CN111560401A

CN111560401A - Molecular breeding method for thickening interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala

Info

Publication number: CN111560401A
Application number: CN202010451893.7A
Authority: CN
Inventors: 鲍宝龙; 车金远
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2020-08-21

Abstract

The invention discloses a molecular breeding method for thickening interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala, 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 Erythroculter ilishaeformis and megalobrama amblycephala are obtained, and mutant individuals with mstn deletion and increased inter-muscular ossification area are obtained through passage. In the invention, a parent method for obtaining erythroculter ilishaeformis and megalobrama amblycephala with increased inter-muscular ossification area by using a gene editing technology is provided for the first time. The method is beneficial to large-scale cultivation of wild erythroculter ilishaeformis and megalobrama amblycephala which have thick and heritable intertillary spurs in production, is different from a transgenic method, can be applied to artificial cultivation, overcomes the difficulty that the intertillary spurs are difficult to process in production, does not worry about the influence of transgenic food on people, is convenient for people to find the intertillary spurs more easily when eating the erythroculter ilishaeformis and the megalobrama amblycephala, and is easy to popularize in production.

Description

Molecular breeding method for thickening interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala

Technical Field

The invention belongs to the technical field of aquatic organism breeding, and particularly relates to a molecular breeding method for thickening interpuscular spurns of erythroculter ilishaeformis and megalobrama amblycephala.

Background

The culter ilishaeformis and the megalobrama amblycephala belong to four major Chinese carps, are main freshwater fish in China, have tender meat and delicious meat taste, are deeply loved by consumers, and have a leading position in freshwater fish culture in China. 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.

Carps all have a certain number of intersomatic spines, i.e., Intersomatic Bones (IB), located in the interval between the muscles on both sides of the spinal vertebrae. The muscle thorns are different from the vertebrae and are scattered in the muscle spaces, which causes great difficulty in further 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 a culter ilishaeformis crispus ' (patent number: CN201110206703.6) ' utilizes dissection to separate the interspinal bones of the culter ilishaeformis from the back meat, 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.

The culter ilishaeformis and megalobrama amblycephala in China have continuously increased culture scale and steadily and continuously increased yield, and are important freshwater culture improved varieties in China. However, a large amount of intermuscular spurs exist in the muscles of the erythroculter ilishaeformis and the megalobrama amblycephala, and influence food processing and eating of people to a certain extent. The shape of the intertuscular spurs is improved on the gene level, new species of erythroculter ilishaeformis and megalobrama amblycephala with increased intertuscular bony area are cultivated, and the intertuscular spurs are easier to remove when people eat or process the erythroculter ilishaeformis and the megalobrama amblycephala. The risk of injury caused by intertuscular spurs is reduced, so that people can eat erythroculter ilishaeformis and megalobrama amblycephala more conveniently, and the consumption of erythroculter ilishaeformis and megalobrama amblycephala in the market is expected to be increased.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for mutating the mstn genes of erythroculter ilishaeformis and megalobrama amblycephala by adopting a CRISPR/Cas9 gene editing method, so as to change the size of the intermuscular spurs and obtain the erythroculter ilishaeformis and the megalobrama amblycephala with large intermuscular bony area.

In order to achieve the purpose, the invention adopts the following technical scheme:

a molecular breeding method for thickening the interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala comprises the following steps:

(1) cloning nucleotide sequences of erythroculter ilishaeformis and megalobrama amblycephala mstn genes;

(2) constructing deletion mutant strains of erythroculter ilishaeformis and megalobrama amblycephala mstn genes by adopting a gene knockout method;

(3) screening individuals with large interspinal spines: selecting Erythroculter ilishaeformis and megalobrama amblycephala which are subjected to gene knockout and have hypertrophy than those of wild species of the same age, respectively hybridizing, collecting fertilized eggs, incubating and culturing to obtain homozygous mutants with stable inheritance, detecting the variation of the interspinal spines, and screening individuals with large interspinal spines.

On the basis of the scheme, the nucleotide sequence of the erythroculter ilishaeformis mstn gene cloned in the step 1) is shown as SEQ ID NO. 1, and the nucleotide sequence of the cloned megalobrama amblycephala mstn gene is shown as SEQ ID NO. 2.

SEQ ID NO 1 Erythroculter ilishaeformis mstn gene nucleotide sequence

Nucleotide sequence of megalobrama amblycephala mstn gene with SEQ ID NO. 2

On the basis of the scheme, the primer pair for cloning the nucleotide sequence of the erythroculter ilishaeformis mstn gene in the step 1) is as follows:

F:5’-CATGTGGTCCAGTGGGTAATG-3’(SEQ ID NO:3)；

R:5’-GTTGATGGGAGACATCTTGGTG-3’(SEQ ID NO:4)；

the primer pair for cloning the nucleotide sequence of the megalobrama amblycephala mstn gene in the step 1) is as follows:

F:5’-GCAAACATCCTCTAGCACGC-3’(SEQ ID NO:5)；

R:5’-CCACAGCGGTCTACTACCA-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 CRISPR/Cas9 gene knockout method is adopted to construct deletion mutant strains of erythroculter ilishaeformis and megalobrama amblycephala mstn genes, and the method specifically comprises the following steps:

designing target spots in exon regions of erythroculter ilishaeformis and megalobrama amblycephala mstn gene sequences, transcribing the designed target spots in vitro to synthesize sgRNA, and mixing the sgRNA with Cas9 mRNA in proportion to prepare a medicament for gene editing injection;

secondly, taking erythroculter ilishaeformis and megalobrama amblycephala with mature gonad development, injecting chorionic gonadotropin (HCG), carrying out artificial insemination after 1-3 days, starting to inject the gene editing injection medicament in the step I after the embryo absorbs water and expands, and accurately injecting the medicament into cells in a cell stage during injection, wherein the injection volume of each embryo is about 2 nL;

randomly extracting three groups of 12 h-injected Erythroculter ilishaeformis and megalobrama amblycephala embryos after injection, shearing the embryos by using scissors, extracting genome DNA, and detecting the knockout efficiency by using a fluorescence capillary electrophoresis method;

and fourthly, feeding the detected effective embryos for 1 month, shearing individual tail fins, extracting genome DNA, carrying out fluorescent STR detection, determining mutant genotypes through molecular cloning, leaving effective mutation F0 with the editing number of being not 3n, and feeding the effectively mutated Erythroculter ilishaeformis and megalobrama amblycephala 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 mstn gene knockout target of the erythroculter ilishaeformis designed in the step I is at least one of the following sequences:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:7)；

exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’(SEQ ID NO:8)；

exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:9)；

exon2-target1:5’-GGGCTAATGCCCGTTACGGA-3’(SEQ ID NO:10)；

exon2-target2:5’-GGACAACCGGAGACCAACTG-3’(SEQ ID NO:11)；

exon2-target3:5’-GGGTCTTCCTCCGTCCGTAA-3(SEQ ID NO:12)；

the sequence of the megalobrama amblycephala mstn gene knockout target point designed in the step I is at least one of the following sequences:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:13)；

exon1-target2:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:14)；

exon1-target3:5’-GGGATCAGTACGACGTTCTG-3’(SEQ ID NO:15)。

on the basis of the scheme, the primers used for detecting the erythroculter ilishaeformis in the third step are as follows:

T1:5’-CATGTGGTCCAGTGGGTAATG-3’(SEQ ID NO:16)；

T2:5’-CGTTTCCCTTCGCGTCATAC-3’(SEQ ID NO:17)；

the primers used for detecting the megalobrama amblycephala in the third step are as follows:

T1:5’-GCAAACATCCTCTAGCACGC-3’(SEQ ID NO:18)；

T2:5’-GATGGTCTCTGTGGTGGCATG-3’(SEQ ID NO:19)。

on the basis of the scheme, the injection dosage of the chorionic gonadotrophin HCG injection in the step II is as follows: 1600-2400U/kg female fish and 800-1200U/kg male fish.

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, distant hybridization and the like, the method can reduce the number of the muscle thorns, but the effect is not obvious. The invention directionally edits the genes of the erythroculter ilishaeformis and the megalobrama amblycephala mstn by a gene editing technology to obtain mutants of the erythroculter ilishaeformis and the megalobrama amblycephala with increased intermuscular ossification area, higher growth speed and stable inheritance, can solve the defect that the intermuscular spurs are difficult to find and then removed in production, can obtain a large amount of stably inherited mutants of the erythroculter ilishaeformis and the megalobrama amblycephala by 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) Cloning of nucleotide partial sequence of erythroculter ilishaeformis mstn gene

Downloading several fish mstn nucleotide sequences which are close to the genetic relationship between Erythroculter ilishaeformis and megalobrama amblycephala 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:

erythroculter ilishaeformis mstn:

F:5’-CATGTGGTCCAGTGGGTAATG-3’(SEQ ID NO:3)；

R:5’-GTTGATGGGAGACATCTTGGTG-3’(SEQ ID NO:4)；

and (3) performing mstn sequence amplification by using the erythroculter ilishaeformis cDNA as a template to obtain a coding region partial sequence of the erythroculter ilishaeformis mstn, wherein the coding region partial sequence is shown as SEQ ID NO. 1.

(2) Construction of deletion mutant strain of erythroculter ilishaeformis mstn gene

The method for obtaining the erythroculter ilishaeformis mstn gene deletion mutant strain by adopting a gene knockout method takes a method for constructing the deletion strain by crusprr/cas 9 as an example, and comprises the following specific steps:

(i) erythroculter ilishaeformis mstn gene knockout target selection

The erythroculter ilishaeformis mstn sequence has three exons: exon1, exon2, exon 3. For more efficient gene editing and base deletion of long fragments, 2 targets were designed on the first exon, with the sequences:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:7)；

exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’(SEQ ID NO:8)；

exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:9)；

in the second exon

exon2-target1:5’-GGGCTAATGCCCGTTACGGA-3’(SEQ ID NO:10)；

exon2-target2:5’-GGACAACCGGAGACCAACTG-3’(SEQ ID NO:11)；

exon2-target3:5’-GGGTCTTCCTCCGTCCGTAA-3(SEQ ID NO:12)；

(ii) Erythroculter ilishaeformis target gene editing

a. Editing is performed by adopting a crispr/cas9 system:

respectively transcribing the designed 6 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, final sgRNA concentration 50 ng/. mu.l.

b. The method comprises the steps of obtaining an erythroculter ilishaeformis with mature gonad, obtaining a female and male fish with a strong physique and no damage, the female-male ratio being 2:1, placing the fish in a canvas cylinder, injecting gonadotropin HCG, the injection amount being 1600 plus 2400U/kg of the female fish, 1200U/kg of the male fish, and the interval being 1-3 days, then carrying out artificial insemination, distributing eggs into a disposable plastic culture dish for convenient microinjection, immediately carrying out microinjection under a microscope after the fertilized eggs absorb water and expand, accurately injecting medicaments into cells in a cell period 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 (the water temperature is 24-27 ℃, and membranes are taken out after about 20 hours).

(iii) Screening of target gene mutant and obtaining of homozygous mutant

Randomly extracting three groups of injected Erythroculter ilishaeformis embryos and non-injected wild embryos respectively (12 h after injection), cutting the embryos with scissors, and extracting genome DNA by an alkaline lysis method.

The knockout efficiency is detected by fluorescence capillary electrophoresis. Feeding the detected effective embryo for 1 month, clipping individual tail fin, extracting genome DNA, carrying out fluorescent STR detection, determining the mutation genotype through molecular cloning, leaving effective mutation F0 with the editing number of not 3n, collecting at least 20 pieces of the Erythroculter ilishaeformis Bleeker with effective mutation, and feeding the Erythroculter ilishaeformis Bleeker to adults.

The detection primer is as follows:

T1:5’-CATGTGGTCCAGTGGGTAATG-3’(SEQ ID NO:16)；

T2:5’-CGTTTCCCTTCGCGTCATAC-3’(SEQ ID NO:17)；

(iv) screening of individuals with large intersomatic spur

Since the topmouth culter can cause muscle hypertrophy after lacking the mstn gene, the topmouth culter which is thicker than the same age wild species is selected for hybridization, fertilized eggs are collected, genome DNA of 10 embryos is randomly extracted after the fertilized eggs develop to the hatching period, STR verification is carried out, and the homozygous mutant F1 which can be stably inherited is obtained. Breeding the F1 generation to about 1cm long, randomly selecting 30 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 megalobrama amblycephala mstn gene

Downloading a megalobrama amblycephala mstn sequence through a gene database, designing a primer, and amplifying a partial CDS region of the mstn sequence, wherein the primer sequence is as follows:

megalobrama amblycephala mstn

F:5’-GCAAACATCCTCTAGCACGC-3’(SEQ ID NO:5)；

R:5’-CCACAGCGGTCTACTACCA-3’(SEQ ID NO:6)；

The amplification of the mstn sequence is carried out by taking the cDNA of the megalobrama amblycephala as a template to obtain the partial sequence of the coding region of the mstn of the megalobrama amblycephala, which is shown as SEQ ID NO. 2.

(2) Construction of deletion mutant strain of megalobrama amblycephala mstn gene

The gene knockout method is adopted to obtain the megalobrama amblycephala mstn gene deletion mutant strain, taking a method of constructing the deletion strain by criespr/cas 9 as an example, the specific steps are as follows:

(i) selection of Megalobrama amblycephala mstn target Gene

The sequence of megalobrama amblycephala mstn has three exons, and in order to carry out gene editing and base deletion of long fragments more efficiently, 3 target points are designed on the first exon, and the sequence is as follows:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:13)；

exon1-target2:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:14)；

exon1-target3:5’-GGGATCAGTACGACGTTCTG-3’(SEQ ID NO:15)。

(ii) megalobrama amblycephala target gene editing

a. Editing is performed by adopting a crispr/cas9 system:

respectively transcribing the designed 3 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, final sgRNA concentration 50 ng/. mu.l.

b. Obtaining megalobrama amblycephala with mature gonad development, namely female and male fishes which are 3-4 winter-old and have more than 1.5kg weight, strong physique and no damage, wherein the ratio of female to male is 2:1, placing the megalobrama amblycephala in a circular canvas cylinder of 5m multiplied by 1.2m, and injecting gonadotropin HCG, wherein the injection amount of female fishes is 1600-2400U/kg, and the injection amount of male fishes is 800-1200U/kg. The interval is 1-3 days, then artificial insemination is carried out, in order to facilitate microinjection, eggs are distributed into a disposable plastic culture dish, detackification is needed if necessary, microinjection is carried out under a microscope immediately after the fertilized eggs absorb water and swell, the medicine is accurately injected into cells in a cell stage during injection, and 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 is 24-27 ℃, membrane appears after about 20 h)

(iii) Screening of target gene mutant and obtaining of homozygous mutant

Three groups of injected megalobrama amblycephala embryos and non-injected wild embryos (after 12h of injection) are respectively randomly extracted, three embryos in each group are cut by scissors, and the genome DNA is extracted by an alkaline cracking method.

The knockout efficiency is detected by fluorescence capillary electrophoresis. Feeding the detected effective embryos for 1 month, shearing individual tail fins, extracting genome DNA, carrying out fluorescent STR detection, determining a mutation genotype through molecular cloning, leaving effective mutation F0 generations with the editing number being not 3n, collecting at least 20 effective mutation megalobrama amblycephala, and feeding the megalobrama amblycephala to adults.

The detection primer is as follows:

T1:5’-GCAAACATCCTCTAGCACGC-3’(SEQ ID NO:18)；

T2:5’-GATGGTCTCTGTGGTGGCATG-3’(SEQ ID NO:19)；

(iv) screening of individuals with large intersomatic spur

As megalobrama amblycephala can cause muscle hypertrophy after lacking the mstn gene, the megalobrama amblycephala with the hypertrophy than that of a wild species in 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 30 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 made to the above embodiments according to the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Sequence listing

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cacagccacg gaggaaagcg agcagtgttc cacatgtgag tttagacaac acagcaagct 180

gatgagactg catgccatca agtcccaaat tcttagcaaa ctccgactca aacaggctcc 240

aaacatcagc cgggacgtgg tcaaacagct gttacccaaa gcaccgcctt tgcaacaact 300

tctggatcag tacgacgttc tgggggatga cagtaaggat ggagctgtgg aagaggatga 360

tgaacatgcc accacagaga ccatcatgac catggccaca gagcccgacc ccatcgttca 420

agtagatcgg aaaccgaagt gttgtttttt ctccttcagt ccgaaaatcc aagcgaaccg 480

gatcgtaaga gcgcagctct gggttcatct gagaccggcg gaagaagcga ccaccgtctc 540

cttacagata tcacggctga tgcccgttac ggacggagga agacacatac gaatacgatc 600

cctgaagatc gatgtgaacg caggagtcac gtcttggcag agtatagacg taaagcaggt 660

gctctcggtg tggttaagac aaccggagac caactggggc atcgagataa acgcgtatga 720

cgcgaaggga aacgacttgg ccgtcacctc agctgaggct ggagaggatg gactgctccc 780

ctttatggag gtgaaaatct cagagggccc aaagcgaatc cggagggact ctggactgga 840

ctgcgacgag aattcctcag agtctcgatg ctgcagatac cctctcactg tggacttcga 900

ggacttcggc tgggactgga ttattgctcc gaaacgctat aaggcgaatt actgttcggg 960

agaatgcgac tacatgcacc tgcagaagta tccccacacc catctggtga acaaggccaa 1020

tccgcgaggc accgccgggc cctgctgcac ccccaccaag atgtctccca tcaacatgct 1080

ttacttcaat ggcaaagagc agatcatcta cggcaagatc ccctcaatgg tagtagaccg 1140

ctgtgg 1146

<210>3

<211>21

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>3

catgtggtcc agtgggtaat g 21

<210>4

<211>22

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>4

gttgatggga gacatcttgg tg 22

<210>5

<211>20

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>5

gcaaacatcc tctagcacgc 20

<210>6

<211>19

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>6

ccacagcggt ctactacca 19

<210>7

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>7

ggttctgggg gatgacagta 20

<210>8

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>8

gggatcagta cgatgttctg 20

<210>9

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>9

ggagccttcc acagccacgg 20

<210>10

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>10

gggctaatgc ccgttacgga 20

<210>11

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>11

ggacaaccgg agaccaactg 20

<210>12

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>12

gggtcttcct ccgtccgtaa 20

<210>13

<211>20

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>13

ggttctgggg gatgacagta 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>14

ggagccttcc acagccacgg 20

<210>15

<211>20

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>15

gggatcagta cgacgttctg 20

<210>16

<211>21

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>16

catgtggtcc agtgggtaat g 21

<210>17

<211>20

<212>DNA

<213> Artificial sequence (Erythroculter ilishaeformis)

<400>17

cgtttccctt cgcgtcatac 20

<210>18

<211>20

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>18

gcaaacatcc tctagcacgc 20

<210>19

<211>21

<212>DNA

<213> Artificial sequence (Megalobrama ambicephala)

<400>19

gatggtctct gtggtggcat g 21

Claims

1. A molecular breeding method for thickening the interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala is characterized by comprising the following steps:

2. The molecular breeding method for the intramuscular thickening of the culter ilishaeformis and the megalobrama amblycephala according to claim 1, wherein the nucleotide sequence of the mstn gene of the culter ilishaeformis cloned in the step 1) is shown in SEQ ID NO. 1, and the nucleotide sequence of the mstn gene of the megalobrama amblycephala cloned is shown in SEQ ID NO. 2.

3. The molecular breeding method for the intramuscular thickening of the culter ilishaeformis and the megalobrama amblycephala according to claim 1, wherein the primer pair for cloning the nucleotide sequence of the mstn gene of the culter ilishaeformis in the step 1) is as follows:

F:5’-CATGTGGTCCAGTGGGTAATG-3’；

R:5’-GTTGATGGGAGACATCTTGGTG-3’；

F:5’-GCAAACATCCTCTAGCACGC-3’；

R:5’-CCACAGCGGTCTACTACCA-3’。

4. the molecular breeding method for the thickening of the interpersonal spurt between the erythroculter ilishaeformis and the megalobrama amblycephala according to claim 1, wherein the gene knockout method in the step (2) is a TALEN method or a crispr/cas9 method.

5. The molecular breeding method for the intertuscular thickening of the erythroculter ilishaeformis and the megalobrama amblycephala according to claim 4, which is characterized in that a CRISPR/Cas9 gene knockout method is adopted to construct a deletion mutant strain of the erythroculter ilishaeformis and the megalobrama amblycephala mstn gene, and specifically comprises the following steps:

6. The molecular breeding method for the intramuscular thickening of the erythroculter ilishaeformis and the megalobrama amblycephala according to claim 5, wherein the drugs injected in the step (i) by gene editing contain Cas9 mRNA with the final concentration of 300ng/μ L and sgRNA with the final concentration of 50ng/μ L.

7. The molecular breeding method for the rough interpersonal stimulation between the erythroculter ilishaeformis and the megalobrama amblycephala according to claim 5, wherein the sequence of the mstn knockout target of the erythroculter ilishaeformis 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’；

exon2-target1:5’-GGGCTAATGCCCGTTACGGA-3’；

exon2-target2:5’-GGACAACCGGAGACCAACTG-3’；

exon2-target3:5’-GGGTCTTCCTCCGTCCGTAA-3；

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’；

exon1-target2:5’-GGAGCCTTCCACAGCCACGG-3’；

exon1-target3:5’-GGGATCAGTACGACGTTCTG-3’。

8. the molecular breeding method for the thickening of the interpersonal spurge between the erythroculter ilishaeformis and the megalobrama amblycephala according to claim 5, wherein the primers used for detecting the erythroculter ilishaeformis in the third step are as follows:

T1:5’-CATGTGGTCCAGTGGGTAATG-3’；

T2:5’-CGTTTCCCTTCGCGTCATAC-3’；

T1:5’-GCAAACATCCTCTAGCACGC-3’；

T2:5’-GATGGTCTCTGTGGTGGCATG-3’。

9. the molecular breeding method for the intramuscular thickening of the erythroculter ilishaeformis and the megalobrama amblycephala according to claim 5, which comprises the following steps: the injection dosage of the chorionic gonadotrophin HCG injection is as follows: 1600-2400U/kg female fish and 800-1200U/kg male fish.

10. The molecular breeding method for thickening between the muscles of an erythroculter ilishaeformis and a megalobrama amblycephala according to any one of claims 1 to 9, which comprises the following steps: 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.