Method for genetic reactivation of individual of carassius auratus and application thereof

Abstract

The application provides a method for genetic reactivation of individual Procyprinus palpebrosus, comprising the following steps: S1, taking the gonadal tissue of Procyprinus palpebrosus, washing, cutting, adding L-15 culture medium containing double antibodies and DPBS buffer solution to centrifuge the gonadal tissue; S2, water-bathing the gonadal tissue to obtain recovered gonadal tissue; S3, digesting the recovered gonadal tissue, filtering, centrifuging and purifying to obtain germ stem cells; S4, injecting morpholino to obtain rare gobi-ocypris rarus with deleted primordial germ cells; S5, transplanting the germ stem cells into the body of the rare gobi-ocypris rarus juvenile to culture, and obtaining the gametes of Procyprinus palpebrosus; and S6, fertilizing the generated gametes of Procyprinus palpebrosus to obtain the individual Procyprinus palpebrosus. The application successfully obtains the sperm of Procyprinus palpebrosus by using the cryopreserved gonadal tissue and the technology of parthenogenesis, and provides a feasible way for the individual genetic reactivation of rare and endangered fish species.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal genetic breeding technology, specifically relating to a method for genetic resurrection of an individual Chinese paddlefish and its application. Background Technology

[0002] Rougefish ( Myxocyprinus asiaticus Also known as bloodletting, it belongs to the order Cypriniformes ( Cypriniformes ), family Corydalis ( Catostomidae The Chinese paddlefish (Pterodon spp.), known as the "Asian Mermaid" for its vibrant colors and graceful form, is a rare freshwater fish endemic to Asia. Primarily distributed in the Yangtze and Minjiang River basins of my country, it is listed as a Class II protected wild animal in China. The wild population of the Chinese paddlefish is even lower than that of other Class I protected species such as the Chinese sturgeon and the Dabry's sturgeon, highlighting the severity of its endangered status. The survival pressure on the Chinese paddlefish also stems from its reproductive characteristics: late sexual maturity (usually requiring 5-6 years), long reproductive cycle, slow embryonic development, and extremely low natural survival rate. These factors collectively make it difficult for the population to recover naturally once damaged. Currently, conservation strategies for the Chinese paddlefish mainly focus on artificial propagation and release. To address the problems of low natural reproductive efficiency and slow population recovery, artificial reproduction, as a highly promising and efficient breeding strategy for fish, is expected to accelerate the reproductive process by utilizing the rapid reproductive characteristics of model fish, thus providing a new technological path for the resource conservation and genetic improvement of rare fish species.

[0003] Surrogacy, or transspecific reproduction, refers to a systematic method of transplanting spermatogonial stem cells (SSCs) or oogonial stem cells (OSCs) from one fish species into the gonads of another, utilizing the germline chimera to produce donor-derived gametes. Surrogacy generally includes the following components: isolation and purification of donor reproductive stem cells, removal of endogenous germ cells from the recipient fish, transplantation of reproductive stem cells, and gametogenesis and gamete production in the recipient fish. The breakthrough of this technology lies in breaking down species reproductive barriers, achieving cross-species gamete production through transspecific surrogacy. This technology has broad application prospects in aquaculture and genetic breeding. First, it can significantly shorten the genetic breeding cycle of fish, greatly improving breeding efficiency. For example, fish with sexual maturity cycles of several years (such as sturgeon and large yellow croaker) can reproduce rapidly using recipient fish with shorter sexual maturity cycles, such as zebrafish and tilapia, increasing breeding efficiency by 5-10 times. Second, this technology can also be used to protect endangered species. For example, the Yangtze River Fisheries Research Institute transplanted spermatogonial stem cells from the paddlefish (Acipenser sinensis), a species belonging to the same family as the Chinese paddlefish, into the Yangtze sturgeon, achieving a recipient chimerism rate of 85.7%. This study suggests that surrogate reproduction technology holds promise for the revival of endangered species. Japan has successfully bred the mountain trout (Acipenser sinensis) through rainbow trout, providing a new approach to the protection of endangered fish species. Furthermore, cryopreserving reproductive stem cells of endangered fish and then using surrogate reproduction to restore populations through recipient fish provides an important experimental model for the diversity of fish reproduction and for studying fish reproductive biology.

[0004] Previous studies have explored the feasibility of combining gene editing with spermatogonial stem cell transplantation between two small model fish species, the rare gudgeon and the zebrafish, as donor and recipient. These studies demonstrated that SSCs from the rare gudgeon can colonize, proliferate, differentiate, and reconstruct spermatogenesis in zebrafish, ultimately leading to the rapid production of donor-derived functional sperm. However, due to the lack of a genetic sex-determining system in zebrafish, their sex differentiation is drastically affected by the number of germ cells, making it nearly impossible to obtain donor eggs when using zebrafish as recipients for surrogate reproduction. Furthermore, the extremely low sperm production of zebrafish further limits their practical application potential in surrogate reproduction technology. Summary of the Invention

[0005] In view of this, the present invention selects the rare Chinese gudgeon, which has a clear genetic sex system, as the recipient and the Chinese paddlefish, which is from a different family, as the donor to carry out surrogate reproduction research, and finally produce sperm from the Chinese paddlefish that has reproductive function.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for genetic resurrection of an individual Chinese paddlefish includes the following steps:

[0008] S1, taking the gonad of the Sinibrama macrops, washing, cutting, adding L-15 culture medium containing double antibodies and DPBS buffer; wherein, when the gonad is testis, repeated centrifugation is needed to remove the mature sperm in the upper layer until the supernatant is clear without sperm, and the obtained sediment is the testis tissue; when the gonad is ovary, the ovum does not need to be removed, and the ovary tissue is obtained;

[0009] S2, water bath of the gonad tissue obtained in S1, dropwise adding L-15 culture medium containing 10% FBS and double antibodies, mixing while adding, centrifuging, removing supernatant, washing, obtaining recovered gonad tissue;

[0010] S3, adding DMEM culture medium containing trypsin and collagenase to the recovered gonad tissue in S2 for digestion, filtering, and purifying the filtrate by Percoll density gradient centrifugation to obtain reproductive stem cells;

[0011] S4, designing morpholino for the dead end gene of the Procypris merus and injecting to obtain the Procypris merus with deleted primordial germ cells and as a recipient fish;

[0012] S5, transplanting the reproductive stem cells obtained in S3 into the juvenile fish of the recipient fish in S4 for culture to obtain Sinibrama macrops sperm;

[0013] S6, fertilizing the Sinibrama macrops sperm obtained in S5 with Sinibrama macrops ovum to obtain Sinibrama macrops individuals.

[0014] Further, the washing in step S1 is performed using L-15 culture medium, and the double antibodies are ampicillin and streptomycin.

[0015] In some specific embodiments, preferably, the dosage ratio of L-15 culture medium containing double antibodies and DPBS buffer in step S1 is 1:3;

[0016] The centrifugation condition is 160-180 rpm for 1 min each time.

[0017] Further, the water bath condition in step S2 is 35℃ for 2-3 min.

[0018] The volume ratio of gonad tissue to L-15 culture medium containing 10% FBS and double antibodies is 1:10.

[0019] The centrifugation condition is 160-180 rpm for 1.5-2.5 min.

[0020] The washing is performed using DMEM culture medium.

[0021] In some specific embodiments, preferably, the trypsin content in each milliliter of DMEM culture medium in step S3 is 0.25%, and the collagenase activity is 0.26 U.

[0022] Digestion condition: 35℃ water bath digestion for 50~80min.

[0023] Filter using a 40µm mesh.

[0024] In some embodiments, preferably, the digestion process also includes blowing several times with a pipette every 15min to fully disperse the cells.

[0025] In some embodiments, preferably, the morpholino sequence in step S4 is: 5'-CTTCATAAGCGGATAACGACATGGA-3'.

[0026] In some embodiments, preferably, the produced sperm is also subjected to PCR detection in step S5, and the amplification primers used in the PCR detection process are as follows:

[0027] Detection of rare gobiocypris rarus sperm primers:

[0028] Gr-ZFH-actin-spe-F: 5'-GGAACTAGGAGGCAAAGT-3';

[0029] Gr-ZFH-actin-spe-R: 5'-GTATGCCCACTGTGATGAG-3';

[0030] Detection of sperm primers of mylopharyngodon piceus:

[0031] Mas-sltm-gene-F: 5'-GATATCTGTGGACAGGGTAGGAGC-3';

[0032] Mas-sltm-gene-R: 5'-GGCTTTATACTGCAGCCAAAGAG-3'.

[0033] The above method is applied in the resurrection and breeding of mylopharyngodon piceus.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] The present application uses gobiocypris rarus with a clear genetic sex system as the recipient, and mylopharyngodon piceus across families as the donor to carry out brood parasitism research, and finally produces sperm with reproductive function and of mylopharyngodon piceus origin. This technology not only solves the problem of low natural reproduction efficiency and slow population recovery of mylopharyngodon piceus, but also fully utilizes the high sperm production characteristics of gobiocypris rarus, and is more suitable for large-scale production practice, thereby improving the breeding efficiency and production quality. This achievement provides a new technical path for the resource protection and genetic improvement of rare fish species. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 A percoll centrifugation chart for the germline stem cells prepared in Example 1.

[0037] Figure 2 A fluorescence chart for the germline stem cells traceable 5 days after transplantation in Example 1.

[0038] Figure 3 A chart of PCR detection results of sperm produced by the Procypris perlicus in Example 1.

[0039] Figure 4 A chart of whole genome sequencing results of sperm of the recipient fish aligned to the Procypris perlicus genome in Example 1.

[0040] Figure 5 A chart of whole genome sequencing results of sperm of the recipient fish aligned to the Myxocyprinus asiaticus genome in Example 1. DETAILED DESCRIPTION

[0041] The present application will be further described in conjunction with specific embodiments so that those skilled in the art better understand the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available.

[0042] Example 1

[0043] The present embodiment provides a method for genetic resurrection of an individual of the rare and endangered fish Myxocyprinus asiaticus, comprising the following steps:

[0044] 1. Obtaining and cryopreserving gonadal tissue of Myxocyprinus asiaticus

[0045] (1) Take the testis of Myxocyprinus asiaticus, weigh and record.

[0046] (2) Wash with L-15 medium, then put it into 500 μL L-15 medium containing double antibodies (ampicillin and streptomycin) and cut the tissue with scissors; then add 1.5 mL pre-cooled DPBS buffer, centrifuge at 180 rpm for 10 s, at this time the tissue block is deposited at the bottom and the sperm is suspended in the supernatant, and 1.5 mL supernatant containing mature sperm is aspirated.

[0047] (3) Repeat step (2) several times until the supernatant is clear and there is no sperm, and try to aspirate the supernatant completely in the last wash, and the sediment is the testis tissue.

[0048] (4) Add 500 μL cryoprotectant to the testis tissue and place it on ice for 50 min to reduce the toxicity of the cryoprotectant to the cells and to balance the testis tissue sample in the cryoprotectant.

[0049] (5) After the testis tissue sample is placed in the programmed cooling box, it is placed in a -80°C refrigerator overnight, and then the testis tissue sample is transferred to liquid nitrogen for long-term storage.

[0050] 2. Revival of the testis tissue of Mylopharyngodon piceus

[0051] (1) The testis tissue sample obtained in the previous step is treated in a water bath at 35°C for 5 min, then transferred to a 5 mL tube, and 10 times the volume of L-15 + 10% FBS medium (with double antibodies) is slowly added dropwise at room temperature, each time by dropwise addition, and mixed after each addition.

[0052] (2) After the dropwise addition is completed and mixed, centrifuge at 180 rpm for 2 min, remove the supernatant, and wash the precipitate twice with L-15 medium containing double antibodies to obtain the revived testis tissue.

[0053] 3. Isolation and purification of Mylopharyngodon piceus germ stem cells

[0054] Add 1 mL of L-15 medium (Sigma-Aldrich) containing 0.25% trypsin (Invitrogen) and collagenase (Roche) with an enzyme activity of 0.26 U to the revived testis tissue, and digest at room temperature for 1 h. During the digestion process, the cells are dispersed by blowing with a 1 mL pipette every 15 min. After digestion, filter with a 40 μm mesh, and then purify the germ stem cells (GSCs) in the filtrate by Percoll density gradient centrifugation method Figure 1 ).

[0055] To determine the enrichment position of GSCs in the Percoll gradient, the interface of each layer from 20% to 60% density was detected by cell viability detection, and the cells in the gradient layer with the most germ cells were selected as donors for GSCs experiments.

[0056] 4. Preparation of fish as a surrogate for parthenogenesis

[0057] In order to completely eliminate its own primordial germ cells (PGCs) and not compete with exogenous PGCs, and to better colonize in early stages, a morpholino (MO: 5'-CTTCATAAGCGGATAACGACATGGA-3') was designed for the dead end gene of Gobiocypris rarus, and the method of early knockdown of dead end was used to eliminate its PGCs (Weidinger et al., 2003, Zhang et al., 2020).

[0058] 5. Obtaining sperm of Mylopharyngodon piceus

[0059] The purified GSCs of the Mylopharyngodon piceus were transplanted into the juvenile Gobiocypris rarus whose endogenous germ cells were effectively eliminated, and the fluorescence of the Mylopharyngodon piceus germ stem cells was tracked at the position of the germ ridge of the recipient on the 5th day after transplantation Figure 2 After 3-4 months of careful feeding, the gametes of the Mylopharyngodon piceus were obtained. Further, in order to verify whether the sperm produced is that of the Mylopharyngodon piceus, the sperm of 3 sexually mature Gobiocypris rarus recipient fish were taken and the genes were extracted, and PCR detection was performed using specific primers, and the results showed that 2 of the 3 sexually mature Gobiocypris rarus recipient fish could produce sperm of the Mylopharyngodon piceus (test results are shown in Figure 3 , wherein Gr, Gr actin represents Gobiocypris rarus sperm, Mas, Mas sltm represents Mylopharyngodon piceus sperm, and M represents Maker).

[0060] The primers for detecting Gobiocypris rarus sperm are as follows:

[0061] Gr-ZFH-actin-spe-F: 5'-GGAACTAGGAGGCAAAGT-3';

[0062] Gr-ZFH-actin-spe-R: 5'-GTATGCCCACTGTGATGAG-3';

[0063] The primers for detecting Mylopharyngodon piceus sperm are as follows:

[0064] Mas-sltm-gene-F: 5'-GATATCTGTGGACAGGGTAGGAGC-3';

[0065] Mas-sltm-gene-R: 5'-GGCTTTATACTGCAGCCAAAGAG-3'.

[0066] The PCR reaction system is as follows:

[0067]

[0068] The PCR reaction program is as follows:

[0069]

[0070] Meanwhile, the genome of the sperm of the Gobiocypris rarus recipient fish was sent to a company for whole genome sequencing, and the sequencing results showed that the similarity of the genome of the sperm of the Gobiocypris rarus recipient fish to the genome of the Gobiocypris rarus was only 58.37% ( Figure 4 ), and the similarity to the genome of the Mylopharyngodon piceus was as high as 99.93% ( Figure 5 ), which indicated that the sperm produced by the recipient fish was the sperm of the Mylopharyngodon piceus.

[0071] 6. Obtaining of Mylopharyngodon piceus individual

[0072] The sperm of the rosy bitterling obtained by the above step is fertilized with the egg of the rosy bitterling, and the rosy bitterling individual is obtained by culture.

[0073] The above series of studies show that the Chinese characteristic experimental fish, the rare gobi-Pracahu, which has a clear genetic sex system, is selected as the receptor, and the cross-species rosy bitterling is selected as the donor to carry out the belly borrowing reproduction research, so that the reproductive cells from the rosy bitterling can be planted and differentiated in the microenvironment of the gonad of the rare gobi-Pracahu, and finally the sperm from the rosy bitterling with reproductive function is produced. Further, the sperm obtained by belly borrowing reproduction is fertilized with the egg of the rosy bitterling to obtain the rosy bitterling individual, which provides a feasible way for the individual genetic revival of rare and endangered fish.

[0074] The specific raw materials in the present application are all existing substances, which can be directly purchased from the market.

[0075] The above is only a preferred embodiment of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for genetic resurrection of an individual of Xiphophorus, characterized in that, The method comprises the following steps: S1, taking the gonad of the Liuyan fish, washing, cutting, adding L-15 culture medium containing double antibodies and DPBS buffer; wherein, when the gonad is testis, repeated centrifugation and removal of the mature sperm in the upper layer are needed until the supernatant is clear and free of sperm, and the obtained sediment is the testis tissue; when the gonad is ovary, the ovum does not need to be removed, and the ovary tissue is obtained; S2, water bath is carried out on the gonad tissue obtained in S1, and L-15 culture medium containing 10% FBS and double antibodies is added drop by drop, mixed, centrifuged, and the supernatant is removed, washed, and the recovered gonad tissue is obtained; S3, DMEM culture medium containing trypsin and collagenase is added to the recovered gonad tissue in S2 for digestion, filtration, and the filtrate is purified by Percoll density gradient centrifugation to obtain the germ stem cells; S4, for gobiocypris rarus dead end Morpholino was designed according to the gene, and injection was performed to obtain gobiocypris rarus with deleted primordial germ cells and as a recipient fish; S5, the germ stem cells obtained in S3 are transplanted into the recipient fish fry in S4, and culture is carried out, and the Liuyan fish sperm is obtained; S6, the Liuyan fish sperm obtained in S5 is used for fertilization with the ovum of the Liuyan fish, and culture is carried out, and the Liuyan fish individual is obtained; The frozen gonad of the Liuyan fish in step S1; The washing in step S1 is carried out by using L-15 culture medium containing double antibodies, and the double antibodies are ampicillin and streptomycin; The dosage ratio of L-15 culture medium containing double antibodies and DPBS buffer in step S1 is 1:3; The centrifugation condition each time is 160-180 rpm for 1 min; The water bath condition in step S2 is 35℃ for 2-3 min; The volume ratio of the gonad tissue to L-15 culture medium containing 10% FBS and double antibodies is 1:10; The centrifugation condition is 160-180 rpm for 1.5-2.5 min; The washing is carried out by using DMEM culture medium; The trypsin content in DMEM culture medium in step S3 is 0.25% per milliliter, and the collagenase activity is 0.26 U; The digestion condition is 35℃ water bath digestion for 50-80 min; The filter uses a sieve with a pore size of 40 µm; The digestion process further comprises blowing several times with a pipette gun every 15 min to fully disperse the cells; The morpholino sequence in step S4 is 5'-CTTCATAAGCGGATAACGACATGGA-3'.

2. The method of claim 1, wherein, The sperm produced in step S5 is further detected by PCR, and the amplification primers used in the PCR detection process are as follows: Detection of rare Gobiocypris rarus sperm primers: Gr-actin-spe-F: 5'-GGAACTAGGAGGCAAAGT-3'; Gr-actin-spe-R: 5'-GTATGCCCACTGTGATGAG-3'; Detection of Liuyan fish sperm primers: Mas-sltm-gene-F: 5'-GATATCTGTGGACAGGGTAGGAGC-3'; Mas-sltm-gene-R: 5'-GGCTTTATACTGCAGCCAAAGAG-3'.

3. Use of the method of any one of claims 1 or 2 in the genetic resurrection of Liuyan fish individuals.

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