CN113444813B - Kit and method for identifying Gansu zokor - Google Patents
Kit and method for identifying Gansu zokor Download PDFInfo
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Abstract
The application provides a kit and a method for identifying Gansu zokor, and belongs to the technical field of molecular identification. It comprises a reagent for detecting any one or more of the following 4 SNP loci in the 12S rRNA gene and the 16S rRNA gene of a species to be detected: the 4 th bit of the conserved motif sequence shown in SEQ ID NO. 5, the 25 th bit of the conserved motif sequence shown in SEQ ID NO. 6, the 22 nd bit and the 31 st bit of the conserved motif sequence shown in SEQ ID NO. 7. The kit for identifying the species of the Gansu zokor simply, conveniently and accurately and the method for identifying the Gansu zokor are provided by the application, so that the problem of zokor species identification through morphology is solved, and the kit has an excellent application prospect in zokor species identification.
Description
Technical Field
The application belongs to the technical field of molecular identification, and particularly relates to a kit and a method for identifying Gansu zokor.
Background
Zokor is a collective term for animals of the subfamily zokorae (myopalacinae), and is mainly distributed in china and south siberian. Existing zokors include the genus pizokor (Eospalax) and the genus pizokor (myspaax), and nearly all of the current zokor species are distributed in our country. The zokor comprises 2-3 species such as grassland zokor (M.aspalax) and northeast zokor (M.psilurus), and the genus of the zokor is distributed only in China, and comprises 6 species such as Chinese zokor (E.fontaniri), stony zokor (E.smithi), rogowski zokor (E.rothskidi), highland zokor (E.baileyi), gansu zokor (E.cansus) and Qinling zokor (E.rufesciens). Zokor almost always lives in a closed underground tunnel system, and unique underground life modes enable zokor to have specificity in aspects of foraging, wedding, breeding and the like, so that the zokor is widely interested by scientists.
Gansu zokor is distributed in Ningxia six-disc mountain area, ningxia south loess hilly area, shanxi Qinling and Shanxi North loess plateau area, gansu Long east and middle area, qinghai east forest grassland, etc. The zokor in Gansu life is hidden under the ground, and occasionally takes food briefly on the ground at night in summer. Nest making is performed in places with deep and loose soil, luxuriant growth of plants, less turning of soil layers and stable environment, and particularly, the density of nest making of young forest lands, alfalfa lands, abandoned lands and farmland ridges for returning to the cultivation is higher. 4-5 months each year is the first peak period of feeding, mating and reproduction activities, and 9-10 months is the second peak period of food accumulation and grain storage activities. The zokor in Gansu is edible and miscellaneous, and mainly gnaws the plant rhizome. The Chinese pine, the pinus sylvestris, the larch, the apple, the hawthorn, the pricklyash peel, the wild apricot and the mountain peach are serious in harm, and the sea buckthorn, the poplar, the willow and the elm are inferior; the crops take green onion, soybean, potato, radish, carrot, angelica and dangshen as the heaviest harmful components, and wheat, millet, corn and the like are used for the times; herbs are the heaviest to be damaged by alfalfa, dandelion and plantain herb, and other herbs are inferior.
Identification of biological species is the basis for understanding and protecting the diversity of organisms, and each organism can be studied in more fields only after being accurately identified. However, since different zokor living environments are similar and the shapes are convergent, species are very difficult to accurately identify through the shapes without professional training.
Therefore, research on a method and a reagent capable of rapidly and accurately identifying species of Gansu zokor has very important significance.
Disclosure of Invention
The application aims to provide a kit and a method for simply, conveniently and accurately identifying the species of Gansu zokor, and solve the problem of zokor species identification through morphology.
The application provides a kit for identifying Gansu zokor, which comprises a reagent for detecting any one or more of the following 4 SNP loci in a 12SrRNA gene and a 16S rRNA gene of a species to be detected:
the 4 th bit of the conserved motif sequence shown in SEQ ID NO. 5, the 25 th bit of the conserved motif sequence shown in SEQ ID NO. 6, the 22 nd bit and the 31 st bit of the conserved motif sequence shown in SEQ ID NO. 7.
Further, the reagent is a reagent for simultaneously detecting the 4 SNP sites.
Further, the bases of the SNP sites are as follows: the 4 th base of the conserved motif sequence shown in SEQ ID NO. 5 is C; the 25 th base of the conserved motif sequence shown in SEQ ID NO. 6 is C; the 22 nd base of the conserved motif sequence shown in SEQ ID NO. 7 is G, and the 31 st base is C.
According to the research, the 4 SNP loci usually exist at the same time, namely when the 4 th base of the conserved motif sequence shown in SEQ ID NO. 5 is C, the 25 th base of the conserved motif sequence shown in SEQ ID NO. 6 is C; the 22 nd base of the conserved motif sequence shown in SEQ ID NO. 7 is G, and the 31 st base is C. Further experiments also find that only the genome of Gansu zokor has the 4 SNP loci, so that whether the species to be detected is Gansu zokor can be identified by detecting any one or more loci of the 4 SNP loci.
Further, the reagent is: reagents for sequencing, reagents for KASP method or reagents for restriction fragment length polymorphism method.
Further, the kit further comprises a reagent for amplifying the conserved motif sequences of the 12S rRNA gene and the 16S rRNA gene; the conserved motif sequence is any one or more sequences shown in SEQ ID NO. 5-7.
Further, the reagent for amplifying the conserved motif sequences of the 12S rRNA gene and the 16S rRNA gene comprises a primer pair shown in SEQ NO. 1-2 and a primer pair shown in SEQ NO. 3-4.
The application also provides the application of the reagent for amplifying the conserved motif sequences of the 12S rRNA gene and the 16S rRNA gene in the kit for identifying Gansu zokor, wherein the conserved motif sequences are any one or more sequences shown in SEQ ID NO 5-7;
preferably, the reagent comprises a primer pair shown in SEQ NO. 1-2 and a primer pair shown in SEQ NO. 3-4.
The application also provides a method for identifying Gansu zokor, which comprises the following steps:
(1) Extracting total genome DNA of a zokor sample to be detected;
(2) Detecting one or more of the following 4 SNP sites in the 12S rRNA gene sequence and the 16S rRNA gene sequence: the 4 th position of a conserved motif sequence shown in SEQ ID NO. 5 in the 12S rRNA gene sequence; the 25 th position of a conserved motif sequence shown in SEQ ID NO. 6 in the 12S rRNA gene sequence; the 22 nd and 31 st positions of a conserved motif sequence shown in SEQ ID NO. 7 in the 16S rRNA gene sequence are analyzed;
preferably, the sample in step (1) is a tissue sample or a blood sample.
Further, the detecting in step (2) includes the following steps:
1) Taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification by using an amplification reagent to obtain an amplification product;
2) Performing agarose gel electrophoresis detection on the PCR amplification product obtained in the step 1);
3) Sequencing the PCR amplification products with bright bands at 490bP and 1450bP in the detection result of the step 2) to obtain a 12S rRNA gene sequence and a 16S rRNA gene sequence;
4) Analyzing one or more of the following 4 SNP loci in the 12S rRNA gene sequence and the 16S rRNA gene sequence obtained in the step 3): the 4 th position of a conserved motif sequence shown in SEQ ID NO. 5 in the 12S rRNA gene sequence; the 25 th position of a conserved motif sequence shown in SEQ ID NO. 6 in the 12S rRNA gene sequence; positions 22 and 31 of the conserved motif sequence shown in SEQ ID NO. 7 in the 16S rRNA gene sequence;
preferably, the amplification reagent in the step 1) comprises a primer pair shown in SEQ NO. 1-2 and a primer pair shown in SEQ NO. 3-4; the annealing temperature of the PCR amplification is 52-56 ℃; the sequencing of step 3) is bidirectional Sanger sequencing.
Further, the detection of step (2) is simultaneous detection of the 4 SNP sites;
preferably, the base of the SNP locus is as follows, and the zokor to be detected is Gansu zokor:
the 4 th base of the conserved motif sequence shown in SEQ ID NO. 5 is C; the 25 th base of the conserved motif sequence shown in SEQ ID NO. 6 is C; the 22 nd base of the conserved motif sequence shown in SEQ ID NO. 7 is G, and the 31 st base is C.
The application has the beneficial effects that:
firstly, the 4 Gansu zokor specific SNP genotypes provided by the application are simultaneously appeared in a 12S rRNA gene segment and a 16S rRNA gene segment (DNA bar code), 1 or more of the SNPs can be detected to judge that the to-be-detected zokor is Gansu zokor, and the requirement on sequencing length is low. According to the application, 4 SNP loci with Gansu zokor specific genotypes are used for zokor species identification, the SNP loci can be mutually verified, and the result is more accurate and reliable.
The key point of the application is that the relation between 4 SNP loci in the conserved motif sequences of the zokor 12S rRNA gene segment and the 16S rRNA gene segment and the species of the Gansu zokor is found, and on the basis, any reagent or equipment capable of detecting the base of any one or a plurality of SNP loci can be used for species identification of the Gansu zokor.
Specifically, the present embodiments provide examples of detecting SNP sites by means of sequencing. The primer pair has good specificity, and the primer pair is used for PCR, so that non-specific amplification is not generated with DNA fragments except the target; the amplification target gene is a mitochondrial single copy gene, cloning is not needed, and the method can be directly used for sequencing and is simple. The application also provides a 3-segment Gansu zokor species conserved motif sequence, which can assist in determining the positions of Gansu zokor species specific SNP loci in 12S rRNA gene segments and 16S rRNA gene segments (DNA barcodes), and is convenient for determining the genotypes of the SNP loci.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
The above-described aspects of the present application will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present application is limited to the following examples only. All techniques implemented based on the above description of the application are within the scope of the application.
Drawings
FIG. 1 is a diagram showing the sequencing peaks (first motif sequence) of the PCR products of the 12S rRNA gene of zokor numbered FFX-5 in example 3.
FIG. 2 is a diagram showing the sequencing peaks (second motif sequence) of the PCR products of the 12S rRNA gene of zokor numbered FFX-5 in example 3.
FIG. 3 is a diagram showing the sequencing peaks of the 16S rRNA gene PCR product of zokor numbered FFX-5 in example 3 (reverse complement of the third motif).
FIG. 4 is a diagram showing the sequencing peaks of the PCR products of the zokor 12S rRNA gene numbered ZX-6 in example 5 (sequence of the position of the first segment of motif).
FIG. 5 is a diagram showing the sequencing peaks of the PCR products of the zokor 12S rRNA gene numbered ZX-6 in example 5 (sequence of the position of the second motif).
FIG. 6 is a diagram showing the sequencing peaks of the 16S rRNA gene PCR product of zokor numbered ZX-6 in example 5 (reverse complement of the position of the third segment motif).
Detailed Description
For a clearer understanding of the present application, the present application will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the application in any way.
Experimental methods for which specific conditions are not noted in the examples are conventional methods and conventional conditions well known in the art, or conditions according to manufacturer's recommendations; the various chemical reagents used in the examples are commercially available and the primers used were designed for the synthesis.
Example 1A kit for identifying Gansu zokor according to the application
The kit comprises the following components:
(1) PCR amplification reagent: a primer pair comprising SEQ NO. 1-2 and a primer pair comprising SEQ NO. 3-4; (2) reagents for sequencing.
Example 2 PCR primer design and verification of SNP site
The applicant compares mitochondrial genome sequences of 121 zokor individuals of 8 zokor species, and finds that 2 Gansu zokor species-specific SNP genotypes exist in the 12S rRNA genes, and 2 Gansu zokor species-specific SNP genotypes exist in the 16S rRNA genes. The conserved sequences are found to exist at the two ends of the 4 SNP loci through comparison, and the conserved sequences can be used for designing PCR primers, so that 12S rRNA and 16S rRNA gene fragments are determined to be DNA barcodes for identifying Gansu zokor species.
8 zokor species 121 zokor individuals: 12 grassland zokors, 10 northeast zokors, 15 Chinese zokors, 18 stoneware zokors, 16 rosis zokors, 14 plateau zokors, 24 Gansu zokors and 12 Qinling zokors.
The 12S rRNA and 16S rRNA and nearby gene sequences were sequenced, and primers containing species-specific SNP genotypes were designed in the conserved regions as follows:
ME12S-1L：AGCACTGAAAATGCTTAGATGG(SEQ ID NO:1)；
ME12S-1R：CGGCTAAGCATAGTGGGGTA(SEQ ID NO:2)。
ME16S-1L：AGAGGAGATAAGTCGTAACAAGGT(SEQ ID NO:3)；
ME16S-1R：TCCTGATCCAACATCGAGGT(SEQ ID NO:4)。
amplifying DNA samples of different zokor species by using the primers, and confirming 4 SNP loci for identifying the Gansu zokor species:
1) The 327 th base of the amplification product of the Gansu zokor 12S rRNA gene (SEQ ID NO:5 conserved motif sequence: ACACCGGCGTAAAGCGTACAA, position 4) genotype is C, and other species have the locus genotype of T;
2) The 428 th base of the amplification product of the Gansu zokor 12S rRNA gene (SEQ ID NO:6 conserved motif sequence: ACTAAAATCAATAACGAAAGTAATCCTA) genotype at position 25) is C, and genotypes at the position of other species are T;
3) 1309 th base of the amplification product of the zokor 16S rRNA gene (SEQ ID NO:7 conserved motif sequence: CCTCCGAATAACAAAACCAAGGCTTACAAGCCAAAGT, position 22) genotype G, and other species, position A genotype;
4) 1318 th base of the amplification product of the 16S rRNA gene of Gansu zokor (SEQ ID NO:7 conserved motif sequence: CCTCCGAATAACAAAACCAAGGCTTACAAGCCAAAGT) is C and the other species is T.
The result proves that the bases of the 4 specific SNP loci are different from other zokors in Gansu zokors, and the Gansu zokors can be identified by detecting the 4 specific SNP loci.
Example 3 identification of species of Gansu zokor
The above 12S rRNA gene fragment primer pair (SEQ ID NO:1 and SEQ ID NO: 2) and 16S rRNA gene fragment primer pair (SEQ ID NO:3 and SEQ ID NO: 4) were first synthesized.
The identification is carried out by adopting the following method:
a) Extracting total genome DNA of zokor muscle Tissue with the number of FFX-5, total genome DNA of zokor liver Tissue with the number of ZX-6 and total genome DNA of zokor liver Tissue with the number of LD-3 respectively by using a Kanji DNeasy Blood & Tissue Kit;
b) Taking zokor genome total DNA numbered FFX-5 and ZX-6 in the step a) as a template, respectively carrying out PCR reaction by using the 12S rRNA and 16S rRNA primers, wherein the reaction system of FFX-5 is 25 mu L, and the annealing temperature is 56 ℃; 25 mu L of ZX-6 reaction system and annealing temperature of 55 ℃; taking the total genome DNA with the number of LD-3 in the step a) as a template, and carrying out PCR reaction by using the 12S rRNA primer pair, wherein the reaction system is 25 mu L, and the annealing temperature is 52 ℃;
c) Detecting the PCR product obtained in the step b) by 1% agarose gel electrophoresis, and observing bright bands of about 490bp and about 1450 bp;
d) Respectively carrying out bidirectional Sanger sequencing on the PCR products in the step c) to obtain 12S rRNA and 16S rRNA gene sequencing peak diagrams;
e) And (3) finding a conserved motif sequence of SEQ ID NO 5-7 in the obtained sequencing peak diagram, and analyzing SNP locus bases.
The results were as follows:
(1) FFX-5 zokor:
the 4 th base in the conserved motif sequence of SEQ ID NO. 5 is C (figure 1);
the 25 th base in the conserved motif sequence of SEQ ID NO. 6 is C (figure 2);
the 22 nd base is G and the 31 st base is C in the conserved motif of SEQ ID NO. 7 (figure 3).
Namely, the sample of the judgment number FFX-5 is Gansu zokor.
(2) ZX-6 zokor:
the 4 th base in the conserved motif sequence of SEQ ID NO. 5 is not C (FIG. 4);
the 25 th base in the conserved motif sequence of SEQ ID NO. 6 is not C (FIG. 5);
the 22 nd base is not G and the 31 st base is not C in the conserved motif of SEQ ID NO. 7 (FIG. 6).
I.e. the sample numbered ZX-6 was judged not to be a gansu zokor.
(3) LD-3 zokor:
the 4 th base in the conserved motif sequence of SEQ ID NO. 5 is C.
Namely, the sample with the number LD-3 is judged to be Gansu zokor.
In addition, zokor numbered FFX-5, LD-3 and ZX-6 were identified by conventional morphological identification: FFX-5 and LD-3 are elliptical, with the tail length exposed or enlarged by thin white hair, cranium occipital bulge, expansion of zygomatic arch, parallel top ridge, fold inwards in the forehead, close to the developed supraorbital ridge, with developed occipital ridge, anterior and maxillary dentition holes surrounded by the third upper molar (M) 3 ) The characteristics of the Chinese zokor are consistent with morphological identification characteristics of the Chinese zokor without backward extension, which indicates that the Chinese zokor is the Chinese zokor.
While ZX-6 individuals have a nasogastric hat shape with dense hair at the tail, a craniocerebral occipital bulge with very extensive zygomatic arches, a significantly wider anterior portion than posterior portion, and a frontal crest relatively close to the center seam; the door tooth holes are surrounded by the anterior jawbone, and these features are consistent with the zokor, indicating that ZX-6 is a zokor, not a Gansu zokor.
Experimental results show that the method for identifying Gansu zokor is accurate and can be practically used for identifying and detecting Gansu zokor species.
In conclusion, the kit for simply, conveniently and accurately identifying the species of Gansu zokor and the method for identifying Gansu zokor are provided, the problem of identification of the species of the zokor through morphology is solved, and the kit has excellent application prospect in species identification of the zokor.
SEQUENCE LISTING
<110> national academy of sciences northwest high Protozoa institute
<120> kit and method for identifying Gansu zokor
<130> GY417-2021P0113585CC
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
agcactgaaa atgcttagat gg 22
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
cggctaagca tagtggggta 20
<210> 3
<211> 24
<212> DNA
<213> artificial sequence
<400> 3
agaggagata agtcgtaaca aggt 24
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
tcctgatcca acatcgaggt 20
<210> 5
<211> 21
<212> DNA
<213> artificial sequence
<400> 5
acaccggcgt aaagcgtaca a 21
<210> 6
<211> 28
<212> DNA
<213> artificial sequence
<400> 6
actaaaatca ataacgaaag taatccta 28
<210> 7
<211> 37
<212> DNA
<213> artificial sequence
<400> 7
cctccgaata acaaaaccaa ggcttacaag ccaaagt 37
Claims (4)
1. A method for identifying gansu zokor, comprising the steps of:
(1) Extracting total genome DNA of a zokor sample to be detected;
(2) The following 4 SNP loci in the 12S rRNA gene sequence and the 16S rRNA gene sequence were detected: the 4 th position of a conserved motif sequence shown in SEQ ID NO. 5 in the 12S rRNA gene sequence; the 25 th position of a conserved motif sequence shown in SEQ ID NO. 6 in the 12S rRNA gene sequence; the 22 nd and 31 st positions of a conserved motif sequence shown in SEQ ID NO. 7 in the 16S rRNA gene sequence are analyzed;
the base of the SNP locus is as follows, and the zokor to be detected is Gansu zokor:
the 4 th base of the conserved motif sequence shown in SEQ ID NO. 5 is C; the 25 th base of the conserved motif sequence shown in SEQ ID NO. 6 is C; the 22 nd base of the conserved motif sequence shown in SEQ ID NO. 7 is G, and the 31 st base is C;
the method can be used for distinguishing Gansu zokor from other zokors; the other zokors are: grassland zokor, northeast zokor, chinese zokor, stoneware zokor, rogowski zokor, plateau zokor or ash-log zokor.
2. The method of claim 1, wherein: the sample in the step (1) is a tissue sample or a blood sample.
3. The method of claim 1, wherein the step of detecting of step (2) is as follows:
1) Taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification by using an amplification reagent to obtain an amplification product;
2) Performing agarose gel electrophoresis detection on the PCR amplification product obtained in the step 1);
3) Sequencing the 12S rRNA gene PCR amplification product with a bright band at 490bp and the 16S rRNA gene PCR amplification product with a bright band at 1450bp in the detection result of the step 2) to obtain a 12S rRNA gene sequence and a 16S rRNA gene sequence;
4) Analyzing the following 4 SNP loci in the 12S rRNA gene sequence and the 16S rRNA gene sequence obtained in the step 3): the 4 th position of a conserved motif sequence shown in SEQ ID NO. 5 in the 12S rRNA gene sequence; the 25 th position of a conserved motif sequence shown in SEQ ID NO. 6 in the 12S rRNA gene sequence; the 16S rRNA gene sequence is shown in SEQ ID NO. 7 at positions 22 and 31 of the conserved motif sequence.
4. A method as claimed in claim 3, wherein: the amplification reagent in the step 1) comprises a primer pair shown in SEQ NO. 1-2 and a primer pair shown in SEQ NO. 3-4; the annealing temperature of the PCR amplification is 52-56 ℃; the sequencing of step 3) is bidirectional Sanger sequencing.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107058492A (en) * | 2017-01-12 | 2017-08-18 | 中国人民解放军第二军医大学 | The method and kit of a kind of pvuii restriction fragment for identifying naked mole mitochondrial cytochrome b genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107058492A (en) * | 2017-01-12 | 2017-08-18 | 中国人民解放军第二军医大学 | The method and kit of a kind of pvuii restriction fragment for identifying naked mole mitochondrial cytochrome b genes |
Non-Patent Citations (3)
| Title |
|---|
| DNA条形码技术在小型兽类鉴定中的探索:以甘肃莲花山为例;何锴等;生物多样性;第21卷(第2期);摘要、方法与结果 * |
| Molecular authentication of the animal crude drug Sailonggu (bone of Myospalax baileyi);Caiquan Zhou等;Biol Pharm Bull;第27卷(第11期);摘要、材料与方法、结果部分、表3和图1 * |
| The validity of different zokor species and the genus Eospalax inferred from mitochondrial gene sequences;Caiquan Zhou等;Integr Zool;第3卷(第4期);第290-298页 * |
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