Chitosan-pDNA nanoparticle and application thereof in induction of pseudomale Atlantic salmon

Abstract

The invention discloses chitosan-pDNA nanoparticles and application thereof in induction of pseudo male salmon of Atlantic salmon, and belongs to the technical field of induction of Atlantic salmon strains. The method comprises the following steps: cloning Amh genes of Atlantic salmon to a pIRES2-EGFP vector to obtain plasmids containing the Amh genes; the preparation method comprises the following steps: dissolving chitosan in a 25mM sodium acetate solution according to the concentration of 0.1% (w / v), and adjusting the pH value to 5.0-5.5 by using acetic acid; diluting the concentration of the obtained plasmid to 200ng / mu l to prepare a plasmid solution containing 0.5% (w / v) sodium sulfate; and respectively preheating the two obtained solutions at 55 DEG C, carrying out isovolumetric mixing, carrying out rapid and violent vortex for 55-60 seconds, and standing at room temperature for more than 30 minutes to obtain the chitosan-pDNA nanoparticles. The chitosan-pDNA nano-particles are used for preparing XX type Atlantic salmon pseudo male fish, mature X type sperms are obtained, the X type sperms are fertilized with normal Atlantic salmon X type ova, and Atlantic salmon all-female offspring seeds are obtained.

Description

Technical Field

[0001] This invention belongs to the field of Atlantic salmon strain induction technology, specifically a chitosan-pDNA nanoparticle and its application in the induction of pseudo-male Atlantic salmon. Background Technology

[0002] Atlantic salmon, commonly known as salmon, belongs to the class Actinopterygii, order Salmoniformes, family Salmonidae, and genus Salmon. It is a vital economic species for global aquaculture. However, my country has long relied on imported breeding stock, facing bottlenecks such as genetic degradation and outdated sex control technology (high risk of hormone-induced residues and low sperm motility in pseudo-males). Overcoming these breeding stock issues is urgently needed. Breeding technology is the core driving force for the development of the Atlantic salmon aquaculture industry. Sex control can optimize the quality of all-female populations, and combined with stress-resistance breeding, it can address the low survival rate of imported seedlings in my country's warm-water environment. Independent breeding not only enables localized seedling production but also has irreplaceable strategic significance for ensuring aquatic food safety and promoting the sustainable development of the industry. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a method for preparing chitosan-pDNA nanoparticles, the product and its application. This invention solves the technical problem of preparing all-female fry of Atlantic salmon by delivering the Amh gene through chitosan nanocarriers.

[0004] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing chitosan-pDNA nanoparticles, the method comprising the following steps: (1) The Amh gene of Atlantic salmon (GenBank accession number: NC_059451) was cloned into the pIRES2-EGFP vector to obtain a plasmid containing the Amh gene; (2) Dissolve chitosan in a 25 mM sodium acetate solution at a concentration of 0.1% (w / v), and adjust the pH to 5.0-5.5 with acetic acid; (3) Dilute the plasmid concentration obtained in (1) to 200 ng / μl to prepare a plasmid solution containing 0.5% (w / v) sodium sulfate; (4) The two solutions obtained in (2) and (3) are preheated at 55°C, mixed in equal volumes, and then rapidly and violently vortexed for 55-60 s. After standing at room temperature for more than 30 minutes, chitosan-pDNA nanoparticles are obtained.

[0005] The present invention also provides chitosan-pDNA nanoparticles prepared by the above method.

[0006] The present invention also provides an application of the above-mentioned chitosan-pDNA nanoparticles, wherein the application is to use the chitosan-pDNA nanoparticles to prepare XX-type Atlantic salmon pseudo-males, obtain mature X-type sperm, and fertilize the X-type sperm with normal Atlantic salmon X-type eggs to obtain all-female Atlantic salmon fry (XX).

[0007] Furthermore, the method for preparing XX-type Atlantic salmon males using chitosan-pDNA nanoparticles involves uniformly spraying the obtained chitosan-pDNA nanoparticles onto the feed at a concentration of 10 mg of chitosan-pDNA nanoparticles per kilogram of feed, drying it in the dark, storing it at low temperature, and feeding the newly hatched female larvae of XX-type Atlantic salmon twice a day with an interval of more than 6 hours. The daily feed amount is 2% of the body weight, and the feeding cycle is the entire sex determination period of Atlantic salmon, that is, 30-120 days after hatching, to obtain pseudo-males with the XX-type sex chromosome karyotype.

[0008] Furthermore, the method for promoting the maturation of pseudo-male fish is as follows: reduce the salinity of 2-year-old pseudo-male fish by 3‰-5‰ every 3 days, adjust the water temperature by decreasing it by 1℃ every day to below 10℃, preferably 5-8℃, provide 200-500 lux of light, and maintain a light cycle of 12L:12D until they are transferred to freshwater for cultivation, and control the light and temperature to promote sexual maturity. After the pseudo-male fish cultivated in freshwater develop secondary sexual characteristics (about 3 months), they are first anesthetized with eugenol at a concentration of 20-40 mg per liter of water, and then artificially induced to spawn with luteinizing hormone-releasing hormone analogue (LHRHa). The dosage of LHRHa is 5 μg / kg, injected into the dorsal muscle.

[0009] The advantages of this invention compared to existing technologies are as follows: This invention provides a method for preparing all-female Atlantic salmon seedlings based on chitosan nanocarrier delivery of the Amh gene. Through key technologies such as integrative sex reversal technology, precise environmental control, and ultra-low temperature cryopreservation of sperm, a highly efficient and stable all-female breeding system is established. This method utilizes chitosan nanocarrier protective plasmids delivered via the digestive tract, achieving non-transgenic sex regulation (induction rate >86.67%), avoiding the environmental pollution risks of traditional hormone-induced methods. Combined with environmental control (light and temperature control), the sexual maturity synchronization rate reaches over 80%, significantly shortening the seedling cultivation cycle by 6-12 months. Addressing the issues of spermatogenesis disorders and asynchronous sexual maturation in pseudo-males, ultra-low temperature cryopreservation technology is used for long-term sperm preservation, greatly improving the utilization rate of pseudo-males. This technology enhances the sperm motility of pseudo-males, achieving a fertilization rate of 90%, and resulting in over 95% all-female offspring. The entire breeding process is completed in a recirculating aquaculture system, achieving standardized production year-round without seasonal limitations through precise control of environmental factors such as temperature, light, and salinity. This system completely avoids the risks of genetic modification, meets green aquaculture standards, and effectively solves the core problems faced by my country's Atlantic salmon farming industry, such as reliance on imported germplasm resources and outdated sex control technology. Attached Figure Description

[0010] Figure 1 This is a flowchart of the method of the present invention; Figure 2 This is a two-factor experimental plot of chitosan-DNA concentration gradient. C1: DNA concentration in the initial solution of the packaging system; C2: DNA concentration in the supernatant of the packaging system after centrifugation to precipitate nanoparticles. Figure 3 Packaging system stability test; M: marker; Lane 1: Untreated plasmid; Lane 2: Untreated chitosan solution; Lane 3: Plasmid + chitosan encapsulated and placed for 24 h without centrifugation; Lane 4: Plasmid + chitosan encapsulated and placed for 24 h, centrifuged and supernatant collected; Lanes 5 and 6: Plasmid + chitosan encapsulated and centrifuged, supernatant discarded, 150 μl of 0.2 M sodium bicarbonate added to the precipitate and placed for 24 h; Figure 4 Transmission electron microscopy image of nanoparticles; Figure 5 Comparison of gonads in vivo between a 250dph female (left) and a pseudo-male (right) (gonad tissue is shown in the red box); Figure 6 Sections of the gonads of female and pseudo-male fish at different stages. Detailed Implementation

[0011] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0012] Example 1 A method for preparing chitosan-pDNA nanoparticles, the method comprising the following steps: (1) The Amh gene of Atlantic salmon (GenBank accession number: NC_059451) was cloned into the pIRES2-EGFP vector and the plasmid containing the Amh gene was obtained by PCR, electrophoresis (1% agarose gel) and sequencing verification. (2) The packaging system for nanoparticles was optimized in terms of efficiency, stability, and feasibility. See details. Figure 2-4 The packaging efficiency EE(%) = [(C1-C2) / C1] × 100%, where C1 is the concentration of DNA in the initial solution of the packaging system, and C2 is the concentration of DNA in the supernatant of the packaging system after centrifugation to precipitate nanoparticles. (3) Dissolve chitosan in a 25mM sodium acetate solution at a concentration of 0.1% (w / v), and adjust the pH to 5.0-5.5 with acetic acid; (4) Dilute the plasmid from step (1) with 0.5% (w / v) sodium sulfate solution to a concentration of 200 ng / μl; (5) The two solutions obtained in (3) and (4) are preheated at 55°C, mixed in equal volumes, and then rapidly and violently vortexed for 55-60 s. After standing at room temperature for more than 30 minutes, chitosan-pDNA nanoparticles are obtained.

[0013] Example 2 The structure of a method for inducing full feminization in Atlantic salmon based on chitosan nanocarrier delivery of the Amh gene is as follows: Figure 1 As shown, it includes the following steps: Step 1: Induction and spawning of pseudo-male parent fish. Purchase all-female, eyed eggs with XX sex chromosome karyotype. After hatching and opening the mouth of the larvae, feed them experimental chitosan-pDNA to induce the production of XX-type pseudo-male fish. Then, silverize the pseudo-male fish individuals to become sea salmon and raise them in seawater until they are 2 years old. Induce them to sexual maturity through environmental regulation. Calculate the induction rate by sectioning gonadal tissue. Kill the fish to obtain all-female sperm with X karyotype, which are used for artificial insemination or cryopreserved in liquid nitrogen in sperm preservation solution. The specific steps for inducing and spawning in pseudo-male parent fish are as follows: (1) Purchase wild-type all-female diploid eyed eggs and hatch them to obtain XX-type larvae; (2) The larvae began to eat about 30 days after hatching. They were fed a chitosan-pDNA experimental diet to induce reversal. The pDNA was cloned from the Atlantic salmon Amh gene (GenBank accession number: NC_059451) into the pIRES2-EGFP vector. The plasmid carrying the Atlantic salmon Amh gene was obtained by PCR, electrophoresis (1% agarose gel) and sequencing verification. Chitosan was dissolved in 25 mM sodium acetate solution at a concentration of 0.1% (w / v), and the pH was adjusted to 5.0-5.5 with acetic acid. The plasmid concentration was then diluted to 200 ng / μl to prepare a solution containing 0.5% (w / v) sodium sulfate. The two solutions were then preheated at 55 °C, mixed in equal volumes, and rapidly and vigorously vortexed for 55-60 s. After standing at room temperature for 30 minutes, chitosan-pDNA nanoparticles were obtained. Finally, the obtained chitosan-pDNA nanoparticles were evenly sprayed onto the feed at a concentration of 10 mg chitosan-pDNA nanoparticles per kilogram of feed, dried in the dark, and stored at low temperature. The newly hatched fry were fed twice a day, with an interval of more than 6 hours, and the daily feed amount was 2% of the body weight. The feeding experiment covered the entire sex determination period of Atlantic salmon (30-120 days after hatching) to obtain XX-type pseudo-male fish. When the fry reached 20 g, they were silvered, and when they reached 100 g, they were transferred to seawater for rearing. The water temperature for seawater rearing was 14-16℃, and the light intensity was 1000 lux and 24 L: 0 D. (3) The induction effect was determined by analyzing the gonadal development morphology of pseudo-male fish through morphological observation and histological sections. (See details below) Figure 5-6 and Table 1; from Figure 6 It can be seen that the gonadal differentiation characteristics of female and pseudo-male fish at 70dph are significantly different; pseudo-male fish at 250dph show obvious seminiferous cell clusters and seminiferous vesicle structures; at 610dp, germ cells at various levels can be observed (dph: days post-hatching); Table 1. Statistics on the proportion of pseudo-male Atlantic salmon at different time periods ; (4) Selected pseudo-male fish were cultured in seawater for 2 years. During the culture period, the gonad development was checked regularly. During the sperm production period, the pseudo-male fish were transferred to freshwater culture under the conditions of 200-500 lux light and 12L:12D light and temperature control to promote sexual maturity. (5) After the pseudo-male fish cultivated in freshwater develops secondary sexual characteristics (about 3 months), they are first anesthetized with eugenol at a concentration of 20-40 mg per liter of water, and then artificially induced to spawn with LHRHa at a dosage of 5 μg / kg, injected into the back muscle. (6) After 4-7 days of induced labor, the male is killed and the sperm is collected. The testes are cut into pieces and the paste-like semen in the vas deferens is carefully picked up with tweezers. A small amount of semen is activated and examined under a microscope. The semen is used for artificial insemination or preserved in a liquid nitrogen tank with sperm preservation solution (10% DMSO segmented cooling) for later use. Vitamin E (0.1mM) and ascorbic acid (1mM) are added to the sperm preservation solution to improve sperm motility.

[0014] Step Two: The specific steps for cultivating and inducing spawning in female parent fish are as follows: (1) Purchase physiological female fish that are 2 years or older and feed them normally, twice a day with an interval of more than 6 hours. The daily feed amount is 2% of their body weight; the water temperature is 14-16℃, and the light intensity is 1000lx and 24L:0D. (2) After feeding the female fish for a period of time, check the gonadal development regularly. When the ovary develops to stage IV and the egg diameter is >5mm, carry out nutritional fortification and reduce the salinity of the female fish by 3‰-5‰ every 3 days, adjust the water temperature by decreasing it by 1℃ every day to below 10℃, with 5-8℃ being ideal, and transfer the female fish to freshwater culture under the conditions of 200-500 lux light and 12L:12D, and control the light and temperature to promote sexual maturity. (3) After the female parent fish develop secondary sexual characteristics (about 6 months), they are first anesthetized with eugenol at a concentration of 20-40 mg per liter of water, and then artificially induced to spawn with LHRHa at a dosage of 5 μg / kg, injected into the back muscle. (4) After 4-7 days of induced spawning, check the spawning status of the female parent fish, and promptly select the parent fish that can spawn for artificial insemination. Then check once every 7 days to select the parent fish that can spawn. The induced spawning group can complete spawning within 1 month and obtain eggs with an X-type sex chromosome karyotype.

[0015] Step 3: The specific steps for preparing all-female seedlings are as follows: (1) Dry insemination: The sperm suspension prepared in step one, which has been genetically identified as type X, is mixed with type X oocytes prepared in step two at a mass ratio of 1:1000. The ratio of male to female parents is controlled at 4:1. The insemination process is carried out in a dark, low-temperature (6–8°C) environment, and the operation time is controlled within 60 seconds. After insemination, the fertilized eggs are rinsed three times with pre-cooled physiological saline, then soaked in iodine solution (effective iodine concentration 50 mg / L) for 5 minutes for disinfection, and then rinsed with sterile water. The treated fertilized eggs are evenly spread in the incubator, and the incubation water temperature is controlled at (6.5 ± 0.5)°C. The water flow rate is maintained at 5–10 L / min to achieve moderate shock, and the dissolved oxygen level is ensured to be no less than 8 mg / L. Strict light protection is maintained throughout the process, and the outside of the incubator is covered with black light-blocking material.

[0016] (2) Hatching and early development management: Fertilized eggs hatch after accumulating a temperature of approximately 500°C per day. Newly hatched fry are transferred to circular rearing tanks and fed 0.2mm salmon-trout formulated feed (crushed) 4–6 times daily. The rearing water temperature is gradually increased to 8–10°C, maintaining dissolved oxygen >7 mg / L and ammonia nitrogen <0.01 mg / L. When the fry enter the silvering stage, the photoperiod is adjusted to 12L:12D, and formulated feed with a particle size of 1.0–1.5 mm is fed.

[0017] (3) Sex ratio verification: Fifty juvenile salmon aged 6 months or older were randomly selected, and their sex was verified by anatomical gonadal histological sections and genetic markers. Statistical results showed that female individuals accounted for ≥95%.

Claims

1. A method for preparing a chitosan-pDNA nanoparticle, characterized by, The method steps are as follows: (1) Cloning of the Atlantic salmon (Salmo salar) IGF-I gene into the pIRES2-EGFP vector to obtain a plasmid containing the IGF-I gene, Amh Amh Amh GenBank Accession No. NC_059451;​​ (2) Dissolve chitosan in 25 mM sodium acetate solution at a concentration of 0.1% (mass / volume), and adjust the pH to 5.0-5.5 with acetic acid; (3) Dilute the plasmid obtained in (1) to a concentration of 200 ng / μl, and prepare a plasmid solution containing 0.5% (mass / volume) sodium sulfate; (4) Preheat the two solutions obtained in (2) and (3) at 55°C, mix them in equal amounts, vortex rapidly and vigorously for 55-60 s, and then stand at room temperature for 30 min or more to obtain chitosan-pDNA nanoparticles.

2. The chitosan-pDNA nanoparticles prepared by the method of claim 1.

3. Use of the chitosan-pDNA nanoparticles according to claim 2, characterized in that, The application is the use of the chitosan-pDNA nanoparticles to prepare XX-type Atlantic salmon pseudo-males, obtain mature X-type sperm, and fertilize normal Atlantic salmon X-type eggs with the X-type sperm to obtain all-female Atlantic salmon fry.

4. Use according to claim 3, characterized in that, The obtained chitosan-pDNA nanoparticles are uniformly sprayed on the feed at a concentration of 10 mg chitosan-pDNA nanoparticles per kg of feed, dried in the dark, and stored at low temperature. The XX-type Atlantic salmon female fry that have just opened their mouths are fed twice a day, with a feeding interval of more than 6 hours, a daily feeding amount of 2% of the body weight, and a feeding period of the entire sex determination period of the Atlantic salmon, i.e. 30-120 days after hatching of the Atlantic salmon, to obtain pseudo-males with sex chromosome karyotype XX.

5. The method of claim 4, wherein, Pseudo-male maturation method: 2-year-old pseudo-males are subjected to a decrease in salinity of 3‰-5‰ every 3 days, a decrease in water temperature of 1°C per day to below 10°C, 200-500 lux of light, and a light cycle of 12L:12D until they are transferred to freshwater culture and sex maturation is promoted by controlling light and temperature; After the pseudo-males cultured in freshwater develop secondary sexual characteristics, they are first anesthetized with eugenol at a concentration of 20-40 mg per liter of water, and then artificially induced to ovulate with LHRHa at a dosage of 5 μg / kg by intramuscular injection.

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