CN118956867A

CN118956867A - A gene editing method and application for constructing hypoxia-resistant Megalobrama agglomerata by targeting mylipb gene

Info

Publication number: CN118956867A
Application number: CN202411034146.8A
Authority: CN
Inventors: 王晶; 肖武汉; 李俊
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2024-07-30
Filing date: 2024-07-30
Publication date: 2024-11-15

Abstract

The invention relates to the technical field of genetic engineering, in particular to a gene editing method for constructing a low-oxygen-resistant megalobrama amblycephala by targeting mylipb genes and application thereof. The invention provides a gRNA of a targeted mylipb gene and a composition consisting of the gRNA and a Cas9 protein, which can be used for knocking out mylipb genes. The invention also provides a gene editing method of the targeting megalobrama amblycephala mylipb gene based on the CRISPR/Cas9 system, which comprises the steps of designing a target sequence, preparing gRNA, and carrying out in-vitro microinjection on a composition of the gRNA and the Cas9 protein to fertilized eggs to obtain F ₀ generation mutants. F ₀ generations are selfed to obtain F ₁ generations, and gene knockout heterozygote megalobrama amblycephala is obtained through genotype confirmation, wherein the heterozygote megalobrama amblycephala is identified as a germplasm material which is more resistant to hypoxia through phenotype. Compared with the traditional method, the breeding method provided by the invention has the remarkable advantages of realizing rapid and accurate directional transformation of biological characters, greatly shortening the breeding period, improving the breeding efficiency and having important application value.

Description

Gene editing method for constructing hypoxia-tolerant megalobrama amblycephala by targeting mylipb genes and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene editing method for constructing a low-oxygen-resistant megalobrama amblycephala by targeting mylipb genes and application thereof.

Background

Megalobrama amblycephala (Megalobrama amblycephala), also called as marchand, is an important representative freshwater aquaculture economic species in important cypridoptera, megalobrama and culter subfamilies in China, and has the important excellent growth characteristics of quick growth, high survival rate, low cost and the like. From the 60 s of the 20 th century, it is popular and increasingly popularized for cultivation. The 2019 yield of megalobrama amblycephala reaches 76.28 ten thousand tons, and the 7 th place of the yield of the freshwater aquaculture fishes in China is one of the most important aquaculture varieties in China.

In aquaculture, fish often experience stress factors such as high temperature, crowding, hypoxia and bacterial attack, sometimes resulting in serious losses, and in addition to these appreciable stress factors, there are also growth-related genetic regulatory mechanisms that limit the annual yield of fish. Compared with other common cultured fishes (carp and crucian carp), the megalobrama amblycephala has the defect of intolerance to hypoxia, and is extremely easy to cause hypoxia death and bring loss to the culture industry. With the occurrence of the inbreeding recession phenomenon, the new germplasm for cultivating the low-oxygen-resistant megalobrama amblycephala has greater application value.

The spontaneous mutation frequency of farmed fish is very low, usually less than 10 ^-6, and needs to exist in a long time or a large population, and even if naturally generated, the farmed fish is difficult to screen. The mutant families with important economic character related genes are often not available, and the breeding period is long, which seriously hinders the pace of fine breed breeding of fish. Therefore, gene editing and transgenesis are important research directions in the field of fish breeding. However, at present, the method for enhancing the hypoxia tolerance of the megalobrama amblycephala by gene editing and constructing new hypoxia tolerance germplasm of the megalobrama amblycephala have not been reported.

Disclosure of Invention

The invention discovers that the mylipb gene for editing the megalobrama amblycephala can enhance the hypoxia tolerance performance in the study for the first time, and based on the method, the invention provides a method for carrying out gene editing on the megalobrama amblycephala mylipb gene by adopting a CRISPR/Cas9 system, and obtains the gene mutation heterozygote megalobrama amblycephala which grows normally and is more hypoxia tolerant, thereby disclosing the key regulation and control effect of the mylipb gene on the hypoxia tolerance of the megalobrama amblycephala.

The technical scheme of the invention is realized as follows:

In a first aspect, the present invention provides a gRNA targeting mylipb genes (NCBI Gene ID: 125243060) that targets a third exon of the mylipb Gene, the gRNA comprising the sequence set forth in SEQ ID NO:1, and a targeting sequence shown in the following formula.

In a second aspect, the invention provides a composition comprising the gcna targeting mylipb gene and Cas9 protein; the composition is used for knocking out mylipb genes.

In a third aspect, the present invention provides a gene editing method for targeting megalobrama amblycephala mylipb gene, comprising the steps of:

(a1) Designing mylipb gene targets, wherein the targeting sequence of the target targets is shown as SEQ ID NO:1 is shown in the specification;

(a2) Designing a specific primer of the gRNA according to mylipb gene target sequences, performing PCR amplification to obtain template DNA, and performing in-vitro transcription by using T7 polymerase to prepare the gRNA of the CRISPR/Cas9 system;

(a3) The gRNA and Cas9 protein composition is injected into fertilized eggs of wild megalobrama amblycephala in an in-vitro manner, and the F ₀ generation of mylipb gene knockout is screened through genotype detection.

In some preferred embodiments, the PCR amplified template of step (a 2) is pUC19-gRNA plasmid, and the primer sequences are set forth in SEQ ID NO: 2-3.

In some preferred embodiments, the wild type megalobrama amblycephala fertilized egg in step (a 3) is an I cell stage fertilized egg.

In some preferred embodiments, the concentration of gRNA in the composition in step (a 3) is 200ng/μl and the concentration of Cas9 protein is 500ng/μl.

In a fourth aspect, the invention provides an application of the gene knockout megalobrama amblycephala constructed according to the gene editing method of the targeting megalobrama amblycephala mylipb gene in researching the biological function of mylipb gene.

In a fifth aspect, the present invention also provides a method for constructing a hypoxia tolerant megalobrama amblycephala by mylipb gene knockout, comprising the steps of:

(b1) Knocking out wild megalobrama amblycephala according to the gene editing method of targeting megalobrama amblycephala mylipb gene

Mylipb genes of the megalobrama amblycephala;

(b2) Mating male and female individuals in the F ₀ generation with mutation to obtain F ₁ generation, wherein megalobrama amblycephala with genotype detected as mylipb gene mutation in F ₁ generation is hypoxia-tolerant megalobrama amblycephala.

In some preferred embodiments, the step (b 2) includes: the heterozygote megalobrama amblycephala with genotype detected as mylipb ⁺/mylipb^- genotype in F1 generation is hypoxia-tolerant megalobrama amblycephala.

In a sixth aspect, the invention provides an application of mylipb genes in improving hypoxia tolerance of megalobrama amblycephala, comprising: the gene knocked-out is carried out on mylipb genes so as to improve the hypoxia tolerance of megalobrama amblycephala.

The beneficial effects of the invention at least comprise the following:

(1) The invention uses CRISPR/Cas9 technology to realize the specific knockout of mylipb genes in megalobrama amblycephala for the first time, and obtains mylipb gene deletion mutant which can normally grow and develop.

(2) The invention discloses that mylipb gene deletion can obviously improve the tolerance of megalobrama amblycephala to low-oxygen environment for the first time, and simultaneously, a mylipb gene knockout heterozygote megalobrama amblycephala new germplasm material with enhanced low-oxygen resistance is successfully cultivated based on a CRISPR/Cas9 technology. Compared with the traditional breeding method, the invention has the remarkable advantages that the high accuracy of gene improvement can be realized, the biological character can be rapidly and accurately directionally transformed, the breeding period is greatly shortened, the breeding efficiency is improved, and the time cost is reduced.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a target for mylipb gene editing by the CRISPR/Cas9 system of the present invention;

FIG. 2 shows agarose gel electrophoresis results of wild megalobrama amblycephala and F ₁ generation heterozygote megalobrama amblycephala;

FIG. 3 shows the result of comparing the gene sequences of the target sites of wild megalobrama amblycephala and F ₁ generation heterozygote megalobrama amblycephala;

FIG. 4 is a control of wild megalobrama amblycephala and F ₁ generation heterozygote megalobrama amblycephala under hypoxia condition;

FIG. 5 is a graph showing survival rate of wild type megalobrama amblycephala and F ₁ generation heterozygote megalobrama amblycephala under normoxic (Nor represents normoxic group) and hypoxic (Hyp represents hypoxic group) conditions, respectively;

FIG. 6 shows the results of detecting transcription level of hypoxia downstream gene in vivo under normoxic and hypoxia conditions, respectively, of wild megalobrama amblycephala and F ₁ generation heterozygote megalobrama amblycephala of the present invention.

Detailed Description

The invention is further illustrated below in conjunction with specific embodiments. It is understood that these examples are only for illustrating the present invention and in no way limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Experimental materials used in the examples of the present invention are available from commercial sources unless otherwise specified. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Experimental materials:

In the invention, the male and female parents of the wild megalobrama amblycephala are fed to the aquatic breeding base of the agricultural university in China. Embryos used for the micro-injection of megalobrama amblycephala are obtained by artificial propagation and spawning of sexually mature male and female parents.

Example 1: CRISPR/Cas9 system construction

(1) Design mylipb Gene target

In the third exon of megalobrama amblycephala mylipb gene, the target point of CRISPR/Cas9 system gene is designed as shown in figure 1.

The targeting sequence of megalobrama amblycephala mylipb gene is 5'-GGGCTCTTGACCGTAGATCT-3' (see SEQ ID NO: 1).

(2) Preparation mylipb gRNA

The upstream and downstream primers for gRNA synthesis were designed based on mylipb gene targets (sequences are shown in the following table). Wherein the upstream primer is gRNA-F, and comprises a T7 promoter and a target sequence of mylipb genes; the downstream primer is a conserved primer gRNA-R. The primers were synthesized by Wuhan Tianhua, a gene technology Co., ltd.

PCR amplification was performed using gRNA-F and gRNA-R as primers and pUC19-gRNAvector as a template. pUC19-gRNAvector in the examples is shown in reference ChangN,Sun C,Gao L,Zhu D,XuX,Zhu X,Xiong JW,Xi JJ.Genome editing with RNA-guided Cas9nucleaseinzebrafish embryos,CellRes,2013,23(4)：465-472)., which is publicly available from the national academy of sciences of aquatic biology, and which is used only for repeated experiments related to the present invention and is not used for other purposes.

The PCR reaction system is as follows:

The reaction conditions are as follows:

The reaction product of 5. Mu.LPCR was subjected to 1.5% agarose gel electrophoresis, and after confirming that the band size was correct, the recovered PCR product was purified, and the concentration of the recovered product was measured using Biophotometer spectrophotometer. Mylipb gRNA was transcribed in vitro according to the TRANSCRIPTAID T7HIGHYIELD TRANSCRIPTIONKIT kit (from Fermentas) and recovered by purification using a lithium chloride precipitation method.

The lithium chloride precipitation method comprises the following steps: adding 1 μl DNase I into mylipb gRNA in vitro transcription system, incubating for 15min to remove residual DNA, adding 2 μl 0.2M EDTA to terminate reaction, adding 2.5ul4M lithium chloride and 75 μl pre-cooled absolute ethanol, and precipitating at-20deg.C for 16h; the precipitate was collected by centrifugation, washed with 50. Mu.l of 70% (V/V) pre-chilled ethanol, centrifuged to remove ethanol, and 25. Mu.l of DEPC water was added to dissolve the precipitate. 1. Mu.L of the RNA concentration was measured, and mylipb gRNA mass% was detected by 1.5% agarose gel electrophoresis, and stored in a-80℃refrigerator for use.

Example 2: construction mylipb Gene knockout megalobrama amblycephala

(1) Microinjection

And simultaneously collecting ovum and sperm of the mature megalobrama amblycephala parent, and carrying out microinjection after in vitro artificial insemination. The injection system was formulated using a mix of gRNA and Cas9 protein (purchased from Novoprotein) with a final concentration of 200ng/μl, cas9 protein of 500ng/μl, with phenol red added as an indicator at a final concentration of 0.2% (M/V). The experimental samples were injected into the fertilized eggs of megalobrama amblycephala in the I-cell stage, spread on a petri dish, using a pressure microinjection apparatus of U.S. WARNERPLI-100A. Injection was completed within 40min after fertilization of the ovum. 1000-2000 fertilized eggs can be injected into every 5ul of mixed reagent. After the injection is completed, the fertilized eggs are placed in oxygenated water with the water temperature of 23-28 ℃ for incubation.

(2) Screening of megalobrama amblycephala mylipb Gene knockout F ₀ generation mutant individuals

Extracting the genomic DNA of the megalobrama amblycephala individuals living after the step (1) is completed, and amplifying nearby gene sequences by adopting target site detection primers (mylipb-F and mylipb-R) through PCR.

Name of the name	SEQIDNO.	Sequence(5’-3’)
			mylipb-F	4	ACATTTGTTTCTGATGCATGTGAAGG
mylipb-R	5	ACCATACACTTCTCAGTGCCCTTAC

The PCR reaction system is as follows:

The reaction conditions are as follows:

after the PCR is completed, the 10 mu LPCR product is taken for 2.5% agarose gel electrophoresis identification and then cut into gel for purification, and target spot detection primers are used for product sequencing identification, as shown in figure 3, the individual with 7 base deletion sequence relative to the wild megalobrama amblycephala is identified as F ₀ generation mylipb gene mutant megalobrama amblycephala.

(3) Analysis of genotype of F ₁ generation mylipb Gene mutant megalobrama amblycephala

After the F ₀ generation mutant megalobrama amblycephala is sexually mature, mating male and female individuals to obtain F ₁ generation individuals. The mylipb mutated F ₁ generation genotype was then analyzed in detail.

After extracting F ₁ generation individual genome DNA, PCR amplification and product sequencing are carried out by using target detection primers (mylipb-F and mylipb-R). The amplified product was ligated to pMD18-T vector (purchased from Takara) and sequenced, and the mylipb gene sequence of the F1 generation mutant individual obtained by sequencing was compared with the mylipb gene sequence of the wild type megalobrama amblycephala.

Sequencing peak diagram results of F ₁ generation mylipb gene mutant megalobrama amblycephala show that double peaks appear at deletion positions, which indicates that the megalobrama amblycephala individual is mylipb gene mutant heterozygote and the genotype is mylipb ⁺/mylipb^-.

Example 3 phenotypic identification

Culturing megalobrama amblycephala F ₁ generation in indoor environment, identifying genotype when F ₁ generation grows to 3months of age (3mpf,3months afterfertilization), and selecting wild type megalobrama amblycephala and mutant heterozygote megalobrama amblycephala with the same weight, wherein the genotypes corresponding to the wild type megalobrama amblycephala and the mutant heterozygote megalobrama amblycephala are mylipb ⁺/mylipb⁺、mylipb⁺/mylipb^- respectively. 400ml of aerated water of saturated oxygen was added to 500ml Erlenmeyer flasks, each containing three fish. A total of 30 fish were placed in 10 replicates per group. The normoxic control group was set at 28 ℃, oxygen concentration 21% vol, and carbon dioxide concentration 5% vol. The hypoxia experimental group was placed in a Ruskinn Invivo2I-400 hypoxia incubator set at 28℃with 10% VOL and 5% VOL and the death of the fish was counted every 15 minutes and recorded by photographing.

The time for generating the floating head phenomenon can intuitively evaluate the hypoxia tolerance of the megalobrama amblycephala. As shown in figure 4, the hypoxia tolerance of the mylipb ⁺/mylipb^- genotype heterozygote megalobrama amblycephala is obviously improved.

The survival rate statistics are shown in fig. 5, and under the condition of low oxygen pressure, the survival rate of the mylipb ⁺/mylipb^- -genotype megalobrama amblycephala is obviously higher than that of the wild type megalobrama amblycephala, and the result shows that the hypoxia tolerance of the mylipb ⁺/mylipb^- -genotype heterozygote megalobrama amblycephala is obviously improved.

Example 4 detection of transcriptional Activity of hypoxia downstream Gene

In this example, by quantitatively determining the difference in transcription levels of the hypoxia downstream genes phd3, glut1, pkd2 and vegfa of the mylipb gene wild type and mutant heterozygotes, these hypoxia downstream genes are closely related to the hypoxia tolerance regulation mechanism, participate in hypoxia tolerance regulation through different mechanisms and pathways, and jointly maintain the survival and adaptation of cells in a hypoxia environment, when the transcription level of the hypoxia downstream genes is higher, the higher hypoxia tolerance is represented, and the detection result indirectly can also reveal whether the mylipb gene is related to the internal regulation pathway of the hypoxia downstream genes.

Culturing megalobrama amblycephala under indoor environment for F ₁ generation, identifying genotype when F ₁ generation grows to 3months old (3mpf,3months afterfertilization), and selecting megalobrama amblycephala with the same weight mylipb ⁺/mylipb⁺、mylipb⁺/mylipb^- genotype. 400ml of aerated water of saturated oxygen was added to 500ml Erlenmeyer flasks, each containing three fish. A total of 30 fish were placed in 10 replicates per group. The normoxic control group was set at 28 ℃, oxygen concentration 21% vol, and carbon dioxide concentration 5% vol. The hypoxia experimental group is placed into a Ruskin Invivo2I-400 hypoxia incubator with the set temperature of 28 ℃, the oxygen concentration of 12% VOL and the carbon dioxide concentration of 5% VOL, fish is taken out and killed after being treated for 3 hours, brain tissues are taken out, RNA in the brain tissues is extracted, cDNA is synthesized through a reverse transcription kit (purchased from Fermentas), hypoxia downstream related genes are amplified through a real-time fluorescence quantitative PCR kit (purchased from Monad), ct values are obtained through real-time detection of fluorescence signal intensity which changes with the amplification, and meanwhile, copy numbers of target genes of samples to be detected can be obtained by using a plurality of standard products with known template concentrations as a reference.

As shown in FIG. 6, the transcription level of the hypoxia downstream gene in mylipb ⁺/mylipb^- genotype megalobrama amblycephala is obviously higher than that of wild type mylipb ⁺/mylipb⁺, which indicates that the mylipb gene is closely related to the internal regulation and control path of the hypoxia downstream gene, and the tolerance and adaptability of mylipb ⁺/mylipb^- genotype heterozygote megalobrama amblycephala to hypoxia are obviously improved.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A gRNA targeting mylipb genes, characterized in that it targets a third exon of mylipb genes, said gRNA comprising the sequence set forth in SEQ ID NO:1, and a targeting sequence shown in the following formula.

2. A composition comprising the gRNA of the targeted mylipb gene of claim 1 and a Cas9 protein; the composition is used for knocking out mylipb genes.

3. A gene editing method of targeting megalobrama amblycephala mylipb gene is characterized by comprising the following steps:

(a2) Designing a specific primer of the gRNA according to mylipb gene targets, performing PCR amplification to obtain template DNA, and performing in-vitro transcription by using T7 polymerase to prepare the gRNA of the CRISPR/Cas9 system;

4. A method according to claim 3, wherein in step (a 2), comprising: the PCR amplified template is pUC19-gRNA plasmid, and primer sequences are respectively shown in SEQ ID NO: 2-3.

5. A method according to claim 3, wherein in step (a 3), comprising: the wild megalobrama amblycephala fertilized egg is an I cell stage fertilized egg.

6. A method according to claim 3, wherein in step (a 3), comprising: in the composition, the concentration of gRNA is 200 ng/. Mu.l, and the concentration of Cas9 protein is 500 ng/. Mu.l.

7. Use of a gene knockout megalobrama amblycephala constructed by the method according to any one of claims 3 to 6 in researching the biological function of mylipb genes.

8. A method for constructing a low-oxygen-resistant megalobrama amblycephala by mylipb gene knockout is characterized by comprising the following steps:

(b1) Mylipb for knocking out wild type megalobrama amblycephala by the method according to any one of claims 3 to 6

A gene;

9. The method of claim 8, wherein in step (b 2), comprising: the heterozygote megalobrama amblycephala with the genotype of mylipb ⁺/mylipb^- is detected as the hypoxia-tolerant megalobrama amblycephala in the F ₁ generation.

An application of mylipb gene in improving hypoxia tolerance of megalobrama amblycephala, which is characterized by comprising the following steps: the gene knocked-out is carried out on mylipb genes so as to improve the hypoxia tolerance of megalobrama amblycephala.