CN111662995B

CN111662995B - DNA bar code, primer, kit, method and application

Info

Publication number: CN111662995B
Application number: CN201910168842.0A
Authority: CN
Inventors: 徐丹洋; 施佳辉; 徐平; 唐蜀昆; 高林瑞; 田飞; 高慧英; 职晓阳; 丁章贵
Original assignee: Yunnan Dayi Microbial Technology Co ltd; Menghai Tea Industry Co ltd
Current assignee: Yunnan Dayi Microbial Technology Co ltd; Menghai Tea Industry Co ltd
Priority date: 2019-03-06
Filing date: 2019-03-06
Publication date: 2024-03-12
Anticipated expiration: 2039-03-06
Also published as: CN111662995A

Abstract

The invention provides a DNA bar code, a primer, a kit, a method and application. The DNA barcode is derived from the genome of the A.niger TMCC 70012 strain and comprises a sequence of at least 500bp selected from the DNA sequences shown as SEQ ID No.4, and the length of the DNA barcode is 500bp-4000bp. The DNA barcode sequence can realize rapid identification and differentiation of the strain in the Aspergillus niger. Therefore, the invention establishes the standard gene sequence and the sample identification method of the puer tea industrial fermentation production strain Aspergillus niger TMCC 70012, and compared with the traditional morphological identification method, the identification efficiency is obviously improved.

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 primer composition, a DNA bar code, a kit, a method and application for identifying puer tea fermentation production strains. Specifically, the puer tea fermentation production strain is Aspergillus niger TMCC 70012.

Background

Pu' er tea is post-fermented tea produced in the geographical mark range of Yunnan, and is prepared by adopting large-leaf green-sun-dried raw tea as a raw material through a series of processes. The traditional puer tea manufacturing process comprises the following steps: the picked fresh tea leaves are rolled and dried to prepare raw material dried green tea, and then the raw material dried green tea leaves are subjected to impurity removal, tidal water, pile fermentation, airing, screening, compression molding and packaging to leave the factory. In the production of puer tea, the pile fermentation process is a main factor for the quality formation of puer tea, and in the process, the content components such as tea polyphenol, caffeine, some polysaccharide substances and the like in the tea are greatly changed, so that the special flavor, taste, quality and various health care effects of puer tea are achieved.

In the traditional puer tea production, the moist heat environment activates enzymes contained in the tea leaves, so that a part of content components contained in the tea leaves are converted into substances which can be utilized by microorganisms; microorganisms grow in a large quantity in the fermentation process of the puer tea to generate abundant intracellular enzymes and extracellular enzymes, and the intracellular enzymes catalyze a series of conversion of the content components in the tea, so that the puer tea has unique quality. The different producing areas, the microorganism species and the differences of community structures, so that the Pu' er tea has special flavor and quality.

Besides the unique flavor and culture of the puer tea, the puer 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 stomach and the like, and is also concerned by people and popular with consumers. The increasing market demand pulls the development of the puer tea industry and promotes the economic growth in Yunnan.

At present, the production of Pu' er tea by most manufacturers is still an empirical fermentation of semi-natural artificial pile fermentation, and although a community mainly comprising dominant common microorganisms including aspergillus niger (Aspergillus niger) is relatively stable in the pile fermentation process, a large improvement space exists for the stability of the product. To further obtain consumer favor and market acceptance, break through foreign trade barriers and promote the market competitiveness of puer tea enterprises, manual control, cleaning and high efficiency of puer tea production must be realized, and the product development and the industry chain extension are not broken. To do this, a new technology must be innovated, and a series of safe, clean, efficient and manually controllable automatic Pu 'er tea new processes are invented to ensure the healthy development of Pu' er tea industry, thereby bringing long-term benefits to the country and people.

The artificial inoculation and the pure fermentation of the puer tea are new development directions of the controllable fermentation of puer tea. In order to protect the fermentation process of the puer tea and the quality microorganism germplasm resources, a quick and accurate identification method for the strains for puer tea fermentation is necessary to be developed.

The DNA bar code technology can rapidly and simply identify and distinguish the similar strains, can provide theoretical basis and technical means for the development of the artificial controllable pure fermentation process and the resource protection of the puer tea, and promote the healthy development of puer tea industry.

Disclosure of Invention

In order to overcome the defect of morphological identification of puer tea fermentation production bacteria, the invention provides a method for identifying puer tea fermentation strain Aspergillus niger (Aspergillus niger) TMCC

The DNA bar code, the primer, the kit, the method and the application of 70012 can accurately identify the Aspergillus niger TMCC 70012 strain from confusing species or complex species, realize rapid identification and differentiation, provide rapid identification and assessment for new fermentation process of puer tea, prevent interference of other miscellaneous bacteria in the fermentation process, and provide evidence method and basis for manual controllable fermentation process of puer tea and strain abuse.

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

(1) A DNA barcode for use in the identification of an aspergillus niger strain, the DNA barcode being derived from the genome of an aspergillus niger TMCC 70012 strain and comprising a sequence selected from at least 500bp in the DNA sequence as shown in SEQ id No.4, and the DNA barcode having a length of 500bp-4000bp, preferably 500bp-900bp.

(2) The DNA barcode according to (1), wherein the nucleotide sequence 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 of (1) or (2).

(4) The primer set according to (3), wherein the nucleotide sequence of the forward primer is the same as that in the genome of the strain A.niger TMCC 70012: the sequence is a sequence from the 1 st position of the nucleotide sequence shown as SEQ ID No.4 to the 3510 th position of the nucleotide sequence shown as SEQ ID No.4 in the genome of the TMCC 70012 strain, and the length of the forward primer is 15-30bp; its reverse primer is reverse complementary to such sequence in the genome of the TMCC 70012 strain: the sequence is from 471 rd position of the nucleotide sequence shown as SEQ ID No.4 to the last position of the nucleotide sequence shown as SEQ ID No.4 in the genome of the TMCC 70012 strain, and the length of the reverse primer is 15-30bp.

(5) The primer set according to (4), wherein the nucleotide sequences of the forward primer and the reverse primer are as follows:

forward primer: 5'-ACGCCTCAGTAGAAGATAGT-3';

reverse primer: 5'-CTTGCCATGGTGGTGGGATA-3'.

(6) A kit for identifying an Aspergillus niger strain, comprising the primer pair according to any one of (3) to (5).

(7) A method for identifying an aspergillus niger strain, comprising the steps of:

a) Providing genomic DNA of a strain to be tested;

b) Performing PCR amplification using the genomic DNA of step a) as a template and the primer set according to any one of (3) to (5) to obtain a PCR product;

c) Detecting PCR products through agarose gel electrophoresis, if no target band exists, judging that the strain to be detected is not the Aspergillus niger TMCC 70012 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 sequence to be detected and the nucleotide sequence of the DNA bar code in the step (1) or the step (2), and judging that the strain to be detected is the aspergillus niger TMCC 70012 strain if the homology is more than 97%.

(8) The use of the DNA barcode according to (1) or (2) for identifying an Aspergillus niger strain.

(9) The use of the primer pair according to any one of (3) to (5) for identifying an A.niger strain.

(10) The use of the kit according to (6) for identifying an Aspergillus niger strain.

Compared with the prior art, the invention has the following advantages and positive effects:

1. the invention adopts protein genomics technology to find a peptide segment which is in the UDP-galactopyranose mutase GLF A gene encoding protein GLF A from the genome of Aspergillus niger TMCC 70012 strain. The invention further develops a DNA bar code based on the GLF A gene through careful research and comparative analysis, and the bar code sequence can realize rapid identification and differentiation of the Aspergillus niger intraspecies.

2. The invention further discovers that the sequence of the GLF A gene (e.g., SEQ ID No. 1) has the characteristics of versatility, easy amplification and easy alignment, and the difference between different strains in Aspergillus niger species is obvious compared with other genes.

3. The invention establishes the standard gene sequence and the sample identification method of the Aspergillus niger TMCC 70012 of the puer tea industrial fermentation production strain, and compared with the traditional morphological identification method, the identification efficiency is obviously improved. The method has low requirements on the integrity of samples, and the identification index can be quantized, so that an effective basis is provided for timely judging the puer tea fermentation process and germplasm resources thereof, and the blank of the puer tea fermentation production strain based on the DNA bar code technology for strain identification is made up. In addition, morphological confusing seeds 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 a conventional molecular identification method.

Drawings

Figure 1 shows a mass spectrum of the newly identified peptide fragment VPSFQDILQGR.

FIG. 2 shows a mass spectrum of synthetic peptide VPSFQDILQGR compared to a mass spectrum of the original identified peptide; the original identification peptide fragment is a peptide fragment obtained by mass spectrometry analysis and identification; the upper part of the figure is the mass spectrum of the original identified peptide fragment, and the lower part is the mass spectrum of the synthesized peptide fragment.

FIG. 3 shows the nucleotide sequence of SEQ ID NO.1, with gray parts being introns.

FIG. 4 shows the correspondence of SEQ ID NO.1 and the amino acid sequence of the protein encoded thereby, with the grey part being the newly identified peptide VPSFQDILQGR.

FIG. 5A shows a SDS-PAGE separation of TMCC 70012 cell whole proteins, bolded bands are positions of GLF A protein; FIG. 5B shows a GLF A protein molecular weight validation chart. The abscissa in FIG. 5B represents the logarithm of the molecular weight MW, and the ordinate represents the ratio of the migration amount of each molecular weight protein of the protein molecular weight markers in SDS-PAGE electrophoresis separation to the total migration amount of SDS-PAGE.

FIG. 6 shows an alignment of sequences having homology thereto by NCBI-BLASTN for barcode sequence SEQ ID NO.1, and the gray line segment under FIG. 6 shows the homologous sequence.

FIG. 7 shows the nucleotide sequence of SEQ ID NO.4, light grey part SEQ ID NO.1, dark grey part introns of SEQ ID NO.1, bolded as primer sequence, underlined as product sequence amplified with this primer, SEQ ID NO.7.

FIG. 8 shows an alignment of sequences of barcode sequence SEQ ID NO.4 having homology thereto by NCBI-BLASTN, and the gray line segment under FIG. 8 shows the homologous sequence.

FIG. 9 shows the result of agarose gel electrophoresis of the product obtained by PCR amplification using the primer designed for the A.niger TMCC 70012GLF A gene of the present invention.

Detailed Description

The invention is further described below by means of the description of specific embodiments and with reference to the accompanying drawings, which are not intended to be limiting, but a person skilled in the art can make various modifications or improvements according to the basic idea of the invention, all without departing from the scope of the invention.

The invention utilizes the systematic protein genomics technology to find a species-specific gene-encoded protein from Aspergillus niger TMCC 70012 strain, which is difficult to find by traditional gene prediction software. Since the gene encodes a protein having high homology with UDP-galactopyranose mutase, it is designated GLF A. The invention discovers that the open reading frame (SEQ ID No. 1) of the gene GLF A encoding the protein can identify the Aspergillus niger TMCC 70012 strain from confusing species, so that the invention can be used for developing DNA bar codes for identifying the Aspergillus niger TMCC 70012 strain produced by the Pu' er tea industrial fermentation. Compared with the prior art, the DNA bar code obtained by the method has higher specificity.

In view of the fact that the DNA barcodes should have a suitable length and sufficient inter-strain specificity, the present invention further provides, through careful research and comparative analysis, a DNA barcode that can accurately and efficiently identify the A.niger TMCC 70012 strain based on the above-mentioned unique DNA sequences.

Thus, in one aspect the present invention provides a DNA barcode for use in the identification of an A.adequasis strain, the DNA barcode being derived from the genome of an A.niger TMCC 70012 strain and comprising a sequence selected from at least 500bp in the DNA sequence shown as SEQ ID No.4 and the DNA barcode being 500bp to 4000bp, preferably 500bp to 900bp in length.

The invention discovers a protein coding sequence which is difficult to be discovered by traditional gene prediction software through systematic protein genomics research, and supports accurate and reliable peptide fragments and relevant proteomics mass spectrum data of the gene coding sequence product; based on the position of the protein coding sequence in the genome, the possible gene sequences (SEQ ID No. 1) and protein sequences (SEQ ID No. 3) encoding the protein were determined. The length of SEQ ID No.1 is 1964bp, and according to comparison and analysis, the SEQ ID No.1 is unique to the genome of the Aspergillus niger TMCC 70012 strain, so that the sequence can be used as a DNA bar code for identifying the Aspergillus niger TMCC 70012 strain. In addition, through theoretical analysis and experimental verification, the invention proves that the specificity of the DNA bar code with longer length and containing the sequence is more ensured. When the length of the DNA barcode is too long (e.g., greater than 4000 bp), it is less desirable for the amplification operation. Therefore, based on the gene sequence, the invention searches a longer DNA barcode sequence with better specificity in the genome of the Aspergillus niger TMCC 70012 strain. On the other hand, in general, the DNA bar code can satisfy the operational requirements of easy amplification and easy alignment even when the length is not less than 500 bp. Therefore, theoretical analysis and experimental verification prove that the sequence of at least 500bp in the DNA sequence shown as SEQ ID No.4 can also realize rapid and accurate identification and differentiation of the strain in the Aspergillus niger.

The barcode sequence can realize rapid and accurate identification and distinction of the strain in the aspergillus niger strain.

In particular, the invention finds that the DNA sequence shown in SEQ ID No.7 contained in SEQ ID No.4 has particularly high specificity among different strains and among different strains in Aspergillus niger TMCC 70012 strain genome. Thus, in a preferred embodiment of the invention, the DNA barcode comprises a sequence selected from the group consisting of at least 500bp in the DNA sequence shown as SEQ ID No.7.

Preferably, the DNA barcode sequence is shown as SEQ ID No.1, SEQ ID No.4 or SEQ ID No.7.

As used herein, the term "misannotation" refers to the inability of gene prediction software (e.g., geneMark, augustus, glimmer, etc.) to predict a gene or protein that is not normally expressed in high amounts under specific conditions after the species has completed genome sequencing, and therefore is difficult to find in research.

The term "DNA barcoding" refers to a novel technique for molecular identification of species using a standard, short DNA fragment within the genome, which allows rapid and accurate species identification.

The term "six-frame translation" is a known term in proteomics and genomics, and is based on the principle that when a DNA encodes a protein, the triplet codon is used to encode the protein, and given a DNA sequence, there are 3 encoding possibilities, plus 3 encoding possibilities on its complementary strand, for a total of 6 encoding possibilities (+1, +2, +3, -3, -2, -1).

In another aspect, the invention provides a primer pair for amplifying a DNA barcode according to the invention.

It will be appreciated by those skilled in the art that, according to the DNA barcode sequences provided herein for identifying the strain of Arthrobacter adequasis, corresponding primer pairs can be readily designed to amplify the desired DNA barcodes.

Preferably, the nucleotide sequence of the forward primer of the primer pair is identical to such sequence in the genome of the A.niger TMCC 70012 strain: the sequence is a sequence from the 1 st position of the nucleotide sequence shown as SEQ ID No.4 to the 3510 th position of the nucleotide sequence shown as SEQ ID No.4 in the genome of the TMCC 70012 strain, and the length of the forward primer is generally 15-30bp; its reverse primer is reverse complementary to such sequence in the genome of the TMCC 70012 strain: the sequence is from 471 rd position of the nucleotide sequence shown as SEQ ID No.4 to the last position of the nucleotide sequence shown as SEQ ID No.4 in the genome of the TMCC 70012 strain, and the length of the reverse primer is generally 15-30bp. The product amplified by the forward and reverse primers is a sequence of at least 500bp selected from the nucleotide sequences shown in SEQ ID No. 4.

In a preferred embodiment, the nucleotide sequence of the forward primer of the primer pair is identical to such sequence in the genome of the A.niger TMCC 70012 strain: the sequence is a sequence in a region from the 1 st position of the nucleotide sequence shown as SEQ ID No.7 to the 369 st position of the nucleotide sequence shown as SEQ ID No.7 in the genome of the TMCC 70012 strain, and the length of the forward primer is generally 15-30bp; its reverse primer is reverse complementary to such sequence in the genome of the TMCC 70012 strain: the sequence is from 471 rd position of the nucleotide sequence shown as SEQ ID No.7 to the last position of the nucleotide sequence shown as SEQ ID No.7 in the genome of the TMCC 70012 strain, and the length of the reverse primer is generally 15-30bp. The product amplified by the forward and reverse primers is a sequence of at least 500bp selected from the nucleotide sequences shown in SEQ ID No.7.

In a more preferred embodiment, the nucleotide sequences of the forward and reverse primers are shown below, respectively:

GLF A-F：5’-ACGCCTCAGTAGAAGATAGT-3’(SEQ ID No.5)；

GLF A-R：5’-GTGGATGGGACGTAAAGCAT-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 aspergillus niger strains, which comprises the primer pair.

In another embodiment, the kit further comprises a DNA barcode according to the 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 means and reagents for experimental manipulation.

In yet another aspect, the invention provides a method for identifying an Aspergillus niger strain, comprising the steps of:

a) Providing genomic DNA of a strain to be tested;

b) Using the genome DNA of the step a) as a template, and carrying out PCR amplification by using the primer pair to obtain a PCR product;

c) Detecting the PCR product by electrophoresis (e.g. agarose gel electrophoresis), if there is no target band, determining that the strain to be detected is not the Aspergillus niger strain TMCC 70012, and if there is a target band, performing 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, and judging that the strain to be detected is the aspergillus niger TMCC 70012 strain if the homology is more than 97% (preferably more than 98%, more preferably more than 99%, and more preferably 100%).

The terms "identity" and "homology" as used herein have the same meaning and are used interchangeably.

In a specific embodiment of the invention, the PCR amplification procedure is: 1) Pre-denaturation at 94 ℃ for 5 min; 2) Denaturation at 94℃for 30 seconds, annealing at 56℃for 30 seconds, elongation at 72℃for 1 minute, wherein the procedure 2) is carried out for 30 cycles; 3) Extension was carried out at 72℃for 10 minutes.

In yet another embodiment of the present invention, the method may further comprise performing a cluster analysis (e.g., phylogenetic tree) of the nucleotide sequence to be tested obtained by sequencing and the DNA barcode of the present invention, and determining that the strain to be tested is the aspergillus niger strain TMCC 70012 if the sequence to be tested and the DNA barcode are clustered together.

In a specific embodiment of the invention, genomic DNA extracted from the strain to be identified is PCR amplified using the primer pair of the invention, followed by agarose gel electrophoresis detection. Identifying strains based on detecting the presence or absence of PCR products: if the strain to be identified does not amplify the corresponding target band, it is indicated that the strain is not TMCC 70012; if the corresponding target band is amplified, it is demonstrated that the strain is likely TMCC 70012. For further identification, the PCR product is sequenced, the DNA sequencing result is subjected to homology comparison with the DNA barcode sequence, so that the similarity (i.e. homology) between the sequences is obtained, and if the sequence homology is less than 97%, the strain to be detected is judged not to be the Aspergillus niger strain TMCC 70012. If the sequence homology is 97% or more, the strain to be tested is judged to be the Aspergillus niger strain TMCC 70012.

If cluster analysis, such as phylogenetic tree, is performed, the DNA bar code is used to construct NJ phylogenetic tree using MEGA 6 or PAUP software together with the DNA sequencing result (i.e., the sequence to be tested) of each strain to be identified. If the test sequence of the strain to be identified is clustered with the DNA barcode of A.niger TMCC 70012, then A.niger TMCC 70012 is identified.

The term "cluster" as used herein refers to a cluster that is in the same branch and has the same evolutionary distance after phylogenetic tree analysis.

The invention also provides application of the DNA bar code in identification of Aspergillus niger strains.

The invention also provides application of the primer pair in identification of Aspergillus niger strains.

The invention also provides application of the kit in identification of Aspergillus niger strains.

Examples

The invention will be further illustrated with reference to specific examples. The methods used in the examples, unless specifically indicated, all employ conventional methods and known tools.

Example 1: GLF A Gene and DNA Bar code acquisition

1. Deep coverage research of a proteome is carried out on Aspergillus niger TMCC 70012 (Aspergillus niger TMCC 70012) of puer tea industrial fermentation bacteria by using a high coverage proteome technology and adopting pFInd and pAnno software, and verification of annotation coding genes is carried out on the genome of the deep coverage research. To find new protein coding regions, a six-frame translation database of Aspergillus niger TMCC 70012 genomic data was obtained using a six-frame translation (Six Frame Translation) strategy in the system protein genomics, the 6 coding possibilities (+1, +2, +3, -1, -2, -3) of the genome were exhausted, and the nucleic acid sequence was referred to as "six-frame translation nucleic acid sequence", and the protein sequence was referred to as "six-frame translation protein sequence". Generally, a six-frame translated nucleic acid sequence is a sequence from one terminator to the next, also referred to herein as a "protein coding frame". Using this database, the identification of new peptides and new proteins was performed using pFind and pAnno software on high coverage proteome mass spectrometry data of total cellular proteins of TMCC 70012 strain.

A peptide VPSFQDILQGR not found in the annotation gene of the Aspergillus niger TMCC 70012 in the prior art is identified, and a mass spectrum is shown in figure 1.

Manual inspection of the mass spectrum results shows that almost all y ion sequences of the peptide VPSFQDILQGR secondary mass spectrum (MS 2) are detected, the matching is good, the signal is strong, and the result is reliable.

To further confirm this identification, the peptide was chemically synthesized according to the amino acid sequence of the newly identified peptide VPSFQDILQGR, and the high energy collision MS2 generated by the synthesized peptide was verified, and both the primary parent ion and the secondary daughter ion were in agreement with theoretical values, indicating that the sequence of the synthesized peptide was correct, see fig. 2.

Based on this, MS of synthetic peptide fragments of the new peptide fragment sequence identified from large-scale proteome data was examined manually ₂ And large-scale identification of a new peptide fragment spectrogram, wherein the two spectrograms are almost completely consistent, and the cosin value obtained by sub-ion similarity is as high as 0.99, so that the identification of the new peptide fragment from Aspergillus niger TMCC 70012 of Pu' er tea industry fermentation bacteria is proved to be correct.

2. Based on the position of the new peptide fragment, a six-frame translated nucleic acid sequence (protein coding frame) SEQ ID NO.2 is obtained, bounded by the region comprised by the previous stop codon and the subsequent stop codon.

3. In order to further determine the coding start and stop sites of the gene encoding the protein, the coding frame of the protein was extended by 1000bp upstream and downstream respectively, gene prediction was performed using GeneMark. The presence of the gene encoding the protein was predicted in this region, the reading frame of which is identical to that of the above protein, the initiator of the gene was found in the ORF sequence, but the terminator of the ORF sequence was actually located in the intron portion, so that according to the prediction of the gene, a true terminator was found in the latter sequence, and thus the possible coding start and termination sites of the gene were determined according to the prediction result, and the complete gene sequence from the initiator to the terminator was SEQ ID NO.1, as shown in FIG. 3.

The corresponding relationship of the amino acid sequences of SEQ ID NO.1 and the protein encoded by the SEQ ID NO.1 is shown in FIG. 4. Peptide VPSFQDILQGR is located near the N-terminus of the protein.

The nucleotide sequence of the gene (SEQ ID NO. 1) comprises a terminator which is 1964bp in total, an intron region is removed, 547 amino acids are coded in total, the theoretical molecular weight is 61.0kDa, and the theoretically coded amino acid sequence is shown as SEQ ID NO. 3.

4. To further determine the correctness of the identified sequence, the molecular weight of the protein encoded by the region in which the identified ORF of VPSFQDILQGR was located, and thus its experimental molecular weight, was determined (fig. 5). The TMCC 70012 strain was cultured in YPD medium and total proteins therein were extracted, SDS-PAGE separation was performed, the electrophoresis gel was stained with Coomassie brilliant blue, and the molecular weight was verified based on the molecular weight characteristics at the time of preparation of proteome samples, and in SDS-PAGE, the ratio of the migration distance of the protein to the migration distance of the dye front was in a relationship with the logarithm of the protein molecular weight, as shown in FIG. 5. The whole lane was cut into 30 strips (fractions) according to molecular weight and protein abundance and subjected to in-gel digestion and mass spectrometry, wherein the GLF A protein was identified in mass spectrometry data of strip 13, matching the predicted molecular weight size. The theoretical molecular weight is 61.0kDa, which is consistent with the position in SDS-PAGE where the gel strip to which the protein belongs. The results showed that the experimental molecular weight of the identified GLF A was between 59.2 and 61.8kDa, consistent with the theoretical molecular weight, confirming the correctness of the identified protein.

5. NCBI-BLASTP analysis is carried out on the amino acid sequence (SEQ ID NO. 3) of the product of the gene theory, so that proteins with higher sequence similarity are retrieved. Blastp shows that GLF A gene product has structural domain HemY superfamily which is not annotated in TMCC-70012 strain detected by us, which shows that the protein formed by the sequence has specific structure and independent function in biological macromolecule. The above blastp comparison results are shown in Table 1.

Table 1 sequence with higher homology to TMCC 70012GLF A protein sequence

TABLE 2 sequence with higher homology to TMCC 70012GLF A Gene sequence

6. The sequence of the identified GLF A gene (i.e., SEQ ID NO. 1) was subjected to NCBI-BLASTN analysis, the results of which are shown in FIG. 6.BLASTN results are summarized in table 2.

The results in Table 2 show that the sequence of the GLF A gene (SEQ ID NO. 1) has a sequence with higher homology with another strain of Aspergillus niger, but has larger difference with other species than Aspergillus niger, and can be used as a DNA bar code for distinguishing TMCC-70012 strain from other strains. In addition, if a high homology alignment screening criterion, such as 99% or more homology, is set, this SEQ ID NO.1 may also be used as a DNA barcode for distinguishing TMCC-70012 strain from other Aspergillus niger strains.

7. Further consideration was given to the sequence of the transcribed spacer before and after the GLF A gene sequence (i.e., SEQ ID NO. 1), resulting in SEQ ID NO.4, as shown in FIG. 7. Homology comparisons were made with SEQ ID NO.4, as shown in FIG. 8. As can be seen from FIG. 8, SEQ ID NO.4 is highly specific in NCBI database, and there is little homology matching of the fragment after 2250bp of SEQ ID NO. 4. SEQ ID NO.4 is effective in distinguishing TMCC-70012 from the near-source strain and thus can be used as a DNA barcode.

Example 2 identification of strains Using DNA barcodes

And judging whether the sample to be detected is the Aspergillus niger TMCC 70012 strain applied in the puer tea industry according to the amplification result of the sample to be detected, the amplification result of the GLF A gene sequence of the Aspergillus niger TMCC 70012 strain and the sequence homology.

(1) Based on SEQ ID NO.4, PCR primers were designed using NCBI primer design tools to obtain forward and reverse primer sequences of:

GLF A-F：5’-ACGCCTCAGTAGAAGATAGT-3’；

GLF A-R：5’-GTGGATGGGACGTAAAGCAT-3’。

the positions of the primers are shown in FIG. 7. The amplified sequence is SEQ ID NO.7 (838 bp).

(2) Bacterial strain origin

TABLE 3 information on relevant strains selected for use

(3) Extracting strain DNA respectively: OMEGA e.z.n.a. was used. ^TM Genomic DNA was extracted from Yeast DNA kit of (A), and the DNA concentration of the sample was diluted to 0.5. Mu.g/. Mu.L with sterilized deionized water.

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

GLF A-F：5’-ACGCCTCAGTAGAAGATAGT-3’；

GLF A-R：5’-GTGGATGGGACGTAAAGCAT-3’。

the PCR reaction system is 50 mu L, and the PCR reagent is Thermo Scientific ^TM Taq DNA Polymerase (recombinant): ddH ₂ O 37.7μL、MgCl ₂ 5. Mu.L of dNTPs 4. Mu.L, 1. Mu.L of forward primer, 1. Mu.L of reverse primer, 1. Mu.L of Taq DNA polymerase 0.3. Mu. L, DNA template, and no dye. The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; next, denaturation at 94℃for 30 seconds, annealing at 56℃for 30 seconds, and extension at 72℃for 1 minute were carried out for a total of 30 cycles; finally, the extension is carried out at 72 ℃ for 10 minutes.

(5) Detection of amplification products: the PCR fragment size was determined by electrophoresis on a 1.0% agarose gel, 1 XTBE, and using a DNA molecular weight marker. If the strain to be detected does not have an expected amplified band with the size of 838bp, the strain is not Aspergillus niger TMCC 70012; if a clear band appears and no band exists, the DNA fragment is sent to a biological sequencing company for sequencing.

(6) The primer can only amplify in Aspergillus niger TMCC 70012, but can not amplify in Aspergillus niger CBS 513.88, CBS 554.65T, CBS 120.49.49, CBS101697, CBS 116681, CBS119557, CBS117984 and CBS118725 of the same species, and the result is shown in FIG. 9. The theoretical amplification sequence of the PCR primer is 838bp, and the actual amplification result is consistent with the expected result. To further verify the sequence of the amplified DNA, sequencing and homologous sequence comparison were performed. This result demonstrates that SEQ ID NO.7 can very specifically and effectively distinguish the A.niger TMCC 70012 strain, thereby acting as a DNA barcode for the A.niger TMCC 70012 strain.

(7) And (3) for the sequencing result of the sequence with the band, firstly checking the quality of the sequence peak diagram obtained after sequencing by using software Chromas, determining that the quality of the peak diagram meets the requirement of data analysis, and then splicing forward and reverse sequences by using SeqMan in DNASTAR software package. And (3) carrying out manual proofreading and sequence splicing on the sequencing result, and judging that the strain to be tested is likely to be the aspergillus niger TMCC 70012 strain if the homology between the DNA fragment of the strain to be tested and the standard DNA bar code of the aspergillus niger TMCC 70012 is more than 97%.

(8) Further cluster analysis, such as phylogenetic tree, may be performed, which may further corroborate the judgment result through the above steps.

SEQUENCE LISTING

<110> Menghai tea industry Limited liability company

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

<130> FI-190367-59:52/C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1964

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 1

atgctcagcc tcgcccgcag gactttgaac cgtgtcccca gctttcaaga tattctacaa 60

ggcaggatga cccaccccga tatgtaagga gacctttccc cctccctcaa acggcaagct 120

tgccttcact acaacaagaa gctcattgga acaattgctg accgttgtct ttgtgcttga 180

attcagctcc gtcgacgttc tcgtcattgg tgccggccct actggtctcg gtgccgcgaa 240

gcgtcttaac cagattgtac gcattgcgcg ccccctatcg tataccttca ctcgaataaa 300

ggctaattgg acaattcgcg ggtgtaggat ggcccttctt ggttgatcgt tgacagcaac 360

gagacccctg gtggtcttgc ttccaccgat gtgacccccg aaggcttcgt atgttgattc 420

ccactttccc ctcaatgggc aggcattgca ctggtcaagc aaacagcgac tgactcgtga 480

caatcacagc tcttcgatgt tggtggtcac gtcatcttct cccactacaa gtacttcgac 540

gactgcatca acgaggctct ccccaaggat gacgactggt acacccacca gcgtatctcc 600

tacgttcgct gccagggcca atgggttccc taccccttcc agaacaacat ctccatgctt 660

cccaagaatg agcaggtccg ctgtatcgat ggcctgatcg acgctgccct tgaggctcgt 720

gtcgccaaca ccaagcccca gaacttcgat gagtggattg ttcgccagat gggtgtcggt 780

atcgccgacc tcttcatgag accctacaac ttcaaggttt gggctgtgcc tacgaccaag 840

gtaagatacg atgtaccaca tggcgagtaa cggcgtgctg attctcatca gatgcaatgt 900

gcctggttgg gtgagcgtgt tgctgcccct aacgtcaagg ccgtgacgac caacgtcatc 960

cttaacaaga ccgctggtaa ctggggtcct aacgctactt tccgtttccc cgcccgtggt 1020

ggtaccggtg gtatctggat cgctgtcgcc gacactatcc ccaaggagaa gactcgcttc 1080

ggtgagaagg gcaaggtcgt caaggtcaat gccaacaaca agaccgtcac cctgggtgat 1140

ggcaccactg tcggctacaa gaagctcgtc tccaccatgg ctgtcgactt cctcgctgag 1200

cagatcggcg accaggagct cgttggcctc accaagcagc tcttctactc ctccactcac 1260

gtcattggtg tcggtatccg tggtacccgc cccgagagaa tcggtgacaa gtgctgggta 1320

agttgacctt ggatggaaac catgaatgac ttcaaaacta acatgctcgt tactagctct 1380

acttccccga ggacaactgc cccttctacc gtgccaccat cttctccaac tactcccccc 1440

acaaccagcc cgagggctcc aagaagcttc ccactctgca gcttgcggat ggctctaagc 1500

cccagagcac tgaggctcag gagggtcctt actggtccat catgttggag gtttccgagt 1560

cttcgatgaa gcctgtcaac cacgagactc tcctggctga ttgcatccag ggtctcgtca 1620

acaccgagat gctgaagccc accgatgaga ttgtctccac ctaccaccgc cgcttcgacc 1680

acggctaccc caccccctcc ctggagcgtg agggtgctct tacccagatc ctgcccagac 1740

tccaggagaa ggacatctgg acccgtggtc gcttcggtag ctggcgctac gaggtcggta 1800

accaggacca ctctttcatg ctcggtgttg aggctgttga caacattgtc aacggcgctg 1860

tcgagctgac ccttaactac cctgacttcg tcaacggtag acagaacacc gagcgtcggc 1920

tggttgacgg cgcccaggtt ttcgccaaga gccaggcgca gtaa 1964

<210> 2

<211> 294

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 2

tgaaaccgat tccaagcgcc ccccaccgct tgcttgtcac gtttcgcttt cccgaccgac 60

cgcaggacgg atacacctat accttcccgc ccatctcccc ttcttgctcc ctccatcacc 120

aacctctctt ctctctctct cttcctcccc tctcatctct ctccttataa ctctgctgtc 180

cctgcagctt cacaaggctc gttttctatg ctcagcctcg cccgcaggac tttgaaccgt 240

gtccccagct ttcaagatat tctacaaggc aggatgaccc accccgatat gtaa 294

<210> 3

<211> 547

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

<400> 3

Met Leu Ser Leu Ala Arg Arg Thr Leu Asn Arg Val Pro Ser Phe Gln

1 5 10 15

Asp Ile Leu Gln Gly Arg Met Thr His Pro Asp Ile Ser Leu Glu Gln

20 25 30

Leu Leu Thr Val Val Phe Val Leu Glu Phe Ser Ser Val Asp Val Leu

35 40 45

Val Ile Gly Ala Gly Pro Thr Gly Leu Gly Ala Ala Lys Arg Leu Asn

50 55 60

Gln Ile Asp Gly Pro Ser Trp Leu Ile Val Asp Ser Asn Glu Thr Pro

65 70 75 80

Gly Gly Leu Ala Ser Thr Asp Val Thr Pro Glu Gly Phe Leu Phe Asp

85 90 95

Val Gly Gly His Val Ile Phe Ser His Tyr Lys Tyr Phe Asp Asp Cys

100 105 110

Ile Asn Glu Ala Leu Pro Lys Asp Asp Asp Trp Tyr Thr His Gln Arg

115 120 125

Ile Ser Tyr Val Arg Cys Gln Gly Gln Trp Val Pro Tyr Pro Phe Gln

130 135 140

Asn Asn Ile Ser Met Leu Pro Lys Asn Glu Gln Val Arg Cys Ile Asp

145 150 155 160

Gly Leu Ile Asp Ala Ala Leu Glu Ala Arg Val Ala Asn Thr Lys Pro

165 170 175

Gln Asn Phe Asp Glu Trp Ile Val Arg Gln Met Gly Val Gly Ile Ala

180 185 190

Asp Leu Phe Met Arg Pro Tyr Asn Phe Lys Val Trp Ala Val Pro Thr

195 200 205

Thr Lys Met Gln Cys Ala Trp Leu Gly Glu Arg Val Ala Ala Pro Asn

210 215 220

Val Lys Ala Val Thr Thr Asn Val Ile Leu Asn Lys Thr Ala Gly Asn

225 230 235 240

Trp Gly Pro Asn Ala Thr Phe Arg Phe Pro Ala Arg Gly Gly Thr Gly

245 250 255

Gly Ile Trp Ile Ala Val Ala Asp Thr Ile Pro Lys Glu Lys Thr Arg

260 265 270

Phe Gly Glu Lys Gly Lys Val Val Lys Val Asn Ala Asn Asn Lys Thr

275 280 285

Val Thr Leu Gly Asp Gly Thr Thr Val Gly Tyr Lys Lys Leu Val Ser

290 295 300

Thr Met Ala Val Asp Phe Leu Ala Glu Gln Ile Gly Asp Gln Glu Leu

305 310 315 320

Val Gly Leu Thr Lys Gln Leu Phe Tyr Ser Ser Thr His Val Ile Gly

325 330 335

Val Gly Ile Arg Gly Thr Arg Pro Glu Arg Ile Gly Asp Lys Cys Trp

340 345 350

Leu Tyr Phe Pro Glu Asp Asn Cys Pro Phe Tyr Arg Ala Thr Ile Phe

355 360 365

Ser Asn Tyr Ser Pro His Asn Gln Pro Glu Gly Ser Lys Lys Leu Pro

370 375 380

Thr Leu Gln Leu Ala Asp Gly Ser Lys Pro Gln Ser Thr Glu Ala Gln

385 390 395 400

Glu Gly Pro Tyr Trp Ser Ile Met Leu Glu Val Ser Glu Ser Ser Met

405 410 415

Lys Pro Val Asn His Glu Thr Leu Leu Ala Asp Cys Ile Gln Gly Leu

420 425 430

Val Asn Thr Glu Met Leu Lys Pro Thr Asp Glu Ile Val Ser Thr Tyr

435 440 445

His Arg Arg Phe Asp His Gly Tyr Pro Thr Pro Ser Leu Glu Arg Glu

450 455 460

Gly Ala Leu Thr Gln Ile Leu Pro Arg Leu Gln Glu Lys Asp Ile Trp

465 470 475 480

Thr Arg Gly Arg Phe Gly Ser Trp Arg Tyr Glu Val Gly Asn Gln Asp

485 490 495

His Ser Phe Met Leu Gly Val Glu Ala Val Asp Asn Ile Val Asn Gly

500 505 510

Ala Val Glu Leu Thr Leu Asn Tyr Pro Asp Phe Val Asn Gly Arg Gln

515 520 525

Asn Thr Glu Arg Arg Leu Val Asp Gly Ala Gln Val Phe Ala Lys Ser

530 535 540

Gln Ala Gln

545

<210> 4

<211> 3979

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 4

atgctcagcc tcgcccgcag gactttgaac cgtgtcccca gctttcaaga tattctacaa 60

ggcaggatga cccaccccga tatgtaagga gacctttccc cctccctcaa acggcaagct 120

tgccttcact acaacaagaa gctcattgga acaattgctg accgttgtct ttgtgcttga 180

attcagctcc gtcgacgttc tcgtcattgg tgccggccct actggtctcg gtgccgcgaa 240

gcgtcttaac cagattgtac gcattgcgcg ccccctatcg tataccttca ctcgaataaa 300

ggctaattgg acaattcgcg ggtgtaggat ggcccttctt ggttgatcgt tgacagcaac 360

gagacccctg gtggtcttgc ttccaccgat gtgacccccg aaggcttcgt atgttgattc 420

ccactttccc ctcaatgggc aggcattgca ctggtcaagc aaacagcgac tgactcgtga 480

caatcacagc tcttcgatgt tggtggtcac gtcatcttct cccactacaa gtacttcgac 540

gactgcatca acgaggctct ccccaaggat gacgactggt acacccacca gcgtatctcc 600

tacgttcgct gccagggcca atgggttccc taccccttcc agaacaacat ctccatgctt 660

cccaagaatg agcaggtccg ctgtatcgat ggcctgatcg acgctgccct tgaggctcgt 720

gtcgccaaca ccaagcccca gaacttcgat gagtggattg ttcgccagat gggtgtcggt 780

atcgccgacc tcttcatgag accctacaac ttcaaggttt gggctgtgcc tacgaccaag 840

gtaagatacg atgtaccaca tggcgagtaa cggcgtgctg attctcatca gatgcaatgt 900

gcctggttgg gtgagcgtgt tgctgcccct aacgtcaagg ccgtgacgac caacgtcatc 960

cttaacaaga ccgctggtaa ctggggtcct aacgctactt tccgtttccc cgcccgtggt 1020

ggtaccggtg gtatctggat cgctgtcgcc gacactatcc ccaaggagaa gactcgcttc 1080

ggtgagaagg gcaaggtcgt caaggtcaat gccaacaaca agaccgtcac cctgggtgat 1140

ggcaccactg tcggctacaa gaagctcgtc tccaccatgg ctgtcgactt cctcgctgag 1200

cagatcggcg accaggagct cgttggcctc accaagcagc tcttctactc ctccactcac 1260

gtcattggtg tcggtatccg tggtacccgc cccgagagaa tcggtgacaa gtgctgggta 1320

agttgacctt ggatggaaac catgaatgac ttcaaaacta acatgctcgt tactagctct 1380

acttccccga ggacaactgc cccttctacc gtgccaccat cttctccaac tactcccccc 1440

acaaccagcc cgagggctcc aagaagcttc ccactctgca gcttgcggat ggctctaagc 1500

cccagagcac tgaggctcag gagggtcctt actggtccat catgttggag gtttccgagt 1560

cttcgatgaa gcctgtcaac cacgagactc tcctggctga ttgcatccag ggtctcgtca 1620

acaccgagat gctgaagccc accgatgaga ttgtctccac ctaccaccgc cgcttcgacc 1680

acggctaccc caccccctcc ctggagcgtg agggtgctct tacccagatc ctgcccagac 1740

tccaggagaa ggacatctgg acccgtggtc gcttcggtag ctggcgctac gaggtcggta 1800

accaggacca ctctttcatg ctcggtgttg aggctgttga caacattgtc aacggcgctg 1860

tcgagctgac ccttaactac cctgacttcg tcaacggtag acagaacacc gagcgtcggc 1920

tggttgacgg cgcccaggtt ttcgccaaga gccaggcgca gtaaagggtt gaaatgttaa 1980

tagaattaca cgggttttaa tttttttctt gaatgattcc ggggggcaaa agatttgttt 2040

agagatctga ttacgatcaa cgggagccat atttcgtcat ttttttactt cttgtcctat 2100

ctatttaact tattttacca attttctttt tgctctttgg ctcattgaaa gcgatgtatg 2160

ggagatgttc ttggcatttt tgtgatgcgt atcatcatcc actttaccag ccgtcttttg 2220

ctccatctac tagacatgaa tagaagttgc ttatcaaacg cgtgtctttc tgttggggac 2280

ttaatttgct gatttacgta tgccggttac atgtctcgcc gcgaccccgt ggagatcgaa 2340

tccagcttca tgacagatga cacatgtatc acccaccgaa gtgaggcagt gtgtgcagca 2400

catcagccca tgcagggcgt ttctaaattc gctcacttct gtcccgatca agtatagcaa 2460

tgtgatatat agcagcatcg caacaactga gtatggagta tggagtatgc agaacggagt 2520

agtatggaat acatcaaacc cacacatatg gagtcatcta ctactacttc gtacttatgt 2580

acaccaatca tctagagaat aaatactact gcagtggtcc aatcggtatt ccgtgtaaga 2640

catactaccg tacgtagtga aacatgggat ctaccgttag ctattaatca atatctgaca 2700

tactatctat ctatcctacc gtgtgataac tccatattct cctcagtact aacccacatc 2760

ccccccatcc acaacccacc cacccgccgt ctcataaggc tgagtgagtg agctcgagct 2820

cgagctcgga gggtatggat gaagaagaga gtgcactgac tatgtcggaa attttcttta 2880

ctcattgttc tactactgta tatctgagta tgctagtaac tactagtagt aatcatgcaa 2940

gtatcccata gttgagcatg tgaattttat atcagtagta gtatcacagc agttactata 3000

tatgatcatg ctgcaggcat acttacatac atatattcac atgtacatac tatggggacc 3060

atcatgtgaa tactacttac atagatacta ctaagtacgt agtacttaac ttagcattcg 3120

cctccaatac tagagaccac cacgcctcag tagaagatag taatttaccg tgataatatt 3180

attaaccacc aaccttacta gtacttactt actatcctcc ccccggaaca tccattaccc 3240

gcaaccaacc atgccaagaa ggggaaccac cccacacacc taaagcggta tagcgcactg 3300

gggcaggacg cgtaagtaga actaaaccca agaaaggaaa aaaaaaaaaa gaatgaaaat 3360

tgggagggag agtcgtgcag aaaaatatgt atccactatt tatccagcat ccacaggcag 3420

aggaaaatta attcccaatg taaaaataga ttttgattat tagtagtagt ctagcccagg 3480

taggacaggt ggtcagaact atcttactct attatttttg cccctcatgg atggccgatg 3540

acgattaaat ggagtgtgat ggcgatgagg gtggtttgtt tcatggatag tagatactct 3600

gtagtactct aggattatgc tagtattgag gtgatactga ggtaataatg gtgatgtcta 3660

atttgttatt tgcagtaatg atagtatctt tcagggaaga gggataccta catacacatg 3720

gttacataac gcaagttggc agctttggat tccatccgct tcagccttat tgtcggtcgg 3780

ttttgctttc ttatttgagc tccccagtct acaccgtcac tcctctacat tacactacac 3840

tacaccatca ttactgaatt ttctgcagct tgcgtgtcga ctgggctcat tgggatggat 3900

gacgaatagc ctcacgagat ctcacccttc attgggtccg gacaccaact ccgcccccca 3960

tgctttacgt cccatccac 3979

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

acgcctcagt agaagatagt 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

gtggatggga cgtaaagcat 20

<210> 7

<211> 838

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 7

acgcctcagt agaagatagt aatttaccgt gataatatta ttaaccacca accttactag 60

tacttactta ctatcctccc cccggaacat ccattacccg caaccaacca tgccaagaag 120

gggaaccacc ccacacacct aaagcggtat agcgcactgg ggcaggacgc gtaagtagaa 180

ctaaacccaa gaaaggaaaa aaaaaaaaag aatgaaaatt gggagggaga gtcgtgcaga 240

aaaatatgta tccactattt atccagcatc cacaggcaga ggaaaattaa ttcccaatgt 300

aaaaatagat tttgattatt agtagtagtc tagcccaggt aggacaggtg gtcagaacta 360

tcttactcta ttatttttgc ccctcatgga tggccgatga cgattaaatg gagtgtgatg 420

gcgatgaggg tggtttgttt catggatagt agatactctg tagtactcta ggattatgct 480

agtattgagg tgatactgag gtaataatgg tgatgtctaa tttgttattt gcagtaatga 540

tagtatcttt cagggaagag ggatacctac atacacatgg ttacataacg caagttggca 600

gctttggatt ccatccgctt cagccttatt gtcggtcggt tttgctttct tatttgagct 660

ccccagtcta caccgtcact cctctacatt acactacact acaccatcat tactgaattt 720

tctgcagctt gcgtgtcgac tgggctcatt gggatggatg acgaatagcc tcacgagatc 780

tcacccttca ttgggtccgg acaccaactc cgccccccat gctttacgtc ccatccac 838

Claims

1. A DNA barcode for use in the identification of an aspergillus niger strain TMCC 70012, characterized in that the DNA barcode is derived from the genome of an aspergillus niger TMCC 70012 strain, the nucleotide sequence of said DNA barcode being selected from the nucleotide sequences set forth in SEQ ID No.4 and comprising the sequence set forth in SEQ ID No.7.

2. The DNA barcode of claim 1, having a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.4 or SEQ ID No.7.

3. A primer pair for amplifying the DNA barcode of claim 1 or 2, wherein the nucleotide sequences of the forward primer and the reverse primer of the primer pair are as follows:

forward primer: 5'-ACGCCTCAGTAGAAGATAGT-3';

reverse primer: 5'-CTTGCCATGGTGGTGGGATA-3'.

4. A kit for identifying aspergillus niger strain TMCC 70012, characterized in that it comprises a primer pair according to claim 3.

5. A method for identifying aspergillus niger strain TMCC 70012, comprising the steps of:

a) Providing genomic DNA of a strain to be tested;

b) Using the genome DNA of the step a) as a template, and carrying out PCR amplification by using the primer pair of the 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 sequence to be detected and the nucleotide sequence of the DNA bar code according to claim 1 or 2, and judging that the strain to be detected is an aspergillus niger TMCC 70012 strain if the homology is more than 97%.

6. Use of a DNA barcode according to claim 1 or 2 for the identification of the aspergillus niger strain TMCC 70012.

7. Use of a primer pair according to claim 3 for the identification of the aspergillus niger strain TMCC 70012.

8. Use of the kit of claim 4 for identifying the aspergillus niger strain TMCC 70012.