CN109385485B

CN109385485B - DNA bar code, primer, kit, method and application

Info

Publication number: CN109385485B
Application number: CN201710674836.3A
Authority: CN
Inventors: 徐平; 田飞; 唐蜀昆; 施佳辉; 周文婧; 高林瑞; 高慧英; 职晓阳; 丁章贵; 刘韬
Original assignee: Yunnan Dayi Microbial Technology Co ltd; Menghai Tea Industry Co ltd
Current assignee: MENGHAI TEA INDUSTRY Co.,Ltd.; Yunnan Dayi Microbial Technology Co., Ltd
Priority date: 2017-08-09
Filing date: 2017-08-09
Publication date: 2021-08-13
Anticipated expiration: 2037-08-09
Also published as: CN109385485A

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 strain of arthrobotrys adenivorus, the DNA barcode being derived from the genome of the strain of arthrobotrys adenivorus TMCC 70007 and comprising at least 500bp of a sequence selected from the DNA sequences shown as SEQ ID No.1, and the DNA barcode having a length of 500bp to 3000bp, preferably 500bp to 1600bp, more preferably 500bp to 850 bp.

(2) The DNA barcode according to (1), wherein the nucleotide sequence thereof is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 7.

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

(4) The primer set according to (3), wherein the nucleotide sequence of the forward primer is identical to such a sequence in the genome of the A.adenine-nodorum 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 a region of the 266 th site of the nucleotide sequence shown as SEQ ID No.1, and the length of the forward primer is 20-30 bp; the reverse primer 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'-AACCGGTCTCCGTCAAACTC-3', respectively;

reverse primer: 5'-AAGATCTCCGCGACAAGCAA-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 (2) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code in the step (1), and if the homology is more than 97%, judging that the strain to be detected is the Arthrospora adenine-feeding TMCC 70007 strain.

(8) 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 LipB from the genome of the Arthrospora adenantha TMCC 70007 strain, and discovers that the coding sequence (SEQ ID No.1) for 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 LipB gene has the characteristics of universality, easy amplification and easy comparison, and the difference of the sequence among different strains in the Nipponica adenine node B yeast is obvious.

4. The invention establishes a standard gene sequence and a sample identification method of the Puer tea industrial fermentation production strain Arthrospora adenini 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. 1A, 1B and 1C show the mass spectra of the newly identified three peptide fragments VTYEQAAEIQDR, ITDNTGVWINDNEK and IAAIGIHVR, respectively.

FIG. 2 shows the mRNA sequence of the protein coding frame and the corresponding map of the protein sequence encoded by it in the region of the peptide stretch, the three new peptide stretches identified being in the grey background.

FIG. 3 shows the corresponding map of the mRNA sequence transcribed by the ORF encoding the newly identified LipB protein and its encoded protein sequence, the grey background region being the intron region.

FIG. 4A shows a SDS-PAGE separation profile of the cellular holoprotein of TMCC 70007, the band shown in bold is the position of the LipB protein; FIG. 4B shows a molecular weight verification chart of the LipB protein, in which the abscissa represents the logarithmic value to 10 of the proteins of different molecular weights in the protein molecular weight markers (marker), and the ordinate represents the ratio of the migration amount of each molecular weight protein in the protein molecular weight markers in SDS-PAGE electrophoretic separation to the total migration amount of SDS-PAGE. Theoretically, the LipB protein has a molecular weight of 26.43kDa, lg (MW) ≈ lg (26.43) ≈ 1.42, and the LipB protein log corresponds to fig. 4B abscissa 1.58, and its mobility corresponds to the value on the y-axis when the mobility curve is x ═ 1.42.

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 amplifying SEQ ID NO.1 in one embodiment of the present invention, the underlined regions in the sequence are the regions where the primers are located, the bold font ATG and TAA are the start and stop sites, and the amplified sequence is SEQ ID NO. 7.

FIG. 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 a protein genomics technology to find a coding protein with missing annotation from the genome of the Aleurospora adephaga TMCC 70007 strain, and the protein is called as a LipB protein because the protein has a structural domain similar to a lipoate-protein ligase-like protein B (LipB) family. The invention obtains the corresponding gene sequence in the TMCC 70007 strain genome from the newly found protein sequence, the gene sequence is called as the LipB gene, and compared with the NCBI nucleic acid database reported at present, in the Alternaria adenini TMCC 70007 strain, the open reading frame (SEQ ID No.1) sequence of the gene for coding the protein has high specificity, and the specificity identification of the Alternaria adenini TMCC 70007 strain can be realized. Therefore, the specific DNA sequence can be used for developing and identifying the DNA bar code of the commercial fermentation production strain of the Puer tea, namely the adenine nodularia cerealis TMCC 70007. 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 1600bp, more preferably 500bp to 850 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 766bp, and because the SEQ ID No.1 is specific to the genome of the Arthrospora adenine-eating strain TMCC 70007, the sequence can be used as a DNA bar code for identifying the Arthrospora adenine-eating strain TMCC 70007. In addition, theoretical analysis and experimental verification prove that the specificity of the DNA barcode containing the sequence and having longer length is better ensured. When the length of the DNA barcode is too long (e.g., greater than 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, or SEQ ID No.2, or 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 a region of the 266 th site of the nucleotide sequence shown as SEQ ID No.1, and the length of the forward primer is 20-30 bp; the reverse primer 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 LipB-F: 5'-AACCGGTCTCCGTCAAACTC-3' (SEQ ID No. 5);

reverse primer LipB-R: 5'-AAGATCTCCGCGACAAGCAA-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;

d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (2) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA barcode in the step (1), and if the homology is more than 97 percent (preferably more than 99 percent), judging that the strain to be detected is the adenine arthrobacter adenine-feeding TMCC 70007 strain.

In a specific embodiment of the present invention, the procedure of PCR amplification is: 1) pre-denaturation at 94-96 deg.C for 8 min; 2) denaturation at 94-96 ℃ for 45 seconds, annealing at 55-57 ℃ for 45 seconds, and extension at 72 ℃ for 1 min 15 seconds, wherein the procedure 2) is performed for 30-35 cycles; 3) extension at 72 ℃ for 10 min.

In another embodiment of the present invention, the method further comprises performing cluster analysis (e.g., phylogenetic tree) on the sequence to be tested obtained from the sequencing result and the DNA barcode of the present invention, and if the sequence to be tested and the DNA barcode are clustered together, determining that the strain to be tested is the alternaria adenini yeast strain TMCC 70007.

In a specific embodiment of the invention, genomic DNA extracted from the strain to be identified is subjected to PCR amplification using the primer pairs of the invention, followed by detection by agarose gel electrophoresis. Identifying the strains based on detecting the presence or absence of PCR products: if the strain to be identified does not amplify a corresponding target band, the strain is not TMCC 70007; if the corresponding target band is amplified, the strain is proved to be possible TMCC 70007. For further identification, the PCR product is sequenced, the DNA sequencing result and the DNA bar code sequence are subjected to homology comparison to obtain the similarity (namely homology) between the sequences, and if the sequence homology is less than 97%, the strain to be detected is judged not to be the adenine node B spore forming yeast strain TMCC 70007. If the sequence homology is greater than or equal to 97% (preferably 99% or more), the test strain is judged to be the Arthrospora adenantha strain TMCC 70007.

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

Proteome and genome analysis methods and mass spectrometry methods used in this example are all known in the art, and therefore detailed descriptions of the procedures are omitted.

1. By utilizing a high-coverage proteome technology, pFind and pAnno software are adopted to carry out deep coverage research on proteome on the Puer tea industrial fermentation bacteria Arthrospora adenine-eating yeast TMCC 70007, and the genome is verified by annotating coding genes. Specifically, in order to find a novel protein coding region, a Six-Frame Translation database of the genome data of the Geobacillus adenini TMCC 70007 was obtained using a Six-Frame Translation (Six Frame Translation) strategy in systematic proteomics, exhaustively listing 6 coding possibilities of the genome (+1, +2, +3, -1, -2, -3), and the 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, three peptide fragments VTYEQAAEIQDR, ITDNTGVWINDNEK and IAAIGIHVR which are not found in the adenine-nodakeslea yeast TMCC 70007 annotated genome in the prior art are found, and the mass spectrograms of the peptide fragments are respectively shown in FIGS. 1A-C.

The results of manual inspection of mass spectrum shows that almost all y ion sequences of secondary mass spectrum (MS2) of three peptide fragments VTYEQAAEIQDR, ITDNTGVWINDNEK and IAAIGIHVR are detected, the matching is good, the signal is strong, and the results are reliable.

2. The six-frame translated nucleic acid sequence (protein coding frame) SEQ ID NO.2 is obtained by taking the region comprising the former stop codon and the latter stop codon as the boundary according to the positions of the three new peptide segments. 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 coding end site of the coding gene (LipB), the coding frame of the protein is respectively expanded by 1000bp upstream and downstream, GeneMark.hmm is adopted for gene prediction, and Schizosaccharomyces pombe (Schizosaccharomyces pombe) is selected as a reference species. The existence of the gene is predicted in the region, the ORF sequence and the protein sequence coded by the gene are obtained, the coding start and termination sites are determined, and the ORF sequence is SEQ ID NO. 1.

The correspondence between the mRNA sequence transcribed from the gene and the amino acid sequence of the protein translated from the mRNA sequence is shown in FIG. 3. Three peptides VTYEQAAEIQDR, ITDNTGVWINDNEK and IAAIGIHVR are located in the middle of the gene.

The nucleotide sequence of the gene is 766bp in total, 235 amino acids are coded, the theoretical molecular weight is 26.43kDa, and the theoretically coded amino acid sequence is shown as SEQ ID NO. 3.

4. To further confirm the correctness of the identified sequences, TMCC 70007 strain was cultured in YPD medium and total protein was extracted therefrom, separated by SDS-PAGE, stained with Coomassie Brilliant blue on electrophoresis gel, and the molecular weight was verified based on the molecular weight characteristics at the time of preparation of the proteome sample, as shown in FIG. 4A. In addition, 25 strips were cut out in total by band for in-gel digestion and mass spectrometry analysis, where the LipB protein was identified in the mass spectrometry data of the 20 th strip, matching the predicted molecular weight size. The theoretical molecular weight calculated based on the amino acid sequence was 26.43kDa, which was consistent with the position of the gel strip to which the protein belongs on SDS-PAGE. The results show that the experimental molecular weight of the identified LipB is basically consistent with the theoretical molecular weight, and the correctness of the identified genes and proteins is verified.

5. The amino acid sequence (SEQ ID NO.3) of the theoretical coding product of the gene (SEQ ID NO.1) was used for NCBI-BLASTP analysis, which has a similar domain to the biotin/thiooctanoyl A/B protein ligase family, a 51% lipoate-protein ligand-like protein B (LipB) similarity and a 79% sequence coverage (Query cover) similarity to Xylonia heveae TC161 strain. The 10 protein sequences with the highest similarity to the protein are shown in Table 1.

The NCBI-BLASTP results show that the detected leaky injection protein is probably a LipB homologous protein, and the sequence of the detected leaky injection protein has certain homology with other protein sequences but also has higher variability. The LipB protein sequence has a certain conservation.

6. When the ORF sequence of the identified LipB gene (i.e., SEQ ID NO.1) was subjected to NCBI-BLASTN and KEGG-BLASTN analyses, no apparently similar sequences were found, indicating that the leaky injection-released gene LipB detected in Arthrospora adenantha has sequence specificity, and although the amino acid sequence partial segment of the LipB protein is conserved, the DNA homology is 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.

7. The region from 1000bp upstream to 1000bp downstream of SEQ ID No.1 was further selected (as shown in SEQ ID No. 4). The sequence of SEQ ID NO.4 was subjected to NCBI-BLASTN analysis, and the Nucleotide collection (nr/nt) database was searched, as a result, see Table 2 below, and it was found that only 1% (about 30 bp) of the sequence had homology with the chromosome 2 and the whole genome sequence (accession No.: HE650822.1) of Saccharomyces cerevisiae (Kazachstania africana) CBS 2517 strain, indicating that SEQ ID NO.4 can be used as a DNA barcode sequence for identifying the adenine-feeding arthrobacter strain TMCC 70007. Furthermore, the sequence of SEQ ID NO.4 was further subjected to NCBI-BLASTN analysis to search the Whole genome sequence of Arthrospora adenine-type yeast (taxonomic ID: taxi: 409370) in the white-genome shotgun contigs (wgs) database, and the specific results are shown in Table 3 below. As a result, it was found that SEQ ID NO.4 has a homology with a partial sequence in the whole genome sequence of the A.adefovea LS3 strain and the degree of similarity was 93% (accession No.: CBZY 010000005.1). Although TMCC 70007 is the same species as LS3 strain, the sequence differences in this region are sufficient to identify TMCC 70007, further indicating that this sequence can be used as a DNA barcode sequence specifically identifying this strain. 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 2

TABLE 3

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.

8. Currently, only the genome of the Arthrospora adenantha LS3 strain has been reported among the species of Arthrospora adenantha. After Local-BLASTN analysis, it was found that the gene sequence (SEQ ID NO.1) had a homologous sequence of 94.26% in the genome of LS3 strain, and that about 40 sites were different among 766 bases of the DNA barcode sequence, although TMCC 70007 and LS3 strain were of the same species (instant Arthrospora adenine, Blastotrys adenoionivorans), 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 an initial site and a termination site of the gene, and the sequences of the obtained positive primer and the negative primer are respectively LipB-F: 5'-AACCGGTCTCCGTCAAACTC-3', respectively; LipB-R: 5'-AAGATCTCCGCGACAAGCAA-3' are provided. The position of the primers is shown in FIG. 6, the theoretical amplification length of the pair of primers is 1579bp, and the amplified sequence is SEQ ID NO. 7.

(2) The source of the strain

TABLE 4 information on the strains selected

(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: LipB-F: 5'-AACCGGTCTCCGTCAAACTC-3', respectively;

reverse primer sequence: LipB-R: 5'-AAGATCTCCGCGACAAGCAA-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. The theoretical amplification fragment of the PCR primer is 1579bp, the sequence of the PCR primer is shown as SEQ ID No.7, and the actual amplification result is consistent with the expectation. To further verify the sequence of the amplified DNA, sequencing and homologous sequence comparison were performed.

(7) For the sequencing result of the sequence with the band, firstly, the quality of a sequence peak image obtained after sequencing is checked by software Chromas, and after the quality of the peak image meets the requirement of data analysis, the forward and reverse sequences are spliced by SeqMan in a DNASTAR software package. And (3) manually proofreading and splicing the sequencing result, and if the homology of the DNA fragment of the strain to be detected and the standard gene sequence of the Arthrospora adenini TMCC 70007 is more than 97%, judging that the strain to be detected can be the Arthrospora adenini TMCC 70007 strain. For example, through DNAMAN, the sequence obtained by sequencing the strain TMCC 70007 is completely consistent with the DNA barcode sequence SEQ ID NO.1 after alignment, and the reliability of the method is further proved. The results are shown in FIG. 8 (the theoretical amplification length is 1579bp, and when sequencing is performed, about 30bp of each end of the amplification product is a low-quality sequence, and the low-quality sequence is removed when the sequences are spliced, so that the sequence shown in FIG. 8 is slightly shorter than the theoretical amplification sequence).

SEQUENCE LISTING

<110> Menghai tea industry, Limited liability company

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

<130> FI-164055-59:52/C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 766

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 1

atgattaaga cgcctttaag gtactttcga cactcctgtc ggggaatcca ggggccagta 60

ctgaggagat ttcaatcttc ctgttcaggg cccgtaattt cacctaaagt tggtgcggat 120

cagaagacgc ttcgagtatt gtatccctca ggtaaggtga cctacgaaca agcagcagaa 180

atacaggaca ggtatgtgcg gaaagcgttg gattccaaag catcaggact atccacgccc 240

cctccaacct tgttgtgttt tgaaatggac ccagtttaca ccatcggccg acgagaacga 300

ggcaagttat cagagactga gagagagcat ttgaaccaca atggcgctca cttggtggag 360

actctccgag gaggccagac tacttttcat ggtcccggac agcttgttgc atatccaatt 420

gtagacctac gagcgctgca tcttccagtt cgttgttacg tgcgaattct ggagaattcg 480

ataattaata ccctcagtaa atacggcatc tcatccaaga taacggacaa cacaggagtg 540

tggattaatg ataacgaaaa gattgctgcc attggcattc atgtgcgacg aagcgtgaca 600

tcgcatggaa tggcccttaa tgtggcaacc gacacagcat ggttcgaccg aattgttgca 660

tgcggactgc ctgacaagag tacaactaca atggccaagc aaggagttga gacctctatc 720

caagaggtgg cccaagtttt ttctgctcaa ttggcatctg cattag 766

<210> 2

<211> 822

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 2

taacccatga ttaagacgcc tttaaggtac tttcgacact cctgtcgggg aatccagggg 60

ccagtactga ggagatttca atcttcctgt tcagggcccg taatttcacc taaagttggt 120

gcggatcaga agacgcttcg agtattgtat ccctcaggta aggtgaccta cgaacaagca 180

gcagaaatac aggacaggta tgtgcggaaa gcgttggatt ccaaagcatc aggactatcc 240

acgccccctc caaccttgtt gtgttttgaa atggacccag tttacaccat cggccgacga 300

gaacgaggca agttatcaga gactgagaga gagcatttga accacaatgg cgctcacttg 360

gtggagactc tccgaggagg ccagactact tttcatggtc ccggacagct tgttgcatat 420

ccaattgtag acctacgagc gctgcatctt ccagttcgtt gttacgtgcg aattctggag 480

aattcgataa ttaataccct cagtaaatac ggcatctcat ccaagataac ggacaacaca 540

ggagtgtgga ttaatgataa cgaaaagatt gctgccattg gcattcatgt gcgacgaagc 600

gtgacatcgc atggaatggc ccttaatgtg gcaaccgaca cagcatggtt cgaccgaatt 660

gttgcatgcg gactgcctga caagagtaca actacaatgg ccaagcaagg agttgagacc 720

tctatccaag aggtggccca agttttttct gctcaattgg catctgcatt agactgcaat 780

aagatagagg agctaccgat ctcgagagac ggaattaatt ag 822

<210> 3

<211> 235

<212> PRT

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 3

Met Ile Lys Thr Pro Leu Arg Tyr Phe Arg His Ser Cys Arg Gly Ile

1 5 10 15

Gln Gly Pro Val Leu Arg Lys Phe Gln Ser Ser Cys Ser Gly Pro Val

20 25 30

Ile Ser Pro Lys Val Gly Ala Asp Gln Lys Thr Leu Arg Val Leu Tyr

35 40 45

Pro Ser Gly Lys Val Thr Tyr Glu Gln Ala Ala Glu Ile Gln Asp Arg

50 55 60

Tyr Val Arg Lys Ala Leu Asp Ser Lys Ala Ser Gly Leu Ser Thr Pro

65 70 75 80

Pro Pro Thr Leu Leu Cys Phe Glu Met Asp Pro Val Tyr Thr Ile Gly

85 90 95

Arg Arg Glu Arg Gly Lys Leu Ser Glu Thr Glu Arg Glu His Leu Asn

100 105 110

His Asn Gly Ala His Leu Val Glu Thr Leu Arg Gly Gly Gln Thr Thr

115 120 125

Phe His Gly Pro Gly Gln Leu Val Ala Tyr Pro Ile Val Asp Leu Arg

130 135 140

Ala Leu His Leu Pro Val Arg Cys Tyr Val Arg Ile Leu Glu Asn Ser

145 150 155 160

Ile Ile Asn Thr Leu Ser Lys Tyr Gly Ile Ser Ser Lys Ile Thr Asp

165 170 175

Asn Thr Gly Val Trp Ile Asn Asp Asn Glu Lys Ile Ala Ala Ile Gly

180 185 190

Ile His Val Arg Arg Ser Val Thr Ser His Gly Met Ala Leu Asn Val

195 200 205

Ala Thr Asp Thr Ala Cys Lys Glu Leu Arg Pro Leu Ser Lys Arg Trp

210 215 220

Pro Lys Phe Phe Leu Leu Asn Trp His Leu His

225 230 235

<210> 4

<211> 2817

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 4

cattagagcg ttctagagta gctgccaatg ccattactgt tcggaatgac ggtccttgca 60

ggtttacagt gaaattgtct ccaccataac ccagctcgtc tagccattca cgctgccgag 120

tactgagatc aatgtgacga gaaatgtcca ctgtgtccca tgtgttaccg gttagaatga 180

acccatactc ggtcagcaaa gtcgagttat cgtgagggcc atagcatagg tacacttctt 240

ctccttcgtc gtaagatacg gtgcttgtca gcgtcagtcc tcggtaatca atcttcactt 300

taacacttgt gtccggggaa tgatttaaaa agtctacaaa tggaaccaag gtcgcatttc 360

ctcgcacgtt catgtagata caacgagtgt tcacacagag ccaagcccat ttgaaccggt 420

ctccgtcaaa ctcggcactg ggatcctctt tagcctctag tcgtcgtaca gttctctctt 480

gctccaggaa ctttgcttcc tggtcctgta caactttttg tacactggca ggcaacccct 540

ggcgccggtc ctgtgtccat gctaaaggta ctccttctaa ctcctcgaga gtgggcaacg 600

tttcaaaaaa gcacttccat ttctcactat catgatactt gtacacatac cacgatagaa 660

gaagggttcc agaaagcatg ttctgttcgt cctgaggcgg tagccggcct tcctttcgaa 720

tggtctcatc gttcaaaagc tgacttcgac tgatatatgc aatcacctcg ccccgctcta 780

tcctatttac acacacaaca ccctttccgg ccccaggaat gtccttcaca cacaccttcg 840

ggcttatccg agctccattt tggtttaacc acgatataag gtccatctgt ctaaggtgac 900

ccgagttgaa atcggggatc acaaatctgc tgtagccgta tgtcatttca ccaaatcctc 960

cccaaacagc acgactcggg aatttcacct ttaccaataa cccatgatta agacgccttt 1020

aaggtacttt cgacactcct gtcggggaat ccaggggcca gtactgagga gatttcaatc 1080

ttcctgttca gggcccgtaa tttcacctaa agttggtgcg gatcagaaga cgcttcgagt 1140

attgtatccc tcaggtaagg tgacctacga acaagcagca gaaatacagg acaggtatgt 1200

gcggaaagcg ttggattcca aagcatcagg actatccacg ccccctccaa ccttgttgtg 1260

ttttgaaatg gacccagttt acaccatcgg ccgacgagaa cgaggcaagt tatcagagac 1320

tgagagagag catttgaacc acaatggcgc tcacttggtg gagactctcc gaggaggcca 1380

gactactttt catggtcccg gacagcttgt tgcatatcca attgtagacc tacgagcgct 1440

gcatcttcca gttcgttgtt acgtgcgaat tctggagaat tcgataatta ataccctcag 1500

taaatacggc atctcatcca agataacgga caacacagga gtgtggatta atgataacga 1560

aaagattgct gccattggca ttcatgtgcg acgaagcgtg acatcgcatg gaatggccct 1620

taatgtggca accgacacag catggttcga ccgaattgtt gcatgcggac tgcctgacaa 1680

gagtacaact acaatggcca agcaaggagt tgagacctct atccaagagg tggcccaagt 1740

tttttctgct caattggcat ctgcattaga ctgcaataag atagaggagc taccgatctc 1800

gagagacgga attaattagc gtcatagccc caattcccag tagaaaaaca cctagcgccg 1860

acgtaatcgt gatagccttg gctgctctgg tggaaatgtt attctttgca tgtgtgtgag 1920

tggtagttgt agtcgtcgaa gtagtagtcg gagcgtcgtt agaattagca tcttgcttgt 1980

cgcggagatc ttggacctgg tccttgatct tgggttcaac ttgtgtgccc ttttcctcta 2040

gtgtcctaac cagttcagca attgtctttg agctattaag agccagttcc tcagccgcca 2100

gagctcggct cttgaataac tcaagttggc atttgacttc ggctagctga gtgctggagt 2160

ccatagagtc attaacgttg ctgtggtctt tattatccac ggagccgttg gtcagatctc 2220

tcaagacggc ttcggcatga gaagaagatg ttgatgatga tgtaggagaa ggcaatgagt 2280

tgatggagtc tcgcttagaa acgggagaag atggctcttt ggaccgatgt gcttcaagct 2340

gttccttctg ttccttaacc agttgttcta attcggaaat gcgagcatct ttttcagcga 2400

gcttttgagc tagaatatga gcagggtcga ttttggcgct ctgtttcttt tgaattccct 2460

gcagtagagt tacaatagag ttgatcttag ctccttcaac gcgggcagag tgaatctgac 2520

taaccagctg tctaactgcg ttttcaaatg caactgatgt ggctccagag gccatgaaca 2580

taccaccgag cgaatttcta gcatgtccca agatgagatc ttccagcgat gcgtctacat 2640

cgccaaacaa agcgatatcc aaagcatccg accgattctg agcacctctc ttccgtagac 2700

caccgggtcc agaaggacca agcgggcctg tcgaagaaga aggggtcact gatgccccgc 2760

cattggtgcc attcacagga ccattagcag ccagagacat taaagatatc ctttcta 2817

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

aaccggtctc cgtcaaactc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

aagatctccg cgacaagcaa 20

<210> 7

<211> 1539

<212> DNA

<213> Arthrospora adenantha (Blastobotrys adeninivorans)

<400> 7

aaccggtctc cgtcaaactc ggcactggga tcctctttag cctctagtcg tcgtacagtt 60

ctctcttgct ccaggaactt tgcttcctgg tcctgtacaa ctttttgtac actggcaggc 120

aacccctggc gccggtcctg tgtccatgct aaaggtactc cttctaactc ctcgagagtg 180

ggcaacgttt caaaaaagca cttccatttc tcactatcat gatacttgta cacataccac 240

gatagaagaa gggttccaga aagcatgttc tgttcgtcct gaggcggtag ccggccttcc 300

tttcgaatgg tctcatcgtt caaaagctga cttcgactga tatatgcaat cacctcgccc 360

cgctctatcc tatttacaca cacaacaccc tttccggccc caggaatgtc cttcacacac 420

accttcgggc ttatccgagc tccattttgg tttaaccacg atataaggtc catctgtcta 480

aggtgacccg agttgaaatc ggggatcaca aatctgctgt agccgtatgt catttcacca 540

aatcctcccc aaacagcacg actcgggaat ttcaccttta ccaataaccc atgattaaga 600

cgcctttaag gtactttcga cactcctgtc ggggaatcca ggggccagta ctgaggagat 660

ttcaatcttc ctgttcaggg cccgtaattt cacctaaagt tggtgcggat cagaagacgc 720

ttcgagtatt gtatccctca ggtaaggtga cctacgaaca agcagcagaa atacaggaca 780

ggtatgtgcg gaaagcgttg gattccaaag catcaggact atccacgccc cctccaacct 840

tgttgtgttt tgaaatggac ccagtttaca ccatcggccg acgagaacga ggcaagttat 900

cagagactga gagagagcat ttgaaccaca atggcgctca cttggtggag actctccgag 960

gaggccagac tacttttcat ggtcccggac agcttgttgc atatccaatt gtagacctac 1020

gagcgctgca tcttccagtt cgttgttacg tgcgaattct ggagaattcg ataattaata 1080

ccctcagtaa atacggcatc tcatccaaga taacggacaa cacaggagtg tggattaatg 1140

ataacgaaaa gattgctgcc attggcattc atgtgcgacg aagcgtgaca tcgcatggaa 1200

tggcccttaa tgtggcaacc gacacagcat ggttcgaccg aattgttgca tgcggactgc 1260

ctgacaagag tacaactaca atggccaagc aaggagttga gacctctatc caagaggtgg 1320

cccaagtttt ttctgctcaa ttggcatctg cattagactg caataagata gaggagctac 1380

cgatctcgag agacggaatt aattagcgtc atagccccaa ttcccagtag aaaaacacct 1440

agcgccgacg taatcgtgat agccttggct gctctggtgg aaatgttatt ctttgcatgt 1500

gtgtgagtgg tagttgtagt cgtcgaagta gtagtcggag cgtcgttaga attagcatct 1560

tgcttgtcgc ggagatctt 1579

Claims

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'-AACCGGTCTCCGTCAAACTC-3', respectively;

reverse primer: 5'-AAGATCTCCGCGACAAGCAA-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 in SEQ ID No.4 and comprises the sequence shown in 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;

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 if the homology is more than 97%, judging that the strain to be detected is the Arthrospora adenine-eating yeast 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.