CN113151521B - Ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof - Google Patents

Ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof Download PDF

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CN113151521B
CN113151521B CN202110455095.6A CN202110455095A CN113151521B CN 113151521 B CN113151521 B CN 113151521B CN 202110455095 A CN202110455095 A CN 202110455095A CN 113151521 B CN113151521 B CN 113151521B
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刘吉平
罗龙辉
孙勋勋
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Guangdong Nongda Assets Management Co ltd
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Abstract

The invention discloses a ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof. The full-length cDNA sequence of the ribosomal RNA gene of the mulberry red rust pathogenic bacteria Puccinia sp is shown as SEQ ID NO.1, and the ribosomal RNA gene of the mulberry red rust pathogenic bacteria Puccinia sp can be applied to detection of the mulberry red rust pathogenic bacteria Puccinia sp or classification of fungal species; a specific primer for detecting the mulberry red rust pathogenic bacteria Puccinia sp is designed based on the gene, the primer has strong specificity and high sensitivity, a method and a detection kit for efficiently, quickly and specifically detecting the mulberry red rust pathogenic bacteria Puccinia sp are established, and the application prospect in detecting the mulberry red rust pathogenic bacteria Puccinia sp is very wide.

Description

Ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof.
Background
3238 Zxft 3238 rust is commonly known as 3262 Zxft 3262 and gold leaf, and is a fungal disease. Mainly comprises tender shoots, young leaves, young shoots, flowers, mulberry and the like which are harmful to the mulberry. The tender buds of the mulberry are damaged by germs and then cause deformity or bending of diseased parts, and the mulberry buds cannot germinate. After the leaves are infected with diseases, round and glossy small points are scattered on the front and back surfaces of the leaves and gradually bulge into a green bubble shape, the color turns yellow (yellow bubble) after the leaves are mature, the epidermis is cracked, orange powdery rust is emitted, and the rust is distributed on the whole leaves (so the name of Jin Sang). After the rust spores are scattered out, the rust apparatus is aged and scorched into 'scorched bubbles'. The disease of young shoots, petioles and veins of leaves develops in a longitudinal strip shape along the vascular bundle direction, the surfaces of the young shoots are all provided with orange rust, disease spots on the young shoots gradually turn black and concave, and the disease of mulberry flowers irregularly expands. The mulberry loses the original luster and turns yellow after being infected with diseases, and orange yellow powder is distributed in the later period. Sang Chi rust is serious in damage, not only causes the reduction of yield of mulberry leaves, but also seriously affects the quality of the mulberry leaves, and results in the failure of silkworm cocoons.
At present, the technology is difficult to separate pathogenic bacteria related to red rust of mulberry, so pathogenic bacteria causing red rust of mulberry are not reported, a method for thoroughly radically treating red rust of mulberry is not provided, and a method for detecting pathogenic bacteria related to red rust of mulberry is not reported. Therefore, a method for detecting and identifying pathogenic bacteria of red rust of mulberry trees is needed, so as to facilitate early warning and prevention and control of red rust of mulberry trees.
Disclosure of Invention
The invention aims to solve the technical problem of providing a ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof, aiming at the defect of detection of mulberry red rust related pathogenic bacteria in the prior art.
A first object of the present invention is to provide a ribosomal RNA gene of the red rust pathogen Puccinia sp.
The second purpose of the invention is to provide the application of the ribosomal RNA gene of the mulberry red rust pathogen Puccinia sp in detecting the mulberry red rust pathogen Puccinia sp or in classifying fungal species.
The third purpose of the invention is to provide a method for detecting mulberry red rust pathogenic bacteria Puccinia sp.
The fourth purpose of the invention is to provide a group of primers for detecting the red rust pathogenic bacteria Puccinia sp.
The fifth purpose of the invention is to provide the application of the primer in detecting mulberry red rust pathogenic bacteria Puccinia sp.
The sixth purpose of the invention is to provide a kit for detecting mulberry red rust pathogenic bacteria Puccinia sp.
The seventh purpose of the invention is to provide the application of any one of the methods or the kit in detecting the red rust pathogenic bacteria Puccinia sp.
An eighth object of the present invention is to provide a method for detecting the red rust pathogen Puccinia sp.
The above purpose of the invention is realized by the following technical scheme:
a ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp, wherein the full-length cDNA sequence of the ribosomal RNA gene is shown as SEQ ID NO. 1.
The ribosomal RNA consists of 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA; the cDNA sequence of the 18S rRNA is a base sequence from 1 st to 1747 th in the sequence shown in SEQ ID NO. 1; the cDNA sequence of ITS1 is the base sequence 1748-1972 in the sequence shown in SEQ ID NO. 1; the cDNA sequence of the 5.8S rRNA is a base sequence 1973 to 2129 in the sequence shown in SEQ ID NO. 1; the cDNA sequence of ITS2 is base sequence 2130-2398 in the sequence shown in SEQ ID NO. 1; the cDNA sequence of the 28S rRNA is the base sequence of 2399-5765 in the sequence shown in SEQ ID NO. 1.
In addition, the application of the ribosomal RNA gene in detecting the red rust pathogenic bacteria Puccinia sp of mulberry or in fungus species classification also belongs to the protection scope of the invention.
The invention also provides a detection method of the mulberry red rust pathogenic bacteria Puccinia sp.
Preferably, 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 cDNA sequence of the ribosomal RNA, and judging whether the sample to be detected contains the pathogenic bacteria Puccinia sp.
More preferably, the method for detecting the red rust pathogenic bacteria Puccinia sp of the mulberry includes the following steps:
s1, collecting red rust disease leaves of a mulberry tree;
s2, extracting total DNA of red rust disease 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: according to the construction process of the Illumina DNA library, the total DNA in the step S2 is constructed into a double-end high-throughput sequencing library with the fragment size of 500 bp.
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 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.
The method for assembling the microbial genome sequence in the step S6 comprises the following steps: and (5) assembling the sequencing data obtained by removing the mulberry genome sequence in the sequencing data 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.
The method for analyzing the ribosome DNA sequence by comparison in the step S8 comprises the following steps: and (3) comparing the complete ribosomal DNA sequence of the step S7 with the nucleotide sequence of the ribosomal RNA gene of the red rust pathogen Puccinia sp of the mulberry tree by using sequence comparison analysis software.
The invention also provides a group of primers for detecting the red rust pathogenic bacteria Pucciia sp. Of the mulberry, and the nucleotide sequence of the primers is shown as SEQ ID NO. 2-3.
The nucleotide sequence of the primer is shown as follows:
CX1791F(SEQ ID NO.2)：5’-GGTGCACTTAGTTGTGGCTC-3’；
CX2269R(SEQ ID NO.3)：5’-CAGTACCAAGGCACACTCCT-3’。
in addition, the application of the primer in detecting the red rust pathogenic bacteria Puccinia sp of the mulberry or preparing a kit for detecting the red rust pathogenic bacteria Puccinia sp of the mulberry also belongs to the protection scope of the invention.
The invention claims a kit for detecting red rust pathogenic bacteria Puccinia sp of mulberry trees, which comprises the primer.
The application of the kit in detecting the red rust pathogenic bacteria Puccinia sp of the mulberry also belongs to the protection scope of the invention.
Based on the primers, the invention also provides another method for detecting the red rust pathogenic bacteria Puccinia sp of the mulberry, which comprises the steps of carrying out PCR amplification by using nucleic acid of a sample to be detected as a template and using the primers as claimed in claim 4, carrying out gel electrophoresis on a product obtained after the PCR amplification, and judging that the sample to be detected contains the red rust pathogenic bacteria Puccinia sp of the mulberry if a strip with the size of 498bp appears.
Preferably, the reaction system of the PCR amplification is: 2 XTaq Master Mix 10. Mu.L, 10. Mu.M primers according to claim 4 each 0.5. Mu.L, nucleic acid template 2. Mu.L, and ddH as a remainder 2 O make up to 20. Mu.L.
Preferably, the reaction conditions for the PCR amplification are: 4min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof. The invention obtains the full-length cDNA sequence of the ribosomal RNA gene of the red rust pathogenic bacteria Puccinia sp for the first time, the length of the full-length cDNA sequence is 5765bp, and the ribosomal RNA gene of the red rust pathogenic bacteria Puccinia sp can be applied to detecting the red rust pathogenic bacteria Puccinia sp of mulberry or classifying fungal species; a specific primer for detecting the mulberry red rust pathogenic bacteria Puccinia sp is designed based on the gene, the primer has strong specificity and high sensitivity, a method and a detection kit for efficiently, quickly and specifically detecting the mulberry red rust pathogenic bacteria Puccinia sp are established, and the application prospect in detecting the mulberry red rust pathogenic bacteria Puccinia sp is very wide.
Drawings
FIG. 1 shows the fungal microorganism classification tree detected from leaves of red rust of mulberry.
FIG. 2 is a specific result of a specific primer for detecting the red rust pathogenic bacteria Puccinia sp of mulberry; wherein, M: takaraDL2000 Marker;1: puccinia sp. (fruit body); 2 and 3 are Sang Chi rust disease leaf total DNA;4: fusarium equiseti (Fusarium equiseti); 5: gibberellasp. (gibberella); 6: alternaria sp. (Alternaria); 7: fusarium graminearum (Fusarium graminearum); 8: clonostachys (spirelospora rosea); 9: fusarium oxysporum (Fusarium oxysporum); 10: mucor circinelloides (Mucor circinelloides); 11: penicillium sp. (Penicillium); 12: phanerochaete chrysosporium (Phanerochaete chrysosporium); 13: mucor racemosus (Mucor racemosus); 14: actinomucor elegans (Actinomucor elegans); 15: blank control (ddH) 2 O)。
FIG. 3 shows the sensitivity results of specific primers for detecting pathogenic bacteria Puccinia sp of red rust of mulberry; wherein lane 1 is 12.5 ng/. Mu.l; lane 2 is 1.25X 10 -1 ng/mul; lane 3 is 1.25X 10 -1 ng/mul; lane 4 is 1.25X 10 - 2 ng/mul; lane 5 is 1.25X 10 -3 ng/mul; lane 6 is 1.25X 10 -4 ng/mul; lane 7 is 1.25X 10 -5 ng/mul; lane 8 is 1.25X 10 -6 ng/mul; lane 9 is 1.25X 10 -7 ng/μl。
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 obtaining of RNA sequence of Rice rust pathogen Puccinia sp. Of mulberry
At present, the technology is difficult to separate pathogenic bacteria related to red rust of mulberry, pathogenic bacteria causing red rust of mulberry are not reported, a method for radically treating red rust of mulberry is not provided, and the acquisition of RNA genome of pathogenic bacteria related to red rust of mulberry is based on sequencing of fruiting bodies at the diseased spots. The inventor determines that pathogenic bacteria Puccinia sp is the main pathogenic bacteria of red rust of mulberry trees by comparing normal leaves with leaves suffering from red rust and combining the infection of pathogenic bacteria of the genus on other species. Therefore, the invention provides a method for detecting and identifying the pathogenic bacteria of the red rust of the mulberry by taking the pathogenic bacteria Puccinia sp as a target strain to obtain the RNA sequence of the pathogenic bacteria.
1. Experimental methods
(1) High throughput sequencing
Randomly searching leaves with typical red rust disease spots of mulberry trees in diseased mulberry trees, collecting, cutting disease spot regions in diseased leaves of mulberry trees, cutting disease spot materials, fully grinding by using liquid nitrogen, extracting total DNA by using a Shanghai industrial fungus genome DNA extraction kit according to the operation instructions, and storing the extracted total DNA at-20 ℃; according to the construction process of an Illumina DNA library, constructing total DNA into a double-end high-throughput sequencing library with the fragment size of 500 bp; and (3) carrying out high-throughput sequencing on the constructed DNA library by using an Illumina Hiseq2500 sequencer, wherein the sequencing strategy is Pair-End 150bp.
(2) Assembling microbial genome sequences
The assembly of the microbial sequences was carried out using Meta Velvet (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. MetaVelvet (v1.2.01) initially assembled sequence tags were broken ribosomal tags and to obtain complete ribosomal DNA sequences, sequence capture and de novo assembly strategies were employed for analysis to assemble complete ribosomal DNA. Selecting a ribosomal DNA sequence containing an ITS sequence of a target pathogenic bacterium as a reference sequence, carrying out mismatch-free 0mismatch and break-free 0gap alignment by adopting bwa (0.7.12-r 1039) software, obtaining a double-end sequencing fragment from sequencing data according to an alignment result, further assembling and extending the sequence by adopting MetaVelvet (v1.2.01) assembly software, and obtaining a complete ribosomal DNA sequence through a plurality of circulation operations.
Sequence tag annotation the assembled sequence tag sequence was aligned to the nt database of NCBI using blastn (2.2.31 +) sequence alignment analysis software, with blastn alignment setting expectation <1e-20, and sequence tags 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 tag annotation result. The average sequencing depth of the ribosomal DNA fragments in the sequencing data was calculated using bwa (0.7.12-r 1039) + samtools (v 1.2) analysis software and used as the relative abundance value for this species.
(3) Assembly of complete ribosomal DNA sequences
Fungal ribosomal DNA consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment. 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.
(4) Comparative analysis of ribosomal DNA sequences
Selecting a ribosomal DNA sequence containing an ITS sequence of a target pathogenic bacterium as a reference sequence, carrying out mismatch-free 0mismatch and break-free 0gap alignment by adopting bwa (0.7.12-r 1039) software, obtaining a double-end sequencing fragment from sequencing data according to an alignment result, further assembling and extending the sequence by adopting MetaVelvet (v1.2.01) assembly software, and obtaining a complete ribosomal DNA sequence through a plurality of circulation operations.
2. Results of the experiment
The nucleotide sequence of the complete ribosomal RNA gene of the mulberry red rust pathogenic bacteria Puccinia sp is shown in SEQ ID No. 1. Wherein, the nucleotide sequence of the 18S rRNA gene is a base sequence from 1 st to 1747 th in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the ITS1 gene is a 1748 th-1972 nd base sequence in a sequence shown by SEQ ID NO. 1; 5.8S rRNA gene nucleotide sequence is 1973-2129 base sequence in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of ITS2 gene is base sequence 2130-2398 in the sequence shown in SEQ ID NO. 1; the nucleotide sequence of the 28S rRNA gene is the base sequence of 2399-5765 in the sequence shown in SEQ ID NO. 1.
Example 2 Classification and identification of Morus alba Chikopsora pathogenic bacteria Puccinia sp
1. Detection method
(1) High throughput sequencing
Randomly searching leaves with typical red rust disease spots in a diseased mulberry garden, collecting, shearing a disease spot area in the mulberry leaves, shearing disease spot materials, fully grinding by using liquid nitrogen, extracting total DNA by using a Kangshi century fungi DNA extraction kit specifically according to an operation instruction, and storing the extracted total DNA at-20 ℃; according to the construction process of an Illumina DNA library, constructing total DNA into a double-end high-throughput sequencing library with the fragment size of 500 bp; the constructed DNA library was subjected to high throughput sequencing using Illumina Hiseq2500 sequencer.
(2) Assembling microbial genome sequences
The assembly of the microbial sequences was carried out using Meta Velvet (v1.2.01) assembly software. Fungal ribosomal DNA consists of an 18S segment, an ITS1 segment, a 5.8S segment, an ITS2 segment and a 28S segment, and the total sequence length is about 5800bp. 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 ribosomal DNA sequence containing an ITS sequence of a target pathogenic bacterium as a reference sequence, carrying out mismatch-free 0mismatch and break-free 0gap alignment by adopting bwa (0.7.12-r 1039) software, obtaining a double-end sequencing fragment from sequencing data according to an alignment result, further assembling and extending the sequence by adopting MetaVelvet (v1.2.01) assembly software, and obtaining a complete ribosomal DNA sequence through a plurality of circulation operations.
Sequence tag annotation the assembled sequence tag sequence was aligned to the nt database of NCBI using blastn (2.2.31 +) sequence alignment analysis software, with blast alignment setting expectation <1e-20, and sequence tags 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 bwa (0.7.12-r 1039) + samtools (v 1.2) analysis software and used as the relative abundance value for this species.
(3) Comparative analysis of ribosomal DNA sequences
Selecting a ribosomal DNA sequence containing an ITS sequence of a target pathogenic bacterium as a reference sequence, carrying out mismatch-free 0mismatch and break-free 0gap comparison by adopting bwa (0.7.12-r 1039) software, obtaining a double-end sequencing fragment from sequencing data according to a comparison result, further assembling and extending the sequence by adopting MetaVelvet (v1.2.01) assembly software, and obtaining a complete ribosomal DNA sequence through a plurality of circulation operations.
2. Results of the experiment
And (4) conjointly consulting the material disease symptoms and related data according to the analysis result of the types and the quantity of the microorganisms, and presuming that the mulberry red rust pathogenic bacteria are the species of the genus Puccinia. 5363A fungus microorganism classification tree detected by diseased leaves of Sang Chi rust disease is shown in figure 1, according to the principle that the higher the numerical value is, the higher the relative abundance of the species is, through the query of the sequence label annotation result and the comparison with the ribosomal RNA sequence of the mulberry red rust disease pathogenic bacteria Puccinia sp.
Example 3 detection kit and detection method for mulberry red rust pathogenic bacteria Puccinia sp
1. Design of primers
In order to further utilize the full-length cDNA sequence of the ribosomal RNA gene of the mulberry red rust pathogenic bacterium Puccinia sp to be applied to the pathogen detection and identification of the mulberry red rust, the invention further designs a pair of specific primer groups CX1791F/CX2269R, and the nucleotide sequence of the specific primer groups CX1791F/CX2269R is shown in Table 1.
TABLE 1 nucleotide sequence of specific primer set CX1791F/CX2269R
Figure BDA0003040208930000081
2. PCR detection kit
The PCR detection kit comprises the following components: specific primer sets CX1791F/CX2269R, 2 XTaq Master Mix, ddH2O.
3. Detection method
(1) PCR amplification reaction
Extracting sample nucleic acid by using the Shanghai artificial fungus genome DNA extraction kit, and performing PCR amplification by using the PCR detection kit in the step 2 by using the extracted sample nucleic acid as a template, wherein the reaction system of the PCR amplification is shown in Table 2;
TABLE 2 reaction System for PCR amplification
Figure BDA0003040208930000082
The conditions of the PCR amplification reaction are as follows: 4min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃.
(2) Detection of PCR amplification reaction products
After the PCR amplification reaction was completed, the 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.
(3) Determination of results
After performing gel electrophoresis on the PCR amplification reaction product, if a band appears, the size of the band is about 498bp and is about 500bp, and the sample to be detected contains mulberry red rust pathogenic bacteria Puccinia sp; or by sequence alignment: and (3) carrying out Sanger sequencing on the recovered PCR product fragment, and then comparing the sequencing result with the full-length cDNA sequence (SEQ ID NO. 1) of the ribosomal RNA gene of the red rust pathogenic bacteria Puccinia sp of the mulberry, thereby determining whether the sample contains the red rust pathogenic bacteria Puccinia sp.
Example 4 specificity assay for detecting Morus alba red rust pathogenic bacteria Puccinia sp
1. Experimental methods
Using total DNA of mulberry tree leaves infected with Sang Chi rust disease as template, and other 11 fungi and ddH 2 And O is negative control, the detection kit and the detection method of the embodiment 3 are applied for detection, and the specificity of the primer is verified.
The 11 fungi are fungi isolated, cultured and stored in the laboratory of the inventor, and species identification is carried out according to international species barcodes, which are respectively as follows: fusarium equiseti (Fusarium equiseti), gibberellasp (Gibberellasp. (Gibberella), alternaria sp. (Alternaria), fusarium graminearum (Fusarium graminearum), clinostasystems (Sporotrichum roseum), fusarium oxysporum (Fusarium oxysporum), mucor circinelloides (Mucor circinelloides), penicillium sp. (Penicillium), phanerochaete chrysosporium (Phanerochaete chrysosporium), mucor racemosus (Mucor racemosus), actinomucor elegans (Mucor yajel).
2. Results of the experiment
As shown in FIG. 2, it can be seen that only lanes 1, 2 and 3 amplify a single bright band, i.e., only the pathogen Puccinia sp. (fruiting body) DNA of red rust of mulberry and the total DNA of leaves with red rust can amplify the target fragment with a size of about 498bp, which is about 500bp, while the other 11 fungi and blank control do not amplify fragments with similar sizes.
The above results illustrate that: the specific primer group CX1791F/CX2269R designed according to the full-length cDNA sequence of ribosomal RNA gene of the mulberry red rust pathogen Puccinia sp can specifically detect the mulberry red rust pathogen Puccinia sp.
Example 5 sensitivity experiment for detecting Morus alba red rust pathogenic bacteria Puccinia sp
1. Experimental methods
The concentration of the total DNA of red rust disease leaves of the mulberry is determined after extraction, and then ten-fold gradient dilution is carried out on the total DNA template, wherein the concentration is respectively 12.5 ng/mu l and 1.25 multiplied by 10 -1 ng/μl、1.25×10 -1 ng/μl、1.25×10 -2 ng/μl、1.25×10 -3 ng/μl、1.25×10 -4 ng/μl、1.25×10 -5 ng/μl、1.25×10 -6 ng/μl、1.25×10 -7 ng/. Mu.l. The total DNA of each concentration was used as a template, and the detection kit and the detection method of example 3 were applied to detect and verify the sensitivity of the primers.
2. Results of the experiment
The results of the sensitivity detection are shown in FIG. 3, and it can be seen that the concentrations were 12.5 ng/. Mu.l, respectively、1.25×10 -1 ng/μl、1.25×10 -1 ng/μl、1.25×10 -2 ng/μl、1.25×10 -3 ng/μl、1.25×10 -4 ng/μl、1.25×10 -5 ng/μl、1.25×10 -6 The ng/mul Sang Chi rust disease total DNA as the template can amplify clear bands with the band size of about 498bp, which shows that the detection limit of the specific primer group designed by the invention is 1.25 multiplied by 10 -6 ng/. Mu.l of the genome of Sang Chi rust pathogen.
The above detailed description is of the preferred embodiment for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, that is, it is not intended that the present invention necessarily depends on the above embodiment for implementation. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Sequence listing
<110> southern China university of agriculture
Ribosomal RNA gene of mulberry red rust pathogenic bacteria Puccinia sp and application thereof
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<213> pathogenic bacteria of red rust of mulberry
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aactgtggta attctagagc taatacatgc tgaaaagccc caacctttgg taggggtgta 180
tttattagat aaaaaaccaa tggctttcgg gtctctttgg tgattcataa taacttctcg 240
aatcgcacag ccttgtgctg gtgatgcttc attcaaatat ctgccctatc aacttttgat 300
ggtaggatag aggcctacca tggtgatgac gggtaacggg gaataagggt tcgattccgg 360
agagagggcc tgagaaacgg ccctcaaatc taaggattgc agcaggcgcg caaattaccc 420
aatcccaaca cggggaggta gtgacaataa ataacaatgt atggctcttt tgggtcttac 480
aattggaatg agtacaattt aaatctctta acgaggatca attggagggc aagtctggtg 540
ccagcagccg cggtaattcc agctccaata gcgtatatta aagttgttga cgttaaaaag 600
ctcgtagtcg aacttcggct tctgacagtt ggtctgcctt ttggtttgta ctgatttgtt 660
ggaggcttac ctcttggtga acttcaatgc actttactgg gtgttgtagg gaaccaggac 720
ttttactttg aaaaaattag agtgttcaaa gcaggctttt gcctgaatac attagcatgg 780
aataatagaa taggacgtgt gatcctattt tgttggtttc taggattacc gtaatgatta 840
atagggtcag ttgggggcat ttgtattaca tcgtcagagg tgaaattctt ggattgatgt 900
aagacaaact actgcgaaag catctgccaa ggatgacttc attgatcaag aacgaaggtt 960
aagggttcaa aaacgatcag ataccgttgt agtcttaaca gtaaactatg ccgactgggg 1020
atcagacaag gattcataat gacttgtttg gcacccaaag ggaaacctga agtttaggtt 1080
cgtgggggag tacggtcaca aggctgaaac ttaaaggaat tgacggaagg gcaccaccag 1140
gcgtggagcc tgcggcttaa tttgactcaa cacggggaaa ctcaccaggt ccagacacaa 1200
taaggattga cagattgata gctttttctt gattttgtgg ttggtggtgc atggccgttc 1260
ttagttggtg gagtgatttg tctggttaat tccgataacg aacgagacct tctcctgcta 1320
aatagtccag ctggctacgg ctggctgctg acttcttaga gggactatca acttttagtt 1380
gatggaagtt ggaggcaata acaggtctgt gatgccctta gatgttctgg gccgcacgcg 1440
cgctacactg accaagccag caagtatatc accttatctg aaaggattgg gtaatcttgt 1500
taaacttggt tgtgatgggg atagagcatt gcaattattg ctcttcaacg aggaatacct 1560
agtaagcgta tgtcatcagc atgcgttgat tacgtccctg ccctttgtac acaccgcccg 1620
tcgctactac cgattggaca tctttgtgag gcgtttggag agcctatgaa gagctggaaa 1680
cagcacttta ctggttcaaa gttctacaaa catggatgtc tagaggaagt aaaagtcgta 1740
acaaggtttc tgtaggtgaa cctgcagaag gatcattatt gaaaatattg ggtgcactta 1800
gttgtggctc aaatttttta aactcacccc attataagcc tctctacatg tttcacaaca 1860
gagtagtttg gcatacgtag taaggttctt caaagacctt attgcattac cccttcatca 1920
tccttatctg ttttactcaa gttttaagaa tgtaaacccc cttaaattaa tacaactttt 1980
aacaatggat ctctaggctc tcacatcgat gaagaacaca gtgaaatgtg ataagtaatg 2040
tgaattgcag atttcagtga atcatcgaat ctttgaacgc accttgcgcc ttttggtatt 2100
ccaaaaggca cacctgtttg agtgtcatga aaccctctca tcattcaatt cttttcatta 2160
aggttgtttg atggatgttg agtgttgctg taattggctc acttcaaata tataagtcct 2220
tttatcattg gttttggaat gacttgatgt gataaactat cttcatcaag gagtgtgcct 2280
tggtactgcc atatctgttg tgataaatag cttctaactt gtcaatgaac aaggcttgcc 2340
ttttcattac acataacatt aagacctcaa atcaggtggg actacccgct gaacttaagc 2400
atatcaataa gcggaggaaa agaaactaac aaggattccc ctagtaacgg cgagtgaaga 2460
gggaaaagcc caaatttgta atctggctct ttcagagtcc gagttgtaat tttaagaact 2520
gttttctgtg ctggaccata tacaagtctg ttgaaaaaca gcatcattga gggtgataat 2580
cccgttcatg atatggacta ccagggcaat atgatacagt ctctaagagt cgagttgttt 2640
gggaatgcag ctcaaagtgg gtggtaaatt ccatctaagg ctaaatactg gtgagagacc 2700
gatagcaaac aagtaccgtg agggaaagat gaaaagaact ttggaaagag agttaacagt 2760
acgtgaaatt gttaaaaggg aaacatttga agttagactt gttattgtta gttcagccct 2820
tccagggtgt attctgatga tcaacagacc agcgtcaatt tttgagtgtt ggataagggt 2880
cttgaaaatg tagcagtctt tgactgtgtt ataatttttg acttggatac aatgcttagg 2940
attgaggatc gcagtaagcc attaatttgg tggaacaatt cattgttctt cttactgagg 3000
atgttggtgc aatagcttta aatgacccgt cttgaaacac ggaccaagga gtctaacatg 3060
cttgcaagta tttgggcgtt gaaaccctta tgcgtaatga aagtaaatgt aaatgggatc 3120
cgtaaaaggt gcaccattga ccagtcctga ttatttatat gaaggtactg agtaagagca 3180
agtatgttgg gacccgaaag atggtgaact atgcctgaat agggtgaagc cagaggaaac 3240
tctggtggaa gctcgtagcg gttctgacgt gcaaatcgat cgtcgaattt gggtataggg 3300
gcgaaagact aatcgaacca tctagtagct ggttcctgcc gaagtttccc tcaggatagc 3360
agagactcgc atcagtttta tgaggtaaag cgaatgatta gaggccttgg ggatgaaaca 3420
tccttaacct attctcaaac tttaaatatg taagacgctc ctgtttctta attgaacgtg 3480
ggcatgcgaa tgagagtctc cagtgggcca tttttggtaa gcagaactgg cgatgcggga 3540
tgaaccgaat gcgaggttaa ggtgccggaa tatacactca tcagacacca caaaaggtgt 3600
tagttcatct agacagccgc acggtggcca tggaagtcgg aatccgctaa ggagtgtgta 3660
acaactcaac ggccgaatga actagccctg aaaatggatg gcgcttaagt gtattaccca 3720
tacctcgcca ttaatattat tcaatatatt aatgagtagg caggcgtgga ggttatgtag 3780
cgaagccttg gcagtgatgc tgggtggaac agcctctagt gcagatcttg gtggaagtag 3840
caaatattca agtgagaacc ttgaagactg aagtggggaa gggttccatg gtaacagcag 3900
ttggacatgg gttagtcgga cctaagagat agggaacttc cgttttaaag aaatgctctt 3960
gttagcatca cctatcgaaa gggaatgtag ttaaaattct acaactggga tacagatttt 4020
gaacggtaac gtatatgaac ttggtgacat ccgcaagggc cctaggaaga gttttctttt 4080
ctccttaaca atctgaaacc ctggaagcga tttattcgga gatagggttt aatgattggt 4140
agagctttac acctctgtag agtctggtgc gtccttgagg atccttgaaa aaccgaggga 4200
ttgaaaaagt cttgtaccca gccgtaccaa tatccgcatc aggtccccaa ggtgatcagc 4260
ctctggtcaa tagaataatg tagataaggg aagtcggcaa aatagatccg taactttggg 4320
aaaaggattg gctcataggg cagggcttgt tgggccctta atagatctgt agggacctga 4380
ctgggactac ttctgggtaa ctggaggtgg acctggatgg gaccttgtag tgatgttttg 4440
ggcagccttt taggcgtctg gcaagcattt aactgtcaac tatgaactgg tacggacaag 4500
gggaatctga ctgtctaatt aaaacatagc attgtgatgg ccagaaagtg gtgttgacac 4560
aatgtgattt ctgcccagtg ctctgaatgt caaagtgaag aaattcaacc aagcgcgggt 4620
aaacggcggg agtaactatg actctcttaa ggtagccaaa tgcctcgtca tctaattagt 4680
gacgcgcatg aatggattaa cgagattccc actgtcccta tctactatct agcgaaacca 4740
cagccaaggg aacgggcttg gcagaatcag cggggaaaga agaccctgtt gagcttgact 4800
ctagtttgac attgtgaaaa gacatagagg gtgtagaata agtgggagcg taagcgccag 4860
tgaaatacca ctacctttat tgtcttttta cttattcaat gaagcagagc tgggattaac 4920
gtcccacttt ttagcattaa ggtctttttt gggccaatct gggttgaaga cattgtcagg 4980
tggggagttt ggctggggcg gcacatctgt taaacaataa cgcaggtgtc ctaaggggga 5040
ctcattgaga acagaaatct caagtagaac aaaagggtaa aagtcccctt gattttgatt 5100
ttcagtgtga atacaaacca tgaaagtgtg gcctatcgat cctttagtcc tttagaattt 5160
aaagctagag gtgccagaaa agttaccaca gggataactg gcttgtggca gccaagcgtt 5220
catagcgacg ttgctttttg atccttcgat gtcggctctt cctatcatac cgaagcagaa 5280
ttcggtaagc gttggattgt tcacccacta atagggaacg tgagctgggt ttagaccgtc 5340
gtgagacagg ttagttttac cctactgatg gagtgtcatt gtaatagtaa ttgaacttag 5400
tacgagagga actgttcatt cacgtaattg gtatttacaa ctgtttgaga ggacaatgtt 5460
gtgaagctac cacgtgttgg attatggctg aacgcctcta agccagaatc cgtgctagaa 5520
acaatgatgt tgtcccgtgc atctaccgtt gagtcaatat agagcttttg cttgtgtacc 5580
atcataagtg agaatgagct ggtcaagtgg aaagacttgg ttggttttct tacatataat 5640
atttgaaata tgtgcggggg taaatccttt gcagacgact tgaatgggaa cggggtactg 5700
taagtggtag agtagccttg ttgctacgat ccactgaggt tcagcccttg ttctaaagat 5760
ttgtt 5765
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtgcactta gttgtggctc 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cagtaccaag gcacactcct 20

Claims (10)

1. The full-length cDNA of ribosomal RNA gene of mulberry red rust pathogen Puccinia 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 full-length cDNA of the ribosomal RNA gene of claim 1 for detecting the red rust pathogen Puccinia sp.
3. A method for detecting red rust pathogenic bacteria Puccinia sp of mulberry trees is characterized in that the full-length cDNA sequence of ribosome RNA of a sample to be detected is compared with the full-length cDNA sequence of the riboribosome RNA gene of claim 1, and whether the red rust pathogenic bacteria Puccinia sp of mulberry trees are contained in the sample to be detected is judged according to the comparison result.
4. A pair of primers for detecting mulberry red rust pathogenic bacteria Puccinia sp is characterized in that the nucleotide sequence is shown as SEQ ID No. 2-3.
5. The application of the primer of claim 4 in detecting mulberry red rust pathogenic bacteria Puccinia sp or preparing a kit for detecting mulberry red rust pathogenic bacteria Puccinia sp.
6. A kit for detecting mulberry red rust pathogen Puccinia sp, which comprises the primer of claim 4.
7. Use of the kit of claim 6 for detecting red rust pathogenic bacteria Puccinia sp.
8. A method for detecting mulberry red rust pathogenic bacteria Puccinia sp is characterized in that nucleic acid of a sample to be detected is used as a template, the primer of claim 4 is used for PCR amplification, the product after PCR amplification is subjected to gel electrophoresis, and if a strip with the size of 498bp appears, the mulberry red rust pathogenic bacteria Puccinia sp is judged to be contained in the sample to be detected.
9. The method of claim 8, wherein the reaction system of the PCR amplification is: 2 XTaq Master Mix 10. Mu.L, 10. Mu.M primers according to claim 4 each 0.5. Mu.L, nucleic acid template 2. Mu.L, and ddH2O for the remainder, for a total of 20. Mu.L.
10. The method of claim 8, wherein the reaction conditions for the PCR amplification are: 4min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃.
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