CN112772468B - Method for breeding new species of normally-developed aponeurosis spinifera - Google Patents
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
A method for breeding a new species of anagen aponeurosis carps relates to a method for breeding a new species of aponeurosis carps. The invention provides a method for removing the muscle thorns without influencing the normal growth of fishes. The invention developsThe method for breeding the new species of the normal aponeurosis carps comprises the following steps: firstly, knocking out bmp6 gene in fertilized egg of original fish variety to obtain F0Generation individuals; two and two pairs F0Carrying out mutant screening and grouping to obtain F1Generation group; three, pair F1Selecting individuals with the same frameshift mutation for mating to construct F2Selecting F2Homozygous mutant individuals of (a); four, pair F2Carrying out expansion propagation on the generation homozygous mutation individual to obtain a new species of the aponeurosis fish. The invention can obtain the strain with the muscle thorns lacked, does not influence the growth, can be stably inherited, and can solve the problems of small muscle thorns, difficult discovery and difficult removal in production.
Description
Technical Field
The invention relates to a method for breeding a new variety of apostichopus japonicus.
Background
According to the report of world food and agriculture organization, 90% of the global fish culture yield comes from 27 kinds of fishes, and 12 kinds of the 27 kinds of fishes contain interspinal spines, and the culture yield accounts for more than 69% of the fish culture yield. The intermuscular spur is a specific skeleton of teleost fish, the small skeleton located between muscle segments, and is formed by ossification of tendons or ligaments. Because the existence of the interspinal stings can cause inconvenience and even body injury of people in the eating process, and simultaneously cause troubles in processing of fish products, the genetic improvement of the interspinal stings becomes one of important target traits of fish genetic breeding.
At present, methods for preparing strains with few intercropping carassius auratus by adopting crossbreeding are adopted, for example, the national invention patent ' an optimized breeding and seedling method for novel intercropping carassius auratus gibelio strains ' (patent number: ZL201010140103.X) and ' a construction method for hybrid megalobrama amblycephala and culter with rapid growth and few intercropping carassius (patent publication number: CN201710788633) obtain novel intercropping carassius auratus strains and hybrid new strains of megalobrama amblycephala and culter with few intercropping spurs. With the development of gene editing technology, abalone dragon and the like (patent application number: 202010451275.2) knock out genes of silver carps and bighead carps by adopting gene editing, and establish a breeding method of new silver carp and bighead carp varieties with coarse inter-muscular stings. Studies of Gaeuxia and the like show that a strain with the quantity of interspinal spines reduced by more than 70 percent can be obtained by knocking out the scxa gene in zebra fish (patent application No. 201911104496.6; Nie et al, 2020), but according to the researches, the zebra fish strain with the quantity of interspinal spines reduced has the abnormal development of main axis skeletons of ribs and the like, and influences the normal growth of fish bodies (Nie et al, 2020; Kague et al, 2019). Although methods for reducing the number of the muscle thorns by adopting a gene editing technology exist at present, the methods all have some defects and influence the normal growth of fishes.
Disclosure of Invention
The invention provides a method for removing the muscle thorns without influencing the normal growth of fishes.
The invention discloses a method for breeding a new species of normally-developing aponeurosis carps, which comprises the following steps:
firstly, knocking out bmp6 gene in fertilized egg of original fish variety to obtain F0Generation individuals;
two and two pairs F0Carrying out mutant screening and grouping to obtain F1Generation group;
three, pair F1Selecting individuals with the same frameshift mutation for mating to construct F2Selecting F2Homozygous mutant individuals of (a);
four, pair F2Carrying out expansion propagation on the generation homozygous mutation individual to obtain a new species of the aponeurosis fish.
The method directionally edits the bmp6 gene of the fish by using a gene editing technology, can obtain the strain with the muscle thorns missing, does not influence the growth, can stably inherit, and can solve the problems of small muscle thorns, difficult discovery and difficult elimination in production. A large number of mutant fishes which can be stably inherited can be obtained by a gene editing method.
Drawings
FIG. 1 is a schematic diagram of the mating strategy for mutation detection in example 1; wild type as Wild type individual, Cas9F0For microinjection F0Generation individuals.
FIG. 2 is a plot of the PCR product sequencing peaks of the wild type colonies of example 1, underlined the target sites and boxed the site of the bmp6 knockout.
FIG. 3 is a plot of the PCR product sequencing peaks of mutant colonies of example 1, underlined as target sites and boxed as the site of the bmp6 knockout.
FIG. 4 is a graph of skeletal staining of wild-type AB line zebrafish in example 1, with obvious intramuscular stinging.
FIG. 5 is a graph of bone staining of a homozygous mutant zebra fish in example 1, with complete loss of the interspinal spine.
FIG. 6 is a graph of bone staining of a homozygous mutant zebra fish in example 1, with complete loss of the interspinal spine and with local labeling.
Fig. 7 is an enlarged view of a portion of fig. 6, with arrows indicating the absence of interspinous processes.
Fig. 8 is an enlarged view of a portion of fig. 6, with arrows indicating the absence of interspinous processes.
FIG. 9 is a comparison graph of the weight growth of the wild type AB line and the homozygous Mutant zebra fish in example 2, WT is the wild type, Mutant is the Mutant, and the weight growth of the homozygous Mutant zebra fish and the wild type AB line zebra fish has no significant difference.
FIG. 10 is a comparison graph of the growth of zebra fish of wild type AB line and homozygous Mutant in example 2, WT is wild type, Mutant is Mutant, and the growth of zebra fish of homozygous Mutant and wild type AB line has no significant difference
FIG. 11 is a comparative observation of the skeletal development of the wild-type AB line and the homozygous mutant zebra fish of example 3 at different developmental stages. FIG. 11A is 8dpf wild type (body length 6.12 mm); b is an 8dpf mutant (body length 5.96 mm); c is 14dpf wild type (body length 7.32 mm); d is a 14dpf mutant (body length 7.92 mm); e is 20dpf wild type (body length 9.87 mm); f is a 20dpf mutant (body length 10.16 mm); g is 26dpf wild type (body length 11.27 mm); h is a 26dpf mutant (11.66 mm in length); i is 32dpf wild type (body length 12.38 mm); j is a 32dpf mutant (body length 12.67 mm); k is 38dpf wild type (body length 15.42 mm); l is a 38dpf mutant (body length 15.07 mm); m is 44dpf wild type (body length 17.02 mm); n is a 44dpf mutant (body length 16.84 mm); o is 50dpf wild type (body length 17.98 mm); p is a 50dpf mutant (body length 18.41 mm); q is 56dpf wild type (body length 19.04 mm); r is a 56dpf mutant (body length 19.85 mm). Af is hip fin; br is gill bone; ct is the vertebral body; df is dorsal fin; fp is a branch fin bone; fr is a fin bone; fro is frontal bone; ga is gill bow; hs is vein acantha; hy is hypopharynx bone; imb is used for treating muscle wound; la is lacrimal bone; na is marrow bow; ns is spinal cord spine; or is orbital bone; op is branchial canopy bone; ot is otolith; pa is parietal bone; pef is pectoral fin; pf is ventral fin; pr is the anterior maxilla; pra is anterior branchia epiphysis; r is a rib; qu is a square bone; sc is the upper occipital bone; so is the upper orbital bone; su is lower gill cap bone; vc is spinal column.
FIG. 12 is a comparison of the reproductive performance of the wild-type AB line zebra fish and the homozygous mutant zebra fish of example 4, and the mutant zebra fish has no significant difference in fertilization rate, hatchability rate and aberration rate compared with the wild-type zebra fish. In the figure, A is fertilization rate, B is hatching rate, C is distortion rate, and a shows that no significant difference exists between wild type (wild type) and mutant type (mutant).
FIG. 13 is a sequence alignment chart of pre-and post-knockout in example 1-pre-knockout of wild-type AB line, post-knockout of heterozygous mutation (variant sequence) -Note: -represents a deletion base.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the method for breeding the new varieties of the normally-developed aponeurosis carps in the embodiment comprises the following steps:
firstly, knocking out bmp6 gene in fertilized egg of original fish variety to obtain F0Generation individuals;
two and two pairs F0Carrying out mutant screening and grouping to obtain F1Generation group;
three, pair F1Selecting mutants with the same frame shiftThe mutated individuals were mated and F was constructed2Selecting F2Homozygous mutant individuals of (a);
four, pair F2Carrying out expansion propagation on the generation homozygous mutation individual to obtain a new species of the aponeurosis fish.
In this embodiment, the bmp6 gene in a fertilized egg of an original fish variety can be knocked out by a gene editing method.
The second embodiment is as follows: the present embodiment differs from the first embodiment in that: step one, knocking out the bmp6 gene by adopting a TALEN or CRISPR/Cas9 method. Other steps and parameters are the same as those in the first embodiment.
The third concrete implementation mode: the present embodiment is different from the first or second embodiment in that: knocking out the bmp6 gene by using a CRISPR/Cas9 method:
designing a target site in the exon region of the bmp6 gene;
secondly, sgRNA containing a target site is obtained by adopting an in vitro transcription method;
③ mixing the sgRNA and the Cas9mRNA or the Cas9 protein in proportion to obtain a gene editing reagent;
and fourthly, injecting the gene editing reagent into the fertilized eggs in the single cell stage obtained by parent pairing propagation in a micro-injection manner. Other steps and parameters are the same as in one or both embodiments.
The fourth concrete implementation mode: the present embodiment differs from the first, second, or third embodiment in that: and the molar concentration of the Cas9mRNA or Cas9 protein and the sgRNA in the third step is 1: 1-1: 5. The other steps and parameters are the same as those in the first, second or third embodiment.
The fifth concrete implementation mode: the present embodiment is different from one of the first to fourth embodiments in that: and fourthly, the injection amount of each fertilized egg gene editing reagent is 1 nL-5 nL. The other steps and parameters are the same as those in one of the first to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the first to fifth embodiments in that: step two microinjection of F of gene editing reagent0Cultivating fertilized eggs until the body length is 2-5 cm, shearing tail fins, extracting genome DNA, detecting mutants, and culturingFeeding the mutant to sexual maturity; then F0Grouping the mutants to obtain F1And (5) embryo generation. Other steps and parameters are the same as those in one of the first to fifth embodiments.
The seventh embodiment: the present embodiment differs from one of the first to sixth embodiments in that: step three:
3.1, mixing F1Culturing embryo to 2-5 cm, cutting tail fin, extracting genome DNA, detecting mutant, performing PCR product sequencing to obtain mutant genotype, maintaining and culturing the individual with frame shift mutation as F2The parent of the generation;
3.2 according to F2Matching the gene types of the generation parents, mixing the parents with the same gene type into a group for propagation to obtain F2Seedling generation;
3.3, mixing F2Culturing the embryo to 2-5 cm, cutting tail fin, extracting genome DNA, detecting mutant, sequencing to obtain F2The genotype of the generation is homozygous mutant individuals. Other steps and parameters are the same as those in one of the first to sixth embodiments.
The specific implementation mode is eight: the present embodiment is different from the first to seventh embodiments in that: step four:
4.1、F2breeding homozygous mutation individuals for 2-4 months, and detecting the quantity of the muscle thorns of the homozygous mutation individuals by adopting a skeletal staining or in vitro separation method;
4.2, carrying out group mating on homozygous mutant individuals without the muscle spines and with the same genotype to obtain a group without the muscle spines which grow normally. Other steps and parameters are the same as those in one of the first to seventh embodiments.
The specific implementation method nine: the present embodiment differs from the first to eighth embodiments in that: the exon region of the bmp6 gene is used for designing N gene knockout target sites. Other steps and parameters are the same as those in one to eight of the embodiments.
In the present embodiment, N is a natural number of 1 or more.
The detailed implementation mode is ten: the present embodiment differs from one of the first to ninth embodiments in that: the upstream primer of the sgRNA is
x in the bmp6 gene knockout target site is 16-20;
the 3 'end of a downstream primer of the sgRNA is added with a thick part of a sequence which is complementary to a sequence at the 5' end of a gRNA framework, and the downstream primer is
Examples 1,
Cultivating a new species of zebra fish with normal development and without interspinal thorns:
wild-type AB line zebra fish (from the Heilongjiang aquatic research institute automatic water circulation system, China aquatic science institute) was used. The ratio of Light to Dark periods is 14h:10h (14h Light:10h Dark); placing sexually mature and healthy zebra fish into a spawning box according to the ratio of male to female being 3:2, and separating the male and female fish by using a baffle. The baffle is taken out before injection next day to finish oviposition and fertilization.
Searching for the GGN of the target site of bmp6 by using ChopChop online (or obtaining the sequence of bmp6 by cloning, if the gene bmp6 has multiple copies due to genome replication, all the copies of the nucleotide sequence are required to be obtained)18NGG (containing PAM sequence) selected 2 target sites (shown in Table 1, target site) in exon 1 of bmp6 geneThe dot-bold part is a PAM sequence).
TABLE 1
Designing a gene target site:
the upstream primer of the sgRNA is
the 3 'end of a downstream primer of the sgRNA is thickened to be a sequence complementary to a sequence at the 5' end of a gRNA framework, and the downstream primer is
Therefore, in this example, the upstream primer sequences of 2 sgrnas are respectively
And
sgrnas of 2 sites were obtained by PCR amplification respectively:
performing PCR amplification with upstream primers of sgRNAs and downstream primers of sgRNAs at a concentration of 10. mu.M (since the gRNA backbone sequences of the upstream and downstream primers are complementary, template-free PCR amplification is possible), wherein the amplification procedure is 95 ℃ denaturation for 3min, 30 cycles of 95 ℃ for 30sec, 58 ℃ for 30sec, and 72 ℃ for 30 sec; extension at 72 ℃ for 5 min. The obtained PCR product was detected by 1.5% agarose gel electrophoresis, and the band was 120 bp. And after detection, purifying and recovering the PCR product by using a PCR product purification and recovery kit, and measuring the concentration for later use.
sgRNA in vitro transcription:
in vitro synthesis of sgRNAs of 2 sites by using a NEB HiScribe T7 rapid high-efficiency RNA synthesis kit (E2050S) and establishment of a 30-mu-l system: includes PCR recovery of gRNA at 1. mu.g, NTP Buffer Mix at 10. mu.l, T7RNA Polymerase Mix at 2. mu.l, and make up to 30. mu.l with enzyme-free water. Transcribing at 37 deg.c for 4 hr, adding 20. mu.l of enzyme-free water after the reaction is completed, mixing, adding 2. mu.l of DNase I, digesting at 37 deg.c for 15min to eliminate unreacted DNA. The sgrnas transcribed in vitro were recovered using an RNA purification kit, and 20 μ l of rnase-free water was added. And (4) measuring the concentration of the recovered product, wherein the concentration of the recovered product is 1000-4000 ng/mu l, and storing the product in a refrigerator at the temperature of-80 ℃ for later use.
Microinjection:
2 in vitro transcribed sgRNA and Cas9 proteins were mixed at a molar ratio of 3:1, incubated at room temperature for 10min, added with 25% phenol red, and injected into zebra fish embryos at the single cell stage. The final concentration of sgRNA was greater than 50 ng/. mu.l, and the control group was injected with 25% phenol red.
Mutation detection strategy:
F0sampling 24-48 h after the generation injection to detect mutation, and calculating the mutation rate. Culturing the embryo until the body length is 2-5 cm, shearing tail fins, and carrying out mutation detection. F0Mating with wild AB line zebra fish after generation mutation individual sexual maturity to obtain F1Detection F1Heterozygous mutant individuals in the generations are selfed to obtain F2And (4) generation. Theoretical F according to Mendelian's law of separation2Homozygous mutant individuals, heterozygous mutant individuals and wild type in the generation were in a ratio of 1: 2: 1. at F2Detection in the generation selects homozygous mutant individuals, and the homozygous mutant individuals are subjected to population crossing to obtain a mutant line capable of being stably inherited (shown in figure 1).
The mutation detection method comprises the following steps:
mu.l of lysate (lysate components: proteinase K0.5 mg/ml, 1M Tris (pH8.0)10mM, KCl 50mM, 0.3% Tween 20, 0.3% NP 40) was added to the embryo or fin sample. The samples were digested with a PCR instrument at 55 ℃ for 50min and 98 ℃ for 10 min. After cracking, Vortex is mixed evenly, centrifugation is carried out for 1-2min at 1000-2000rpm, and supernatant is taken as a PCR amplification template. Taking 2 mul of supernatant as a template, 20 mul of an amplification system, and performing the same amplification procedure as PCR amplification of sgRNA. Detecting the mutation by 8% polyacrylamide gel electrophoresis, and subjecting the obtained mutation F1And (3) carrying out TA cloning on the generation individual, carrying out sequencing after colony PCR detection, wherein the PCR primer is a detection primer, and carrying out sequencing to obtain a mutation sequence.
Knock-out results:
wild-type AB lines (FIG. 2) and heterozygous mutations F for 2 target sites of exon 11Generation mutant colonies were PCR sequenced (fig. 3). The result of the comparative sequencing yielded 1 variant, which had a variation in both the 1 target site (italic) and the 2 target site (bold), 16bp (underlined) at the 1 target site was changed to 3bp, AA at the 2 target site was changed to G (underlined), and 1bp (FIG. 13) was deleted.
And (3) bone staining observation:
bone double staining was performed with alcian blue and red, at F2In the generation individuals, 10 tails of wild type AB lines (+/+) and homozygous mutant individuals (-/-) adult fishes are randomly extracted, skeletal staining is carried out, and the influence of gene function loss on the development of the interspinal spines is observed through a microscope.
Skeletal staining shows that the wild AB line zebra fish has interspinal spines (as shown in figure 4), the homozygous mutant zebra fish has complete loss of the interspinal spines (as shown in figures 5-8), and the rib and other main axis skeletal development of the homozygous mutant zebra fish strain is normal and has no influence.
Example 2
Observation of growth and development of homozygous mutant type:
the growth of 14 wild-type AB line zebra fish families and 13 homozygous mutant line families was continuously observed and measured, and the body weight and the body length of homozygous mutant zebra fish were found to have no significant difference from the wild-type AB line in 5 developmental stages of 15dph, 30dph, 45dph, 60dph and 70dph (as shown in FIG. 9 and FIG. 10).
F was constructed by crossing the homozygous mutant obtained in example 1 with the wild-type AB line and selfing the progeny of the cross2Family generation, detection F2Generation of individuals, F2Wild type in generations: heterozygous mutation: the homozygous mutation ratio was 1: 2: 1. in this example, 14 wild-type AB family zebra fish are selected from F2Wild type construction in generations. In this example, 13 homozygous mutant lines were also F2Construction of homozygous mutations in generations. The method for constructing the family can avoid the interference of genetic background on the growth and development contrast of wild type and homozygous mutant.
Example 3
Homozygous mutant skeletal development observations:
skeletal development observation of 3 wild-type AB lines and 3 homozygous mutant zebra fish families was performed at 9 developmental stages, and it was found that skeletal development of homozygous mutant zebra fish was substantially identical to that of wild-type zebra fish except for the intermuscular spurs by alcian blue and red skeletal staining (as shown in FIG. 11).
F was constructed by crossing the mutant homozygous line obtained in example 1 with the wild-type AB line and selfing the progeny of the cross2Family generation, detection F2Generation of individuals, F2Wild type in generations: heterozygous mutation: the homozygous mutation ratio was 1: 2: 1. in this example 3 wild-type AB family zebra fish are selected from F2Wild type construction in generations. In this example 3 mutant homozygous families, also from F2Mutant homozygotes in the generation were constructed.
Example 4
Analysis of homozygous mutant reproductive capacity:
comparing the reproductive performance of 14 wild-type AB-line zebra fish families and 13 homozygous mutant-type zebra fish families, the homozygous mutant-type zebra fish is found to have no significant difference in fertilization rate, hatchability rate and aberration rate from the wild-type AB-line zebra fish families (as shown in FIG. 12).
F was constructed by crossing the homozygous mutant obtained in example 1 with the wild-type AB line and selfing the progeny of the cross2Family generation, detection F2Generation of individuals, F2Wild type in generations: heterozygous mutation: the homozygous mutation ratio is1: 2: 1. in this example, 14 wild-type AB family zebra fish were bred from F2Wild type construction in generations. In this example, 13 homozygous mutant lines were derived from F2Mutant homozygotes in the generation were constructed.
Although the invention has been described above by way of general illustration and specific embodiments, it is within the scope of the invention as claimed that modifications and improvements may be made thereto without departing from the spirit of the invention.
Sequence listing
<110> institute of aquatic products of Heilongjiang, China institute of aquatic science
<120> cultivation method of new species of normally-developed aponeurosis spinifera
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<141> 2020-11-27
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Claims (8)
1. The method for breeding a new species of normally-developed amateus spinosus without interspinal muscles is characterized by comprising the following steps of:
firstly, knocking out bmp6 gene in fertilized egg of original zebra fish variety to obtain F0Generation individuals;
two and two pairs F0Selection of bmp6 Gene mutant0The generation bmp6 gene mutation is subjected to sexual maturity and then is mated with wild AB line zebra fish to obtain F1Generation;
three, pair F1Selecting bmp6 gene mutant from generation group, selecting individuals with same frame shift mutation, mating, and constructing F2Selecting F2Homozygous mutant individuals of (a);
four, pair F2Carrying out expanding propagation on the generation homozygous mutation individual to obtain a new species of the zebra fish without the interspinal stinging;
wherein 2 gene knockout target sites are designed in exon 1 of the zebra fish bmp6 gene in the first step, and are GGCAGGTAGCAGTTCTGTACTGG and GGTTACGGTCTCAGGAGAAGAGG respectively;
the sequence of the bmp6 heterozygous mutant gene is AGTGAAGAACCTATGGCGTGATTAAGAAGAGGACAGAACATTGTTTTGGAAGTGATTTAGAAGTGCACCCTAATCATAATCAACAGAGACTGGATGTAAATATGACGAGCGCCTGGTTTGCTCTGCTGAGCTTCTTGTGGAGCTGTTGTTTGGCAGGTAGCAGTTCCAGGAGCTTCAGTCCAACTTCATACATCGGAGGTTACGGTCTCAGGAGGGAGGGAGATGCAGAAAGAGATTCTGTCCATACTGGGACTGAACCACAGA.
2. The method for breeding a new variety of normally-developing amanita zebrafish without interspinous insertions as claimed in claim 1, wherein the first step is to knock out bmp6 gene by TALEN or CRISPR/Cas9 method.
3. The method for breeding a new variety of normally-developing amanita zebrafish without interspinous spines as claimed in claim 2, wherein the method for knocking out bmp6 gene by CRISPR/Cas9 is adopted:
designing a target site in the exon region of the bmp6 gene;
secondly, sgRNA containing a target site is obtained by adopting an in vitro transcription method;
③ mixing the sgRNA with the Cas9mRNA or the Cas9 protein in proportion to obtain a gene editing reagent;
and fourthly, injecting the gene editing reagent into the fertilized eggs in the single cell stage obtained by parent pairing propagation in a micro-injection manner.
4. The breeding method of the new species of normally-developing amateus fasciatus without interspinal stinging according to claim 3, wherein the molar concentration of Cas9mRNA or Cas9 protein and sgRNA in the third generation is 1: 1-1: 5; and fourthly, the injection amount of each fertilized egg gene editing reagent is 1 nL-5 nL.
5. The method for breeding a new variety of normally-developing amateus fasciatus without interspinal needling according to claim 1 or 4, wherein step two comprises microinjecting F of a gene editing reagent0Cultivating the fertilized eggs until the body length is 2-5 cm, shearing tail fins, extracting genome DNA, detecting the bmp6 gene mutant, and feeding the bmp6 gene mutant to sexual maturity.
6. The method for breeding a new species of normally developing amateus fasciatus without interspinal stinging according to claim 1 or 4, which is characterized by comprising the following steps:
3.1, mixing F1Culturing the embryo to 2-5 cm in length, cutting tail fin, extracting genome DNA, detecting bmp6 gene mutant, carrying out PCR product sequencing on the bmp6 gene mutant to obtain genotype of the bmp6 gene mutant, reserving and culturing the individual with frameshift mutation to sexual maturity as F2The parent of the generation;
3.2 according to F2Matching the gene types of the generation parents, mixing the parents with the same gene type into a group for propagation to obtain F2Seedling generation;
3.3, mixing F2Culturing the embryo to 2-5 cm, cutting tail fin, extracting genome DNA, detecting bmp6 gene mutant, sequencing bmp6 gene mutant to obtain F2The genotype of the generation is homozygous mutant individuals.
7. The method for breeding a new species of normally developing amateus fasciatus without interspinal stinging according to claim 1 or 4, which is characterized by comprising the following steps:
4.1、F2breeding homozygous mutation individuals for 2-4 months, and detecting the quantity of the muscle thorns of the homozygous mutation individuals by adopting a skeletal staining or in vitro separation method;
4.2, carrying out group mating on homozygous mutant individuals without the muscle spines and with the same genotype to obtain a group without the muscle spines which grow normally.
8. The method for breeding a new species of normally-developing amateus fasciatus without interspinal stinging according to claim 3, wherein an upstream primer of the sgRNA is
GATCACTAATACGACTCACTATA+GGNx+ GTTTTAGAGCTAGAAATAGC wherein GGNxThe sequence GATCACTAATACGACTCACTATA is a T7 promoter sequence, and the sequence GTTTTAGAGCTAGAAATAGC is a gRNA framework 5' end sequence, which is a bmp6 gene knockout target site;
the 3 'end GCTATTTCTAGCTCTAAAAC sequence of the downstream primer of the sgRNA is a sequence complementary to the 5' end sequence of the gRNA backbone, and the downstream primer is GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC.
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| PCT/CN2021/124375 WO2022111124A1 (en) | 2020-11-27 | 2021-10-18 | Method for breeding novel species of normally developed fish without intermuscular bones |
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| CN113151361A (en) * | 2021-04-30 | 2021-07-23 | 中国水产科学研究院黑龙江水产研究所 | Method for cultivating crucian carp strain without muscle intermingled bones |
| CN115943930B (en) * | 2022-12-30 | 2024-03-19 | 中国科学院水生生物研究所 | Method for creating crucian carp without intramuscular thorns |
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