Accelerator for up-regulating key gene of scapharca broughtonii steroid hormone synthesis pathway and application thereof

Abstract

The invention relates to an accelerant for up-regulating key genes of a steroid hormone synthesis pathway of scapharca broughtonii and application of the accelerant, and belongs to the technical field of aquaculture, the accelerant is prepared from the following components in parts by mass: 15-20 parts of cholesterol, 8-10 parts of linolenic acid, 10-12 parts of phosphatidylcholine, 0.5-0.8 part of zinc sulfate, 30-40 parts of L-arginine and 0.5-0.75 * 10 <-3 > part of vitamin D3. Core genes (SF-1, StAR, 17 beta-HSD14 and CYP17) of a steroid hormone synthesis pathway of scapharca broughtonii are specifically focused, synergistic up-regulation of the genes is realized through a gonad maturation accelerator, a core molecular pathway of gonad development is directly activated, and the regulation effect is accurate and stable.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a promoter that upregulates key genes in the steroid hormone synthesis pathway of cockle and its application. Background Technology

[0002] Kuihan ( Scapharca broughtonii Clams are an important marine economic shellfish in my country, prized for their delicious and nutritious meat, possessing both edible and economic value, and holding a vital position in the marine aquaculture industry. In recent years, with the continuous expansion of artificial clam farming, the supply and demand imbalance for high-quality seedlings has become increasingly prominent. The core of seedling quality depends on the synchronicity, maturity, and development speed of the parent shellfish's gonads. Parent shellfish with incomplete gonad development and asynchronous maturation will experience low spawning rates, poor fertilization rates, and low larval survival rates, severely restricting the large-scale and intensive development of the clam farming industry.

[0003] However, existing technologies for regulating gonadal development in cockles have significant limitations: traditional methods often rely on environmental factors (such as temperature regulation and nutritional fortification) or non-specific hormone addition, lacking precise targeting of the molecular mechanisms of gonadal development, resulting in unstable regulatory effects, poor synchronization of gonadal development, and long maturation cycles. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide a gonadal maturation promoter that upregulates key genes in the steroid hormone synthesis pathway of cockle.

[0005] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned gonadal maturation promotion method in the breeding of cockle seedlings, in contrast to the prior art.

[0006] The core technical principle of this invention is based on the core logic of "exogenous precursor supply - endogenous pathway activation - gonadal development regulation": By adding a gonadal maturation promoter containing natural steroid hormone precursors to the aquaculture water, the precursor substances enter the body of the cockle through gill respiration, surface osmosis, and feeding. After being absorbed by the hepatopancreas and gonadal tissues, they serve as substrates to initiate the steroid hormone synthesis pathway. On the one hand, this directly provides raw materials for the enzymatic reaction of cholesterol → pregnenolone → sex hormones (estrogen and androgen); on the other hand, it specifically induces... SF-1 (transcription regulatory factors) StAR (cholesterol transporter) CYP17 (Sex hormone precursor synthase) 17β-HSD14 The synergistic upregulation of key genes such as (sex hormone-converting enzymes) accelerates the synthesis and release of active sex hormones; sex hormones further act on the liver and pancreas, promoting... Vg (Vitrinogen) gene transcription and Vg Protein synthesis, VgProteins are transported to the gonads via body fluids, providing essential nutrients for oocyte proliferation and differentiation, ultimately achieving rapid and synchronous maturation of the gonads; simultaneously... Vg Gene expression levels serve as a quantitative indicator of gonadal maturity, ensuring the accuracy of parent shellfish selection.

[0007] The technical solution adopted by the present invention to solve the first technical problem mentioned above is as follows: A promoter that upregulates key genes in the steroid hormone synthesis pathway of cockle, the promoter comprising the following components and parts by weight: 15-20 parts cholesterol, 8-10 parts linolenic acid, 10-12 parts phosphatidylcholine, 0.5-0.8 parts zinc sulfate, 30-40 parts L-arginine, and 0.5-0.75 × 10⁻⁶ vitamin D3. -3 share.

[0008] One of the specific promoters is cholesterol 1.5-2.0 g, linolenic acid 0.8-1.0 g, phosphatidylcholine 1.0-1.2 g, zinc sulfate 50-80 mg, L-arginine 3.0-4.0 g, and vitamin D3 50-75 μg.

[0009] Preferably, the promoter is: cholesterol 2.0 g, linolenic acid 1.0 g, phosphatidylcholine 1.1 g, zinc sulfate 50 mg, L-arginine 4.0 g, and vitamin D3 50 μg.

[0010] Preferably, the promoter is: cholesterol 1.8 g, linolenic acid 0.8 g, phosphatidylcholine 1.1 g, zinc sulfate 50 mg, L-arginine 4.0 g, and vitamin D3 50 μg.

[0011] Application of the promoter in promoting the gonadal maturation of cockles: Starting 20 days before the planned spawning date, the promoter is evenly sprinkled into the water body for cultivating cockles in the form of an additive after the parent cockles are transferred to the tank every day, so that the final concentration of the promoter in the water body is 4 ppm.

[0012] This invention also provides key genes of the promoter in the upregulation of steroid hormone synthesis pathway and Vg In its application, the key gene is SF-1 , StAR , 17β-HSD14 , CYP17 The application concentration is 4 ppm.

[0013] The application scenarios of the gonadal maturation promoter of the present invention specifically include: 1. Large-scale cultivation of cockle broodstock: Suitable for the synchronous development and regulation of gonadal development in factory-style hatcheries and offshore raft aquaculture areas, solving the problem of asynchronous maturation of batches of broodstock; 2. Precise selection of high-quality parent oysters: Vg The expression level is a non-invasive molecular marker, which can replace the traditional dissection method to achieve rapid and accurate screening of mature parent shellfish and reduce the damage rate of parent shellfish; 3. Improvement in seedling yield and quality: By enhancing the gonadal maturity and reproductive performance of parent shellfish, the supply of high-quality seedlings can be increased, seedling costs can be reduced, and the intensive development of the cockle farming industry can be supported. 4. Technology transfer to similar shellfish: Its core principles can be transferred to other shellfish such as mud clams and hairy clams, or to the gonadal development regulation of marine shellfish such as scallops and oysters, and have a wide range of applications.

[0014] The beneficial effects of this invention compared to existing technologies: It specifically targets the core genes of the steroid hormone synthesis pathway in cockles ( SF-1 , StAR , 17β-HSD14 , CYP17 This method achieves synergistic upregulation of the aforementioned genes through gonadal maturation promoters, directly activating core molecular pathways in gonadal development. The regulatory effect is precise and stable, overcoming the drawbacks of traditional methods' "blind regulation." It also establishes a... Vg The quantitative correlation standard of "expression level - gonadal maturity" enables non-invasive and accurate detection of gonadal maturity, avoiding damage to broodstock caused by traditional dissection and observation, while improving the accuracy and efficiency of broodstock screening. The gonadal maturity promoter is easy to apply (add to water), the optimal concentration (4 ppm) is easy to control, has no adverse effects on the aquaculture environment, and is suitable for different scales of cockle farming scenarios (laboratory seedling cultivation, factory farming, marine raft farming, etc.), and has strong practicality and promotion value. Attached Figure Description

[0015] Figure 1 To investigate the effects of different concentrations of promoters on the ovaries of cockles SF-1 , StAR , 17β-HSD14 , CYP17 and Vg The effect of relative gene expression levels, A represents... SF-1 Gene, B is StAR Gene, C for 17β-HSD14 Gene, D is CYP17 Gene, E is Vg Gene. Detailed Implementation

[0016] To fully illustrate the technical solution of the present invention, the following detailed description is provided in conjunction with embodiments.

[0017] Example 1: A method for using a gonadal maturation promoter that upregulates key genes in the steroid hormone synthesis pathway of cockles. The gonadal maturation promoter is composed of the following components: cholesterol 2.0 g, linolenic acid 1.0 g, phosphatidylcholine 1.1 g, zinc sulfate 50 mg, L-arginine 4.0 g, and vitamin D3 50 μg. After mixing, it dissolves rapidly in seawater (dissolution time ≤ 5 minutes) without significant precipitation. The optimal final concentration is 4 ppm, i.e., 4 grams of gonadal maturation promoter per cubic meter of aquaculture water.

[0018] Example 2: Application test of a promoter to improve the gonadal maturation of parent cockles Experimental group: The gonadal maturation promoter prepared in Example 1 was fed to the broodstock of cockles. Specifically, the experimental group was fed the gonadal maturation promoter 20 days before spawning. After the broodstock were transferred to the pond daily, the feeding dosage was 4 ppm and the feeding method was to sprinkle it into the whole pond.

[0019] Control group: This group was not fed gonadal maturation promoters, and all other breeding conditions were the same as those of the experimental group.

[0020] All groups were tested in culture ponds of the same size, and three identical culture ponds were set up for each group, with the same amount of cockles stocked in each pond. A gradual temperature increase method was used for temporary rearing to promote maturity. The feed consisted mainly of *Phaeodactylum tricornutum*, supplemented with *Isorrhiza glomerata* (…). Isochrysis galbana ) and Spirulina ( Spirulina Nutritional fortification was performed, and females and males were separated after at least two rigorous selection processes. Female cockles were induced to lay eggs in the pond using a method of air-drying, warming, and intermittent aeration, while male cockles were placed in a glass tank to release sperm. After fertilization, each egg was surrounded by 3-4 sperm cells. Samples were continuously taken to observe embryonic development until it developed into D-shaped larvae. During the experiment, water quality management was maintained, with dissolved oxygen at 5-6 mg / L, water temperature at 19-20 ℃, salinity at 29‰-31‰, and pH at 7.5-8.0.

[0021] According to the above plan, after 20 days of continuous feeding, spawning was induced. The egg-laying rate, sperm-release rate, and hatching rate of the parent plants in the control group and the feeding experimental group were statistically analyzed. The experimental results are shown in Table 1.

[0022] Table 1. Effects of feeding promoters on oviposition rate, sperm release rate, and larval hatching rate of adult cockles. ; Note: Data in the same column without a letter or with the same letter above the header indicates that the difference is not significant. P >0.05), different lowercase letters indicate significant differences ( P <0.05).

[0023] As shown in Table 1, compared with the control group, the egg-laying rate of the fed group of adult cockles was significantly increased by approximately 14.8% ( P <0.05%, the ejaculation rate increased by approximately 19.4% ( P <0.05, the hatching rate increased slightly, but there was no significant difference ( P >0.05).

[0024] Example 3: Application test of a promoter to improve the gonadal maturation of parent cockles The clams used in the experiment were collected from wild populations in the waters off Qingdao, Shandong Province. After sampling, all clams were transported back to the laboratory and temporarily kept in filtered seawater for one week. They were fed with Chlorella twice a day and the water was changed once a day. A growth promoter was started 20 days before the experiment. The gonadal tissue of all samples was sampled, flash-frozen in liquid nitrogen, and stored at -80 ℃ for subsequent experiments.

[0025] Experimental groups: Specifically, they include experimental group 1, experimental group 2 and experimental group 3, where the concentration of the accelerator in the water is 2 ppm in experimental group 1, 4 ppm in experimental group 2 and 6 ppm in experimental group 3. Control group: This group was not given any growth promoters, and all other breeding conditions were the same as those of the experimental group.

[0026] RNA extraction was performed on three cockle ovary samples randomly selected from both the experimental and control groups. The RNA extraction method used was the Trizol (Solarbio, China) method. The integrity and concentration of the extracted RNA were tested, and the OD (Organic Oxygen Demand) was also assessed. 260 / OD 280 The ratio should be in the range of 1.8 to 2.2.

[0027] Steroid-producing factor 1 (SPF1) was obtained from the transcriptome library of *Cynodon dactylon* based on whole transcriptome sequencing results. Steroidgenic factor 1 , SF-1 ), acute regulatory protein of steroid hormone synthesis ( steroidogenic acute regulatory protein , StAR ), 17β-hydroxysteroid dehydrogenase 14 ( 17β-hydroxysteroid dehydrogenase 14 , 17 β-HSD14 ), Cytochrome P450 17 ( Cytochrome P450 17 , CYP17 Four genes related to the synthesis of sex steroid hormones and vitellogenin ( Vitellogenin , Vg The cDNA fragment was analyzed using Primer 3.0 according to... SF-1 , StAR , 17β- HSD14, CYP17 and VgSpecific primers were designed based on the gene sequence, and the core fragment validation primers are shown in Table 2. The RNA from the ovaries of *Cycas revoluta* was reverse transcribed into cDNA using the Evo M-MLVPlus cDNA synthesis kit (Accurate, China), and then the target gene was amplified using 2 × TapPlus Master Mix II (Dye Plus) (Vazyme, China). After detecting the PCR products, the gel was excised and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction kit (TaKaRa, China). The target fragment was ligated into DH5α chemicompetent cells (Vazyme, China) using the 5 min TA / Blunt-Zero Cloning kit (Vazyme, China), and after overnight culture, positive colonies were selected and sent to the company for sequencing.

[0028] Table 2. Primer sequences for core fragment validation .

[0029] Three cockle ovary samples were randomly selected from both the experimental and control groups. RNA was reverse transcribed into cDNA using the HiScript® III RT SuperMix for qPCR kit (Vazyme, China), followed by qRT-PCR using the ChamQ SYBR Color qPCRMaster Mix kit (Vazyme, China). The RL15 gene was used as an internal control gene. Primers used for qRT-PCR are shown in Table 3. All data were analyzed using one-way ANOVA in SPSS 22.0. Quantitative data are expressed as mean ± standard deviation (SD), and the significance level for statistical analysis was set at 0.5%. P < 0.05.

[0030] Table 3 Primer sequences for qRT-PCR experiments .

[0031] Depend on Figure 1 The results showed that, compared with the control group, each treatment group SF-1 Expression levels were all significantly upregulated. p < 0.05). The expression level in experimental group 2 was the highest and significantly higher than that in experimental groups 1 and 3. p < 0.05), indicating SF-1 Expression peaked at 4 ppm, decreased at 6 ppm, but was still significantly higher than the control group. StAR Expression levels were also significantly increased in all treatment groups. p < 0.05, the expression level in experimental group 2 was the highest and significantly higher than that in the other two experimental groups (p The value is <0.05, indicating that its effect is strongest at 4 ppm and slightly weakens at 6 ppm. 17β-HSD14 Expression levels were significantly upregulated in all treatment groups. p <0.05), the expression level in experimental group 2 was the highest, significantly higher than that in experimental groups 1 and 3 ( p <0.05), indicating that the gene reaches its maximum expression at 4 ppm and shows a decreasing trend at 6 ppm. CYP17 Expression levels were significantly upregulated in all treatment groups. p <0.01), the expression level in experimental group 2 was the highest, and significantly higher than that in experimental groups 1 and 3. p The value <0.05 indicates that its activation is most significant at 4 ppm, and although it decreases at 6 ppm, it is still much higher than that of the control group. Vg Expression levels were also significantly increased in all treatment groups. p <0.05, the expression level in group 2 was the highest and significantly better than other treatment groups ( p <0.05, indicating that its inducing effect is strongest at 4 ppm and slightly weakens at 6 ppm. These results demonstrate that the promoter of this invention can significantly upregulate key genes in the steroid hormone synthesis pathway (…). SF- 1 , StAR , 17β-HSD14 , CYP17 )and Vg The expression of [the substance] promotes the development and maturation of the gonads of cockle, with 4 ppm being the optimal concentration.

Claims

1. A promoter that upregulates key genes in the biosynthesis pathway of cockle steroid hormones, characterized in that, The promoter is composed of the following components and their weight percentages: cholesterol 15-20 parts, linolenic acid 8-10 parts, phosphatidylcholine 10-12 parts, zinc sulfate 0.5-0.8 parts, L-arginine 30-40 parts, and vitamin D3 0.5-0.75 × 10⁻⁶. -3 share.

2. The specific accelerator according to claim 1, characterized in that, Cholesterol 1.5-2.0 g, linolenic acid 0.8-1.0 g, phosphatidylcholine 1.0-1.2 g, zinc sulfate 50-80 mg, L-arginine 3.0-4.0 g, vitamin D3 50-75 μg.

3. A specific accelerator according to claim 2, characterized in that, The promoters are: cholesterol 2.0g, linolenic acid 1.0g, phosphatidylcholine 1.1g, zinc sulfate 50mg, L-arginine 4.0g, and vitamin D3 50μg.

4. A specific accelerator according to claim 2, characterized in that, The promoters are: cholesterol 1.8g, linolenic acid 0.8g, phosphatidylcholine 1.1g, zinc sulfate 50mg, L-arginine 4.0g, and vitamin D3 50μg.

5. The application of the promoter according to any one of claims 1-4 in promoting the gonadal maturation of cockles, characterized in that, starting 20 days before the planned spawning date, the promoter is evenly sprinkled into the water body for cultivating cockles in the form of an additive after the cockles are transferred to the tank each day, so that the final concentration of the promoter in the water body is 4 ppm.

6. The promoter according to any one of claims 1-4 in upregulating key genes in the steroid hormone synthesis pathway and Vg Its application is characterized by the fact that the key gene is SF-1 , StAR , 17β-HSD14 , CYP17 The application concentration is 4 ppm.

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