CN109402279B - DNA bar code, primer, kit, method and application - Google Patents

Abstract

The invention belongs to the field of species and strain identification, and particularly relates to a DNA bar code, a primer, a kit, a method and application for identifying a desmospora adephagi strain. The DNA bar code can accurately identify the Pu' er tea fermentation strain, namely, the adenine arthrobotrys yeast TMCC 70007, and can quickly and accurately identify the strain from confusable strains or other strains in the same species.

Description

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.

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 TMCC 70007 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 TMCC 70007 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 3000bp, 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 or SEQ ID No.4 or SEQ ID No. 7.

(3) A primer pair for amplifying the DNA barcode according to (1).

(4) The primer set according to (3), wherein the nucleotide sequence of the forward primer is identical to such a sequence in the genome of the A.adenine-nodorum TMCC 70007 strain: the sequence is a sequence from 1000bp upstream of the 1 st site of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC 70007 strain to the 882 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 TMCC 70007 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 TMCC 70007 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'-ACTCGAAAACCCTATACACCGA-3', respectively;

reverse primer: 5'-CTTGCACTTGTTTAGTGCCCA-3' are provided.

(6) A kit for identifying a strain of Arthrospora adenantha comprising the primer pair according to (3).

(7) A method for identifying a strain of alternaria adefovea comprising the steps of:

a) providing the genome DNA of a strain to be tested;

b) performing PCR amplification by using the genomic DNA in the step a) as a template and using the primer pair in the step (3) to obtain a PCR product;

c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC 70007 strain, and if the target band exists, performing the step d);

d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code of claim 1, and if the homology is more than 99%, judging that the strain to be detected is the adenine arthrobacter adenine-burning TMCC 70007 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 TMCC 70007 strain, and discovers that the open reading frame (SEQ ID No.1) of the gene coding the protein is unique to the genome of the Arthrospora adenantha TMCC 70007 strain. The invention further develops the specific DNA sequence into a DNA bar code through careful research and comparative analysis, and the DNA bar code is used as an effective tool for identifying the strain Alternaria adenini TMCC 70007 produced by the industrial fermentation of the Pu' er tea. The 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 B.

4. The invention establishes a standard gene sequence and a sample identification method of the Puer tea industrial fermentation production strain Arthrospora adenini TMCC 70007, and compared with the traditional morphological identification method, the identification efficiency is obviously improved. The method has low requirement on the integrity of the sample, and the identification index can be quantized, which provides effective basis for timely judging Pu' er tea and germplasm resources thereof. In addition, morphological confusing species are further added, and 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 NAFAYFVQR.

FIG. 2 shows the mRNA sequence of the protein coding frame and the corresponding map of the encoded protein sequence of the region in which the peptide fragment is located, the grey background part being the new peptide fragment identified.

FIG. 3 shows a corresponding plot of the mRNA sequence transcribed by the ORF encoding the newly identified MRPS9 protein and its encoded protein sequence, with the new peptide fragment identified in the grey background section.

FIG. 4A shows an alignment of the barcode sequence SEQ ID NO.1 with homology thereto by NCBI-BLASTN, with 1 homology sequence shown in the lower short grey segment of FIG. 4A; FIG. 4B shows SEQ ID NO.1 in combination with yeast (Sugiyamaella lignohabinans) CBS 10342^TAlignment of homologous sequences (accession number: XM-004200814.1) in the strains.

FIG. 5 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. 6 shows the positions of primers used for amplification of SEQ ID NO.1 in one embodiment of the present invention, the underlined regions in the sequence are the regions where the primers are located, the bold font ATG and TAG are the start and stop sites, the gray background region is the intron region of the gene, and the amplified sequence is SEQ ID NO. 7.

FIG. 7 shows the result of agarose gel electrophoresis of PCR amplification products of test strains obtained using the primers of the present invention.

FIG. 8 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 the protein genomics technology to find the coding protein with missing annotation from the genome of the Alternaria adenini TMCC 70007 strain, and the protein has homology with the mitochondrion 37S ribosomal protein MRPS9, so the protein is called as MRPS9 protein. The invention obtains the corresponding gene sequence in the TMCC 70007 strain genome from the newly found protein sequence, the gene sequence is called MRPS9 gene, and the gene sequence (SEQ ID No.1) for coding the protein is found to have higher specificity compared with the reported sequence in NCBI database and the nucleic acid sequence of similar strains thereof, and can be used for developing DNA bar codes for specifically identifying the strain TMCC 70007 of the industrial fermentation production strain of the puerh tea. Compared with the prior art, the DNA barcode obtained by the method has higher specificity.

Considering that the DNA barcode should have a suitable length and sufficient specificity among strains, the present invention further obtains a DNA barcode that can accurately and efficiently identify the strain of Arthrospora adenine-feeding TMCC 70007 based on the above-mentioned characteristic DNA sequence through careful study and comparative analysis.

Thus, in one aspect the present invention provides a DNA barcode for identifying a strain of arthrospora adephaga yeast, the DNA barcode being derived from the genome of the strain of arthrospora adenosylsis TMCC 70007 and comprising at least 500bp of a sequence selected from the DNA sequences as shown in SEQ ID No.1, and the DNA barcode having a length of 500bp to 3000bp, 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 1382bp, and because the SEQ ID No.1 is specific to the genome of the Arthrospora adenine-feeding TMCC 70007 strain, the sequence can be used as a DNA bar code for identifying the Arthrospora adenine-feeding TMCC 70007 strain. 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 3000bp), 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.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 TMCC 70007 strain to the 882 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 TMCC 70007 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 TMCC 70007 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 MRPS 9-F: 5'-ACTCGAAAACCCTATACACCGA-3' (SEQ ID No. 5);

reverse primer MRPS 9-R: 5'-CTTGCACTTGTTTAGTGCCCA-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) taking the genome DNA of the step a) as a template, and carrying out PCR amplification by using the primer pair according to the invention to obtain a PCR product;

c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC 70007 strain, and if the target band exists, performing the step d);

d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code of claim 1, and if the homology is more than 99%, judging that the strain to be detected is the adenine arthrobacter adenine-burning TMCC 70007 strain.

In a specific embodiment of the present invention, the procedure of PCR amplification is: 1) pre-denaturation at 94-96 deg.C for 8 min; 2) denaturation at 94-96 ℃ for 45 seconds, annealing at 55-57 ℃ for 45 seconds, and extension at 72 ℃ for 1 min 15 seconds, wherein the procedure 2) is performed for 32-35 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 99 percent, the strain to be detected is judged not to be the adenine node B spore forming yeast strain TMCC 70007. And if the sequence homology is more than or equal to 99 percent, judging that the strain to be detected is the adenine-node B.adephagus strain TMCC 70007.

If a clustering analysis is performed, such as a phylogenetic tree, the DNA barcode is used with the DNA sequencing results (i.e., the sequences to be tested) of each strain to be identified to construct an NJ phylogenetic tree using MEGA 6 or PAUP software. If the sequence to be tested of the strain to be identified is clustered with the DNA bar code of the Arthrospora adenini TMCC 70007, the strain is identified as the Arthrospora adenini TMCC 70007.

The term "cluster" as used herein means that the 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 MRPS9 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 zymocyte Arthrospora adenine-eating yeast TMCC 70007 in the Pu' er tea industry, and the genome is verified by annotating coding genes. Specifically, in order to find a novel protein coding region, a Six-Frame Translation database of the genome data of the Geobacillus adenini TMCC 70007 was obtained using a Six-Frame Translation (Six Frame Translation) strategy in systematic proteomics, exhaustively listing 6 coding possibilities of the genome (+1, +2, +3, -1, -2, -3), and the 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 TMCC 70007 strain.

Through identification, a peptide segment NAFAYFVQR which is not found in the adenine node B.adenine-eating yeast TMCC 70007 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 NAFAYFVQR are detected, the matching is good, the signal is strong, and the result is more reliable.

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. 2.

3. In order to further determine the coding start site and the termination site of the gene coding for the protein and to obtain a DNA barcode of more suitable length and sufficient specificity, the coding frame of the protein is extended by 1000bp upstream and downstream, respectively, and genetic Mark.hmm is used for gene prediction, and the reference species is Schizosaccharomyces pombe (Schizosaccharomyces pombe). The presence of the gene was predicted in this region, and its coding start and stop sites were determined, and its ORF sequence was SEQ ID NO. 1. The 935-th 1110 th site of SEQ ID NO.1 is an intron of the gene.

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. 3. Peptide segment NAFAYFVQR is located downstream of the gene.

The nucleotide sequence of the ORF is 1382bp in total, 401 amino acids are coded, the theoretical molecular weight is 45.41kDa, and the theoretically coded amino acid sequence is shown as SEQ ID NO. 3.

4. The amino acid sequence of the theoretical encoded product of this gene was analyzed by blastp, and the homology of the sequence to mitochondrial 37S ribosomal protein (MRPS9) of yeast (Sugiyamaella lignohabitans) was 45%. The blastp alignment results are summarized in table 1.

The blastp result shows that the detected leakage-release protein is a MRPS9 homologous gene product, and the sequence of the leakage-release protein has certain conservation.

5. The ORF sequence of the identified MRPS9 gene (i.e., SEQ ID NO.1) was subjected to NCBI-BLASTN analysis, the results of which are shown in FIG. 4A. The BLASTN results are summarized in table 2. The results show that, in the NCBI database, only a partial sequence of SEQ ID No.1 is present in the sequence of Sugiyamaella lignohabitans CBS 10342^TThe chromosome A of the gene has homology, but only about 70bp, which shows that the leaky-expulsive gene MRPS9 detected in the adenine-node B yeast has sequence specificity, and although the partial segment of the amino acid sequence of the MRPS9 protein is relatively conservative, 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. SEQ ID NO.1 and CBS 10342^TThe genomic homology sequence was analyzed by DNAMAN and showed 49.08% identity, and the sequence alignment is shown in FIG. 4B.

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

6. The region from 1000bp upstream to 1000bp downstream of SEQ ID No.1 was further selected (as shown in SEQ ID No. 4). The sequence of SEQ ID NO.4 was subjected to NCBI-BLASTN analysis, and the Nucleotide collection (nr/nt) database was searched, and as a result, it was found that only 2% of the sequence (about 70bp) had homology to part of mRNA (accession No.: XM-018878203.1) of mitochondrial 37S ribosomal protein MRPS9 of the yeast Sugiyamaella lignohabinans, and the results of the alignment 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, we further performed NCBI-BLASTN analysis of the sequence of SEQ ID NO.4 and searched the Whole genome sequence of Arthrospora adenine-saccharomycete (taxonomic ID: taxi: 409370) in the white-genome shotgun contigs (wgs) database. As a result, it was found that the homology of SEQ ID NO.4 to the entire gene sequence Arad1A _ contig _1(CBZY010000006.1) of Arad1, a sequence coverage of 99% of the Alternaria adenini LS3 strain was 93%, and that the homologous region was different by about 180 bases although TMCC 70007 and LS3 strains were the same species. The alignment results are summarized in table 4. From this, it is found that the Arthrospora adenine-feeding yeast TMCC 70007 SEQ ID NO.4 has sufficient discrimination with the homologous sequence in the genome of Arthrospora adenine-feeding yeast LS3, and can be used as a DNA barcode sequence. In conclusion, the sequence from upstream 1000bp to downstream 1000bp of SEQ ID NO.1 (SEQ ID NO.4) can be used as a DNA barcode for identifying the strain of Arthrospora adenantha.

TABLE 3

TABLE 4

7. 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 91.64%, although TMCC 70007 and LS3 strains were of the same species (instant Arthromyces adenine) but 111 sites were different in 1382 bases of the DNA barcode sequence, as shown in FIG. 5. This further demonstrates that the use of this DNA barcode sequence can effectively distinguish TMCC 70007 strain from other strains within the species.

Example 2 identification of strains Using DNA barcodes

And judging whether the sample to be detected is the bacterial strain TMCC 70007 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 TMCC 70007 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 MRPS 9-F: 5'-ACTCGAAAACCCTATACACCGA-3', respectively; MRPS 9-R: 5'-CTTGCACTTGTTTAGTGCCCA-3' are provided. The position of the primers is shown in FIG. 6. The amplified sequence is SEQ ID NO. 7.

(2) The source of the strain

TABLE 5 information on selected related strains

(3) Respectively extracting strain DNA: OMEGA e.z.n.a was used.^TMYeast genomic DNA was extracted using the Yeast DNA kit (Bio Rad laboratories) and the DNA concentration of the sample was diluted to 0.5. mu.g/. mu.L with sterile deionized water.

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

forward primer sequence: MRPS 9-F: 5'-ACTCGAAAACCCTATACACCGA-3', respectively;

reverse primer sequence: HSP 40-R: MRPS 9-R: 5'-CTTGCACTTGTTTAGTGCCCA-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 TMCC 70007, 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. 7. 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 adenantha TMCC 70007 is more than 99%, judging that the strain to be detected is possible to be the Arthrospora adenantha TMCC 70007 strain. For example, through DNAMAN comparison and analysis, the sequence obtained by sequencing the strain TMCC 70007 is completely consistent with the DNA barcode sequence SEQ ID NO.1 after comparison, and the reliability of the method is further proved. The results are shown in table 4 and fig. 8.

TABLE 6

SEQUENCE LISTING

<110> Menghai tea industry, Limited liability company

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

<130> FI-163710-59:52/C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1382

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 1

atgagagcat tgcgggctct ggtgcccaga cgcccactgg gccgccagct cctgggcatg 60

cgattctatt cggacgttcc aaagccatca tcgcccattg caaacccctt tgagcagcga 120

ctgcagggac tggtcaagga gtatgaaaaa tttaacaaca ttggctccca ggtgtctgcc 180

gacgaagctc gcgacgctga gggtgtacag tttaaagacg agaggcacct ggcccctgac 240

ctcagccgac tgagagttgt gccccgagaa cgggcctttt tcatgggcat gcctgctcat 300

gaagagatta tgcgaacctt aaacgacctt acgatgaaga accagactat tccccggtta 360

ggtcgatcag agattgaagt ccctacctgg ctgtctctta acgaatacag agacatgttg 420

ggtgctaagc actttaagca aaagtactac cgggagctcc ttacttctct gaatgacctg 480

gccctcatgg acccccagct tgtaccttca tctgtgcctg atgcactatc tcgatttgta 540

tctcttcgct ctgacggttt cattgccaag aagccaaaga ctcttgacga agtaggacga 600

gccattgcag ttggccgacg caagaccgcc agtgctcggg ttcagctcgt caagggatct 660

ggactagctc tagttaacgg aaagcctctg gatgaagcct ttgaacgtcc tgctgaccgt 720

gatgccatgt tgtaccctct caaggtcgta gctggtgaac aatcctacaa catttttgct 780

actgtcactg gaggaggaaa gaccggacag gctagtgcca ttgctcacgg aatcgctcag 840

gccctagtca ttcacaaccc tctgctactg tcccgactag ctagagccaa gtgcctcaag 900

agagatcctc gtgtcaagga gagaaagaag cctggtaagg ttaaggcccg aaaatcctac 960

acctgggtca agcgttaatg tacatatata tatacacgat tcagtcattc cattcgtcca 1020

ttcatttcgt tgtcttaatc actttgtatc tgttatgcta ctttgaactg tctgtacgtc 1080

gtttgtcgtt tgcctactgc tgcactccag atacttctct tacttcccac tttgattccg 1140

atcaccgatt cctaaataac tctcccatga acccaaagaa cgctttcgca tactttgttc 1200

aaagagccca ggctagaagc ttggtacgct acgcattccg cactgcgtac caagtacgtg 1260

atgttaatac tcgcaaggaa ctgataacat gggccagaca agagtttgaa cgaaatcgaa 1320

acgtcgaaga cccggtatgt ggacaggagg tgaaaagttt tactttgcag tcgctaactt 1380

ag 1382

<210> 2

<211> 441

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 2

taaggcccga aaatcctaca cctgggtcaa gcgttaatgt acatatatat atacacgatt 60

cagtcattcc attcgtccat tcatttcgtt gtcttaatca ctttgtatct gttatgctac 120

tttgaactgt ctgtacgtcg tttgtcgttt gcctactgct gcactccaga tacttctctt 180

acttcccact ttgattccga tcaccgattc ctaaataact ctcccatgaa cccaaagaac 240

gctttcgcat actttgttca aagagcccag gctagaagct tggtacgcta cgcattccgc 300

actgcgtacc aagtacgtga tgttaatact cgcaaggaac tgataacatg ggccagacaa 360

gagtttgaac gaaatcgaaa cgtcgaagac ccggtatgtg gacaggaggt gaaaagtttt 420

actttgcagt cgctaactta g 441

<210> 3

<211> 401

<212> PRT

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 3

Met Arg Ala Leu Arg Ala Leu Val Pro Arg Arg Pro Leu Gly Arg Gln

1 5 10 15

Leu Leu Gly Met Arg Phe Tyr Ser Asp Val Pro Lys Pro Ser Ser Pro

20 25 30

Ile Ala Asn Pro Phe Glu Gln Arg Leu Gln Gly Leu Val Lys Glu Tyr

35 40 45

Glu Lys Phe Asn Asn Ile Gly Ser Gln Val Ser Ala Asp Glu Ala Arg

50 55 60

Asp Ala Glu Gly Val Gln Phe Lys Asp Glu Arg His Leu Ala Pro Asp

65 70 75 80

Leu Ser Arg Leu Arg Val Val Pro Arg Glu Arg Ala Phe Phe Met Gly

85 90 95

Met Pro Ala His Glu Glu Ile Met Arg Thr Leu Asn Asp Leu Thr Met

100 105 110

Lys Asn Gln Thr Ile Pro Arg Leu Gly Arg Ser Glu Ile Glu Val Pro

115 120 125

Thr Trp Leu Ser Leu Asn Glu Tyr Arg Asp Met Leu Gly Ala Lys His

130 135 140

Phe Lys Gln Lys Tyr Tyr Arg Glu Leu Leu Thr Ser Leu Asn Asp Leu

145 150 155 160

Ala Leu Met Asp Pro Gln Leu Val Pro Ser Ser Val Pro Asp Ala Leu

165 170 175

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

180 185 190

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

195 200 205

Thr Ala Ser Ala Arg Val Gln Leu Val Lys Gly Ser Gly Leu Ala Leu

210 215 220

Val Asn Gly Lys Pro Leu Asp Glu Ala Phe Glu Arg Pro Ala Asp Arg

225 230 235 240

Asp Ala Met Leu Tyr Pro Leu Lys Val Val Ala Gly Glu Gln Ser Tyr

245 250 255

Asn Ile Phe Ala Thr Val Thr Gly Gly Gly Lys Thr Gly Gln Ala Ser

260 265 270

Ala Ile Ala His Gly Ile Ala Gln Ala Leu Val Ile His Asn Pro Leu

275 280 285

Leu Leu Ser Arg Leu Ala Arg Ala Lys Cys Leu Lys Arg Asp Pro Arg

290 295 300

Val Lys Glu Arg Lys Lys Pro Asp Thr Ser Leu Thr Ser His Phe Asp

305 310 315 320

Ser Asp His Arg Phe Leu Asn Asn Ser Pro Met Asn Pro Lys Asn Ala

325 330 335

Phe Ala Tyr Phe Val Gln Arg Ala Gln Ala Arg Ser Leu Val Arg Tyr

340 345 350

Ala Phe Arg Thr Ala Tyr Gln Val Arg Asp Val Asn Thr Arg Lys Glu

355 360 365

Leu Ile Thr Trp Ala Arg Gln Glu Phe Glu Arg Asn Arg Asn Val Glu

370 375 380

Asp Pro Val Cys Gly Gln Glu Val Lys Ser Phe Thr Leu Gln Ser Leu

385 390 395 400

Thr

<210> 4

<211> 2436

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 4

ggttaccgat tttcactttt cactttgaaa aactcgaaaa ccctatacac cgaaatcatg 60

agagcattgc gggctctggt gcccagacgc ccactgggcc gccagctcct gggcatgcga 120

ttctattcgg acgttccaaa gccatcatcg cccattgcaa acccctttga gcagcgactg 180

cagggactgg tcaaggagta tgaaaaattt aacaacattg gctcccaggt gtctgccgac 240

gaagctcgcg acgctgaggg tgtacagttt aaagacgaga ggcacctggc ccctgacctc 300

agccgactga gagttgtgcc ccgagaacgg gcctttttca tgggcatgcc tgctcatgaa 360

gagattatgc gaaccttaaa cgaccttacg atgaagaacc agactattcc ccggttaggt 420

cgatcagaga ttgaagtccc tacctggctg tctcttaacg aatacagaga catgttgggt 480

gctaagcact ttaagcaaaa gtactaccgg gagctcctta cttctctgaa tgacctggcc 540

ctcatggacc cccagcttgt accttcatct gtgcctgatg cactatctcg atttgtatct 600

cttcgctctg acggtttcat tgccaagaag ccaaagactc ttgacgaagt aggacgagcc 660

attgcagttg gccgacgcaa gaccgccagt gctcgggttc agctcgtcaa gggatctgga 720

ctagctctag ttaacggaaa gcctctggat gaagcctttg aacgtcctgc tgaccgtgat 780

gccatgttgt accctctcaa ggtcgtagct ggtgaacaat cctacaacat ttttgctact 840

gtcactggag gaggaaagac cggacaggct agtgccattg ctcacggaat cgctcaggcc 900

ctagtcattc acaaccctct gctactgtcc cgactagcta gagccaagtg cctcaagaga 960

gatcctcgtg tcaaggagag aaagaagcct ggtaaggtta aggcccgaaa atcctacacc 1020

tgggtcaagc gttaatgtac atatatatat acacgattca gtcattccat tcgtccattc 1080

atttcgttgt cttaatcact ttgtatctgt tatgctactt tgaactgtct gtacgtcgtt 1140

tgtcgtttgc ctactgctgc actccagata cttctcttac ttcccacttt gattccgatc 1200

accgattcct aaataactct cccatgaacc caaagaacgc tttcgcatac tttgttcaaa 1260

gagcccaggc tagaagcttg gtacgctacg cattccgcac tgcgtaccaa gtacgtgatg 1320

ttaatactcg caaggaactg ataacatggg ccagacaaga gtttgaacga aatcgaaacg 1380

tcgaagaccc ggtatgtgga caggaggtga aaagttttac tttgcagtcg ctaacttagg 1440

aacaaatgag atatctaatt gctatgggca ctaaacaagt gcaagaaatg gcaaagacta 1500

ttcactaggt acactattat ggagtcacgc agttgcggag tacggtgatg gacttgtcgg 1560

acgctgcgca gatgccgtcc actacttcct ctaccagaga agcagaaatt ccattctcct 1620

cagcccaagg gctcagctgg ccagtgagat gctggaatgt ctggttgcgt gagtttttca 1680

cttgcaatgc ctgttccttg acattcttgg tcatggcact aactgcgctt ctcagggctt 1740

cgctagcatc caaattggcc tttacctgat tcctattagt tttgagacga atcatgtagc 1800

ctgatacaac attgcaagtc tccaaaattg taaatgctct gtggaaccat ttccattggc 1860

cggtctgttg ctcatcgagc gccatgcttg acagcttttg ttcaacagca gtgtatccct 1920

ccttactctc cttggcctgg gcgctggcgg ctaccagcac aatagtttcc agagcccata 1980

actcttcatc tgtaaactgg ctggagtgat gcttaataat gtcctcaaga gctttcacca 2040

agccaatgag ctcgtcaccg ccaggaacaa gcttgcggat gattttttct cggatctgga 2100

acgcatgagc ccactcggac ttctgaacgg ggcccactgt caagacctga gataagggga 2160

tctcatcagg gccattacag ttccacattg agtcaaaatc tcggttgtct tcgagggcca 2220

cagacgtatt cgtagcttcg atgaactttg tgtttgcatt gtcagtagtc aatcgctcta 2280

gtttgactgt ttccaagtcc accagtgcct tgttgagact gtgcgaaaac ttgtaatgaa 2340

ggtccataaa cccttcaatc tgagtgtagg ctccattgta ggcaaacttg atgtagttga 2400

caaagtcaga tgcctgccgg gcaatttcat aagtag 2436

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence

<400> 5

actcgaaaac cctatacacc ga 22

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<400> 6

cttgcacttg tttagtgccc a 21

<210> 7

<211> 1454

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 7

actcgaaaac cctatacacc gaaatcatga gagcattgcg ggctctggtg cccagacgcc 60

cactgggccg ccagctcctg ggcatgcgat tctattcgga cgttccaaag ccatcatcgc 120

ccattgcaaa cccctttgag cagcgactgc agggactggt caaggagtat gaaaaattta 180

acaacattgg ctcccaggtg tctgccgacg aagctcgcga cgctgagggt gtacagttta 240

aagacgagag gcacctggcc cctgacctca gccgactgag agttgtgccc cgagaacggg 300

cctttttcat gggcatgcct gctcatgaag agattatgcg aaccttaaac gaccttacga 360

tgaagaacca gactattccc cggttaggtc gatcagagat tgaagtccct acctggctgt 420

ctcttaacga atacagagac atgttgggtg ctaagcactt taagcaaaag tactaccggg 480

agctccttac ttctctgaat gacctggccc tcatggaccc ccagcttgta ccttcatctg 540

tgcctgatgc actatctcga tttgtatctc ttcgctctga cggtttcatt gccaagaagc 600

caaagactct tgacgaagta ggacgagcca ttgcagttgg ccgacgcaag accgccagtg 660

ctcgggttca gctcgtcaag ggatctggac tagctctagt taacggaaag cctctggatg 720

aagcctttga acgtcctgct gaccgtgatg ccatgttgta ccctctcaag gtcgtagctg 780

gtgaacaatc ctacaacatt tttgctactg tcactggagg aggaaagacc ggacaggcta 840

gtgccattgc tcacggaatc gctcaggccc tagtcattca caaccctctg ctactgtccc 900

gactagctag agccaagtgc ctcaagagag atcctcgtgt caaggagaga aagaagcctg 960

gtaaggttaa ggcccgaaaa tcctacacct gggtcaagcg ttaatgtaca tatatatata 1020

cacgattcag tcattccatt cgtccattca tttcgttgtc ttaatcactt tgtatctgtt 1080

atgctacttt gaactgtctg tacgtcgttt gtcgtttgcc tactgctgca ctccagatac 1140

ttctcttact tcccactttg attccgatca ccgattccta aataactctc ccatgaaccc 1200

aaagaacgct ttcgcatact ttgttcaaag agcccaggct agaagcttgg tacgctacgc 1260

attccgcact gcgtaccaag tacgtgatgt taatactcgc aaggaactga taacatgggc 1320

cagacaagag tttgaacgaa atcgaaacgt cgaagacccg gtatgtggac aggaggtgaa 1380

aagttttact ttgcagtcgc taacttagga acaaatgaga tatctaattg ctatgggcac 1440

taaacaagtg caag 1454

Claims (5)

1. A primer pair for amplifying DNA barcodes for identifying the adenine node B.adenine node B yeast TMCC 70007 strain comprises the nucleotide sequences of a forward primer and a reverse primer which are respectively shown as follows:

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

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

wherein the DNA barcode is derived from the genome of the Arthrospora adenantha TMCC 70007 strain, and the nucleotide sequence is selected from the sequences shown as SEQ ID No.4 and comprises the sequence shown as SEQ ID No. 1.

2. A kit for identifying a nodospora adevorans TMCC 70007 strain comprising the primer pair according to claim 1.

3. A method for identifying a strain of the nodospora adephagi TMCC 70007, comprising the steps of:

a) providing the genome DNA of a strain to be tested;

b) performing PCR amplification by using the genomic DNA of the step a) as a template and using the primer pair of claim 1 to obtain a PCR product;

c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC 70007 strain, and if the target band exists, performing the step d);

d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code of claim 1, and if the homology is more than 99%, judging that the strain to be detected is the adenine arthrobacter adenine-burning TMCC 70007 strain.

4. The use of the primer pair according to claim 1 for identifying the strain of the Arthrospora adenantha TMCC 70007.

5. Use of the kit according to claim 2 for identifying the strain of Arthrospora adenantha TMCC 70007.

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