CN109385486B - DNA bar code, primer, kit, method and application - Google Patents
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Abstract
The invention belongs to the field of species and strain identification, and particularly relates to a DNA bar code, a primer, a kit, a method and application for identifying a desmospora adephagi strain. The DNA bar code can accurately identify the Pu' er tea fermentation strain, namely, the adenine arthrobotrys yeast TMCC 70007, and can quickly and accurately identify the strain from confusable strains or other strains in the same species.
Description
Technical Field
The invention belongs to the field of species and strain identification, and particularly relates to a DNA bar code, a primer, a kit, a method and application.
Background
Pu' er tea is post-fermented tea with geographical identification of Yunnan, which is prepared by a series of processes by adopting big-leaf sun-cured green tea as a raw material. The traditional Pu' er tea preparation process comprises the following steps: the picked fresh tea leaves are rolled, dried in the sun, purified, moistened, piled, dried in the air, screened, pressed and formed, and packaged for delivery. In the production of Pu ' er tea, the pile fermentation process is a main factor for the formation of the Pu ' er tea, and in the process, tea polyphenol, caffeine, some polysaccharide substances and other contained components in the tea are greatly changed, so that the special flavor, taste, quality and various health-care effects of the Pu ' er tea are achieved.
In the traditional Pu 'er tea production, enzymes contained in tea leaves are activated by a damp and hot environment, a part of contained components in the tea leaves are converted into substances which can be utilized by microorganisms, the microorganisms are bred in a large quantity to generate abundant intracellular enzymes and extracellular enzymes, the components contained in the tea leaves are catalyzed to generate a series of conversion, and other factors such as damp and hot are added to gradually form the Pu' er tea with different qualities. Different origins, flavors and qualities are also characterized due to the different microbial community structures.
Besides the unique flavor and culture of Pu' er tea, the health care effects of losing weight, reducing blood sugar and blood fat, preventing and improving cardiovascular diseases, resisting aging, resisting cancer, diminishing inflammation, promoting digestion, nourishing the stomach and the like are also concerned by people. In recent years, Pu 'er tea is more and more popular, and the development of the Pu' er tea industry is pulled by the increasing market demand, so that the economic growth of Yunnan areas is promoted.
However, Pu' er tea also faces some dilemmas, such as unstable product quality, existence of a certain safety hazard, long production period, too high labor input, excessive microorganism quantity, breeding of mites and the like. With the improvement of living standard of people, consumers pay more and more attention to the problems of food such as sanitation, safety and the like. In recent years, food quality safety events are frequent, the safety problem of tea quality is increasingly highlighted, and besides the problem of pesticide residues, microorganisms in the pile fermentation process can also become important factors influencing the tea quality.
At present, the production of Pu' er tea by most manufacturers is still the empirical fermentation of semi-natural artificial pile fermentation, although communities mainly comprising dominant common microorganisms such as adenine-nodulospora adeninivorans (Blastobotrys adeninivorans) are relatively stable in the pile fermentation process, the stability of the product still has a larger space for improving. Certain potential safety hazards inevitably exist in the production process, and in order to further obtain the favor of consumers and the acceptance of the market, break through the foreign trade barrier and improve the market competitiveness of Pu 'er tea enterprises, the manual control, cleaning and high efficiency of the Pu' er tea production process must be realized, new products are not developed, and the industrial chain is extended. In order to achieve the purpose, technology must be innovated, a series of safe, clean, efficient, artificial, controllable and automatic Pu 'er tea new processes are invented, the healthy development of the Pu' er tea industry can be ensured, and long-term benefits are brought to the nation and people.
With further development of Pu ' er tea scientific research, more and more people begin to explore the artificial Pu ' er tea inoculation and fermentation, and at present, a plurality of bacterial strains are applied to the artificial controllable Pu ' er tea fermentation. In recent years, due to the increase of market demands, a plurality of small-scale manufacturers lack systematic and deep research on Pu 'er tea, and greatly abuse a plurality of other people's patents to profit, for example, according to a plurality of patent methods for inoculating and fermenting Pu 'er tea, a plurality of strains are adopted to ferment Pu' er tea, and a series of methods such as Pu 'er tea processing and production are carried out, so that the economic benefits of Pu' er tea production enterprises with related patents are greatly damaged.
In order to ensure that the germplasm resources of Pu 'er tea fermentation microorganisms are effectively protected, prevent the occurrence of the infringement behavior of abusing Pu' er tea fermentation microorganisms and solve the problem that the strains are difficult to prove when infringement occurs in the manual controllable fermentation process of Pu 'er tea, the development of a method for quickly and accurately identifying the strains for Pu' er tea fermentation is imperative, a molecular identification method of the strains is urgently needed to be established, and a method for accurately identifying and distinguishing similar strains by a DNA bar code technology is developed so as to solve the problems of identification and identification of industrial production strains by a method combining morphological characteristics and molecular data analysis, so that a theoretical basis is provided for the development of the controllable industrial fermentation process and resource protection of Pu 'er tea, and the healthy and prosperous development of the Pu' er tea industry is promoted.
Disclosure of Invention
In order to overcome the defects of morphological identification of the bacterial strain of the Arthrospora adenine saccharomycete for fermentation production of the Pu ' er tea, the invention provides a DNA bar code, a primer, a kit, a method and application for identifying the bacterial strain of the Arthrospora adenine saccharomycete, so that the bacterial strain of the Arthrospora adenine saccharomycete TMCC 70007 can be quickly identified and distinguished, quick evaluation can be provided for a new fermentation process of the Pu ' er tea, interference of other mixed bacteria in the fermentation process can be prevented, and a proof-raising method and basis can be provided for an artificial controllable fermentation process of the Pu ' er tea and illegal abuse of the bacterial strain.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) a DNA barcode for identifying an Arthrospora adenantha strain, the DNA barcode is derived from the genome of the Arthrospora adenantha strain TMCC 70007 strain and comprises at least 600bp of sequence selected from the DNA sequences shown as SEQ ID No.1, and the length of the DNA barcode is 600bp-3000bp, preferably 600bp-2200bp, and more preferably 600bp-1500 bp.
(2) The DNA barcode according to (1), wherein the nucleotide sequence thereof is shown as SEQ ID No.1, SEQ ID No.4 or SEQ ID No. 7.
(3) A primer pair for amplifying the DNA barcode according to (1).
(4) The primer set according to (3), wherein the nucleotide sequence of the forward primer is identical to such a sequence in the genome of the A.adenine-nodorum TMCC 70007 strain: the sequence is a sequence from 1000bp upstream of the 1 st position of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC 70007 strain to the 589 th position of the nucleotide sequence shown as SEQ ID No.1, and the length of the forward primer is 20-30 bp; the reverse primer of the strain is reversely complementary to a sequence in the genome of the TMCC 70007 strain: the sequence is a sequence in a region from 601 th site of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC 70007 strain to 1000bp downstream of the last site of the nucleotide sequence shown as SEQ ID No.1, and the length of the reverse primer is 20-30 bp.
(5) The primer pair according to (4), wherein the nucleotide sequences of the forward primer and the reverse primer are respectively as follows:
a forward primer: 5'-GCTCAACTGCCAATGGTTCA-3', respectively;
reverse primer: 5'-CTTGCCATGGTGGTGGGATA-3' are provided.
(6) A kit for identifying a strain of Arthrospora adenantha comprising the primer pair according to (3).
(7) A method for identifying a strain of alternaria adefovea comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA in the step a) as a template and using the primer pair in the step (3) to obtain a PCR product;
c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC 70007 strain, and if the target band exists, performing the step d);
d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA barcode according to claim 1, and if the homology is more than 97%, judging that the strain to be detected is the Arthrospora adenine-feeding TMCC 70007 strain.
(8) The application of the DNA bar code in the identification of the adenine node B yeast strain according to the (1).
(9) The application of the primer pair in (3) in identifying the adenine nodospora spaying yeast strain.
(10) The application of the kit in (6) in identifying the adenine nodospora spacicola strains.
Compared with the prior art, the invention has the following advantages and positive effects:
1. the invention adopts a protein genomics technology to discover a leakage injection coding protein from the genome of the Arthrospora adenantha TMCC 70007 strain, and discovers that the open reading frame (SEQ ID No.1) of the gene coding the protein is unique to the genome of the Arthrospora adenantha TMCC 70007 strain. The invention further develops the specific DNA sequence into a DNA bar code through careful research and comparative analysis, and the DNA bar code is used as an effective tool for identifying the strain Alternaria adenini TMCC 70007 produced by the industrial fermentation of the Pu' er tea. The barcode sequence can realize the rapid and accurate identification and differentiation of the strains in the adenine-feeding node spore yeast.
2. Compared with the prior art, the specificity of the DNA bar code discovered by adopting the protein genomics technology is better.
3. The present invention further found that the sequence of ARC19 gene (e.g. SEQ ID No.1) is versatile, easily amplified, easily aligned compared to other genes, and is significantly different between different strains within the species saccharomyces pombe.
4. The invention establishes a standard gene sequence and a sample identification method of the Puer tea industrial fermentation production strain Arthrospora adenini TMCC 70007, and compared with the traditional morphological identification method, the identification efficiency is obviously improved. The method has low requirement on the integrity of the sample, and the identification index can be quantized, which provides effective basis for timely judging Pu' er tea and germplasm resources thereof. In addition, morphological confusing species are further added, and identification rules are established by using a phylogenetic tree method based on cluster analysis for identification, so that the reliability and accuracy of identification are greatly superior to those of the conventional molecular identification method, and the blank of strain identification of the Pu' er fermented tea production strain based on the DNA barcode technology is filled.
Drawings
Fig. 1 shows the mass spectrum of newly identified peptide fragment SQSLRPYLMAVR.
FIG. 2 shows a comparison of the mass spectrum of the as-identified peptide fragment with the mass spectrum of the synthetic peptide fragment of chemically synthesized peptide fragment SQSLRPYLMAVR; the original identified peptide segment is obtained by mass spectrometry and identification; the upper part of the figure is a mass spectrogram of the original identified peptide fragment, and the lower part is a mass spectrogram of the synthesized peptide fragment.
FIG. 3 shows a map of the mRNA sequence of the protein coding frame and its encoded protein sequence of the region in which the peptide fragment is located, the grey background part being the new peptide fragment identified.
FIG. 4 shows a map of the mRNA sequence transcribed from the gene encoding ARC19 and its encoded protein sequence, with the grey background region being the intron region.
FIG. 5 shows an alignment of the barcode sequence SEQ ID NO.1 with homology thereto by NCBI-BLASTN, the 9 homologous sequences being shown in the lower 9 short grey dashes in FIG. 5.
FIG. 6 shows a comparison of the bar code sequence SEQ ID NO.1 of C.adenini 70007 with the homologous sequence of C.adenini LS3 strain;
FIG. 7 shows the positions of primers used for amplifying SEQ ID NO.1 in one embodiment of the present invention, the underlined regions in the sequence are the regions where the primers are located, the bold font ATG and TAA are the start and stop sites, and the amplified sequence is SEQ ID NO. 7.
FIG. 8 shows the result of agarose gel electrophoresis of the PCR amplification product of the test strain obtained using the primers of the present invention.
FIG. 9 shows a comparison of the sequence of SEQ ID NO.1 with the PCR amplified sequence of the test strain.
Detailed Description
The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.
The invention adopts a protein genomics technology to find a coding protein with missing annotation from the genome of the adenine arthrobacter TMCC 70007 strain, and the ARC 3878 gene protein sequence is called as ARC19 protein because the similarity of the ARC19 gene protein sequence and the ARC19 subunit (accession number: CDO53724.1) of the ARP2/3 complex of Geotrichum candidum CLIB 918 reaches 91 percent. The invention obtains the corresponding gene sequence in the TMCC 70007 strain genome from the newly found protein sequence, the corresponding gene sequence is called ARC19 gene, and the open reading frame (SEQ ID No.1) of the gene for coding the protein is found to have good specificity relative to the homologous gene reported in the existing database, and the specific DNA sequence can be used for developing and identifying the DNA bar code of the strain TMCC 70007 of the industrial fermentation production strain of the puerh tea. Compared with the prior art, the DNA barcode obtained by the method has higher specificity.
Considering that the DNA barcode should have a suitable length and sufficient specificity among strains, the present invention further obtains a DNA barcode that can accurately and efficiently identify the Arthrospora adenine-feeding strain TMCC 70007 based on the above-mentioned characteristic DNA sequence through careful study and comparative analysis.
Thus, in one aspect the present invention provides a DNA barcode for identifying a strain of nodospora adenosylvestris, the DNA barcode being derived from the genome of the strain of nodospora adenosylvestris TMCC 70007 and comprising a sequence of at least 600bp selected from the DNA sequences shown as SEQ ID No.1, and the DNA barcode having a length of 600bp to 3000bp, preferably 600bp to 2200bp, more preferably 600bp to 1500 bp.
The bar code sequence of the invention can realize the rapid and accurate identification and differentiation of the strains in the adenine-feeding node spore yeast.
The length of the SEQ ID No.1 is 1189bp, and the sequence can be used as a DNA bar code for identifying the adenine nodospora cerealis TMCC 70007 strain because the SEQ ID No.1 is specific to the genome of the adenine nodospora cerealis TMCC 70007 strain. In addition, theoretical analysis and experimental verification prove that the specificity of the DNA bar code which contains the sequence and is longer in length is better ensured. When the length of the DNA barcode is too long (e.g., greater than 3000bp), it is less desirable for the amplification procedure. On the other hand, the sequence selected from the sequences not less than 600bp can meet the requirement of specificity and the operation requirement of easy amplification and easy comparison. Therefore, theoretical analysis and experimental verification prove that at least 600bp sequence selected from the DNA sequence shown as SEQ ID No.1 can realize the rapid and accurate identification and differentiation of the strains in the adenine-node-B yeast.
In a preferred embodiment of the invention, the DNA barcode sequence is as shown in SEQ ID No.1, SEQ ID No.4 or SEQ ID No. 7.
In this context, the term "missing-release gene" means that after the genome of a species is sequenced, the gene cannot be predicted by using a gene prediction software (e.g., GeneMark, Augustus, Glimer, etc.), and the gene is generally expressed in a low amount under specific conditions and is therefore difficult to be found in research.
The term "DNA barcoding" refers to a new molecular identification technology for identifying species using a standard, short DNA fragment in the genome, which allows rapid and accurate species identification.
The term "six-frame translation" is a known term in proteomics and genomics, and is briefly described on the principle that when a DNA encodes a protein, the protein is encoded using triplet codons, giving a DNA sequence with 3 coding possibilities, plus 3 coding possibilities on its complementary strand, for a total of 6 coding possibilities (+1, +2, +3, -3, -2, -1).
Another embodiment of the present invention provides a primer pair for amplifying the DNA barcode according to the present invention.
Preferably, the nucleotide sequence of its forward primer is identical to such a sequence in the genome of the strain Arthrospora adevorans TMCC 70007: the sequence is a sequence from 1000bp upstream of the 1 st site of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC 70007 strain to the 589 th site of the nucleotide sequence shown as SEQ ID No.1, and the length of the forward primer is 20-30 bp; the reverse primer is reversely complementary with the sequence in the genome of the TMCC 70007 bacterial strain: the sequence is a sequence in a region from 601 th site of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC 70007 strain to 1000bp downstream of the last site of the nucleotide sequence shown as SEQ ID No.1, and the length of the reverse primer is 20-30 bp.
In a more preferred embodiment, the nucleotide sequences of the forward primer and the reverse primer are as follows:
forward primer ARC 19-F: 5'-GCTCAACTGCCAATGGTTCA-3' (SEQ ID No. 5);
reverse primer ARC 19-R: 5'-CTTGCCATGGTGGTGGGATA-3' (SEQ ID No. 6).
The primers of the invention enable specific amplification of the DNA barcode sequence.
The invention also provides a kit for identifying the adenine node B yeast strain, which comprises the primer pair.
In another embodiment, the kit further comprises a DNA barcode according to the present invention. The DNA barcode may be present on a recording medium. The recording medium is, for example, an optical disc. The kit may also comprise any tools and reagents for the experimental procedure.
In yet another aspect of the invention, a method for identifying a strain of nodospora adenine dinucleotide is provided, comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA of the step a) as a template and using the primer pair of claim 3 to obtain a PCR product;
c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC 70007 strain, and if the target band exists, performing the step d);
d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (2) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA barcode according to claim 1, and if the homology is more than 97 percent (preferably more than 99 percent), judging that the strain to be detected is the adenine arthrobacter adenine-burning TMCC 70007 strain.
In a specific embodiment of the present invention, the procedure of PCR amplification is: 1) pre-denaturation at 94-96 deg.C for 8 min; 2) denaturation at 94-96 ℃ for 45 seconds, annealing at 55-57 ℃ for 45 seconds, and extension at 72 ℃ for 1 min 15 seconds, wherein the procedure 2) is performed for 30-35 cycles; 3) extension at 72 ℃ for 10 min.
In another embodiment of the present invention, the method further comprises performing cluster analysis (e.g., phylogenetic tree) on the sequence to be tested obtained from the sequencing result and the DNA barcode of the present invention, and if the sequence to be tested and the DNA barcode are clustered together, determining that the strain to be tested is the alternaria adenini yeast strain TMCC 70007.
In a particular embodiment of the invention, the genomic DNA extracted from the strain to be identified is amplified by PCR using the primer pairs of the invention and then detected by agarose gel electrophoresis. Identifying the strains based on detecting the presence or absence of PCR products: if the strain to be identified does not amplify a corresponding target band, the strain is not TMCC 70007; if the corresponding target band is amplified, the strain is proved to be possible TMCC 70007. For further identification, the PCR product is sequenced, the DNA sequencing result and the DNA bar code sequence are subjected to homology comparison to obtain the similarity (namely homology) between the sequences, and if the sequence homology is less than 97%, the strain to be detected is judged not to be the adenine node B spore forming yeast strain TMCC 70007. If the sequence homology is greater than or equal to 97% (preferably 99% or more), the test strain is judged to be the Arthrospora adenantha strain TMCC 70007.
If a clustering analysis is performed, such as a phylogenetic tree, the DNA barcode is used with the DNA sequencing results (i.e., the sequences to be tested) of each strain to be identified to construct an NJ phylogenetic tree using MEGA 6 or PAUP software. If the sequence to be tested of the strain to be identified is clustered with the DNA bar code of the Arthrospora adenini TMCC 70007, the strain is identified as the Arthrospora adenini TMCC 70007.
The term "cluster" as used herein means that the clusters are in the same branch and have the same evolutionary distance after phylogenetic tree analysis.
The invention also provides application of the DNA bar code in identifying the alternaria adephaga yeast strain.
The invention also provides application of the primer pair in identifying the Arthrospora adenanthaticus strain.
Examples
The present invention is further illustrated by the following specific examples. The methods used in the examples, unless otherwise specified, were carried out using conventional methods and known tools.
Example 1: ARC19 Gene and DNA barcode Generation
Proteome and genome analysis methods and mass spectrometry methods used in this example are all known in the art, and therefore detailed descriptions of the procedures are omitted.
1. By utilizing a high-coverage proteome technology, pFind and pAnno software are adopted to carry out deep coverage research on proteome on the Puer tea industrial fermentation bacteria Arthrospora adenine-eating TMCC 70007, and the genome is verified by annotating coding genes. Specifically, in order to find a novel protein coding region, a Six-Frame Translation database of the genome data of the Geobacillus adenini TMCC 70007 was obtained using a Six-Frame Translation (Six Frame Translation) strategy in systematic proteomics, exhaustively listing 6 coding possibilities of the genome (+1, +2, +3, -1, -2, -3), and the nucleic acid sequence was referred to as a "Six-Frame Translation nucleic acid sequence" and the protein sequence was referred to as a "Six-Frame Translation protein sequence". In general, a six-frame translational nucleic acid sequence is a sequence from one terminator to the next, which is also referred to herein as a "protein coding frame". The discovery and identification of new peptide fragments and new proteins was performed using this six-box translation database, using pFind and pAnno software to compare with whole protein mass spectral data of TMCC 70007 strain.
Through identification, a peptide segment SQSLRPYLMAVR which is not found in the annotated genome of the adenine-eating arthrobotrys yeast TMCC 70007 in the prior art is found, and a mass spectrum is shown in figure 1.
The manual inspection of the mass spectrum result shows that the secondary Mass Spectrum (MS) of the peptide segment SQSLRPYLMAVR is detected2) Almost all the y ion sequences are well matched, the signals are strong, and the results are reliable. To further confirm this identification, the peptide fragment was chemically synthesized according to the amino acid sequence of newly identified peptide fragment SQSLRPYLMAVR, and utilized the aboveSQSLRPYLMAVR, the mass spectrometry conditions produced a secondary spectrum of the synthesized peptide fragment, see FIG. 2.
Next, high energy collision MS on the synthetic peptide fragments2Verification is carried out, and the primary parent ion and the secondary daughter ion both accord with theoretical values, so that the sequence of the synthesized peptide fragment is correct (figure 2). On the basis, the MS of the peptide fragment synthesized by manually checking new peptide fragment sequences identified according to large-scale proteome data2And a new peptide fragment spectrogram is identified on a large scale, the two spectrograms are almost completely consistent, and a cosin value obtained by the similarity of the sub-ions is as high as 0.99, so that the new peptide fragment identified from the industrial fermentation bacteria of the Pu' er tea by the adenine nodospora gladiata TMCC 70007 is correct and error-free.
2. The six-frame translated nucleic acid sequence (protein coding frame) SEQ ID NO.2 is obtained by defining the region between the former stop codon and the latter stop codon, depending on the position of the novel peptide fragment. The mRNA corresponding to the sequence of SEQ ID NO.2 and the amino acid sequence encoded by the mRNA are shown in FIG. 3.
3. In order to further determine the coding start site and the coding end site of the gene for coding the protein, the coding frame of the protein is respectively expanded by 1000bp upstream and downstream, GeneMark.hmm is adopted for gene prediction, and Schizosaccharomyces pombe (Schizosaccharomyces pombe) is selected as a reference species. The gene is predicted in the region, the coding frame of the gene is consistent with the sequence of the coding frame of the protein, the coding start and termination sites of the gene are determined, and the ORF of the gene is SEQ ID NO. 1. Positions 37-127, 455-507 and 697-1024 of SEQ ID NO.1 are introns of the gene.
The correspondence between the mRNA sequence transcribed from the encoding gene (ARC19) and the amino acid sequence of the protein translated therefrom is shown in FIG. 4. Peptide SQSLRPYLMAVR was located upstream of the gene.
The nucleotide sequence of the coding gene (ARC19) is 1189bp, the intron region is removed, 232 amino acids are coded, the theoretical molecular weight is 26.77kDa, and the theoretical coding amino acid sequence is shown in SEQ ID NO. 3.
4. The amino acid sequence (SEQ ID NO.3) of the theoretical coding product of the gene is used for carrying out NCBI-BLASTP analysis, the sequence has a structural domain similar to that of ARPC4 super family protein, the protein family is a composition of a plurality of eukaryotic ARP2/3 complex subunits, and ARP2/3 complex is involved in intracellular actin polymerization. The identified ARC19 protein sequence was 91% similar to the ARC19 subunit (accession number: CDO53724.1) of the ARP2/3 complex of Geotrichum candidum (Geotrichum candidum) CLIB 918 and the protein BLASTP sequence query coverage was 71%. The above blastp alignment results are summarized in table 1.
The NCBI-BLASTP results show that the missing injection protein detected by the people is ARC19 homologous gene product, and the sequence of the missing injection protein has certain conservation.
5. The ORF sequence of the identified ARC19 gene (i.e., SEQ ID NO.1) was subjected to NCBI-BLASTN analysis, and the results are shown in FIGS. 5 and 7. The BLASTN results are summarized in table 2.
The result shows that only a part of the sequence of the gene has 85 percent of homology with 13 percent of the mRNA sequence of the subunit No.4 of the ARP2/3 complex of the Pyrenophora tritici-repentis (Pyrenophora tritici-repentis) Pt-1C-BFP strain, and the homologous segment has only about 160bp, which shows that the missed-injection gene ARC19 detected in the adenine-node spore-feeding yeast has better sequence specificity compared with the existing sequences in the NCBI nr database, and the DNA homology is very low although the amino acid sequence part segment of the ARC19 protein is conserved. The result proves that the ORF sequence of the gene can be used for developing the DNA bar code of the Puer tea fermentation strain Alternaria adefovea TMCC 70007.
6. The region from 1000bp upstream to 1000bp downstream of SEQ ID No.1 was further selected (as shown in SEQ ID No. 4). The sequence of SEQ ID NO.4 is subjected to NCBI-BLASTN analysis, a Nucleotide collection (nr/nt) database is searched, and as a result, see the following table 3, the sequence is found to have 85% identity with a 6% sequence (about 160bp long) of Pyrenophora tritici-repentis Pt-1C-BFP strain and actin related protein 2/3 complex subunit 4mRNA sequence, and further shows that the sequence has better specificity and can be used as a DNA bar code for specifically identifying the adenine-nodulospora yeast TMCC 70007 strain. Furthermore, the sequence of SEQ ID NO.4 was further subjected to NCBI-BLASTN analysis, and the Whole genome sequence of Arthrospora adenine-type yeast (taxonomy ID: taxi: 409370) in the white-genome shotgun contigs (wgs) database was searched, and the search results are summarized in Table 4. As a result, the similarity of the SEQ ID NO.4 and the complete genome shotgun sequencing sequence Arad1D chromosome contig 1 of the Alternaria adenini LS3 strain is 91%, and the sequence query coverage rate is 72%. Although TMCC 70007 and LS3 strains are the same species, the sequences of TMCC 70007 and LS3 strains have obvious sequence difference in the homologous regions, and the sequence difference is enough to distinguish the two strains, so that the sequences have better specificity, and can be used as DNA barcode sequences for specifically identifying the strains. In conclusion, the sequence from upstream 1000bp to downstream 1000bp of SEQ ID NO.1 (SEQ ID NO.4) can be used as a DNA barcode for identifying the strain of Arthrospora adenantha.
7. Currently, only the genome of the Arthrospora adenantha LS3 strain has been reported among the species of Arthrospora adenantha. After analysis by Local-BLASTN, the gene sequence SEQ ID NO.1 was found to have a homologous sequence in the genome of LS3 strain. After DNAMAN analysis, the homology of the two sequences was 78.69%, although TMCC 70007 and LS3 strains were of the same species (instant Arthromyces adenine, Blastotrys adenoninivorans), about 30% of the sites were different in 1189 bases of the DNA barcode sequence, and the results are shown in FIG. 6. This further demonstrates that the use of this DNA barcode sequence can effectively distinguish TMCC 70007 strain from other strains within the species.
Example 2 identification of strains Using DNA barcodes
And judging whether the sample to be detected is the industrial application strain of the Puer tea, namely the Arthropoda adenine-eating yeast TMCC 70007 according to the amplification result of the sample to be detected and the sequence homology of the sample to be detected and the strain SEQ ID NO.1 of the Arthropoda adenine-eating yeast TMCC 70007.
(1) Based on SEQ ID NO.1, PCR primers are designed at two ends of the gene by adopting an NCBI primer design tool, an amplification product needs to comprise an initial site and a termination site of the gene, and the obtained forward and reverse primer sequences are respectively
Forward primer sequence: ARC 19-F: 5'-GCTCAACTGCCAATGGTTCA-3', respectively;
reverse primer sequence: ARC 19-R: 5'-CTTGCCATGGTGGTGGGATA-3' are provided.
The position of the primers is shown in FIG. 7. The amplified sequence is SEQ ID NO. 7.
(2) The source of the strain
TABLE 5 information on selected related strains
| Sample numbering | Name of scholars | | Habitat | |
| 1 | | TMCC | 70007 | Pu' er tea pile fermentation, China |
| 2 | Arthrospora adevora | CBS 8244T | Soil, the Netherlands | |
| 3 | | CBS | 7350 | Corn fodder stored in cellar, Holland |
| 4 | | CBS | 7370 | Soil, south Africa |
| 5 | | CBS | 8335 | Clay, Italy |
| 6 | Blastobotrys raffinosifermentans | CBS 6800T | Is unknown |
(3) Respectively extracting strain DNA: OMEGA e.z.n.a was used.TMYeast genomic DNA was extracted using the Yeast DNA kit (Bio Rad laboratories) and the DNA concentration of the sample was diluted to 0.5. mu.g/. mu.L with sterile deionized water.
(4) Amplifying DNA fragments, and carrying out Polymerase Chain Reaction (PCR), wherein the sequences of the primers are as follows:
forward primer sequence: ARC 19-F: 5'-GCTCAACTGCCAATGGTTCA-3', respectively;
reverse primer sequence: ARC 19-R: 5'-CTTGCCATGGTGGTGGGATA-3' are provided.
The PCR reaction system is 50 mu L, and the PCR reagent is Thermo ScientificTMTaq DNA Polymerase (recombinant) of (1): ddH2O 37.7μL、MgCl2mu.L, dNTPs 4. mu.L, forward primer 1. mu.L, reverse primer 1. mu.L, Taq DNA polymerase 0.3. mu. L, DNA template 1. mu.L, without dye. The amplification procedure was: pre-denaturation at 94 ℃ for 8 min; then denaturation at 94 ℃ for 45 seconds, annealing at 56 ℃ for 45 seconds, and extension at 72 ℃ for 1 minute and 15 seconds are carried out for 32-35 cycles in total; final extension at 72 ℃ for 10 min.
(5) Detection of amplification products: the PCR fragment size was detected by electrophoresis on 1.0% agarose gel, 1 XTBE, and DNA marker. If the strain to be detected has no amplified band, the strain is not the adenine node B spore feeding yeast TMCC 70007; if a clear band appears and no miscellaneous band exists, the DNA fragment is sent to a biological sequencing company for DNA fragment sequencing.
(6) The primer can only realize amplification in the Arthrospora adenini TMCC 70007, and can only realize amplification in the same species of the Arthrospora adenini CBS 8244T CBS 7350, CBS 7370, CBS 8335 and Blastotrys raffinositifications CBS 6800TAmplification cannot be achieved. The results are shown in FIG. 8. The theoretical amplification fragment of the PCR primer is 1380bp, and the actual amplification result is consistent with the expectation. To further verify the sequence of the amplified DNA, sequencing and homologous sequence comparison were performed.
(7) For the sequencing result of the sequence with the band, firstly, the quality of a sequence peak image obtained after sequencing is checked by software Chromas, after the quality of the peak image is determined to meet the requirement of data analysis, the forward and reverse sequences are spliced by Seqman in a DNASTAR software package. And (3) manually proofreading and splicing the sequence, and if the homology of the DNA fragment of the strain to be detected and the standard gene sequence of the adenine node B.sp.tmcc 70007 is more than 97%, judging that the strain to be detected can be the adenine node B.sp.tmcc 70007 strain. For example, through DNAMAN, the sequence obtained by sequencing the strain TMCC 70007 is completely consistent with the DNA barcode sequence SEQ ID NO.1 after alignment, and the reliability of the method is further proved. The results are shown in FIG. 9.
SEQUENCE LISTING
<110> Menghai tea industry, Limited liability company
<120> DNA barcodes, primers, kits, methods and uses
<130> FI-164052-59:52/C
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1189
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 1
atgatacccc attctatagc cgttgaaatg gacagggtga cgctcagcca cgcttcacca 60
ctcacaacat ggtaggtaga gacaagaggc actaaaggat agctggggaa atggtgatac 120
taactagtct caatcgctac gcccgtatct tatggcggtg cgccagtcgt tgacggcggc 180
actttgcctg gagaactttg cttctcaggt ggtggaacga cacaataacc ctgaggtgga 240
gtccagaaag acccccgaag cgctgctcaa cccactgact attgctcgaa atgaaaacga 300
aaaggtgctt atcgagcctt ctatcaactc cgtgcgagtt tctatcaaga tcaagcaggc 360
cgatgagatt gagaccattc tggtgcatca gttcacgcgg ttcttgaccg gacgagccga 420
gagcttttac attctacgac gaaagcctat tgatgtgagt tggattggac attgggtgaa 480
tatgtgttga gagtactaac tgtttagggc tatgatattt cgtttttgat cacaaacttt 540
cacactgagc agatgctcaa gcataagcta gtggacttta ttattgagtt tatggaggag 600
gttgacaagg agatttcgga aatgaagttg ttccttaacg ctcgagctcg attcgtggcc 660
gagtcatatt tgacaccggt aagttgattg gtctttgacg atttccatac ctaggatgct 720
aacggatagt ttgattagaa gtgcgggaca agcgctgtac atgtatacat gtattcaatt 780
agatgacata cgattatagt ccttgactag cgttcattag ctattaatag tctataattg 840
atcaaactcg tcggtttcta tcccttcaaa gagccattga gaaccattcc gggggcgtcc 900
agctcatgtt atatcaagaa aaaatcactt tttatacaaa actgtttagt gattagactt 960
gcgagactgt gaaagcccaa gtagacattc agactgcttg gatatctgtg ttctcgaccc 1020
ttagctcccg agtagtggac gagcgagaga tttcccagaa gcattgcgtc tggacaaatt 1080
tctgccccaa gtagcggatg tattaccatg gctcgaagag acagggcttc aactcatggc 1140
gctcgctctt tcccttcgta ttgaagaaca taatatctgt aagtgttag 1189
<210> 2
<211> 366
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 2
tagtctcaat cgctacgccc gtatcttatg gcggtgcgcc agtcgttgac ggcggcactt 60
tgcctggaga actttgcttc tcaggtggtg gaacgacaca ataaccctga ggtggagtcc 120
agaaagaccc ccgaagcgct gctcaaccca ctgactattg ctcgaaatga aaacgaaaag 180
gtgcttatcg agccttctat caactccgtg cgagtttcta tcaagatcaa gcaggccgat 240
gagattgaga ccattctggt gcatcagttc acgcggttct tgaccggacg agccgagagc 300
ttttacattc tacgacgaaa gcctattgat gtgagttgga ttggacattg ggtgaatatg 360
<210> 3
<211> 232
<212> PRT
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 3
Met Ile Pro His Ser Ile Ala Val Glu Met Asp Arg Ser Gln Ser Leu
1 5 10 15
Arg Pro Tyr Leu Met Ala Val Arg Gln Ser Leu Thr Ala Ala Leu Cys
20 25 30
Leu Glu Asn Phe Ala Ser Gln Val Val Glu Arg His Asn Asn Pro Glu
35 40 45
Val Glu Ser Arg Lys Thr Pro Lys Ala Met Leu Asn Pro Leu Thr Ile
50 55 60
Ala Arg Asn Glu Asn Glu Lys Val Leu Ile Glu Pro Ser Ile Asn Ser
65 70 75 80
Val Arg Val Ser Ile Lys Ile Lys Gln Ala Asp Glu Ile Glu Thr Ile
85 90 95
Leu Val His Gln Phe Thr Arg Phe Leu Thr Gly Arg Ala Lys Ser Phe
100 105 110
Tyr Ile Leu Arg Arg Lys Pro Ile Asp Gly Tyr Asp Ile Ser Phe Leu
115 120 125
Ile Thr Asn Phe His Thr Glu Gln Met Leu Lys His Lys Leu Val Asp
130 135 140
Phe Ile Ile Glu Phe Met Glu Glu Val Asp Lys Glu Ile Ser Glu Met
145 150 155 160
Lys Leu Phe Leu Asn Ala Arg Ala Arg Phe Val Ala Glu Ser Tyr Leu
165 170 175
Thr Pro Leu Pro Ser Ser Gly Arg Ala Arg Asp Phe Pro Glu Ala Leu
180 185 190
Arg Leu Asp Lys Phe Leu Pro Gln Val Ala Asp Val Leu Pro Trp Leu
195 200 205
Glu Glu Thr Gly Leu Gln Leu Met Ala Leu Ala Leu Ser Leu Arg Ile
210 215 220
Glu Glu His Asn Ile Cys Lys Cys
225 230
<210> 4
<211> 2361
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 4
tcgtcatcct tttggcgtac atagtcgtta aatatttcct gtcggcggac tggatcctta 60
atgacccaat attctggttt ggttactaac gctttcatag catcggtcca tgaccaagat 120
atgtctaccc ctacctgttc aagcattgct cggtaatcct caattgcatc ttccttggac 180
ttgtaccatc gctcatgaga aggattgaga aggctttggc tagcgaaggg atgattttca 240
gttcctgaac cactgctaaa cacatacctt tcggatgact gcgctgttga ctgagcataa 300
cctccattgg ctgacccggg cgcggtacca gtcgaagtct ttgagtgctc attgatgatc 360
cgttcaactt caggaggggt ctcccatact gactgtccag tctgtgagtt gtaccagtaa 420
tggtgtcccc catcggccac gtacttccgc cagttgacct ccagaaatgc ccgctccaaa 480
tcagtgaaaa gctcttccgg cttatcccaa gtggtttcta acgtctccgt attgtaataa 540
tacaccgctc cattatcctc tacctcttgc cagggcatgg tcgtgccctt tgcttatcaa 600
tcaattagtt attgaaaaag gaacaaatat caccgcaaac ttatcttgca acaattcaat 660
ggatactgct gctcgatata tttcgagaag aaatcagtac tgtaagcgag tcacgtgact 720
gtacttcttg gggaacgtac aggttgctca actgccaatg gttcatggag ttttttaggt 780
caattataga ataaccatta tggagtttta tttaattata aaaaccgcca cttttttatt 840
gttgtttccc cgagaaatgc ttcatttttc aacatgatac cccattctat agccgttgaa 900
atggacaggg tgacgctcag ccacgcttca ccactcacaa catggtaggt agagacaaga 960
ggcactaaag gatagctggg gaaatggtga tactaactag tctcaatcgc tacgcccgta 1020
tcttatggcg gtgcgccagt cgttgacggc ggcactttgc ctggagaact ttgcttctca 1080
ggtggtggaa cgacacaata accctgaggt ggagtccaga aagacccccg aagcgctgct 1140
caacccactg actattgctc gaaatgaaaa cgaaaaggtg cttatcgagc cttctatcaa 1200
ctccgtgcga gtttctatca agatcaagca ggccgatgag attgagacca ttctggtgca 1260
tcagttcacg cggttcttga ccggacgagc cgagagcttt tacattctac gacgaaagcc 1320
tattgatgtg agttggattg gacattgggt gaatatgtgt tgagagtact aactgtttag 1380
ggctatgata tttcgttttt gatcacaaac tttcacactg agcagatgct caagcataag 1440
ctagtggact ttattattga gtttatggag gaggttgaca aggagatttc ggaaatgaag 1500
ttgttcctta acgctcgagc tcgattcgtg gccgagtcat atttgacacc ggtaagttga 1560
ttggtctttg acgatttcca tacctaggat gctaacggat agtttgatta gaagtgcggg 1620
acaagcgctg tacatgtata catgtattca attagatgac atacgattat agtccttgac 1680
tagcgttcat tagctattaa tagtctataa ttgatcaaac tcgtcggttt ctatcccttc 1740
aaagagccat tgagaaccat tccgggggcg tccagctcat gttatatcaa gaaaaaatca 1800
ctttttatac aaaactgttt agtgattaga cttgcgagac tgtgaaagcc caagtagaca 1860
ttcagactgc ttggatatct gtgttctcga cccttagctc ccgagtagtg gacgagcgag 1920
agatttccca gaagcattgc gtctggacaa atttctgccc caagtagcgg atgtattacc 1980
atggctcgaa gagacagggc ttcaactcat ggcgctcgct ctttcccttc gtattgaaga 2040
acataatatc tgtaagtgtt agataggagc gtacaattgc agcacatatc cttatgccag 2100
cgtgttatcc caccaccatg gcaaggtggt tatcgatgaa acggtccgcc tctctcttca 2160
gcatcaagat acagctcgat acaacaaacg cctctacagt ctgggggtcg gtatgcttat 2220
cttcgttcca catacgcaga tcgatgaaca aacggccccc attgccgcct gagtacttct 2280
tggtgccggg ggtacgatac tggtcattgc gaatgagatg tgcgacgcgg gcgccctttt 2340
tactgcgtcc accgccgggt a 2361
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
<210> 7
<211> 1380
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 7
gctcaactgc caatggttca tggagttttt taggtcaatt atagaataac cattatggag 60
ttttatttaa ttataaaaac cgccactttt ttattgttgt ttccccgaga aatgcttcat 120
ttttcaacat gataccccat tctatagccg ttgaaatgga cagggtgacg ctcagccacg 180
cttcaccact cacaacatgg taggtagaga caagaggcac taaaggatag ctggggaaat 240
ggtgatacta actagtctca atcgctacgc ccgtatctta tggcggtgcg ccagtcgttg 300
acggcggcac tttgcctgga gaactttgct tctcaggtgg tggaacgaca caataaccct 360
gaggtggagt ccagaaagac ccccgaagcg ctgctcaacc cactgactat tgctcgaaat 420
gaaaacgaaa aggtgcttat cgagccttct atcaactccg tgcgagtttc tatcaagatc 480
aagcaggccg atgagattga gaccattctg gtgcatcagt tcacgcggtt cttgaccgga 540
cgagccgaga gcttttacat tctacgacga aagcctattg atgtgagttg gattggacat 600
tgggtgaata tgtgttgaga gtactaactg tttagggcta tgatatttcg tttttgatca 660
caaactttca cactgagcag atgctcaagc ataagctagt ggactttatt attgagttta 720
tggaggaggt tgacaaggag atttcggaaa tgaagttgtt ccttaacgct cgagctcgat 780
tcgtggccga gtcatatttg acaccggtaa gttgattggt ctttgacgat ttccatacct 840
aggatgctaa cggatagttt gattagaagt gcgggacaag cgctgtacat gtatacatgt 900
attcaattag atgacatacg attatagtcc ttgactagcg ttcattagct attaatagtc 960
tataattgat caaactcgtc ggtttctatc ccttcaaaga gccattgaga accattccgg 1020
gggcgtccag ctcatgttat atcaagaaaa aatcactttt tatacaaaac tgtttagtga 1080
ttagacttgc gagactgtga aagcccaagt agacattcag actgcttgga tatctgtgtt 1140
ctcgaccctt agctcccgag tagtggacga gcgagagatt tcccagaagc attgcgtctg 1200
gacaaatttc tgccccaagt agcggatgta ttaccatggc tcgaagagac agggcttcaa 1260
ctcatggcgc tcgctctttc ccttcgtatt gaagaacata atatctgtaa gtgttagata 1320
ggagcgtaca attgcagcac atatccttat gccagcgtgt tatcccacca ccatggcaag 1380
Claims (5)
1. A primer pair for amplifying DNA barcodes for identifying the adenine node B.adenine node B yeast TMCC 70007 strain comprises the nucleotide sequences of a forward primer and a reverse primer which are respectively shown as follows:
a forward primer: 5'-GCTCAACTGCCAATGGTTCA-3', respectively;
reverse primer: 5'-CTTGCCATGGTGGTGGGATA-3', respectively;
wherein the DNA barcode is derived from the genome of the Arthrospora adenantha TMCC 70007 strain, and the nucleotide sequence is selected from the sequences shown as SEQ ID No.4 and comprises the sequence shown as SEQ ID No. 1.
2. A kit for identifying a nodospora adevorans TMCC 70007 strain comprising the primer pair according to claim 1.
3. A method for identifying a strain of the nodospora adephagi TMCC 70007, comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA of the step a) as a template and using the primer pair of claim 1 to obtain a PCR product;
c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC 70007 strain, and if the target band exists, performing the step d);
d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code of claim 1, and if the homology is more than 97%, judging that the strain to be detected is the adenine arthrobacter adenine-burning TMCC 70007 strain.
4. The use of the primer pair according to claim 1 for identifying the strain of the Arthrospora adenantha TMCC 70007.
5. Use of the kit according to claim 2 for identifying the strain of Arthrospora adenantha TMCC 70007.
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Non-Patent Citations (1)
| Title |
|---|
| The complete genome of Blastobotrys(Arxula) adeninivorans LS3- a yeast of biotechnological interest;Gotthard Kunze et al.;《Biotechnology for Biofuels》;20141231;第7卷(第66期);全文 * |
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