CN115720874A - Creating method and application of inonotus spiny germplasm for cultured economic fishes - Google Patents

Abstract

The invention belongs to the field of aquatic organism breeding, and particularly relates to a method for creating a non-muscle-stabbing germplasm for cultivating economic fishes and application thereofrunx2bThe gene is used as a target gene to carry out gene editing on the economic fish megalobrama amblycephala, F0 generation small fish with mutation is selected to be cultured into parent fish, F1 generation is generated by mating and breeding female and male parents of F0 generation mutant, and the megalobrama amblycephala F1 without interspinal stings is screened out and cultured to adult fish for culturing the new germplasm of the megalobrama amblycephala without interspinal stings. The novel germplasm of the Megalobrama amblycephala for the non-intermuscular thorn breeding economic fish is obtained by using a gene mutation method, an improvement method is provided for solving the problem that the eating and processing of important breeding economic fish such as the grass, the silver carp, the bighead carp, the Megalobrama amblycephala and the like in China are obviously influenced by the intermuscular thorn existence in muscles, a basis is provided for the subsequent research of a molecular forming mechanism of the intermuscular thorn, and the novel germplasm has important scientific research value and industrial application valueThe value is obtained.

Description

Non-muscle interspinous seeding creation method for cultured economic fishes and application

Technical Field

The invention belongs to the field of aquatic organism breeding, and particularly relates to a creation method of an economic fish culture stinging-free germplasm and application.

Background

Fish internus spina (intermuscular bone) is a small and hard bony spur derived from ossification of the muscle tendon between the diaphragms (Patterson and Johnson,1995 nie et al, 2021. The main cultured fishes in China basically contain a certain amount of intermuscular spines, such as cyprinus carpio (Mylopharyngodon piceus), grass carp (Ctenophagogon idellus), silver carp (Hypoph thyyhiths molitrix), bighead carp (H.nobilis), megalobrama amblycephala and other cyprinus carpio, which bring great inconvenience to the eating of fresh fishes and the processing of minced fillet products, and seriously affect the high-quality development of the fish breeding industry and the aquaculture industry (Malachio et al, 2012 Nie et al, 2020. Therefore, if the thornless fresh water fish can be cultivated, the method plays an important role in promoting the development of aquaculture breeding in China.

Gene Editing technology (Gene Editing) enables humans to "edit" a target Gene of a species, thereby achieving operations such as knock-out and knock-in of a specific DNA fragment (wang et al, 2018). Nie et al. (2021) obtained scxa gene mutant zebrafish by CRISPR/Cas9 gene editing technique, the mutant had no intersomatic spurs on the back compared to wild-type zebrafish, only the tail contained a few intersomatic spurs, and the total number was reduced by about 70% compared to wild-type. Xu et al (2022) obtained a zebra fish mutant line of bm p6 by gene editing and showed complete deletion of the intermuscular spiny spine, and the patent of "breeding method for a new variety of anaplastic intermuscular spiny fish" obtained the patent grant of the national invention (patent grant No.: ZL 202011352379.4). Nie et al. (2022) constructed zebrafish runx2b gene mutation strain, the mutation body muscle thorns were completely deleted, and the growth, the swimming ability, the amino acid and fatty acid content in the muscle and the like of the mutant strain were not significantly different from the wild type strain. The above studies on the remarkable improvement effect on the intramuscular stimulation property have been limited to model fish zebra fish. Furthermore, the effectiveness of gene editing is closely related to the position of the gene mutation, and the resulting phenotype may also vary significantly (Xu et al, 2022) even without the phenotype.

In the aspect of improving the interspinal thorn character of important cultured economic fishes, researchers have conducted some researches by using methods such as selective breeding, crossbreeding, polyploidy and gynogenesis breeding (wang xu dong et al, 2021), but the effect is not very remarkable. In the aspect of gene editing breeding, zhong et al. (2016) uses CRISPR/Cas9 gene editing technology to knock out carp sp7a gene, and the result shows that sp7a gene ^-/- The mutant has a phenomenon that the intersomatic puncture is obviously shortened. Baobaolon and the like (patent application No.: CN202010451893.7, CN202010451894.1, CN202010451275.2 and CN 202010451274.8) knock out the mstn gene of fishes such as erythroculter ilishaeformis, megalobrama amblycephala and grass carp by gene editing, and establish a molecular breeding method for thickening the intermuscular spurs of the fishes. At present, no formal report is found on a method for obtaining a new species without interspinal stinging completely by genetic improvement on economic fishes.

Disclosure of Invention

The invention aims to construct a new germplasm creating method without intersomatic spurs for cultured economic fishes, which is to mutate runx2b genes of the cultured economic fishes.

The invention also aims to apply the method for creating the novel non-intramuscular-stinging germplasm of the economic fish, and the method is used for breeding the economic fish without intramuscular-stinging.

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

a method for creating a novel non-intramuscular-thorn germplasm for cultured economic fishes, which is characterized in that a runx2b gene of the cultured economic fishes is mutated.

In the above method, preferably, the farmed economic fish is megalobrama amblycephala;

in the above method, preferably, the mutation method is CRISPR/Cas9;

in the above-described method, preferably, the target gene of CRISPR/Cas9 is SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3.

In the above method, preferably, the mutated amateur megalobrama amblycephala has a gene represented by SEQ ID No.4 or SEQ ID No.5 or SEQ ID No. 6.

The protection content of the invention also comprises: the method is applied to breeding of economic fish without muscle prick.

Compared with the prior art, the invention has the following advantages:

the method can be used for obtaining the novel germ plasm of the megalobrama amblycephala without the intermuscular spurs. The method is simple and easy to implement and simple to operate. The megalobrama amblycephala strain without the intermuscular spurs is obtained by using a gene mutation method, and has the basic value and the popularization and application value of scientific research for researching the intermuscular spurs. The method can become an efficient technical means for obtaining the new germplasm of the economic fish cultured without the intersomatic spurs.

Drawings

FIG. 1 is a schematic sequence diagram of the target site and mutation site of the megalobrama amblycephala runx2b gene;

yellow indicates the runx2b gene target site, green indicates the PAM sequence, and red arrow indicates the runx2b gene target get mutation site.

FIG. 2 is a schematic diagram of the red skeletal staining of alizarin in whole body of wild-type megalobrama amblycephala (45 days after hatching), and the arrows indicate the internus muscularis.

FIG. 3 is a schematic diagram of X-ray imaging of wild type megalobrama amblycephala (135 days after hatching), with arrows indicating interspinal stings.

FIG. 4 is a schematic diagram showing the staining of the whole alizarin red bone of a mutant megalobrama amblycephala (45 days after hatching).

FIG. 5 is a schematic diagram of X-ray imaging of mutant megalobrama amblycephala (135 days after hatching).

Detailed Description

The technical scheme of the invention is a conventional mode in the field if not specifically stated; the reagents or the materials used are selected from,

all are commercially available, unless otherwise specified. In this embodiment, a CRISPR/Cas9 method is used to mutate runx2b gene of megalobrama amblycephala to prepare a new germplasm of the inonotus megalobrama amblycephala, and other cultured economic fishes or other gene editing modes in the field can successfully prepare the new germplasm of the inonotus cultured economic fishes as long as the mutation is made for the corresponding runx2b gene.

Example 1:

a method for creating a novel non-muscle-stab germplasm for culturing economic fishes comprises the following steps:

1.1 Experimental materials

Taking an economic fish megalobrama amblycephala as an example, the wild megalobrama amblycephala is raised in a breeding base with water of one hundred volumes in Huanggang city of Hubei province. The embryo for megalobrama amblycephala microinjection is obtained by the artificial propagation and spawning of sexually mature male and female parents.

1.2 Experimental methods

1.2.1 determination of sgRNA target sites

The target sites of 3 runx2b sgrnas were selected, respectively:

runx2b-1target:5‘GGGCTCCTACCAGTTCTCCA3’，

runx2b-2target:5‘GGGCTCAATCTTCCCCACCT’3，

runx2b-3target:5‘GGCCGACCACCCCGCCGAAC’3

1.2.2 in vitro Synthesis of sgRNA

The sgRNA is formed by a conserved downstream primer Scaffold (GATCCGCACCGATCGCCGTGTCCACTTTTC AAGTTGATAACGGACTAGCCCTTATTTAACTGTATAAC) and an upstream primer which is provided with a T7 promoter and specifically comprises a target sequence

Performing overlap PCR amplification. The PCR system is as follows: prime r star Mix 10.5. Mu.L, scaffold 5. Mu.L, sgRNA 5. Mu.L, ddH ₂ O4.5. Mu.L. PCR conditions of pre-denaturation at 98 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 15s,45 cycles, and re-extension at 72 ℃ for 5min. mu.L of the PCR product was subjected to 2% agarose gel electrophoresis, and the PCR product was recovered by purification after the band size was verified to be correct, and measured by Nanodro p 2000 (Thermo Scientific, USA). RNA was transcribed in vitro according to the (Th organom Scientific, USA) kit procedure, then sg RNA was purified and recovered using lithium chloride precipitation method, 1. Mu.L of the RNA concentration was taken out and assayed by 1.5% agarose gel electrophoresisAnd after the quality of the RNA is detected by electrophoresis, placing the RNA in a refrigerator at the temperature of-80 ℃ for storage, and obtaining the sgRNA.

1.2.3 in vitro transcription of ZCAS9mRNA

The pT3TS-nCas9n plasmid was linearized with XbaI (NEB, USA) restriction enzyme, and recovered with Gel Extraction Kit (Omega, USA) liquid purification Kit after completion of the linearization as confirmed by 1% agarose Gel electrophoresis. The zCas9mRNA was transcribed in vitro according to the T3 mms message mcmahine (Invitrogen, usa), purified using lithium chloride precipitation to recover zCas9mRNA, dissolved in water without enzyme and measured for concentration using Nanodrop 2000 (Thermo Scientific, usa), the mass of the transcribed mRNA was checked by 1.5% agarose gel electrophoresis and stored at-80 ℃ for future use.

1.2.4 microinjection

The male and female megalobrama amblycephala is divided into different cement ponds or culture barrels according to the proportion of 3 to 1 or 5 in the evening, the female parent is injected with a first injection of luteinizing hormone releasing hormone A2 (1 microgram/kg) at the evening, the needle pitch is about 12 hours (the time is properly shortened or prolonged according to different water temperatures), and then the second injection is carried out, wherein the male parent does not inject the first injection, and the second injection is half of the injection amount of the female parent. The effect time after the injection of the oxytocic is generally 6 hours (the effect time is closely related to the water temperature, and the time is properly prolonged according to different water temperatures). Megalobrama amblycephala eggs were then manually peeled into dry centrifuge tubes, while male fish sperm were manually peeled into centrifuge tubes containing 10mL Hank's solution for in vitro fertilization. Microinjection was started 10min after artificial insemination. Three sgRNAs and ZCAS9mRNA are mixed to prepare an injection system, wherein the final concentration of the sgRNAs is 100-200 ng/. Mu.L, the final concentration of the ZCAS9mRNA is 500-1000 ng/. Mu.L, and phenol red with the final concentration of 0.2% is added as an indicator. Experimental samples were injected into megalobrama amblycephala embryos at the 1-cell stage using a Picoiter Microinjector injector (Warner, PL-100A, USA). Culturing megalobrama amblycephala embryos by using methylene blue culture solution after injection, culturing in water with the water temperature of 20-25 ℃, and timely replacing embryo water.

1.2.5 detection of target mutation Rate

Selecting 30 embryos which are 48 hours after injection, and rapidly extracting megalobrama amblycephala embryos by a cracking methodFetal genomic DNA. The embryos to be lysed were placed in a 96 well 200. Mu.L well plate and 50. Mu.L lysis buffer (10 mmol/L Tris +50mmol/L KCl +1.5mmol/L MgCl) was added ₂ +0.3% Tween-20+0.3% Nonident P-40) 94 ℃ PCR reaction for 20min, and 55 ℃ to end the reaction; placing on ice, adding 5. Mu.L Pk enzyme (10 mg/ml), mixing by vortex, performing PCR reaction at 55 ℃ for 60min, performing PCR reaction at 94 ℃ for 20min, and finishing the reaction at 16 ℃.

A target-adjacent gene sequence was amplified with a target amplification detection primer (runx 2 b-1F; 5 TGTCGGTGAAGAATGAATGA/R: 5 CTCCAAT GAAGGCAAAA; runx 2b-2F; 5 TTTACCTGCGAGTGTGTTGC/R: 5 AGAGCGGAGTGGGGTGGAGT; 5 AGCGGACCTTCCAGCA/R: 5 CGGGAGGTTCGGATT GA).

The PCR reaction system is as follows:

PCR Master Mix (assist in san Jose, shanghai) 10. Mu.L, upstream and downstream primers 0.5. Mu.L each, genomic DNA template 2. Mu.L, sterile water 7. Mu.L.

The PCR reaction conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 30s,35 cycles, and re-extension at 72 ℃ for 5min. And (3) carrying out 3% agarose gel electrophoresis on 10 mu L of PCR products, and selecting and culturing an individual F0 with double bands and double peaks at sequencing target sites near the PCR products after detection until adult fish.

1.2.6 obtaining Megalobrama amblycephala F1 without interspinal thrust

The F1 generation is bred by hybridizing a female individual and a male individual of the F0 generation of the mutated megalobrama amblycephala, the embryo DNA of the F1 generation is extracted according to the above, PCR product amplification is carried out by using a target detection primer, and the heterozygote F1 with the mutation is screened out by using 3% agarose gel electrophoresis and sequencing (Protechinics, inc., wuhan). The sequence of each individual was analyzed and the mutant was analyzed for mutant target sequences as compared to the wild normal gene sequence.

1.2.7 genotype and phenotype analysis of new germplasm of Megalobrama amblycephala without intermuscular thorn

The results of comparing the target site gene sequences of wild-type megalobrama amblycephala and the amateur variant megalobrama amblycephala are shown in fig. 1: the sequence shown by the wild target site SEQ ID NO.1 is mutated into the sequence shown by SEQ ID NO. 4; a sequence shown in a wild type target site SEQ ID NO.2 is mutated into a sequence shown in SEQ ID NO. 5; the sequence shown in the wild target site SEQ ID NO.1 is mutated into the sequence shown in the SEQ ID NO. 6.

In order to observe the intramuscular pricking phenotype of the megalobrama amblycephala F1 generation mutant, the intramuscular pricking phenotype of the megalobrama amblycephala is observed by an alizarin red integral skeleton staining method. The integral intermuscular thorn phenotype of the wild megalobrama amblycephala is shown in figure 2, and the number of intermuscular thorns is obvious; the integral intramuscular prick phenotype of the runx2b mutant megalobrama amblycephala (the megalobrama amblycephala with mutant genotypes shown by SEQ ID NO. 4-SEQ ID NO. 6) is shown in figure 4, and the intramuscular pricks of the mutant with three genotypes are completely disappeared.

The alizarin red whole bone staining method comprises the following steps: fixing 45dph of megalobrama amblycephala in 4% paraformaldehyde for 48h, and then fixing in ddH ₂ Rinsing in O water overnight; then at 3% H ₂ O ₂ Bleaching for 4h with 1% KOH in the same volume of mixed solution; in ddH ₂ Rinsing in O for 30min; treating in 30% saturated borax solution for 12 hr; ddH ₂ Rinsing for 30min by O; staining in a 1% alizarin red S (Sigma) and 1% KOH mixed solution for 12h;1% washing excess alizarin red dye solution in KOH solution; rinsing in mixed solution of 1% trypsin (Solarbio) and 2% saturated borax for 3-5 days to remove impurities; gradient transparency in 50%,100% glycerol, and storage.

In order to obtain and store Megalobrama amblycephala parents without intermuscular spurs, young Megalobrama amblycephala parents of the F1 generation are cultivated to about four months (the body length is 10-15 cm), a portable X-ray imager (Version 90, orlon, china) is used for scanning the intermuscular spurs of the Megalobrama amblycephala, the Megalobrama amblycephala with normal intermuscular spurs is shown in a figure 3, the Megalobrama amblycephala with mutation and without intermuscular spurs is shown in a figure 5, the Megalobrama amblycephala without intermuscular spurs and normal activities are screened out and continuously cultivated until sexual maturity, and the next generation is bred.

Claims (6)

1. A method for creating new germplasm without intersomatic spurs for culturing economic fishesrunx2bThe gene is mutated.

2. The method of claim 1, wherein the farmed economic fish is megalobrama amblycephala.

3. The method of claim 2, wherein the mutation method is CRISPR/Cas9.

4. The method of claim 3, the target gene of CRISPR/Cas9 is SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3.

5. The method of claim 2, wherein the mutated Megalobrama amblycephala has a gene of SEQ ID No.4 or SEQ ID No.5 or SEQ ID No. 6.

6. Use of the method of claim 1 for breeding economic fish without muscle stab.

Images