CN107794262B - Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof - Google Patents
Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof Download PDFInfo
- Publication number
- CN107794262B CN107794262B CN201710064069.4A CN201710064069A CN107794262B CN 107794262 B CN107794262 B CN 107794262B CN 201710064069 A CN201710064069 A CN 201710064069A CN 107794262 B CN107794262 B CN 107794262B
- Authority
- CN
- China
- Prior art keywords
- sequence
- mulberry
- software
- dna
- ribosomal rna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a mulberry black spot pathogenic bacterium for the first timePseudocercospora moriThe full-length cDNA sequence of ribosomal RNA of (1) and use thereof.Pseudocercospora moriThe full-length cDNA sequence of the ribosomal RNA is shown in SEQ.ID.NO1, the segmented cDNA sequence is shown in SEQ.ID.NO2, and the sequence of 2 pairs of primers contained in the sequence is shown in SEQ.ID.NO3. The invention is toPseudocercospora moriThe cDNA sequence of ribosomal RNA of (1) is applied to the detectionPseudocercospora moriIn the bacteria, qualitative and quantitative results can be obtained simultaneously. The results show that in the study on the fungus on the black spot disease leaves of the mulberry, the fungus with the highest relative abundance isPseudocercosporaBelongs to pathogenic bacteria. In addition to this, the present invention is,Pseudocercospora morithe cDNA sequence of ribosomal RNA of (a) can be applied to the research of fungal species classification.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a ribosomal RNA sequence of mulberry black spot pathogen Pseudocercospora mori and application thereof.
Background
In the existing methods for fungal research, sequencing alignment is often performed by ribosomal DNA (rDNA) sequence for identification of fungi. Ribosomes have important functions in cells, and many of the rDNA-encoded genes are closely involved in the reaction process of protein synthesis and play a decisive role in protein biosynthesis. The rDNA sequence is divided into a transcription region and a non-transcription region, the transcription region is composed of genes encoding ribosome 5.8S, 18S and 28S protein structures and 2 transcription spacers (ITS) ITS1 and ITS2 among the genes, and a transcription unit is formed by the two regions.
rDNA sequences encoding 5.8S, 18S and 28S are conserved in rDNA, and can be used as molecular markers for analyzing blood series research between families or higher order elements of natural species classification. 5.8SrDNA is short in sequence and highly conserved, so that the method is difficult to be used for phylogeny and molecular identification of fungi; the 18SrDNA fragment is long, and a conservative region and a variable region exist in the fragment, so that in the existing research, after a certain domain fragment is amplified by selecting different specific amplification primers, the sequencing and the analysis and comparison of the sequencing result can be used for the research of class order of fungoid, family, genus and the like. However, rDNA sequences based on 5.8S, 18S and 28S are difficult to fully reflect the molecular characteristics of the fungal species classification, and even the pathogenic fungal species cannot be determined effectively, but the ITS sequences are often used for species (speces) level identification. However, the high variability of ITS segment has many problems in application, so that the classification and identification research of fungal species by using transcriptomics technology to obtain the full-length sequence of rRNA and integrating the advantages and disadvantages of each gene region is a necessary choice for the development of modern biotechnology.
Disclosure of Invention
The technical problem to be solved by the invention is to make up for the blank of the prior art and provide a full-length cDNA sequence of ribosomal RNA of mulberry black spot pathogen Pseudocercospora mori.
Another object of the present invention is to provide the use of ribosomal RNA sequences of mulberry black spot pathogen Pseudocercospora mori for the quantitative detection of mulberry black spot pathogen Pseudocercospora mori.
Still another object of the present invention is to provide a method for quantitatively detecting the application of ribosomal RNA sequence of mulberry black spot pathogen Pseudocercospora mori.
It is still another object of the present invention to provide the use of ribosomal RNA sequences of the mulberry black spot pathogen Pseudocercospora mori for the classification of fungal species.
The purpose of the invention is realized by the following technical scheme:
the invention provides a full-length cDNA sequence of ribosomal RNA of mulberry black spot pathogen Pseudocercospora mori, which is shown in SEQ.ID.NO1.
The ribosomal RNA consists of 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA; the cDNA sequence of the 18S rRNA is shown as the base sequences 1 to 1726 in the sequence shown in SEQ.ID.NO1; the cDNA sequence of the ITS1 is 1727-1876 in the sequence shown in SEQ.ID.NO1, the cDNA sequence of the 5.8S rRNA is 1877-2034 in the sequence shown in SEQ.ID.NO1, the cDNA sequence of the ITS2 is 2035-2183 in the sequence shown in SEQ.ID.NO1, and the cDNA sequence of the 28S rRNA is 2184-5469 in the sequence shown in SEQ.ID.NO1.
The full-length cDNA sequence of the ribosomal RNA comprises 2 pairs of primer sequences which are respectively shown as SEQ.ID.NO2-SEQ.ID.NO5.
The invention also provides application of the cDNA sequence of the ribosomal RNA of the mulberry black spot pathogen Pseudocercospora mori in quantitative detection of the mulberry black spot pathogen Pseudocercospora mori.
The invention also provides a method for applying the cDNA sequence of the ribosomal RNA of the mulberry black spot pathogen Pseudocercospora mori in quantitative detection of the mulberry black spot pathogen Pseudocercospora mori, which comprises the following steps:
s1, collecting diseased leaves of the mulberry;
s2, extracting total DNA of diseased leaves of the mulberry;
s3, constructing an Illumina DNA library;
s4, Illumina high-throughput sequencing;
s5, removing a mulberry genome sequence in the sequencing data;
s6, assembling a microbial genome sequence;
s7, assembling a complete ribosome DNA sequence;
s8, comparing and analyzing ribosome DNA sequences;
the method for constructing the Illumina DNA library in the step S3 comprises the following steps: constructing the total DNA in the step S2 into a double-ended high-throughput sequencing library with the fragment size of 500bp according to an Illumina library construction flow;
the method for removing the mulberry genome sequence in the sequencing data in the step S5 comprises the following steps: and comparing and analyzing the high-throughput sequencing data in the step S4 by using comparison software. And selecting an alignment algorithm, aligning the sequencing data with a mulberry reference genome, and judging the sequencing data of the aligned reference genome as a mulberry genome sequence. Mulberry genomic sequences were removed from the sequencing data using a written computer program.
S5 the reference genome sequence selected for removing the genome DNA sequence of the mulberry is as follows:
morus (Morus nodabis) whole genome sequence (GCA _000414095.2) and chloroplast genome sequence (NC _ 027110.1).
Preferably, the alignment software is bwa (0.7.12-r1039) software;
preferably, the alignment algorithm is a mem alignment algorithm;
preferably, the alignment of the sequencing data to the mulberry reference genome is selected from the default parameters of the double-ended alignment method and bwa (0.7.12-r1039) software;
preferably, the written computer program is written in the python computer language.
The method for assembling the microbial genome sequence in the step S6 comprises the following steps: assembling the sequencing data with the mulberry genome sequence removed in the step S5 by using assembling software.
Preferably, the assembly software is metavelvetet (v1.2.01).
The method for assembling the complete ribosomal DNA sequence described in step S7 is: comparing the assembled sequence by adopting comparison software, acquiring a double-end sequencing fragment from sequencing data according to a comparison result, assembling and extending the sequence by using the assembly software, and performing multiple cycle operations until a complete ribosome DNA sequence is obtained;
preferably, the alignment software is bwa (0.7.12-r1039) software;
preferably, the alignment method employs a mismatch-free 0mismatch and break-free 0gap alignment;
preferably, the assembly software is MetaVelvet (v1.2.01) software.
The invention also provides the application of the cDNA sequence of ribosomal RNA of the mulberry black spot pathogen Pseudocercospora mori in the classification of fungal species.
The invention has the following beneficial effects:
the invention provides a cDNA sequence of ribosomal RNA of mulberry black spot pathogen Pseudocercospora mori for the first time, establishes a novel fungus species classification method based on the sequence, and particularly has good application in quantitative detection of the mulberry black spot Pseudocercospora mori.
Drawings
FIG. 1 shows the electrophoretogram of two pairs of primers and the universal primer ITS1/ITS4 for verifying the ribosome assembly result.
FIG. 2 shows the classification of fungal microorganisms detected from leaves of black speck of mulberry leaves.
FIG. 3 is a graph showing the result of PCR detection and identification of pathogen of mulberry leaf blight using specific primer sequences.
Detailed Description
The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are biochemical reagent raw materials which are conventionally commercially available or commercially available, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.
Example 1
Randomly finding leaves with typical black spot disease spots in a diseased mulberry field, collecting, shearing the disease spot area in the mulberry leaves, fully grinding the sheared disease spot material by using liquid nitrogen, and extracting the total RNA of the diseased mulberry leaves; the extracted total RNA was stored at-80 ℃. Reverse transcribing the total RNA to a cDNA library; the designed primer sequence and the universal primer sequences Its1 and Its4 are respectively shown as SEQ.ID.NO2-SEQ.ID.NO7, and are shown in Table 1. Carrying out PCR amplification by taking the cDNA library as a template; the PCR reaction system is shown in Table 2.
TABLE 1 PCR verification primer sequence Listing
TABLE 2 PCR reaction System (20. mu.L)
Universal primer ITS primer set PCR program: pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; 10min at 72 ℃.
And (3) verifying the reaction conditions of the P1-F/R primer group and the P2-F/R primer group PCR reaction system (20 mu L) of the ribosome assembly result: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 2 min; 7min at 72 ℃. mu.L of the PCR amplification product was detected by electrophoresis on 1.2% agarose gel (EB staining). Recovering PCR product fragments with corresponding sizes by agarose gel electrophoresis, as shown in figure 1, wherein in figure 1, M is Takara DL2000 Marker; 1. fungal universal primers ITS1 and ITS 4; 2. verifying ribosome assembly result primers P1-F and P1-R; 3. verifying ribosome assembly result primers P2-F and P2-R; 4. blank water control.
Performing Sanger sequencing on the recovered fragments; the sequencing results were then aligned with the full-length cDNA sequence of ribosomal RNA of the mulberry black spot pathogen Pseudocercospora mori to thereby determine whether or not there is a black spot pathogen Pseudocercospora mori on the leaves, and from this, it was presumed whether or not the black spot pathogen Pseudocercospora mori is likely to be a pathogenic bacterium.
As can be seen from FIG. 1, the results in lane 1 show that the template DNAs amplified with the fungal universal primers ITS1 and ITS4 are fungal DNAs, and the PCR results of the P1 primer set and P1 primer set for verifying the ribosome assembly result (lanes 2 and 3), respectively, are amplified to corresponding target bands, and the results after further sequencing are highly consistent with the assembly result.
Example 2 application experiment
Randomly finding leaves with typical black spot disease spots in a diseased mulberry field, collecting, shearing the disease spot area in the mulberry leaves, fully grinding the sheared disease spot material by using liquid nitrogen, extracting total DNA by using the Kangji fungus DNA extraction kit according to the operation instructions, and storing the extracted total DNA at-20 ℃. According to the construction process of the Illumina library, total DNA is constructed into a double-end high-throughput sequencing library with the fragment size of 500bp, an Illumina Hiseq2500 sequencer is used for carrying out high-throughput sequencing on the constructed DNA library, 18.56M pairs of sequencing fragments are measured in total, the sequencing reading length is 125bp at the double ends, and the total sequencing data volume is 4.64 Gb.
Because the DNA extraction process comprises leaf materials, in order to reduce the influence of mulberry genome data in sequencing data on the assembly of a microbial sequence, the genome DNA sequence of the mulberry is removed before the assembly of the microbial sequence. The Morus notubis whole genome sequence (GCA _000414095.2) and the chloroplast genome sequence (NC _027110.1) are selected as reference genome sequences, and data alignment analysis is carried out by using bwa (0.7.12-r1039) alignment software. And (3) selecting a mem alignment algorithm by alignment, using a double-end alignment method and default parameters of software, aligning the sequencing data with the mulberry reference genome, and judging the sequencing fragment of the aligned reference genome as a mulberry genome sequence. The mulberry sequencing data was removed from the fastq sequencing data using a computer program written in python, and then re-entered into microbial sequence assembly. The assembly of the microbial sequences was performed using MetaVelvet (v1.2.01) assembly software.
Ribosomal DNA of the target pathogenic fungus consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment, and the total length of the sequence is about 6 Kb. MetaVelvet (v1.2.01) initially assembled sequence tags were broken ribosomal tags and to obtain complete ribosomal DNA sequences, the analysis used sequence capture and de novo assembly strategies to assemble complete ribosomal DNA. Selecting a ribosome DNA sequence containing an ITS sequence of a target pathogen as a reference sequence, carrying out 0mismatch and 0gap alignment by adopting bwa (0.7.12-r1039) software, obtaining a double-end sequencing fragment from sequencing data according to the alignment result, further assembling and extending the sequence by adopting MetaVelvet (v1.2.01) assembly software, and obtaining a complete ribosome DNA sequence through a plurality of circulation operations.
Sequence tag annotation Using blastn (2.2.31+) sequence alignment analysis software, the assembled sequence tag sequences were aligned to the nt database of NCBI with blastn alignment setting expectation <1e-20, and sequence tags were annotated according to the alignment results. Ribosomal DNA sequences are the most common important molecular markers for bacterial and fungal identification, and therefore species classification and quantification takes ribosomal DNA as the main molecular marker. And selecting a ribosome DNA sequence as a microorganism identification and quantitative analysis basis according to the sequence label annotation result. The average sequencing depth of the ribosomal DNA fragments in the sequencing data was calculated using the bwa (0.7.12-r1039) + samtools (v1.2) analysis software and used as the abundance value for that species.
The results showed that 261 rRNA sequence tags were co-injected, and the data contained 232 bacterial sequence tags and 29 fungal sequence tags, corresponding to 16 fungi. Through query of sequence tag annotation results and comparison with the full-length cDNA sequence of ribosomal RNA of mulberry black spot pathogen Pseudocercospora mori, the fungus with the highest relative abundance is pathogenic bacteria of the genus Pseudocercospora, which is shown in figure 2.
Example 3 validation test
The Pseudocercospora mori ribosomal DNA can be quickly detected and identified by extracting the mulberry leaf (lesion) total DNA infected with diseases without involving an RNA experiment with higher requirements. In order to further verify and utilize the whole gene sequence of the Pseudocercospora mori ribosomal RNA to be applied to the pathogen detection and identification of mulberry leaf blight, the result is positive. The invention further designs a pair of specific primer sequences 4W1724F/4W2196R which are respectively shown in SEQ.ID.NO8 and SEQ.ID.NO9, and the sequences are shown in Table 3:
TABLE 3A pair of specific primer sequences designed based on the complete gene sequence of Pseudocercospora mori ribosomal RNA
The size of the target fragment to be amplified is about 473 bp. The electrophoresis results are shown in FIG. 3, wherein M: takara DL1000 Marker; 1. mulberry leaf disease fruiting body DNA; cladosporium cladosporioides (Mycospora bronchiseptica); cladosporium perangustum (Cladosporium minutum); cladosporium oxysporum (Cladosporum oxysporum); penicillium verruculosum (Penicillium verrucosum); penicillium citrinum, Aspergillus, 7 Aspergillus; phanerochaete chrysosporium (Phanerochaete chrysosporium); ceriporia mellea (Ceriporiopsis melissa); 10; beauveria bassiana (Beauveria bassiana); schizophyllum commune (Schizophyllum commune); candida mucina (Candida); lasiodipidipsideadothieobromae (longan. pythium aphanidermatum); pseudomonas aeruginosa; alcaligenes faecalis 16. Phyllantia moricola (Mulberry powdery mildew pathogen-Mulberry needle shell); ciboria carunculoides (Morganella sclerotiorum pathogen-P.carunculus); 18. positive control (lesion DNA of morus alternatus pathogen); 19, negative control (mulberry DNA); 20. blank control (water). 1-15 are fungi and bacteria isolated and cultured in the laboratory of the invention and species identification was performed according to the international species barcode, as described above. Wherein, 1-13 are various fungi; 14-15 are bacteria; 16-17 are other mulberry mycosis pathogens.
As a result of electrophoresis shown in FIG. 3, it was found that only the DNA of the lesion or pathogen fruiting body template of the pathogenic bacteria of the mulberry leaf disease amplified the target fragment, the size of which is about 473bp and is between 400 and 500bp, and as seen from the figure, no other bacteria have amplified fragments with similar sizes, and the target fragment amplified by the pathogenic bacteria of the mulberry leaf disease has clear brightness and is far brighter than Marker.
SEQUENCE LISTING
<110> southern China university of agriculture
Ribosomal RNA sequence of <120> mulberry black spot pathogenic bacteria and application thereof
<130>
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 5469
<212> DNA
<213> full-Length cDNA sequence of ribosomal RNA
<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 sequence
<400> 2
<210> 3
<211> 20
<212> DNA
<213> primer P1-R sequence
<400> 3
tccctacctg atccgaggtc 20
<210> 4
<211> 20
<212> DNA
<213> primer P2-F sequence
<400> 4
<210> 5
<211> 20
<212> DNA
<213> primer P2-R sequence
<400> 5
<210> 6
<211> 19
<212> DNA
<213> Universal primer ITS1 sequence
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Universal primer ITS4 sequence
<400> 7
<210> 8
<211> 21
<212> DNA
<213> primer sequence 4w1724F sequence
<400> 8
gctacactga cagagccaac g 21
<210> 9
<211> 19
<212> DNA
<213> primer sequence 4W2196R sequence
<400> 9
Claims (7)
1. The mulberry black spot pathogen Pseudocercosporammori ribosomal RNA is characterized in that the full-length cDNA sequence of the ribosomal RNA is shown in SEQ.ID.NO1.
2. The mulberry black spot pathogen pseudosporisorium ribosomal RNA according to claim 1, wherein the ribosomal RNA consists of 18SrRNA, ITS1, 5.8SrRNA, ITS2, 28 SrRNA; the cDNA sequence of the 18SrRNA is shown as base sequences 1-1726 in a sequence shown in SEQ.ID.NO1; the cDNA sequence of the ITS1 is 1727-1876 in the sequence shown in SEQ.ID.NO1, the cDNA sequence of the 5.8SrRNA is 1877-2034 in the sequence shown in SEQ.ID.NO1, the cDNA sequence of the ITS2 is 2035-2183 in the sequence shown in SEQ.ID.NO1, and the cDNA sequence of the 28SrRNA is 2184-5469 in the sequence shown in SEQ.ID.NO1.
3. Use of the ribosomal RNA sequence of the mulberry black spot pathogen Pseudocercosporammori according to claim 1 or 2 for the quantitative detection of the mulberry black spot pathogen Pseudocercosporammori.
4. The application according to claim 3, characterized in that the method of application comprises the following steps:
s1, collecting diseased leaves of the mulberry;
s2, extracting total DNA of diseased leaves of the mulberry;
s3, constructing an Illumina DNA library;
s4, Illumina high-throughput sequencing;
s5, removing a mulberry genome sequence in the sequencing data;
s6, assembling a microbial genome sequence;
s7, assembling a complete ribosome DNA sequence
S8, comparing and analyzing ribosome DNA sequences;
the method for constructing the Illumina DNA library in the step S3 comprises the following steps: constructing the total DNA in the step S2 into a double-ended high-throughput sequencing library with the fragment size of 500bp according to an Illumina library construction flow;
the method for comparative analysis of ribosomal DNA sequences as described in step S8 is: and (3) comparing the complete ribosomal DNA sequence in the step S7 with the full-length cDNA sequence of rRNA of the pathogenic bacteria of mulberry leaf smut/black spot by using sequence comparison analysis software.
5. The use of claim 4, wherein the method for removing the mulberry genome sequence in the sequencing data of step S5 is: performing data comparison analysis on the high-throughput sequencing data in the step S4 by using comparison software, selecting a comparison algorithm, comparing the sequencing data with a mulberry reference genome, judging the sequencing data of the compared reference genome as a mulberry genome sequence, and removing the mulberry genome sequence from the sequencing data by using a programmed computer program;
s5, the reference genome sequence selected for removing the mulberry genome sequence in the sequencing data is as follows:
morus morusinobilis whole genome sequence GCA _000414095.2 and chloroplast genome sequence NC _ 027110.1;
the method for assembling the microbial genome sequence in the step S6 comprises the following steps: assembling the sequencing data of the mulberry genome sequence removed obtained in the step S5 by using assembling software;
the method for assembling the complete ribosomal DNA sequence described in step S7 is: and comparing the assembled sequence by adopting comparison software, acquiring a double-end sequencing fragment from sequencing data according to a comparison result, assembling and extending the sequence by using the assembly software, and performing multiple cycles of operation until a complete ribosome DNA sequence is obtained.
6. The use of claim 5, wherein the alignment software of step S5 is the software bwa0.7.12-r 1039; step S5, the comparison algorithm is a mem comparison algorithm; the alignment of the sequencing data to the mulberry reference genome described in step S5 is the default parameter for the double-ended alignment method and the bwa0.7.12-r1039 software; the written computer program of step S5 is written in python computer language; step S6, the assembly software is MetaVelvetv1.2.01; the comparison software of the step S7 is bwa0.7.12-r1039 software; step S7, the comparison method adopts mismatch-free 0mismatch and fracture-free 0gap comparison; the assembly software in step S7 is metavelvetv1.2.01 software.
7. Use of the ribosomal RNA sequence of the mulberry black spot pathogen Pseudocercosporammori according to claim 1 or 2 for the classification of fungal species.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710064069.4A CN107794262B (en) | 2017-01-24 | 2017-01-24 | Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710064069.4A CN107794262B (en) | 2017-01-24 | 2017-01-24 | Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107794262A CN107794262A (en) | 2018-03-13 |
| CN107794262B true CN107794262B (en) | 2021-02-19 |
Family
ID=61530133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710064069.4A Active CN107794262B (en) | 2017-01-24 | 2017-01-24 | Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107794262B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112553219B (en) * | 2020-12-29 | 2022-09-13 | 华南农业大学 | Method for detecting alternaria leaf spot based on ribosome 28s gene |
-
2017
- 2017-01-24 CN CN201710064069.4A patent/CN107794262B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| and 28S ribosomal RNA gene, partial sequence.《GeneBank》.2016,第1-2页. * |
| Crous PW等.Pseudocercospora pallida strain CPC 10776 18S ribosomal RNA gene, internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence * |
| Phylogenetic lineages in Pseudocercospora;Crous PW等;《Studies in Mycology》;20120522;第75卷;第37–114页 * |
| 我国假尾孢属真菌9个省级新记录种;翟凤艳等;《河南农业科学》;20111231;第40卷(第3期);第96-98页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107794262A (en) | 2018-03-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106868116B (en) | Mulberry pathogenic bacterium high-throughput identification and species classification method and application thereof | |
| CN109652580B (en) | Ribosomal RNA sequence of pathogenic bacteria Septoria sp of early-baking Oak and application thereof | |
| Mota-Gutierrez et al. | Metataxonomic comparison between internal transcribed spacer and 26S ribosomal large subunit (LSU) rDNA gene | |
| Miller et al. | Metabarcoding of fungal communities associated with bark beetles | |
| JP2019537780A (en) | Pathogen identification and antibiotic characterization in metagenomic samples | |
| Thodberg et al. | Comprehensive profiling of the fission yeast transcription start site activity during stress and media response | |
| CN111197050B (en) | Ribosomal RNA gene of mulberry pseudo-blight pathogen and application thereof | |
| Weig et al. | Diagnostic assessment of mycodiversity in environmental samples by fungal ITS1 rDNA length polymorphism | |
| CN107794262B (en) | Ribosomal RNA sequence of mulberry black spot pathogenic bacteria and application thereof | |
| Sting et al. | Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing | |
| CN106801053B (en) | Ribosomal RNA sequence of mulberry powdery mildew pathogenic bacteria Phyllactinia mori and application thereof | |
| CN107164471B (en) | Molecular identification method for rapidly identifying truth of beauveria bassiana in traditional Chinese medicine stiff silkworm | |
| CN112331268A (en) | Method for obtaining specific sequence of target species and method for detecting target species | |
| Meydan et al. | Identification of translation start sites in bacterial genomes | |
| Urban et al. | Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions | |
| CN113936740A (en) | High-throughput detection method and system for pathogenic bacteria virulence factor in environmental sample | |
| Shinohara et al. | Nanopore based sequencing enables easy and accurate identification of yeasts in breweries | |
| CN113151521B (en) | Ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof | |
| CN109486836B (en) | Oak powdery mildew pathogen Erysipheitoides ribosomal RNA gene and application thereof | |
| KR20170134624A (en) | Microbial total analysis system, determination system, microbial total analysis method and judgment method | |
| CN112779274B (en) | Ribosomal RNA gene of mulberry plaster disease pathogenic bacteria and application thereof | |
| Rangkuti et al. | DNA Barcoding of Rattan (Arecaceae) From Gunung Walat Education Forest, Sukabumi-West Java | |
| CN112553219B (en) | Method for detecting alternaria leaf spot based on ribosome 28s gene | |
| CN109504688B (en) | Application of ITS1 gene in detection of powdery mildew pathogen Erysiphe alpithioides | |
| KR102597906B1 (en) | Primer set for detecting bacteroides and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |