CN111197050A - Ribosomal RNA gene of mulberry pseudoblight pathogenic bacteria and application thereof - Google Patents
Ribosomal RNA gene of mulberry pseudoblight pathogenic bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses a ribosomal RNA gene of mulberry pseudoblight pathogenic bacteria and application thereof. The full-length cDNA sequence of the ribosomal RNA gene of the mulberry pseudoblight pathogenic bacteria is shown as SEQ ID NO.1, and the ribosomal RNA gene of the mulberry pseudoblight pathogenic bacteria Fusarium sp can be applied to detection of the mulberry pseudoblight pathogenic bacteria Fusarium sp or classification of fungal species; based on the gene, a specific primer capable of specifically detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp is designed, a method and a detection kit for efficiently, quickly and specifically detecting the mulberry pseudostem blight pathogenic bacteria Fusarium sp are established, and the application prospect in detecting the mulberry pseudostem blight pathogenic bacteria Fusarium sp is very wide.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, relates to a ribosomal RNA gene of mulberry rhizoctonia solani pathogenic bacteria and application thereof.
Background
Mulberry pseudostem blight (Fusarium sp) is widely distributed, and both main stems and branches can cause diseases. Generally, pathogenic bacteria of the mulberry tree trunk blight live through the winter on diseased branches, and the pathogenic bacteria spread with wind, rain and insects in the next 4-5 months, and continuously invade branches which are weak in growth, young in mulberry trees and damaged by insects. When pathogenic bacteria of the pseudostem blight of the mulberry are harmful branches, most of the pathogenic bacteria of the pseudostem blight of the mulberry are on half branches, usually winter buds are taken as centers, brown oblong disease spots are formed, the disease spots are in an edema shape when wet, and the disease spots shrink after being dried, are easily peeled off by a harmful cortex, and are densely grown black spots under the skin. If the scab surrounds the branch for a circle, the branch withers. In recent years, the disease area and the damage degree of the mulberry pseudoblight in mulberry fields are increased year by year, and the yield and the quality of the mulberry are seriously influenced. Therefore, the detection of the pathogenic bacteria of the mulberry pseudoblight disease, especially the early detection, is extremely important.
In the existing methods for studying fungi, sequencing alignment is usually 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 for phylogeny between analytical families or higher order elements. 5.8S rDNA sequences are short and highly conserved, so that the sequences are difficult to be used for phylogeny and molecular identification of fungi; whereas the 18S rDNA fragment is long, and both the conserved and variable regions are present in the fragment. Therefore, in the existing research, after a certain domain fragment is amplified by selecting different specific amplification primers, sequencing and analyzing and comparing sequencing results can be used for researching the class order of fungoid, family, genus and the like. However, classification of fungal species based on the rDNA sequences of 5.8S, 18S, and 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 the study.
At present, no relevant report is provided on a detection method of pathogenic bacteria of the mulberry pseudoblight, so that the establishment of the method capable of quickly and specifically detecting the pathogenic bacteria of the mulberry pseudoblight has important significance for controlling the morbidity and the harm of the mulberry pseudoblight.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the blank of the existing oak early baking disease pathogenic bacteria detection technology and provides a ribosomal RNA gene of mulberry pseudostem blight pathogenic bacteria Fusarium sp and application thereof.
The first purpose of the invention is to provide a ribosomal RNA gene of mulberry rhizoctonia solani pathogenic bacterium Fusarium sp.
The second purpose of the invention is to provide the application of the ribosomal RNA gene of the mulberry pseudostem blight pathogenic bacterium Fusarium sp in detecting the mulberry pseudostem blight pathogenic bacterium Fusarium sp or in fungus species classification.
The third purpose of the invention is to provide a method for detecting Fusarium sp which is a pathogen of rhizoctonia solani of mulberry.
The fourth purpose of the invention is to provide a group of primers for detecting the Fusarium sp which is a pathogen of the mulberry rhizoctonia solani.
The fifth purpose of the invention is to provide the application of the primer in detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp and/or preparing a kit for detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp.
The sixth purpose of the invention is to provide a method for detecting Fusarium sp which is a pathogen of rhizoctonia solani of mulberry.
The seventh purpose of the invention is to provide a kit for detecting mulberry rhizoctonia solani pathogenic bacteria Fusarium sp.
The eighth purpose of the invention is to provide the application of the method or the kit in detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a ribosomal RNA gene of mulberry bacterial wilt-like pathogen Fusarium sp, and the full-length cDNA sequence of the ribosomal RNA gene is shown in SEQ ID NO. 1.
The invention obtains the full-length cDNA sequence of the ribosomal RNA gene of the mulberry pseudostem blight pathogenic bacterium Fusarium sp for the first time, and the length of the full-length cDNA sequence is 7894 bp. 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 a base sequence from 1 st to 1796 th in a sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS1 gene is the 1797 th to 1946 th base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 5.8SrRNA gene is a base sequence of 1947-2104 in a sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS2 gene is a 2105-2267 th base sequence in a sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 28S rRNA gene is the base sequence 2268-5608 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 the base sequence of 5609-7894 in the sequence shown in SEQ ID NO. 1.
The application of the ribosomal RNA gene of the mulberry pseudostem blight pathogenic bacterium Fusarium sp in detecting the mulberry pseudostem blight pathogenic bacterium Fusarium sp or in classifying fungal species also belongs to the protection scope of the invention.
The invention also provides a method for detecting the mulberry pseudostem blight pathogenic bacteria Fusarium sp, which is characterized by comparing the full-length cDNA sequence of the ribosome 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 mulberry pseudostem blight pathogenic bacteria Fusarium sp according to the comparison result.
Preferably, the method is: and taking the DNA of a sample to be detected as a template, performing library construction, high-throughput sequencing and assembly to obtain complete ribosomal DNA, then comparing the complete ribosomal DNA with the full-length cDNA sequence of the ribosomal RNA gene, and judging whether the sample to be detected contains Fusarium sp which is a pathogen of the mulberry pseudostem blight or not according to the comparison result.
Particularly preferably, the method comprises the following steps:
s1, collecting mulberry branch of paraquat disease;
s2, extracting total DNA of the branches of the mulberry tree with the disease of paraquat;
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: the total DNA described in step S2 was constructed as a double-ended high-throughput sequencing library with a fragment size of 350bp according to the Illumina DNA library construction protocol.
The method for analyzing the ribosomal DNA sequences in an alignment manner in step S8 comprises the following steps: and (3) comparing the complete ribosomal DNA sequence in the step S7 with the full-length cDNA sequence of the ribosomal RNA gene of the mulberry rhizoctonia solani pathogenic bacterium Fusarium sp by using sequence comparison analysis software.
The method for removing the mulberry genome sequence in the sequencing data in the step S5 comprises the following steps: comparing and analyzing the high-throughput sequencing data obtained in the step S4 by using comparison software; 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.
Preferably, the alignment software is software bwa 0.7.17-r 1188.
Preferably, the alignment algorithm is a mem alignment algorithm.
Preferably, the written computer program is a python language program.
The method for assembling the microbial genome sequence in the step S6 comprises the following steps: 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: the sequence tags initially assembled by adopting SPAdes v.3.5.0 software are broken ribosome tags, and in order to obtain a complete ribosome DNA sequence, a sequence capture and de novo assembly strategy is adopted for analysis so as to assemble the complete ribosome DNA; selecting a ribosome DNA sequence containing a suspected target pathogenic bacterium ITS sequence as a reference sequence, performing double-end sequence comparison by adopting bwa 0.7.17-r1188 software, obtaining 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 a complete ribosome DNA sequence through a plurality of circulation 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 the mulberry bakanae disease-like pathogenic bacterium Fusarium sp, and the primers can amplify the sequence shown in SEQ ID NO.1 or the fragment 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', respectively;
nucleotide sequence of primer ZHR1RF (SEQ ID NO. 3): 5'-GCGCCTACCCTGTAGCAAAT-3' are provided.
The application of the primer in detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp and/or preparing a kit for detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp also belongs to the protection scope of the invention.
Based on the primers, the invention also provides a method for detecting the mulberry bacterial wilt-like pathogen Fusarium sp, wherein the method is used for detecting whether the ribosomal RNA gene exists in the nucleic acid of the sample to be detected, and if so, the sample to be detected is positive to the mulberry bacterial wilt-like pathogen Fusarium sp.
Preferably, the method is: and (3) performing PCR amplification by using the nucleic acid of the sample to be detected as a template and using the primer, wherein if the amplification result is positive, the sample to be detected is positive for the pathogenic bacterium Fusarium sp of the mulberry tree rhizoctonia solani.
Preferably, the method for determining is: and (3) carrying out gel electrophoresis on the reaction product obtained by the PCR amplification, and if a strip appears and the size of the strip is 200bp, judging that the sample to be detected contains Fusarium sp which is pathogenic bacteria of the mulberry tree 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, balance ddH2O make up to 20. mu.L.
Preferably, the reaction conditions for the PCR amplification are: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 2min at 72 ℃ and 35 cycles; 5min at 72 ℃.
The invention also provides a kit for detecting the pathogenic bacteria Fusarium sp of the mulberry bacterial wilt-like disease, and the kit comprises the primer.
In addition, the application of the method or the kit in detecting the mulberry rhizoctonia solani pathogenic bacterium Fusarium sp also belongs to the protection scope of the invention.
The invention has the following beneficial effects:
the invention provides a ribosomal RNA gene of mulberry pseudoblight pathogenic bacteria and application thereof. The invention obtains the full-length cDNA sequence of the ribosomal RNA gene of the mulberry pseudostem blight pathogenic bacterium Fusarium sp for the first time, the length of the full-length cDNA sequence is 7894bp, and the ribosomal RNA gene of the mulberry pseudostem blight pathogenic bacterium Fusarium sp can be applied to detection of the mulberry pseudostem blight pathogenic bacterium Fusarium sp or classification of fungal species; based on the gene, a specific primer capable of specifically detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp is designed, a method and a detection kit for efficiently, quickly and specifically detecting the mulberry pseudostem blight pathogenic bacteria Fusarium sp are established, and the application prospect in detecting the mulberry pseudostem blight pathogenic bacteria Fusarium sp is very wide.
Drawings
FIG. 1 shows the fungal microorganism classification detected from diseased mulberry shoots.
FIG. 2 is a diagram showing the result of specific detection of Fusarium sp, a pathogen of pseudostem blight of mulberry; wherein, M: TakaraDL2000 Marker; 1: mucor racemosu (Mucor racemosus); 2: actinomucor elegans (Actinomucor elegans); 3: cladosporium perangustum (Cladosporium pernyu); 4: fusarium oxysporum (Fusarium oxysporum); 5: auricularia polytricha (Auricularia polytricha); 6: fusarium equiseti (Fusarium equiseti); 7: trichoderma (Trichoderma); 8: mucor circinelloides (Mucor circinelloides); 9: ralstonia solanacearum (lakerea solanacearum); 10: enterobacter cloacae (Enterobacter cloacae); 11: klebsiella sp (Klebsiella sp); 12: blank spaceControl (ddH)2O); 13 and 14: fusarium sp DNA of mulberry pseudoblight pathogenic bacteria; 15 and 16: total DNA of diseased branches with mulberry pseudoblight disease.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 detection of ribosomal RNA Gene of Fusarium sp, a pathogen of Mulberry bacterial wilt
1. Experimental methods
(1) High throughput sequencing
Randomly searching diseased branches with typical mulberry paraquat disease symptoms in a diseased mulberry garden, collecting, cleaning the surfaces with sterile water, cutting off lesion materials, fully grinding with liquid nitrogen, extracting total DNA by using a Shanghai bio-fungus genome DNA extraction kit according to the operation instructions, and storing the extracted total DNA at-20 ℃; randomly breaking the qualified DNA sample into fragments with the length of about 350bp by adopting a Covaris ultrasonic disruptor (Covaris, USA), constructing a small fragment genomic DNA library, and referring to NEB in the library construction process
Ultra DNA Library Prep Kit for
(NEB, USA) library construction kit instructions. Quality control was carried out after library construction by the qPCR method and Agilent2100Bioanalyzer (Agilent Technologies, USA). Sequencing the DNA library qualified by 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 quantity of each sample is not less than 2 Gb.
(2) Assembling microbial genome sequences
The microbial sequence assembly is carried out by SPAdes v.3.5.0 software; the ribosomal DNA sequence of the target fungus consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment. The sequence tags initially assembled by SPAdes v.3.5.0 software were fragmented ribosomal tags and in order to obtain the complete ribosomal DNA sequence, the analysis used sequence capture and de novo assembly strategies to assemble the complete ribosomal DNA. Selecting a ribosome DNA sequence containing a suspected target pathogen ITS sequence as a reference sequence, performing double-end sequence comparison by adopting bwa 0.7.17-r1188 software, obtaining 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 a complete ribosome DNA sequence through a plurality of circulation operations. For the assembly uncertainty region, PCR amplification and Sanger sequencing were performed to verify the correctness of the assembly.
Sequence tag annotation the sequencing sequence tags were analyzed in alignment with the bacterial and fungal libraries of the nt database of NCBI, respectively, 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 and identification and quantification use ribosomal DNA as the main molecular marker. And other species select a conserved sequence as a judgment basis according to the published sequence information. According to multiple dimensions of sequence alignment similarity, alignment sequence length, sequence integrity and the like, selecting an optimal alignment result to annotate the Tag sequence through a similarity evaluation algorithm, and analyzing the diversity of microorganisms in tissues. And calculating the average sequencing depth value of the sequence tag according to the statistic of the sequence tag, and taking the average sequencing depth value as the relative abundance value of the species.
2. Results of the experiment
The full-length cDNA sequence of the ribosomal RNA gene of mulberry bacterial wilt-like pathogen Fusarium sp is shown in SEQ ID NO.1, and the ribosomal RNA gene consists of 18S rRNA gene, ITS1 gene, 5.8S rRNA gene, ITS2 gene and 28S rRNA gene; the nucleotide sequence of the 18S rRNA gene is a base sequence from 1 st to 1796 th in a sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS1 gene is the 1797 th to 1946 th base sequence in the sequence shown in SEQ ID NO. 1; 5.8 the nucleotide sequence of the SrRNA gene is the base sequence of 1947-2104 in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS2 gene is a 2105-2267 th base sequence in a sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 28S rRNA gene is the 2268-5608 base sequence 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 the base sequence of 5609-7894 in the sequence shown in SEQ ID NO. 1.
Example 2 method for detecting Fusarium sp of mulberry pseudostem blight pathogenic bacteria
1. Experimental methods
(1) High throughput sequencing
Randomly searching diseased branches with typical mulberry paraquat disease symptoms in a diseased mulberry garden, collecting, cleaning the surfaces with sterile water, cutting off lesion materials, fully grinding with liquid nitrogen, extracting total DNA by using a Shanghai bio-fungus genome DNA extraction kit according to the operation instructions, and storing the extracted total DNA at-20 ℃; randomly breaking the qualified DNA sample into fragments with the length of about 350bp by adopting a Covaris ultrasonic disruptor (Covaris, USA), constructing a small fragment genomic DNA library, and referring to NEB in the library construction process
Ultra DNA Library Prep Kit for
(NEB, USA) library construction kit instructions. Quality control was carried out after library construction by the qPCR method and Agilent2100Bioanalyzer (Agilent Technologies, USA). Sequencing the DNA library qualified by 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 quantity of each sample is not less than 2 Gb.
(2) Assembling microbial genome sequences
The microbial sequence assembly is carried out by SPAdes v.3.5.0 software; the ribosomal DNA sequence of the target fungus consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment. The sequence tags initially assembled by SPAdes v.3.5.0 software were fragmented ribosomal tags and in order to obtain the complete ribosomal DNA sequence, the analysis used sequence capture and de novo assembly strategies to assemble the complete ribosomal DNA. Selecting a ribosome DNA sequence containing a suspected target pathogen ITS sequence as a reference sequence, performing double-end sequence comparison by adopting bwa 0.7.17-r1188 software, obtaining 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 a complete ribosome DNA sequence through a plurality of circulation operations. For the assembly uncertainty region, PCR amplification and Sanger sequencing were performed to verify the correctness of the assembly.
Sequence tag annotation the sequencing sequence tags were analyzed in alignment with the bacterial and fungal libraries of the nt database of NCBI, respectively, 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 and identification and quantification use ribosomal DNA as the main molecular marker. And other species select a conserved sequence as a judgment basis according to the published sequence information. According to multiple dimensions of sequence alignment similarity, alignment sequence length, sequence integrity and the like, selecting an optimal alignment result to annotate the Tag sequence through a similarity evaluation algorithm, and analyzing the diversity of microorganisms in tissues. And calculating the average sequencing depth value of the sequence tag according to the statistic of the sequence tag, and taking the average sequencing depth value as the relative abundance value of the species.
(3) Sequence annotation and genetic evolution analysis
And assembling according to the high-throughput sequencing data and experimental verification results to obtain a complete ribosomal DNA sequence. The annotation of rRNA sequences is performed by adopting a blastn alignment strategy based on the rRNA structures of closely related species in a nt database, and the lengths and GC proportions of intervals of 18S, ITS1, 5.8S, ITS2 and 28S are respectively counted. The analysis of the species and quantity of bacteria and fungi was visualized using MEGAN (Version 6.4.0). Bacteria identification conditions: the length of the sequence is more than 500bp, the similarity with the aligned sequence in the nt database is more than 97%, and the sequence coverage is more than 80%. And (3) fungus identification conditions: the length of the sequence is more than 500bp, the similarity with the aligned sequence in the nt database is more than 75%, and the sequence coverage is more than 80%.
2. Results of the experiment
According to the analysis result of the types and the quantity of the microorganisms, the suspected pathogen is the Fusarium species by combining the examination of the material symptoms and the related data.
As shown in figure 1, according to the principle that the higher the numerical value is, the higher the relative abundance of the species is, the fungi with the highest relative abundance is found to be pathogenic bacteria of the genus Fusarium by inquiring the annotation result of the sequence tags and comparing the annotation result with the full-length cDNA sequence of the ribosomal RNA gene of the pathogenic bacteria Fusarium sp of the pseudoblight of the mulberry.
Example 3 method for detecting Fusarium sp, a pathogen of phomopsis
1. Experimental methods
(1) PCR amplification reaction
In order to further utilize the full-length cDNA sequence of the ribosomal RNA gene of the mulberry pseudostem blight pathogenic bacterium Fusarium sp to be applied to the pathogen detection and identification of the mulberry pseudostem blight, the invention further designs a pair of specific primer groups ZHR1RF/ZHR1RF, and the nucleotide sequence of the primer groups is shown in Table 1;
then, using the total DNA of the infected mulberry tree rhizoctonia solani as a template, and using other 8 fungi, 3 bacteria and ddH2And O is a negative control, the primer group ZHR1RF/ZHR1R is used for PCR amplification, a PCR amplification reaction system is shown in Table 2, and the size of a target fragment to be amplified is about 200 bp.
TABLE 1 nucleotide sequences of the specific primer set ZHR1RF/ZHR1RF
TABLE 2PCR amplification reaction System
(2) Conditions for PCR amplification reactions
The conditions for verifying the PCR amplification reaction specific for the primer set ZHR1RF/ZHR1R were: 5min at 94 ℃; 30s at 94 ℃, 30s at 47 ℃, 2min at 72 ℃ and 35 cycles; 5min at 72 ℃.
(3) Detection of PCR amplification reaction product
After the PCR reaction was completed, 4. mu.L of each PCR amplification product was detected by electrophoresis using 1.0% agarose gel (EB staining), and PCR product fragments corresponding in size were recovered by agarose gel electrophoresis.
(4) Sequence alignment
And performing Sanger sequencing on the PCR product fragment, and then comparing the sequencing result with the full-length cDNA sequence (SEQ ID NO.1) of ribosomal RNA gene of mulberry pseudostem blight pathogen Fusarium sp, thereby determining whether the mulberry pseudostem blight pathogen Fusarium sp exists on the blade, and thus presuming whether the mulberry pseudostem blight pathogen Fusarium sp is possibly a pathogenic bacterium.
2. Results of the experiment
The specific detection result of mulberry pseudostem blight pathogenic bacterium Fusarium sp is shown in FIG. 2, and it can be seen that only 13-16 lanes amplify a single bright band, that is, only the mulberry pseudostem blight pathogenic bacterium Fusarium sp DNA (fruiting body) and the total DNA of the branches with mulberry pseudostem blight amplify target fragments, the size of the target fragments is about 200bp and is between 200-250 bp, and other 8 fungi, 3 bacteria and ddH2None of the negative controls for O amplified a fragment of similar size.
The above results illustrate that: the specific primer group ZHR1RF/ZHR1RF designed according to the full-length cDNA sequence of ribosomal RNA gene of mulberry pseudostem blight pathogenic bacterium Fusarium sp can specifically detect the mulberry pseudostem blight pathogenic bacterium Fusarium sp.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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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
tggtcatcaacgacccgtct 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 catagttaag7260
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 (10)
1. A ribosomal RNA gene of mulberry bacterial wilt-like pathogen Fusarium sp is characterized in that the full-length cDNA sequence of the ribosomal RNA gene is shown in SEQ ID NO. 1.
2. The use of the ribosomal RNA gene of mulberry pseudostem blight pathogen Fusarium sp as claimed in claim 1 for detecting or classifying fungi species.
3. A method for detecting mulberry pseudostem blight pathogenic bacteria Fusarium sp is characterized in that a full-length cDNA sequence of ribosome RNA genes of a sample to be detected is compared with a sequence shown in SEQ ID NO.1, and whether the sample to be detected contains the mulberry pseudostem blight pathogenic bacteria Fusarium sp is judged according to a comparison result.
4. A group of primers for detecting mulberry rhizoctonia solani pathogenic bacteria Fusarium sp is characterized in that the primers can amplify a sequence shown in SEQ ID NO.1 or a fragment thereof.
5. The primer according to claim 4, wherein the primer is a primer group ZHR1RF/ZHR1RF, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 2-3.
6. The use of the primer of claim 5 in detecting mulberry pseudoblight pathogenic bacterium Fusarium sp and/or in preparing a kit for detecting mulberry pseudoblight pathogenic bacterium Fusarium sp.
7. A method for detecting mulberry bacterial wilt-like pathogen Fusarium sp, which is characterized in that the method is used for detecting whether the ribosomal RNA gene of claim 1 exists in nucleic acid of a sample to be detected, and if the ribosomal RNA gene exists, the sample to be detected is positive to the mulberry bacterial wilt-like pathogen Fusarium sp.
8. The method according to claim 7, wherein the nucleic acid of the sample is used as a template, the primer according to claim 4 or 5 is used for PCR amplification, and if the amplification result is positive, the sample is positive for Fusarium sp.
9. A kit for detecting Fusarium sp, a pathogen of rhizoctonia solani of mulberry, which is characterized in that the kit comprises the primer of claim 4 or 5.
10. Use of the method according to any one of claims 7 to 8 or the kit according to claim 9 for detecting Fusarium sp.
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| 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 |
| CN112779274A (en) * | 2021-01-11 | 2021-05-11 | 华南农业大学 | Ribosomal RNA gene of mulberry plaster disease pathogenic bacteria and application thereof |
| CN113151521A (en) * | 2021-04-26 | 2021-07-23 | 华南农业大学 | Ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof |
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