CN111549031A - Molecular breeding method for thickening muscle of grass carp and black carp - Google Patents
Molecular breeding method for thickening muscle of grass carp and black carp Download PDFInfo
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Abstract
The invention discloses a molecular breeding method for thickening thorns between muscles of grass carps and black carps, and belongs to the technical field of aquatic organism breeding. The molecular breeding method is based on a technical means of gene editing, F0 generations of mstn genes of targeted mutation grass carp and black carp are obtained, and mutant individuals with mstn deletion and increased inter-musculus ossification area are obtained through passage. In the invention, a parent method for obtaining grass carp and black carp with increased inter-muscular ossification area by using a gene editing technology is proposed for the first time. The method is beneficial to large-scale cultivation of wild grass carp and black carp with thick and heritable interspinal spines in production, is different from a transgenic method, can be applied to artificial cultivation, overcomes the difficulty that the interspinal spines are small and difficult to process in production, does not worry about the influence of transgenic food on people, is convenient for people to find the interspinal spines more easily when eating the grass carp and the black carp, and is easy to popularize in production.
Description
Technical Field
The invention belongs to the technical field of aquatic organism breeding, and particularly relates to a molecular breeding method for thickening thorns between muscles of grass carps and black carps.
Background
Grass carp and black carp belong to four major Chinese carps, have the characteristics of short growth cycle, high survival rate and the like in the culture process, are the main freshwater cultured fishes in China, have tender meat quality, have delicious meat taste and occupy the leading position in the freshwater fish culture in China, are mass aquatic products which are popular in the market and are deeply loved by consumers. The gene editing is an important technical means for fish character breeding, and specific genes can be edited in a targeted mode through the gene editing technology so as to obtain a new variety with excellent characters, so that the method has important significance for breeding a variety with excellent culture performance in aquaculture. At present, many researchers at home and abroad develop molecular breeding work for researching body color, disease resistance, gonad development and the like of cultured fishes through a gene editing technology, and the molecular breeding work has a positive effect on cultivating excellent varieties.
Most freshwater fishes, especially cyprinid fishes, have a certain number of intermuscular spines, and the intermuscular spines (IB) are intermuscular bones which are located in the interval between two muscles of the vertebra and are membranous hard bones only existing in low-grade bonito. The muscle spines are different from the vertebrae and are scattered in the muscle spaces, which causes great difficulty in processing and eating of fish meat. At present, the invention of the national invention patent ' a method for separating the back meat and the spine meat of the grass carp with crisp meat ' (patent number: CN201110206703.6) ' separates the interspinal bones of the grass carp from the back meat by dissection, but a lot of fish meat is lost; the invention discloses a method for optimally breeding and breeding carassius auratus gibelio new strains (patent number: ZL201010140103.X), which utilizes Xingguo red carp sperms as heterogenous sperms to stimulate gynogenesis and reproduction of carassius auratus gibelio gynogenesis to generate full female offspring, and the muscle thorns of the obtained carassius auratus gibelio new strains are reduced; the invention discloses a construction method of a hybrid bream and culter with rapid growth and less intertillary spurs (patent number CN201710788633), which utilizes triangular bream as a female parent to perform distant hybridization with a culter alburnus male parent, and the obtained hybrid shows excellent properties of rapid growth, excellent shape, less intertillary spurs, simple shape and the like. However, the variety obtained by the traditional breeding method has a general effect on reducing the number of the muscle thorns. Myostatin, mstn (myostatin), is a class of glycoproteins that are widely expressed in skeletal muscle and are negative regulators of muscle growth. At present, no document reports that the mstn gene can influence the ossification area of fish intermuscular spurs at home and abroad.
Grass carp and black carp are common freshwater economic fishes in China, have fresh and tender meat quality and rich nutrition, and are always favored by consumers in China. The culture scale of grass carp and black carp in China is continuously increased, the annual total yield of grass carp is over 300 ten thousand tons, the annual yield of black carp is over 200 ten thousand tons, and the yield is steadily and continuously increased, so that the method is an important fresh water culture fine breed in China. However, the muscles of grass carp and black carp have a lot of muscle thorns, which affect food processing and eating of people to a certain extent. The shape of the silver carp and the black carp is improved from the gene level, and the area of the thorns between the grass carp and the black carp is increased, so that the thorns between the muscles can be removed more easily when people eat or process the grass carp and the black carp. The risk of injury caused by the muscle thorns is reduced, so that people can eat the grass carps and the black carps more conveniently, and the consumption of the grass carps and the black carps in the market is expected to be improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for mutating the mstn gene of grass carp and black carp by using a CRISPR/Cas9 gene editing method, so as to change the size of the interspinal thorn and obtain the grass carp and the black carp with large interspinal thorn ossification area.
In order to achieve the purpose, the invention adopts the following technical scheme:
a molecular breeding method for thickening the muscle of grass carp and black carp comprises the following steps:
(1) cloning the nucleotide sequence of mstn gene of grass carp and black carp;
(2) constructing deletion mutant strains of mstn genes of grass carps and black carps by adopting a gene knockout method;
(3) screening individuals with large interspinal spines: selecting grass carp and black carp which are subjected to gene knockout and have hypertrophy than wild species of the same age, respectively hybridizing, collecting fertilized eggs, incubating and cultivating to obtain homozygous mutant with stable inheritance, detecting the change of the interspinal spines and screening individuals with large interspinal spines.
On the basis of the scheme, the nucleotide sequence of the grass carp mstn gene cloned in the step 1) is shown as SEQ ID NO. 1, and the nucleotide sequence of the cloned black carp mstn gene is shown as SEQ ID NO. 2.
SEQ ID NO 1 grass carp mstn gene nucleotide sequence
2 herring mstn gene nucleotide sequence of SEQ ID NO
On the basis of the scheme, the primer pair for cloning the grass carp mstn gene nucleotide sequence in the step 1) is as follows:
F:5’-GTGTATTAATTGCATGTGGTCC-3’(SEQ ID NO:3)；
R:5’-GCTGTTACAGCAAAGATATAAATG-3’(SEQ ID NO:4)；
the primer pair for cloning the nucleotide sequence of the herring mstn gene in the step 1) is as follows:
F:5’-TGCATGTGGTCCAGTGGGTTATG-3’(SEQ ID NO:5)；
R:5’-CTCTTTGCCGTTGAAGTAAAG-3’(SEQ ID NO:6)；
on the basis of the scheme, the gene knockout method in the step (2) is a TALEN method or a crispr/cas9 method.
On the basis of the scheme, a method for constructing a deletion mutant strain of mstn genes of grass carps and black carps by using a CRISPR/Cas9 gene knockout method specifically comprises the following steps:
designing target spots in exon regions of mstn gene sequences of grass carps and black carps, transcribing the designed target spots in vitro into sgRNA, mixing the sgRNA with Cas9 mRNA in proportion, and preparing a medicament for gene editing injection;
secondly, taking grass carp and black carp with mature gonad development, injecting luteinizing hormone-releasing hormone (LRH) -A, carrying out artificial insemination after 1-3 days, starting to inject the gene editing injection medicine in the first step after the embryo absorbs water and swells, and accurately injecting the medicine into cells in a cell period during injection, wherein the injection volume of each embryo is about 2 nL;
randomly extracting three groups of embryos of the grass carps and the black carps which are injected for 12 hours after the injection, cutting the embryos by scissors, extracting genome DNA, and detecting the knockout efficiency by a fluorescence capillary electrophoresis method, wherein each group comprises three embryos;
and fourthly, feeding the detected effective embryos for 1 month, shearing individual tail fins, extracting genome DNA, carrying out fluorescent STR detection, determining a mutant genotype through molecular cloning, reserving the effective mutant F0 with the editing number being not 3n, and feeding the effectively mutated grass carp and black carp to adults.
On the basis of the scheme, the medicine injected by gene editing in the step (i) contains Cas9 mRNA with the final concentration of 300 ng/. mu.L and sgRNA with the final concentration of 50 ng/. mu.L.
On the basis of the scheme, the sequence of the grass carp mstn gene knockout target point designed in the step I is at least one of the following sequences:
4 targets designed on the first exon, the sequences are:
exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:7)；
exon1-target2:5’-GGTCATGATGGTCTCTGTGG-3’(SEQ ID NO:8)；
exon1-target3:5’-GGGTTGTCTGAACTCACATG-3’(SEQ ID NO:9)；
exon1-target4:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:10)；
3 targets designed on the second exon, the sequences are:
exon2-target1:5’-GGGTGGTGTGATCAGATGTC-3’(SEQ ID NO:11)；
exon2-target2:5’-GGTGGTGTGATCAGATGTCC-3’(SEQ ID NO:12)；
exon2-target3:5’-GGGACGCTCACTCACTCTCT-3’(SEQ ID NO:13)；
the mstn gene knockout target sequence of the black carp designed in the step I is at least one of the following sequences:
4 targets designed on the first exon, the sequences are:
exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:14)；
exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’(SEQ ID NO:15)；
exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:16)；
exon1-target4:5’-GGTCATGATGGTCTCTGTGG-3’(SEQ ID NO:17)。
on the basis of the scheme, the primers used for detecting the Chinese herbal fish in the third step are as follows:
T1:5’-GTGTATTAATTGCATGTGGTCC-3’(SEQ ID NO:18)；
T2:5’-GCTGTTACAGCAAAGATATAAATG-3’(SEQ ID NO:19)；
the primer used for detecting the black carp in the third step is as follows:
T1:5’-TGCATGTGGTCCAGTGGGTTATG-3’(SEQ ID NO:20)；
T2:5’-GCTTCCATGTTCAGCGTGC-3’(SEQ ID NO:21)。
on the basis of the scheme, the injection dosage of the luteinizing hormone releasing hormone analogue LRH-A in the step (II) is as follows: 10 ug/kg.
On the basis of the scheme, the method for detecting the change of the interspinal stings in the step (3) comprises fish hard bone staining and in-vitro muscle stab separation, wherein the hard bone staining adopts an alizarin red staining method.
The invention has the beneficial effects that:
compared with the traditional breeding methods such as gynogenesis and the like, the method reduces the number of the interspinal spines, directionally edits the mstn genes of the grass carps and the black carps by using a gene editing technology to obtain the mutant grass carps and the black carps with increased interspinal spines ossification area, higher growth speed and stable inheritance, can overcome the defect that the interspinal spines are small and difficult to find in production so as to remove, can obtain a large number of stably inherited mutant grass carps and black carps by using a gene knockout method, and is more popular with the public.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
(1) Grass carp mstn gene nucleotide partial sequence clone
Downloading several fish mstn nucleotide sequences which are close to the genetic relationship with grass carps in a gene database NCBI, carrying out homologous comparison, selecting a relatively conserved region to design a PCR amplification primer, wherein the primer sequence is as follows:
grass carp mstn:
F:5’-GTGTATTAATTGCATGTGGTCC-3’(SEQ ID NO:3)；
R:5’-GCTGTTACAGCAAAGATATAAATG-3’(SEQ ID NO:4)；
the mstn sequence is amplified by taking grass carp cDNA as a template to obtain a partial sequence of a coding region of grass carp mstn, which is shown as SEQ ID NO: 1.
(2) Construction of deletion mutant strain of grass carp mstn gene
A gene knockout method is adopted to obtain a grass carp mstn gene deletion mutant strain, taking a method for constructing a deletion strain by criprpr/cas 9 as an example, the specific steps are as follows:
(i) grass carp mstn gene knockout target selection
The grass carp mstn sequence has three exons: exon1, exon2, exon 3. For more efficient gene editing and base deletion of long fragments, 4 targets were designed on the first exon, with the sequences:
exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:7)；
exon1-target2:5’-GGTCATGATGGTCTCTGTGG-3’(SEQ ID NO:8)；
exon1-target3:5’-GGGTTGTCTGAACTCACATG-3’(SEQ ID NO:9)；
exon1-target4:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:10)；
designing 3 targets on the second exon, wherein the sequences are as follows:
exon2-target1:5’-GGGTGGTGTGATCAGATGTC-3’(SEQ ID NO:11)；
exon2-target2:5’-GGTGGTGTGATCAGATGTCC-3’(SEQ ID NO:12)；
exon2-target3:5’-GGGACGCTCACTCACTCTCT-3’(SEQ ID NO:13)；
(ii) grass carp target gene editing
a. Editing is performed by adopting a crispr/cas9 system:
respectively transcribing the designed 7 target spots in vitro into sgRNAs, respectively mixing the sgRNAs and Cas9 mRNA into a tube according to a proportion, and preparing the sgRNAs into a medicament for gene editing injection, wherein the medicament comprises: cas9 mRNA final concentration 300 ng/. mu.L, sgRNA final concentration 50 ng/. mu.L.
b. Obtaining grass carp with mature gonad, placing the grass carp in a canvas cylinder, injecting LRH-A twice at an interval of 1:1 in a male-female ratio of 10ug/kg for each time, carrying out artificial insemination, distributing ovum into a disposable plastic culture dish for convenient microinjection, immediately carrying out microinjection under a microscope after the fertilized ovum absorbs water and expands, and accurately injecting the medicine into cells at a cell stage during injection, wherein the injection volume of each embryo is about 2 nL. And (3) timely feeding the injected and non-injected fertilized eggs into an incubator, and incubating at room temperature (water temperature is 25 ℃, and membranes are taken out after about 24 hours).
(iii) Screening of target gene mutant and obtaining of homozygous mutant
Three groups of injected grass carp embryos and non-injected wild embryos (after 12h of injection) are randomly extracted respectively, three embryos in each group are cut by scissors, and the genome DNA is extracted by an alkaline lysis method.
The knockout efficiency is detected by fluorescence capillary electrophoresis. Feeding the detected effective embryos for 1 month, clipping individual tail fins, extracting genome DNA, carrying out fluorescent STR detection, determining a mutant genotype through molecular cloning, leaving effective mutant F0 generations with the editing number of not 3n, collecting at least 20 grass carps with effective mutation, and feeding the grass carps to adults.
The detection primer is as follows:
F:5’-GTGTATTAATTGCATGTGGTCC-3’(SEQ ID NO:18)；
R:5’-GCTGTTACAGCAAAGATATAAATG-3’(SEQ ID NO:19)；
(iv) screening of individuals with large intersomatic spur
Because the grass carp loses the mstn gene and then can cause muscle hypertrophy, the grass carp with the body type hypertrophy compared with that of a wild species with the same age is selected for hybridization, fertilized eggs are collected, genome DNA of 10 embryos is randomly extracted after the grass carp develops to the hatching period, STR verification is carried out, and the homozygous mutant F1 capable of being inherited stably is obtained. Breeding the F1 generation to about 1cm long, randomly selecting 100 individuals, respectively shearing partial tails, extracting genomic DNA, carrying out STR detection and molecular cloning verification until obtaining a homozygous mutant F1 with effective mutation and stable inheritance, breeding to adults, verifying the variation of the interspinal spine, and selecting the individuals with large interspinal spine by adopting living CT scanning, wherein the individuals can be used for stable passage.
The method for detecting the change of the interspinal stings comprises the steps of dyeing the hard bones of the fishes and separating the interspinal stings in vitro, wherein the dyeing of the hard bones adopts an alizarin red dyeing method.
Example 2
(1) Cloning of nucleotide sequence of herring mstn gene
Downloading a herring mstn sequence through a gene database, designing a primer, and amplifying a coding region partial sequence of mstn, wherein the primer sequence is as follows:
black carp mstn
F:5’-TGCATGTGGTCCAGTGGGTTATG-3’(SEQ ID NO:5)；
R:5’-CTCTTTGCCGTTGAAGTAAAG-3’(SEQ ID NO:6)；
And (2) performing mstn sequence amplification by using black carp cDNA as a template to obtain a partial sequence of a coding region of black carp mstn, wherein the partial sequence is shown as SEQ ID NO. 2.
(2) Construction of deletion mutant strain of herring mstn gene
A gene knockout method is adopted to obtain a herring mstn gene deletion mutant strain, taking a method for constructing a deletion strain by criprpr/cas 9 as an example, the specific steps are as follows:
(i) selection of Mylopharyngodon Piceus mstn target Gene
The herring mstn sequence has three exons: exon1, exon2, exon 3. For more efficient gene editing and base deletion of long fragments, 4 targets were designed on the first exon, with the sequences:
exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:14)；
exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’(SEQ ID NO:15)；
exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:16)；
exon1-target4:5’-GGTCATGATGGTCTCTGTGG-3’(SEQ ID NO:17)；
(ii) herring target gene editing
a. Editing is performed by adopting a crispr/cas9 system:
respectively transcribing the designed 4 target spots in vitro into sgRNAs, respectively mixing the sgRNAs with Cas9 mRNA in a certain proportion together to form a tube, and preparing the drug for gene editing injection, wherein the drug comprises: cas9 mRNA final concentration 300 ng/. mu.l, sgRNA final concentration 50 ng/. mu.l.
b. Obtaining black carps with mature gonads, placing the black carps in a canvas cylinder, injecting LRH-A twice at an interval of 1:1 in a male-female ratio of 10ug/kg for each time, carrying out artificial insemination, distributing eggs into a disposable plastic culture dish for convenient microinjection, if necessary, debonding, immediately carrying out microinjection under a microscope after the fertilized eggs absorb water and swell, and accurately injecting medicaments into cells in a cell stage during injection, wherein the injection volume of each embryo is about 2 nL. Timely feeding the injected and un-injected fertilized eggs into an incubator, and incubating at room temperature (water temperature 25 ℃, membrane emergence for about 24 h)
(iii) Screening of target gene mutant and obtaining of homozygous mutant
Three groups of injected black carp embryos and non-injected wild embryos (after 12h of injection) are randomly extracted respectively, three embryos in each group are cut by scissors, and the genome DNA is extracted by an alkaline lysis method.
The knockout efficiency is detected by fluorescence capillary electrophoresis. Feeding the detected effective embryos for 1 month, cutting individual tail fins, extracting genome DNA, carrying out fluorescent STR detection, determining a mutant genotype through molecular cloning, leaving effective mutant F0 generations with the editing number of not 3n, collecting at least 20 fishes of the effectively mutated herrings, and feeding the fishes to adults.
The detection primer is as follows:
T1:5’-TGCATGTGGTCCAGTGGGTTATG-3’(SEQ ID NO:20)；
T2:5’-GCTTCCATGTTCAGCGTGC-3’(SEQ ID NO:21)；
(iv) screening of individuals with large intersomatic spur
Because the black carp loses the mstn gene and then can cause muscle hypertrophy, the black carp with the hypertrophy compared with the wild herring with the same age is selected for hybridization, fertilized eggs are collected, after the fertilized eggs are developed to the hatching period, genome DNA of 10 embryos is randomly extracted, STR verification is carried out, and the homozygous mutant F1 capable of being inherited stably is obtained. Breeding the F1 generation to about 1cm long, randomly selecting 100 individuals, respectively shearing partial tails, extracting genomic DNA, carrying out STR detection and molecular cloning verification until obtaining a homozygous mutant F1 with effective mutation and stable inheritance, breeding to adults, verifying the variation of the interspinal spine, and selecting the individuals with large interspinal spine by adopting living CT scanning, wherein the individuals can be used for stable passage.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Sequence listing
<110> Shanghai ocean university
<120> molecular breeding method for thickening thorns between muscles of grass carp and black carp
<160>21
<170>SIPOSequenceListing 1.0
<210>1
<211>1487
<212>DNA
<213> grass carp (Ctenophaggodon idellus)
<400>1
gtgtattaat tgcatgtggt ccagtgggta atggagatat aacggcgcac cagcagcctt 60
ccacagccac ggaggaaagc gagcagtgtt ccacatgtga gttcagacaa cacagcaagc 120
tgatgagact gcatgccatc aagtcccaaa ttcttagcaa actccgactc aaacaggctc 180
caaacatcag ccgggacgtg gtcaagcagc tgttacccaa agcaccgcct ttgcaacaac 240
ttctggatca gtacgatgtt ctgggggatg acagtaagga tggagctttg gaagaggatg 300
atgaacatgc caccacagag accatcatga ccatggccac agagcctgac cccatcgttc 360
aagtagatcg gaaaccgaag tgttgctttt tctccttcag tccgaaaatc caagcgaacc 420
ggatcgtaag agcgcagctc tgggttcatc tgagaccggc ggaagaagcg accaccgtct 480
tcttacagat atcacggctg atgcccgtta cggacggagg aagacacata cgaatacgat 540
ccctgaagat cgatgtgaac gcaggagtca cgtcttggca gagtatagac gtaaagcagg 600
tgctctcggt gtggttaaga caaccggaga ccaactgggg catcgagata aacgcgtatg 660
acgcgaaggg aaacgacttg gccatcacct cagctgaggc tggagaggat ggactgctcc 720
cctttatgga ggtgaaaatc tcagagggcc caaagcgaat ccggagggac tctggactgg 780
actgcgacga gaattcctcg gagtctcgat gctgcagata ccctctcact gtggacttcg 840
aggacttcgg ctgggactgg attattgctc cgaaacgcta taaggcgaat tactgttcgg 900
gagaatgcga ctacatgcac ctgcagaagt atccccacac ccatctggtg aacaaggcca 960
atccgcgagg caccgccggg ccctgctgca cccccaccaa gatgtctccc atcaacatgc 1020
tttacttcaa cggcaaagaa cagatcatct acggcaagat cccctcaatg gtagtagacc 1080
gctgtggctg ctcgtgaacc agtgcccaga caggacttga tccgtctcaa agacccggac 1140
atctgatcac accacccacc atccattatc agtgctttcc gcaagacact gtgcaataga 1200
aggacgctca ctcactctct gggcactgct tcatttgact atgttttttg tcattttcct 1260
ctaaatcagtatctctgcca caggagtcca atgtcacaag gatatactaa aggaatgtct 1320
actggctgga cttgggaatg gacactattg aaatggacga cattctctgc tttatttcat 1380
gttttcacct tgtcagaata ctctcattag gatacgcaga caacatacaa aaatcgtatt 1440
atgcaaccac tccaaaatac aatcatttat atctttgctg taacagc 1487
<210>2
<211>2658
<212>DNA
<213> Black carp (Mylopharyngodon piceus)
<400>2
tgcatgtggt ccagtgggta atggagatat aacggcgcac cagcagcctt ccacagccac 60
ggaggaaagc gagcagtgtt ccacatgtga gtttagacaa cacagcaagc tgatgagact 120
gcatgccatc aagtcccaaa ttcttagcaa actccgactc aaacaggctc gaaacatcag 180
ccgggacgtg gtcaagcagc tgttacccaa agcaccgcct ttgcaacaac ttctggatca 240
gtacgatgtt ctgggggatg acagtaagga tggagctatg gaagaggatg atgaacatgc 300
caccacagag accatcatga ccatggccac agagcgtaag gattgtctat ttcaaagatt 360
cattcatgta aatgtatcct cttaacaatg gacaaagaat atgtctactt ggagagaccc 420
tttacgcagt ctttgtgccc aagccagttg cacccacaag gcagctactg ggcataaaat 480
gcacagttaa gatgtataaa gacgattcct gtttttttga ggctttgtgc ccctcttttt 540
tttctcagcc ttgctaaagc aattttacgc acgctgaaca tggaagcgcg gcaaaagcgc 600
accagcgtcg gtcggaagaa aatgttttca cctttaccac acgtgatagg ctataaactg 660
tagtttaaat tggttcatat cttagccaat cttcaggtta aaatgtgtag cctattatgt 720
gtaatgcctt ttatatgtag tgtctaaata tagatattat atctgtagcc gatccattcg 780
tttttaaagg cttctgcaat gccgtaaagt atatgacttg catgtctttt tcacttttaa 840
aataactacg tgcgtcattt catggacctc tttaataggc taataaaact gtcactgaaa 900
accagtagat cccgtataat cactttgatt tactttctct tctttgtttt tcagctgacc 960
ccatcgttca agtagatcgg aaaccgaagt gttgtttttt ctccttcagt ccgaaaatcc 1020
aagcgaaccg aatcgtaaaa gcgcagctct gggttcatct gagaccggcg gaagaagcga 1080
ccaccgtctt cttacagata tcacggctga tgcccgttac ggacggagga agacacatac 1140
gaatacgatc cctgaagatc gatgtgaacg caggtgtcac gtcttggcag agtatagacg 1200
taaagcaggt gctctcggtg tggttaaaac aaccggagac caactggggc atcgagataa 1260
acgcgtatga cgcgaaggga aacgacttgg ccatcacctc agctgaggct ggagaggatg 1320
gactggtgag ttgagctgtt gtgttaccaa atgtgcgttt tttacacaat acaaccgctt 1380
ttagacagag ctctgccagc agaaatcgac aatatcaaga aatgatatgg tctagttata 1440
caaaattctg tctcccaaaa atttgtgcac attttcatta attgcatgtt tcggagcgcg 1500
caacacaaca gagtttagag ctcgaaacat tcgggacccg atacctgaac gaatgattct 1560
ttcaatccgg ttcttttgag tgaatcaaaa gcgaacagcg cgaccactgt aattcggttt 1620
tagaactaac gattcttttg acctagttct tgtaatgaat caacagcatt caccacctta 1680
gtagtacagt ccgattcccg aaccagtgac tcttattggc cggttctttt tcgtgaaaaa 1740
cacttatgaa tcaatcgaaa cgcttacaga attaaactga ctcaaagaat cgcaagttac 1800
tttcgccata tctgactcga aacaaactga aaaaaattat gttaaggctc ctgaaactta 1860
aatcagagta gtagttagtg gttattcaca tgacaacttg tcttgtaatc agtggtacat 1920
tataaaaata atgtcttagt atttttaaca taaatataat gcaaagtgtg agaatagtca 1980
ttaaaaaagc agaaattgta ttgcctgtat ttctgtaggc ctatattggt tctgctgaaa 2040
tctgaaatga tctgatgatt tgctgggggc agtaaatgca atatgaattg aatgaatttc 2100
ttttttttcc tgttttacta gaagaatcct taatggcgcc attaaggaat gggaattgtt 2160
aagtggaatc gtaaccagaa tcattaaatt cacatctgtt ttggctagct gtagcattcg 2220
ttaatggttg gacaaagcaa ttttgaggtc acatgagagt tgttgatagc ccatagatat 2280
gaaagtacca agagtaagct gaagcactgg ttctttgggt tctttctctc acagctcccc 2340
tttatggagg tgaaaatctc agagggccca aagcgaatcc ggagggactc tggactggac 2400
tgcgatgaga attcctcaga gtctcgatgc tgcagatacc ctctcactgt ggacttcgag 2460
gacttcggct gggactggat tattgctccg aaacgctata aggcgaatta ctgttcggga 2520
gaatgcgact acatgcacct gcagaagtat ccccacaccc atctggtgaa caaggccaat 2580
ccgcgaggca ccgccgggcc ctgctgcacc cccaccaaga tgtctcccat caacatgctt 2640
tacttcaacg gcaaagag 2658
<210>3
<211>22
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>3
gtgtattaat tgcatgtggt cc 22
<210>4
<211>24
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>4
gctgttacag caaagatata aatg 24
<210>5
<211>23
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>5
tgcatgtggt ccagtgggtt atg 23
<210>6
<211>21
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>6
ctctttgccg ttgaagtaaa g 21
<210>7
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>7
ggttctgggg gatgacagta 20
<210>8
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>8
ggtcatgatg gtctctgtgg 20
<210>9
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>9
gggttgtctg aactcacatg 20
<210>10
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>10
ggagccttcc acagccacgg 20
<210>11
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>11
gggtggtgtg atcagatgtc 20
<210>12
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>12
ggtggtgtga tcagatgtcc 20
<210>13
<211>20
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>13
gggacgctca ctcactctct 20
<210>14
<211>20
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>14
ggttctgggg gatgacagta 20
<210>15
<211>20
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>15
gggatcagta cgatgttctg 20
<210>16
<211>20
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>16
ggagccttcc acagccacgg 20
<210>17
<211>20
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>17
ggtcatgatg gtctctgtgg 20
<210>18
<211>22
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>18
gtgtattaat tgcatgtggt cc 22
<210>19
<211>24
<212>DNA
<213> Artificial sequence (Ctenophagogon idellus)
<400>19
gctgttacag caaagatata aatg 24
<210>20
<211>23
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>20
tgcatgtggt ccagtgggtt atg 23
<210>21
<211>19
<212>DNA
<213> Artificial sequence (Mylopharyngodon piceus)
<400>21
gcttccatgt tcagcgtgc 19
Claims (10)
1. A molecular breeding method for thickening the muscle of grass carp and black carp is characterized by comprising the following steps:
(1) cloning the nucleotide sequence of mstn gene of grass carp and black carp;
(2) constructing deletion mutant strains of mstn genes of grass carps and black carps by adopting a gene knockout method;
(3) screening individuals with large interspinal spines: selecting grass carp and black carp which are subjected to gene knockout and have hypertrophy than wild species of the same age, respectively hybridizing, collecting fertilized eggs, incubating and cultivating to obtain homozygous mutant with stable inheritance, detecting the change of the interspinal spines and screening individuals with large interspinal spines.
2. The molecular breeding method for the rough penetration between the muscles of the grass carps and the black carps as claimed in claim 1, wherein the nucleotide sequence of the grass carp mstn gene cloned in the step 1) is shown as SEQ ID NO. 1, and the nucleotide sequence of the cloned black carp mstn gene is shown as SEQ ID NO. 2.
3. The molecular breeding method for the rough interpupillary needling between grass carp and black carp according to claim 1, wherein the primer pair for cloning the nucleotide sequence of the mstn gene of grass carp in the step 1) is as follows:
F:5’-GTGTATTAATTGCATGTGGTCC-3’；
R:5’-GCTGTTACAGCAAAGATATAAATG-3’；
the primer pair for cloning the nucleotide sequence of the herring mstn gene in the step 1) is as follows:
F:5’-TGCATGTGGTCCAGTGGGTTATG-3’；
R:5’-CTCTTTGCCGTTGAAGTAAAG-3’。
4. the method for molecular breeding with rough insertions between muscles of grass carp and black carp according to claim 1, wherein the gene knockout method in step (2) is TALEN method or crispr/cas9 method.
5. The molecular breeding method for the thickening of the interpupillary bones between grass carp and black carp muscles according to claim 4, which is characterized in that a method for constructing a deletion mutant strain of mstn genes of grass carp and black carp by using a CRISPR/Cas9 gene knockout method comprises the following steps:
designing target spots in exon regions of mstn gene sequences of grass carps and black carps, transcribing the designed target spots in vitro into sgRNA, mixing the sgRNA with Cas9 mRNA in proportion, and preparing a medicament for gene editing injection;
secondly, taking grass carp and black carp with mature gonad development, injecting luteinizing hormone-releasing hormone (LRH) -A, carrying out artificial insemination after 1-3 days, starting to inject the gene editing injection medicine in the first step after the embryo absorbs water and swells, and accurately injecting the medicine into cells in a cell period during injection, wherein the injection volume of each embryo is about 2 nL;
randomly extracting three groups of embryos of the grass carps and the black carps which are injected for 12 hours after the injection, cutting the embryos by scissors, extracting genome DNA, and detecting the knockout efficiency by a fluorescence capillary electrophoresis method, wherein each group comprises three embryos;
and fourthly, feeding the detected effective embryos for 1 month, shearing individual tail fins, extracting genome DNA, carrying out fluorescent STR detection, determining a mutant genotype through molecular cloning, reserving the effective mutant F0 with the editing number being not 3n, and feeding the effectively mutated grass carp and black carp to adults.
6. The molecular breeding method for the thickening between the muscles of the grass carp and the black carp, according to claim 5, characterized in that in the step (r), the medicine injected by gene editing contains Cas9 mRNA with a final concentration of 300ng/μ L and sgRNA with a final concentration of 50ng/μ L.
7. The molecular breeding method for the rough stabbing between the muscles of the grass carps and the black carps as claimed in claim 5, wherein the mstn gene knockout target sequence of the grass carps designed in the step (r) is at least one of the following sequences:
exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’；
exon1-target2:5’-GGTCATGATGGTCTCTGTGG-3’；
exon1-target3:5’-GGGTTGTCTGAACTCACATG-3’；
exon1-target4:5’-GGAGCCTTCCACAGCCACGG-3’；
exon2-target1:5’-GGGTGGTGTGATCAGATGTC-3’；
exon2-target2:5’-GGTGGTGTGATCAGATGTCC-3’；
exon2-target3:5’-GGGACGCTCACTCACTCTCT-3’；
the mstn gene knockout target sequence of the black carp designed in the step I is at least one of the following sequences:
exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’；
exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’；
exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’；
exon1-target4:5’-GGTCATGATGGTCTCTGTGG-3’。
8. the molecular breeding method for the rough interpupillary pricking between the muscles of the grass carp and the black carp as claimed in claim 5, wherein the primers used for detecting the grass carp in the third step are as follows:
T1:5’-GTGTATTAATTGCATGTGGTCC-3’；
T2:5’-GCTGTTACAGCAAAGATATAAATG-3’；
the primer used for detecting the black carp in the third step is as follows:
T1:5’-TGCATGTGGTCCAGTGGGTTATG-3’；
T2:5’-GCTTCCATGTTCAGCGTGC-3’。
9. the molecular breeding method for the thickening of the muscle between the grass carp and the black carp, according to claim 5, wherein: the injection dosage of the luteinizing hormone releasing hormone analog LRH-A is as follows: 10 ug/kg.
10. The molecular breeding method for the thickening of the interpupillary bones between the muscles of grass carp and black carp according to any one of claims 1 to 9, which comprises: the method for detecting the change of the interspinal stings in the step (3) comprises the steps of dyeing the hard bones of the fishes and separating the interspinal stings in vitro, wherein the dyeing of the hard bones adopts an alizarin red dyeing method.
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| WO2022227692A1 (en) | 2021-04-30 | 2022-11-03 | 中国水产科学研究院黑龙江水产研究所 | Intermuscular bone-free crucian strain and cultivation method therefor |
| CN117265017A (en) * | 2023-09-16 | 2023-12-22 | 华中农业大学 | Method for creating myotonin-free grass carp based on runx2b gene editing and application |
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