CN117165695A - Introgression gene, primer, method and application for identifying Changfeng silver carp - Google Patents
Introgression gene, primer, method and application for identifying Changfeng silver carp Download PDFInfo
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- CN117165695A CN117165695A CN202311173958.6A CN202311173958A CN117165695A CN 117165695 A CN117165695 A CN 117165695A CN 202311173958 A CN202311173958 A CN 202311173958A CN 117165695 A CN117165695 A CN 117165695A
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- 241000252234 Hypophthalmichthys nobilis Species 0.000 title claims abstract description 74
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 19
- 241000252233 Cyprinus carpio Species 0.000 claims abstract description 22
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 238000001764 infiltration Methods 0.000 claims abstract description 6
- 230000008595 infiltration Effects 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000001502 gel electrophoresis Methods 0.000 claims description 7
- 241000252210 Cyprinidae Species 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 abstract description 10
- 230000003321 amplification Effects 0.000 abstract description 7
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 238000012408 PCR amplification Methods 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 17
- 241000720946 Hypophthalmichthys molitrix Species 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the technical field of silver carp, in particular to an introgression gene, primer, method and application for identifying silver carp. The introgression gene is shown as SEQ ID NO.1 and is derived from the carp genome. The infiltration gene can be used as a long-chain identification gene and an identification nucleic acid sequence, and the amplification product is subjected to electrophoresis detection by designing a primer and carrying out PCR amplification, so that whether the electrophoresis band is a 302bp band or not can be determined, the long-chain identification gene provides support for molecular identification of the long-chain silver carp, and provides powerful guarantee for seed counterfeiting and healthy development of seed industry. The identification method is simple and feasible, convenient to operate and high in identification success rate.
Description
Technical Field
The application relates to the technical field of silver carp, in particular to an introgression gene, primer, method and application for identifying silver carp.
Background
The Changfeng silver carp is the first new variety of four large-scale Chinese carps cultivated by adopting a comprehensive breeding technology combining artificial gynogenesis, molecular marker assistance and group breeding in the water production research institute of China aquatic science research institute, and is also one of the important popularization varieties of the national mass freshwater fish industry technology system. The appearance characteristics of the Changfeng silver carp are basically consistent with those of the common silver carp, the silver carp has larger head, very low eye position, flat and slightly high body type side, spindle shape, dark gray back, white sides and abdomen, fine scales, and the median cutin edge of the abdomen extends from the lower part of the pectoral fin to the anus, the pectoral fin does not exceed the base of the pectoral fin, and each fin has grey color. Compared with the common silver carp, the silver carp has shorter, thicker and stronger body shape.
In the practical process, the identification of the Changfeng silver carp is particularly important, and the identification accuracy can be provided by providing a molecular biology identification means.
Disclosure of Invention
In view of the above, the embodiment of the application provides an introgression gene, a primer, a method and application for identifying the silver carp. Therefore, the embodiment of the application at least discloses the following technical scheme:
(1) And identifying the nucleic acid molecule of the Changfeng silver carp, wherein the nucleic acid molecule is shown as SEQ ID NO. 1.
(2) And identifying the infiltration gene of the Changfeng silver carp, wherein the infiltration gene is derived from the carp genome as shown in SEQ ID NO. 1.
(3) And identifying a primer pair of an introgression gene in the Changfeng silver carp, wherein the introgression gene is derived from a carp genome, and the primer pair comprises a DNA molecule shown as SEQ ID NO. 2-3.
(4) The kit for identifying the introgression genes in the Changfeng silver carp comprises the primer pair and reagents required by PCR.
(5) A method of identifying a long-spotted silver carp, comprising:
obtaining a DNA sample to be detected, wherein the DNA sample to be detected is from at least one of silver carps, silver carps and carps;
taking the DNA as a template, and adopting the primer pair to perform PCR reaction or the kit to perform PCR reaction;
the resulting PCR reaction product is detected for gel electrophoresis, and the origin of the DNA sample is confirmed from the bands of the gel electrophoresis.
(6) The embodiment of the application discloses application of the nucleic acid molecule (1), the introgression gene (2), the primer pair (3) or the kit (4) in the detection of the introgression gene of the silver carp, and the identification of the silver carp, the common silver carp and the carp.
Compared with the prior art, the embodiment provided by the embodiment provides the infiltration gene, the primer, the method and the application for identifying the Changfeng silver carp, and has at least one of the following beneficial effects:
the introgression gene can be used as a long-chain identification gene and an identification nucleic acid sequence, and by designing a primer and carrying out PCR (polymerase chain reaction) amplification on the primer, an amplification product is subjected to electrophoresis detection, and whether the amplification product is a long-chain or not can be determined according to whether an electrophoresis band is a 302bp band. Therefore, the DNA sample of the silver carp can be judged whether to contain the introgression genes or not, and the silver carp, the ordinary silver carp and the carp can be effectively distinguished. The introgression gene, the primer, the method and the application for identifying the Changfeng silver carp can provide support for molecular identification of the Changfeng silver carp, provide powerful guarantee for seed counterfeiting and healthy development of seed industry, and have the advantages of simplicity, easiness in implementation, convenience in operation and high identification success rate.
Drawings
FIG. 1 is a diagram of electrophoresis results provided by an embodiment of the present application; the Changfeng silver carp is marked as Hypophthalmichthys molitrix Changfeng variety (CF), the common silver carp is marked as Hypophthalmichthys Molitrix (HM), the carp is marked as Cyprinus Carpio (CC), and the lane M is a band of 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
The application deeply digs the genome of the Changfeng silver carp to find that the gene sequence with strong specificity is an introgression gene, the introgression gene can be used as an identification gene and an identification nucleic acid sequence of the Changfeng chain, and the application can determine whether the Changfeng silver carp is the Changfeng chain according to whether the electrophoresis band is 302bp band or not by designing a primer and carrying out PCR (polymerase chain reaction) amplification on the primer and carrying out electrophoresis detection on the amplified product. Therefore, the DNA sample of the silver carp can be judged whether to contain the introgression genes or not, and the silver carp, the ordinary silver carp and the carp can be effectively distinguished. The introgression gene, the primer, the method and the application for identifying the Changfeng silver carp can provide support for molecular identification of the Changfeng silver carp, provide powerful guarantee for seed counterfeiting and healthy development of seed industry, and have the advantages of simplicity, easiness in implementation, convenience in operation and high identification success rate.
Based on this, the example discloses a nucleic acid molecule for identifying the long-spotted silver carp, which is shown as SEQ ID NO. 1.
Based on this, the example discloses the identification of the introgression gene of the Changfeng silver carp, which is derived from the carp genome, as shown in SEQ ID NO. 1.
Based on this, the examples disclose primer pairs for identifying introgression genes in Changfeng silver carp, which introgression genes are derived from the carp genome, comprising DNA molecules having the sequences shown in SEQ ID No. 2-3.
Based on this, the examples disclose a kit for identifying introgression genes in Changfeng silver carp, which comprises the primer pair and the reagents required for performing PCR.
In some embodiments, the reagents required to perform PCR include DNA polymerase, mg 2+ One or more of dNTPs, PCR Buffer, and sterile water. Wherein the DNA polymerase is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V, such as Taq polymerase or rTap enzyme.
Based on this, the embodiment discloses a method for identifying the silver carp, which comprises the following steps:
obtaining a DNA sample to be detected, wherein the DNA sample to be detected is from at least one of silver carps, silver carps and carps;
taking the DNA as a template, and adopting the primer pair to perform PCR reaction or the kit to perform PCR reaction;
the resulting PCR reaction product is detected for gel electrophoresis, and the origin of the DNA sample is confirmed from the bands of the gel electrophoresis.
In some embodiments, the reaction system of the PCR reaction comprises, in terms of 50. Mu.L, 25. Mu.L of a 2 XPCR buffer mix, 1.5. Mu.L of a 10. Mu.M upstream primer as set forth in SEQ ID NO.2, 1.5. Mu.L of a 10. Mu.M upstream primer as set forth in SEQ ID NO.3, 1. Mu.L of a 100 ng/. Mu.L template (i.e., the DNA sample to be tested in the above embodiments), and the balance ddH 2 O。
Based on this, the examples disclose the application of the nucleic acid molecule, the introgression gene, the primer pair or the kit in the detection of the introgression gene of the silver carp, and the identification of the silver carp, the common silver carp and the carp.
In some embodiments, there is provided a method of identifying a Changfeng silver carp, or a method of introgressing a gene or a method of identifying Changfeng silver carp, a plain silver carp and a carp, comprising:
1. collection of authentication samples
Fin bar samples of individual silver carps, ordinary silver carps and carp are collected respectively and stored in absolute ethyl alcohol, the silver carps are marked as Hypophthalmichthys molitrix Changfeng variety (CF), the ordinary silver carps are marked as Hypophthalmichthys Molitrix (HM), the carp is marked as Cyprinus Carpio (CC), and the silver carps are brought back to a laboratory.
2. Genomic DNA extraction
(1) About 0.5g of fin tissue is taken, absolute ethyl alcohol on the surface of the tissue is repeatedly washed by ultrapure water, and the tissue is fully sheared and then placed into a centrifuge tube with the volume of 1.5 mL.
(2) To the centrifuge tube, 10-15. Mu.L of proteinase K and 500. Mu.L of HOM buffer were added and digested at 55℃for 4-6 hours.
(3) 500. Mu.L of 4.5M NaCL solution and 300. Mu.L of chloroform were added, and after thoroughly mixing for 20min, the mixture was centrifuged at 13000rpm for 10min.
(4) The supernatant was transferred to another sterilized fresh tube, 600. Mu.L of anhydrous isopropanol was added, shaken well for 20min, and centrifuged at 13000rpm for 10min.
(5) The supernatant was discarded, 500. Mu.L of 75% ethanol was added, and the mixture was digested at 55℃for 5 minutes, followed by centrifugation at 13000rpm for 5 minutes.
(6) The supernatant was discarded, and the tube was inverted on absorbent paper and dried naturally at room temperature.
(7) 100. Mu.L of sterilized double distilled water was added to dissolve DNA at 4℃overnight.
(8) DNA detection was performed on a 1% agarose gel and stored at-20℃until use.
3. Amplification of introgressed gene-specific fragments in Changfeng silver carp
(1) Primer information
14270-3F: GGAGAGAGTCCTTTCTCATG as shown in SEQ ID NO.2
14270-4R: CTCAAAGCACACGTGAGATC as shown in SEQ ID NO.3
(2) PCR reaction system
TABLE 1
| System components | Volume of |
| 2×PCR buffer mix | 25μL |
| Upstream primer (10. Mu.M) | 1.5μL |
| Downstream primer (10. Mu.M) | 1.5μL |
| DNA template (100 ng/. Mu.L) | 1μL |
| ddH 2 O | 21μL |
| Total system | 50μL |
(3) PCR reaction procedure
Pre-denaturation at 95℃for 3min,32 cycles (denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 15 s), extension at 72℃for 5min, and storage at 16 ℃.
4. Electrophoresis of penetration gene specific fragments of Changfeng silver carp
After amplification, 5. Mu.L of PCR amplification product was sampled on 2.0% agarose gel, and the leftmost sample was DL2000marker,120V, and the power was turned off for 30min, and then the electrophoresis results were observed with a gel imaging system.
5. Screening and identifying genes of introgression in Changfeng silver carp, ordinary silver carp and carp
As shown in FIG. 1, the electrophoresis result shows that a 302bp obvious band (shown as SEQ ID NO. 1) appears in the Changfeng silver carp, a 399bp obvious band (shown as SEQ ID NO. 4) appears in the carp, and no band in the ordinary silver carp is the introgression gene fragment, so that the Changfeng chain and the ordinary chain can be identified, and the specific DNA molecule is derived from a lithium genome.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.
Claims (8)
1. And identifying the nucleic acid molecule of the Changfeng silver carp, wherein the nucleic acid molecule is shown as SEQ ID NO. 1.
2. And identifying the infiltration gene of the Changfeng silver carp, wherein the infiltration gene is derived from the carp genome as shown in SEQ ID NO. 1.
3. And identifying a primer pair of an introgression gene in the Changfeng silver carp, wherein the introgression gene is derived from a carp genome, and the primer pair comprises a DNA molecule shown as SEQ ID NO. 2-3.
4. A kit for identifying introgression genes in long-spotted silver carp comprising the primer pair of claim 3 and reagents required for performing PCR.
5. The kit according to claim 3, wherein the reagents required for performing PCR include DNA polymerase, mg 2+ dNTP, PCRBuffer and sterile water.
6. A method of identifying a long-spotted silver carp, comprising:
obtaining a DNA sample to be detected, wherein the DNA sample to be detected is from at least one of silver carps, silver carps and carps;
performing a PCR reaction using the primer set of claim 3 or the kit of claim 4 or 5 using the DNA as a template;
the resulting PCR reaction product is detected for gel electrophoresis, and the origin of the DNA sample is confirmed from the bands of the gel electrophoresis.
7. The method of claim 7, wherein the source of interpreting the DNA sample if the gel electrophoresis results in a 302bp band comprises at least a long-dashed strand.
8. Use of the nucleic acid molecule of claim 1, the introgression gene of claim 2, the primer pair of claim 3 or the kit of claim 4 or 5 in the detection of the introgression gene of the long-spotted silver carp, the identification of the long-spotted silver carp, the plain silver carp and the carp.
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| CN202311173958.6A CN117165695A (en) | 2023-09-12 | 2023-09-12 | Introgression gene, primer, method and application for identifying Changfeng silver carp |
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| CN112725465A (en) * | 2021-02-02 | 2021-04-30 | 中国水产科学研究院长江水产研究所 | Precise Changfeng silver carp identification primer, identification method and application thereof |
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-
2023
- 2023-09-12 CN CN202311173958.6A patent/CN117165695A/en active Pending
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