. 4C). The dominant genera containing the l.adhs gene remained almost identical for different fragment lengths of the same Daqu, but there was a difference in the source of the l.adhs gene fragments among the 3 Daqus. No l.adhs gene fragments from Bacillus (Bacillus) and Enterobacter (Enterobacter) were detected in HT master batch, but Bacillus (Bacillus) and Enterobacter (Enterobacter) were detected in SC and SD master batches and were the dominant microbial genera of l.adhs origin in SC and SD master batches, respectively.
By further analysis, 81.31% of the microorganisms containing the l.adhs gene fragment were found to be able to be annotated to the species level, with a total of 50 species. SD-1 Daqu had lower numbers of OTUs and coverage (Table 3). Overall, the results of the abundance information of the l.adhs gene fragment at the species level were similar to those at the genus level. There were 15 species of microorganisms with relative abundance of greater than 1% at the species level. Among the major dominant microorganisms are Aspergillus fumigatus (Aspergillus fumigatus), Bacillus amylothermophilus (Bacillus thermoamylovorans), Enterobacter cloacae (Enterobacter cloacae) and Enterobacter hollisae (Enterobacter hormichei).
The difference of the level and the composition of the potential higher alcohol-producing microorganism species in the 3 types of the Daqu is more obvious, wherein the Aspergillus fumigatus (Aspergillus fumigatus) accounts for a smaller proportion of SC-2, SC-3 and SD-3 Daqu and accounts for a larger proportion of HT-2, HT-3 and SD-2 Daqu (the relative abundance is more than 50%). Bacillus amyloliquefaciens (Bacillus thermoamylovorans) was detected only in SC-2 and SC-3 master batches and the relative abundance was greater than 50%. Enterobacter cloacae (Enterobacter cloacae) and Enterobacter hohnsonii (Enterobacter hormaechei) were not detected in both HT-2 and HT-3 koji, and were detected in all other koji. Enterobacter hollandii (Enterobacter hormaechei) is more abundant than Enterobacter cloacae (Enterobacter cloacae), the abundance of the Enterobacter hollandii (Enterobacter hormaechei) in the SD-3 Daqu is the greatest and the relative abundance is more than 40%, and the abundance of the Enterobacter cloacae (Enterobacter cloacae) in the SD-2 Daqu is the least (FIG. 5). The sum of the relative abundances of the 4 dominant microorganisms on the species level of the ADHs gene fragment information exceeds 60% in each Daqu, and the sum of the relative abundances of the SC-3 Daqu is as high as 95%. Thus, Aspergillus fumigatus (Aspergillus fumigatus), Bacillus amylothermophilus (Bacillus thermoamylovorans) and Enterobacter cloacae (Enterobacter cloacae) and Enterobacter hollisae (Enterobacter hormaechei) are the dominant microorganisms for the ADHs gene fragment of the 3 types of Daqu L..
Based on the results, the designed degenerate primers can amplify the L.ADHs gene segments, and the dominant microorganisms containing the L.ADHs gene segments in the yeast in different regions have differences. Thus, the types of the microorganisms which potentially produce higher alcohol in the yeast for making hard liquor in different areas are different. Therefore, potential higher alcohol-producing microorganisms can be researched by researching the composition of Daqu L.ADHs gene fragments in different regions.
Claims (5)
1. A degenerate primer for amplifying a microbial alcohol dehydrogenase gene fragment, the degenerate primer having a nucleotide sequence as set forth in:
L.ADHs-F：5′ATAGAATACTGCGGCgtntgyca3′
L.ADHs-R：5′TTTGTACGCTGTTACnccngcrca3′。
2. a detection method for amplifying a microbial alcohol dehydrogenase gene fragment by using degenerate primers, characterized by comprising the steps of:
(1) extracting total DNA of the microorganism of a sample to be detected;
(2) carrying out 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 amplification product A1;
(3) purifying the amplification product A1 to obtain a purified amplification product B1;
(4) and (3) carrying out high-throughput sequencing on the purified amplification product B1, and judging whether the microorganisms of the sample to be detected contain alcohol dehydrogenase or not by obtaining an operation classification unit whether a sequencing result contains the alcohol dehydrogenase and GroES-like protein or not.
3. The detection method according to claim 2, wherein the step (4) comprises:
after the high-throughput sequencing result is spliced by FLASH, using Usearch to perform operation classification unit OTU division on the sequencing result with 95% -100% similarity, performing 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 annotation information is the alcohol dehydrogenase gene OTU.
4. The method for detecting amplified microorganism alcohol dehydrogenase gene segment according to claim 2, wherein the PCR amplification reaction in step (2) is performed under the following conditions:
the PCR amplification procedure was: pre-denaturation at 94-95 ℃ for 3-5min, denaturation at 94-95 ℃ for 30-60 s, annealing at 55-60 ℃ for 30-60 s, extension at 72 ℃ for 30-60 s, cycle number of 30-35, and final extension at 72 ℃ for 10 min.
5. Use of the degenerate primer of claim 1 for detecting whether a microorganism contains a segment of an alcohol dehydrogenase gene.
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