CN107794262A - The ribosomal RNA sequences of mulberry tree Alternaria alternata caused occurrence and its application - Google Patents

The ribosomal RNA sequences of mulberry tree Alternaria alternata caused occurrence and its application Download PDF

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CN107794262A
CN107794262A CN201710064069.4A CN201710064069A CN107794262A CN 107794262 A CN107794262 A CN 107794262A CN 201710064069 A CN201710064069 A CN 201710064069A CN 107794262 A CN107794262 A CN 107794262A
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刘吉平
刘希
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South China Agricultural University
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Abstract

Present invention firstly discloses a kind of mulberry tree Alternaria alternata caused occurrencePseudocercospora moriRRNA full length cDNA sequence and its application.Pseudocercospora moriRRNA full length cDNA sequence as shown in SEQ.ID.NO1, its be segmented cDNA sequence as shown in SEQ.ID.NO2, the 2 pairs of primer sequences included in its sequence are as shown in SEQ.ID.NO3.The present invention willPseudocercospora moriRRNA cDNA sequence be applied to detectionPseudocercospora moriIn bacterium, qualitative and quantitative result can be obtained simultaneously.As a result show, on to mulberry tree black spot disease leaf in the research of fungi, relative abundance highest fungi isPseudocercosporaBelong to pathogen.In addition,Pseudocercospora moriThe cDNA sequence of rRNA can be applied in the research in terms of fungi kind classification.

Description

The ribosomal RNA sequences of mulberry tree Alternaria alternata caused occurrence and its application
Technical field
The invention belongs to biological technical field, more particularly to a kind of mulberry tree black spot cause of disease Pseudocercospora Mori ribosomal RNA sequences and its application.
Background technology
In existing fungal studies method, it is sequenced often through rDNA (ribosomeDNA, rDNA) sequence Compare the identification for fungi.Ribosomes has critical function in cell, and the gene many of rDNA codings is all closed with protein Into course of reaction it is closely related, played a decisive role in the biosynthesis of protein.RDNA sequences are divided into transcriptional domain and non-turn Record area, gene and intergenic 2 transcribed spacer of the transcriptional domain by encoding ribosomal 5.8S, 18S, 28S protein structure (InternalTranscribed Spacer, ITS) ITS1 and ITS2 compositions, collectively constitute a transcript unit.
It is more conservative that 5.8S, 18S, 28S rDNA sequences are encoded in rDNA, can be used for analyzing nature species taxonomy The molecular labeling of blood system research between section or between higher level rank member.Because 5.8SrDNA sequences are short and highly conserved, it is difficult to be used for The systematic growth of fungi and Molecular Identification；And 18SrDNA fragment is longer, conserved region and variable region in fragment be present, it is therefore, existing Have in research, after selecting different specificity amplification primers to expand a certain domain fragment, by sequencing and to surveying The analyses and comparison of sequence result, the research available for taxonomic categories such as eubacteriales, section, category.But based on 5.8S, 18S, 28S RDNA sequences are difficult to characterization of molecules of the reflection comprehensively to the classification of fungi kind, or even can not effectively determine the kind of disease fungus Category, and kind of (species) horizontal discriminating is often carried out using ITS sequence.But the high variability of ITS sections, applying In there are many problems, therefore, using transcription group technology obtain rRNA full length sequence, each gene region it is excellent lack Point integrates, carry out the classification of fungal species and identification research be modern biotechnology development inevitable choice.
The content of the invention
The technical problem to be solved in the present invention is to make up the blank of prior art, there is provided a kind of mulberry tree Alternaria alternata caused occurrence The full length cDNA sequence of Pseudocercospora mori rRNA.
It is another object of the present invention to provide mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori ribose Application of the body RNA sequence in quantitative detection mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori.
It is still another object of the present invention to provide mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori ribosomes The method that RNA sequence is applied in quantitative detection mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori.
Another object of the present invention is to provide mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori cores Application of the sugared body RNA sequence in terms of fungi kind classification.
The purpose of the present invention is achieved by the following technical programs：
The invention provides a kind of total length of mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori rRNAs CDNA sequence, as shown in SEQ.ID.NO1.
The rRNA is made up of 18S rRNA, ITS1,5.8S rRNA, ITS2,28S rRNA；The 18S rRNA CDNA sequence be in sequence shown in SEQ.ID.NO1 shown in the 1st~1726 base sequence；Shown ITS1 cDNA sequence is The 1727th~1876 in sequence shown in SEQ.ID.NO1, the cDNA sequence of the 5.8S rRNA is sequence shown in SEQ.ID.NO1 In the 1877th~2034, the cDNA sequence of the ITS2 is the 28S the 2035th~2183 in sequence shown in SEQ.ID.NO1 RRNA cDNA sequence is the 2184th~5469 in sequence shown in SEQ.ID.NO1.
Include 2 pairs of primer sequences in the full length cDNA sequence of the rRNA, the primer sequence is respectively such as Shown in SEQ.ID.NO2~SEQ.ID.NO5.
Present invention also offers the mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori rRNAs Application of the cDNA sequence in quantitative detection mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori.
Present invention also offers the mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori rRNAs The method that cDNA sequence is applied in quantitative detection mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori, including following step Suddenly：
S1. mulberry tree disease leaf is collected；
S2. the STb gene of mulberry tree disease leaf is extracted；
S3.IlluminaDNA library constructions；
S4.Illumina high-flux sequences；
S5. mulberry tree genome sequence in sequencing data is removed；
S6. microbial genome sequence is assembled；
S7. complete ribosomal dna sequence is assembled；
S8. comparative analysis ribosomal dna sequence；
The method of Illumina DNA libraries structure is described in step S3：According to Illumina library construction flows, by institute State double end high-throughput sequencing libraries that STb gene described in step S2 is configured to clip size 500bp；
The method of mulberry tree genome sequence is in removal sequencing data described in step S5：Using comparing software in step S4 The high-flux sequence data carry out comparing analysis.Alignment algorithm is selected, by the sequencing data and mulberry tree reference gene Group is compared, and the sequencing data for comparing upper reference gene group is determined as into mulberry tree genome sequence.Use what is write Computer program removes mulberry tree genome sequence from the sequencing data.
Described in S5 remove mulberry tree genomic dna sequence select reference gene group sequence be：
Morus (Morus notabilis) whole genome sequence (GCA_000414095.2) and Chloroplast gene sequence (NC_027110.1)。
Preferably, the comparison software is bwa (0.7.12-r1039) software；
Preferably, the alignment algorithm is mem alignment algorithms；
Preferably, it is described by sequencing data and mulberry tree reference gene group selection is compared be double end comparison methods and The default parameters of bwa (0.7.12-r1039) software；
Preferably, the computer program write is write with python computer languages.
The method of assembling microbial genome sequence is described in step S6：Using composite software to having been removed described in step S5 The sequencing data of mulberry tree genome sequence is assembled.
Preferably, the composite software is MetaVelvet (v1.2.01).
The method of the complete ribosomal dna sequence of assembling is described in step S7：Using software is compared, the assembling sequence is entered Row compares, and obtains double end sequencing fragments from sequencing data according to comparison result, sequence is assembled using composite software And extension, through multiple circulate operations, until obtaining complete ribosomal dna sequence；
Preferably, the comparison software is bwa (0.7.12-r1039) software；
Preferably, the comparison method is used without mispairing 0mismatch and compared without fracture 0gap；
Preferably, the composite software is MetaVelvet (v1.2.01) software.
The rRNA for additionally providing mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori of the present invention Application of the cDNA sequence in terms of fungi kind classification.
The invention has the advantages that：
The present invention provides the rRNA of the mulberry tree Alternaria alternata caused occurrence Pseudocercospora mori first CDNA sequence, a kind of new fungi kind sorting technique is established based on the sequence, especially it is quantitative detection mulberry tree it is black Pinta pathogen Pseudocercospora mori have good application.
Brief description of the drawings
The two pairs of primers and universal primer ITS1/ITS4 electrophoretograms of Fig. 1 checking ribosomes assembling results.
Fungi microbe classification tree detected by Fig. 2 mulberry leaf blackspot blades.
The special primer sequence of Fig. 3 checkings is used for cause of disease PCR detections and the qualification result of mulberry tree dirt leaf disease.
Embodiment
The application process of the present invention is further illustrated with reference to specific embodiment.Following embodiments and accompanying drawing are only used for showing Example property explanation, it is impossible to be interpreted as limitation of the present invention.Unless stated otherwise, the reagent raw material used in following embodiments is normal Advise the biochemical reagents raw material that purchased in market or commercial sources obtain, unless stated otherwise, the method and apparatus used in following embodiments For method and apparatus commonly used in the art.
Embodiment 1
The blade with typical black spot scab searched out at random in the mulberry field of morbidity, collect, the disease in clip mulberry leaf Spot region, the scab materials'use liquid nitrogen cut are fully ground, and extract the total serum IgE of mulberry tree disease leaf；The total serum IgE of extraction is stored in- 80℃.By total serum IgE reverse transcription into cDNA library；Design primer sequence and universal primer sequence Its1, Its4 respectively such as Shown in SEQ.ID.NO2~SEQ.ID.NO7,1 is shown in Table.Enter performing PCR amplification by template of cDNA library；PCR reaction systems are shown in Table 2 It is shown.
The PCR of table 1 verifies primer sequence table
The PCR reaction systems of table 2 (20 μ L)
Universal primer ITS primer sets PCR programs：94 DEG C of 5min of pre-degeneration；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 30 Circulation；72℃10min.
Verify the P1-F/R primer sets and P2-F/R primer sets PCR reaction systems (20 μ L) reaction bar of ribosomes assembling result Part：94℃5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 2min, 30 circulations；72℃7min.5 μ L pcr amplification product is taken to use 1.2% Ago-Gel (EB dyeing) electrophoresis detection.PCR primer piece corresponding to size is reclaimed by agarose gel electrophoresis Section, as shown in accompanying drawing 1, in Fig. 1, M:Takara DL2000 Marker；1. fungi universal primer ITS1 and ITS4；2. verify core Sugared body assembling result primer P1-F and P1-R；3. verify ribosomes assembling result primer P2-F and P2-R；4. blank water compares.
The fragment of recovery is subjected to Sanger sequencings；Then by sequencing result and mulberry tree Alternaria alternata caused occurrence The full length cDNA sequence of Pseudocercospora mori rRNA is compared, thereby determine that on blade whether have it is black Pinta pathogen Pseudocercospora mori, and can thus speculate Alternaria alternata caused occurrence Pseudocercospora mori Whether it is probably pathogenic bacteria.
As shown in Figure 1, the result of swimming lane 1 illustrates that with the template DNA of fungi universal primer ITS1 and ITS4 amplification be to belong to The DNA of fungi, and verify that the P1 primer sets of ribosomes assembling result and the PCR results of P1 primer sets are (swimming lane 2 and 3) respectively, Corresponding purpose band is arrived in amplification, and further the result after sequencing and the result assembled are highly consistent.
The application experiment of embodiment 2
The blade with typical black spot scab searched out at random in the mulberry field of morbidity, collect, the disease in clip mulberry leaf Spot region, the scab materials'use liquid nitrogen cut are fully ground, and it is century fungal DNA extracts reagent that the extraction of STb gene, which uses health, Box, is specifically carried out according to its operating instruction, and the STb gene after extraction is stored in -20 DEG C.According to Illumina library construction flows, STb gene is configured to clip size 500bp double end high-throughput sequencing libraries, uses Illumina Hiseq2500 sequenators High-flux sequence is carried out to the DNA library built, it is double end 125bp to fragment, sequencing reading length is sequenced to measure 18.56M altogether, Total sequencing data amount 4.64Gb.
Due to including blade material in DNA extraction process, be reduce sequencing data in mulberry tree genomic data to microorganism The influence of sequence assembling, remove the genomic dna sequence of mulberry tree first before microorganism sequence assembling is carried out.Choose Morus Notabilis whole genome sequences (GCA_000414095.2) and Chloroplast gene sequence (NC_027110.1) are as reference Genome sequence, software is compared using bwa (0.7.12-r1039) and carries out comparing analysis.Selection mem alignment algorithms are compared, Using double end comparison methods and the default parameters of software, sequencing data is compared with mulberry tree reference gene group, and will compare Mulberry tree genome sequence is determined as to the sequencing fragment of upper reference gene group.Using the computer program that python writes from Mulberry tree sequencing data is removed in fastq sequencing datas, then enters back into microorganism sequence assembling.The assembling of microorganism sequence uses MetaVelvet (v1.2.01) composite software is carried out.
The rDNA of target disease fungus is by 18S sections, ITS1 sections, 5.8S sections, ITS2 sections and 28S sections Composition, sequence total length is about 6Kb.The sequence label that MetaVelvet (v1.2.01) is initially assembled is the ribosomes mark of fracture Label, to obtain complete ribosomal dna sequence, analysis uses sequence capturing and from the beginning packaging strategy, to assemble complete ribose Body DNA.Ribosomal dna sequence of the selection comprising target pathogenic bacteria ITS sequence is reference sequences, using bwa (0.7.12- R1039) software carries out 0mismatch and 0gap comparisons, obtains double end sequencing fragments from sequencing data according to comparison result, Further sequence is assembled and extended using MetaVelvet (v1.2.01) composite software, through multiple circulate operations, is obtained Complete ribosomal dna sequence.
Sequence label annotation use blastn (2.2.31+) sequence alignment analysis software, the sequence label sequence of assembling and NCBI nt databases are compared, and blastn compares setting desired value<1e-20, sequence label is carried out according to comparison result Annotation.Ribosomal dna sequence is the important the most frequently used molecular labeling of bacterium and Fungal identification, thus species taxonomy identification and Quantify using rDNA as main molecular labeling.Result is annotated according to sequence label, selects ribosomal dna sequence as micro- life Thing is identified and quantitative analysis foundation.Using bwa (0.7.12-r1039)+samtools (v1.2) analysis software, sequencing number is calculated According to the average sequencing depth of middle ribosomes DNA fragmentation, and in this, as the Abundances of the species.
As a result show, 61 rRNA sequence labels of note 2, packet contain 232 bacterial sequences labels, 29 fungi sequences altogether Column label, corresponding 16 kinds of fungies.By to sequence label annotate result inquiry and with mulberry tree Alternaria alternata caused occurrence The comparison of the full length cDNA sequence of Pseudocercospora mori rRNAs, discovery relative abundance highest fungi are Pseudocercospora belongs to pathogen, as shown in accompanying drawing 2.
The checking test of embodiment 3
Mulberry vacation tail spore Pseudocercospora mori rDNAs are total by the mulberry leaf (scab) for extracting introduced disease DNA can quickly carry out Testing and appraisal, the RNA experiment higher without being related to requirement.For further checking and utilize institute of the present invention State the Pathogen test and mirror applied to mulberry tree dirt leaf disease of the complete genome sequence of Pseudocercospora mori rRNAs Fixed, result is affirmative.The present invention further devises a pair of special primer sequence 4w1724F/4W2196R respectively such as Shown in SEQ.ID.NO8 and SEQ.ID.NO9,3 are shown in Table：
A pair of special primers of complete genome sequence design of the table 3 based on Pseudocercospora mori rRNAs Sequence
The target fragment size for intending amplification is about 473bp.Shown in electrophoresis result as accompanying drawing 3, in accompanying drawing 3, M：Takara DL1000Marker；1. mulberry dirt leaf disease DNA of fruiting body；2.Cladosporium cladosporioides (branch spore sample branch spores It is mould)；3.Cladosporiumperangustum (thin spore branch spore)；4.Cladosporium oxysporum (sharp spore cladosporium)； 5.Penicillium verruculosum (penicillium verruculosum)；6.Penicillium citrinum (Penicillium citrinum), 7.Aspergillus (aspergillus)；8.Phanerochaete chrysosporium (Phanerochaete chrysosporium)；9.Ceriporia Mellea (honey color wax bacterium)；10；Beauveria bassiana (beauveria bassiana)；11.Schizophyllum Commune (schizophyllum commune)；12.Candida mucifera (Candida)； 13.Lasiodiplodiapseudotheobromae (longan Jiao's maize ear rot bacterium)；14.Pseudomonas aeruginosa (verdigris Pseudomonad)；15.Alcaligenesfaecalis (Alcaligenes faecalis) 16.Phyllactinia moricola (powdery mildews in mulberry Pathogen-mulberry ball pin shell)；17.Ciboria carunculoides (mulberry sclerotiniose pathogen-caruncula shape cup cup fungi)；18. sun Property control (the scab DNA of mulberry dirt leaf disease pathogen)；19, negative control (mulberry tree DNA)；20. blank control (water).1-15 is this The fungi and bacterium that invention laboratory is separately cultured, and species identification has been carried out according to international species bar code, as above.Wherein, 1-13 is various fungies；14-15 is bacterium；16-17 is other mulberry tree nosomycosis pathogens.
Fig. 3 electrophoresis result, find only have the scab of mulberry dirt leaf disease pathogen or cause of disease fructification template DNA to amplify mesh Tap section, size is about 473bp, and between 400 and 500, from figure, other bacterium are without the amplification piece of similar size Section, and the target fragment brightness that mulberry dirt leaf disease pathogen is expanded is clear, and it is remote bright in Marker.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>The ribosomal RNA sequences of mulberry tree Alternaria alternata caused occurrence and its application
<130>
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 5469
<212> DNA
<213>The full length cDNA sequence of rRNA
<400> 1
ctatacggtg aaactgcgaa tggctcatta aatcagttat cgtttatttg atagtacctt 60
actacatgga taaccgtggt aattctagag ctaatacatg ctaaaaaccc caacttcgga 120
aggggtgtat ttattagata aaaaaccaat gcccttcggg gctccttggt gaatcataat 180
aacttcacga atcgcatggc cttgcgccgg cgatggttca ttcaaatttc tgccctatca 240
actttcgatg gtaggataga ggcctaccat ggtttcaacg ggtaacgggg aattagggtt 300
cgactccgga gagggagcct gagaaacggc taccacatcc aaggaaggca gcaggcgcgc 360
aaattaccca atcccgacac ggggaggtag tgacaataaa tactgataca gggctctttt 420
gggtcttgta attggaatga gtacaattta aatcccttaa cgaggaacaa ttggagggca 480
agtctggtgc cagcagccgc ggtaattcca gctccaatag cgtatattaa agttgttgca 540
gttaaaaagc tcgtagttga accttgggcc tggctggccg gtccgcctca ccgcgtgtac 600
tggtccggcc gggcctttcc ttctggggag cctcatgccc ttcactgggc gtgttgggga 660
accaggactt ttactttgaa aaaattagag tgttcaaagc aggcctttgc tcgaatacat 720
tagcatggaa taatagaata ggacgtgtgg ttctattttg ttggtttcta ggaccaccgt 780
aatgattaat agggacagtc gggggcatca gtattccgtt gtcagaggtg aaattcttgg 840
atttacggaa gactaactac tgcgaaagca tttgccaagg atgttttcat taatcaggaa 900
cgaaagttag gggatcgaag acgatcagat accgtcgtag tcttaaccat aaactatgcc 960
gactagggat cggtggatgt tatctttttg actccatcgg caccttacga gaaatcaaag 1020
tttttgggtt ctggggggag tatggtcgca aggctgaaac ttaaagaaat tgacggaagg 1080
gcaccaccag gcgtggagcc tgcggcttaa tttgactcaa cacggggaaa ctcaccaggt 1140
ccagacacaa gtaggattga cagattgaga gctctttctt gattttgtgg gtggtggtgc 1200
atggccgttc ttagttggtg gagtgatttg tctgcttaat tgcgataacg aacgagacct 1260
taacctgcta aatagccagg cccgctttgg cgggtcgccg gcttcttaga gggactatcg 1320
gctcaagccg atggaagttt gaggcaataa caggtctgtg atgcccttag atgttctggg 1380
ccgcacgcgc gctacactga cagagccaac gagttcatca ccttggccgg aaggtctggg 1440
taatcttgtt aaactctgtc gtgctgggga tagagcattg caattattgc tcttcaacga 1500
ggaatgccta gtaagcgcat gtcatcagca tgcgttgatt acgtccctgc cctttgtaca 1560
caccgcccgt cgctactacc gattgaatgg ctcagtgagg cctccggact ggcccaggga 1620
ggtcggcaac gaccacccag ggccggaaag ttggtcaaac tcggtcattt agaggaagta 1680
aaagtcgtaa caaggtctcc gtaggtgaac ctgcggaggg atcattactg agtgagggct 1740
cacgcccgac ctccaaccct ttgtgaacca aacttgttgc ttcgggggcg accctgccga 1800
cgactccgtc gccgggcgcc cccggaggtc ttctaaacac tgcatctttg cgtcggagtt 1860
tcaaacaaat gaaacaaaac tttcaacaac ggatctcttg gttctggcat cgatgaagaa 1920
cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga 1980
acgcacattg cgccctttgg tattccgaag ggcatgcctg ttcgagcgtc atttcaccac 2040
tcaagcctgg cttggtattg ggcgccgcgg tgtttccgcg cgcctgaaag tcttccggct 2100
gagctgtccg tctctaagcg ttgtggattt ttcaattcgc ttcggagtgc gggcggccgc 2160
ggccgttaaa tctttattca aaggttgacc tcggatcagg tagggatacc cgctgaactt 2220
aagcatatca ataagcggag gaaaagaaac caacagggat tgccctagta acggcgagtg 2280
aagcggcaac agctcaaatt tgaaatctgg cgtaagcccg agttgtaatt tgtagaggat 2340
gcttctgggt agcggccggt ctaagttcct tggaacagga cgtcatagag ggtgagaatc 2400
ccgtatgtga ctggcttgca ccctccacgt agctccttcg acgagtcgag ttgtttggga 2460
atgcagctct aaatgggagg taaatttctt ctaaagctaa ataccggcca gagaccgata 2520
gcgcacaagt agagtgatcg aaagatgaaa agcactttgg aaagagagtt aaaaagcacg 2580
tgaaattgtt gaaagggaag cgcccgcaac cagactttgc ggcggtgttc ggccggtctt 2640
ctgaccggtt tactcgccgc cgtgaggcca tcatcgtctg ggaccgctgg ataagacctg 2700
aggaatgtag ctcccttcgg ggtgtgttat agcctctggt gatgcagcgc gtctcgggcg 2760
aggtccgcgc ttcggcaagg atgatggcgt aatggttgtc ggcggcccgt cttgaaacac 2820
ggaccaagga gtctaacatc tatgcgagtg ttcgggtgtc aaacccctac gcgtaatgaa 2880
agtgaacgga ggtgggaact ttttgtgcac catcgaccga tcctgatgtc ctcggatgga 2940
tttgagtaag agcatagctg ttgggacccg aaagatggtg aactatgcct gaatagggtg 3000
aagccagagg aaactctggt ggaggctcgc agcggttctg acgtgcaaat cgatcgtcaa 3060
atttgggtat aggggcgaaa gactaatcga accatctagt agctggttcc tgccgaagtt 3120
tccctcagga tagcagtaac gttttcagtt ttatgaggta aagcgaatga ttagaggcct 3180
tggggttgaa acaaccttaa cctattctca aactttaaat atgtaagaag tccttgttac 3240
ttagttgaac gtggacattt gaatgtaccg ttactagtgg gccatttttg gtaagcagaa 3300
ctggcgatgc gggatgaacc gaacgcgagg ttaaggtgcc ggaatatacg ctcatcagac 3360
accacaaaag gtgttagttc atctagacag caggacggtg gccatggaag tcggaatccg 3420
ctaaggagtg tgtaacaact cacctgccga atgaactagc cctgaaaatg gatggcgctt 3480
aagcgtatta cccatacctc gccgccaggg tagaaacgat gccctggcga gtaggcaggc 3540
gtggaggctc gtgacgaagc cttcggagtg atccggggta gaacagcctc tagtgcagat 3600
cttggtggta gtagcaaata ctcaaatgag aactttgagg actgaagtgg ggaaaggttc 3660
cgtgtgaaca gcagttggac acgggttagt cgatcctaag ccatagggaa gttccgtttt 3720
aaagtgtgcg ctccgcaccg cctggcgaaa gggaagccgg ttaacattcc ggcacctcga 3780
tgtggattat ccgcggcaac gcaactgaag gtggagacgt cggcgggggc cccgggaaga 3840
gttctctttt cttcttaacg gtccatcacc ctgaaatcgg tttgtccgga gctagggttt 3900
aacgaccggt agagcggcac acctttgtgc cgtccggtgc gctcccgacg acccttgaaa 3960
atccgccgga aggaatgatt ttcacgcgag gtcgtactca taaccgcagc aggtctccaa 4020
ggtgaacagc ctctagttga tagaacaatg tagataaggg aagtcggcaa aatagatccg 4080
taacttcggg aaaaggattg gctctaaggg ttgggcgcgt tgggccttgg gcagattccc 4140
cgggagcagg tcggcactag cttcacggcc ggcgccttcc agcacccggt ggcggacgcc 4200
cttggcaggc ttcggccgtc cggcgcgcgc ttaacaacca acttagaact ggtacggaca 4260
aggggaatct gactgtctaa ttaaaacata gcattgcgat ggtcagaaag tgatgttgac 4320
gcaatgtgat ttctgcccag tgctctgaat gtcaaagtga agaaattcaa ccaagcgcgg 4380
gtaaacggcg ggagtaacta tgactctctt aaggtagcca aatgcctcgt catctaatta 4440
gtgacgcgca tgaatggatt aacgagattc ccactgtccc tatctactat ctagcgaaac 4500
cacagccaag ggaacgggct tggcagaatc agcggggaaa gaagaccctg ttgagcttga 4560
ctctagtttg acattgtgaa aagacatagg gggtgtagaa taggtgggag cttcggcgcc 4620
ggtgaaatac cactaccctt atcgtttttt tacttaatca atgaagcgga actggtcttc 4680
accgaccatt ttctggcgtt aaggtccttc gcgggccgat ccgggttgat gacattgtca 4740
ggtggggagt ttggctgggg cggcacatct gttaaaccat aacgcaggtg tcctaagggg 4800
gactcatgga gaacagaaat ctccagtaga gcaaaagggc aaaagtcccc ttgattttga 4860
ttttcagtgt gaatacaaac catgaaagtg tggcctatcg atcctttagt ccctcgaaat 4920
ttgaggctag aggtgccaga aaagttacca cagggataac tggcttgtgg cagccaagcg 4980
ttcatagcga cgttgctttt tgatccttcg atgtcggctc ttcctatcat accgaagcag 5040
aattcggtaa gcgttggatt gttcacccac taatagggaa cgtgagctgg gtttagaccg 5100
tcgtgagaca ggttagtttt accctactga tgaccgtcgt cccaatggta ataccgctta 5160
gtacgagagg aaccgcggtt tcagataatt ggtttttgcg gctgtccgac cgggcagtgc 5220
cgcgaagcta ccatctgctg gattatggct gaacgcctct aagtcagaat ccatgccaga 5280
acgggacgat cctctctagc acgccttagg cggataagaa taggcactgc cagtacccgg 5340
gaccctctca tctcttgcag gacacgcaag agcgaagggc gtatcgtaat ttaatcgcgc 5400
gctaggatga atcccttgca gacgacttgg acgtctgacc gggtcgtgta agcagtcgag 5460
tagccttgt 5469
<210> 2
<211> 20
<212> DNA
<213>Primer P1-F sequences
<400> 2
gtttcaacgg gtaacgggga 20
<210> 3
<211> 20
<212> DNA
<213>Primer P1-R sequences
<400> 3
tccctacctg atccgaggtc 20
<210> 4
<211> 20
<212> DNA
<213>Primer P2-F sequences
<400> 4
gcatgcgttg attacgtccc 20
<210> 5
<211> 20
<212> DNA
<213>Primer P2-R sequences
<400> 5
ggtgaagacc agttccgctt 20
<210> 6
<211> 19
<212> DNA
<213>Universal primer ITS1 sequences
<400> 6
tccgtaggtg aacctgcgg 19
<210> 7
<211> 20
<212> DNA
<213>Universal primer ITS4 sequences
<400> 7
tcctccgctt attgatatgc 20
<210> 8
<211> 21
<212> DNA
<213>Primer sequence 4w1724F sequences
<400> 8
gctacactga cagagccaac g 21
<210> 9
<211> 19
<212> DNA
<213>Primer sequence 4W2196R sequences
<400> 9
tgaaactccg acgcaaaga 19

Claims (8)

  1. A kind of 1. mulberry tree Alternaria alternata caused occurrencePseudocercospora mori RRNA, it is characterised in that the ribose Body RNA full length cDNA sequence is as shown in SEQ.ID.NO1.
  2. 2. mulberry tree Alternaria alternata caused occurrence according to claim 1Pseudocercospora mori RRNA, its feature It is, the rRNA is made up of 18S rRNA, ITS1,5.8S rRNA, ITS2,28S rRNA；The 18S rRNA's CDNA sequence is shown in the 1st~1726 base sequence in sequence shown in SEQ.ID.NO1；Shown ITS1 cDNA sequence is The 1727th~1876 in sequence shown in SEQ.ID.NO1, the cDNA sequence of the 5.8S rRNA is sequence shown in SEQ.ID.NO1 In the 1877th~2034, the cDNA sequence of the ITS2 is the 28S the 2035th~2183 in sequence shown in SEQ.ID.NO1 RRNA cDNA sequence is the 2184th~5469 in sequence shown in SEQ.ID.NO1.
  3. 3. mulberry tree Alternaria alternata caused occurrence according to claim 1Pseudocercospora mori RRNA, its feature It is, includes 2 pairs of primer sequences in the full length cDNA sequence of the rRNA, the primer sequence is respectively such as Shown in SEQ.ID.NO2~SEQ.ID.NO5.
  4. 4. mulberry tree Alternaria alternata caused occurrence according to claim 1 or claim 2Pseudocercospora mori RRNA sequence It is listed in quantitative detection mulberry tree Alternaria alternata caused occurrencePseudocercospora moriApplication.
  5. 5. application according to claim 4, it is characterised in that the method for the application comprises the following steps：
    S1. mulberry tree disease leaf is collected；
    S2. the STb gene of mulberry tree disease leaf is extracted；
    S3. IlluminaDNA library constructions；
    S4. Illumina high-flux sequences；
    S5. mulberry tree genome sequence in sequencing data is removed；
    S6. microbial genome sequence is assembled；
    S7. complete ribosomal dna sequence is assembled
    S8. comparative analysis ribosomal dna sequence；
    The method of Illumina DNA libraries structure is described in step S3：According to Illumina library construction flows, by the step STb gene is configured to clip size 500bp double end high-throughput sequencing libraries described in rapid S2；
    It is to the method for comparative analysis ribosomal dna sequence described in step S9：Using sequence alignment analysis software, by step S7 institutes The full length cDNA sequence that complete ribosomal dna sequence is stated with mulberry tree dirt leaf disease/Alternaria alternata caused occurrence rRNA is compared.
  6. 6. according to the method for claim 5, it is characterised in that mulberry tree genome in sequencing data is removed described in step S15 The method of sequence is：Comparing analysis is carried out to high-flux sequence data described in step S14 using software is compared, selects ratio To algorithm, the sequencing data is compared with mulberry tree reference gene group, and the sequencing that upper reference gene group will be compared Data judging is mulberry tree genome sequence, and mulberry tree genome sequence is removed from the sequencing data using the computer program write Row；
    Described in S15 remove mulberry tree genomic dna sequence select reference gene group sequence be：
    Morus morus notabili（Morus notabilis）Whole genome sequence（GCA_000414095.2）With Chloroplast gene sequence （NC_027110.1）；
    The method of assembling microbial genome sequence is described in step S16：Using composite software to having removed mulberry described in step S15 The sequencing data of tree genome sequence is assembled；
    The method of the complete ribosomal dna sequence of assembling is described in step S17：Using software is compared, the assembling sequence is carried out Compare, double end sequencing fragments obtained from sequencing data according to comparison result, sequence is carried out using composite software assembling with Extension, through multiple circulate operations, until obtaining complete ribosomal dna sequence.
  7. 7. according to the method for claim 6, it is characterised in that it is bwa that software is compared described in step S15（0.7.12- r1039）Software；Alignment algorithm described in step S15 is mem alignment algorithms；Sequencing data and mulberry tree are referred into base described in step S15 It is double end comparison methods and bwa because group is compared（0.7.12-r1039）The default parameters of software；Write described in step S15 Computer program preferably write with python computer languages；Composite software described in step S16 is MetaVelvet （v1.2.01）；It is bwa that software is compared described in step S17（0.7.12-r1039）Software；Comparison method described in step S17 uses Compared without the mismatch of mispairing 0 and without 0 gap of fracture；Composite software described in step S17 is MetaVelvet（v1.2.01）It is soft Part.
  8. 8. mulberry tree Alternaria alternata caused occurrence according to claim 1 or claim 2Pseudocercospora mori RRNA sequence It is listed in the application in terms of fungi kind classification.
CN201710064069.4A 2017-01-24 2017-01-24 Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof Active CN107794262B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197050A (en) * 2020-01-08 2020-05-26 华南农业大学 Ribosomal RNA gene of mulberry pseudoblight pathogenic bacteria and application thereof
CN112553219A (en) * 2020-12-29 2021-03-26 华南农业大学 Method for detecting alternaria leaf spot based on ribosome 28s gene

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197050A (en) * 2020-01-08 2020-05-26 华南农业大学 Ribosomal RNA gene of mulberry pseudoblight pathogenic bacteria and application thereof
CN111197050B (en) * 2020-01-08 2023-08-18 华南农业大学 Ribosomal RNA gene of mulberry pseudo-blight pathogen and application thereof
CN112553219A (en) * 2020-12-29 2021-03-26 华南农业大学 Method for detecting alternaria leaf spot based on ribosome 28s gene

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