CN109006693B - Method for inducing gene mutation of paralichthys olivaceus - Google Patents
Method for inducing gene mutation of paralichthys olivaceus Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 241000269979 Paralichthys olivaceus Species 0.000 title claims abstract description 26
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 8
- 230000001939 inductive effect Effects 0.000 title claims abstract description 8
- 235000013601 eggs Nutrition 0.000 claims abstract description 32
- 210000004681 ovum Anatomy 0.000 claims description 31
- 230000035772 mutation Effects 0.000 claims description 20
- 239000013535 sea water Substances 0.000 claims description 18
- 108010000912 Egg Proteins Proteins 0.000 claims description 16
- 102000002322 Egg Proteins Human genes 0.000 claims description 16
- 238000011534 incubation Methods 0.000 claims description 16
- 230000001678 irradiating effect Effects 0.000 claims description 11
- 238000007865 diluting Methods 0.000 claims description 8
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- 241000694873 Paralichthyidae Species 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 230000009027 insemination Effects 0.000 claims description 4
- 241000251468 Actinopterygii Species 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 231100000310 mutation rate increase Toxicity 0.000 abstract description 2
- 231100000350 mutagenesis Toxicity 0.000 description 11
- 238000002703 mutagenesis Methods 0.000 description 10
- 230000004720 fertilization Effects 0.000 description 9
- 241000269908 Platichthys flesus Species 0.000 description 7
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- 208000031320 Teratogenesis Diseases 0.000 description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention relates to a method for inducing gene mutation of paralichthys olivaceus, which adopts normal-temperature plasma to irradiate the sperms or the fertilized eggs of the paralichthys olivaceus. Preferably, the irradiation power of the normal temperature plasma is 120-200W, the irradiation time is 1.5-30 min, and the irradiation distance is less than or equal to 2 mm. The invention adopts normal temperature plasma to mutate the fertilized eggs or sperms of the paralichthys olivaceus, and mutant individuals with a certain proportion can be generated. The method is simple and convenient to operate, safe and reliable, can be successfully used for breeding of paralichthys olivaceus and other fishes, greatly increases mutation rate, and improves the efficiency of creating new germplasm resources.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a method for inducing gene mutation of paralichthys olivaceus.
Background
The flounder is an important marine culture fish in China, has the characteristics of tender and delicious meat, high economic value and the like, and is deeply loved by consumers. The improved variety is an important basic guarantee for the healthy and sustainable development of aquaculture industry, and a plurality of excellent new varieties of paralichthys olivaceus are bred by Chinese breeding workers, so that provenance support is provided for the development of the paralichthys olivaceus aquaculture industry. In recent years, along with over fishing and environmental pollution, natural resources of the paralichthys olivaceus are attenuated, so that the number and genetic diversity of germplasm resources for breeding are greatly reduced. Therefore, in order to select and breed the improved variety of the paralichthys olivaceus and the sustainable development of the breeding industry, the germplasm resources of the paralichthys olivaceus must be created efficiently.
The mutagenesis method is an effective means for germplasm resource innovation. The mutagenesis methods currently used for fish are mainly ray mutagenesis and chemical mutagenesis, and the mutagenesis sources mainly used include gamma rays, X rays and N-ethyl-N-nitrosourea (ENU). Among them, gamma rays and X rays are radioactive, equipment is expensive, and special protection facilities are required to ensure safety. ENU is a chemical mutagen that induces mutations in DNA by alkylating modifications of bases in genomic DNA, which induce mismatches in DNA replication. The substance is a 2A carcinogen and can cause great harm to the health of human bodies. Meanwhile, the mutagenesis efficiency of the above several mutagenesis sources is low. Therefore, it is necessary to provide a safe and reliable mutagenesis method with a high mutagenesis rate for Paralichthys olivaceus.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for inducing the gene mutation of paralichthys olivaceus.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a method for inducing gene mutation of paralichthys olivaceus, which adopts normal-temperature plasma to irradiate the sperms or the fertilized eggs of the paralichthys olivaceus.
Preferably, the irradiation power of the normal temperature plasma is 120-200W, the irradiation time is 1.5-30 min, and the irradiation distance is less than or equal to 2 mm.
Preferably, when the fertilized egg is irradiated, the irradiation power of the plasma is 120-160W, and the irradiation time is 20-25 min.
Preferably, when the sperm is irradiated, the irradiation power of the plasma is 160-200W, and the irradiation time is 2-10 min.
Preferably, when irradiating the fertilized egg, the method comprises the steps of:
1) collecting fresh lefteye flounder sperm and ovum, and placing in different containers;
2) diluting the sperms by 6-40 times by using a diluent;
3) mixing the diluted sperms and the ova, stirring uniformly, adding seawater to obtain a sperm-egg mixture activated by the seawater, and incubating the sperm-egg mixture in the seawater for 60 minutes to obtain fertilized ova;
4) and transferring the fertilized eggs into a culture dish, irradiating the fertilized eggs by adopting plasma at normal temperature, and transferring the fertilized eggs into an incubation jar for incubation.
Preferably, the incubation temperature in step 3) and step 4) is 15-20 ℃, preferably 17 ℃.
Preferably, when the sperm cells are irradiated, the method comprises the steps of:
1) collecting fresh lefteye flounder sperm and ovum, and placing in different containers;
2) diluting the sperm by 6-40 times by using a diluent, transferring the diluted sperm into a culture dish, and irradiating the sperm by using plasma at normal temperature;
3) mixing the irradiated sperms and the ova, adding seawater after uniformly stirring, and activating the ova to complete artificial fertilization to obtain fertilized ova;
4) and transferring the fertilized eggs into an incubation jar for incubation.
Preferably, the two methods further comprise step 4) mutation rate detection: and obtaining fries after the fertilized eggs are hatched, cutting fin lines of the fries at the age of 3 months, obtaining the SNP number and the InDel number of each fry individual by a high-throughput whole genome re-sequencing method, and dividing the sum of the SNP number and the InDel number by the whole genome base number to obtain the mutation rate.
The invention has the beneficial effects that:
the invention adopts normal temperature plasma to mutate the fertilized eggs or sperms of the paralichthys olivaceus, and mutant individuals with a certain proportion can be generated. The method is simple and convenient to operate, safe and reliable, can be successfully used for breeding of paralichthys olivaceus and other fishes, greatly increases mutation rate, and improves the efficiency of creating new germplasm resources.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are only a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The invention relates to a method for inducing gene mutation of paralichthys olivaceus, which adopts normal-temperature plasma to irradiate the sperms or the fertilized eggs of the paralichthys olivaceus.
Normal Temperature Plasma (ARTP) is a novel method for mutation breeding, and has the characteristics of simple operation, no environmental pollution and harm, and the like. The ARTP emission source used in the present invention is manufactured by Luoyang Huaqingtianmu Biotech Co., Ltd, and is of type ARTP-P.
In one embodiment of the invention, the irradiation power of the normal temperature plasma is 120-200W, the irradiation time is 1.5-30 min, and the irradiation distance is less than or equal to 2 mm. Generally, the higher the irradiation power of the normal temperature plasma, the larger the distortion rate and mutation rate of the irradiation target. However, too high an irradiation power may cause a mortality rate of the irradiation object to rise straight. The irradiation time has the above-described influence law on the irradiation target. Therefore, the relationship between the irradiation power and the irradiation time needs to be further defined.
In a preferred embodiment of the invention, when the fertilized egg is irradiated, the irradiation power of the plasma is 120-160W, and the irradiation time is 20-25 min; when the sperm is irradiated, the irradiation time is 2-10 min when the irradiation power of the plasma is 160-200W. Thus, relatively high teratogenesis rate and mutation rate can be obtained, and the death rate of the fertilized eggs of the paralichthys olivaceus can be accepted.
In one embodiment of the present invention, if the fertilized egg is irradiated with the normal temperature plasma, the method comprises the steps of:
1) collecting fresh sperm and ovum of Paralichthys olivaceus, and placing in different containers.
2) And (4) diluting the sperm by 6-40 times by using a diluent.
The invention selects Ringer's Solution as diluent, and the formula is as follows: each 1000ml of distilled water is added with 8.22g of NaCl, 0.39g of KCl and CaCl2·2H2O 0.72，MgCl2·6H2O 0.23g，NaH2PO4·2H2O 0.28g，NaHCO30.20g and 1.00g of glucose, and adjusting the pH value to 6.5-7.2.
3) Artificial insemination: and mixing the sperm and the ovum diluted by the ringer's solution, stirring uniformly, adding seawater to obtain a sperm-ovum mixture activated by the seawater, and incubating the sperm-ovum mixture in the seawater at 15-20 ℃, preferably 17 ℃ for 60 minutes to obtain a fertilized ovum.
4) Normal temperature plasma treatment: and transferring the fertilized eggs into a culture dish, irradiating by adopting plasma at normal temperature for the duration and power of the irradiation, transferring the fertilized eggs into an incubation jar, and incubating under a running water condition.
In one embodiment of the present invention, if normal temperature plasma is used to irradiate the lefteye flounder sperm, the method comprises the following steps:
1) collecting fresh sperm and ovum of Paralichthys olivaceus, and placing in different containers.
2) Plasma treatment: diluting the sperm by 6-40 times by using a diluent, preferably ringer's solution, transferring the diluted sperm to a culture dish, and irradiating the sperm by using plasma at normal temperature.
3) Artificial insemination: and mixing the irradiated sperms and the ova, stirring uniformly, adding seawater, activating the ova to complete artificial fertilization, and thus obtaining fertilized ova.
4) And transferring the fertilized eggs into an incubation jar for incubation.
The invention also provides a mutation rate detection method after mutagenesis, which is carried out when the fry is 3 months old, and the specific process comprises the following steps:
irradiating the flounder sperms or the flounder oosperms by adopting normal-temperature plasma, and then hatching according to the routine to obtain the fry. Cutting fin rays when the fries are 3 months old, and obtaining the SNP number and the InDel number of each fry through a high-throughput whole genome re-sequencing method. Since the number of SNPs and InDel can represent almost all types of variation, the mutation rate was obtained by dividing the sum of the two by the number of total genomic bases.
Among them, the High-throughput sequencing technology (High-throughput sequencing) is marked by the capability of sequencing several hundred thousand to several million DNA molecules at a time in parallel and the short reading length. In the invention, Illumina Hiseq1500 is adopted for sequencing.
Single Nucleotide Polymorphisms (SNPs for short) refer to genetic markers formed by variation of a Single Nucleotide on a genome. Refers to variation of a single nucleotide in the genome, including substitutions, transversions, deletions and insertions. It is abundant in number and polymorphism. Insertion-deletion markers (InDel) refer to the difference in the genome-wide in both parents. One parent has a certain number of nucleotide insertions or deletions in its genome relative to the other parent.
Example 1
Irradiating fertilized ovum with plasma at normal temperature
1) Collection of sperm and ovum: collecting fresh male flounder sperm and female flounder ovum, and storing in dark place;
2) artificial insemination: diluting the collected sperms with ringer's solution, and mixing the diluted semen with ovum. After uniformly stirring, adding seawater for activation, wherein the temperature of the seawater is 17 ℃ to obtain a seawater-activated sperm-egg mixture, and incubating the sperm-egg mixture in the seawater at 17 ℃ for 60 minutes;
3) normal temperature plasma irradiation treatment: and after fertilization for 60 minutes, transferring the fertilized eggs into a culture dish, flatly laying a layer, reserving a little seawater, and carrying out normal-temperature plasma irradiation with the irradiation interval of 2 mm. After the irradiation is finished, transferring the fertilized eggs to a hatching cylinder for hatching by flowing water;
4) and (3) mutation rate detection: at 3 months of age, cutting fin rays of 3 fish, obtaining the SNP number and the InDel number of each individual by using a high-throughput genome-wide re-sequencing method, and dividing the sum of the SNP number and the InDel number by the number of bases of the genome-wide to obtain the mutation rate.
Wherein, the volume ratio of the ringer's solution to the sperms, the volume ratio of the semen to the ovum, the plasma power and the irradiation time at normal temperature, and the test results are shown in table 1.
TABLE 1
In table 1, the fertilization rate is the percentage of the number of floating embryos in the gastral phase to the total number of eggs used for induction;
the hatchability is the percentage of the number of hatched fries in the number of fertilized eggs;
the deformity rate is the percentage of the number of the malformed fries to the number of the hatched fries;
mutation rate is the percentage of the number of SNPs and InDel to the number of bases in the whole genome.
As can be seen from table 1, the fertilization rate and the hatching rate gradually decreased and the teratogenesis rate and the mutation rate gradually increased with the increase of the irradiation time under the condition that the plasma irradiation power was not changed. When the irradiation time is 30min, the hatchability is reduced to below 40 percent, which is not beneficial to seed selection.
Example 2
Subjecting the sperm of Paralichthys olivaceus to normal temperature plasma irradiation
1) Collection of sperm and ovum: collecting fresh sperm of male flounder and ovum of female flounder, and storing in dark place;
2) normal temperature plasma irradiation treatment: diluting the sperms by using ringer's solution, spreading the diluted sperms in a culture dish for normal-temperature plasma irradiation, wherein the irradiation interval is 2 mm;
3) artificial fertilization: storing the irradiated sperms in dark, mixing the sperms with the ova when needed, adding seawater after uniformly stirring, and activating the ova to complete artificial fertilization. Transferring the fertilized eggs to a hatching jar for hatching by flowing water;
4) and (3) mutation rate detection: at 3 months of age, cutting fin rays of 3 fish, obtaining the SNP number and the InDel number of each individual by using a high-throughput genome-wide re-sequencing method, and dividing the sum of the SNP number and the InDel number by the number of bases of the genome-wide to obtain the mutation rate.
Wherein, the volume ratio of the ringer's solution to the sperms, the volume ratio of the semen to the ovum, the plasma power and the irradiation time at normal temperature, and the test results are shown in table 2.
TABLE 2
In table 2, fertilization rate, hatchability, teratogenicity rate and mutation rate are defined as in table 1.
As can be seen from table 2, the fertilization rate and the hatching rate gradually decreased and the teratogenesis rate and the mutation rate gradually increased with the increase of the irradiation time under the condition that the plasma irradiation power was not changed. When the irradiation time is 12min, the hatchability is reduced to about 24.8 percent, which is not beneficial to seed selection.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (9)
1. A method for inducing the gene mutation of the paralichthys olivaceus is characterized in that normal temperature plasma is adopted to irradiate the sperms or the fertilized eggs of the paralichthys olivaceus,
when the fertilized eggs are irradiated, the irradiation power of the plasma is 120-160W, the irradiation time is 20-25 min, and the irradiation interval is less than or equal to 2 mm;
when the sperms are irradiated, the irradiation power of the plasma is 160-200W, the irradiation time is 4-10 min, and the irradiation distance is less than or equal to 2 mm.
2. The method according to claim 1, wherein when irradiating the fertilized egg, the method comprises the steps of:
1) collecting fresh lefteye flounder sperm and ovum, and placing in different containers;
2) diluting the sperms by 6-40 times by using a diluent;
3) mixing the diluted sperms and the ova, stirring uniformly, adding seawater to obtain a sperm-egg mixture activated by the seawater, and incubating the sperm-egg mixture in the seawater for 60 minutes to obtain fertilized ova;
4) and transferring the fertilized eggs into a culture dish, irradiating the fertilized eggs by adopting plasma at normal temperature, and transferring the fertilized eggs into an incubation jar for incubation.
3. The method according to claim 2, wherein the incubation temperature in step 3) and step 4) is 15-20 ℃.
4. The method according to claim 3, wherein the incubation temperature in step 3) and step 4) is 17 ℃.
5. The method of claim 2, further comprising the step of 4) mutation rate detection: and obtaining fries after the fertilized eggs are hatched, cutting fin lines of the fries at the age of 3 months, obtaining the SNP number and the InDel number of each fry individual by a high-throughput whole genome re-sequencing method, and dividing the sum of the SNP number and the InDel number by the whole genome base number to obtain the mutation rate.
6. The method of claim 1, wherein when irradiating the sperm, the method comprises the steps of:
1) collecting fresh lefteye flounder sperm and ovum, and placing in different containers;
2) diluting the sperm by 6-40 times by using a diluent, transferring the diluted sperm into a culture dish, and irradiating the sperm by using plasma at normal temperature;
3) mixing the irradiated sperms and the ova, adding seawater after uniformly stirring, and activating the ova to finish artificial insemination to obtain fertilized ova;
4) and transferring the fertilized eggs into an incubation jar for incubation.
7. The method according to claim 6, wherein the incubation temperature in step 3) and step 4) is 15-20 ℃.
8. The method according to claim 7, wherein the incubation temperature in step 3) and step 4) is 17 ℃.
9. The method of claim 6, further comprising the step of 4) mutation rate detection: and obtaining fries after the fertilized eggs are hatched, cutting fin lines of the fries at the age of 3 months, obtaining the SNP number and the InDel number of each fry individual by a high-throughput whole genome re-sequencing method, and dividing the sum of the SNP number and the InDel number by the whole genome base number to obtain the mutation rate.
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| 低温等离子体改性变异山羊绒纤维上色率研究;李倩;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20130815(第08期);第B024-80页 * |
| 常压室温等离子体技术诱变选育鲅鱼抗氧化肽发酵菌株;艾冰花等;《海洋科学》;20161130(第11期);第28-33页 * |
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