CN109385483B

CN109385483B - DNA bar code, primer, kit, method and application

Info

Publication number: CN109385483B
Application number: CN201710656060.2A
Authority: CN
Inventors: 田飞; 徐平; 唐蜀昆; 施佳辉; 高林瑞; 高慧英; 职晓阳; 丁章贵
Original assignee: Yunnan Dayi Microbial Technology Co ltd; Menghai Tea Industry Co ltd
Current assignee: MENGHAI TEA INDUSTRY Co.,Ltd.; Yunnan Dayi Microbial Technology Co., Ltd
Priority date: 2017-08-03
Filing date: 2017-08-03
Publication date: 2021-07-27
Anticipated expiration: 2037-08-03
Also published as: CN109385483A

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 TMCC70007, and can quickly and accurately identify the strain from confusable strains or other strains in the same species.

Description

DNA bar code, primer, kit, method and application

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-dried raw 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, 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 the Pu' er tea with different qualities is gradually formed by adding other factors such as damp and hot. Different origins, because of the different microbial community structures, have different flavors and qualities.

Besides the unique flavor and culture of Pu 'er tea, the Pu' er tea has the health care effects of losing weight, reducing blood sugar and blood fat, preventing and improving cardiovascular diseases, resisting aging, resisting cancer, diminishing inflammation, helping digestion, nourishing the stomach and the like and is 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, certain potential safety hazard, long production period, too high labor input, excessive microorganism quantity, mite breeding 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-nodulation yeast (Saccharomyces adeninivorans) and the like in the pile fermentation process are relatively stable, 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 the further development of Pu ' er tea scientific research, more and more people begin to explore the artificial inoculation and fermentation of Pu ' er tea, and at present, a plurality of bacterial strains are applied to the artificial controllable fermentation of Pu ' er tea. 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 infringement behavior of abusing the Pu ' er tea fermentation microorganisms and solve the difficult problem of difficult demonstration in the process of artificially controllable fermentation of Pu ' er tea during the infringement of strains, the development of a method for quickly and accurately identifying the Pu ' er tea fermentation strains is imperative, a molecular identification method of the Pu ' er tea fermentation strains needs to be urgently established, a method for accurately identifying and distinguishing the similar strains by a DNA bar code technology is developed, so that the problems of identification and identification of industrial production strains are solved by a method combining morphological characteristics and molecular data analysis, a theoretical basis is provided for the development of the controlled industrial fermentation process of Pu ' er tea and resource protection, 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 alternaria adegua strain for fermentation production of Pu ' er tea, the invention provides a DNA bar code, a primer, a kit, a method and application for identifying the alternaria adegua strain, so that the alternaria adegua strain TMCC70007 strain is 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-lifting method and basis can be provided for an artificial controllable fermentation process of the Pu ' er tea and illegal abuse of the strain.

In order to achieve the purpose, the invention adopts the following technical scheme:

(1) a DNA barcode for identifying a desmodium adenanthus yeast strain, the DNA barcode being derived from the genome of the desmodium adenanthus TMCC70007 strain and comprising at least 500bp of a sequence selected from the DNA sequences shown as SEQ ID No.1, and the length of the DNA barcode being 500bp to 3500bp, preferably 500bp to 2200bp, more preferably 500bp to 1500 bp.

(2) The DNA barcode according to (1), wherein the nucleotide sequence thereof is shown as SEQ ID No.1, SEQ ID No.2, 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 TMCC70007 strain: 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 TMCC70007 strain to a 535 nd 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 of the strain is reversely complementary to a sequence in the genome of the TMCC70007 strain: the sequence is a sequence in a region from the 501 st bit of the nucleotide sequence shown as SEQ ID No.1 to the downstream 1000bp of the last bit of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC70007 strain, 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'-CGACCGAGAACGACAGGAAT-3', respectively;

reverse primer: 5'-ACACGTCAACAGGCGATCAA-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 TMCC70007 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 TMCC70007 strain.

(8) Use of the DNA barcode according to (1) for identifying a strain of Alternaria adefovea.

(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 TMCC70007 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 TMCC70007 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 TMCC70007 produced by the industrial fermentation of the Pu' er tea. The bar code sequence can realize the rapid and accurate identification and differentiation of the strains in the adenine node spore feeding 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 invention further discovers that compared with other genes, the sequence (such as SEQ ID No.1) in the missed-release gene has the characteristics of universality, easiness in amplification and easiness in comparison, and the difference of the sequence among different strains in the adenine-dependent node spore yeast is obvious.

4. The invention establishes a standard gene sequence and a sample identification method of the Puer tea industrial fermentation production strain Arthrospora adenini TMCC70007, 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 an identification rule can be established for identification by using a phylogenetic tree method based on cluster analysis, so that the reliability and accuracy of the identification are greatly superior to those of the conventional molecular identification method, and the blank of the 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 DYTPAQMAAVNR.

FIG. 2 shows a comparison of the mass spectrum of chemically synthesized peptide DYTPAQMAAVNR with the mass spectrum of the original identified peptide; 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.

Figure 4 shows a corresponding map of the mRNA sequence transcribed by the ORF encoding the newly identified HSP40 protein and its encoded protein sequence, with the new peptide fragment identified in the grey background section.

FIG. 5A shows a SDS-PAGE separation of the cellular holoprotein of TMCC70007, the bold band is the position of HSP40 protein; fig. 5B shows a molecular weight validation graph of HSP40 protein. In FIG. 5B, the abscissa represents the logarithmic value of the protein molecular weight marker (marker) with the base 10 for the different molecular weights, and the ordinate represents the ratio of the migration amount of each molecular weight protein in the protein molecular weight marker in SDS-PAGE electrophoretic separation to the total migration amount of SDS-PAGE. Theoretically, the molecular weight MW of HSP40 protein is 37.7kDa, lg (MW) lg (37.7) ≈ 1.58, and the molecular logarithm of HSP40 corresponds to fig. 5B abscissa 1.58, and its mobility corresponds to the value on the y-axis when the mobility curve is x 1.58.

FIG. 6 shows an alignment of the barcode sequence SEQ ID NO.1 with homology thereto by NCBI-BLASTN, the three short grey segments below FIG. 6 showing 3 homologous sequences.

FIG. 7 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. 8 shows the positions of primers used to amplify SEQ ID NO.1, the underlined regions in the sequence being the regions where the primers are located, the bold font ATG and TAA being the start and stop sites, and the amplified sequence being SEQ ID NO.7, in one embodiment of the invention.

FIG. 9 shows the results of agarose gel electrophoresis of PCR amplification products of test strains obtained using the primers of the present invention.

FIG. 10 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 misannotated coding protein from the genome of the Alternaria adenini TMCC70007 strain, and the protein is called HSP40 protein because the protein has a structural domain of HSP40 family. The invention obtains the corresponding gene sequence in the TMCC70007 strain genome from the newly found protein sequence, the gene sequence is called HSP40 gene, and the gene sequence (SEQ ID No.1) for coding the protein in the Alternaria adenini TMCC70007 strain 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 Alternaria adenini TMCC70007 strain produced by the industrial fermentation of Pu' er 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 strain of Arthrospora adenine-feeding TMCC70007 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 arthrobacter adenivorus, the DNA barcode being derived from the genome of the strain of arthrobacter adenivorus TMCC70007 and comprising at least 500bp of a sequence selected from the DNA sequences shown as SEQ ID No.1, and the DNA barcode having a length of 500bp to 3500bp, preferably 500bp to 2200bp, more preferably 500bp 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 SEQ ID No.1 is 1035bp, and the sequence itself can be used as a DNA bar code for identifying the strain of the nodospora adenine-eating yeast TMCC70007 because the SEQ ID No.1 is specific to the genome of the strain of the nodospora adenine-eating yeast TMCC 70007. In addition, theoretical analysis and experimental verification prove that the specificity of the DNA barcode containing the sequence and having longer length is better ensured. When the length of the DNA barcode is too long (e.g., greater than 3500bp), it is less desirable for the amplification procedure. On the other hand, when the length of the DNA bar code is not less than 500bp, the operation requirements of easy amplification and easy comparison can be met; theoretical analysis and experimental verification prove that at least 500bp 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-adenine-B-yeast.

In a preferred embodiment of the invention, the DNA barcode sequence is as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 7.

In this context, the term "missing-release gene" means that after the species has completed the genome sequencing, 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 thus is difficult to be found in the study.

The term "DNA barcoding" refers to a new molecular identification technique 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, there are 3 coding possibilities given to a DNA sequence, using triplet codons to encode the protein, 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 TMCC70007 strain to a 535 nd 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 of the strain is reversely complementary to a sequence in the genome of the TMCC70007 strain: the sequence is a sequence in a region from the 501 st bit of the nucleotide sequence shown as SEQ ID No.1 to the downstream 1000bp of the last bit of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC70007 strain, 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 HSP 40-F: 5'-CGACCGAGAACGACAGGAAT-3' (SEQ ID No. 5);

reverse primer HSP 40-R: 5'-ACACGTCAACAGGCGATCAA-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;

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% (preferably more than 99%), judging that the strain to be detected is the adenine nodospora glaucomatosa TMCC70007 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 specific embodiment of the invention, genomic DNA extracted from the strain to be identified is subjected to PCR amplification using the primer pairs of the invention, followed by detection 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 TMCC70007, the strain is identified as the Arthrospora adenini TMCC 70007.

The term "cluster" as used herein means that the branches are located at 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 adenine node B saccharomycetes strains.

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: acquisition of HSP40 Gene and DNA Bar codes

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 high-coverage proteomics research on the Puer tea industrial fermentation strain Arthrospora adenine-eating yeast TMCC70007, 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 TMCC70007 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 protein sequence was referred to as a "Six-Frame Translation protein sequence", and the corresponding nucleic acid sequence was referred to as a "Six-Frame Translation nucleic acid sequence". Generally, 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 TMCC70007 strain.

Through identification, a peptide segment DYTPAQMAAVNR which is not found in the adenine node B.adenine-eating yeast TMCC70007 annotated genome in the prior art is found, and the mass spectrum of the peptide segment is shown in figure 1.

The results of manual inspection of mass spectrum spectrogram show that almost all y ion sequences of the secondary mass spectrum spectrogram (MS2) of the peptide segment DYTPAQMAAVNR are detected, the matching is good, the signal is strong, and the result is more reliable. To further confirm this identification, the peptide fragment was chemically synthesized according to the amino acid sequence of newly identified peptide fragment DYTPAQMAAVNR, and a secondary spectrum of the synthesized peptide fragment was generated using the mass spectrometry conditions of DYTPAQMAAVNR as described above, see fig. 2.

Next, the high energy collision MS2 generated by the synthesized peptide fragment was verified, and both the primary parent ion and the secondary daughter ion were in agreement with the theoretical values, indicating that the sequence of the synthesized peptide fragment was correct (fig. 2). On the basis, the MS2 of the peptide section synthesized by the new peptide section sequence identified according to the large-scale proteome data and the spectrogram of the large-scale identified new peptide section are manually checked, the two are almost completely consistent, and the Cosin value obtained by the similarity of the daughter ions is as high as 0.96, thereby proving that the new peptide section identified from the Pu' er tea industry fermentation bacteria of the adenine arthrobacter adeguas TMCC70007 is correct.

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 existence of the gene is predicted in the region, the coding frame of the gene is consistent with the coding frame sequence of the protein, the coding start and termination sites of the gene are determined, and the ORF sequence of the gene is SEQ ID NO. 1.

The correspondence between the mRNA sequence transcribed from the gene and the amino acid sequence of the protein translated from the mRNA sequence is shown in FIG. 4. Peptide DYTPAQMAAVNR was located upstream of the gene.

The nucleotide sequence of the gene totally 1035bp, totally 344 amino acids and theoretical molecular weight of 37.67kDa, and the theoretical coding amino acid sequence is shown in SEQ ID NO. 3.

4. To further confirm the correctness of the identified sequences, TMCC70007 strain was cultured in YPD medium and total protein was extracted therefrom, separated by SDS-PAGE, stained with Coomassie Brilliant blue on electrophoresis gel, and the molecular weight was verified based on the molecular weight characteristics at the time of preparation of the proteome sample, as shown in FIG. 5. In addition, 25 strips were cut together in bands for in-gel digestion and mass spectrometry analysis, where HSP40 protein was identified in the mass spectrometry data of the 17 th strip, matching the predicted molecular weight size. The theoretical molecular weight calculated based on the amino acid sequence was 37.7kDa, which was consistent with the position of the gel strip to which the protein belongs in SDS-PAGE. The results showed that the experimental molecular weight of the identified HSP40 was about 33.6 ± 1.9kDa, which substantially coincides with the theoretical molecular weight, confirming the correctness of the identified protein.

5. The amino acid sequence of the theoretical coding product of the gene is subjected to blastp analysis, the sequence has similar structural domains of a DnaJ super family, and DnaJ/Hsp40(heat shock protein40 ) is a conserved protein family and is closely related to protein translation, folding, reverse folding, transfer and degradation. The identified HSP40 protein sequence was compared to yeast (Sugiyamaella lignohabinans) CBS 10342^TThe Hlj1p protein (accession number ANB12940.1) of the strain had the highest similarity of 62% and the sequence coverage was 99%.

The blastp result indicates that the detected leakage injection protein is likely to be HSP40 homologous protein, and the sequence of the protein has certain conservation.

6. The ORF sequence of the identified HSP40 gene (i.e., SEQ ID NO.1) was subjected to NCBI-BLASTN analysis, and the results are shown in FIG. 6. The BLASTN results are summarized in table 2. The result shows that SEQ ID NO.1 has no sequence with higher homology in NCBI database, and only 2% -3% of about 30bp base sequences in 1035 bases are relatively conserved, which shows that the missed-release gene HSP40 detected in the adenine arthrobacter adephaga has sequence specificity, and although the amino acid sequence part segment of HSP40 protein is relatively conserved, the DNA homology is very low. The ORF sequence of the gene can be used for developing the DNA bar code of the Puer tea fermentation strain Arthrospora adenini TMCC 70007.

TABLE 2 list of nucleic acid sequences with higher similarity to SEQ ID NO.1

7. 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 was subjected to NCBI-BLASTN analysis, and the Nucleotide collection (nr/nt) database was searched to find that only 1% of the sequence (38bp) had homology to mRNA of member 12(DNAJB12) of the DnaJ (Hsp40) homologous protein B subfamily of the horse hedgehog. The alignment results are summarized in table 3. From the above results, it can be considered that SEQ ID NO.4 can serve as a DNA barcode sequence for identifying a strain of Alternaria adephaga strain TMCC 70007. Furthermore, the sequence of SEQ ID NO.4 was further subjected to NCBI-BLASTN analysis to search for the shotgun genome sequence of Arthrospora adephaga (taxonomy ID: 409370) in the white-genome shotgun contigs (wgs) database. As a result, it was found that SEQ ID NO.4 has 90% homology (about 300 bases are different) with a partial sequence of the entire Arad1B chromosome of the Alternaria adenini LS3 gene sequence, the query sequence coverage was 99%, and the alignment results are summarized in Table 4. This suggests that although TMCC70007 and LS3 strains are of the same species, homologous sequences in their genomes are sufficiently distinguishable that SEQ ID NO.4 can serve as a DNA barcode sequence. In conclusion, the sequence (SEQ ID NO.4) between the upstream 1000bp and the downstream 1000bp of SEQ ID NO.1 can be used as a DNA barcode for specifically identifying the adenine-node-adenine-spore yeast strain TMCC 70007.

TABLE 3

TABLE 4

8. Currently, of the species Arthrospora adenantha, only the genome of the strain Arthrospora adenantha LS3 has been reported (Kunze et al Biotechnology for Biofuels2014,7: 66). 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 91.70%, although TMCC70007 and LS3 strains were of the same species (instant Arthromyces adenine, Blastotrys adenonivorans), 84 sites were different in 1035 bases of the DNA barcode sequence, and the results are shown in FIG. 7. This further demonstrates that the use of this DNA barcode sequence can effectively distinguish TMCC70007 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 bacterial strain TMCC70007 applied to the Pu' er tea industry or not according to the amplification result of the sample to be detected and the sequence homology of the bacterial strain SEQ ID NO.1 of the bacterial strain TMCC70007 of the Arthrospora adenini.

(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 the initial and termination sites of the gene, and the sequences of the obtained positive and negative primers are respectively HSP 40-F: 5'-CGACCGAGAACGACAGGAAT-3', respectively; HSP 40-R: 5'-ACACGTCAACAGGCGATCAA-3' are provided. The position of the primers is shown in FIG. 8. 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	Strain number	Habitat
					1	Arthrospora adevora	TMCC	70007	Pu' er tea pile fermentation, China
2	Arthrospora adevora	CBS	8244^T	Soil, the Netherlands
					3	Arthrospora adevora	CBS	7350	Corn fodder stored in cellar, Holland
4	Arthrospora adevora	CBS	7370	Soil, south Africa
					5	Arthrospora adevora	CBS	8335	Clay, Italy
6	Blastobotrys raffinosifermentans	CBS 6800^T	Is unknown

(3) Respectively extracting strain DNA: OMEGA e.z.n.a was used.^TMYeast DNA kit extractYeast genomic DNA was taken and the DNA concentration of the sample was diluted to 0.5. mu.g/. mu.L with sterilized deionized water.

(4) Amplifying DNA fragments, and carrying out Polymerase Chain Reaction (PCR), wherein the sequences of the primers are respectively as follows:

forward primer sequence: HSP 40-F: 5'-CGACCGAGAACGACAGGAAT-3', respectively;

reverse primer sequence: HSP 40-R: 5'-ACACGTCAACAGGCGATCAA-3' are provided.

The PCR reaction system is 50 mu L, and the PCR reagent is Thermo Scientific^TMTaq 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 adenantha TMCC70007, and the Arthrospora adenantha CBS 8244 of the same species^TCBS 7350, CBS 7370, CBS 8335 and Blastotrys raffinositifications CBS 6800^TAmplification cannot be achieved. The results are shown in FIG. 9. The theoretical amplification fragment of the PCR primer is 1418bp, 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, and after the quality of the peak image meets 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 sequencing result, and if the homology of the DNA fragment of the strain to be detected and the standard gene sequence of the Arthrospora adenini TMCC70007 is more than 97%, judging that the strain to be detected can be the Arthrospora adenini TMCC70007 strain. For example, through DNAMAN, the sequence obtained by sequencing the strain TMCC70007 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. 10.

SEQUENCE LISTING

<110> Menghai tea industry, Limited liability company

<120> DNA barcodes, primers, kits, methods and uses

<130> FI-163691-59:52/C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1035

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 1

atgtctacgt catcagaacg ctcttctaca aaggctgaga gccgagatca caagcagggg 60

cgccagaaca gggactatac ccctgctcag atggcagcgg taaacagagt gcgaaagtgc 120

agggtaactg attattatgc tattctagat attgaatcgc cttcatcaga ggcggaaatt 180

cgcaaagcct atcgtaagtt ggcactgatt atgcatcccg acaagaacgg ggcccctgga 240

gcagacgaag cttttaagat ggtgtcaaag gcattccagg ttctgtctga tagcgacaag 300

aagaggatat tcgatcaaac aggagcggat cccgactcta gaggaggtgg tggaggtatg 360

ggatcgtttg ctcgtggagc aggaggacca ggcatgcagt ttggaggggg aggagatatt 420

tctcctgaag acctgttcaa catgttcttt ggtggcggtg gtggaagccc gttccaggcc 480

cagtttgggg gctttggtgg gccaggcatt agagtacaca catttggcgg gggctctccg 540

ttttctgcgt ttggtaacac tgctggcgcc cagcgcagac gggctgctgc tgctcaacaa 600

gaagactttt cactgcgaaa cttagtgcaa atgctaccgc ttctattact attcggactg 660

ccttggctgt tatccctgtt cggggactcg cctggtgcag gggccgtacc aagctttcga 720

ttcaaccaaa gacccccctt catggaggag aggctcacat cccggtacag cattccgtat 780

tttgtcaatc cccaggatgt gcagaatttg gccgaccgaa agctggctca attggaccgc 840

caggcagaag tatcctacat ccagtctatg agacgtcagt gcaacactga agtggaatac 900

aagcagcaga agatttcgga tgcccgagga tggttctttg tcgacgaaga ggcttacgac 960

gcagcgcgta agatacccct tcctagttgc gagtttttag atggactggg gatcacgcat 1020

gagagggtct tataa 1035

<210> 2

<211> 1071

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 2

tagaaagaac caaagaaaaa aagttttcga ataggcatgt ctacgtcatc agaacgctct 60

tctacaaagg ctgagagccg agatcacaag caggggcgcc agaacaggga ctatacccct 120

gctcagatgg cagcggtaaa cagagtgcga aagtgcaggg taactgatta ttatgctatt 180

ctagatattg aatcgccttc atcagaggcg gaaattcgca aagcctatcg taagttggca 240

ctgattatgc atcccgacaa gaacggggcc cctggagcag acgaagcttt taagatggtg 300

tcaaaggcat tccaggttct gtctgatagc gacaagaaga ggatattcga tcaaacagga 360

gcggatcccg actctagagg aggtggtgga ggtatgggat cgtttgctcg tggagcagga 420

ggaccaggca tgcagtttgg agggggagga gatatttctc ctgaagacct gttcaacatg 480

ttctttggtg gcggtggtgg aagcccgttc caggcccagt ttgggggctt tggtgggcca 540

ggcattagag tacacacatt tggcgggggc tctccgtttt ctgcgtttgg taacactgct 600

ggcgcccagc gcagacgggc tgctgctgct caacaagaag acttttcact gcgaaactta 660

gtgcaaatgc taccgcttct attactattc ggactgcctt ggctgttatc cctgttcggg 720

gactcgcctg gtgcaggggc cgtaccaagc tttcgattca accaaagacc ccccttcatg 780

gaggagaggc tcacatcccg gtacagcatt ccgtattttg tcaatcccca ggatgtgcag 840

aatttggccg accgaaagct ggctcaattg gaccgccagg cagaagtatc ctacatccag 900

tctatgagac gtcagtgcaa cactgaagtg gaatacaagc agcagaagat ttcggatgcc 960

cgaggatggt tctttgtcga cgaagaggct tacgacgcag cgcgtaagat accccttcct 1020

agttgcgagt ttttagatgg actggggatc acgcatgaga gggtcttata a 1071

<210> 3

<211> 344

<212> PRT

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 3

Met Ser Thr Ser Ser Glu Arg Ser Ser Thr Lys Ala Glu Ser Arg Asp

1 5 10 15

His Lys Gln Gly Arg Gln Asn Arg Asp Tyr Thr Pro Ala Gln Met Ala

20 25 30

Ala Val Asn Arg Val Arg Lys Cys Arg Val Thr Asp Tyr Tyr Ala Ile

35 40 45

Leu Asp Ile Glu Ser Pro Ser Ser Glu Ala Glu Ile Arg Lys Ala Tyr

50 55 60

Arg Lys Leu Ala Leu Ile Met His Pro Asp Lys Asn Gly Ala Pro Gly

65 70 75 80

Ala Asp Glu Ala Phe Lys Met Val Ser Lys Ala Phe Gln Val Leu Ser

85 90 95

Asp Ser Asp Lys Lys Arg Ile Phe Asp Gln Thr Gly Ala Asp Pro Asp

100 105 110

Ser Arg Gly Gly Gly Gly Gly Met Gly Ser Phe Ala Arg Gly Ala Gly

115 120 125

Gly Pro Gly Met Gln Phe Gly Gly Gly Gly Asp Ile Ser Pro Glu Asp

130 135 140

Leu Phe Asn Met Phe Phe Gly Gly Gly Gly Gly Ser Pro Phe Gln Ala

145 150 155 160

Gln Phe Gly Gly Phe Gly Gly Pro Gly Ile Arg Val His Thr Phe Gly

165 170 175

Gly Gly Ser Pro Phe Ser Ala Phe Gly Asn Thr Ala Gly Ala Gln Arg

180 185 190

Arg Arg Ala Ala Ala Ala Gln Gln Glu Asp Phe Ser Leu Arg Asn Leu

195 200 205

Val Gln Met Leu Pro Leu Leu Leu Leu Phe Gly Leu Pro Trp Leu Leu

210 215 220

Ser Leu Phe Gly Asp Ser Pro Gly Ala Gly Ala Val Pro Ser Phe Arg

225 230 235 240

Phe Asn Gln Arg Pro Pro Phe Met Glu Glu Arg Leu Thr Ser Arg Tyr

245 250 255

Ser Ile Pro Tyr Phe Val Asn Pro Gln Asp Val Gln Asn Leu Ala Asp

260 265 270

Arg Lys Leu Ala Gln Leu Asp Arg Gln Ala Glu Val Ser Tyr Ile Gln

275 280 285

Ser Met Arg Arg Gln Cys Asn Thr Glu Val Glu Tyr Lys Gln Gln Lys

290 295 300

Ile Ser Asp Ala Arg Gly Trp Phe Phe Val Asp Glu Glu Ala Tyr Asp

305 310 315 320

Ala Ala Arg Lys Ile Pro Leu Pro Ser Cys Glu Phe Leu Asp Gly Leu

325 330 335

Gly Ile Thr His Glu Arg Val Leu

340

<210> 4

<211> 3066

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 4

cccagtgaaa ttgactgtac cattctcaaa tgctgagcga atgcggccaa tatggcgagg 60

atcagacaca acaattttta aaaagggcga ctttgcttgt ccattatacc cccacagaga 120

ctctttcatg caaatttcaa tatgatccac ttggcccgag aaagcagaat ccaaggcgtc 180

aatgagacca ggaatgtttt cttctcgaaa tcccacaggt gctggaacat aaaagtaatg 240

aaggaaatta gtaacgttgc agcacaccgt gtttccttca tccgtgacac caaatagacg 300

aatcactgac tctccttgca tcattgcttc ttcagcatca atctgttgaa agttgatatc 360

tcgctcataa gcagtaaaat ctgcgagctt aggtctggtc cacttttgat ctgtttctgc 420

tccgcctgaa tttgttagtt acaatggact tgaagcctac aattcactta ccgccttcca 480

tttggttgat ctcttgagtc aaccgagtca aatcttgctc gaaggaagtg ccctcgcttt 540

tagctcgctt tgcagggcca tcatcaatag ctgggcgttt cacgccctga tctaaggctt 600

caggtgccat tttagtttcc acttcgcagt cctacttcaa tgcaagtgac gcgataacta 660

gttcaagacg cgttttccgg atctaaatta catatcttcc agcctcaatc ggtgtggctg 720

tgagccccat acggtgcggt gcggttccag aatattttgc attggggatc ttcccgcccc 780

tgactgtcaa tagaggacac aaaagcatag aaaccggtga tccacgattt tattaaaggc 840

tgggtctgac ctacagctgg caaacaacgg ggcatcttgg taatattgga cgtccagact 900

gagcgatcat aacacaaata gctcgttttc actcccttgg aagcatttac agacgaccga 960

gaacgacagg aatagccagt gctgtgcacg gatcaggtta gaaagaacca aagaaaaaaa 1020

gttttcgaat aggcatgtct acgtcatcag aacgctcttc tacaaaggct gagagccgag 1080

atcacaagca ggggcgccag aacagggact atacccctgc tcagatggca gcggtaaaca 1140

gagtgcgaaa gtgcagggta actgattatt atgctattct agatattgaa tcgccttcat 1200

cagaggcgga aattcgcaaa gcctatcgta agttggcact gattatgcat cccgacaaga 1260

acggggcccc tggagcagac gaagctttta agatggtgtc aaaggcattc caggttctgt 1320

ctgatagcga caagaagagg atattcgatc aaacaggagc ggatcccgac tctagaggag 1380

gtggtggagg tatgggatcg tttgctcgtg gagcaggagg accaggcatg cagtttggag 1440

ggggaggaga tatttctcct gaagacctgt tcaacatgtt ctttggtggc ggtggtggaa 1500

gcccgttcca ggcccagttt gggggctttg gtgggccagg cattagagta cacacatttg 1560

gcgggggctc tccgttttct gcgtttggta acactgctgg cgcccagcgc agacgggctg 1620

ctgctgctca acaagaagac ttttcactgc gaaacttagt gcaaatgcta ccgcttctat 1680

tactattcgg actgccttgg ctgttatccc tgttcgggga ctcgcctggt gcaggggccg 1740

taccaagctt tcgattcaac caaagacccc ccttcatgga ggagaggctc acatcccggt 1800

acagcattcc gtattttgtc aatccccagg atgtgcagaa tttggccgac cgaaagctgg 1860

ctcaattgga ccgccaggca gaagtatcct acatccagtc tatgagacgt cagtgcaaca 1920

ctgaagtgga atacaagcag cagaagattt cggatgcccg aggatggttc tttgtcgacg 1980

aagaggctta cgacgcagcg cgtaagatac cccttcctag ttgcgagttt ttagatggac 2040

tggggatcac gcatgagagg gtcttataaa actgcatact aacgaagcct aaaattttgt 2100

ctcatagggt gttttctaga agttaatttt tttccccgtt tatttagaca atgtgtttac 2160

tcgacttaga agacctagac taacacgctc tttttttgca atgttttgca tgatattttg 2220

cggggccggg tttgtttgtg tctattatga catcgtaccg cctatgatca acagttactg 2280

actgcaggtg gtggtgcgtc aagcaaggag gaggaggggc cgagggatcc agccactacc 2340

cgatggcttg cttgatcgcc tgttgacgtg ttcaattgac aggttattcc cggcgcggtg 2400

gcgcttctta gctgtgtatt gtccgtcaaa aattaaactc taaccccctt ttacgggctc 2460

tcggtggggc cctggtcttt attttgtttc gcatcttgtc atcgtaagat cgtcaacgtc 2520

gtgacgatct tgcccgcaac cggtccaagg gggtgctata tttgcatcca gccacatcaa 2580

gaagagtgcg catataaagt ttggaatatc cttgaaagtg gacaaggcag ccggtggatt 2640

ttgctcagta gaatgaaaag tgtaggatta gcgctgctga gcttggtgtc ggtgtcgtgg 2700

gcacagacat tcccgttgag gtcggtaaag ggcaccgggg tttccaggtt tctggggcag 2760

tctaaacagc tcaagacttc cgaatccagt ataaaggcaa caagcgacgg agacagcgac 2820

aaggatggaa cagagtccgt ggagactttt agtagtgatg tcgcgtacgt gatggatgtg 2880

tcgctggggt ctgatgagca gaactttact ctgattgttg acactggttc gtactataca 2940

tgggtgtacg ggtccaagtg tacggagagc gtgtgccaaa agcaccaaca gttcaacccc 3000

acaaactcga catcattcca caacacgtcg gctacttttg acatctcctt tacttcagga 3060

agcatt 3066

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

cgaccgagaa cgacaggaat 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

acacgtcaac aggcgatcaa 20

<210> 7

<211> 1418

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 7

cgaccgagaa cgacaggaat agccagtgct gtgcacggat caggttagaa agaaccaaag 60

aaaaaaagtt ttcgaatagg catgtctacg tcatcagaac gctcttctac aaaggctgag 120

agccgagatc acaagcaggg gcgccagaac agggactata cccctgctca gatggcagcg 180

gtaaacagag tgcgaaagtg cagggtaact gattattatg ctattctaga tattgaatcg 240

ccttcatcag aggcggaaat tcgcaaagcc tatcgtaagt tggcactgat tatgcatccc 300

gacaagaacg gggcccctgg agcagacgaa gcttttaaga tggtgtcaaa ggcattccag 360

gttctgtctg atagcgacaa gaagaggata ttcgatcaaa caggagcgga tcccgactct 420

agaggaggtg gtggaggtat gggatcgttt gctcgtggag caggaggacc aggcatgcag 480

tttggagggg gaggagatat ttctcctgaa gacctgttca acatgttctt tggtggcggt 540

ggtggaagcc cgttccaggc ccagtttggg ggctttggtg ggccaggcat tagagtacac 600

acatttggcg ggggctctcc gttttctgcg tttggtaaca ctgctggcgc ccagcgcaga 660

cgggctgctg ctgctcaaca agaagacttt tcactgcgaa acttagtgca aatgctaccg 720

cttctattac tattcggact gccttggctg ttatccctgt tcggggactc gcctggtgca 780

ggggccgtac caagctttcg attcaaccaa agacccccct tcatggagga gaggctcaca 840

tcccggtaca gcattccgta ttttgtcaat ccccaggatg tgcagaattt ggccgaccga 900

aagctggctc aattggaccg ccaggcagaa gtatcctaca tccagtctat gagacgtcag 960

tgcaacactg aagtggaata caagcagcag aagatttcgg atgcccgagg atggttcttt 1020

gtcgacgaag aggcttacga cgcagcgcgt aagatacccc ttcctagttg cgagttttta 1080

gatggactgg ggatcacgca tgagagggtc ttataaaact gcatactaac gaagcctaaa 1140

attttgtctc atagggtgtt ttctagaagt taattttttt ccccgtttat ttagacaatg 1200

tgtttactcg acttagaaga cctagactaa cacgctcttt ttttgcaatg ttttgcatga 1260

tattttgcgg ggccgggttt gtttgtgtct attatgacat cgtaccgcct atgatcaaca 1320

gttactgact gcaggtggtg gtgcgtcaag caaggaggag gaggggccga gggatccagc 1380

cactacccga tggcttgctt gatcgcctgt tgacgtgt 1418

Claims

1. A primer pair for amplifying DNA barcodes for identifying the adenine node B.adenine node B yeast TMCC70007 strain comprises the nucleotide sequences of a forward primer and a reverse primer which are respectively shown as follows:

a forward primer: 5'-CGACCGAGAACGACAGGAAT-3', respectively;

reverse primer: 5'-ACACGTCAACAGGCGATCAA-3', respectively;

wherein the DNA barcode is derived from the genome of the Arthrospora adenantha TMCC70007 strain, and the nucleotide sequence is selected from the sequences shown in SEQ ID No.4 and comprises the sequence shown in SEQ ID No. 1.

2. A kit for identifying a nodospora adevorans TMCC70007 strain comprising the primer pair according to claim 1.

3. A method for identifying a strain of the nodospora adephagi TMCC70007, 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;

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.