CN109402278B - DNA bar code, primer, kit, method and application - Google Patents
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Abstract
The invention belongs to the field of species and strain identification, and particularly relates to a DNA bar code, a primer, a kit, a method and application for identifying a desmospora adephagi strain. The DNA bar code can accurately identify the Pu' er tea fermentation strain, namely, the adenine arthrobotrys yeast TMCC70007, and can quickly and accurately identify the strain from confusable strains or other strains in the same species.
Description
Technical Field
The invention belongs to the field of species and strain identification, and particularly relates to a DNA bar code, a primer, a kit, a method and application.
Background
Pu' er tea is post-fermented tea with geographical identification of Yunnan, which is prepared by a series of processes by adopting big-leaf sun-dried raw tea as a raw material. The traditional Pu' er tea preparation process comprises the following steps: the picked fresh tea leaves are rolled, dried in the sun, purified, moistened, piled, dried in the air, screened, pressed and formed, and packaged for delivery. In the production of Pu ' er tea, the pile fermentation process is a main factor for the formation of the Pu ' er tea, and in the process, tea polyphenol, caffeine, polysaccharide substances and other contained components in the tea are greatly changed, so that the special flavor, taste, quality and various health-care effects of the Pu ' er tea are achieved.
In the traditional Pu 'er tea production, enzymes contained in tea leaves are activated by a damp and hot environment, a part of contained components in the tea leaves are converted into substances which can be utilized by microorganisms, the microorganisms are bred in a large quantity to generate abundant intracellular enzymes and extracellular enzymes, the components contained in the tea leaves are catalyzed to generate a series of conversion, and the Pu' er tea with different qualities is gradually formed by adding other factors such as damp and hot. Different origins, because of the different microbial community structures, have different flavors and qualities.
Besides the unique flavor and culture of Pu 'er tea, the Pu' er tea has the health care effects of losing weight, reducing blood sugar and blood fat, preventing and improving cardiovascular diseases, resisting aging, resisting cancer, diminishing inflammation, helping digestion, nourishing the stomach and the like and is also concerned by people. In recent years, Pu 'er tea is more and more popular, and the development of the Pu' er tea industry is pulled by the increasing market demand, so that the economic growth of Yunnan areas is promoted.
However, Pu' er tea also faces some dilemmas, such as unstable product quality, certain potential safety hazard, long production period, too high labor input, excessive microorganism quantity, mite breeding and the like. With the improvement of living standard of people, consumers pay more and more attention to the problems of food such as sanitation, safety and the like. In recent years, food quality safety events are frequent, the safety problem of tea quality is increasingly highlighted, and besides the problem of pesticide residues, microorganisms in the pile fermentation process can also become important factors influencing the tea quality.
At present, the production of Pu' er tea by most manufacturers is still the empirical fermentation of semi-natural artificial pile fermentation, although communities mainly comprising dominant common microorganisms such as adenine-nodulation yeast (Saccharomyces adeninivorans) and the like in the pile fermentation process are relatively stable, the stability of the product still has a larger space for improving. Certain potential safety hazards inevitably exist in the production process, and in order to further obtain the favor of consumers and the acceptance of the market, break through the foreign trade barrier and improve the market competitiveness of Pu 'er tea enterprises, the manual control, cleaning and high efficiency of the Pu' er tea production process must be realized, new products are not developed, and the industrial chain is extended. In order to achieve the purpose, technology must be innovated, a series of safe, clean, efficient, artificial, controllable and automatic Pu 'er tea new processes are invented, the healthy development of the Pu' er tea industry can be ensured, and long-term benefits are brought to the nation and people.
With the further development of Pu ' er tea scientific research, more and more people begin to explore the artificial inoculation and fermentation of Pu ' er tea, and at present, a plurality of bacterial strains are applied to the artificial controllable fermentation of Pu ' er tea. In recent years, due to the increase of market demands, a plurality of small-scale manufacturers lack systematic and deep research on Pu 'er tea, and greatly abuse a plurality of other people's patents to profit, for example, according to a plurality of patent methods for inoculating and fermenting Pu 'er tea, a plurality of strains are adopted to ferment Pu' er tea, and a series of methods such as Pu 'er tea processing and production are carried out, so that the economic benefits of Pu' er tea production enterprises with related patents are greatly damaged.
In order to ensure that the germplasm resources of Pu ' er tea fermentation microorganisms are effectively protected, prevent the infringement behavior of abusing the Pu ' er tea fermentation microorganisms and solve the difficult problem of difficult demonstration in the process of artificially controllable fermentation of Pu ' er tea during the infringement of strains, the development of a method for quickly and accurately identifying the Pu ' er tea fermentation strains is imperative, a molecular identification method of the Pu ' er tea fermentation strains needs to be urgently established, a method for accurately identifying and distinguishing the similar strains by a DNA bar code technology is developed, so that the problems of identification and identification of industrial production strains are solved by a method combining morphological characteristics and molecular data analysis, a theoretical basis is provided for the development of the controlled industrial fermentation process of Pu ' er tea and resource protection, and the healthy and prosperous development of the Pu ' er tea industry is promoted.
Disclosure of Invention
In order to overcome the defects of morphological identification of the alternaria adegua strain for fermentation production of Pu ' er tea, the invention provides a DNA bar code, a primer, a kit, a method and application for identifying the alternaria adegua strain, so that the alternaria adegua strain TMCC70007 strain is quickly identified and distinguished, quick evaluation can be provided for a new fermentation process of the Pu ' er tea, interference of other mixed bacteria in the fermentation process can be prevented, and a proof-lifting method and basis can be provided for an artificial controllable fermentation process of the Pu ' er tea and illegal abuse of the strain.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) a DNA barcode for identifying a desmodium adenanthus yeast strain, the DNA barcode being derived from the genome of the desmodium adenanthus TMCC70007 strain and comprising at least 500bp of a sequence selected from the DNA sequences shown as SEQ ID No.1, and the length of the DNA barcode being 500bp to 3500bp, preferably 500bp to 2200bp, more preferably 500bp to 1500 bp.
(2) The DNA barcode according to (1), wherein the nucleotide sequence thereof is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 7.
(3) A primer pair for amplifying the DNA barcode according to (1).
(4) The primer set according to (3), wherein the nucleotide sequence of the forward primer is identical to such a sequence in the genome of the A.adenine-nodorum TMCC70007 strain: the sequence is a sequence from 1000bp upstream of the 1 st site of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC70007 strain to a 535 nd site of the nucleotide sequence shown as SEQ ID No.1, and the length of the forward primer is 20-30 bp; the reverse primer of the strain is reversely complementary to a sequence in the genome of the TMCC70007 strain: the sequence is a sequence in a region from the 501 st bit of the nucleotide sequence shown as SEQ ID No.1 to the downstream 1000bp of the last bit of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC70007 strain, and the length of the reverse primer is 20-30 bp.
(5) The primer pair according to (4), wherein the nucleotide sequences of the forward primer and the reverse primer are respectively as follows:
a forward primer: 5'-CGTAGCAGCTTGTCCGGTAT-3', respectively;
reverse primer: 5'-CTTGCCGTTTGTGTTCTGGG-3' are provided.
(6) A kit for identifying a strain of Arthrospora adenantha comprising the primer pair according to (3).
(7) A method for identifying a strain of alternaria adefovea comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA in the step a) as a template and using the primer pair in the step (3) to obtain a PCR product;
c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC70007 strain, and if the target band exists, performing the step d);
d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code of claim 1, and if the homology is more than 97%, judging that the strain to be detected is the adenine arthrobacter adenine-burning TMCC70007 strain.
(8) Use of the DNA barcode according to (1) for identifying a strain of Alternaria adefovea.
(9) The application of the primer pair in (3) in identifying the adenine nodospora spaying yeast strain.
(10) The application of the kit in (6) in identifying the adenine nodospora spacicola strains.
Compared with the prior art, the invention has the following advantages and positive effects:
1. the invention adopts a protein genomics technology to discover a leakage injection coding protein from the genome of the Arthrospora adenantha TMCC70007 strain, and discovers that the open reading frame (SEQ ID No.1) of the gene coding the protein is unique to the genome of the Arthrospora adenantha TMCC70007 strain. The invention further develops the specific DNA sequence into a DNA bar code through careful research and comparative analysis, and the DNA bar code is used as an effective tool for identifying the strain Alternaria adenini TMCC70007 produced by the industrial fermentation of the Pu' er tea. The bar code sequence can realize the rapid and accurate identification and differentiation of the strains in the adenine node spore feeding yeast.
2. Compared with the prior art, the specificity of the DNA bar code discovered by adopting the protein genomics technology is better.
3. The invention further discovers that compared with other genes, the sequence (such as SEQ ID No.1) in the gene for missed injection has the characteristics of universality, easy amplification and easy comparison, and the difference of the sequence among different strains in the B.adeninivorans strain of the adenine node B is obvious.
4. The invention establishes a standard gene sequence and a sample identification method of the Puer tea industrial fermentation production strain Arthrospora adenini TMCC70007, and compared with the traditional morphological identification method, the identification efficiency is obviously improved. The method has low requirement on the integrity of the sample, and the identification index can be quantized, which provides effective basis for timely judging Pu' er tea and germplasm resources thereof. In addition, morphological confusing species are further added, and an identification rule can be established for identification by using a phylogenetic tree method based on cluster analysis, so that the reliability and accuracy of the identification are greatly superior to those of the conventional molecular identification method, and the blank of the strain identification of the Pu' er fermented tea production strain based on the DNA barcode technology is filled.
Drawings
Fig. 1 shows the mass spectrum of newly identified peptide fragment LFEPIEPSHR.
FIG. 2 shows a comparison of the mass spectrum of the as-identified peptide fragment with the mass spectrum of the synthetic peptide fragment of chemically synthesized peptide fragment LFEPIEPSHR; the original identified peptide segment is obtained by mass spectrometry and identification; the upper part of the figure is a mass spectrogram of the original identified peptide fragment, and the lower part is a mass spectrogram of the synthesized peptide fragment.
FIG. 3 shows a map of the mRNA sequence of the protein coding frame and its encoded protein sequence of the region in which the peptide fragment is located, the grey background part being the new peptide fragment identified.
Figure 4 shows a corresponding plot of the transcribed mRNA sequence encoding the newly identified RRI1 protein gene and its encoded protein sequence, with the new peptide fragment identified in the grey background section.
FIG. 5A shows a SDS-PAGE separation profile of the cellular holoprotein of TMCC70007, the band shown in bold is the location of the RRI1 protein; fig. 5B shows a molecular weight validation graph of RRI1 protein. In FIG. 5B, the abscissa represents the logarithmic value of the protein molecular weight marker with a base of 10 for different molecular weights, and the ordinate represents the ratio of the migration amount of each molecular weight protein of the protein molecular weight marker in SDS-PAGE electrophoretic separation to the total migration amount of SDS-PAGE. Theoretically, the molecular weight of HSP40 protein MW 39.09kDa, lg (MW) lg (39.09) approximately equals 1.59, and the molecular logarithm of HSP40 protein corresponds to the abscissa 1.58 of fig. 5B, and its mobility corresponds to the value on the y-axis when the mobility curve is x 1.59.
FIG. 6 shows an alignment of the barcode sequence SEQ ID NO.1 with homology thereto by NCBI-BLASTN, the homology sequence being shown in the lower gray bar of FIG. 6.
FIG. 7 shows a comparison of the bar code sequence SEQ ID No.1 of the C.adenine-nodorum barcode sequence with the homologous sequence of the A.adenine-nodorum LS3 strain.
FIG. 8 shows the positions of primers used to amplify SEQ ID NO.1, the underlined regions in the sequence being the regions where the primers are located, the bold font ATG and TAA being the start and stop sites, and the amplified sequence being SEQ ID NO.7, in one embodiment of the invention.
FIG. 9 shows the results of agarose gel electrophoresis of PCR amplification products of test strains obtained using the primers of the present invention.
FIG. 10 shows a comparison of the sequence of SEQ ID NO.1 with the PCR amplified sequence of the test strain.
Detailed Description
The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.
The invention adopts the protein genomics technology to discover the leakage injection release from the genome of the adenine arthrospora cereus TMCC70007 strainDue to its interaction with yeast (Sugiyaella lignohabinans) CBS10342TThe Rri1p protein of the strain was 55% similar and was therefore designated Rri1 protein. The invention obtains the corresponding gene sequence in the TMCC70007 strain genome from the newly found protein sequence, the gene sequence is called RRI1 gene, and the gene sequence (SEQ ID No.1) of the gene for coding the protein is found to be unique to the genome of the Alternaria adephaga TMCC70007 strain. The special DNA sequence can be used for developing and identifying DNA bar codes of the strain Alternaria adefovea TMCC70007 produced by industrial fermentation of Pu' er tea. Compared with the prior art, the DNA barcode obtained by the method has higher specificity.
Considering that the DNA barcode should have a suitable length and sufficient specificity among strains, the present invention further obtains a DNA barcode that can accurately and efficiently identify the strain of Arthrospora adenine-feeding TMCC70007 based on the above-mentioned characteristic DNA sequence through careful study and comparative analysis.
Thus, in one aspect the present invention provides a DNA barcode for identifying a strain of arthrobacter adenivorus, the DNA barcode being derived from the genome of the strain of arthrobacter adenivorus TMCC70007 and comprising at least 500bp of a sequence selected from the DNA sequences shown as SEQ ID No.1, and the DNA barcode having a length of 500bp to 3500bp, preferably 500bp to 2200bp, more preferably 500bp to 1500 bp.
The bar code sequence of the invention can realize the rapid and accurate identification and differentiation of the strains in the adenine-feeding node spore yeast.
The length of SEQ ID No.1 is 1035bp, and the sequence itself can be used as a DNA bar code for identifying the strain of the nodospora adenine-eating yeast TMCC70007 because the SEQ ID No.1 is specific to the genome of the strain of the nodospora adenine-eating yeast TMCC 70007. In addition, theoretical analysis and experimental verification prove that the specificity of the DNA barcode containing the sequence and having longer length is better ensured. When the length of the DNA barcode is too long (e.g., greater than 3500bp), it is less desirable for the amplification procedure. On the other hand, when the length of the DNA bar code is not less than 500bp, the operation requirements of easy amplification and easy comparison can be met; theoretical analysis and experimental verification prove that at least 500bp sequence selected from the DNA sequence shown as SEQ ID No.1 can realize the rapid and accurate identification and differentiation of the strains in the adenine-node-adenine-B-yeast.
In a preferred embodiment of the invention, the DNA barcode sequence is as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 7.
In this context, the term "missing-release gene" means that after the species has completed the genome sequencing, the gene cannot be predicted by using a gene prediction software (e.g., GeneMark, Augustus, Glimer, etc.), and the gene is generally expressed in a low amount under specific conditions and thus is difficult to be found in the study.
The term "DNA barcoding" refers to a new molecular identification technique for identifying species using a standard, short DNA fragment in the genome, which allows rapid and accurate species identification.
The term "six-frame translation" is a known term in proteomics and genomics, and is briefly described on the principle that when a DNA encodes a protein, there are 3 coding possibilities given to a DNA sequence, using triplet codons to encode the protein, plus 3 coding possibilities on its complementary strand, for a total of 6 coding possibilities (+1, +2, +3, -3, -2, -1).
Another embodiment of the present invention provides a primer pair for amplifying the DNA barcode according to the present invention.
Preferably, the nucleotide sequence of its forward primer is identical to such a sequence in the genome of the strain Arthrospora adevorans TMCC 70007: the sequence is a sequence from 1000bp upstream of the 1 st site of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC70007 strain to a 535 nd site of the nucleotide sequence shown as SEQ ID No.1, and the length of the forward primer is 20-30 bp; the reverse primer of the strain is reversely complementary to a sequence in the genome of the TMCC70007 strain: the sequence is a sequence in a region from the 501 st bit of the nucleotide sequence shown as SEQ ID No.1 to the downstream 1000bp of the last bit of the nucleotide sequence shown as SEQ ID No.1 in the genome of the TMCC70007 strain, and the length of the reverse primer is 20-30 bp.
In a more preferred embodiment, the nucleotide sequences of the forward primer and the reverse primer are as follows:
forward primer RRI 1-F: 5'-CGTAGCAGCTTGTCCGGTAT-3' (SEQ ID No. 5);
reverse primer RRI 1-R: 5'-CTTGCCGTTTGTGTTCTGGG-3' (SEQ ID No. 6).
The primers of the invention enable specific amplification of the DNA barcode sequence.
The invention also provides a kit for identifying the adenine node B yeast strain, which comprises the primer pair.
In another embodiment, the kit further comprises a DNA barcode according to the present invention. The DNA barcode may be present on a recording medium. The recording medium is, for example, an optical disc. The kit may also comprise any tools and reagents for the experimental procedure.
In yet another aspect of the invention, a method for identifying a strain of nodospora adenine dinucleotide is provided, comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA of the step a) as a template and using the primer pair of claim 3 to obtain a PCR product;
c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC70007 strain, and if the target band exists, performing the step d);
d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA barcode according to claim 1, and if the homology is more than 97% (preferably more than 99%), judging that the strain to be detected is the adenine nodospora glaucomatosa TMCC70007 strain.
In a specific embodiment of the present invention, the procedure of PCR amplification is: 1) pre-denaturation at 94-96 deg.C for 8 min; 2) denaturation at 94-96 ℃ for 45 seconds, annealing at 55-57 ℃ for 45 seconds, and extension at 72 ℃ for 1 min 15 seconds, wherein the procedure 2) is performed for 30-35 cycles; 3) extension at 72 ℃ for 10 min.
In another embodiment of the present invention, the method further comprises performing cluster analysis (e.g., phylogenetic tree) on the sequence to be tested obtained from the sequencing result and the DNA barcode of the present invention, and if the sequence to be tested and the DNA barcode are clustered together, determining that the strain to be tested is the alternaria adenini yeast strain TMCC 70007.
In a specific embodiment of the invention, genomic DNA extracted from the strain to be identified is subjected to PCR amplification using the primer pairs of the invention, followed by detection by agarose gel electrophoresis. Identifying the strains based on detecting the presence or absence of PCR products: if the strain to be identified does not amplify a corresponding target band, the strain is not TMCC 70007; if the corresponding target band is amplified, the strain is proved to be possible TMCC 70007. For further identification, the PCR product is sequenced, the DNA sequencing result and the DNA bar code sequence are subjected to homology comparison to obtain the similarity (namely homology) between the sequences, and if the sequence homology is less than 97%, the strain to be detected is judged not to be the adenine node B spore forming yeast strain TMCC 70007. If the sequence homology is greater than or equal to 97% (preferably 99% or more), the test strain is judged to be the Arthrospora adenantha strain TMCC 70007.
If a clustering analysis is performed, such as a phylogenetic tree, the DNA barcode is used with the DNA sequencing results (i.e., the sequences to be tested) of each strain to be identified to construct an NJ phylogenetic tree using MEGA 6 or PAUP software. If the sequence to be tested of the strain to be identified is clustered with the DNA bar code of the Arthrospora adenini TMCC70007, the strain is identified as the Arthrospora adenini TMCC 70007.
The term "cluster" as used herein means that the branches are located at the same branch and have the same evolutionary distance after phylogenetic tree analysis.
The invention also provides application of the DNA bar code in identifying the alternaria adephaga yeast strain.
The invention also provides application of the primer pair in identifying the adenine node B saccharomycetes strains.
Examples
The present invention is further illustrated by the following specific examples. The methods used in the examples, unless otherwise specified, were carried out using conventional methods and known tools.
Example 1: acquisition of RRI1 Gene and DNA barcodes
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 TMCC70007, and the genome is verified by annotating coding genes. Specifically, in order to find a novel protein coding region, a Six-Frame Translation database of the genome data of the Geobacillus adenini TMCC70007 was obtained using a Six-Frame Translation (Six Frame Translation) strategy in systematic proteomics, exhaustively listing 6 coding possibilities of the genome (+1, +2, +3, -1, -2, -3), and the protein sequence was referred to as a "Six-Frame Translation protein sequence", and the corresponding nucleic acid sequence was referred to as a "Six-Frame Translation nucleic acid sequence". Generally, a six-frame translational nucleic acid sequence is a sequence from one terminator to the next, which is also referred to herein as a "protein coding frame". The discovery and identification of new peptide fragments and new proteins was performed using this six-box translation database, using pFind and pAnno software to compare with whole protein mass spectral data of TMCC70007 strain.
Through identification, a peptide segment LFEPIEPSHR which is not found in the adenine node B.adenine-eating yeast TMCC70007 annotated genome in the prior art is found, and the mass spectrum of the peptide segment is shown in figure 1.
The results of manual inspection of mass spectrum spectrogram show that almost all y ion sequences of the secondary mass spectrum spectrogram (MS2) of the peptide segment LFEPIEPSHR are detected, the matching is good, the signal is strong, and the result is more reliable. To further confirm this identification, the peptide fragment was chemically synthesized according to the amino acid sequence of newly identified peptide fragment LFEPIEPSHR, and a secondary spectrum of the synthesized peptide fragment was generated using the mass spectrometry conditions of LFEPIEPSHR as described above, see fig. 2.
Next, the high energy collision MS2 generated by the synthesized peptide fragment was verified, and both the primary parent ion and the secondary daughter ion were in agreement with the theoretical values, indicating that the sequence of the synthesized peptide fragment was correct (fig. 2). On the basis, the MS2 of the peptide segment synthesized by the new peptide segment sequence identified according to the large-scale proteome data and the spectrogram of the large-scale identified new peptide segment are manually checked, the two are almost completely consistent, and the Cosin value obtained by the similarity of the daughter ions is 0.82, thereby proving that the new peptide segment identified from the Pu' er tea industrial fermentation bacteria of the adenine arthrobacter adefovea TMCC70007 is correct.
2. The six-frame translated nucleic acid sequence (protein coding frame) SEQ ID NO.2 is obtained by defining the region between the former stop codon and the latter stop codon, depending on the position of the novel peptide fragment. The mRNA corresponding to the sequence of SEQ ID NO.2 and the amino acid sequence encoded by the mRNA are shown in FIG. 3.
3. In order to further determine the coding start site and the coding end site of the gene for coding the protein, the coding frame of the protein is respectively expanded by 1000bp upstream and downstream, GeneMark.hmm is adopted for gene prediction, and Schizosaccharomyces pombe (Schizosaccharomyces pombe) is selected as a reference species. The existence of the gene is predicted in the region, the coding frame of the gene is consistent with the coding frame sequence of the protein, the coding start and termination sites of the gene are determined, and the ORF sequence of the gene is SEQ ID NO. 1.
The correspondence between the mRNA sequence transcribed from the gene and the amino acid sequence of the protein translated from the mRNA sequence is shown in FIG. 4. Peptide segment LFEPIEPSHR is located downstream of the gene.
The nucleotide sequence of the gene totally 1035bp, totally 344 amino acids and the theoretical molecular weight of 39.09kDa, and the theoretically encoded amino acid sequence is shown in SEQ ID NO. 3.
4. To further confirm the correctness of the identified sequences, TMCC70007 strain was cultured in YPD medium and total protein was extracted therefrom, separated by SDS-PAGE, stained with Coomassie Brilliant blue on electrophoresis gel, and the molecular weight was verified based on the molecular weight characteristics at the time of preparation of the proteome sample, as shown in FIG. 5. In addition, 25 strips were cut together per band for in-gel digestion and mass spectrometry analysis, where the RRI1 protein was identified in the mass spectrometry data of the 15 th strip, matching the predicted molecular weight size. The theoretical molecular weight calculated based on the amino acid sequence was 39.09kDa, which was consistent with the position of the gel strip to which the protein belongs in SDS-PAGE. The results show that the experimental molecular weight of the identified RRI1 is about 36.5-39.0kDa, which is substantially consistent with the theoretical molecular weight, confirming the correctness of the identified protein.
5. The amino acid sequence of the theoretical coding product of the gene is used for performing blastp analysis, the sequence has a similar structural domain of an MPN super family, and the Mov34/MPN/PAD-1 protein super family mainly comprises a proteasome regulatory protein Rpn11 signal corpuscle complex CSN5 subunit. The identified RRI1 protein sequence was compared to yeast (Sugiyamaella lignohabinans) CBS10342TThe Rri1p protein (accession number ANB11586.1) similarity of the strain was 55%, and the sequence coverage was 97%. The above blastp alignment results are summarized in table 1.
The blastp results indicated that the detected leaky-expulsive gene product was RRI1, which has a sequence with more homologous sequences. The similarity with the homologous sequence of the previous 50 proteins is basically between 50 and 62 percent, so that the RRI1 protein sequence has enough specificity.
6. The ORF sequence of the identified RRI1 gene (i.e., SEQ ID NO.1) was subjected to NCBI-BLASTN analysis, the results of which are shown in FIG. 6. The BLASTN results are summarized in table 2. The results showed that SEQ ID NO.1 has no sequence with higher homology in NCBI database, only 40 bases (only 3% of the total length of SEQ ID NO.1 sequence) at position 380-420 among 1035 bases are conserved, and the sequence has homology with a small part of genome sequence (sequence accession number: LK978268, site: 447 214214487) of Meloidogyne incognita (Dracuculus medinensis). This shows that the leaky-expulsive gene RRI1 detected in Arthrospora adenantha has better sequence specificity compared with the existing sequence in NCBI nr database, and although the amino acid sequence segment of RRI1 protein is conservative, its DNA homology is very low. The result proves that the ORF sequence of the gene can be used for developing the DNA bar code of the Puer tea fermentation strain Alternaria adefovea TMCC 70007.
TABLE 2 list of nucleic acid sequences with higher similarity to SEQ ID NO.1
7. The region from 1000bp upstream to 1000bp downstream of SEQ ID No.1 was further selected (as shown in SEQ ID No. 4). The sequence of SEQ ID NO.4 was subjected to NCBI-BLASTN analysis, and the Nucleotide collection (nr/nt) database was searched, and as a result, it was found that only 1% (about 40bp) of the sequence had 90% homology with the genomic sequence of C.mobaraensis. The alignment results are summarized in table 3. From the above results, it can be considered that SEQ ID NO.4 can serve as a DNA barcode sequence for identifying a strain of Alternaria adephaga strain TMCC 70007. Furthermore, the sequence of SEQ ID NO.4 was further subjected to NCBI-BLASTN analysis to retrieve the Whole genome shotgun sequencing sequence of Arthrospora adephaga (taxonomic ID: taxi: 409370) in the white-genome shotgun contigs (wgs) database. As a result, it was found that SEQ ID NO.4 has 89% homology with the entire gene sequence Arad1B chromosome sequence contig1 of A.adenini LS3 and the sequence coverage is 81%, which indicates that TMCC70007 and LS3 strains have sufficient discrimination in this region to distinguish TMCC70007 from LS3 strains, although they are the same species. The alignment results are summarized in table 4. Therefore, the Arthrospora adenantha TMCC70007 SEQ ID NO.4 has sufficient discrimination with the homologous sequence in the genome of Arthrospora adenantha 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
8. Currently, of the species Arthrospora adenantha, only the genome of the strain Arthrospora adenantha LS3 has been reported (Kunze et al Biotechnology for Biofuels2014,7: 66). After analysis by Local-BLASTN, the gene sequence SEQ ID NO.1 was found to have a homologous sequence in the genome of LS3 strain. After DNAMAN analysis, the homology of the two sequences was 93.82%, although TMCC70007 and LS3 strains were of the same species (instant Arthrospora adenine, Blastotrys adenonivorans), 63 sites were different in 1035 bases of the DNA barcode sequence, and the results are shown in FIG. 7. This further demonstrates that the use of this DNA barcode sequence can effectively distinguish TMCC70007 strain from other strains within the species.
Example 2 identification of strains Using DNA barcodes
And judging whether the sample to be detected is the bacterial strain TMCC70007 applied to the Pu' er tea industry or not according to the amplification result of the sample to be detected and the sequence homology of the bacterial strain SEQ ID NO.1 of the bacterial strain TMCC70007 of the Arthrospora adenini.
(1) Based on SEQ ID NO.1, PCR primers are designed at two ends of the gene by adopting an NCBI primer design tool, an amplification product needs to comprise an initial site and a termination site of the gene, and the sequences of the obtained positive primer and negative primer are respectively RRI 1-F: 5'-CGTAGCAGCTTGTCCGGTAT-3', respectively; RRI 1-R: 5'-CTTGCCGTTTGTGTTCTGGG-3' are provided. The position of the primers is shown in FIG. 8. The amplified sequence is SEQ ID NO. 7.
(2) The source of the strain
TABLE 5 information on selected related strains
| Sample numbering | Name of scholars | | Habitat | |
| 1 | | TMCC | 70007 | Pu' er tea pile fermentation, China |
| 2 | | CBS | 8244T | Soil, the Netherlands |
| 3 | | CBS | 7350 | Corn fodder stored in cellar, Holland |
| 4 | | CBS | 7370 | Soil, south Africa |
| 5 | | CBS | 8335 | Clay, Italy |
| 6 | Blastobotrys raffinosifermentans | CBS 6800T | Is unknown |
(3) Respectively extracting strain DNA: OMEGA e.z.n.a was used.TMYeast genomic DNA was extracted using the Yeast DNA kit (Bio Rad laboratories) and the DNA concentration of the sample was diluted to 0.5. mu.g/. mu.L with sterile deionized water.
(4) Amplifying DNA fragments, and carrying out Polymerase Chain Reaction (PCR), wherein the sequences of the primers are respectively as follows:
forward primer sequence: RRI 1-F: 5'-CGTAGCAGCTTGTCCGGTAT-3', respectively;
reverse primer sequence: RRI 1-R: 5'-CTTGCCGTTTGTGTTCTGGG-3' are provided.
The PCR reaction system is 50 mu L, and the PCR reagent is Thermo ScientificTMTaq DNA Polymerase (recombinant) of (1): ddH2O 37.7μL、MgCl2mu.L, dNTPs 4. mu.L, forward primer 1. mu.L, reverse primer 1. mu.L, Taq DNA polymerase 0.3. mu. L, DNA template 1. mu.L, without dye. The amplification procedure was: pre-denaturation at 94 ℃ for 8 min; then denaturation at 94 ℃ for 45 seconds, annealing at 56 ℃ for 45 seconds, and extension at 72 ℃ for 1 minute and 15 seconds are carried out for 32-35 cycles in total; final extension at 72 ℃ for 10 min.
(5) Detection of amplification products: the PCR fragment size was detected by electrophoresis on 1.0% agarose gel, 1 XTBE, and DNA marker. If the strain to be detected has no amplified band, the strain is not the adenine node B spore feeding yeast TMCC 70007; if a clear band appears and no miscellaneous band exists, the DNA fragment is sent to a biological sequencing company for DNA fragment sequencing.
(6) The primer can only realize amplification in the Arthrospora adenantha TMCC70007, and the Arthrospora adenantha CBS 8244 of the same speciesTCBS 7350, CBS 7370, CBS 8335 and Blastotrys raffinositifications CBS 6800TAmplification cannot be achieved. The results are shown in FIG. 9. The theoretical amplification fragment of the PCR primer is 1485bp, and the actual amplification result is consistent with the expectation. To further verify the sequence of the amplified DNA, sequencing was performedAnd comparing with homologous sequences.
(7) For the sequencing result of the sequence with the band, firstly, the quality of a sequence peak image obtained after sequencing is checked by software Chromas, and after the quality of the peak image meets the requirement of data analysis, the forward and reverse sequences are spliced by SeqMan in a DNASTAR software package. And (3) manually proofreading and splicing the sequencing result, and if the homology of the DNA fragment of the strain to be detected and the standard gene sequence of the Arthrospora adenini TMCC70007 is more than 97%, judging that the strain to be detected can be the Arthrospora adenini TMCC70007 strain. For example, through DNAMAN, the sequence obtained by sequencing the strain TMCC70007 is completely consistent with the DNA barcode sequence SEQ ID NO.1 after alignment, and the reliability of the method is further proved. The results are shown in FIG. 10.
SEQUENCE LISTING
<110> Menghai tea industry, Limited liability company
<120> DNA barcodes, primers, kits, methods and uses
<130> FI-163716-59:52/C
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1035
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 1
atggctcaga aggcgtttga actagagaat gacataaagc tcgtagggga ggatgacaag 60
ttatactacc agtataataa ggaggagcag cagaagctgt tacgagagcg gccctggaag 120
gacgatcctc actatttcaa gcgagtgagg atatcagcta ttgccctgct gcgaatggcc 180
attcatgccc gtgcgggagg atcaattgaa gtgatgggag tgatgactgg caagattctg 240
ccaaacgagt ttgttatcat ggatacgtat cccttgccag tggaaggtac cgaaactaga 300
gtgaacgcac tgggagaagc ttatgagtac atggtagatt acctcaacag tcttcaggca 360
gtgggccgaa gtgagaacat tgtcggatgg tatcactcgc atccagggta tgggtgttgg 420
ctgagcggaa ttgatgtagg tactcaggcc cagaatcagc agtttcagga tccatttctg 480
gccattgtgg tggaccctaa tcgcaccatt tcagccggaa aagtggacat tggtgcgttt 540
cgaacatatc ccgaggggta taagccatcc aaggaggagc aggaggagat tatccagggc 600
attccatttg ccaagatgga agactttgga gtgcatgctg atcggtacta tccgctggag 660
atttcatatt tcaagtcaac attggacgct aagctgctag aaattctatg gaacaagtat 720
tgggcttcta ctctgtcaca atcgccgctt ctcaccaata tggagtataa tactaaccag 780
atgagggatc tagtatcaaa aattgacaac gtgcgagatt ctgtcggtgc ccgaaagtgc 840
cctaatcttt cttcgctgag tggagttaag agtatttccc agcttccagc agctgcaact 900
agtaaaaaca gatccatggc cgaccttgtg gaggaatctc gtaagattgg cgctgagcaa 960
attaggggcc tggttactcg agagcttaac agccggctgt ttgagcccat tgaaccatct 1020
<210> 2
<211> 1071
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 2
tgatcaatct ccatattaac gtacagagct ggaacgatgg ctcagaaggc gtttgaacta 60
gagaatgaca taaagctcgt aggggaggat gacaagttat actaccagta taataaggag 120
gagcagcaga agctgttacg agagcggccc tggaaggacg atcctcacta tttcaagcga 180
gtgaggatat cagctattgc cctgctgcga atggccattc atgcccgtgc gggaggatca 240
attgaagtga tgggagtgat gactggcaag attctgccaa acgagtttgt tatcatggat 300
acgtatccct tgccagtgga aggtaccgaa actagagtga acgcactggg agaagcttat 360
gagtacatgg tagattacct caacagtctt caggcagtgg gccgaagtga gaacattgtc 420
ggatggtatc actcgcatcc agggtatggg tgttggctga gcggaattga tgtaggtact 480
caggcccaga atcagcagtt tcaggatcca tttctggcca ttgtggtgga ccctaatcgc 540
accatttcag ccggaaaagt ggacattggt gcgtttcgaa catatcccga ggggtataag 600
ccatccaagg aggagcagga ggagattatc cagggcattc catttgccaa gatggaagac 660
tttggagtgc atgctgatcg gtactatccg ctggagattt catatttcaa gtcaacattg 720
gacgctaagc tgctagaaat tctatggaac aagtattggg cttctactct gtcacaatcg 780
ccgcttctca ccaatatgga gtataatact aaccagatga gggatctagt atcaaaaatt 840
gacaacgtgc gagattctgt cggtgcccga aagtgcccta atctttcttc gctgagtgga 900
gttaagagta tttcccagct tccagcagct gcaactagta aaaacagatc catggccgac 960
cttgtggagg aatctcgtaa gattggcgct gagcaaatta ggggcctggt tactcgagag 1020
cttaacagcc ggctgtttga gcccattgaa ccatctcatc gcatgacata g 1071
<210> 3
<211> 344
<212> PRT
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 3
Met Ala Gln Lys Ala Phe Glu Leu Glu Asn Asp Ile Lys Leu Val Gly
1 5 10 15
Glu Asp Asp Lys Leu Tyr Tyr Gln Tyr Asn Lys Glu Glu Gln Gln Lys
20 25 30
Leu Leu Arg Glu Arg Pro Trp Lys Asp Asp Pro His Tyr Phe Lys Arg
35 40 45
Val Arg Ile Ser Ala Ile Ala Leu Leu Arg Met Ala Ile His Ala Arg
50 55 60
Ala Gly Gly Ser Ile Glu Val Met Gly Val Met Thr Gly Lys Ile Leu
65 70 75 80
Pro Asn Glu Phe Val Ile Met Asp Thr Tyr Pro Leu Pro Val Glu Gly
85 90 95
Thr Glu Thr Arg Val Asn Ala Leu Gly Glu Ala Tyr Glu Tyr Met Val
100 105 110
Asp Tyr Leu Asn Ser Leu Gln Ala Val Gly Arg Ser Glu Asn Ile Val
115 120 125
Gly Trp Tyr His Ser His Pro Gly Tyr Gly Cys Trp Leu Ser Gly Ile
130 135 140
Asp Val Gly Thr Gln Ala Gln Asn Gln Gln Phe Gln Asp Pro Phe Leu
145 150 155 160
Ala Ile Val Val Asp Pro Asn Arg Thr Ile Ser Ala Gly Lys Val Asp
165 170 175
Ile Gly Ala Phe Arg Thr Tyr Pro Glu Gly Tyr Lys Pro Ser Lys Glu
180 185 190
Glu Gln Glu Glu Ile Ile Gln Gly Ile Pro Phe Ala Lys Met Glu Asp
195 200 205
Phe Gly Val His Ala Asp Arg Tyr Tyr Pro Leu Glu Ile Ser Tyr Phe
210 215 220
Lys Ser Thr Leu Asp Ala Lys Leu Leu Glu Ile Leu Trp Asn Lys Tyr
225 230 235 240
Trp Ala Ser Thr Leu Ser Gln Ser Pro Leu Leu Thr Asn Met Glu Tyr
245 250 255
Asn Thr Asn Gln Met Arg Asp Leu Val Ser Lys Ile Asp Asn Val Arg
260 265 270
Asp Ser Val Gly Ala Arg Lys Cys Pro Asn Leu Ser Ser Leu Ser Gly
275 280 285
Val Lys Ser Ile Ser Gln Leu Pro Ala Ala Ala Thr Ser Lys Asn Arg
290 295 300
Ser Met Ala Asp Leu Val Glu Glu Ser Arg Lys Ile Gly Ala Glu Gln
305 310 315 320
Ile Arg Gly Leu Val Thr Arg Glu Leu Asn Ser Arg Leu Phe Glu Pro
325 330 335
Ile Glu Pro Ser His Arg Met Thr
340
<210> 4
<211> 3066
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 4
aatccgtgac taacagcact gtcaagactt ctacacagac tcctcagact cagcataatt 60
tcacaatgac tccattgcct tcagtcaccg aggctccctg ttcgaccgaa aagggtgtgt 120
cgtcattgac ccagactaac caggtttcaa ttgcttcgca atcgtgccct actgtcactg 180
tgactaacgt ttctggcacg ccatccacaa agaccgtgac cgtgacctgc tccgaggccg 240
tttgccagac ccctactccg agtcaagaga ccgtctctgg taccgtcacc gttactgtcc 300
catgcgtcac cgttactgag actgtaaagt gcacaaagaa cgtatgccag gttcattcca 360
ctacccaatt gtcaaacact cagcctggta ctaccaaatc ctcggagacc cagcctgatg 420
tgaccactac tatcacttct gagatcacag tagtactaac tgagtcttgc tccgagtctc 480
agtccgcctc tcagtccact cagtccgctc catccactca gcccactcag tccactcagt 540
ccactcagtc cgctcagtcc gctgagtccg cttctagccc tgtctctcag tcgcctagca 600
ctcgaaccac tattgtgaga gagacttcca ccattactaa cgtaaacttt gccacttcta 660
ctattgtcgg cactgtatcc tcctcatcct cttccaatgc tgagatgccc cagcactccc 720
actcttctga ggtctcccag gttcctgccc cttcgcaagt gcccgctcag tcctctatca 780
accaggtgaa tggcgcttcg accactaagc tatctactgt ggcagttgtg ctgtccgcca 840
tgctcatgat tttgtaaata ctgtgattgt tataaatgca actttaacct ttaataattg 900
atttgttctt ctttttgtct gttgttgcac atttcctacc tattttgccc cgtagcagct 960
tgtccggtat catttatata gccgtgggcc tcattattga tcaatctcca tattaacgta 1020
cagagctgga acgatggctc agaaggcgtt tgaactagag aatgacataa agctcgtagg 1080
ggaggatgac aagttatact accagtataa taaggaggag cagcagaagc tgttacgaga 1140
gcggccctgg aaggacgatc ctcactattt caagcgagtg aggatatcag ctattgccct 1200
gctgcgaatg gccattcatg cccgtgcggg aggatcaatt gaagtgatgg gagtgatgac 1260
tggcaagatt ctgccaaacg agtttgttat catggatacg tatcccttgc cagtggaagg 1320
taccgaaact agagtgaacg cactgggaga agcttatgag tacatggtag attacctcaa 1380
cagtcttcag gcagtgggcc gaagtgagaa cattgtcgga tggtatcact cgcatccagg 1440
gtatgggtgt tggctgagcg gaattgatgt aggtactcag gcccagaatc agcagtttca 1500
ggatccattt ctggccattg tggtggaccc taatcgcacc atttcagccg gaaaagtgga 1560
cattggtgcg tttcgaacat atcccgaggg gtataagcca tccaaggagg agcaggagga 1620
gattatccag ggcattccat ttgccaagat ggaagacttt ggagtgcatg ctgatcggta 1680
ctatccgctg gagatttcat atttcaagtc aacattggac gctaagctgc tagaaattct 1740
atggaacaag tattgggctt ctactctgtc acaatcgccg cttctcacca atatggagta 1800
taatactaac cagatgaggg atctagtatc aaaaattgac aacgtgcgag attctgtcgg 1860
tgcccgaaag tgccctaatc tttcttcgct gagtggagtt aagagtattt cccagcttcc 1920
agcagctgca actagtaaaa acagatccat ggccgacctt gtggaggaat ctcgtaagat 1980
tggcgctgag caaattaggg gcctggttac tcgagagctt aacagccggc tgtttgagcc 2040
cattgaacca tctcatcgca tgacatagca ttaacacatt aatatataaa ctatccttac 2100
attgtctcta ttgtgtcttt ttaaccttgt cttgcttctt ttccttaggc tgctttggct 2160
tggtgagcag gaccataacc aggtaaagga atccgccaag cactgcaact acagttgcca 2220
attgaacaaa gccaaagctc tgaacagggg ccacaggaat gctgtaaggt gaagcaacgc 2280
tttcgggctc agaagaggga gtctcagaat aggggcttcc tgggatacgg taaaacacag 2340
tgtttggtgg gaacaggctt tcataggcca ccgctggata ctctccagcc agcggatctt 2400
gaatgttagc acaggcccag aacacaaacg gcaagggcat tgcgtgatcc actttggtat 2460
caccttcctg agggacctcg taccgggaat gcatgggcag ggtaaagtta aagtcctggc 2520
cgctggtgtc aggaggatag atctgaacaa ccgcttcgga tccccaactg tgcacttccc 2580
atacaggagc ctcaagatca gtctctcccc atactccaat tagctttccc gacgcgggag 2640
ttgaaggggt ttctcgttcc agatcttgca gctggtactt gtcgaggaat agggagcgag 2700
gaatgttata ctttgcaaat agtttgcacg atggcaaatt aggagccctg actccggaaa 2760
gagtgagctt gtgtcggggg tgaagaccca atggctgctc aaagcccccg gtgaatgagc 2820
tgttgtgcga gtaccgcgaa agcaacgata ctgctaccag aacctgcttg tattcttctt 2880
cctcattgtg gcccactttt cttataagac cagacagagg gaattcgtgc ctttcaacgt 2940
aataatccgg gttaagagcc agcattccga cttcaacggt tcgatcaatt tgttcagtac 3000
gctggggagg gataatcgaa gtgcccagct ccgctgccat gcgttgtcta gagggtcctg 3060
accagt 3066
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
<210> 7
<211> 1485
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 7
cgtagcagct tgtccggtat catttatata gccgtgggcc tcattattga tcaatctcca 60
tattaacgta cagagctgga acgatggctc agaaggcgtt tgaactagag aatgacataa 120
agctcgtagg ggaggatgac aagttatact accagtataa taaggaggag cagcagaagc 180
tgttacgaga gcggccctgg aaggacgatc ctcactattt caagcgagtg aggatatcag 240
ctattgccct gctgcgaatg gccattcatg cccgtgcggg aggatcaatt gaagtgatgg 300
gagtgatgac tggcaagatt ctgccaaacg agtttgttat catggatacg tatcccttgc 360
cagtggaagg taccgaaact agagtgaacg cactgggaga agcttatgag tacatggtag 420
attacctcaa cagtcttcag gcagtgggcc gaagtgagaa cattgtcgga tggtatcact 480
cgcatccagg gtatgggtgt tggctgagcg gaattgatgt aggtactcag gcccagaatc 540
agcagtttca ggatccattt ctggccattg tggtggaccc taatcgcacc atttcagccg 600
gaaaagtgga cattggtgcg tttcgaacat atcccgaggg gtataagcca tccaaggagg 660
agcaggagga gattatccag ggcattccat ttgccaagat ggaagacttt ggagtgcatg 720
ctgatcggta ctatccgctg gagatttcat atttcaagtc aacattggac gctaagctgc 780
tagaaattct atggaacaag tattgggctt ctactctgtc acaatcgccg cttctcacca 840
atatggagta taatactaac cagatgaggg atctagtatc aaaaattgac aacgtgcgag 900
attctgtcgg tgcccgaaag tgccctaatc tttcttcgct gagtggagtt aagagtattt 960
cccagcttcc agcagctgca actagtaaaa acagatccat ggccgacctt gtggaggaat 1020
ctcgtaagat tggcgctgag caaattaggg gcctggttac tcgagagctt aacagccggc 1080
tgtttgagcc cattgaacca tctcatcgca tgacatagca ttaacacatt aatatataaa 1140
ctatccttac attgtctcta ttgtgtcttt ttaaccttgt cttgcttctt ttccttaggc 1200
tgctttggct tggtgagcag gaccataacc aggtaaagga atccgccaag cactgcaact 1260
acagttgcca attgaacaaa gccaaagctc tgaacagggg ccacaggaat gctgtaaggt 1320
gaagcaacgc tttcgggctc agaagaggga gtctcagaat aggggcttcc tgggatacgg 1380
taaaacacag tgtttggtgg gaacaggctt tcataggcca ccgctggata ctctccagcc 1440
agcggatctt gaatgttagc acaggcccag aacacaaacg gcaag 1485
Claims (5)
1. A primer pair for amplifying DNA barcodes for identifying the adenine node B.adenine node B yeast TMCC70007 strain comprises the nucleotide sequences of a forward primer and a reverse primer which are respectively shown as follows:
a forward primer: 5'-CGTAGCAGCTTGTCCGGTAT-3', respectively;
reverse primer: 5'-CTTGCCGTTTGTGTTCTGGG-3', respectively;
wherein the DNA barcode is derived from the genome of the Arthrospora adenantha TMCC70007 strain, and the nucleotide sequence is selected from the sequences shown in SEQ ID No.4 and comprises the sequence shown in SEQ ID No. 1.
2. A kit for identifying a nodospora adevorans TMCC70007 strain comprising the primer pair according to claim 1.
3. A method for identifying a strain of the nodospora adephagi TMCC70007, comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA of the step a) as a template and using the primer pair of claim 1 to obtain a PCR product;
c) detecting the PCR product by electrophoresis, if no target band exists, judging that the strain to be detected is not the adenine arthrobacter adevorans TMCC70007 strain, and if the target band exists, performing the step d);
d) sequencing the obtained PCR product to obtain a nucleotide sequence to be detected; and (3) carrying out homology comparison on the nucleotide sequence to be detected and the nucleotide sequence of the DNA bar code, and if the homology is more than 97%, judging that the strain to be detected is the Arthrospora adenine-eating yeast TMCC70007 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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| Title |
|---|
| The complete genome of Blastobotrys(Arxula) adeninivorans LS3- a yeast of biotechnological interest;Gotthard Kunze et al.;《Biotechnology for Biofuels》;20141231;第7卷(第66期);全文 * |
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