CN111197050B - Ribosomal RNA gene of mulberry pseudo-blight pathogen and application thereof - Google Patents
Ribosomal RNA gene of mulberry pseudo-blight pathogen and application thereof Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses a ribosomal RNA gene of mulberry pseudo-blight pathogen and application thereof. The full-length cDNA sequence of the ribosomal RNA gene of the mulberry pseudo-blight pathogen is shown as SEQ ID NO.1, and the ribosomal RNA gene of the mulberry pseudo-blight pathogen Fusarium sp can be applied to detection of the mulberry pseudo-blight pathogen Fusarium sp or fungus species classification; based on the gene, a specific primer capable of specifically detecting the Fusarium sp of the mulberry pseudo-blight pathogen is designed, a method and a detection kit for efficiently, rapidly and specifically detecting the Fusarium sp of the mulberry pseudo-blight pathogen are established, and the method and the kit have a very wide application prospect in detecting the Fusarium sp of the mulberry pseudo-blight pathogen.
Description
Technical Field
The invention belongs to the field of biotechnology. More specifically, it relates to a ribosomal RNA gene of a mulberry pseudodry disease pathogen and its use.
Background
The mulberry quasi-blight (Fusarium sp) is widely distributed, and both main stems and branches can be ill. The pathogenic bacteria of the mulberry quasi-dry disease normally overwhelm on the diseased branches, and the pathogenic bacteria spread along with wind, rain and insects in the next 4-5 months continuously invade the branches of the mulberry and insect injury which are weak in growth and young. When pathogenic bacteria of the pseudo-blight of the mulberry are harmful branches, the pathogenic bacteria mostly occur on half branches, often take winter buds as the centers, form brown oblong disease spots, the disease spots are edema-shaped when wet, shrink after drying, the harmful cortex is easy to peel off, and dark spots are densely generated under the skin. If the disease spots surround the branches in a circle, the branches die. In recent years, the disease area and the hazard degree of the pseudo-blight of the mulberry are increased year by year, and the yield and the quality of the mulberry are seriously affected. Therefore, detection of the pathogenic bacteria of Mulberry pseudo-blight is extremely important, especially in early detection.
In the existing fungus research method, sequencing alignment is often performed by a ribosomal DNA (rDNA) sequence for fungus identification. Ribosomes have important functions in cells, and many genes encoded by rDNA are closely related to the reaction process of protein synthesis and play a decisive role in protein biosynthesis. The rDNA sequence is divided into a transcribed region and a non-transcribed region, the transcribed region consists of a gene encoding the ribosomal 5.8S, 18S, 28S protein structure and 2 transcribed spacer (Internal Tanscribed Spacer, ITS) ITS1 and ITS2 regions between the genes, which together form a transcriptional unit. The rDNA sequences of the rDNA, which code for 5.8S, 18S and 28S, are relatively conservative, and can be used for analyzing phylogenetic development among the plants or higher order elements. Because 5.8S rDNA sequences are short and highly conserved, they are difficult to use for phylogenetic and molecular identification of fungi; whereas the 18S rDNA fragment is longer, there are conserved regions and variable regions in the fragment. Therefore, in the existing research, after a domain fragment is amplified by selecting different specific amplification primers, the method can be used for researching classification order elements of fungi orders, families, genera and the like through sequencing and analysis comparison of sequencing results. However, classification of fungal species based on rDNA sequences of 5.8S, 18S, 28S is difficult to study and the species of pathogenic fungi cannot be determined. Therefore, it is necessary to use the full-length sequence of rDNA for research.
At present, no relevant report on a detection method of the mulberry quasipunculus is provided, so that a method capable of rapidly and specifically detecting the mulberry quasipunculus is established, and the method has important significance for controlling the morbidity and the hazard of the mulberry quasipunculus.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the blank of the existing oak early-drying disease pathogenic bacteria detection technology and providing a ribosomal RNA gene of a mulberry bacterial pathogen Fusarium sp and application thereof.
The first object of the present invention is to provide a ribosomal RNA gene of Fusarium sp which is a pathogenic bacterium of Mulberry pseudodry disease.
The second object of the invention is to provide the application of the ribosomal RNA gene of the Fusarium sp of the mulberry quasipunculus in detecting the Fusarium sp of the mulberry quasipunculus or in classifying fungus species.
The third object of the invention is to provide a method for detecting Fusarium sp which is a pathogenic bacterium of the quasipunculus of the mulberry.
The fourth object of the invention is to provide a set of primers for detecting Fusarium sp which is a pathogenic bacterium of the bacterial wilt of mulberry.
The fifth object of the invention is to provide application of the primer in detecting Fusarium sp which is a pathogenic bacterium of the mulberry, and/or preparing a kit for detecting Fusarium sp which is a pathogenic bacterium of the mulberry.
The sixth object of the invention is to provide a method for detecting Fusarium sp which is a pathogenic bacterium of the quasipunculus of the mulberry.
The seventh object of the invention is to provide a kit for detecting Fusarium sp which is a pathogenic bacterium of the pseudo-blight of mulberry.
The eighth object of the invention is to provide the application of the method or the kit in detecting Fusarium sp which is a pathogenic bacterium of the Mulberry sheath blight.
The above object of the present invention is achieved by the following technical scheme:
the invention firstly provides a ribosomal RNA gene of Fusarium sp of a mulberry pseudodry disease pathogen, and the full-length cDNA sequence of the ribosomal RNA gene is shown as SEQ ID NO. 1.
The invention obtains the full-length cDNA sequence of the ribosomal RNA gene of the Fusarium sp of the mulberry pseudodry disease pathogen for the first time, and the length of the full-length cDNA sequence is 7894bp. The ribosomal RNA gene consists of an 18S rRNA gene, an ITS1 gene, a 5.8S rRNA gene, an ITS2 gene and a 28S rRNA gene; the nucleotide sequence of the 18S rRNA gene is 1 st to 1796 th base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS1 gene is 1797 to 1946 base sequences in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 5.8SrRNA gene is 1947 th to 2104 th base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS2 gene is 2105 th to 2267 th base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 28S rRNA gene is 2268 to 5608 base sequences in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the IGS region (the interval region between the IGS region and the next 18s rDNA gene) is 5609 th to 7894 th base sequence in the sequence shown in SEQ ID NO. 1.
The application of the ribosomal RNA gene of the mulberry quasipunculus sp in detecting the mulberry quasipunculus sp or in fungus species classification is also within the protection scope of the invention.
The invention also provides a method for detecting the Fusarium sp of the mulberry quasipunculus, which comprises the steps of comparing the full-length cDNA sequence of the ribosomal RNA gene of the sample to be detected with the sequence shown in SEQ ID NO.1, and judging whether the sample to be detected contains the Fusarium sp of the mulberry quasipunculus according to the comparison result.
Preferably, the method is as follows: and (3) taking the DNA of the sample to be detected as a template, carrying out library construction, high-throughput sequencing and assembly to obtain complete ribosomal DNA, then comparing with the full-length cDNA sequence of the ribosomal RNA gene, and judging whether the sample to be detected contains Fusarium sp of the mulberry bacterial wilt-like pathogen according to the comparison result.
Particularly preferably, the method comprises the steps of:
s1, collecting the ramulus mori pseudo-blight disease;
s2, extracting total DNA of the ramulus mori pseudo-blight disease;
s3, constructing an illumina DNA library;
s4, illumina high-throughput sequencing;
s5, removing the mulberry genome sequence in the sequencing data;
s6, assembling a microorganism genome sequence;
s7, assembling a complete ribosome DNA sequence;
s8, comparing and analyzing the ribosome DNA sequence.
The method for constructing the Illumina DNA library in the step S3 comprises the following steps: and (3) constructing the total DNA in the step S2 into a double-end high-throughput sequencing library with the fragment size of 350bp according to an Illumina DNA library construction flow.
The method for aligning and analyzing the ribosomal DNA sequence in the step S8 comprises the following steps: the complete ribosomal DNA sequence of step S7 was aligned with the full-length cDNA sequence of the ribosomal RNA gene of Fusarium sp, a. Mulberosum pathogen, using sequence alignment analysis software.
The method for removing the mulberry genome sequence in the sequencing data in the step S5 comprises the following steps: 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, and judging the sequencing data of the compared reference genome as a mulberry genome sequence; the mulberry genomic sequence was removed from the sequencing data using a written computer program.
Preferably, the alignment software is bwa 0.7.17-r1188 software.
Preferably, the alignment algorithm is a mem alignment algorithm.
Preferably, the written computer program is a python language program.
The method for assembling the genome sequence of the microorganism in the step S6 comprises the following steps: and (3) assembling the sequencing data obtained by removing the mulberry genome sequence in the sequencing data in the step S5 by using assembling software.
Preferably, the assembly software is SPADes v.3.5.0 software.
The method for assembling the complete ribosomal DNA sequence described in step S7 is as follows: the sequence tag initially assembled by adopting SPades v.3.5.0 software is a broken ribosome tag, and sequence capturing and de-head assembling strategies are adopted for analysis to assemble complete ribosome DNA sequence; selecting a ribosome DNA sequence containing an ITS sequence of suspected target pathogenic bacteria as a reference sequence, adopting bwa 0.7.17-r1188 software to carry out double-end sequence comparison, acquiring a double-end sequencing fragment from sequencing data according to a comparison result by using a self-written python language program, and then adopting SPADes v.3.5.0 software to assemble and extend the sequence to obtain a complete ribosome DNA sequence through a plurality of cyclic operations; for the assembly uncertainty region, PCR amplification and Sanger sequencing were performed to verify the correctness of the assembly.
The invention also provides a group of primers for detecting Fusarium sp which is a pathogenic bacterium of the mulberry pseudo-blight, and the primers can amplify a sequence shown as SEQ ID NO.1 or fragments thereof.
Preferably, the primer is a primer group ZHR1RF/ZHR1RF, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 2-3:
nucleotide sequence of primer ZHR1RF (SEQ ID No. 2): 5'-GCCAGTCCGTACTTGTTCGT-3';
nucleotide sequence of primer ZHR1RF (SEQ ID No. 3): 5'-GCGCCTACCCTGTAGCAAAT-3'.
The application of the primer in detecting the Fusarium sp of the mulberry pseudotrunk disease and/or preparing a kit for detecting the Fusarium sp of the mulberry pseudotrunk disease shall also be within the protection scope of the invention.
Based on the primer, the invention also provides a method for detecting the Fusarium sp of the bacterial wilt-like disease of the mulberry, which is to detect whether the ribosomal RNA gene exists in the nucleic acid of a sample to be detected, and if the ribosomal RNA gene exists, the sample to be detected is positive to the Fusarium sp of the bacterial wilt-like disease of the mulberry.
Preferably, the method is as follows: and (3) taking nucleic acid of the sample to be detected as a template, carrying out PCR amplification by using the primer, and if the amplification result is positive, determining that the sample to be detected is positive to Fusarium sp of the bacterial wilt-like disease of the mulberry.
Preferably, the method for judging is as follows: and (3) carrying out gel electrophoresis on the PCR amplified reaction product, and if a band appears and the size of the band is 200bp, judging that the sample to be detected contains Fusarium sp which is a pathogenic bacterium of the mulberry bacterial wilt.
Preferably, the reaction system of the PCR amplification is: 2 XTaq Master Mix 10. Mu.L, primer sets ZHR1RF/ZHR1RF each 0.5. Mu.L (10. Mu.M), template DNA 1. Mu.L, the remainder ddH 2 O is added up to 20 mu L.
Preferably, the reaction conditions for the PCR amplification are: 94 ℃ for 5min;94 ℃ for 30s,55 ℃ for 30s,72 ℃ for 2min,35 cycles; and at 72℃for 5min.
The invention also provides a kit for detecting Fusarium sp which is a pathogenic bacterium of the pseudo-blight of mulberry, and the kit comprises the primer.
In addition, the application of the method or the kit in detecting the Fusarium sp of the bacterial wilt-like disease of the mulberry is also in the protection scope of the invention.
The invention has the following beneficial effects:
the invention provides a ribosomal RNA gene of mulberry pseudo-blight pathogen and application thereof. The invention obtains the full-length cDNA sequence of the ribosomal RNA gene of the Fusarium sp of the mulberry quasi-trunk disease pathogen for the first time, the length of the full-length cDNA sequence is 7894bp, and the ribosomal RNA gene of the Fusarium sp of the mulberry quasi-trunk disease pathogen can be applied to detection of Fusarium sp or fungus species classification of the mulberry quasi-trunk disease pathogen; based on the gene, a specific primer capable of specifically detecting the Fusarium sp of the mulberry pseudo-blight pathogen is designed, a method and a detection kit for efficiently, rapidly and specifically detecting the Fusarium sp of the mulberry pseudo-blight pathogen are established, and the method and the kit have a very wide application prospect in detecting the Fusarium sp of the mulberry pseudo-blight pathogen.
Drawings
FIG. 1 is a classification tree of fungal microorganisms detected by diseased shoots of mulberry.
FIG. 2 is a graph showing the results of specific detection of Fusarium sp, a pathogenic bacterium of Mulberry pseudo-blight; wherein M: takaraDL2000 Marker;1: mucor racemosus (Mucor total); 2: actinomucor elegans (Mucor elegans); 3: cladosporium perangustum (Cladosporium tenuis); 4: fusarium oxysporum (Fusarium oxysporum); 5: auricularia polytricha (Auricularia polytricha); 6: fusarium equiseti (Fusarium equisetum); 7: trichoderma (Trichoderma); 8: mucor circinelloides (Mucor circinelloides); 9: ralstonia solanacearum (Lawsonia solanaceae); 10: enterobacter cloacae (enterobacter cloacae); 11: klebsiella sp (Klebsiella sp); 12: blank control (ddH) 2 O); 13 and 14: fusarium sp DNA of the pathogenic bacteria of the pseudo-blight of mulberry; 15 and 16: total DNA of the disease branch with mulberry quasi-dry disease.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 detection of the acquisition of ribosomal RNA Gene of Fusarium sp, a pathogen of Mulberry
1. Experimental method
(1) High throughput sequencing
Random search for conditions with typical dry-off in morbid mulberry gardensCollecting the disease branch, cleaning the surface with sterile water, cutting off disease spot material, fully grinding with liquid nitrogen, extracting total DNA with Shanghai fungus genome DNA extraction kit, specifically according to its operation instruction, and storing the extracted total DNA at-20deg.C; randomly breaking the qualified DNA sample into fragments with the length of about 350bp by adopting a covarias ultrasonic breaker (covarias, USA), constructing a small fragment genome DNA library, and referring to NEB in the library construction processUltra DNA Library Prep Kit for(NEB, USA) library construction kit instructions. Quality control was performed after library construction was completed by qPCR method and Agilent 2100Bioanalyzer (Agilent Technologies, USA). And (3) sequencing the DNA library qualified in quality inspection by adopting an Illumina Hiseq 4000 (Illumina, USA) high-throughput sequencing platform, wherein the sequencing strategy is PE150 (Pair-End 150), and the sequencing data volume of each sample is not less than 2Gb.
(2) Assembly of microbial genome sequences
Microorganism sequence assembly was performed using SPADes v.3.5.0 software; the ribosomal DNA sequence of the fungus of interest consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment. The sequence tag initially assembled by the SPAdes v.3.5.0 software is a broken ribosomal tag and, to obtain the complete ribosomal DNA sequence, the analysis uses sequence capture and de novo assembly strategies to assemble the complete ribosomal DNA. And selecting a ribosome DNA sequence containing an ITS sequence of suspected target pathogenic bacteria as a reference sequence, comparing the double-end sequences by adopting bwa 0.7.17-r1188 software, acquiring a double-end sequencing fragment from sequencing data by using a self-written python language program according to a comparison result, assembling and extending the sequence by adopting SPADes v.3.5.0 software, and obtaining the complete ribosome DNA sequence through a plurality of cyclic operations. For the assembly uncertainty region, PCR amplification and Sanger sequencing were performed to verify the correctness of the assembly.
Sequence tag annotation sequencing sequence tags were aligned to bacterial and fungal libraries, respectively, of the nt database of NCBI using a blastn (v2.5.0+) alignment analysis strategy. Since ribosomal DNA sequences are important and most commonly used molecular markers for bacterial and fungal identification and databases contain abundant species sequence information, species classification identification and quantification uses ribosomal DNA as the primary molecular marker. Other species select a conserved sequence as a judgment basis according to the published sequence information. According to the sequence comparison similarity, comparing a plurality of dimensions such as sequence length, sequence integrity and the like, selecting an optimal comparison result to annotate the Tag sequence through a similarity evaluation algorithm, and analyzing the diversity of microorganisms in a tissue. Based on the statistics of the sequence tags, an average sequencing depth value of the sequence tags is calculated and used as a relative abundance value of the species.
2. Experimental results
The full-length cDNA sequence of the ribosomal RNA gene of the mulberry bacterial pseudodry pathogen Fusarium sp is shown as SEQ ID NO.1, and the ribosomal RNA gene consists of an 18S rRNA gene, an ITS1 gene, a 5.8S rRNA gene, an ITS2 gene and a 28S rRNA gene; the nucleotide sequence of the 18S rRNA gene is 1 st to 1796 th base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS1 gene is 1797 to 1946 base sequences in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 5.8S rRNA gene is 1947 to 2104 base sequences in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS2 gene is 2105 th to 2267 th base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 28S rRNA gene is 2268 to 5608 base sequences in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the IGS region (the spacer region between the IGS region and the next 18s rDNA gene) is 5609 th to 7894 th base sequence in the sequence shown in SEQ ID NO. 1.
Example 2 method for detecting Fusarium sp, a pathogenic bacterium of Mulberry pseudo-blight
1. Experimental method
(1) High throughput sequencing
Randomly searching for disease branch with typical symptoms of Morus pseudodry disease in morbid mulberry garden, collecting, cleaning surface with sterile water, cutting off disease spot material, grinding with liquid nitrogen, and extracting total DNA with ShanghaiThe kit for extracting the genome DNA of the engineering fungus is specifically carried out according to the operation instruction, and the extracted total DNA is stored at-20 ℃; randomly breaking the qualified DNA sample into fragments with the length of about 350bp by adopting a covarias ultrasonic breaker (covarias, USA), constructing a small fragment genome DNA library, and referring to NEB in the library construction processUltra DNA Library Prep Kit for/>(NEB, USA) library construction kit instructions. Quality control was performed after library construction was completed by qPCR method and Agilent 2100Bioanalyzer (Agilent Technologies, USA). And (3) sequencing the DNA library qualified in quality inspection by adopting an Illumina Hiseq 4000 (Illumina, USA) high-throughput sequencing platform, wherein the sequencing strategy is PE150 (Pair-End 150), and the sequencing data volume of each sample is not less than 2Gb.
(2) Assembly of microbial genome sequences
Microorganism sequence assembly was performed using SPADes v.3.5.0 software; the ribosomal DNA sequence of the fungus of interest consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment. The sequence tag initially assembled by the SPAdes v.3.5.0 software is a broken ribosomal tag and, to obtain the complete ribosomal DNA sequence, the analysis uses sequence capture and de novo assembly strategies to assemble the complete ribosomal DNA. And selecting a ribosome DNA sequence containing an ITS sequence of suspected target pathogenic bacteria as a reference sequence, comparing the double-end sequences by adopting bwa 0.7.17-r1188 software, acquiring a double-end sequencing fragment from sequencing data by using a self-written python language program according to a comparison result, assembling and extending the sequence by adopting SPADes v.3.5.0 software, and obtaining the complete ribosome DNA sequence through a plurality of cyclic operations. For the assembly uncertainty region, PCR amplification and Sanger sequencing were performed to verify the correctness of the assembly.
Sequence tag annotation sequencing sequence tags were aligned to bacterial and fungal libraries, respectively, of the nt database of NCBI using a blastn (v2.5.0+) alignment analysis strategy. Since ribosomal DNA sequences are important and most commonly used molecular markers for bacterial and fungal identification and databases contain abundant species sequence information, species classification identification and quantification uses ribosomal DNA as the primary molecular marker. Other species select a conserved sequence as a judgment basis according to the published sequence information. According to the sequence comparison similarity, comparing a plurality of dimensions such as sequence length, sequence integrity and the like, selecting an optimal comparison result to annotate the Tag sequence through a similarity evaluation algorithm, and analyzing the diversity of microorganisms in a tissue. Based on the statistics of the sequence tags, an average sequencing depth value of the sequence tags is calculated and used as a relative abundance value of the species.
(3) Sequence annotation and genetic evolution analysis
And assembling according to high-throughput sequencing data and experimental verification results to obtain a complete ribosome DNA sequence. The rRNA sequence was annotated using a blastn comparison strategy, based on the rRNA structure of closely related species in the nt database, and the length and GC ratios of the 18S, ITS1,5.8S, ITS2, 28S intervals were counted, respectively. Species and number analyses of bacteria and fungi were visualized using MEGAN (Version 6.4.0). Bacterial identification conditions: the sequence length is more than 500bp, the similarity with the comparison sequence in the nt database is more than 97%, and the sequence coverage is more than 80%. Fungus identification conditions: the sequence length is more than 500bp, the similarity with the comparison sequence in the nt database is more than 75%, and the sequence coverage is more than 80%.
2. Experimental results
And according to the analysis results of the types and the amounts of the microorganisms, the suspected pathogen is presumed to be Fusarium species by consulting the signs of the binding materials and related data.
As shown in FIG. 1, the fungus microorganism classification tree detected by the mulberry diseased branches is shown as a figure, and according to the principle that the higher the numerical value is, the higher the relative abundance of the species is, the fungus with the highest relative abundance is found to be the Fusarium pathogen by inquiring the sequence tag annotation result and comparing the sequence tag annotation result with the full-length cDNA sequence of the ribosomal RNA gene of Fusarium sp of the Fusarium.
Example 3 method for detecting Fusarium sp, a pathogen of Mulberry pseudodry disease
1. Experimental method
(1) PCR amplification reaction
In order to further utilize the full-length cDNA sequence of the ribosomal RNA gene of the mulberry quasicarum sp to be applied to pathogen detection and identification of the mulberry quasicarum sp, the invention further designs a pair of specific primer sets ZHR1RF/ZHR1RF, and the nucleotide sequence of the primer sets are shown in a table 1;
then, the total DNA of the mulberry pseudo-blight infected with the disease was used as a template, and other 8 fungi, 3 bacteria and ddH were used 2 O is a negative control, PCR amplification is carried out by using the primer set ZHR1RF/ZHR1R, a PCR amplification reaction system is shown in a table 2, and the size of a target fragment to be amplified is about 200bp.
TABLE 1 nucleotide sequences of specific primer sets ZHR1RF/ZHR1RF
TABLE 2PCR amplification reaction System
(2) Conditions of PCR amplification reaction
The conditions for validating the PCR amplification reaction specific for primer set ZHR1RF/ZHR1R are: 94 ℃ for 5min;94℃for 30s,47℃for 30s,72℃for 2min,35 cycles; and at 72℃for 5min.
(3) Detection of PCR amplification reaction products
After the PCR reaction was completed, 4. Mu.L of each PCR amplified product was subjected to electrophoresis detection by using 1.0% agarose gel (EB staining), and a PCR product fragment corresponding in size was recovered by agarose gel electrophoresis.
(4) Sequence alignment
The PCR product fragment was subjected to Sanger sequencing, and then the sequencing result was aligned with the full-length cDNA sequence (SEQ ID NO. 1) of the ribosomal RNA gene of the Fusarium sp of the mulberry, thereby determining whether the Fusarium sp of the mulberry exists on the leaf, and it was presumed that whether the Fusarium sp of the mulberry is likely to be pathogenic.
2. Experimental results
As shown in FIG. 2, only 13-16 lanes amplify single bright bands, namely only Fusarium sp DNA (fruiting body) of the pathogenic bacteria of the Mulberry pseudodry and total DNA of the disease branch with the Mulberry pseudodry are amplified to obtain target fragments, the size is about 200bp and is between 200 and 250bp, and other 8 fungi, 3 bacteria and ddH 2 None of the negative controls of O amplified fragments of similar size.
The above results illustrate: the specific primer set ZHR1RF/ZHR1RF designed according to the full-length cDNA sequence of the ribosomal RNA gene of the Fusarium sp of the mulberry can specifically detect the Fusarium sp of the mulberry.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> ribosomal RNA genes of Morus pseudodrynaria and use thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7894
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tacctggttg attctgccag tagtcatatg cttgtctcaa agattaagcc atgcatgtct 60
aagtataagc aattatacag cgaaactgcg aatggctcat tatataagtt atcgtttatt 120
tgatagtacc ttactacttg gataaccgtg gtaattctag agctaataca tgctaaaaat 180
cccgacttcg gaagggatgt atttattaga ttaaaaacca atgcccttcg gggctcactg 240
gtgattcatg ataactcctc gaatcgcatg gccttgtgcc ggcgatggtt cattcaaatt 300
tcttccctat caactttcga tgtttgggta ttggccaaac atggttgcaa cgggtaacgg 360
agggttaggg ctcgaccccg gagaaggagc ctgagaaacg gctactacat ccaaggaagg 420
cagcaggcgc gcaaattacc caatcccgac acggggaggt agtgacaata aatactgata 480
cagggctctt ttgggtcttg taattggaat gagtacaatt taaatccctt aacgaggaac 540
aattggaggg caagtctggt gccagcagcc gcggtaattc cagctccaat agcgtatatt 600
aaagttgttg tggttaaaaa gctcgtagtt gaaccttggg cctggctggc cggtccgcct 660
caccgcgtgt actggtccgg ccgggccttt ccctctgtgg aaccccatgc ccttcactgg 720
gtgtggcggg gaaacaggac ttttactgtg aaaaaattag agtgctccag gcaggcctat 780
gctcgaatac attagcatgg aataatagaa taggacgtgt ggttctattt tgttggtttc 840
taggaccgcc gtaatgatta atagggacag tcgggggcat cagtattcaa ttgtcagagg 900
tgaaattctt ggatttattg aagactaact actgcgaaag catttgccaa ggatgttttc 960
attaatcagg aacgaaagtt aggggatcga agacgatcag ataccgtcgt agtcttaacc 1020
ataaactatg ccgactaggg atcggacggt gttatttttt gacccgttcg gcaccttacg 1080
agaaatcaaa gtgcttgggc tccaggggga gtatggtcgc aaggctgaaa cttaaagaaa 1140
ttgacggaag ggcaccacca ggggtggagc ctgcggctta atttgactca acacggggaa 1200
actcaccagg tccagacaca atgaggattg acagattgag agctctttct tgattttgtg 1260
ggtggtggtg catggccgtt cttagttggt ggagtgattt gtctgcttaa ttgcgataac 1320
gaacgagacc ttaacctgct aaatagcccg tattgctttg gcagtacgct ggcttcttag 1380
agggactatc ggctcaagcc gatggaagtt tgaggcaata acaggtctgt gatgccctta 1440
gatgttctgg gccgcacgcg cgctacactg acggagccag cgagtacttc cttgtccgaa 1500
aggtccgggt aatcttgtta aactccgtcg tgctggggat agagcattgc aattattgct 1560
cttcaacgag gaatccctag taagcgcaag tcatcagctt gcgttgatta cgtccctgcc 1620
ctttgtacac accgcccgtc gctactaccg attgaatggc tcagtgaggc gtccggactg 1680
gcccagagag gtgggcaact accactcagg gccggaaagc tctccaaact cggtcattta 1740
gaggaagtaa aagtcgtaac aaggtctccg ttggtgaacc agcggaggga tcattaccga 1800
gtttacaact cccaaacccc tgtgaacata ccttaatgtt gcctcggcgg atcagcccgc 1860
gccctgtaaa acgggacggc ccgccagagg acccaaactc taatgtttct tattgtaact 1920
tctgagtaaa acaaacaaat aaatcaaaac tttcaacaac ggatctcttg gttctggcat 1980
cgatgaagaa cgcagcaaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc 2040
gaatctttga acgcacattg cgcccgctgg tattccggcg ggcatgcctg ttcgagcgtc 2100
atttcaaccc tcaagccccc gggtttggtg ttggggatcg gctctgcctc acggcggtgc 2160
cgcccccgaa atacattggc ggtctcgctg cagcctccat tgcgtagtag ctaacacctc 2220
gcaactggaa cgcggcgcgg ccatgccgta aaaccccaac ttctgaatgt tgacctcgga 2280
tcaggtagga atacccgctg aacttaagca tatcaataag cggaggaaaa gaaaccaaca 2340
gggattgccc tagtaacggc gagtgaagcg gcaacagctc aaatttgaaa tctggctttc 2400
gggcccgagt tgtaatttgt agaggatact tttgatgcgg tgccttccga gttccctgga 2460
acgggacgcc atagagggtg agagccccgt ctggttggat gccaaatctc tgtaaagttc 2520
cttcgacgag tcgagtagtt tgggaatgct gctctaaatg ggaggtatat gtcttctaaa 2580
gctaaatatt ggccagagac cgatagcgca caagtagagt gatcgaaaga tgaaaagcac 2640
tttgaaaaga gagttaaaaa gtacgtgaaa ttgttgaaag ggaagcgttt atgaccagac 2700
ttggacttgg ttaatcatct ggggttctcc ccagtgcact tttccagttc aggccagcat 2760
cagttttcgc cgggggataa aggcttcggg aatgtagctc tcttcgggga gtgttatagc 2820
ccgttgtgta ataccctggc ggagactgag gttcgcgctt ctgcaaggat gctggcgtaa 2880
tggtcatcaa cgacccgtct tgaaacacgg accaaggagt cgtcttcgta tgcgagtgtt 2940
cgggtgtcaa acccctacgc gtaatgaaag tgaacgcagg tgagagcttc ggcgcatcat 3000
cgaccgatcc tgatgttctc ggatggattt gagtaagagc atacggggcc ggacccgaaa 3060
gaaggtgaac tatgcctgta tagggtgaag ccagaggaaa ctctggtgga ggctcgcagc 3120
ggttctgacg tgcaaatcga tcgtcaaata tgggcatggg ggcgaaagac taatcgaacc 3180
ttctagtagc tggtttccgc cgaagtttcc ctcaggatag cagtgttgaa ctcagtttta 3240
tgaggtaaag cgaatgatta gggactcggg ggcgctatat tgccttcatc cattctcaaa 3300
ctttaaatat gtaagaagcc cttgttgctt aattgaacgt gggcattcga atgtatcaac 3360
actagtgggc catttttggt aagcagaact ggcgatgcgg gatgaaccga acgcgaggtt 3420
aaggtgccag agtagacgct catcagacac cacaaaaggt gttagtacat cttgacagca 3480
ggacggtggc catggaagtc ggaatccgct aaggactgtg taacaactca cctgccgaat 3540
gtactagccc tgaaaatgga tggcgctcaa gcgtctcacc catacctcgc cctcagggta 3600
gaaacgatgc cctgaggagt aggcggacgt ggaggtcagt gacgaagcct agggcgtgag 3660
cccgggttga acggcctcta gtgcagatct tggtggtagt agcaaatact tcaatgagaa 3720
cttgaaggac cgaagtgggg aaaggttcca tgtgaacagc ggttggacat gggttagtcg 3780
atcctaagct atagggaagt tccgtttcaa aggtgcactt tgcaccgtct agcgaaaggg 3840
gagccggtca atattccggc acctggatgt gggttttgcg cggcaacgca actgaacgtg 3900
gagacgacgg cgggggcccc gggcagagtt ctcttttctt cttaacagtc tatcaccctg 3960
aaatcggttt gtccggagct agggtttaat ggctggaaga gcccagcacc tctgctgggt 4020
ccggtgcgct ctcgacgtcc cttgaaaatc cacgggaagg aataattctc acgccagttc 4080
gtactcataa ccgcagcagg tctccaaggt gaacagcctc tggttgatag aacaatgtag 4140
ataagggaag tcggcaaaat agatccgtaa cttcgggata aggattggct ctaagggttg 4200
ggcacgttgg gccttgggcg gacgccttgg gagcaggctg ccactagtcg ggcaaccgac 4260
cggcggcggc cagcatccga gtgttgatgc ccttggcagg cttcggccgt ccggcgtgcg 4320
gttaacaacc aacttagaac tggtacggac aaggggaatc tgactgtcta attaaaacat 4380
agcattgcga tggccagaaa gtggtgttga cgcaatgtga tttctgccca gtgctctgaa 4440
tgtcaaagtg aagtaattca accaagcgcg ggtaaacggc gggagtaact atgactctct 4500
taaggtagcc aaatgcctcg tcatctaatt agtgacgcgc atgaatggat taacgagatt 4560
cccactgtcc ctatctacta tctagcgaaa ccacagccaa gggaacgggc ttggcagaat 4620
cagcggggaa agaagaccct gttgagcttg actctagttt gacattgtga aaagacatag 4680
gaggtgtaga ataggtggga gcttcggcgc cggtgaaata ccactactcc tattgttttt 4740
ttacttattc aatgaagcgg ggctggattt acgtccaact tctggtttta aggtccttcg 4800
cgggccgacc cgggttgaag acattgtcag gtggggagtt tggctggggc ggcacatctg 4860
ttaaaccata acgcaggtgt cctaaggggg gctcatggag aacagaaatc tccagtagaa 4920
caaaagggta aaagtcccct tgattttgat tttcagtgtg aatacaaacc atgaaagtgt 4980
ggcctatcga tcctttagtc cctcgacatt tgaggctaga ggtgccagaa aagttaccac 5040
agggataact ggcttgtggc ggccaagcgt tcatagcgac gtcgcttttt gatccttcga 5100
tgtcggctct tcctatcata ccgaagcaga attcggtaag cgttggattg ttcacccact 5160
aatagggaac gtgagctggg tttagaccgt cgtgagacag gttagtttta ccctactgat 5220
gacctcaccg caatggtaat tcagcttagt acgagaggaa ccgctgattc agataattgg 5280
tttttgcggc tgtccgaccg ggcagtgccg cgaagctacc atctgctgga taatggctga 5340
acgcctctaa gtcagaatcc atgccagaac gcggtgatac cacccgcacg tatagatgga 5400
caagaatagg cctcggctta gcgtcttagc aggcgattct tccacggcgc tcgaagcgtg 5460
tcgtggtatt tcgcgtattg taattttaac acgagcgggg tcaaatcctt tgcagacgac 5520
ttagctgtgc gaaacggtcc tgtaagcagt agagtagcct tgttgttacg atctgctgag 5580
ggtaagccgt ccttcgcctc gatttcccca atgatttctc tgagcaattc agggagttgt 5640
aggggttgtg ggttttgggt gcttttgatg tgtcgtctcc gggcggcggg tgcagggtaa 5700
gcaggtttga cttggtgaaa atcgttcgag cattgggagc ccggtcttgc gctgctgcgt 5760
gctttggggg gtgcagggta ggctggttga actttgtcga tttgggtgtc ggcttggggg 5820
tttacctatg cgggtggtct ggtatagggt aggctacttc ctaggtgagc gcgtagctgt 5880
ctgtttcctc cggctaccgg gtaggcagct ttgccttggc cgattcagcg gctcgaggtg 5940
cagggtaggc aggttagatc cagctctagg gtaggtacag ggtaggcagt cctatatagc 6000
gtcttgcggg tgtagggtag gcgagtatag ggtaggcagc tttaatcgag cggattgcac 6060
tactggcata accagtctga atccttgagg tctagggtag gagtctaggg taggtgcggc 6120
atacattatg gtttggccgt gagtcgattt ttttgttttc ccatagttaa gaatattgcg 6180
gaaaatcaaa agtggcccgg gaacccggtc tggcgtgcgg ccgtctcgaa tcctcccgga 6240
cggtatgtga gaggtgaacc atcggcccgg cgggggccca ggggcagcct cagggtaggt 6300
aaaatcaaaa aagttgttaa gaggcgcggt gtcggtgtgc ttgtactgag gttagagatt 6360
tatttacagg gtactgtggg gcagcggatg gacctgagct ctgtgggctg ggaacttttt 6420
tttcggtggg tggtggggtg tagggtagct gagcaggtct agggtaggta ctatataggg 6480
taggtacata gtaagtggta gacagtatag ggtagctcta tttagggtat agagtaaata 6540
ggggtatagg gtaagtagtt agaaagaggg tggcctaaag aattatttta aaattaatta 6600
gtagtataga caggtttggc tggttttagg gtatagggta ggctatactt actatagggt 6660
aagctataaa aggtctaggg taggttaaaa agaggtaaaa taactaagga attgttaata 6720
tgggtgattt gatatggttg agaggaaaaa gtgcgagagg ttactgagtg aaatgttgaa 6780
aaagtgatgt gatagactgc aagtccggcg cagagatata gtaacagatc gacctggata 6840
ccaaaagacg cgtcttttct cgacgagtcc agcggtagtg gcctcgggct gaacgggtga 6900
ctgtgtgagg agccgtggct gttggaaggt cggtgctggt ctggggggtg gtatagggta 6960
ggctgtcggg tatagggtag gcacctctta ttggccagaa tcggctctgc tatagggtaa 7020
gctcattgtg actatagggt agctacaggg taggccactt tcctgcagag agccggctac 7080
cctacagcca acttcgatcg ccctttgcgg cggccacaga cctcgcacgg ggtcgggacc 7140
accaccatta gactcgccgc ggcctattta gtcggttggg agacttttga aaaaatgcgt 7200
gcaaaatggt tttgtggttt ggtggccgtg agtcgatttt tttgttttcc catagttaag 7260
aattttgcgg aaaataaaaa gtggcccacg agccggttct ggcgtgcggc ccactaaaac 7320
ggtctcggag ggtatatgag aagggggcaa agccgcccgg cctgaaaggg tcggacaaag 7380
cggcggcgca tccctctcag tacctgatct tgcagacttc cactgcgtgt ccctctgtac 7440
agctttgaag gccccggcct cggcagcggg gggttcatag tggcggtcga cctccacgaa 7500
accgcacgct ccggcatgac ggcgtactgg ggatgcctgc gttacggcag ctagggcttg 7560
ctctggctgc cagcagatgg gctctgtgga tgactggccg ctggctagac ctgaaactag 7620
agcatcggga ggtaacctca cgctgcggac accgaaatgg tagaagcagt gtgctgcgtc 7680
ctcctcctgg ggcccccaag ccacaccacc cacagcggga ccggtgcggc ggacggaagc 7740
cctggggaac ttagaggggg aaagcggatt gcctgtcacg gcgcggttgg cctctgccga 7800
acgtgctgcg accggcgcga cctcagtgtc gccaccagta acttatttct ccggcgcctt 7860
cgggcgctgg tggccaaccc cggcacacga tagt 7894
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gccagtccgt acttgttcgt 20
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gcgcctaccc tgtagcaaat 20
Claims (2)
1. The application of the ribosomal RNA gene of the mulberry quasipunculus sp in detecting the mulberry quasipunculus sp is provided, and the full-length cDNA sequence of the ribosomal RNA gene is shown as SEQ ID NO. 1.
2. A method for detecting Fusarium sp which is a pathogenic bacterium of the mulberry tree is characterized by detecting whether a ribosome RNA gene of the Fusarium sp which is a pathogenic bacterium of the mulberry tree exists in nucleic acid of a sample to be detected, if so, the sample to be detected is positive to the Fusarium sp which is a pathogenic bacterium of the mulberry tree, and the full-length cDNA sequence of the ribosome RNA gene is shown as SEQ ID NO. 1.
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| CN112779274B (en) * | 2021-01-11 | 2023-05-09 | 华南农业大学 | Ribosomal RNA gene of mulberry plaster disease pathogenic bacteria and application thereof |
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