Low-temperature breeding method of large yellow croaker

Abstract

The invention belongs to the technical field of aquaculture breeding, and particularly discloses a low-temperature breeding method for large yellow croakers. The method comprises the following steps: selecting healthy adult fishes from a plurality of sampling areas, and performing baseline evaluation and genome DNA extraction; by detecting a specific site of a low-temperature-resistant dominant gene, screening an individual carrying a dominant genotype, and performing phenotype verification under a low-temperature condition; optimizing the target gene by using a gene editing technology, injecting fertilized eggs, and hatching to obtain an F1 generation; gradient cooling treatment is conducted on the F1 generation, and functional feed is fed; healthy large yellow croakers F1 are obtained through genotype verification and low-temperature stress screening; taking the F1 as parent fish, repeating the steps to perform multi-generation breeding, and screening a low-temperature-resistant index gt in combination with LASSO regression and a machine learning model; the number of the individuals is 0.7. According to the method, the survival rate and the growth performance of the large yellow croaker in the low-temperature environment are remarkably improved, the breeding period is shortened, the breeding cost is reduced, and an efficient breeding scheme is provided for large yellow croaker breeding in the low-temperature environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture breeding, and specifically relates to a low-temperature breeding method for large yellow croaker (Pseudosciaena crocea). Background Art

[0002] The large yellow croaker (Pseudosciaena crocea) is one of the important marine economic fish species in China. Its meat is delicious and rich in nutrition, with high economic value. However, the breeding environment of large yellow croaker has a significant impact on its growth and survival, especially in low-temperature environments. During the low-temperature period in winter, the growth rate of large yellow croaker will decrease significantly, and even a large number of deaths may occur, which not only limits the breeding cycle of large yellow croaker, but also increases the breeding cost and reduces the breeding efficiency.

[0003] Traditional large yellow croaker breeding mainly relies on the natural environment and conventional breeding techniques, lacking a systematic selection for cold tolerance. At present, although some studies focus on the cold adaptation of large yellow croaker, most of them are concentrated on short-term physiological responses or simple environmental regulation, lacking a comprehensive solution from the gene level to systematic breeding. In addition, the existing breeding methods are often inefficient and difficult to quickly screen out excellent individuals with significant cold tolerance.

[0004] In terms of gene research, although there have been some studies on cold-related genes in large yellow croaker, a systematic breeding system combining genotype and phenotype has not been formed. For example, the FADS2 gene is related to the activity of fatty acid desaturase, affecting the fluidity of cell membranes and thus the membrane structure stability of large yellow croaker at low temperatures; the UCP1 gene is related to the expression of mitochondrial uncoupling protein, affecting the cold-induced thermogenesis efficiency. However, the potential roles of these genes have not been fully utilized in the low-temperature breeding of large yellow croaker.

[0005] In addition, although the existing aquaculture environment regulation technologies can alleviate the impact of low temperature on large yellow croaker to a certain extent, they cannot fundamentally improve the cold tolerance of large yellow croaker. For example, simple water temperature regulation can maintain a certain breeding temperature, but it cannot simulate the temperature changes in the natural environment, nor can it enhance the cold adaptation ability of large yellow croaker itself. Summary of the Invention

[0006] The present invention aims to provide a low-temperature breeding method for large yellow croaker, which significantly improves the survival rate and growth performance of large yellow croaker in a low-temperature environment, shortens the breeding cycle, and reduces the breeding cost.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A low-temperature breeding method for large yellow croaker, comprising the following steps:

[0009] S1. Initial population construction: Select healthy adult fish from multiple sampling areas. After excluding parasite infections, they are temporarily cultured in a recirculating water system. After 7 days of adaptation, a baseline assessment is carried out, and then they are transferred to a cement pond and temporarily cultured for 7 days. Then, they enter the experimental period, and gradient cooling treatment is carried out to screen out low-temperature tolerant individuals and low-temperature sensitive individuals, and genomic DNA is extracted.

[0010] S2. Screening for low-temperature tolerance potential: Detect specific loci of the low-temperature tolerance dominant genes FADS2, UCP1, and HSF1, screen individuals carrying low-temperature tolerance dominant genotypes, and conduct phenotypic verification under low-temperature conditions. The screening threshold is set as individuals with a 24-hour survival rate > 40% and a hepatic glycogen content > 25 mg / g.

[0011] S3. Use the individuals screened in S2 as parents for artificial breeding, and obtain fertilized eggs by heat-preserving induced spawning.

[0012] S4. Perform gene editing on the FADS2 and UCP1 genes to obtain sgRNA. Mix the Cas9 protein with sgRNA and inject it into the fertilized eggs in S3, and conduct artificial hatching to obtain the F1 generation.

[0013] S5. Gradient cooling acclimation and nutritional co-regulation: For the F1 generation obtained in S4, gradient cooling treatment is carried out during the fry stage and the adult stage respectively, and functional feed is fed.

[0014] S6. Screening: Conduct genotype verification through sequencing and low-temperature stress screening, eliminate abnormal individuals, and screen out surviving and healthy large yellow croaker F1.

[0015] S7. Multi-generation selective breeding and optimization: Use the large yellow croaker F1 screened in S6 as broodstock, and repeat S2 - S6 again to screen large yellow croaker F2. Use large yellow croaker F2 as broodstock and repeat S2 - S6 again to screen large yellow croaker F3. Repeat this step to obtain large yellow croaker F n generation, n ≥ 3. Collect phenotypic, environmental, and behavioral data, screen key features through LASSO regression, use XGBoost and 1D-CNN to construct a selective breeding model, and screen individuals with a low-temperature tolerance index > 0.7.

[0016] Preferably, in S1, the healthy adult fish has a body length of 14.71 - 15.71 cm and a body weight of 56.69 - 64.75 g; the water temperature of the recirculating water system is 17.5 - 18.5 °C, the salinity is 27 - 29 ‰, and the dissolved oxygen ≥ 6 mg / L.

[0017] Preferably, in S1, it is transferred to a cement pond and temporarily cultured for 7 days at a water temperature of 14.5 - 15.5 °C, then enters the test period, and gradient cooling treatment is carried out at a rate of 0.5 °C / 48 h until 6 °C, and it lasts for 5 d. Low-temperature sensitive individuals that cannot tolerate low temperature when the temperature drops to ≥9 °C and low-temperature tolerant individuals that can still survive well after the temperature drops to 6 °C and is maintained for 5 d are screened out.

[0018] Preferably, in S2, the specific loci of the low-temperature tolerance advantage genes FADS2, UCP1, and HSF1 are rs235671, rs108931, and rs204576 respectively.

[0019] Preferably, in S4, the nucleotide sequence of the FADS2 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the UCP1 gene is as shown in SEQ ID NO.2; for the gene editing, cytosine at the rs235671 locus of the FADS2 gene is replaced with thymine, and a low-temperature response element is inserted into the promoter region of the UCP1 gene.

[0020] Preferably, in S4, the mixing molar ratio of the Cas9 protein to the sgRNA is 1:3.

[0021] Preferably, in S5, the gradient cooling treatment during the fry stage is specifically: the water temperature is reduced from 18 °C to 10 °C at a rate of 1 °C / week; the gradient cooling treatment during the adult fish stage is specifically: a low-temperature shock at 8 °C is carried out once at 9:00 and 21:00 every day, and each time it lasts for 2 h.

[0022] Preferably, in S5, the functional feed includes the following components: dihydromyricetin 200 mg / kg, coated vitamin C / E complex 0.2%, Bacillus sp. BC - 01 1×10 8 CFU / g.

[0023] Preferably, in S6, the low-temperature stress temperature is 10 °C, and the low-temperature stress time is 48 h.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] The present invention discloses a low-temperature breeding method for large yellow croaker, which significantly improves the low-temperature tolerance and breeding efficiency of large yellow croaker. Through genotype screening and phenotypic verification, excellent individuals with the potential for low-temperature tolerance are quickly locked, and the survival rate of the selected population exceeds 70% under the condition of 6°C. Gene editing technology further enhances the low-temperature tolerance. Combined with the gradient cooling and acclimation technology, the survival rate of fry reaches 86%. During the adult fish stage, the LDH activity is significantly increased and the liver glycogen consumption rate is reduced through low-temperature shock. Combining the above technologies, the present invention significantly improves the survival rate and growth performance of large yellow croaker in a low-temperature environment, shortens the breeding cycle, reduces the breeding cost, and provides a systematic solution for the rapid breeding and industrial application of low-temperature tolerance traits.

[0026] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and examples. Description of the Drawings

[0027] Figure 1 It is a scatter plot of the correlation between the low-temperature survival rate and liver glycogen content of the initial population;

[0028] Figure 2 It is the dynamic statistical result of gradient cooling and survival rate during the fry stage;

[0029] Figure 3 It is the comparison result of growth performance between the functional feed group and the control group in Example 1;

[0030] Figure 4 It is the sampling area map of the initial population sample in Example 1. Detailed Embodiments

[0031] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0032] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0033] In the present invention, unless otherwise specified, other test materials and instrument equipment are all conventional test materials in the art and can be obtained through commercial channels.

[0034] Example 1

[0035] In the present invention, unless otherwise specified, other test materials and instrument equipment are all conventional test materials in the art and can be obtained through commercial channels.

[0036] Dihydromyricetin (feed grade) was purchased from Changsha Watts Biotechnology Co., Ltd.; the coated vitamin C / E complex was a coated VC / VE composite microcapsule (feed grade) purchased from Zhejiang NHU Co., Ltd., and Bacillus sp. BC-01 was purchased from Beijing Keyto Biotechnology Co., Ltd.

[0037] The basic feed ingredients include: 50% fish meal, 15% soybean meal, 20% flour, 6% fish oil, 2% calcium dihydrogen phosphate, 1% vitamin premix, 1% mineral premix, 0.02% antioxidant (ethoxyquin), and 4.98% others.

[0038] Example 1

[0039] A low-temperature breeding method for large yellow croaker includes the following steps:

[0040] S1. Initial population construction: Select 500 healthy adult fish with a body length of 15.21 ± 0.5 cm and a body weight of 60.72 ± 4.03 g from sampling areas in Zhujiajian, Liuhang, Changzhi Island, Daishan, and Dengbu. After excluding parasite infections, they are temporarily cultured in a recirculating water system of model Haili HL-2000. The water temperature is set at 17.5 - 18.5 °C, the salinity is 27 - 29‰, and the dissolved oxygen ≥ 6 mg / L. After 7 days of adaptation, a baseline assessment is carried out, and then they are transferred to a cement pool of 0.86 m × 1.3 m. Under the condition of a water temperature of 14.5 - 15.5 °C, they are temporarily cultured for 7 days and enter the experimental period to avoid interference from other stress factors on the experiment. A THD-1006 type low-temperature constant temperature bath (Ningbo Tianheng Instrument Factory) is used for cooling and temperature control, and gradient cooling treatment is carried out at a rate of 0.5 °C / 48 h until 6 °C and maintained for 5 d. Screen out low-temperature sensitive individuals that cannot tolerate low temperatures when the temperature drops to ≥ 9 °C and low-temperature tolerant individuals that can still survive well after the temperature drops to 6 °C and is maintained for 5 d. Cut the fin rays and quickly freeze them in liquid nitrogen and store them at -80 °C. Genomic DNA is extracted by the conventional phenol / chloroform method. Among them, the culture water is sand-filtered seawater from the large yellow croaker culture area of Xiangshan Port. During the temporary culture and experiment of the fish, the water is changed once in the morning and once in the evening, and the water change volume each time is 1 / 3. The new water is pre-cooled before water change, and the temperature difference before and after water change is kept within the range of ± 0.2 °C.

[0041] S2. Screening for low-temperature tolerance potential: Use a fluorescence quantitative PCR SYBR quantitative detection kit (TaKaRa) to detect the rs235671, rs108931, and rs204576 loci of the low-temperature tolerance dominant genes FADS2, UCP1, and HSF1 respectively, screen out individuals carrying low-temperature tolerance dominant genotypes, and place the fish in a 10 °C circulating water tank (model: Haisheng HS-T300), and use a colorimetric method to detect the glycogen content in the liver (the kit is A043). The screening threshold is set as individuals with a 24-hour survival rate > 40% and a glycogen content in the liver > 25 mg / g;

[0042] S3. Use the individuals selected in S2 as parents for artificial breeding. The breeding water temperature is 20-22°C, and gonadotropin-releasing hormone analogue (GnRHa) and dopamine inhibitor (domperidone, DOM) are injected to induce spawning of the parent fish to obtain fertilized eggs. The injection protocol is as follows: First injection of GnRHa (5 μg / kg) + DOM (5 mg / kg); after 12 hours, a second injection of GnRHa (5 μg / kg) is carried out.

[0043] S4. Replace cytosine with thymine at the rs235671 locus of the FADS2 gene, and insert a cold-responsive element CANNTG (E-box) into the promoter region of the UCP1 gene to obtain sgRNA. Mix Cas9 protein (Thermo Fisher TrueCut TM Cas9, 10 μg / μL) and sgRNA at a molar ratio of 1:3 and inject them into the fertilized eggs in S3 (4 nL / egg) through a Nanoject III microinjector (Drummond). Verify the editing efficiency > 80% by T7E1 digestion and perform artificial hatching to obtain the F1 generation. The nucleotide sequence of the FADS2 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the UCP1 gene is as shown in SEQ ID NO.2.

[0044] SEQ ID NO.1: 5'-GACGTGCTCCGCTACTACGA-3';

[0045] SEQ ID NO.2: 5'-CAGCTACGTGATCTACGACT-3'.

[0046] S5. Gradient cooling acclimation and nutritional co-regulation: For the F1 generation obtained in S4, during the fry stage, the water temperature is decreased from 18°C to 10°C at a rate of 1°C / week. During the adult stage, a low-temperature shock of 8°C is carried out at 9:00 and 21:00 every day for a gradient cooling treatment lasting 2 hours each time. Add functional feed to the basic feed. During the low-temperature period (10°C), feed at 2.5% of the body weight and increase the feeding amount by 20%, 4 times a day (04:00, 10:00, 16:00, 22:00), and evenly distribute the feeding amount each time. The functional feed includes dihydromyricetin 200 mg / kg, coated vitamin C / E complex 0.2%, Bacillus sp. BC-01 1×10 8 CFU / g;

[0047] S6. Screening: Confirm the FADS2 C→T mutation by Sanger sequencing, detect the expression level of UCP1 by qPCR (ΔΔCt ≤ -2.0), screen at 10°C under low-temperature stress for 48 h, and select individuals with a survival rate > 70% for subsequent breeding. Abnormal individuals with a food intake < 1.5 g / d are excluded, and healthy surviving large yellow croakers F1 are selected.

[0048] S7. Multi-generation breeding optimization: Use the large yellow croaker F1 obtained by screening in S6 as broodstock, repeat S2 - S6 again to screen large yellow croaker F2, use large yellow croaker F2 as broodstock, repeat S2 - S6 again to screen large yellow croaker F3, and repeat this step to obtain large yellow croaker F n generation, n ≥ 3. Collect phenotypic data, weigh at 08:00 every day (electronic balance Sartorius CPA225D), and calculate the daily weight gain (g / d); randomly select 100 individuals from each batch for an 8°C stress test, and record the 24h / 48h LT50. Environmental data such as water temperature, salinity, and dissolved oxygen are automatically recorded every 15 minutes (YSI Pro DSS sensor), and the data is uploaded to the MySQL database; through LASSO regression (α = 0.01), retain 15 key features such as FADS2 genotype, 8°C food intake, and HSF1 expression level (contribution > 5%), as shown in Table 1 below. Use XGBoost (learning rate 0.15, tree depth 6) to fuse 1D-CNN (process 24-hour water temperature time series data), with a 10-fold cross-validation AUC = 0.89 and a recall rate > 85%. Breeding decision: Select individuals with a low-temperature tolerance index (LT Index) > 0.7 in each generation, and preferentially edit the high-contribution genes identified by the model.

[0049] Table 1 Feature contribution ranking and model interpretation

[0050]

[0051]

[0052] As can be seen from Table 1, 1. Overall feature contribution distribution: Among the 15 key features screened by LASSO regression, genotype features (4 items) and environmental features (3 items) have the highest prediction contribution to the low-temperature tolerance trait, ranking among the top three in the total contribution.

[0053] 2. Key feature analysis:

[0054] (1) Genotype features dominate low-temperature tolerance

[0055] UCP1 promoter mutation (contribution 0.980): This gene editing significantly enhances the expression of mitochondrial uncoupling protein by inserting a low-temperature response element (E-box), optimizing the energy metabolism efficiency at low temperatures, and is the core driving factor for the low-temperature tolerance trait.

[0056] FADS2 gene (rs235671) (contribution 0.123) and HSF1 expression (contribution 0.850): The C→T mutation of FADS2 enhances the unsaturation of membrane phospholipids and maintains the fluidity of cell membranes under low temperatures; HSF1 upregulates the expression of heat shock proteins (such as HSP70) and enhances protein repair ability.

[0057] (2) Synergistic effects of environment and physiological phenotype

[0058] Water temperature fluctuation variance (contribution 0.860): drastic temperature fluctuations (such as ±2℃ / d) significantly increase metabolic pressure, which needs to be alleviated through gradient cooling acclimatization; gill Na+ / K+-ATPase activity (contribution 0.790) and liver glycogen content (contribution 0.101): high enzyme activity ensures ion balance and avoids low temperature osmotic stress; liver glycogen>25mg / g is the screening threshold, reflecting energy reserve capacity.

[0059] (3) Behavioral and secondary physiological indicators

[0060] Deviation of circadian feeding rhythm (contribution 0.620) and group swimming activity (contribution 0.540): Low temperature leads to feeding disorder (deviation > 15%) and reduced activity (activity < 10 times / minute), suggesting that the feeding strategy needs to be optimized; serum cortisol (contribution 0.650) and LDH activity (contribution 0.650):

[0061] Increased cortisol reflects a chronic stress state; increased LDH activity indicates enhanced anaerobic metabolism.

[0062] Genotype and environmental characteristics: The combined contribution of the two reached 4.913 (accounting for 58.3%), indicating that the low-temperature tolerance trait is jointly dominated by genetic potential and environmental regulation; Physiological and behavioral characteristics: reflect phenotypic plasticity, but the contribution is relatively low (a total of 4.631, accounting for 41.7%).

[0063] Comparative Example 1

[0064] The test method was the same as that in Example 1, except that gene editing of S4 and gradient cooling acclimation of S5 were not performed, the water temperature was maintained at 17.5-18.5°C throughout the process, and basic feed was fed at a daily feeding amount of 2.5% of the fish body weight.

[0065] The effects of the above Example 1 and Comparative Example 1 were verified by the following experiments.

[0066] 1. The correlation analysis between the initial population low-temperature survival rate and liver glycogen content in Example 1S2 was performed. The results are as follows: Figure 1 ;

[0067] Depend on Figure 1It can be seen that there is a significant positive correlation between the displayed survival rate and the hepatic glycogen content (R 2 = 0.67, p < 0.001), and the screening threshold is set as the survival rate > 40% and the hepatic glycogen > 25 mg / g.

[0068] 2. In Example 1 and Comparative Example 1, the hepatic glycogen reserve in the fry stage and the gill Na + / K + -ATPase activity in S5 were detected by HPLC, and the LDH activity and the hepatic glycogen consumption rate in the adult fish stage were detected. The results are shown in Table 1. The gradient cooling survival rate of the fry stage under different dynamic temperature changes was measured, and the results are as Figure 2 .

[0069] Table 1 Measurement results of each index in the fry stage and adult fish stage of Comparative Example 1 and Example 1

[0070]

[0071] As can be seen from Table 1, the hepatic glycogen reserve in the fry stage increased by 30%, and the gill Na+ / K+-ATPase activity was maintained at ≥ 8 U / mg.

[0072] In the adult fish stage, the LDH activity increased by 25%, and the hepatic glycogen consumption rate decreased by 46.8%.

[0073] From Figure 2 it can be seen that when gradually decreasing from 18°C to 10°C (1°C decrease per week), the survival rate in the 7th week was 86%, which was significantly higher than that of Comparative Example 1.

[0074] 3. Randomly select 200 tails from the F1 generation of Example 1 and divide them into two groups: Experimental group: Add functional feed (containing 200 mg / kg of dihydromyricetin, 0.2% of coated vitamin C / E, 1×10 8 CFU / g of Bacillus) to the basal feed; Control group: Feed the basal feed. Feeding management: During the low temperature period (10°C), both groups were fed at 3% of the body weight, and the experimental group additionally increased the feeding amount by 20%. The individual daily weight gain, 48h survival rate and hepatic glycogen consumption rate of the experimental group and the control group were measured. The results are shown in Table 2 and Figure 3 .

[0075] Table 2 Measurement results of individual indexes under different feed feeding conditions

[0076] Index Experimental group Control group Average daily weight gain (g / d) 2.5±0.3 1.8±0.2 48h survival rate (%) 88 52 Rate of liver glycogen consumption (mg / (gh)) 1.3±0.2 2.1±0.3

[0077] As can be seen from Table 2, the average daily weight gain of the experimental group (2.5 ± 0.3 g / d) was significantly higher than that of the control group (1.8 ± 0.2 g / d), indicating that the experimental diet was more conducive to individual growth; the 48-hour survival rate of the experimental group (88%) was significantly higher than that of the control group (52%), showing an obvious advantage in stress resistance; the liver glycogen consumption rate of the experimental group (1.3 ± 0.2 mg / (g·h)) was significantly lower than that of the control group (2.1 ± 0.3 mg / (g·h)), indicating that the experimental diet might improve the individual physiological state by alleviating the energy metabolism stress.

[0078] As Figure 3 can be seen, the average daily weight gain of the experimental group was 2.5 g, with an error bar of ±0.3 g (n = 100); the average daily weight gain of the control group was 1.8 g, with an error bar of ±0.2 g (n = 100); the statistical difference result of the t-test showed p < 0.05, indicating that the functional diet significantly promoted growth during the low-temperature period.

[0079] 4. Measure Example 1 and Comparative Example 1 respectively, and count the number of surviving individuals and the average liver glycogen content after 48 h of stress at 10°C.

[0080] In Comparative Example 1, the number of surviving individuals was 110 tails, and the survival rate was 22%.

[0081] In Example 1, the number of surviving individuals was 352 tails, and the survival rate was 70.4%.

[0082] The average liver glycogen content of Comparative Example 1 was 20.5 ± 3.2 mg / g, which was significantly lower than that of Example 1 (32.5 ± 2.8 mg / g, p < 0.01).

[0083] In summary, genotype characteristics (especially UCP1 promoter editing) are the core determinants of cold tolerance traits, and environmental stresses such as water temperature fluctuations need to be alleviated through precise regulation. This contribution ranking provides a theoretical basis for multi-generation breeding, and the prediction accuracy can be further improved by enhancing the gene-environment interaction (G×E) model in the future.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for low-temperature breeding of large yellow croaker, characterized in that, It includes the following steps: S1. Initial population construction: Select healthy adult fish from multiple sampling areas. After excluding parasite infections, they are temporarily cultured in a recirculating water system. After 7 days of adaptation, a baseline assessment is carried out, then they are transferred to a cement pond, temporarily cultured for 7 days, enter the experimental period, and gradient cooling treatment is carried out to screen out cold-tolerant individuals and cold-sensitive individuals, and genomic DNA is extracted; S2. Screening of cold tolerance potential: Detect specific loci of cold tolerance dominant genes FADS2, UCP1, and HSF1, screen individuals carrying cold tolerance dominant genotypes, and conduct phenotypic verification under low temperature conditions. The screening threshold is set as individuals with a 24-hour survival rate > 40% and a liver glycogen content > 25 mg / g; S3. Use the individuals screened in S2 as parents for artificial breeding, and obtain fertilized eggs by heat preservation and inducing spawning; S4. Gene editing is carried out on the FADS2 and UCP1 genes to obtain sgRNA. Cas9 protein is mixed with sgRNA and injected into the fertilized eggs in S3 for artificial hatching to obtain the F1 generation; S5. Gradient cooling acclimation and nutritional co-regulation: For the F1 generation obtained in S4, gradient cooling treatment is carried out during the fry stage and the adult fish stage respectively, and functional feed is fed; S6. Screening: Genotype verification is carried out by sequencing, and cold stress screening is carried out to eliminate abnormal individuals, and healthy surviving large yellow croaker F1 is screened; S7. Multi-generation breeding optimization: Using the large yellow croaker F1 screened in S6 as broodstock, repeat S2 - S6 again to screen large yellow croaker F2. Then use large yellow croaker F2 as broodstock and repeat S2 - S6 again to screen large yellow croaker F3. Repeat this step to obtain large yellow croaker F n generation, where n ≥ 3. Collect phenotypic, environmental, and behavioral data, screen key features through LASSO regression, use XGBoost and 1D-CNN to construct a breeding model, and screen individuals with a low-temperature tolerance index > 0.

7.

2. The method for low-temperature breeding of large yellow croaker according to claim 1, characterized in that, In S1, the healthy adult fish have a body length of 14.71 - 15.71 cm and a body weight of 56.69 - 64.75 g; the water temperature of the recirculating water system is 17.5 - 18.5 °C, the salinity is 27 - 29 ‰, and the dissolved oxygen ≥ 6 mg / L.

3. The method for low-temperature selection and breeding of large yellow croaker according to claim 1, characterized in that, In S1, when transferred to the cement pond, under the condition of a water temperature of 14.5 - 15.5 °C, they are temporarily cultured for 7 days, enter the experimental period, and gradient cooling treatment is carried out at a rate of 0.5 °C / 48 h until 6 °C, lasting for 5 d, to screen out cold-sensitive individuals that cannot tolerate low temperature when the temperature drops to ≥ 9 °C and cold-tolerant individuals that can still survive well after the temperature drops to 6 °C and is maintained for 5 d.

4. The method for low-temperature selection and breeding of large yellow croaker according to claim 1, characterized in that In S2, the specific loci of the cold tolerance dominant genes FADS2, UCP1, and HSF1 are rs235671, rs108931, and rs204576 respectively.

5. The method for low-temperature selection and breeding of large yellow croaker according to claim 1, characterized in that, In S4, the nucleotide sequence of the FADS2 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the UCP1 gene is as shown in SEQ ID NO.2; the gene editing is to replace cytosine with thymine at the rs235671 locus of the FADS2 gene and insert a cold response element in the promoter region of the UCP1 gene.

6. The low-temperature breeding method of large yellow croaker according to claim 1, characterized in that In S4, the molar ratio of the Cas9 protein to the sgRNA mixture is 1:

3.

7. The method for low-temperature breeding of large yellow croaker according to claim 1, characterized in that, In S5, the gradient cooling treatment during the fry stage is specifically: the water temperature is reduced from 18 °C to 10 °C at a rate of 1 °C / week; the gradient cooling treatment during the adult fish stage is specifically: a low temperature shock of 8 °C is carried out once at 9:00 and 21:00 every day, each lasting for 2 h.

8. The low-temperature selective breeding method for large yellow croaker according to claim 1, characterized in that In S5, the functional feed includes the following components: dihydromyricetin 200 mg / kg, coated vitamin C / E complex 0.2%, Bacillus sp. BC-01 1×10 8 CFU / g.

9. The method for low-temperature breeding of large yellow croaker according to claim 1, characterized in that, In S6, the cold stress temperature is 10 °C and the cold stress time is 48 h.

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