CN117327740A - Production method of meat red tilapia with ornamental and edible values - Google Patents

Production method of meat red tilapia with ornamental and edible values Download PDF

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CN117327740A
CN117327740A CN202311238746.1A CN202311238746A CN117327740A CN 117327740 A CN117327740 A CN 117327740A CN 202311238746 A CN202311238746 A CN 202311238746A CN 117327740 A CN117327740 A CN 117327740A
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tilapia
hps5
cells
red
gene
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王德寿
王晨旭
陶文静
李明辉
柳兴永
郑树清
孙丽娜
魏静
魏玲
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Western Chongqing Science City Germplasm Creation Science Center
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Abstract

The invention belongs to the field of aquaculture, and particularly relates to a production method of a meat red tilapia with ornamental and edible values. According to the invention, through CRISPR/Cas9 gene editing breeding technology, a key gene hps5 for synthesizing melanin is directionally mutated in wild black tilapia, and a red commercial tilapia is created. The body color of the tilapia mossambica is mainly caused by the increase and enlargement of yellow pigment cells, the deletion of melanin and the remarkable reduction of iridescent cells (complete deletion of early iridescent cells). The iris part of the eye of the nile tilapia with the hps5 homozygous mutation is partially restored from 90dpf due to iridescent cells but unevenly distributed, and meanwhile, melanin is deleted to present special patterns which have not been reported so far, and the patterns can further improve the overall ornamental value of the red tilapia.

Description

Production method of meat red tilapia with ornamental and edible values
Technical Field
The invention belongs to the field of aquaculture, and particularly relates to a production method of a meat red tilapia with ornamental and edible values.
Background
Traditional selective breeding aiming at important economic characters such as body color and the like of cultured fishes often adopts a cross breeding mode, and is quite time-consuming and labor-consuming. Among them, the most notable goldfish and koi have undergone thousands of years of artificial breeding history.
The CRISPR/Cas9 gene editing breeding technology is used as a high-efficiency and convenient molecular breeding means and is widely applied to genetic analysis of important economic traits of animals and plants and subsequent genetic breeding work. Four basic types of pigment cells (melanocytes, yellow pigment cells, iridescent cells and red pigment cells) are the basis of tilapia body color and fringe formation, and applicant has created tilapia as a good model for studying the body color formation mechanism of vertebrates by mutating tens of key genes for pigment cell differentiation and pigment synthesis and metabolism in nile tilapia by means of genetic editing breeding techniques in earlier studies (Wang, c, lu, b, li, t, liang, g, xu, m, liu, x, et al, 2021.Nile tilapia:a model for studying teleost color patterns.Journal of Heredity 112 (5), 469-484). The applicant then produced by homozygous mutations pmela and pmelb (Wang, C., xu, J., kocher, T.D., li, M., wang, D.,2022a.CRISPR knockouts of pmela and pmelb engineered agolden tilapia by regulating relative pigment cell abundance.Journal of Heredity 113 (4), 398-413), hps4 (Wang, C., kocher, T.D., lu, B., xu, J., wang, D.,2022b.Knockout of Hermansky-Pudlak Syndrome 4 (hps 4) lead to silver-white tilapia lacking melanomes.Aquaculture 559,738420), mitfa and mitfb (Wang, C., kocher, T.D., wu, J., li, P., liang, G., lu, B., xu, J., chen, X., wang, D.,2023.Knockout of microphthalmia-associated transcription factor (confers a red and yellow tilapia with few pigmented meophres, aquaculture), XU.74, B., lung, M., X., U.D., J., U.S., U.M., tan, d., tao, w., et al, 2022.Production of all male amelanotic red tilapia by combining MAS-GMT and tyrb mutation.aquaculture 546,737327) and establish a corresponding stable family to successfully create improved tilapia having a color such as gold, silver, red-yellow, and full-white, which is excellent in body color, and has both ornamental and edible value, or which can serve aquaculture for good ornamental and edible fish (Chen Songlin, wang Deshou, basket fries, cui Zhongkai, li Minghui.2023. Current and existing problems and prospects for chinese fish genome editing breeding).
Meanwhile, a natural red mutant also exists in tilapia, and the mutant is bred and promoted for over 80 years, wherein the price of pure red black spot-free tilapia can reach 1.5-2 times of that of wild tilapia in a plurality of countries and regions worldwide. However, as black spots are mostly generated in the breeding process of the red tilapia, the body color of the red tilapia is impure, so that the red tilapia with commodity specifications cannot be sold at a price higher than that of the wild black tilapia, the feed cost and the manpower resource are wasted greatly, and the development of the red tilapia industry is severely restricted. Although the applicant has created the optimized tilapia with golden, silvery, reddish yellow and other colors in the tilapia, the body color of the optimized tilapia is still relatively single compared with the various body colors and specks of other beautiful fish.
Disclosure of Invention
In view of the above, the invention creates a novel body-color-improved tilapia which is reddish and has ornamental and edible values by means of a gene editing breeding technology, and solves the key problems of nonuniform body color and single body color of red tilapia.
According to the invention, through CRISPR/Cas9 gene editing breeding technology, a key gene hps5 for synthesizing melanin is directionally mutated in wild black tilapia, and a red commercial tilapia is created. The body color of the tilapia mossambica is mainly caused by the increase and enlargement of yellow pigment cells, the deletion of melanin and the remarkable reduction of iridescent cells (complete deletion of early iridescent cells).
Since it has been confirmed in earlier studies that red and yellow of tilapia are mainly caused by the relative number and dimensional changes of yellow pigment cells and red pigment cells (Wang, c., kocher, t.d., wu, j., li, p., liang, g., lu, b., xu, j., chen, x., wang, d.,2023.Knockout of microphthalmia-associated transcription factor (mitf) confers a red and yellow tilapia with few pigmented melanophores.aquaculture 565,739151), the inability of the body colors of wild-type tilapia and red tilapia to appear as a flesh red is mainly caused by dense coverage of body surface iridescent cells, this rule of elimination of the relative amounts of several pigment cells and pigment synthesis ability of tilapia would theoretically create a completely new body color (e.g., flesh red) of tilapia. In the process of researching hps5, the inventor finds that the gene mutation not only can cause early body color transparency (iridescent cell deficiency, obvious reduction of melanocyte, insufficient melanin synthesis, and increase of yellow pigment cell), viscera, muscle and other tissues and organs of the tilapia, but also can change the tilapia mutant into stable meat red along with individual development even due to body wall thickening, melanin deficiency, increase of yellow pigment cell and small amount of restoration of iridescent cell. Meanwhile, the iris part of the eye of the nile tilapia with the hps5 homozygous mutation is partially restored from 90dpf but unevenly distributed due to iridescent cells, and meanwhile, melanin is deleted to present special patterns which have not been reported so far, and the patterns can further improve the overall ornamental value of the red tilapia.
The red tilapia created by the invention not only solves the problems of more black spots and impure body color on the surface of the red tilapia in the traditional sense, so that the red character of the tilapia is enhanced and purified, but also further increases the diversity of the body color of the tilapia, and the tilapia can serve as an excellent ornamental fish and edible fish for aquaculture. In addition, the tilapia is transparent in body color in early development, and can be used as a good pharmacological, pathological and developmental biology research model to serve basic medical research and development of related teaching research.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a production method of a meat red tilapia with ornamental and edible values, which comprises the following steps:
step 1: preparing hps5gRNA and Cas9 mRNA; the sequence of the hps5 gene is shown as SEQ ID NO. 1; wherein, the target sequence of hps5gRNA is GGTGCACCTGATTCAGAGGGAGGAGG is used as Palm recognition area.
The hps5gRNA was amplified using a plasmid (pMD 19-T gRNA scaffold vector) (Chang, N., sun, C, gao, L., zhu, D, xu, X, zhu, X, et al, 2013.Genome editing with RNA-guide Cas9 nuclease in zebrafish embryos. Cell Res. 23:465-472) as a template and hps5-F and Cas9-R as primers; hps5-F: TAATACGACTCACTATAGGTGCACCTGATTCAGAGGGGTTTTAGAGCTAGAAATAGC; cas9-R: AGCACCGACTCGGTGCCAC.
Step 2: the mixture of hps5gRNA and Cas9 mRNA is injected into fertilized eggs of tilapia, so as to knock out the hps5 of the tilapia. hps5gRNA and Cas9 mRNA were mixed at 500 ng/. Mu.L and 150 ng/. Mu.L concentrations in a 1:1 ratio by volume. In addition, when the fertilized egg grows to 1 cell stage, the mixture of hps5gRNA and Cas9 mRNA is injected into the fertilized egg of tilapia, so as to knock out hps5 of tilapia;
step 3: mating F0 generation hps5 mutant tilapia with wild tilapia to obtain F1 generation individual, selecting F1 male and female individuals with the same mutation type to obtain F2 generation individual, and screening from F2 generation individual to obtain hps5 -/- Mutants. hps5 -/- The mutants appeared to appear as a flesh red-translucent body color from 90dpf,at the same time, a stable, as yet unreported, special pattern of motifs is present.
The invention also provides application of the hps5 gene in regulating melanin synthesis and survival and growth of iridescent cells of tilapia and regulating growth and size of yellow pigment cells.
The invention also provides the application of the hps5 gene in regulating the coloring of retinal pigment epithelium or the color and pattern of iris.
The invention has the beneficial effects that:
according to the invention, through the CRISPR/Cas9 gene editing breeding technology in the tilapia, the key gene hps5 for controlling the survival of iridescent cells and melanin generation is successfully mutated, and the genetically stable body color optimized tilapia which is reddish in meat and has special pattern-like patterns on eyes is obtained. The tilapia can serve as potential ornamental fish and edible fish for aquaculture, and is also an important supplement for the situation of single body color of the tilapia. The red tilapia which reaches the commodity specification has little melanin except the base part of the dorsal fin, and other parts have no melanin at all, have uniform body color, have a certain degree of similarity with the natural red tilapia without melanin, and can solve or replace the difficult problem that black spots appear in the breeding process of the red tilapia so as to severely restrict the development of the tilapia industry. The tilapia with the meat red color can be used as a new bred fish fine variety to be promoted in a large area, and the breeding population is driven to increase income and become rich.
In addition, it is worth mentioning that the meat red mutant shows the whole transparent phenotype in early development stage due to the deficiency of iridescent cells and insufficient melanin synthesis, and the transparent tilapia in this stage has a small size, is highly similar to the phenotype of the model organisms such as transparent zebra fish, green and the like, and can be used as a research model of good developmental biology, pathology, toxicology and experimental pharmacology, and serve for genetic analysis of related problems in basic science research.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.
Drawings
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:
FIG. 1 shows the structure of tilapia hps5 gene and the process of creating homozygous mutant of the gene. African tilapia hps5 gene structure, target sequence and enzyme cleavage site information. The gene has 22 exons and 21 introns. The knockout target selected by the invention is positioned in the second exon region, and Hpy188I is taken as an enzyme cutting site. And B, schematic diagram of the cleavage effect of the hps5 target sequence of the restriction endonuclease. And C, a Morganal sequencing result of the base mutation type of the tilapia hps5 knock-down group. Wherein the orange region is a PAM region, the minus sign indicates the missing base, and WT is the wild type sequence. dHps 5 -/- PAGE electrophoretic screening of mutants. Wherein blue triangles represent mutant bands and blue arrows represent wild-type bands. Homozygous mutants do not possess wild-type bands, whereas heterozygous individuals possess both mutant and wild-type bands. E, establishing a line flow of the homozygous mutant. First, F0 positive fish is mated with wild type to obtain F1, and then F1 female and male individuals with the same mutation type are selected to obtain F2 through selfing. F, complete structural domain of tilapia HPS5 protein and incomplete structure of homozygous mutant. Wild-type tilapia has two WD40 domains (shown in green boxes), four low complexity regions (shown in yellow boxes) and one coiled-coil domain (shown in brown boxes). The complete tilapia HPS5 protein has 1122 amino acid sequences, and the tilapia HPS5 created by the invention -/- Mutants can only produce truncated proteins with 134 amino acid residues.
FIG. 2 shows tilapia hps5 -/- The mutant early (12 dpf) appears transparent due to insufficient melanin synthesis and the deletion of iridescent cells. A-C is wild tilapia. D-J hps5 -/- Mutants. The whole fish detects a complete transparent body color, and the head top has a small amount of melanin cells with insufficient pigment synthesis and no iridescent cells.At the same time iridescent cells are also absent from the trunk, iris and fin. K, N and O hps5 -/- Statistical analysis of the number of mutant and wild type overhead melanocytes, relative content of iris melanin, and relative content of iris iridescent cells. L, M hps5 -/- Quantitative analysis of the number and size of mutant and wild-type parietal yellow pigment cells. Data in K-O are represented by mean±sd (n=5). The difference between wild type and mutant was measured by two-stable Student's t-test, P<0.001。
FIG. 3 is 30dpf tilapia hps5 -/- The mutants appeared clear-red in body color due to the absence of iridescent cells and melanin. A-C is wild tilapia. D-F hps5 -/- Mutants. G, H, wild-type and hps5 -/- Statistical analysis of the number of melanocytes in the mutant striation and spacer striation. Wild type and hps5 -/- Statistical analysis of the relative content of iridescent cells in the mutant fringe areas and the spacer fringe areas. Data in G-J are represented by mean±sd (n=5). The difference between wild type and mutant was measured by two-stable Student's t-test, P<0.001。
FIG. 4 is a3 month old hps5 with ontogenesis -/- The iridescent cell part of the mutant is restored, the body color is in a meat red-semitransparent shape, and meanwhile, the iris shows a special pattern. A, A ', C, D, C ' and D ' are wild tilapia. B, B ', E, F, E ' and F ': hps5 -/- Mutants.
FIG. 5 is 5 months of age hps5 -/- The tilapia mutant shows stable flesh red character due to restoration of white iridescent cells and deletion of melanin. A, C, D, D' and E are wild type tilapia. B, F, G, G' and H: hps5 -/- Mutants. I-K wild-type and hps5 -/- Statistical analysis of the relative content of pigmented melanocytes, red and yellow pigment cells and iridescent cells in the mutant tail fins. L and M wild type and hps5 -/- Statistical analysis of the number of pigmented melanocytes and yellow pigment cells in the back scale of the mutant. N wild-type and hps5 -/- Statistical analysis of melanin content in the mutant dorsal skin. Data in I-M are represented by mean±sd (n=5). The difference between wild type and mutant was through two-linkedStudent's t-test, P<0.001, ns, the difference is not significant.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Example 1
1. Amplification of fragments of interest in tilapia hps5 DNA
Tilapia hps5 DNA sequence comprising 12829 bases as shown in SEQ ID NO. 1 was downloaded in NCBI and Ensemble public databases. The tilapia hps5 gene has 22 exons and 21 introns, as shown in fig. 1A; the gene encodes 1122 amino acids, with two WD40 domains, four low complexity domains and one coiled-coil domain.
Designing website through NCBI on-line primerhttps://www.ncbi.nlm.nih.gov/tools/primer- blast/index.cgi? LINK_LOC=BlastHome) is introduced into a target fragment sequence containing a knockout target point in tilapia hps5 DNA (the target sequence is positioned in the center of the target sequence), a specific amplification primer is designed, the F primer sequence is GCCACATGAACCGCACTTTG, and the R primer sequence is GCTAACCTTGTTGCAACCGC.
Shearing the tail fin end of wild nile tilapia, mashing, digesting with 20-30 mu L of trypsin, separating, extracting and purifying genome by using a DNA extraction kit according to the specification steps, drying, and adding 100 mu L of enzyme-free water for dissolving for later use. And genome amplification is carried out by utilizing the F/R primer sequence designed in the above way, so that a target segment including a target point in wild tilapia hps5 DNA is obtained, and sequencing and sequence comparison of mutants and wild individuals are facilitated for the target segment in the wild hps5 DNA.
2. Preparation of hps5gRNA and Cas9 mRNA
SEQ ID NO:1 in zifit.parts. Org/ZiFiT/ChoiceMenu. AspxThe hps5 gene sequences shown were targeted for gene knockout site selection and target sequence design. Selected hps5 exon 2 (exon) design knockout target with target sequence GGTGCACCTGATTCAGAGGGAGGAGG is used as Palm recognition area. Plasmid (pMD 19-T gRNA scaffold vector) template amplification was performed in a 60. Mu.L system. Wherein the forward primer (hps 5-F: TAATACGACTCACTATAGGTGCACCTGATTCAGAGGGGTTTTAGAGCTAGAAATAGC) comprises a T7 polymerase binding site, a 20bp hps5gRNA target sequence and a partial sequence of a gRNA scaffold; the reverse universal primer (Cas 9-R: AGCACCGACTCGGTGCCAC) is located at the 3' end of the gRNA scaffold. The amplification reaction solution is prepared according to the following system:
2 XTaq enzyme 30μL
hps5-F 1.2μL
Cas9-R 1.2μL
Universal plasmid template (pMD 19-TgRNA scaffold vector) 800ng
DDW up to 20.0μL
And then agarose gel electrophoresis is adopted to detect whether the plasmid template is successfully amplified, and if so, the gel recovery of the target fragment is carried out. The mass of the gel recovered product was measured, in vitro transcription was performed using Megascript T7 kit (Ambion, USA), and 500ng of the purified target fragment (gel recovered product) was added as a template at 37℃and reacted for 4 hours to obtain hps5gRNA.
The Cas9 nuclease expression vector pcdna3.1 (+) (Invitrogen) is used for in vitro transcription of Cas9 messenger RNA (mRNA). Plasmid templates were prepared using plasmid midi kit, linearized with XbaI and purified by ethanol precipitation. Cas9 mRNA was produced by in vitro transcription of 1 μg DNA using T7 mMESSAGE mMACHINE kit (Ambion, USA) according to the manufacturer's instructions. The resulting mRNA was purified using MegaClear kit (Ambion, USA), suspended in RNase-free water, and quantified using NanoDrop-2000.
3.Knockout of tilapia hps5
The synthesized hps5gRNA and Cas9 mRNA were mixed at concentrations of 500 ng/. Mu.L and 150 ng/. Mu.L in a volume ratio (v 1: v 2) of 1:1, and 0.5% phenol red was added as an indicator.
After spawning of nile tilapia, tilapia total female (XX) and total male (XY) fertilized eggs were divided into two groups (each group containing both total female and total male fertilized eggs), and when developed to the 1-cell stage, the first group was injected with a phenol red-added mixture of hps5gRNA and Cas9 mRNA knockdown hps5 under a Nikon microinjection system, and the other group was injected with a synthetic pCS2 empty mRNA (Chang, n., sun, c., gao, l., zhu, d., xu, x., zhu, x., et al, 2013.Genome editing with RNA-guiided Cas9 nuclease in zebrafish embryos.cell res.23:465-472 and Cong, l., ran, f.a., cox, d., lin, s., barretto, r., habib, n., et al, 2013.Multiplex genome engineering using CRISPR/Cas science.819-823) as negative controls. The fertilized eggs after injection are hatched in a constant temperature circulating water hatching system at 26 ℃. 72h after injection, respectively collecting 20 knocked-out groups, a control group and wild tilapia embryos in a centrifuge tube, removing yolk, respectively adding 600 mu L of STNE lysate and 20 mu L of proteinase K, and performing overnight pyrolysis at 55 ℃ and 180 rpm; extracting genome DNA by adopting a phenol/chloroform/isoamyl alcohol method, and performing CRISPR/Cas9 activity verification; PCR amplification was performed in a 60. Mu.L format using specific primers (F/R) for target site detection designed on and downstream of the hps5 target site. Specific primers for target site detection were designed via www.ncbi.nlm.nih.gov/tools/primer-blast/index. Cgilink_loc=blasthome website, and amplified fragment length was 477bp. F primer is 20bp long and the sequence is GCCACATGAACCGCACTTTG; the R primer is 20bp long and has a sequence of GCTAACCTTGTTGCAACCGC, and is identical to the primer for amplifying the target fragment in the hps5 DNA.
Subjecting the amplified fragment to 1.5% agarose gel electrophoresis (180V, 20 min); cutting gel, purifying and recovering DNA target fragment; the DNA fragments of the knockdown group, the control group and the wild tilapia are digested with specific endonuclease Hpy I (the digestion site is positioned on the target sequence), and the digestion conditions are as follows: and the enzyme digestion is carried out for 4 hours at 37 ℃. The enzyme digestion reaction liquid is prepared according to the following system:
CutSmart Buffer 2.0μL
Hpy188I(2000U/mL) 0.3μL
DNA Fragment 400.0ng
DDW up to 20.0μL
and then detecting whether an uncut strip with enzyme cutting site mutation exists by agarose gel electrophoresis, wherein the presence of enzyme cutting site mutation is the knockout group. As shown in fig. 1B, the simultaneous addition of hps5gRNA and Cas9 mRNA group successfully achieved hps5 gene editing.
The uncut strips in the results of the enzyme digestion detection by gel electrophoresis were cut, recovered, ligated, subcloned into the usual vector pMD-19T, and positive clones were identified by the universal primer M13 +/-colony PCR, the colony PCR system was as follows:
2×Taq Enzyme 7.5μL
M13+ 0.3μL
M13- 0.3μL
DDW 6.9μL
designing a PCR program: stage 1 (1 cycle): 95 ℃ for 3min; stage 2 (28 cycles): 95 ℃ for 30s;60 ℃ for 30s;72 ℃,30s; stage 3 (1 cycle) 72℃for 10min; amplifying at 4 ℃ for 10min; and (3) electrophoresis, selecting 10 positive clones respectively, and carrying out gene sequencing to determine the mutation type of the target site sequence. As shown in FIG. 1C, multiple mutation types are detected in the positive clone of tilapia, a-4 bp non-frameshift mutation male and female individual with the most mutation types in F1 is selected, selfed and screened to obtain hps5 with 134 truncated proteins only -/- Mutants are shown in FIGS. 1D to 1F.
By passing tilapia hps5 -/- Mutant and wild tilapia observations:
(1) 12dpf tilapia hps5 -/- The mutant pigment cells were significantly different from the wild type individuals.
The trunk and head of wild tilapia detect a large number of uniformly distributed melanocytes; retinal pigment epithelium (retinal pigment epithelium, RPE) is black, while a large number of melanocyte and iridescent cell distributions are detected in the iris (fig. 2A); the schematic enlarged top head of wild tilapia shows giant melanocytes (macro-melanophores) and iridescent cells (fig. 2B); wild-type tilapia RPE has a large number of melanosomes in its RPE, while iris also has a large number of melanocytes and iridescent cells in its iris (fig. 2C).
Unlike the wild type, 12dpf hps5 -/- The mutant whole fish had significantly increased and enlarged yellow pigment cells (fig. 2D); an enlarged schematic view of the head top of the whole fish shows that melanocytes are greatly reduced and show a significant lack of melanin synthesis (shown by black triangles) while yellow pigment cells are significantly increased and enlarged (shown by yellow triangles) (fig. 2E); the RPE melanin synthesis was inadequate, dark red, but still had a small number of melanosomes, which were also detected by the iris but less than the wild-type (fig. 2F); hps5 -/- The mutant exhibits a systemic transparent character while the peritoneum is also highly transparent, thus exhibiting color and location including internal organs such as liver and intestinal tract (fig. 2G and 2H); interestingly, 12dpf hps5 -/- Mutants showed more accurate intestinal morphology and orientation after open ingestion (figures 2I and 2J).
hps5 -/- Statistical analysis of mutant and wild type head-top melanocyte numbers, iris melanin relative content, and iris iridescent cell relative content is shown in FIGS. 2K, 2N and 2O, hps5 -/- Quantitative analysis of the number and size of mutant and wild-type parietal yellow pigment cells is shown in FIGS. 2L and 2M.
(2) As shown in FIG. 3, 30dpf tilapia hps5 -/- Mutant melanocytes and iridescent cells were significantly reduced.
A large number of melanocytes were detected in wild-type tilapia, wherein the number of melanocytes in black vertical lines was greater than in light-colored spacer areas; at the same time, a large number of iridescent cells were detected in both the vertical and the spacer areas (fig. 3a, b); the RPE is filled with melanin while a large number of yellow pigment cells and iridescent cells are also detected in the iris (fig. 3C).
30dpf hps5 -/- The mutant exhibited a completely transparent body color, while hypertrophied swimming bladder structures (indicated by arrows) were observed (fig. 3D); none of the torso and fin is detectedMelanocytes and iridescent cells of color (fig. 3E); the RPE has no melanin and appears dark red, but the iris has a large number of melanosomes and appears black; simultaneous hps5 -/- The presence of the dark red RPE and black iris in the mutant has not been observed to date in any other tilapia body color mutant (fig. 3F).
Wild-type and hps5 -/- The results of the statistical analysis of the numbers of melanocytes in the mutant striation regions and the spacer striation regions are shown in fig. 3G and 3H. Wild-type and hps5 -/- The results of statistical analysis of the relative amounts of iridescent cells in the mutant streak regions and the spacer streak regions are shown in FIGS. 3I and 3J.
(3) As shown in FIG. 4, 90dpf tilapia hps5 -/- The mutant part of iridescent cells are restored, and the eyes are provided with special floral patterns.
As shown in fig. 4a, a ', C, D, C ' and D ', wild tilapia exhibits dark vertical lines and light spacing lines; a large number of mature melanosomes are detected by both RPE and iris; whether or not the eye moves, the entire eye always appears stable black, suggesting that there is a large number of mature melanosomes in the RPE.
As shown in fig. 4b, b ', E, F, E ' and F ', 90dpf hps5 -/- Mutants exhibited a reddish-translucent body color due to a small restoration of iridescent cells and a gradual thickening of the body wall. The RPE is dark red, small amount of melanosomes can be detected, large amount of white iridescent cells are detected in the iris, and a stable, as yet unreported special pattern appears. The whole eye is thus in a particular state of coloration. Although the RPE is a condition in which a small amount of melanin is recovered with ontogenesis, even slight eye movements may cause the RPE to exhibit a "red" to "black" or a "black" to "red" color "transition due to a small amount of melanin and maldistribution.
(4) 150dpf tilapia hps5 -/- The mutant and the wild type have significant differences in the composition and size of the pigment cells.
As shown in fig. 5a, c, d' and E, wild tilapia exhibited stable dark vertical lines and corresponding light spacing lines; the RPE coloration deepens while a large number of melanocytes are detected in the iris; a large number of melanocytes and yellow pigment cells were detected in the tail fin and scale.
As shown in FIGS. 5B, F, G' and H, 150dpf hps5 -/- The mutants showed a reddish body due to a small restoration of iridescent cells, thickening of the body wall and loss of melanin; whereas the RPE appears black due to slow recovery of melanosomes; no pigmented melanocytes but white iridescent cells were detected in the tail fin, while distinct white streaks and reddish fin edges were observed; the number of the yellow pigment cells in the scales is obviously increased compared with that of the wild type, meanwhile, the interval between the yellow pigment cells is obviously reduced (shown by a purple dotted line), and even part of the yellow pigment cells are in a pair aggregation distribution (shown by a purple triangle).
Wild-type and hps5 -/- The results of statistical analysis of the relative amounts of pigmented melanocytes, red and yellow pigment cells and iridescent cells in the mutant tail fins are shown in fig. 5I-K. Wild-type and hps5 -/- The results of the quantitative analysis of the pigmented melanocytes and yellow pigment cells in the back scales of the mutants are shown in fig. 5L and 5M. Wild-type and hps5 -/- The results of the statistical analysis of melanin content in the mutant dorsal skin are shown in figure N, which shows that: there was sufficient melanin in the wild type tilapia dorsal skin, whereas no melanin was detected in the mutant.
Overall, the invention discovers that tilapia hps5 has the function of regulating melanin synthesis and survival and multiple functions of iridescent cells. Meanwhile, the hps5 gene in tilapia can regulate the size and the number of the yellow pigment cells. Knocking out the gene leads to insufficient synthesis of early melanin of tilapia, and the later period is completely free of melanin, but conversely, yellow pigment cells are obviously increased and enlarged; at the same time, with the restoration of part of white iridescent cells by the ontogenesis mutation, the whole fish finally shows a meat red phenotype. It should be noted that hps5 not only regulates the pigmentation of the retinal pigment epithelium, but also regulates the color and even the pattern of the iris. The gene is characterized in that the homozygote mutant of the tilapia mossambica has insufficient melanin synthesis of retinal pigment epithelium, and the iris shows partial iridescent cell recovery along with the development of individuals, and simultaneously has uniform special patterns due to no melanin. The pattern is a novel special pattern which has not been reported so far, and the formation mechanism of the pattern is still to be further analyzed. Also based on the above results, it is speculated that if iridescent cells are unable to recover during development, the body color of tilapia will be determined by the yellow pigment cells and the red pigment cells, plus a thickened red muscle layer, and whole fish will eventually exhibit a uniform bright red or semi-transparent-bright red color.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.

Claims (6)

1. The production method of the meat red tilapia with ornamental and edible values is characterized by comprising the following steps of:
step 1: preparing hps5gRNA and Cas9 mRNA; the sequence of the hps5 gene is shown as SEQ ID NO. 1;
step 2: injecting the mixture of hps5gRNA and Cas9 mRNA into fertilized eggs of tilapia, and knocking out the hps5 of the tilapia;
step 3: mating F0 generation hps5 mutant tilapia with wild tilapia to obtain F1 generation individual, selecting F1 male and female individuals with the same mutation type to obtain F2 generation individual, and screening from F2 generation individual to obtain hps5 -/- Mutants.
2. The method of claim 1, wherein in step 1, the target sequence of hps5gRNA is GGTGCACCTGATTCAGAGGGAGGAGG is used as Palm recognition area.
3. The method of claim 1, wherein in step 2, the hps5gRNA and Cas9 mRNA are mixed at a concentration of 500ng/μl and 150ng/μl in a ratio of 1:1 by volume.
4. The method according to claim 1, wherein in step 2, the mixture of hps5gRNA and Cas9 mRNA is injected into the fertilized eggs of tilapia, and the knockdown of hps5 of tilapia is performed when the fertilized eggs develop to 1 cell stage.
The hps5 gene is applied to regulating melanin synthesis and survival and multiple of iridescent cells of tilapia and regulating multiple and size of yellow pigment cells, and the sequence of the hps5 gene is shown as SEQ ID NO. 1.
The application of hps5 gene in regulating and controlling the coloring of retinal pigment epithelium or the color and pattern of iris, and the sequence of hps5 gene is shown as SEQ ID NO. 1.
CN202311238746.1A 2023-09-22 2023-09-22 Production method of meat red tilapia with ornamental and edible values Pending CN117327740A (en)

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