CN116574814A - A molecular marker related to the growth traits of bream and its application - Google Patents

Abstract

The invention discloses a molecular marker related to the growth traits of bream and its application. The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1, located in the first exon of the PI3K gene, starting from ATG There is a G/A mutation site at the 319th base of . The present invention verifies that the growth performance such as crude protein, crude fat content and individual body weight are significantly decreased by constructing the PI3K gene knockout mutant of the bream, and the expression of genes downstream of the PI3K pathway is significantly changed, which proves that the PI3K gene is directly related to the growth phenotype of the bream. The overexpression of the PI3K gene leads to a significant increase in the expression level without changing the normal body shape of the embryo. Applying the molecular markers and PI3K genes related to the growth traits of the round head bream to the molecular assisted breeding of new strains of the round head bream can speed up the screening And cultivate fast-growing bream improved species, greatly increasing the aquaculture output.

Description

A molecular marker related to growth traits of bream and its application

technical field

The invention belongs to the technical field of fish genetics and breeding, and in particular relates to a molecular marker related to the growth traits of bream and its application.

Background technique

Group head bream, also known as Wuchang fish, will have a national aquaculture output of 760,000 tons in 2021. At present, there are still the following problems in the industry system of bream: under the condition of breeding, bream is not resistant to hypoxia, is prone to hypoxic stress death, and is prone to inbreeding, resulting in germplasm decline. The improved breed of bream with characteristics will be beneficial to the development of bream aquaculture.

The PI3K/AKT pathway is a signaling pathway with complex functions, which is crucial for cell growth and development. Insulin-like growth factor IGF can activate PI3K/AKT signaling pathway, further activate mTOR pathway to mediate growth factor signaling in body growth and key cellular processes, lipid metabolism, protein synthesis and apoptosis. Thus, the PI3K/AKT pathway plays an important role in protein synthesis and muscle growth. Although the PI3K-AKT signaling pathway is complex and diverse, it can be found that PI3K genes have certain effects on growth mechanisms. In the prior art, it has been found that the PI3K gene of roundhead bream is related to growth, and growth leads to changes in the expression of PI3K gene, but it cannot be used for phenotypic and functional identification. There is no relevant application of the growth-related gene PI3K in roundhead bream in breeding. reports.

Contents of the invention

The main purpose of the present invention is to provide a molecular marker related to the growth traits of bream. By screening the mutation sites of the PI3K gene, it is found that the base mutation of the PI3K gene is located in the first exon, starting from ATG There is a G/A mutation site at the 319th base of the protein, and the secondary space structure of the protein has not changed.

Another object of the present invention is to provide the application of the molecular markers related to the growth traits of bream, combining gene function and phenotype of bream for assisted breeding of new breeds of bream, which is helpful for screening bream Fast growing strain.

To achieve the above object, the present invention adopts the following technical solutions:

The invention provides a molecular marker related to the growth traits of bream. The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1, which is located in the first exon of the PI3K gene, starting from the ATG There is a G/A mutation site at base 319, and the sequence is as follows:

ATGCCTCCAAGACCCTCCTCGGGAGAACTATGGGGCATCCATTTGATGCCCCCCAGAATACTGGTGGACTGTTTACTGCCCAATGGCATGATCCTGACACTAGAGTGCCTCCGGGAGGCTACACTCATCACAATCAAACATGAACTGTTTAAGGAGGCAAGGAAGTATCCCCTCCACCATCTTCTGCAGGAGGAGACCTCCTATATTTTCGTCAGCGTTA CGCAAGAGGCGGAGCGTGAGGAGTTCTACGATGAGACGAGAAGACTTTGCGACCTTCGGCTATTCCAGCCCTTCCTAAAGGTCATTGAACCTGTAGGA A (SEQ ID NO: 1, where the bold underline indicates the mutation site).

Preferably, the growth traits of the bream include crude protein, crude fat content and individual body weight.

The invention also provides the application of the molecular markers related to the growth traits of the bream in assisted breeding of new fast-growing varieties of the bream.

Preferably, the bream is "Pujiang No. 2" bream.

The present invention also provides a method for cultivating fast-growing bream species, comprising: injecting linear PI3K mRNA into 1-2 cell stages of fertilized eggs of bream by microinjection, and the injection volume of a single embryo is 1 nL, and then cultured normally As for the adult fish, the adult fish whose PI3K gene overexpression is detected is the parent of the fast-growing bream, which is subcultured until it is stable, and then obtained.

Preferably, the primer sequences for synthesizing the linear PI3K mRNA are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

Preferably, the primer sequences for detecting the linear PI3K mRNA are shown in SEQ ID NO:4 and SEQ ID NO:5.

Preferably, the adult fish overexpressing the PI3K gene is detected by qRT-PCR, and the specific primer sequences used are shown in SEQ ID NO: 8 and SEQ ID NO: 9.

The invention also provides the application of PI3K gene in screening fast-growing bream species.

Preferably, the fast-growing group bream species are screened by detecting the expression content of the group bream PI3K gene, wherein the group group bream overexpressed with the PI3K gene is a fast-growing group group bream variety.

Compared with prior art, the beneficial effect of the present invention is:

1. Through animal experiments, it was found that after knockout of the PI3K gene in bream, the crude protein, crude fat content and individual body weight were significantly reduced, the growth performance was significantly reduced, and the expression of genes downstream of the PI3K pathway was significantly changed, which proved that the PI3K gene and bream The growth phenotype is directly related, and the overexpression of the PI3K gene leads to a significant increase in the expression level without changing the normal body shape of the embryo. According to the analysis of the different genotypes of the aforementioned mutation sites, the molecular markers related to the growth traits of the group head bream were applied. Breeding new strains of bream will help to select fast-growing strains of bream.

2. The PI3K gene is used for molecular assisted breeding of fast-growing strains of bream, and the fast-growing breed of bream is selected. At the same time, it is verified that the overexpression of the PI3K gene will not affect the normal body shape of the embryo. Combined with the transposon insertion technology, the The PI3K gene is overexpressed, so as to obtain an excellent strain of bream with significantly improved growth performance, which is helpful to improve the aquaculture output of bream.

Description of drawings

Fig. 1 is a schematic diagram of the design of the PI3K gene target point of the bream in the embodiment.

Fig. 2 is the target position of the bream PI3K gene in the embodiment.

Fig. 3 is the position of the knockout target point of the bream PI3K gene in the embodiment.

Figure 4 is a comparison of randomly selected individuals between the PI3K gene knockout mutant (PI3K ^+/- ) and the control group in the example; A: knockout mutant PI3K ^+/- ; B: control group.

Figure 5 shows the basic nutritional components of the PI3K gene knockout mutant (PI3K ^+/- ) and the control group in the example; A: crude protein; B: crude ash; C: crude fat; D: moisture.

Fig. 6 is a schematic diagram of the PI3K pathway and partial gene expression of the PI3K gene knockout mutant (PI3K ^+/- ) pathway in the embodiment.

Fig. 7 shows the relative expression level of PI3K gene overexpression 72h embryo and PI3K gene in the embodiment; A: embryo of control group; B: PI3K overexpression embryo; C: relative expression level of PI3K gene.

Detailed ways

The present invention will be further explained below in conjunction with the accompanying drawings and embodiments.

In the following examples, the association analysis between the PI3K gene and the growth traits of the bream was carried out, and the verification and analysis were mainly carried out from the following three aspects: 1. The CRISPR/Cas9 gene knockout technology was combined with the PI3K gene (https://www. ncbi.nlm.nih.gov/search/all/?term=XM_048171881.1); 2. By inserting linear mRNA to verify that the overexpression of PI3K gene will not affect the normal growth body shape; 3. Part of the PI3K gene Deletion and overexpression combined with growth phenotypes in bream.

Example 1 Verification of the function of the growth-related gene PI3K in bream

1.1 Material method

1.1.1 Experimental fish

The experimental fish is "Pujiang No. 2" bream. The parents used in the experiment are both 2 years old. They are sexually mature and have good growth and development. They are preserved in the Bream Genetics and Breeding Center of Shanghai Ocean University. Microinjected embryos are one-cell embryos after artificial insemination. After microinjection, they are incubated at room temperature (~25°C). During incubation, the water is changed every 4 hours to prevent embryo hypoxia. They are used to extract and detect overexpressed embryos After sampling, put them in liquid nitrogen for rapid freezing, and then store them in a -80°C refrigerator. Three individuals from the knockout mutant and the control group were hatched for about a week, and then the fry were placed in a pond (6×4×1.5m, length:width:height), where there were enough rotifers to prepare for the fry. The water depth of each pond is 0.5m, and the temperature is 24-26°C. After half a month, add water to the pond to 1.2m, feed the fish twice a day, and detect mutants after three months of breeding.

1.1.2 Cas9 target selection and gRNA synthesis

Use Ensembl genome browser to query the exon, intron and start codon position of PI3K gene, and then use ZiFiT to design the target position of PI3K gene knockout. Use T7 promoter, the 5' end of T7 is GG to improve the synthesis efficiency and the third base is preferably G/A, the 3' end sequence is the PAM region composed of NGG, the target is unique to avoid off-target, Moreover, it is best to design the target after the ATG in the first three regions of the CDS. If the intron region is short, the target can be designed across the intron. The target includes 20 bases in total, and the distance between the target and the upstream and downstream primers is equal. If it is greater than 100bp and the absolute value of the difference between the two distances is greater than 100bp, the amplified product should be less than 500bp. The electrophoresis band of the screening target should be bright and single. The general formula of the T7 promoter target is 5'-GG-(N)18 -NGG-3, as shown in Figure 1.

According to the preserved pMD19-gRNAscaffold plasmid, carry out the expansion culture of shaking bacteria, as follows:

(1) Add 5 μL of pMD19-gRNA scaffold plasmid to 100 μL of DH5α competent cells for transformation, place on ice (~20°C) for 30 minutes, heat shock at 42°C for 45 seconds, and immediately place on ice for 1 minute.

(2) Add 900 mL of LB medium (Amp-), incubate on a shaking table for 60 min (37° C.), and centrifuge at 1800 rpm for 6 min.

(3) Discard 700uL of supernatant, evenly spread 250uL of bacterial solution in LB medium (Amp+), and incubate overnight at 37°C.

(4) After 12 hours, it is advisable to pick colonies of uniform size and luster on the ultra-clean workbench. If the growth is not good, continue to culture in a constant temperature incubator to extract the pMD19-gRNAscaffold plasmid.

Preparation of gRNA Using the pMD19-gRNAscaffold plasmid template, PCR amplification was performed with upstream and downstream primers T7-target-sfd and tracrrev (Table 1), and the PCR product was purified and recovered with a purification kit to obtain high-purity DNA, which was passed through T7 in vitro transcription reagent The cassette was transcribed into linear gRNA and the concentration and quality were determined, see in vitro transcription reaction system (Table 2).

Table 1: gRNA reaction system and procedure for high-fidelity enzyme amplification of PI3K

Table 2: In vitro transcription reaction system

reaction system Sample volume (μL) Purified DNA Fragments x = 1μL/c 10m/nATP/CTP/GTP/UTP Each 1 10×transcription buffer 2 T7 enzyme MIX 2 RNase-free H ₂ o 12-x

1.1.3 Preparation of linear PI3K mRNA

Primers were designed and synthesized according to the ORF region of the bream PI3K gene (Table 3). The PCR product was recovered from rubber tapping and ligated and transformed. The steps were the same as 1.1.2(1)-(4).

Table 3: Primer sequences

1.1.4 Microinjection and embryo screening

The microinjection process of PI3K gene knockout is as follows: about 100 fertilized bream eggs are adhered to each glass petri dish, and Cas9 mRNA (900ng/uL) and PI3K gene gRNA (100ng/uL) are mixed at a ratio of 9:1. After the volume ratio is mixed, it is added to a microinjection needle for injection. It is injected into the yolk at the 1-2 cell stage of fertilized egg development. The injection volume remains the same. About 1000 fertilized eggs are injected into each target point, and normal fertilized eggs are used as a control.

Microinjected embryos with overexpression were one-cell embryos after artificial insemination, which were incubated at room temperature (~25°C) after microinjection. The water was changed every 4 hours during incubation to prevent hypoxia of the embryos. The embryos used to extract and detect the overexpression of the PI3K gene were sampled and placed in liquid nitrogen for quick freezing, and then stored in a -80°C refrigerator, followed by quantitative fluorescence detection.

1.1.5 Screening knockout mutants

Microinjected embryos of PI3K gene are one-cell embryos after artificial insemination. After microinjection, they are incubated at room temperature (~25°C). During incubation, the water is changed every 4 hours to ensure the dissolved oxygen content in the water and prevent embryo hypoxia. Dead, after about 7 days of hatching, put the fry into ponds (6×4×1.5m), where there are enough rotifers to prepare for fry, the water depth of each pond is 0.5m, and the temperature is 24-26°C. Half a month later, add water to the pond to 1.2m, feed the fish twice a day, detect mutants after three months of breeding, cut fins at about 20g each, and culture them separately in small tanks. After DNA extraction by alkaline cracking, use T7E1 ( T7endonuclease I) endonuclease digestion for genotype detection, samples with potential bands were sequenced, F0 heterozygotes were screened out, mutation types were determined, and at least three mutants and corresponding number of control groups were randomly selected to measure growth data.

1.1.6qRT-PCR analysis

Total RNA content was isolated using TRIzol reagent from the gills of PI3K knockout mutant F0 and control fish, which was normal bream without knockout, with three samples for each gene. After DNase treatment, it was reverse-transcribed into cDNA using the Prime Script RT kit, and a stable housekeeping gene was used as a control. Use SYBR Green Premix Ex Taq (qRT-PCR) ^TM real-time PCR detection system on CFX96Touch, PI3K gene and 18s qRT PCR primer pair (Table 4).

Table 4: Genes and specific primers used in qRT-PCR

1.1.7 Basic nutrients

40 g of dorsal muscle was taken from the knockout mutant and the control group, and three fish were taken from each group. The moisture content was measured by freeze-vacuum drying, the crude ash content was measured by burning in a muffle furnace at 550 °C to constant weight, the crude fat was extracted by the chloroform-methanol method, and the crude protein content in the sample was measured by a Kjeldahl nitrogen analyzer.

1.1.8 Growth measurement

The growth data of the knockout mutant PI3K ^+/- and the control group were measured and counted, including body weight, body length, total length, and body thickness.

1.1.9 Statistical Analysis

All data were statistically analyzed (SPSS17.0), and the difference was statistically significant (p<0.05).

2.2 Results

2.1.1 Target selection and sequence amplification

Through comparison, screening and uniqueness, two targets for PI3K gene knockout are selected. Target 1 and target 2 are both antisense targets. Target 1 is located in the second exon, and target 2 is located in the third exon. The PI3K gene mutation site is located at the 319th base G/A at the beginning of ATG, and the target one is located upstream of this position (Figure 2 and Table 5).

Table 5: Targets and Validation Primer Sequences

2.2.2 Knockout mutant screening

997 larvae of PI3K gene knockout group bream grew and survived, and were sampled and stored in batches using fin clipping markers to identify knockout mutants. Genomic DNA was extracted by alkaline cleavage as a template. With the help of primers and reaction procedures, the sequence near the target was obtained. After T7EI digestion and screening of mutants, it was found through sequencing that there were a large number of gene mutations or deletions after the target knockout of the gene, which seriously affected protein function. Among the 997 microinjected fishes, a total of 285 had confirmed or mutated around the target site, and target 1 and target 2 each had 6 main mutation types, and the mutation efficiency was 28.59% (Fig. 4).

Table 6: Sequences of the PI3K gene knockout mutants of bream

Note: A single dash indicates a changed base, and a double dash indicates a missing base.

2.2.3 Knockout mutant PI3K ^+/- phenotype

Randomly selected knockout mutant PI3K ^+/- and the control group showed significant differences in growth-related traits (p<0.05), and the knockout mutant PI3K ^+/- individual size was significantly smaller than the control group (Fig. 4, Table 7 ).

Table 7: Growth data of knockout mutant PI3K ^+/- and control group

weight(g) Body length (cm) Full length (cm) Body thickness (cm) Mutant PI3K ^+/- 31.14±2.59 ^a 11.02±0.83 ^a 12.87±0.77 ^a 1.43±0.14 ^a control group 34.23± ^2.31b 11.88± ^0.76b 13.46± ^0.39b 1.52± ^0.09b

2.2.4 Differences in protein content

The crude protein and crude fat contents of the knockout mutant PI3K ^+/- were significantly lower than those of the control group (p<0.05), the water content was significantly higher than that of the control group (p<0.05), while the crude ash content was not significantly different (p>0.05 )(Figure 5).

2.2.5 Expression of PI3K gene and downstream pathway genes in knockout mutant PI3K ^+/-

The knockout mutant PI3K ^+/- was significantly (p<0.05) lower than the control group, and the expression levels of downstream pathway genes in the knockout mutant PI3K ^+/- were significantly (p<0.05) lower than eIF4E in the control group (Figure 6).

2.2.6 PI3K mRNA overexpression

Using microinjection technique, linear PI3K mRNA was injected in the 1-2 cell stage of bream fertilized eggs, and the injection volume of each embryo was 1 nL. After 72 hours, the collected embryos and qPCR expression analysis showed that the expression level of PI3K gene injection group was significantly higher In the control group, compared with the control group, the embryos in the overexpression group injected for 72 hours developed normally and no deformities occurred (Fig. 7).

In summary, the growth rate of PI3K ^+/- knockout mutants in bream was significantly lower than that of normal bream, and the expression of each gene in the downstream pathway was correspondingly increased or decreased, which affected the synthesis and production of protein in fish. Cell growth and proliferation, resulting in a reduced growth rate of the mutant. The nutritional value of muscle is mainly related to fat and protein. The crude protein and crude fat content of the bream knockout mutant PI3K ^+/- are lower than that of the control group, and the water content is higher than that of the control group. It can be seen from the test of the protein content The PI3K gene has an important influence on the growth of bream.

In the present invention, by verifying the function and phenotype of the PI3K gene, and using the CRISPR/Cas9 gene knockout technology to knock out the PI3K gene, a PI3K ^+/- mutant is obtained, the growth performance of the PI3K ^+/- mutant is significantly reduced, and the PI3K gene Overexpression will not affect the normal body shape of the embryo, and promote the application of PI3K gene in the molecular assisted breeding process of bream, and use transposon insertion of PI3K gene to obtain bream with significantly improved growth performance, and accelerate the development of excellent strains of bream Breeding process to promote the development of bream seed industry.

The above-mentioned description of the embodiments is for those of ordinary skill in the technical field to understand and use the invention, but the present invention is not limited to the above-mentioned embodiments, those skilled in the art can make according to the technical scheme of the present invention without departing from the scope of the present invention. All improvements and modifications should be within the protection scope of the present invention.

Claims (10)

1. A molecular marker related to megalobrama amblycephala growth traits, which is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO:1, which is located at the first exon of the PI3K gene, there is a G/A mutation site at the 319 th base from ATG.

2. The molecular marker related to the megalobrama amblycephala growth trait according to claim 1, wherein the megalobrama amblycephala growth trait comprises crude protein, crude fat content and individual body weight.

3. The use of the molecular marker related to the growth traits of megalobrama amblycephala according to claim 1 or 2 in auxiliary breeding of new megalobrama amblycephala fast growth varieties.

4. The use according to claim 3, wherein said megalobrama amblycephala is "Pujiang No. 2" megalobrama amblycephala.

5. A cultivation method of a fast-growing megalobrama amblycephala variety is characterized by comprising the following steps: and (3) injecting linear PI3KmRNA into the 1-2 cell period of the fertilized eggs of the megalobrama amblycephala by microinjection, wherein the injection amount of a single embryo is 1nL, then normally culturing to adult fish, detecting the adult fish over-expressed by PI3K gene as a parent of the megalobrama amblycephala growing rapidly, and carrying out subculture until the adult fish is stable.

6. The cultivation method of the quick-growing megalobrama amblycephala variety according to claim 5, wherein the primer sequence for synthesizing the linear PI3KmRNA is shown as SEQ ID NO:2 and SEQ ID NO: 3.

7. The cultivation method of the megalobrama amblycephala variety with rapid growth according to claim 5, wherein the primer sequence for detecting the linear PI3KmRNA is shown as SEQ ID NO:4 and SEQ ID NO: shown at 5.

8. The cultivation method of the quick-growing megalobrama amblycephala variety according to claim 5, wherein the qRT-PCR detection of adult fish over-expressed by PI3K gene adopts a specific primer sequence shown in SEQ ID NO:8 and SEQ ID NO: shown at 9.

The PI3K gene is applied to screening of fast-growing megalobrama amblycephala varieties.

10. The use according to claim 9, wherein the fast growing megalobrama amblycephala variety is selected by detecting the expression level of the PI3K gene of megalobrama amblycephala, wherein the megalobrama amblycephala with over-expressed PI3K gene is the fast growing megalobrama amblycephala variety.