CN111269943B - Method for increasing growth speed of zebra fish through gene knockout technology - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a method for increasing the growth speed of zebra fish by a gene knockout technology, which comprises the following steps: searching mRNA sequence and genome sequence of zebra fish actRIIB gene and alk4 gene through NCBI online database; designing knockout target sites of actRIIB gene and alk4 gene; in vitro transcription to obtain Cas9mRNA; cas9mRNA and actRIIB and alk4 gene gRNA microinjection; screening F1 generation heterozygous mutant individuals and determining the mutation type; obtaining an F2 generation homozygous mutant individual, and selfing the F1 generation individual to obtain an F2 generation after the F1 generation heterozygous mutant individual is mature; individuals with homozygous mutations were screened from F2 according to mendelian's law. The invention discovers that the actRIIB and alk4 of the zebra fish are knocked out together, so that the zebra fish can grow rapidly.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for increasing the growth speed of zebra fish through a gene knockout technology.
Background
Currently, the closest prior art:
the growth rate of the fishes is improved, so that the time to market can be shortened, the culture yield in unit time is improved, the incidence of the fishes can be reduced to a certain extent, and the culture risk is reduced. The growth speed of the fishes is directly related to the culture popularization and culture benefits of the fishes, so how to increase the growth speed of the fishes is always a key point and a hot spot of related researches on the fishes. Most of the fast-growing transgenic fish development technologies are gene overexpression technologies at present, and the selected target genes are growth hormone genes, such as transgenic yellow river carps developed by the Zhu Yangtze academy team and transgenic salmon approved to be marketed in the United states at present. However, in some fishes, the growth hormone gene is overexpressed, and the growth of the fishes is not obviously promoted, for example, the growth speed of the fishes is not accelerated by overexpressing the growth hormone gene in domesticated rainbow trout, so that the application range of the method has certain limitation, and transgenic fishes generated by the overexpressed gene possibly have certain environmental hazards and are difficult to popularize and breed. The method for obtaining the fast-growing fish strain by using the gene editing technology CRISPR/Cas9 to silence gene expression is a hot point of research in recent years, the technology also needs search and screening of target genes, the most researched target genes are somatostatin genes at present, but the genes are knocked out in some fishes, the fast-growing fish strain cannot be obtained, and the application range still has certain limitation. The actRIIB and alk4 genes are knocked out together, so that zebra fish with remarkably accelerated growth speed can be obtained, the technology is expected to be popularized to economic fish, and the economic benefit of fish culture is increased to a certain extent.
Myostatin, also known as growth and differentiation factor 8 (gdf-8), plays a negative regulatory role in the growth of many biological muscles. For example, in cattle, mice, dogs and humans, a complete or partial loss of function of the gene results in a significant increase in muscle mass. According to the sequence analysis and homology analysis, myostatin is classified as a member of Transforming growth factor superfamily (TGF), and as a cytokine signaling protein, its specific main receptor for signal transmission is Activin type IIB receptor (activib), and then binds to another common receptor with weaker specificity, activin-like 4 (alk 4), through which signal is conducted to intracellular signaling pathway.
The negative regulation effect of the myostatin on muscle growth is realized by the activin receptor, and in the transgenic mice of the activin receptor actRIIB with skeletal muscle specificity expressing the significant inhibitory type, the activin receptor expressing the significant inhibitory type losing the signaling protein group is combined with the myostatin in vivo in a large amount, so that the pathway of the activin receptor for transmitting the myostatin signal is prevented, and the transgenic mice also show the abnormal proliferation of muscle tissues. As can be seen, the research aiming at actRIIB and alk4 is mostly concentrated in model organisms such as mice, is a basic theory research, and is not applied at present. In the fishes, the research on actRIIB and alk4 is very little, and the research is still in the starting stage, so that the research finds that the gene of actRIIB and alk4 is knocked out together, a fast-growing zebra fish strain can be obtained, and the research is expected to be popularized to economic fishes to promote the development of fishery.
In summary, the problems of the prior art are as follows: the studies on actRIIB and alk4 in fish are currently in the infancy.
The difficulty of solving the technical problems is as follows:
1, the efficiency of knocking out two genes simultaneously by using the CRIPR/Cas9 technology is low, and a large number of F1 generation individuals need to be screened.
2, the homozygous individuals can be obtained only by 3 passages, and the proportion of the homozygous individuals in the F2 is only 6.25 percent, so the screening workload is large.
The significance of solving the technical problems is as follows:
the growth rate of the fishes is improved, so that the time to market can be shortened, the culture yield in unit time is improved, the incidence of the fishes can be reduced to a certain extent, and the culture risk is reduced. The growth speed of the fishes is directly related to the culture popularization and the culture benefit. We find that the actRIIB and alk4 genes are knocked out together to obtain fast-growing zebra fish strains, and the selection range for developing the target genes of the fast-growing fish strains is increased to a certain extent.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for increasing the growth speed of zebra fish by gene knockout technology.
The invention is realized in such a way that a method for increasing the growth speed of zebra fish by a gene knockout technology comprises the following steps:
firstly, searching mRNA sequences and genome sequences of an actRIIB gene and an alk4 gene of the zebra fish through an NCBI (national center of Biotechnology information) online database;
step two, actRIIB gene and alk4 gene gRNA are obtained;
thirdly, cas9mRNA is obtained through in vitro transcription;
fourthly, the micro co-injection of Cas9mRNA, actRIIB and alk4 gene gRNA;
fifthly, screening the F1 generation heterozygous mutant individuals and determining the mutation types;
sixthly, obtaining F2 generation homozygous mutation individuals, and selfing the F1 generation individuals to obtain F2 generation after the F1 generation heterozygous mutation individuals mature; individuals with homozygous mutations were screened from F2 according to mendelian's law.
Further, the mRNA sequence and the genome sequence of zebra fish actRIIB gene and alk4 gene are searched for through an NCBI online database;
actRIIB gene mRNA sequence NM-131210.3;
actRIIB gene genome sequence NC-007135.7;
designing a knockout target site on a first exon of the gene through sequence comparison, wherein the sequence of the target site is SEQ ID NO:1;
alk4 gene mRNA sequence: NM _130990.1;
alk4 gene genomic sequence: NC _007134.7;
designing a knockout target site on a first exon of the gene through sequence comparison, wherein the sequence of the target site is SEQ ID NO:2.
further, the second step of obtaining actRIIB gene and alk4 gene gRNA: using gRNA-plasmid as a model, and using actRIIB-gRNA-F/gRNA-R and alk4-gRNA-F/gRNA-R as primers respectively:
performing PCR amplification by using KOD Plus high-fidelity enzyme, tapping and recovering an amplification product, transforming competent escherichia coli after the recovered product is connected with a pMD18-T vector, selecting 5 escherichia coli for monoclonal amplification culture, and extracting plasmid DNA; then plasmid DNA assaySequence verification is carried out, and plasmid DNA is extracted after the correct sequence is cloned and amplified for culture and is used as a template for next step of transcription of gRNA; reuse of
The T7Transcriptionkit carries out in-vitro reverse transcription on the constructed plasmid to obtain a 20ul reaction system.
Further, the reverse transcription 20ul reaction system is:
10x ReactionBuffer 2ul；
Plasmid DNA4ul，1ug；
T7Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul。
further, the third step of in vitro transcription obtains Cas9mRNA: cas9 plasmid from Addgene TM Use of
T7/T3Transcriptionkit, cas9mRNA synthesis, system and reaction conditions:
10x ReactionBuffer 2ul；
Plasmid DNA4ul，1ug；
T7Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul。
further, the fourth step of micro-co-injection of Cas9mRNA and actRIIB and alk4 gene gRNA: the microinjection system was first configured as follows:
Cas9mRNA300ng/ul；
actRIIBgRNA 30ng/ul；
alk4gRNA 30ng/ul；
Phenol-red 0.2ul；
DEPC Waterup to 2ul；
and (3) after the solution is prepared and uniformly mixed, injecting the mixed solution into zebra fish fertilized eggs in a unicellular period in a microinjection mode, and detecting the mutation efficiency of actRIIB and alk4 genes after culturing for 24 hours.
Further, the screening and mutation type determination of the heterozygous mutant individuals of the fifth generation F1 comprises the following steps:
(1) After the zebra fish fertilized eggs are cultured for 3 months to be sexually mature after microinjection, the zebra fish fertilized eggs are hybridized with wild individuals to obtain F1 filial generation, after the F1 generation individuals are cultured for 2 months, part of tail fin tissues are cut, genome DNA is extracted, and F-actRIIB/R-actRIIBB are respectively used as primers to detect the mutation efficiency of actRIIB genes;
(2) F-alk4/R-alk4 is used as a primer to detect the mutation efficiency of the alk4 gene;
(3) KOD PLUS high-fidelity enzyme is used for PCR amplification, PCR products are connected with a pMD18-T vector and then transformed into competent escherichia coli, 10 escherichia coli are respectively selected for monoclonal amplification culture, plasmid DNA is extracted, and F1 generation individuals with mutant actRIIB genes and alk4 genes are obtained through screening after plasmid DNA sequencing comparison.
In summary, the advantages and positive effects of the invention are: at present, fast-growing fishes are obtained by a gene editing technology, operated genes are single genes, and the fact that the multiple genes are knocked out together can obtain better fast-growing characters is found, so that the invention firstly provides a strategy. Secondly, there are few target genes currently available for developing fast-growing fishes, and the present invention increases the number of target genes. Finally, the invention discovers that the co-knockout of the actRIIB and alk4 of the zebra fish can cause the zebra fish to grow rapidly, and is expected to be popularized to economic fish.
Drawings
FIG. 1 is a flowchart of a method for increasing the growth rate of zebra fish by gene knockout technology according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of the screening of mutation types of actRIIB gene and alk4 gene provided by the embodiment of the invention.
FIG. 3 is a schematic diagram of the significantly increased growth rate of heterozygotes and homozygotes of the co-knock-out actRIIB and alk4 genes zebra fish provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Aiming at the problems in the prior art, the invention provides a method for increasing the growth rate of zebra fish by gene knockout technology, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for increasing the growth rate of zebra fish by gene knockout technology provided by the embodiment of the invention comprises the following steps:
s101: searching mRNA sequence and genome sequence of actRIIB gene and alk4 gene of zebra fish through NCBI online database;
s102: obtaining actRIIB gene and alk4 gene gRNA;
s103: in vitro transcription to obtain Cas9mRNA;
s104: cas9mRNA and actRIIB and alk4 gene gRNA microinjection;
s105: screening F1 generation heterozygous mutant individuals and determining the mutation types;
s106: obtaining an F2 generation homozygous mutant individual, selfing the F1 generation individual to obtain an F2 generation after the F1 generation heterozygous mutant individual is sexually mature, and screening the homozygous mutant individual from the F2 according to the Mendel's law.
The technical scheme of the invention is further described in the following with reference to the attached drawings.
The invention discloses a method for knocking out zebra fish actRIIB gene and alk4 gene together by CRISPR/Cas9 gene editing technology, which comprises the following steps:
(1) Searching mRNA sequence and genome sequence of actRIIB gene and alk4 gene of zebra fish through NCBI online database;
actRIIB Gene mRNA sequence: NM _131210.3;
actRIIB Gene genomic sequence: NC _007135.7;
designing a knockout target site on a first exon of the gene through sequence comparison, wherein the sequence of the target site is SEQ ID NO:1:
GTTCGCTTCTCTGCTCACTTTGG(ii) a (TGG is PAM sequence)
alk4 gene mRNA sequence: NM _130990.1;
alk4 gene genomic sequence: NC _007134.7;
designing a knockout target site on a first exon of the gene through sequence comparison, wherein the sequence of the target site is SEQ ID NO:2:
GCTACAGCAGTTCGTCGAGGAGG(ii) a (AGG is a PAM sequence).
(2) acquisition of actRIIB gene and alk4 gene gRNA:
taking gRNA-plasmid as a template, and taking actRIIB-gRNA-F/gRNA-R and alk4-gRNA-F/gRNA-R as primers respectively:
SEQ ID NO：3actRIIB-gRNA-F：
GTTCGCTTCTCTGCTCACTTTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG；
SEQ ID NO：4alk4-gRNA-F：
GCTACAGCAGTTCGTCGAGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG；
SEQ ID NO：5gRNA-R：AGCACCGACTCGGTGCCACT；
performing PCR amplification by using KOD Plus (TAKARA) high-fidelity enzyme, tapping and recovering an amplification product, converting competent escherichia coli after the recovered product is connected with a pMD18-T vector (TAKARA), extracting plasmid DNA after selecting 5 escherichia coli monoclonally for amplification culture, then sequencing the plasmid DNA for sequence verification, and extracting the plasmid DNA as a template for next step of transcription of gRNA after cloning and amplification culture with correct sequence; reuse of
T7Transcription Kit(Invitrogen TM ) The constructed plasmid was reverse transcribed in vitro to 20ul reaction system as follows:
10x ReactionBuffer 2ul；
plasmid DNA4ul (about 1 ug);
T7Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul；
reacting the system at 37 ℃ for one hour, and purifying for later use;
(3) In vitro transcription to obtain Cas9mRNA: cas9 plasmid from Addgene TM (# 63154), use
T7/T3Transcription Kit(Invitrogen TM ) Cas9mRNA is synthesized, and the system and the reaction conditions are the same as those in (2);
(4) Cas9mRNA and actRIIB and alk4 gene gRNA microinjection: the microinjection system was first configured as follows:
Cas9mRNA300ng/ul；
actRIIBgRNA 30ng/ul；
alk4gRNA 30ng/ul；
Phenol-red 0.2ul；
DEPC Waterup to 2ul；
after the solution is prepared and mixed evenly, injecting the mixed solution into zebra fish fertilized eggs in a unicellular stage in a microinjection way, and detecting the mutation efficiency of actRIIB and alk4 genes after culturing for 24 hours;
(5) Screening of F1 generation heterozygous mutant individuals and determination of mutation types:
after (4) culturing the zebra fish fertilized eggs subjected to microinjection for 3 months until sexual maturity, hybridizing the fertilized eggs with wild individuals to obtain hybridized F1 generation, culturing F1 generation individuals for 2 months, shearing part of tail fin tissues, extracting genome DNA, and detecting mutation efficiency of actRIIB genes by respectively taking F-actRIIB/R-actRIIBB as primers;
SEQ ID NO：6F-actRIIB：5’-GTGTGAGTGTGTGATCGGTT-3’；
SEQ ID NO：6R-actRIIB：5’-TCTGACGCGTTCAAAACGAG-3’；
f-alk4/R-alk4 is used as a primer to detect the mutation efficiency of the alk4 gene;
SEQ ID NO：8F-alk4：5’-GGTTGGTCGGTTTGTGGTTG-3’；
SEQ ID NO：9R-alk4；5’-CGCCGACCGCTAGTAATCCA-3’；
performing PCR amplification by using KOD PLUS high fidelity enzyme (TAKARA), transforming competent escherichia coli after connecting a PCR product with a pMD18-T vector (TAKARA), extracting plasmid DNA after respectively selecting 10 escherichia coli monoclonals for amplification culture, and screening to obtain F1 generation individuals with mutant actRIIB genes and alk4 genes after sequencing and comparison of the plasmid DNA;
(6) Obtaining of F2 generation homozygous mutant individuals: after the heterozygous mutation individuals of the F1 generation mature, selfing the individuals of the F1 generation to obtain an F2 generation, and screening out homozygous mutation individuals from the F2 according to the Mendel's law;
as a result: FIG. 2actRIIB Gene and alk4 Gene mutation type screening
Through the detection of the F1 generation individuals, an F1 generation individual is screened, wherein actRIIB and alk4 genes are mutated and are both frame shift mutations (the dotted line boxes mark parts).
The technical effects of the present invention will be described in detail below with reference to the accompanying drawings.
FIG. 3 shows that the growth speed of heterozygote and homozygote of common knockout actRIIB and alk4 genes zebra fish is remarkably increased, and the growth speed of dpf: days post-fertilization (Days post fertilization); actRIIB +/ -/alk4 +/- : actRIIB and alk4 gene co-mutation heterozygotes; actRIIB -/- /alk4 -/- : actRIIB and alk4 gene co-mutant homozygotes;
the weight records of control fish, heterozygote and homozygote 20, 40, 60, 80 and 100 days after fertilization are carried out, and the heterozygote and the homozygote are found to be remarkably increased relative to the control fish, and the growth speed of the homozygote is remarkably increased relative to that of the heterozygote.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Sequence listing
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<120> a method for increasing the growth rate of zebra fish by gene knockout technology
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<213> Artificial Sequence (Artificial Sequence)
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Claims (6)
1. A method for increasing the growth rate of zebra fish by using a gene knockout technology, which is characterized by comprising the following steps of:
firstly, searching mRNA sequences and genome sequences of an actRIIB gene and an alk4 gene of the zebra fish through an NCBI (national center of Biotechnology information) online database;
step two, actRIIB gene and alk4 gene gRNA are obtained;
thirdly, cas9mRNA is obtained through in vitro transcription;
fourthly, the micro co-injection of Cas9mRNA, actRIIB and alk4 gene gRNA;
fifthly, screening the F1 generation heterozygous mutant individuals and determining the mutation types;
sixthly, obtaining an F2 generation homozygous mutation individual, and selfing the F1 generation individual to obtain an F2 generation after the F1 generation heterozygous mutation individual is mature; screening homozygous mutant individuals from the F2 according to Mendel's law;
the first step is to search mRNA sequences and genome sequences of an actRIIB gene and an alk4 gene of the zebra fish through an NCBI online database;
actRIIB gene mRNA sequence NM-131210.3;
the actRIIB gene genome sequence is NC-007135.7;
designing a knockout site on a first exon of the gene through sequence comparison, wherein the sequence of the target site is SEQ ID NO:1;
the alk4 gene mRNA sequence is NM-130990.1;
alk4 gene genome sequence NC _007134.7
Designing a knockout site on a first exon of the gene through sequence comparison, wherein the sequence of the target site is SEQ ID NO:2.
2. the method for increasing the growth rate of zebrafish through gene knockout technology in claim 1, wherein the second step of obtaining actRIIB gene and alk4 gene gRNA: taking gRNA-plasmid as a template, and taking actRIIB-gRNA-F/gRNA-R and alk4-gRNA-F/gRNA-R as primers respectively:
performing PCR amplification by using KOD Plus high-fidelity enzyme, tapping and recovering an amplification product, connecting the recovered product with a pMD18-T vector, then transforming competent escherichia coli, selecting 5 escherichia coli for monoclonal amplification culture, and then extracting plasmid DNA; then, sequencing plasmid DNA for sequence verification, and extracting the plasmid DNA after cloning and amplifying culture with correct sequence to be used as a template for next step of transcribing gRNA; reuse of
T7Transcriptionkit carries out in-vitro reverse transcription on the constructed plasmid to obtain a 20ul reaction system;
the sequence of actRIIB-gRNA-F is SEQ ID NO:3;
the sequence of alk4-gRNA-F is SEQ ID NO:4;
the sequence of gRNA-R is SEQ ID NO:5.
3. the method for increasing the growth rate of zebrafish through gene knockout technology according to claim 2, wherein the reverse transcription 20ul reaction system is:
10x ReactionBuffer 2ul；
Plasmid DNA4ul，1ug；
T7 Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul。
4. the method for increasing the growth rate of zebrafish through gene knockout technology as claimed in claim 2, wherein the third step of in vitro transcription obtains Cas9mRNA: cas9 plasmid from Addgene TM Use of
T7/T3Transcriptionkit, cas9mRNA synthesis, system and reaction conditions:
10x ReactionBuffer 2ul；
Plasmid DNA4ul，1ug；
T7 Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul。
5. the method for increasing the growth rate of zebrafish through gene knockout technology of claim 2, wherein the fourth step of microinjection of Cas9mRNA with actRIIB and alk4 gene gRNA: the microinjection system was first configured as follows:
Cas9 mRNA300 ng/ul；
actRIIBgRNA 30ng/ul；
alk4 gRNA 30ng/ul；
Phenol-red 0.2ul；
DEPC Waterup to 2ul；
cas9mRNA, actRIIBgRNA, alk4 gRNA, phenol-red and DEPC Waterup are mixed and mixed evenly, zebra fish fertilized eggs at a unicellular stage are injected microscopically, and the mutation efficiency of actRIIB and alk4 genes is detected after 24 hours of culture.
6. The method for increasing the growth rate of zebrafish through gene knockout technology as claimed in claim 2, wherein the screening of heterozygous mutant individuals of the fifth F1 generation and the determination of the mutation type comprise:
(1) After culturing the zebra fish fertilized eggs after microinjection for 3 months until sexual maturity, hybridizing with wild individuals to obtain hybridized F1 generation, culturing F1 generation individuals for 2 months, shearing part of tail fin tissues, extracting genome DNA, and detecting mutation efficiency of actRIIB gene by respectively taking F-actRIIB/R-actRIIBB as primers;
(2) F-alk4/R-alk4 is used as a primer to detect the mutation efficiency of the alk4 gene;
(3) Performing PCR amplification by using KOD PLUS high-fidelity enzyme, transforming competent escherichia coli after connecting a PCR product with a pMD18-T vector, extracting plasmid DNA after respectively selecting 10 escherichia coli monoclonally amplified cultures, and screening to obtain F1 generation individuals with mutant actRIIB genes and alk4 genes after plasmid DNA sequencing comparison;
the F-actRIIB/R-actRIIBB sequence is SEQ ID NO:6 and SEQ ID NO:7;
the sequences of F-alk4/R-alk4 are respectively SEQ ID NO:8 and SEQ ID NO:9.
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| CN110004183A (en) * | 2019-04-08 | 2019-07-12 | 湖南师范大学 | A kind of large fragment stat1a/stat1b Gene Double mutation deletion form zebra fish |
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| CN103045623A (en) * | 2012-12-21 | 2013-04-17 | 中山大学 | Preparation method of tilapia activin receptor IIB recombinant protein and application thereof |
| CN110004183A (en) * | 2019-04-08 | 2019-07-12 | 湖南师范大学 | A kind of large fragment stat1a/stat1b Gene Double mutation deletion form zebra fish |
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