Establishment method of Nile tilapia mstnb homozygous knockout line and rapid growth line obtained by same

A Nile tilapia, homozygous technology, applied to other methods of inserting foreign genetic materials, transforming growth factors, chemical instruments and methods, etc., can solve the problem that there is no homozygous mutant line

Pending Publication Date: 2022-05-06
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no report on the homozygous mutant line obtained by knocking out the mstn gene in Nile tilapia

Method used

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  • Establishment method of Nile tilapia mstnb homozygous knockout line and rapid growth line obtained by same
  • Establishment method of Nile tilapia mstnb homozygous knockout line and rapid growth line obtained by same
  • Establishment method of Nile tilapia mstnb homozygous knockout line and rapid growth line obtained by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Using CRISPR / Cas9 gene editing technology to effectively target the mstnb gene of Nile tilapia

[0051] 1. Target design

[0052] Target site design is carried out on Zifit (http: / / zifit.partners.org / zifit / Disclaimer.aspx). The design principles and steps can be found in Li Minghui’s paper (Establishment of Tilapia Gene Knockout Technology and Its Role in Sex Determination and Differentiation) Application in Research, Li Minghui, 2014) The gRNA primer sequence is (SEQ ID NO:2): 5'-TAATACGACTCACTATA GCA GCCTTCCGTCAGCACC GTTTTAGAGCTAGAAATAGC-3'. The underlined sequence is the target site (SEQ ID NO:1), the 5' end is the T7 promoter sequence, and the 3' end is the gRNA template plasmid binding sequence. Primers were synthesized by BGI.

[0053] 2. gRNA and Cas9 synthesis and microinjection

[0054] 2.1 gRNA synthesis

[0055] 1) Using the gRNA plasmid as a template (see Chang et al., 2013 for plasmid information), use the designed F primer (SEQID NO: 2) ...

Embodiment 2

[0087] Embodiment 2: Passage establishment based on F0 generation positive fish

[0088] 1. Obtain the F1 generation of heterozygous mstnb mutation;

[0089] When the male fishes were sexually mature (about 6 months old), they were mated with wild-type female fishes to obtain F1 generation fish with different mutation types. The mutation types were identified by detection primer amplification combined with sequencing technology, and the same mutation types ( And the bases are not multiple deletions of 3) male and female tilapia as the F1 generation broodstock. When the F1 broodstock is sexually mature, the two can be mated to obtain the F2 fish containing the homozygous deletion.

[0090] 2. Obtain the F2 generation of homozygous mstnb mutation

[0091] Homozygous mutations were detected by polyacrylamide gel electrophoresis (PAGE) combined with sequencing technology. Firstly, the genomic DNA of the F2 generation fish was extracted, amplified using PAGE primers as a templat...

Embodiment 3

[0094] Example 3 Based on the phenotypic identification and muscle histological observation of mstnb homozygous mutant fish

[0095] assessment method:

[0096] 1. Growth performance evaluation

[0097] 1.1 Body weight;

[0098] 1.2 Body length;

[0099] 1.3 Body height;

[0100] 1.4 Body width;

[0101] 1.5 weight gain rate (weight gain rate, WGR, %) = 100 × (W t -W 0 ) / W 0 ;

[0102] 1.6 Fullness (condition factor, CF,%) = 100×Wt / L 3 t;

[0103] 1.7 Specific growth rate (specific growth rate, SGR, % / d) = 100 × (ln W t -ln W 0 ) / t;

[0104] Note: W 0 is the initial body weight of the fish (g); W t is the final body weight of the fish (g); L t The final body length of the test fish (cm); t is the number of days of the test (d).

[0105] 2. Significance analysis

[0106] After the data was sorted on WPS Excel 2020, the difference between groups was analyzed on SPASS 20.0, the significance level was set at 0.05, and the experimental data was expressed as mean ± ...

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Abstract

The invention provides an establishment method of a nile tilapia mstnb homozygous knockout line and a rapid growth strain obtained by the same, the method comprises the following steps: (1) mixing nile tilapia mstn gene gRNA with Cas9 mRNA, and incubating at room temperature to obtain a compound of gRNA and Cas9 mRNA; adding a small molecule dye into the compound, and uniformly mixing to obtain the CRISPR/Cas9 gene edited microinjection; (2) enabling fertilized eggs of the Nile tilapia in a cell stage after artificial insemination to uniformly lie flat in a culture dish; (3) transferring the microinjection into a micro needle tube and injecting the microinjection into the cell fertilized eggs fixed in the step (2) by using a microinjector; and (4) transferring the fertilized eggs into a 26 +/-2 DEG C constant-temperature circulating water hatching system for hatching after injection. The weight of the Nile tilapia mossambica with the mstnb mutation obtained by the method is increased by about 50% compared with that of wild tilapia mossambica at the age of seven months, important support is provided for increasing the breeding yield of the Nile tilapia mossambica, and the method has a good application prospect.

Description

technical field [0001] The invention relates to a fishery breeding and breeding method, in particular to a method for establishing a homozygous knockout line of Nile tilapia mstnb and a fast-growing line obtained therefrom. Background technique [0002] Hybridization and selective breeding are the main methods of fish breeding. However, due to the existence of interspecific reproductive isolation, it is difficult to form fertile lines through distant hybridization. And due to the lack of genetic and reproductive rules for reference and reference, it is difficult to predict the types of offspring that may appear in the offspring of distant hybrids. If blind breeding is carried out solely on the basis of complementary phenotypic advantages, it will easily lead to adverse results such as death of hybrid offspring, lack of hybrid advantage, and difficulty in forming strains. Therefore, although hybrid breeding is the most conventional breeding method for fish, it still has man...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/55C12N15/12A01K67/027
CPCC12N15/89C12N9/22C07K14/495A01K67/0276A01K2217/075A01K2217/15A01K2227/40A01K2267/02
Inventor 吴忧周林燕
Owner SOUTHWEST UNIVERSITY
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