CN109402279A - DNA bar code, primer, kit, methods and applications - Google Patents
DNA bar code, primer, kit, methods and applications Download PDFInfo
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Abstract
The invention belongs to species and bacterial strain to identify field, and in particular to a kind of for identifying the DNA bar code for eating adenine section spore yeast strain, primer, kit, methods and applications.The DNA bar code can accurately identify Pu-erh tea fermentation bacterial strain food adenine section spore yeast TMCC 70007, can rapidly and accurately come by the bacterial strain from confusing bacterial strain or with identifying in other bacterial strains in species.
Description
Technical field
The invention belongs to species and bacterial strains to identify field, and in particular to DNA bar code, primer, kit, method and answer
With.
Background technique
Pu'er tea is a kind of post-fermented tea with Yunnan geographical mark, uses large leaf solar dried green tea as raw material,
It is made by series of process.Traditional Pu'er tea manufacturing process are as follows: the fresh tea leaf of picking rubbed, sundrying, removal of impurities,
Tidewater pile fermentation, is dried, is sieved, compression moulding, packaging factory.In the production of Pu'er tea, pile-fermentation process is Pu'er tea product
The principal element that matter is formed, in the process, the component contents such as tea polyphenols, caffeine and some polysaccharide materials in tealeaves
Great variety has occurred, has achieved flavour, mouthfeel, quality and the various health-care efficacies of Pu'er tea.
In traditional Pu'er production, the contained enzyme of damp and hot Context Activation tealeaves itself, a part contained in tealeaves
Component content is converted to the substance that microorganism can utilize, and microorganism largely breeds, and generates endocellular enzyme and ectoenzyme abundant, catalysis
A series of transformations occur for the ingredient for including in tealeaves, along with the other factors such as damp and hot, gradually form the Pu'er tea of different qualities.
The different places of production, because of the difference of its biological community structure, flavor and quality are also with their own characteristics.
Pu'er tea is other than its unique flavor and culture, and such as weight-reducing, reducing blood sugar and blood lipid, prevention and improvement are cardiovascular
The health-care efficacies such as disease, anti-aging, anticancer, anti-inflammatory, aid digestion and nourishing the stomach are also paid close attention to by people.In recent years, Pu'er tea is got over
Come more welcome, the market demand gradually increased has pulled the development of Pu-Erh Tea industry, promotes Yunnan Province economic growth.
However, Pu'er tea is also faced with some predicaments, such as unstable product quality, there are certain security risks, production
Period is long, labour's investment is excessively high, micro organism quantity is exceeded and acarid is bred etc..As the improvement of people's living standards, consumption
Health of the person to food, security problems pay attention to day by day.Safety of Food Quality event took place frequently in recent years, and the safety of tea quality is asked
Topic also becomes increasingly conspicuous, and microorganism, which is also possible to become, other than the residue problem of pesticide, during pile-fermentation influences tealeaves
An important factor for quality.
The Pu'er tea production of majority producer is still the empirical formula fermentation of semi-natural artificial pile fermentation at present, although pile fermentation process
In to eat the Community Facies based on the advantages general character microorganisms such as adenine section spore yeast (Blastobotrys adeninivorans)
To stabilization, but there is also biggish rooms for promotion for the stability of product.Also inevitably there is certain in production process
Security risk, in order to further obtain the favor of consumer and the approval in market, breakthrough foreign trade barrier, promote Pu'er tea enterprise
The market competitiveness, it is necessary to realize the human controllableization of Pu'er tea production technology, clean and efficient, and continually develop new production
Product, extension industry chain.Accomplish these, must just innovate technologies, invent it is a series of it is safe and clean, efficient, human controllable is automatic
The Pu'er tea new process of change, just can ensure that the sound development of Pu-Erh Tea industry, bring long-term interest to the country and people.
With further going deep into for Pu'er tea scientific research, more and more people have started the spy of manual fermentation Pu'er tea
Rope, there are many bacterial strains at present ferments applied to Pu'er tea human controllable.In recent years due to the market demand increase, many scales compared with
Small producer lacks system in-depth study to Pu'er tea, abuses other people many patents without restraint and makes profit, for example, according to some
The patented method of inoculation fermentation Pu'er tea carries out the serial of methods such as Pu-erh tea fermentation using some strains, carries out Pu'er tea and adds
The business activities such as work production, compromise the economic interests of some Pu'er tea manufacturing enterprises with related patents significantly.
To ensure that Pu-erh tea fermentation microorganism germ plasma resource is effectively protected, abuse Pu-erh tea fermentation microorganism is prevented
Abuse occurs, and solves the problems, such as to be difficult to put to the proof when strain is encroached right in Pu'er tea human controllable's fermentation process, develop to general
The fermentation of Pu'er tea tea with bacterial strain carry out fast accurate mirror method for distinguishing it is imperative, need to establish its method for identifying molecules, exploitation passes through
DNA bar code technology accurately identifies and distinguishes the method with its similar strain, to reach through morphological feature and molecular data point
Phase separation in conjunction with method come solve the problems, such as industrial producing strain identify and identification, for Pu'er tea have control industrial fermentation process exploitation and
Protection of resources provides theoretical foundation, promotes the prosperity and development of Pu-Erh Tea industry health.
Summary of the invention
In order to overcome present in food adenine section spore yeast strain of the Morphological Identification for Pu-erh tea fermentation production not
Foot, the present invention provides it is a kind of for identifies eat the DNA bar code of adenine section spore yeast strain, primer, kit, method and
Using, thus to food 70007 bacterial strain of adenine section spore yeast TMCC quickly identified and distinguished, can be Pu'er tea newly ferment
Technique provides rapid evaluation, prevents the interference of other miscellaneous bacterias in fermentation process, and for Pu'er tea human controllable zymotechnique and
Strain, which is illegally abused, provides the means of proof and foundation.
To achieve the goals above, the present invention adopts the following technical scheme:
(1) a kind of for identifying the DNA bar code of food adenine section spore yeast strain, the DNA bar code is from food gland
The genome of 70007 bacterial strain of purine section spore yeast TMCC, and include in the DNA sequence dna as shown in SEQ ID No.1
At least sequence of 500bp, and the length of the DNA bar code be 500bp-3000bp, preferably 500bp-2200bp, more preferably
For 500bp-1500bp.
(2) DNA bar code according to (1), nucleotide sequence such as SEQ ID No.1 or SEQ ID No.4 or SEQ
Shown in ID No.7.
(3) a kind of for expanding the primer pair of the DNA bar code according to (1).
(4) primer pair according to (3), the nucleotide sequence and food adenine section spore yeast TMCC of forward primer
Such sequence in 70007 strain gene groups is identical: the sequence is from the 70007 strain gene group of TMCC such as SEQ
1st upstream 1000bp of nucleotide sequence shown in ID No.1 to the nucleotide sequence as shown in SEQ ID No.1
Sequence in 882 regions, and the length of the forward primer is 20-30bp；Its reverse primer and the TMCC 70007
Such sequence reverse complemental in strain gene group: the sequence is from the 70007 strain gene group of TMCC such as SEQ ID
The 501st of nucleotide sequence shown in No.1 arrives last downstream of the nucleotide sequence as shown in SEQ ID No.1
Sequence in the region of 1000bp, and the length of the reverse primer is 20-30bp.
(5) the nucleotide sequence difference of the primer pair according to (4), forward primer and reverse primer is as follows:
Forward primer: 5 '-ACTCGAAAACCCTATACACCGA-3 '；
Reverse primer: 5 '-CTTGCACTTGTTTAGTGCCCA-3 '.
(6) a kind of for identifying the kit of food adenine section spore yeast strain, it include the primer pair according to (3).
It is (7) a kind of for identifying the method for food adenine section spore yeast strain, comprising the following steps:
A) genomic DNA of strain to be tested is provided；
B) using genomic DNA described in step a) as template, PCR amplification is carried out using the primer pair according to (3), is obtained
To PCR product；
C) electrophoresis detection PCR product, if determining strain to be tested not is food adenine section spore yeast without target stripe
70007 bacterial strain of TMCC then carries out step d) if there is target stripe；
D) gained PCR product is sequenced, obtains nucleotide sequence to be measured；By the nucleotide sequence to be measured and right
It is required that the nucleotide sequence of DNA bar code described in 1 carries out sequence analysis, if homology determines to be measured 99% or more
Bacterial strain is food 70007 bacterial strain of adenine section spore yeast TMCC.
(8) DNA bar code according to (1) is identifying the application in food adenine section spore yeast strain.
(9) primer pair according to (3) is identifying the application in food adenine section spore yeast strain.
(10) kit according to (6) is identifying the application in food adenine section spore yeast strain.
Compared with the prior art, the present invention has the following advantages and good effect:
1, the present invention uses protein gene omics technology from the gene of food 70007 bacterial strain of adenine section spore yeast TMCC
A leakage annotation coding albumen is had found in group, and it was found that encode open reading frame (the SEQ ID of the gene of the albumen
No.1) peculiar to eat the genome of 70007 bacterial strain of adenine section spore yeast TMCC.The present invention further passes through careful grind
Study carefully and comparative analysis, by distinctive DNA sequence dna exploitation for DNA bar code, produces bacterium as Pu'er tea industrial fermentation is identified
The effective tool of strain food adenine section spore yeast TMCC 70007.The bar code sequence is able to achieve in food adenine section spore yeast kind
The quick and precisely identification and differentiation of bacterial strain.
2, the specificity for the DNA bar code that compared with prior art, the present invention is had found using protein gene omics technology
More preferably.
3, the present invention is it has furthermore been found that compared to other genes, which annotates sequence (such as the SEQ ID in gene
No.1) have the characteristics that general, easy amplification, easily compare, difference is bright between different strains in B.adeninivorans kind
It is aobvious.
4, the present invention establishes the standard of Pu'er tea industrial fermentation production bacterial strain food adenine section spore yeast TMCC 70007
Gene order and sample discrimination method significantly improve determination rates compared to traditional Morphological Identification method.This method pair
Low in the integrated degree requirement of sample, identification beacon can quantify, this for determining that Pu'er tea and its germ plasm resource provide in time
Effective foundation.Obscure kind in addition, further joined morphology and the phylogenetic tree based on clustering can be used
Method sets up identification rule and is identified that the reliability and accuracy of identification are also significantly better than conventional method for identifying molecules, more
The blank that such Pu'er fermented tea production bacterial strain is identified based on the bacterial strain of DNA bar code technology is mended.
Detailed description of the invention
Fig. 1 shows the mass spectrogram of new identification peptide fragment NAFAYFVQR.
Fig. 2 shows the mRNA sequence of peptide fragment region protein coding frame and its correspondences of the protein sequence of coding
Figure, gray background part are the new peptide fragment identified.
Fig. 3 shows the protein sequence for encoding mRNA sequence and its coding that the ORF of new identification of M RPS9 albumen is transcribed
Corresponding diagram, gray background part are the new peptide fragment identified.
Fig. 4 A shows the sequence therewith with homology that bar code sequence SEQ ID NO.1 is obtained by NCBI-BLASTN
Column comparison chart, a short gray segments below Fig. 4 A show 1 homology sequence；Fig. 4 B show SEQ ID NO.1 with
Yeast (Sugiyamaella lignohabitans) CBS 10342THomologous sequence (accession number: XM_ in bacterial strain
004200814.1) comparison chart.
Fig. 5 shows the 70007 bar code sequence SEQ ID NO.1 of TMCC and food adenine section of food adenine section spore yeast
The comparison diagram of spore yeast LS3 bacterial strain homologous sequence；
Fig. 6 is shown for expanding the position of the primer of SEQ ID NO.1 in one embodiment of the invention, under having in sequence
The region of scribing line is primer region, and the ATG and TAG of overstriking font are starting and termination site, gray background region
Subregion is included for gene, the sequence expanded is SEQ ID NO.7.
Fig. 7, which is shown, carries out agarose gel electrophoresis to the strain to be tested pcr amplification product obtained using primer of the invention
Result.
Fig. 8 shows the comparison diagram of SEQ ID NO.1 sequence Yu strain to be tested PCR amplification sequence.
Specific embodiment
Description below by way of specific embodiment and the invention will be further described referring to attached drawing, but it is pair that this, which is not,
Limitation of the invention, those skilled in the art's basic thought according to the present invention, can make various modifications or improvements, but only
Basic thought of the invention is not departed from, is all within the scope of the present invention.
The present invention uses protein gene omics technology from the genome of food 70007 bacterial strain of adenine section spore yeast TMCC
In have found the coding albumen of leakage annotation, since the albumen and mitochondria 37S ribosomal protein MRPS9 have homology,
It is referred to as MRPS9 albumen.The present invention has obtained pair in 70007 strain gene group of TMCC by the newfound protein sequence
Answer gene order, be referred to as MRPS9 gene, and it was found that encode the gene order (SEQ ID No.1) of the albumen relative to
In ncbi database it has been reported that sequence and its similar strain nucleic acid sequence have compared with high specific, can be used for developing spy
The opposite sex identifies the DNA bar code of Pu'er tea industrial fermentation production bacterial strain food adenine section spore yeast TMCC 70007.With existing skill
Art is compared, and the present invention is higher by the specificity for the DNA bar code that the above method obtains.
In view of DNA bar code should have specificity, the present invention between suitable length and enough bacterial strains further to lead to
Careful research and comparative analysis are crossed, food adenine section can accurately and efficiently be identified by obtaining based on above-mentioned distinctive DNA sequence dna
The DNA bar code of 70007 bacterial strain of spore yeast TMCC.
One aspect of the present invention provides a kind of for identifying the DNA bar shaped of food adenine section spore yeast strain as a result,
Code, the DNA bar code derive from the genome of food 70007 bacterial strain of adenine section spore yeast TMCC, and include selected from such as SEQ
At least sequence of 500bp in DNA sequence dna shown in ID No.1, and the length of the DNA bar code is 500bp-3000bp,
Preferably 500bp-2200bp, more preferably 500bp-1500bp.
Bar code sequence of the invention is able to achieve the quick and precisely identification of bacterial strain in food adenine section spore yeast kind and distinguishes.
The length of SEQ ID No.1 is 1382bp, since SEQ ID No.1 is food adenine section spore yeast TMCC 70007
Strain gene group institute is peculiar, therefore the sequence itself can be used as identifying food 70007 bacterial strain of adenine section spore yeast TMCC
DNA bar code.In addition, by theory analysis and experimental verification, present invention demonstrates that including the sequence, the longer DNA bar code of length
Specificity be more guaranteed.(3000bp is greater than) when the length of DNA bar code is too long, for amplification operation
It is less desirable.On the other hand, in general, when the length of DNA bar code is not less than 500bp, it also can satisfy easily amplification, easily compare
Operation requires；The present invention passes through theory analysis and experimental verification, it was demonstrated that in the DNA sequence dna as shown in SEQ ID No.1
At least the sequence of 500bp also can be realized the quick and precisely identification of bacterial strain in food adenine section spore yeast kind and distinguish.
In a preferred embodiment of the present invention, the DNA bar code sequence such as SEQ ID No.1, SEQ ID No.4
Or shown in SEQ ID No.7.
Herein, after term " leakage annotation gene " refers to that species complete gene order-checking, using predictive genes software
(such as GeneMark, Augustus, Glimmer etc.) fails to come out the predictive genes, these gene General Expression amounts are high,
It just expresses under given conditions, so being difficult to be found under study for action.
Term " DNA bar code technology (DNA barcoding) " refers to a segment standard, short DNA piece in genome
Section identifies a Molecular Identification new technology of species, can fast and accurately carry out species identification.
Term " translation of six frames " is the known term in proteomics and genomics, and sketching its principle is DNA encoding
When protein, using triplet codon coding protein, a DNA sequence dna is given, there are 3 kinds of codifiabilities, in addition it is mutually
The 3 kinds of codings mended on chain are possible, share 6 kinds of codifiabilities (+1 ,+2 ,+3, -3, -2, -1).
Another embodiment of the invention provides a kind of for expanding the primer of DNA bar code of the present invention
It is right.
Preferably, in the nucleotide sequence of forward primer and food 70007 strain gene group of adenine section spore yeast TMCC
Such sequence it is identical: the sequence be from the 70007 strain gene group of TMCC the nucleosides as shown in SEQ ID No.1
Sequence in the region of the 1st upstream 1000bp to the 882nd of the nucleotide sequence as shown in SEQ ID No.1 of acid sequence
Column, and the length of the forward primer is 20-30bp；In its reverse primer and the 70007 strain gene group of TMCC in this way
Sequence reverse complemental: the sequence be from the 70007 strain gene group of TMCC the nucleotide as shown in SEQ ID No.1
The 501st of sequence is into the region of last downstream 1000bp of the nucleotide sequence as shown in SEQ ID No.1
Sequence, and the length of the reverse primer is 20-30bp.
In a more preferred embodiment, the nucleotide sequence of the forward primer and reverse primer difference is as follows:
Forward primer MRPS9-F:5 '-ACTCGAAAACCCTATACACCGA-3 ' (SEQ ID No.5)；
Reverse primer MRPS9-R:5 '-CTTGCACTTGTTTAGTGCCCA-3 ' (SEQ ID No.6).
Primer of the invention is able to achieve the specific amplification to the DNA bar code sequence.
The present invention also provides a kind of for identifying the kit of food adenine section spore yeast strain, comprising according to the present invention
The primer pair.
In another embodiment, the kit also includes DNA bar code according to the present invention.The DNA item
Shape code may be present in recording medium.The recording medium is, for example, CD.The kit can also be comprising being used for experimental implementation
Any tool and reagent.
It is yet another aspect of the present invention to provide a kind of for identifying the method for food adenine section spore yeast strain, including following
Step:
A) genomic DNA of strain to be tested is provided；
B) using genomic DNA described in step a) as template, PCR expansion is carried out using primer pair according to the present invention
Increase, obtains PCR product；
C) electrophoresis detection PCR product, if determining strain to be tested not is food adenine section spore yeast without target stripe
70007 bacterial strain of TMCC then carries out step d) if there is target stripe；
D) gained PCR product is sequenced, obtains nucleotide sequence to be measured；By the nucleotide sequence to be measured and right
It is required that the nucleotide sequence of DNA bar code described in 1 carries out sequence analysis, if homology determines to be measured 99% or more
Bacterial strain is food 70007 bacterial strain of adenine section spore yeast TMCC.
In one embodiment of the invention, the program of the PCR amplification are as follows: 1) 94 DEG C of -96 DEG C of initial denaturations 8 are divided
Clock；2) it is denaturalized 45 seconds for 94 DEG C -96 DEG C, 55 DEG C -57 DEG C are annealed 45 seconds, and 72 DEG C extend 1 point 15 seconds, wherein the program 2) carry out 32-
35 circulations；3) extend 10 minutes for 72 DEG C.
In another embodiment of the invention, the method also includes the sequences to be measured for obtaining sequencing result and this hair
Bright DNA bar code carries out clustering (such as phylogenetic tree), if sequence to be measured and DNA bar code cluster together,
Then determine that strain to be tested is food adenine section spore yeast strain TMCC 70007.
In a specific embodiment of the invention, using primer pair of the invention to extracting from bacterial strain to be identified
Genomic DNA carries out PCR amplification, then carries out agarose gel electrophoresis detection.Identified based on detecting whether that there are PCR products
Bacterial strain: if bacterial strain to be identified does not amplify corresponding target stripe, illustrating the bacterial strain not is TMCC 70007；If
Corresponding target stripe is amplified, then proves that the bacterial strain may be TMCC 70007.For further progress identification, PCR is produced
Object is sequenced, and DNA sequencing result and DNA bar code sequence are carried out sequence analysis, and the similitude obtained between sequence is (i.e. same
Source property), if it is food adenine section spore yeast strain TMCC that sequence homology, which less than 99%, determines strain to be tested not,
70007.If sequence homology is greater than or equal to 99%, determine that strain to be tested is food adenine section spore yeast strain TMCC
70007。
If carrying out clustering, such as phylogenetic tree, then by the DNA of the DNA bar code and each bacterial strain to be identified
Sequencing result (sequence i.e. to be measured) applies MEGA 6 or PAUP software building NJ phylogenetic tree together.If bacterial strain to be identified
Sequence to be measured and the DNA bar code cluster of food adenine section spore yeast TMCC 70007 together, are then accredited as food adenine section
Spore yeast TMCC 70007.
Term " cluster " used herein refers to after phylogenetic tree is analyzed, and is in the same branch, and evolve away from
From identical.
The present invention also provides DNA bar codes of the present invention to identify answering in food adenine section spore yeast strain
With.
The present invention also provides primer pairs of the present invention to identify the application in food adenine section spore yeast strain.
Embodiment
Below with reference to specific embodiment, the present invention will be further described.Method therefor is not illustrating in embodiment
In the case where, it is all made of conventional method and known means.
The acquisition of embodiment 1:MRPS9 gene and DNA bar code
Protein group used in the embodiment and genome analytical method and mass spectrometry method are the known of this field
Method, therefore specific operation process repeats no more.
1, protein technique is covered using height, it is fast to Pu'er tea industrial fermentation bacterium food gland using pFind and pAnno software
Purine section spore yeast TMCC 70007 has carried out high covering proteomics research, and has carried out annotation encoding gene to its genome
Verifying.Specifically, translating (Six using six frames in protein mapping genomics to find new protein-coding region
Frame Translation) strategy, obtain the six frames translation number of food 70007 genomic data of adenine section spore yeast TMCC
According to library, 6 kinds of codings of exhaustion genome may (+1 ,+2 ,+3, -1, -2, -3), and protein sequence is known as " six frames translation
Protein sequence ", corresponding nucleic acid sequence are known as " six frames translate nucleic acid sequence ".Typically, six frames translation nucleic acid sequence be from
One terminator to next terminator sequence, herein, also referred to as " encoding histone frame ".It is turned over using this six frame
Database is translated, is compared using pFind and pAnno software with the holoprotein modal data of 70007 bacterial strain of TMCC, thus into
The discovery and identification of row new peptide fragment and novel protein.
It is identified, it was found that eat in the annotation genome of adenine section spore yeast TMCC 70007 for one and do not send out in the prior art
Existing peptide fragment NAFAYFVQR, mass spectrogram are as shown in Figure 1.
Check mass spectrogram the results show that detecting the several of the second order ms spectrogram (MS2) of peptide fragment NAFAYFVQR by hand
All y ion sequences, match, and signal is strong, as a result more credible.
2, according to the position where new peptide fragment, the region for including with previous terminator codon and the latter terminator codon
For boundary, six frames translation nucleic acid sequence (encoding histone frame) SEQ ID NO.2 is obtained.The corresponding mRNA of SEQ ID NO.2 sequence and
Its encoded amino acid sequence is as shown in Figure 2.
3, in order to further determine the coding initiation site and termination site of the gene for encoding the albumen, and in order to obtain
Length is more suitable and has the DNA bar code of enough specificity, and above-mentioned encoding histone frame is upstream expanded with downstream respectively
1000bp is opened up, predictive genes are carried out using GeneMark.hmm, selects schizosaccharomyces pombe with reference to species
(Schizosaccharomyces pombe).The presence of the gene has been arrived in the regional prediction, it is determined that it encodes starting and end
Stop bit point, ORF sequence are SEQ ID NO.1.935-1110 of SEQ ID NO.1 are the introne of the gene.
The corresponding relationship of the amino acid sequence of the mRNA sequence of the genetic transcription and the albumen of its translation is as shown in Figure 3.Peptide
Section NAFAYFVQR is located at the downstream of the gene.
The total 1382bp of the nucleotide sequence of the ORF encodes 401 amino acid, theoretical molecular weight 45.41kDa, reason altogether
By the amino acid sequence of coding as shown in SEQ ID NO.3.
4, blastp analysis, sequence and yeast are carried out with the amino acid sequence of the gene theory coded product
The homology of (Sugiyamaella lignohabitans) mitochondria 37S ribosomal protein (MRPS9) is 45%.Blastp ratio
Result is summarised in table 1.
The result shows that detected leakage annotation albumen is MRPS9 homologous gene product, sequence has certain blastp
Conservative.
5, the ORF sequence (i.e. SEQ ID NO.1) for the MRPS9 gene identified is subjected to NCBI-BLASTN analysis, as a result
See Fig. 4 A.BLASTN result is summarised in table 2.The result shows that in ncbi database, SEQ ID NO.1 only a fraction sequence
Column and Sugiyamaella lignohabitans CBS 10342TChromosome A have a homology, but only 70bp or so,
This shows that the leakage annotation gene M RPS9 detected in food adenine section spore yeast has sequence-specific, although MRPS9 albumen
Amino acid sequence partial sector it is more conservative, but its DNA homology is very low.This ORF sequence for having proved the gene can be with
For developing the DNA bar code of Pu-erh tea fermentation bacterial strain food adenine section spore yeast TMCC 70007.SEQ ID NO.1 and CBS
10342TGenome homologous sequence is after DNAMAN is analyzed, and consistency 49.08%, alignment is as shown in Figure 4 B.
Table 2 and SEQ ID NO.1 have the nucleic acid sequence list of higher similarity
6, region (such as SEQ ID between SEQ ID NO.1 upstream 1000bp to downstream 1000bp has further been selected
Shown in NO.4).SEQ ID NO.4 sequence is subjected to NCBI-BLASTN analysis, retrieves Nucleotide collection (nr/
Nt) database, as a result, finding its sequence (about 70bp) and yeast Sugiyamaella for only having 2%
Lignohabitans species mitochondria 37S ribosomal protein MRPS9 part mRNA (accession number: XM_018878203.1) has
Homology, comparison result are summarised in table 3.According to the above results, it is believed that SEQ ID NO.4 can be used as identification food adenine
Save the DNA bar code sequence of spore yeast strain TMCC 70007.In addition, we further carry out SEQ ID NO.4 sequence
NCBI-BLASTN analysis, retrieves and eats adenine section spore yeast in Whole-genome shotgun contigs (wgs) database
The whole genome sequence of (taxology ID:taxid:409370).As a result, it has been found that SEQ ID NO.4 and food adenine section spore yeast
LS3 complete genome sequence Arad1A_contig_1 (CBZY010000006.1) homology is 93%, and sequential covering rate is 99%, to the greatest extent
Pipe TMCC 70007 and LS3 bacterial strain are same species, but homology segment has a base about more than 180 different.Comparison result is summarized
In table 4.It follows that food 70007 SEQ ID NO.4 of adenine section spore yeast TMCC and food adenine section spore yeast LS3
Homologous sequence in genome has enough discriminations, can be used as DNA bar code sequence.In conclusion SEQ ID NO.1
Sequence (SEQ ID NO.4) between upstream 1000bp to downstream 1000bp can be used as identification food adenine section spore yeast strain
DNA bar code.
Table 3
Table 4
7, currently, only having eaten the genome of adenine section spore yeast LS3 bacterial strain in food adenine section spore yeast species
Be reported (Kunze et al.Biotechnology for Biofuels2014,7:66).After Local-BLASTN is analyzed,
It was found that gene order SEQ ID NO.1 is in LS3 strain gene group, there are homologous sequences.After DNAMAN is analyzed, two sequences
The homology of column is 91.64%, although TMCC 70007 and LS3 bacterial strain be the same species (instant adenine section spore yeast,
Blastobotrys adeninivorans), but in 1382 bases of the DNA bar code sequence, 111 sites are not
The same, as a result as shown in Figure 5.This further demonstrates can effectively distinguish TMCC using the DNA bar code sequence
Other bacterial strains in 70007 bacterial strains and kind.
Embodiment 2. utilizes DNA bar code strain identification
According to the amplification of sample to be tested and with food 70007 bacterial strain SEQ ID NO.1's of adenine section spore yeast TMCC
Sequence homology, to determine whether sample to be tested is Pu'er tea industrial application bacterial strain food adenine section spore yeast TMCC 70007.
(1) it is based on SEQ ID NO.1, PCR primer is designed at the gene both ends using NCBI design of primers tool, amplification produces
Object must include starting and the termination site of gene, and obtaining positive and negative primer sequence is respectively MRPS9-F:5 '-
ACTCGAAAACCCTATACACCGA-3'；MRPS9-R:5 '-CTTGCACTTGTTTAGTGCCCA-3 '.Figure is seen in the position of primer
6.The sequence expanded is SEQ ID NO.7.
(2) bacterium source
The related strain information that table 5 is selected
(3) bacterial strain DNA is extracted respectively: using OMEGA E.Z.N.A.TMYeast DNA kit extract Yeast genome
DNA, and the DNA concentration of sample is diluted to 0.5 μ g/ μ L with the deionized water of sterilizing.
(4) amplification of DNA fragments, carries out polymerase chain (PCR) reaction, and the primer sequence is respectively as follows:
Forward primer sequence: MRPS9-F:5 '-ACTCGAAAACCCTATACACCGA-3 '；
Reverse primer sequences: HSP40-R:MRPS9-R:5 '-CTTGCACTTGTTTAGTGCCCA-3 '.
PCR reaction system is 50 μ L, and PCR reagent is Thermo ScientificTMTaq DNA Polymerase (weight
Group): ddH2O 37.7μL、MgCl25 μ L, dNTPs 4 μ L, 1 μ L of forward primer, 1 μ L, Taq archaeal dna polymerase of reverse primer, 0.3 μ
L, 1 μ L of DNA profiling is free of dyestuff.Amplification program are as follows: 94 DEG C initial denaturation 8 minutes；Following 94 DEG C are denaturalized 45 seconds, 56 DEG C of annealing
45 seconds, 72 DEG C extended 1 point 15 seconds, carried out 32-35 circulation altogether；Last 72 DEG C extend 10 minutes.
(5) amplified production detects: with 1.0% Ago-Gel, 1 × TBE electrophoresis liquid electrophoresis detection, using DNA
Marker detects PCR fragment size.If strain to be tested does not have amplified band, illustrating the bacterial strain not is food adenine section spore yeast
TMCC 70007；If there is obvious clearly band, and without miscellaneous band, biological order-checking company is sent to carry out DNA fragmentation sequencing.
(6) primer is only capable of realizing amplification in food adenine section spore yeast TMCC 70007, fast in infraspecific food gland
Purine section spore yeast CBS 8244T, CBS 7350, CBS 7370, CBS 8335 and Blastobotrys
raffinosifermentans CBS 6800TIn then can not achieve amplification.As a result as shown in Figure 7.In order to further verify expansion
The sequence of the DNA of increasing has carried out sequencing and sequence alignment.
(7) for having the sequencing result of the sequence of band, the sequence peak obtained after checking sequencing with software Chromas first
Plot quality is carried out forward and reverse after determining the requirement that peak figure quality reaches data analysis with the SeqMan in DNASTAR software package
The splicing of sequence.Sequencing result is subjected to manual check and correction, sequence assembly, if strain to be tested DNA fragmentation and food adenine section spore
The homology of 70007 standard gene sequence of yeast TMCC can determine whether that the strain to be tested may be food gland 99% or more
70007 bacterial strain of purine section spore yeast TMCC.It is analyzed for example, being compared by DNAMAN, the sequencing of bacterial strain TMCC 70007 is obtained into sequence
Column compare rear completely the same with DNA bar code sequence SEQ ID NO.1, further demonstrate the reliability of this method.As a result
As shown in table 4 and Fig. 8.
Table 6
SEQUENCE LISTING
<110>Menghai Tea Co., Ltd.
<120>DNA bar code, primer, kit, methods and applications
<130> FI-163710-59:52/C
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1382
<212> DNA
<213>adenine section spore yeast (Blastobotrys adeninivorans) is eaten
<400> 1
atgagagcat tgcgggctct ggtgcccaga cgcccactgg gccgccagct cctgggcatg 60
cgattctatt cggacgttcc aaagccatca tcgcccattg caaacccctt tgagcagcga 120
ctgcagggac tggtcaagga gtatgaaaaa tttaacaaca ttggctccca ggtgtctgcc 180
gacgaagctc gcgacgctga gggtgtacag tttaaagacg agaggcacct ggcccctgac 240
ctcagccgac tgagagttgt gccccgagaa cgggcctttt tcatgggcat gcctgctcat 300
gaagagatta tgcgaacctt aaacgacctt acgatgaaga accagactat tccccggtta 360
ggtcgatcag agattgaagt ccctacctgg ctgtctctta acgaatacag agacatgttg 420
ggtgctaagc actttaagca aaagtactac cgggagctcc ttacttctct gaatgacctg 480
gccctcatgg acccccagct tgtaccttca tctgtgcctg atgcactatc tcgatttgta 540
tctcttcgct ctgacggttt cattgccaag aagccaaaga ctcttgacga agtaggacga 600
gccattgcag ttggccgacg caagaccgcc agtgctcggg ttcagctcgt caagggatct 660
ggactagctc tagttaacgg aaagcctctg gatgaagcct ttgaacgtcc tgctgaccgt 720
gatgccatgt tgtaccctct caaggtcgta gctggtgaac aatcctacaa catttttgct 780
actgtcactg gaggaggaaa gaccggacag gctagtgcca ttgctcacgg aatcgctcag 840
gccctagtca ttcacaaccc tctgctactg tcccgactag ctagagccaa gtgcctcaag 900
agagatcctc gtgtcaagga gagaaagaag cctggtaagg ttaaggcccg aaaatcctac 960
acctgggtca agcgttaatg tacatatata tatacacgat tcagtcattc cattcgtcca 1020
ttcatttcgt tgtcttaatc actttgtatc tgttatgcta ctttgaactg tctgtacgtc 1080
gtttgtcgtt tgcctactgc tgcactccag atacttctct tacttcccac tttgattccg 1140
atcaccgatt cctaaataac tctcccatga acccaaagaa cgctttcgca tactttgttc 1200
aaagagccca ggctagaagc ttggtacgct acgcattccg cactgcgtac caagtacgtg 1260
atgttaatac tcgcaaggaa ctgataacat gggccagaca agagtttgaa cgaaatcgaa 1320
acgtcgaaga cccggtatgt ggacaggagg tgaaaagttt tactttgcag tcgctaactt 1380
ag 1382
<210> 2
<211> 441
<212> DNA
<213>adenine section spore yeast (Blastobotrys adeninivorans) is eaten
<400> 2
taaggcccga aaatcctaca cctgggtcaa gcgttaatgt acatatatat atacacgatt 60
cagtcattcc attcgtccat tcatttcgtt gtcttaatca ctttgtatct gttatgctac 120
tttgaactgt ctgtacgtcg tttgtcgttt gcctactgct gcactccaga tacttctctt 180
acttcccact ttgattccga tcaccgattc ctaaataact ctcccatgaa cccaaagaac 240
gctttcgcat actttgttca aagagcccag gctagaagct tggtacgcta cgcattccgc 300
actgcgtacc aagtacgtga tgttaatact cgcaaggaac tgataacatg ggccagacaa 360
gagtttgaac gaaatcgaaa cgtcgaagac ccggtatgtg gacaggaggt gaaaagtttt 420
actttgcagt cgctaactta g 441
<210> 3
<211> 401
<212> PRT
<213>adenine section spore yeast (Blastobotrys adeninivorans) is eaten
<400> 3
Met Arg Ala Leu Arg Ala Leu Val Pro Arg Arg Pro Leu Gly Arg Gln
1 5 10 15
Leu Leu Gly Met Arg Phe Tyr Ser Asp Val Pro Lys Pro Ser Ser Pro
20 25 30
Ile Ala Asn Pro Phe Glu Gln Arg Leu Gln Gly Leu Val Lys Glu Tyr
35 40 45
Glu Lys Phe Asn Asn Ile Gly Ser Gln Val Ser Ala Asp Glu Ala Arg
50 55 60
Asp Ala Glu Gly Val Gln Phe Lys Asp Glu Arg His Leu Ala Pro Asp
65 70 75 80
Leu Ser Arg Leu Arg Val Val Pro Arg Glu Arg Ala Phe Phe Met Gly
85 90 95
Met Pro Ala His Glu Glu Ile Met Arg Thr Leu Asn Asp Leu Thr Met
100 105 110
Lys Asn Gln Thr Ile Pro Arg Leu Gly Arg Ser Glu Ile Glu Val Pro
115 120 125
Thr Trp Leu Ser Leu Asn Glu Tyr Arg Asp Met Leu Gly Ala Lys His
130 135 140
Phe Lys Gln Lys Tyr Tyr Arg Glu Leu Leu Thr Ser Leu Asn Asp Leu
145 150 155 160
Ala Leu Met Asp Pro Gln Leu Val Pro Ser Ser Val Pro Asp Ala Leu
165 170 175
Ser Arg Phe Val Ser Leu Arg Ser Asp Gly Phe Ile Ala Lys Lys Pro
180 185 190
Lys Thr Leu Asp Glu Val Gly Arg Ala Ile Ala Val Gly Arg Arg Lys
195 200 205
Thr Ala Ser Ala Arg Val Gln Leu Val Lys Gly Ser Gly Leu Ala Leu
210 215 220
Val Asn Gly Lys Pro Leu Asp Glu Ala Phe Glu Arg Pro Ala Asp Arg
225 230 235 240
Asp Ala Met Leu Tyr Pro Leu Lys Val Val Ala Gly Glu Gln Ser Tyr
245 250 255
Asn Ile Phe Ala Thr Val Thr Gly Gly Gly Lys Thr Gly Gln Ala Ser
260 265 270
Ala Ile Ala His Gly Ile Ala Gln Ala Leu Val Ile His Asn Pro Leu
275 280 285
Leu Leu Ser Arg Leu Ala Arg Ala Lys Cys Leu Lys Arg Asp Pro Arg
290 295 300
Val Lys Glu Arg Lys Lys Pro Asp Thr Ser Leu Thr Ser His Phe Asp
305 310 315 320
Ser Asp His Arg Phe Leu Asn Asn Ser Pro Met Asn Pro Lys Asn Ala
325 330 335
Phe Ala Tyr Phe Val Gln Arg Ala Gln Ala Arg Ser Leu Val Arg Tyr
340 345 350
Ala Phe Arg Thr Ala Tyr Gln Val Arg Asp Val Asn Thr Arg Lys Glu
355 360 365
Leu Ile Thr Trp Ala Arg Gln Glu Phe Glu Arg Asn Arg Asn Val Glu
370 375 380
Asp Pro Val Cys Gly Gln Glu Val Lys Ser Phe Thr Leu Gln Ser Leu
385 390 395 400
Thr
<210> 4
<211> 2436
<212> DNA
<213>adenine section spore yeast (Blastobotrys adeninivorans) is eaten
<400> 4
ggttaccgat tttcactttt cactttgaaa aactcgaaaa ccctatacac cgaaatcatg 60
agagcattgc gggctctggt gcccagacgc ccactgggcc gccagctcct gggcatgcga 120
ttctattcgg acgttccaaa gccatcatcg cccattgcaa acccctttga gcagcgactg 180
cagggactgg tcaaggagta tgaaaaattt aacaacattg gctcccaggt gtctgccgac 240
gaagctcgcg acgctgaggg tgtacagttt aaagacgaga ggcacctggc ccctgacctc 300
agccgactga gagttgtgcc ccgagaacgg gcctttttca tgggcatgcc tgctcatgaa 360
gagattatgc gaaccttaaa cgaccttacg atgaagaacc agactattcc ccggttaggt 420
cgatcagaga ttgaagtccc tacctggctg tctcttaacg aatacagaga catgttgggt 480
gctaagcact ttaagcaaaa gtactaccgg gagctcctta cttctctgaa tgacctggcc 540
ctcatggacc cccagcttgt accttcatct gtgcctgatg cactatctcg atttgtatct 600
cttcgctctg acggtttcat tgccaagaag ccaaagactc ttgacgaagt aggacgagcc 660
attgcagttg gccgacgcaa gaccgccagt gctcgggttc agctcgtcaa gggatctgga 720
ctagctctag ttaacggaaa gcctctggat gaagcctttg aacgtcctgc tgaccgtgat 780
gccatgttgt accctctcaa ggtcgtagct ggtgaacaat cctacaacat ttttgctact 840
gtcactggag gaggaaagac cggacaggct agtgccattg ctcacggaat cgctcaggcc 900
ctagtcattc acaaccctct gctactgtcc cgactagcta gagccaagtg cctcaagaga 960
gatcctcgtg tcaaggagag aaagaagcct ggtaaggtta aggcccgaaa atcctacacc 1020
tgggtcaagc gttaatgtac atatatatat acacgattca gtcattccat tcgtccattc 1080
atttcgttgt cttaatcact ttgtatctgt tatgctactt tgaactgtct gtacgtcgtt 1140
tgtcgtttgc ctactgctgc actccagata cttctcttac ttcccacttt gattccgatc 1200
accgattcct aaataactct cccatgaacc caaagaacgc tttcgcatac tttgttcaaa 1260
gagcccaggc tagaagcttg gtacgctacg cattccgcac tgcgtaccaa gtacgtgatg 1320
ttaatactcg caaggaactg ataacatggg ccagacaaga gtttgaacga aatcgaaacg 1380
tcgaagaccc ggtatgtgga caggaggtga aaagttttac tttgcagtcg ctaacttagg 1440
aacaaatgag atatctaatt gctatgggca ctaaacaagt gcaagaaatg gcaaagacta 1500
ttcactaggt acactattat ggagtcacgc agttgcggag tacggtgatg gacttgtcgg 1560
acgctgcgca gatgccgtcc actacttcct ctaccagaga agcagaaatt ccattctcct 1620
cagcccaagg gctcagctgg ccagtgagat gctggaatgt ctggttgcgt gagtttttca 1680
cttgcaatgc ctgttccttg acattcttgg tcatggcact aactgcgctt ctcagggctt 1740
cgctagcatc caaattggcc tttacctgat tcctattagt tttgagacga atcatgtagc 1800
ctgatacaac attgcaagtc tccaaaattg taaatgctct gtggaaccat ttccattggc 1860
cggtctgttg ctcatcgagc gccatgcttg acagcttttg ttcaacagca gtgtatccct 1920
ccttactctc cttggcctgg gcgctggcgg ctaccagcac aatagtttcc agagcccata 1980
actcttcatc tgtaaactgg ctggagtgat gcttaataat gtcctcaaga gctttcacca 2040
agccaatgag ctcgtcaccg ccaggaacaa gcttgcggat gattttttct cggatctgga 2100
acgcatgagc ccactcggac ttctgaacgg ggcccactgt caagacctga gataagggga 2160
tctcatcagg gccattacag ttccacattg agtcaaaatc tcggttgtct tcgagggcca 2220
cagacgtatt cgtagcttcg atgaactttg tgtttgcatt gtcagtagtc aatcgctcta 2280
gtttgactgt ttccaagtcc accagtgcct tgttgagact gtgcgaaaac ttgtaatgaa 2340
ggtccataaa cccttcaatc tgagtgtagg ctccattgta ggcaaacttg atgtagttga 2400
caaagtcaga tgcctgccgg gcaatttcat aagtag 2436
<210> 5
<211> 22
<212> DNA
<213>artificial sequence
<400> 5
actcgaaaac cctatacacc ga 22
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
cttgcacttg tttagtgccc a 21
<210> 7
<211> 1454
<212> DNA
<213>adenine section spore yeast (Blastobotrys adeninivorans) is eaten
<400> 7
actcgaaaac cctatacacc gaaatcatga gagcattgcg ggctctggtg cccagacgcc 60
cactgggccg ccagctcctg ggcatgcgat tctattcgga cgttccaaag ccatcatcgc 120
ccattgcaaa cccctttgag cagcgactgc agggactggt caaggagtat gaaaaattta 180
acaacattgg ctcccaggtg tctgccgacg aagctcgcga cgctgagggt gtacagttta 240
aagacgagag gcacctggcc cctgacctca gccgactgag agttgtgccc cgagaacggg 300
cctttttcat gggcatgcct gctcatgaag agattatgcg aaccttaaac gaccttacga 360
tgaagaacca gactattccc cggttaggtc gatcagagat tgaagtccct acctggctgt 420
ctcttaacga atacagagac atgttgggtg ctaagcactt taagcaaaag tactaccggg 480
agctccttac ttctctgaat gacctggccc tcatggaccc ccagcttgta ccttcatctg 540
tgcctgatgc actatctcga tttgtatctc ttcgctctga cggtttcatt gccaagaagc 600
caaagactct tgacgaagta ggacgagcca ttgcagttgg ccgacgcaag accgccagtg 660
ctcgggttca gctcgtcaag ggatctggac tagctctagt taacggaaag cctctggatg 720
aagcctttga acgtcctgct gaccgtgatg ccatgttgta ccctctcaag gtcgtagctg 780
gtgaacaatc ctacaacatt tttgctactg tcactggagg aggaaagacc ggacaggcta 840
gtgccattgc tcacggaatc gctcaggccc tagtcattca caaccctctg ctactgtccc 900
gactagctag agccaagtgc ctcaagagag atcctcgtgt caaggagaga aagaagcctg 960
gtaaggttaa ggcccgaaaa tcctacacct gggtcaagcg ttaatgtaca tatatatata 1020
cacgattcag tcattccatt cgtccattca tttcgttgtc ttaatcactt tgtatctgtt 1080
atgctacttt gaactgtctg tacgtcgttt gtcgtttgcc tactgctgca ctccagatac 1140
ttctcttact tcccactttg attccgatca ccgattccta aataactctc ccatgaaccc 1200
aaagaacgct ttcgcatact ttgttcaaag agcccaggct agaagcttgg tacgctacgc 1260
attccgcact gcgtaccaag tacgtgatgt taatactcgc aaggaactga taacatgggc 1320
cagacaagag tttgaacgaa atcgaaacgt cgaagacccg gtatgtggac aggaggtgaa 1380
aagttttact ttgcagtcgc taacttagga acaaatgaga tatctaattg ctatgggcac 1440
taaacaagtg caag 1454
Claims (10)
1. a kind of for identifying the DNA bar code of food adenine section spore yeast strain, the DNA bar code is from food adenine section
The genome of 70007 bacterial strain of spore yeast TMCC, and include in the DNA sequence dna as shown in SEQ ID No.1 at least
The sequence of 500bp, and the length of the DNA bar code be 500bp-3000bp, preferably 500bp-2200bp, more preferably
500bp-1500bp。
2. DNA bar code according to claim 1, nucleotide sequence such as SEQ ID No.1, SEQ ID No.4 or SEQ
Shown in ID No.7.
3. a kind of for expanding the primer pair of DNA bar code according to claim 1.
4. primer pair according to claim 3, the nucleotide sequence and food adenine section spore yeast TMCC of forward primer
Such sequence in 70007 strain gene groups is identical: the sequence is from the 70007 strain gene group of TMCC such as SEQ
1st upstream 1000bp of nucleotide sequence shown in ID No.1 to the nucleotide sequence as shown in SEQ ID No.1
Sequence in 882 regions, and the length of the forward primer is 20-30bp；Its reverse primer and the TMCC 70007
Such sequence reverse complemental in strain gene group: the sequence is from the 70007 strain gene group of TMCC such as SEQ ID
The 501st of nucleotide sequence shown in No.1 arrives last downstream of the nucleotide sequence as shown in SEQ ID No.1
Sequence in the region of 1000bp, and the length of the reverse primer is 20-30bp.
5. the nucleotide sequence difference of primer pair according to claim 4, forward primer and reverse primer is as follows:
Forward primer: 5 '-ACTCGAAAACCCTATACACCGA-3 '；
Reverse primer: 5 '-CTTGCACTTGTTTAGTGCCCA-3 '.
6. it is a kind of for identifying the kit of food adenine section spore yeast strain, it include primer pair according to claim 3.
7. a kind of for identifying the method for food adenine section spore yeast strain, comprising the following steps:
A) genomic DNA of strain to be tested is provided；
B) using genomic DNA described in step a) as template, PCR amplification is carried out using primer pair according to claim 3,
Obtain PCR product；
C) electrophoresis detection PCR product, if determining strain to be tested not is food adenine section spore yeast TMCC without target stripe
70007 bacterial strains then carry out step d) if there is target stripe；
D) gained PCR product is sequenced, obtains nucleotide sequence to be measured；By the nucleotide sequence to be measured and claim
The nucleotide sequence of DNA bar code described in 1 carries out sequence analysis, if homology determines strain to be tested 99% or more
It is food 70007 bacterial strain of adenine section spore yeast TMCC.
8. DNA bar code according to claim 1 is identifying the application in food adenine section spore yeast strain.
9. primer pair according to claim 3 is identifying the application in food adenine section spore yeast strain.
10. kit according to claim 6 is identifying the application in food adenine section spore yeast strain.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105779297A (en) * | 2014-12-16 | 2016-07-20 | 勐海茶业有限责任公司 | Strain of Arxula adeninivorans for producing high activity polyphenoloxidase and application thereof to production of Pu'er tea |
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Non-Patent Citations (1)
| Title |
|---|
| GOTTHARD KUNZE ET AL.: "The complete genome of Blastobotrys(Arxula) adeninivorans LS3- a yeast of biotechnological interest", 《BIOTECHNOLOGY FOR BIOFUELS》 * |
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