Primer for detecting hypoxia marker HIF-1alpha of epinephelus micropunctatus as well as design method and application of primer

Abstract

The invention belongs to the technical field of biological genetic genes and aquaculture, and discloses a primer for detecting epinephelus microsporoides HIF-1alpha as well as a design method and application thereof, and the design method comprises the following steps: S1, performing anoxic domestication on epinephelus microsporoides domestication; s2, low-oxygen treatment and sample collection; and S3, primer design. According to the invention, the qRT-PCR primer suitable for the detection of the Epinephelus microarus HIF-1alpha gene is designed based on modes of low-oxygen domestication, conservative structural domain, contrast verification and the like, the system can accurately detect the expression level of HIF-1alpha mRNA in a blood tissue sample, and the amplification efficiency reaches 96.06%; the method has the advantages of high sensitivity, high sensitivity and the like, has extremely high significance on hypoxia stress of the epinephelus microcarpa, has the advantages of rapidness, high specificity, high sensitivity and the like, is suitable for low-cost and large-scale sample analysis and is applied to domestication, breeding and other works of the epinephelus microcarpa in a large scale, and the whole detection process is completed in only 70 minutes.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetics and aquaculture, and particularly relates to a primer for detecting HIF-1α, a hypoxia marker of Epinephelus gracilis, a design method and an application thereof. Background Art

[0002] In recent years, the rapid decline in dissolved oxygen (DO) content in water bodies caused by intensive aquaculture and drastic weather changes has become a common problem in aquaculture. The low oxygen environment will significantly affect the immune and disease resistance of farmed fish. In severe cases, it will lead to large-scale deaths of fish, causing huge economic losses to the aquaculture industry. Therefore, it has become one of the scientific problems that urgently need to be solved in the aquaculture industry.

[0003] The fine-spot grouper (scientific name: fine-spot grouper, commonly known as blue melon seed grouper) is a high-value aquatic species belonging to the genus Epinephelus of the family Serranidae in the order Perciformes. It is named because of the densely covered small black spots on its sides and its laterally flattened body like melon seeds. It is mainly distributed in Southeast Asia and the western Pacific region. Its ecological habits are warm-water reef-dwelling carnivorous fish. It lives in coral reefs or rocky reefs and feeds on small fish and crustaceans. The fine-spot grouper itself lacks auxiliary respiratory organs like freshwater fish (such as the labyrinth of mangrove), so it mainly relies on dissolved oxygen in water, and has a low tolerance threshold for low oxygen in water. It is generally believed that its critical asphyxiation point is less than 0.33 mg / L, which is much lower than that of freshwater grouper (the critical asphyxiation point is generally greater than 3 mg / L).

[0004] With the scale-up of Epinephelus, intensive aquaculture and deep-sea aquaculture have become key research areas. Since traditionally farmed Epinephelus has a weak tolerance to low oxygen, insufficient dissolved oxygen in the water has become a major obstacle to the development of Epinephelus aquaculture. At the same time, the accumulation of feces during high-density aquaculture will also enrich pathogens in the water and increase the stress on Epinephelus. Therefore, detecting the expression changes of HIF-1α in Epinephelus is of great significance in intensive aquaculture and deep-sea aquaculture of Epinephelus.

[0005] According to existing research results, hypoxia-inducible factor 1α (HIF-1α) is a central factor in the hypoxia regulation pathway. Under hypoxic conditions, the stability of HIF-1α increases and it gradually accumulates and translocates to the nucleus, promoting the transcription of specific genes, so HIF-1α can be used as a hypoxia marker. In addition, HIF-1α can activate a variety of cells (B cells, macrophages, neutrophils, etc.) to exert immune responses by regulating the expression of a variety of cytokines (chemokines, inflammatory factors, and antimicrobial peptides, etc.). Therefore, by accurately detecting the change level of the fish hypoxia marker HIF-1α, it can help observe the fish's hypoxia tolerance; however, since the gene sequence of HIF-1α is different for each fish, there are many technical difficulties in quickly and accurately detecting the change level of the fish hypoxia marker HIF-1α.

[0006] Quantitative Real-time polymerase chain reaction (qRT-PCR) is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. It is a method for quantitatively analyzing specific DNA sequences in the sample to be tested by using internal or external reference methods. It combines PCR technology and real-time fluorescence detection technology, and can monitor the amplification of DNA in real time during the PCR reaction, thereby achieving quantitative analysis of the target DNA. The specificity of qRT-PCR depends on oligonucleotide primers that are complementary to both ends of the target sequence. However, due to many specific technical difficulties, there are no universal primers and specific methods for PCR detection of HIF-1α, a hypoxia marker in various fish, so far, especially primers with high sensitivity and strong specificity for marine fish have not been reported so far.

[0007] CN110894229A discloses a goldfish hypoxia-inducible factor HIF-1α gene, cloning method and application thereof. The nucleotide sequence of the goldfish hypoxia-inducible factor HIF-1α gene provided is SEQ ID NO.1. This invention application discloses the nucleotide sequence and amino acid sequence of the goldfish hypoxia-inducible factor HIF-1α gene, and verifies the role played by this gene under hypoxic stress in goldfish through qPCR technology. The expression patterns of goldfish hypoxia-inducible factor HIF-1α in liver, brain, heart and gill tissues are analyzed by real-time fluorescence quantitative qPCR. The results show that under hypoxic stress conditions in goldfish, the expression patterns of the HIF-1α gene in the four tissues are different, which can provide basic data and references for the hypoxic adaptation mechanism of fish, the hypoxic signal transduction pathway and the cultivation of new varieties of hypoxia-tolerant fish. However, this technical solution does not provide primers that can be used for rapid detection of HIF-1α in other fish. If a comprehensive detection of the nucleotide sequences and amino acid sequences of the hypoxia-inducible factor HIF-1α genes of different fish is carried out, a large number of instruments and consumables are required, and the detection and analysis steps are complex, with a high comprehensive cost, making it difficult to meet the industrial requirements.

[0008] CN118028485A discloses a primer for detecting HIF-1α of Hexagram otaki based on PCR. The DNA sequence of the primer is as follows: upstream primer: F 5'-GCTGGGTGACATAAGAGAGATG-3'; downstream primer: R 5'-TGAAGGCAGCAGAAGTATGG-3'. It has the advantages of high sensitivity and strong specificity, and can detect the relative expression level of HIF-1α in the blood of Hexagram otaki, so as to accurately evaluate the hypoxia stress or pathogen infection in the breeding of Hexagram otaki, and provide basic data for preventing hypoxia and disease in the high-density breeding of Hexagram otaki. Existing research results show that Hexagrammos otakii (scientific name: Hexagrammos otakii) is a cold-temperature nearshore bottom fish that shows strong adaptability to low-oxygen environments (dissolved oxygen concentration below 2 mg / L), and its suffocation point is about 0.71-0.84 mg / L. Although Hexagrammos otakii's tolerance to low oxygen is relatively weak compared to freshwater fish, the comprehensive ecological habits, data on related species and low oxygen response mechanisms show that Hexagrammos otakii's tolerance to low oxygen is still significantly higher than that of Epinephelus spicata. Hexagrammos otakii can survive for several hours in dissolved oxygen below 1.5 mg / L through low metabolic rate, HIF-1α activation and enhanced antioxidant system; while Epinephelus spicata, as a tropical coral reef fish, has a lower critical suffocation point (the critical suffocation point is less than 0.33 mg / L), and is more sensitive to hypoxia. Currently, there is no research on the hypoxia tolerance of fine-spotted grouper. Fine-spotted grouper itself lacks the auxiliary respiratory organs of other fish, and its hypoxia tolerance mechanism is significantly different from that of other fish. In addition, the primer region designed by CN118028485A for otaki six-line fish is in a non-conserved region (amino acid range 737-745), which is quite different from many bony fish, especially fine-spotted grouper. Based on these significant differences, CN118028485A obviously cannot solve the problem of hypoxia tolerance detection of fine-spotted grouper, and the document does not specifically record that its primers can be applied to the detection of other fish, let alone how to apply its detection results to the breeding activities of other fish.

[0009] Therefore, for the fine-spot grouper, whose hypoxia tolerance mechanism is significantly different from that of conventional fish and has extremely low oxygen tolerance, how to design a highly sensitive and specific primer, and then quickly detect the changes in the relative amount of HIF-1α expression in the fine-spot grouper based on PCR, and further apply it in the breeding, domestication and large-scale farming of fine-spot grouper, significantly improve the detection speed and reduce the cost, is of great industrial significance. Summary of the invention

[0010] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a primer, a design method and an application thereof for detecting HIF-1α, a hypoxia marker of fine-spot grouper. Hypoxia domestication is adopted in combination with a scientific design method, and qRT-PCR primers suitable for HIF-1α gene detection of fine-spot grouper are designed based on conservative domains and comparative verification, filling the gap in the technical field of hypoxia-inducible factor detection of this species. Based on the specific primer pairs designed and verified by conservative domains, the system can accurately detect the expression level of HIF-1α mRNA in blood tissue samples, and its amplification efficiency reaches 96.06% after melting curve analysis. It has extremely strong significance for hypoxia stress of fine-spot grouper, and has the advantages of rapidity, strong specificity and high sensitivity, and the entire detection process only takes 70 minutes to complete, which is suitable for low-cost large-scale sample analysis, and can be applied on a large scale in the domestication and breeding of fine-spot grouper.

[0011] To achieve the above object, the present invention adopts the following technical solutions: A method for designing primers for detecting HIF-1α, a hypoxia marker of Epinephelus spiculus, is characterized in that it comprises the following steps: S1: Hypoxia acclimation of Epinephelus gracilis Multiple groups of healthy groupers were raised in multiple tanks, fed once in the morning and once in the evening every day, the water temperature was controlled at 22±0.5℃, and cultured in anoxic circulating water for 21 consecutive days; S2: Hypoxia treatment and sample collection After acclimation, nitrogen was added to the water tank to maintain the dissolved oxygen concentration at 0.5 ± 0.1 mg / L, and the dissolved oxygen concentration was tested. Three fish were randomly selected from each group at 0 h, 1 h, 3 h, 6 h, and 9 h, anesthetized, and venous blood was obtained using a syringe; S3: Primer Design Based on the translation of the nucleotide sequence of HIF-1α into the amino acid sequence, the domain structure of HIF-1α was predicted using the domain online website; Based on the NCBI database, 4 pairs of primers were designed in the structural domain of HIF-1α, specifically: The first pair: qHIF-1αF1 5'-TGGGAAGGAGTCTGAGGTGT -3', qHIF-1αR1 5′-TCCGCATGAGCAGTTTCCT; The product length is 138 bp, and the binding position is HLH; The second pair: qHIF-1αF2 5'-GTGTGGGTGGAAACACAAG -3', qHIF-1αR2 5'-CCTGGATGCCACTTAGCACA-3'; The product length is 97 bp, and the binding position is PAC; The third pair: qHIF-1αF3 5'-TGAGGACACTTCCAATGGGC -3', qHIF-1αR3 5' -CAACAGGTCGTCTGGGTCAT -3'; The product length is 199 bp, and the binding site is PAS; The fourth pair: qHIF-1αF4 5'-AGGCCAAGGAACCAAACACA -3', qHIF-1αR4 5'-GCCCATTGGAAGTGTCCTCA-3'; The product length is 165 bp, and the binding site is a non-structured domain position between two PASs; S4: Primer Validation S4-1 extracts mRNA from blood of Epinephelus gracilis; S4-2 reverse transcribes blood mRNA of Epinephelus spicata into cDNA; S4-3 respectively uses the four pairs of primers to perform qRT-PCR amplification on the blood cDNA of Epinephelus gracilis; S4-4 After qRT-PCR, the performance of each primer pair was observed through melting curve analysis. A single peak indicated that the amplification effect of the primer was good.

[0012] S5: Agarose gel electrophoresis S5-1 Take agarose and add it to 1×TAE, then heat it in a microwave oven to dissolve it into 1% agarose gel, add nucleic acid dye; seal both ends of the electrophoresis template, pour in the agarose gel solution, and insert the comb; after condensation, pull out the comb and put the electrophoresis gel into the electrophoresis tank; S5-2 Take 10 μL of the amplified product, add the loading buffer, mix well, and add to the sample well. At the same time, add 5 μL of Maker to the first lane; maintain the power at 130V, and stop the electrophoresis until the bromophenol blue gun reaches 2 / 3 of the bottom of the gel; After S5-3 electrophoresis, the agarose gel was taken out and then placed in a gel imager for observation and photography to screen out the best pair of primers.

[0013] A primer for detecting HIF-1α, a hypoxia marker of Epinephelus spicata, is obtained by the aforementioned design method, and is a qRT-PCR primer pair for detecting HIF-1α gene of Epinephelus spicata based on real-time fluorescence quantitative PCR, comprising an upstream primer and a downstream primer, and the DNA sequences are: Upstream primer: F 5'-TGGGAAGGAGTCTGAGGTGT-3', Downstream primer: R 5′-TCCGCATTGAGCAGTTTCCT-3′.

[0014] Application of a primer for detecting HIF-1α, a hypoxia marker of Epinephelus gracilis, in breeding of Epinephelus gracilis, comprising the following steps: The primers are used to perform real-time fluorescence quantification on blood cDNA of multiple groups of fine-spot grouper samples after hypoxia acclimation treatment for 0h, 1h, 3h, 6h and 9h, and the relative expression of HIF-1α after different hypoxia stress time is detected. After analyzing and comparing the change trends, the relative expression at 0h (that is, the relative expression of HIF-1α in the blood of healthy fine-spot grouper) is selected as the standard for detecting whether the fine-spot grouper is subjected to hypoxia stress. A value higher than this value indicates that it is subjected to hypoxia stress, and a value close to this value indicates that it is not subjected to hypoxia stress. Fish whose relative expression value does not change or does not change significantly after hypoxia stress are used as breeding fish.

[0015] When the primers are used to perform real-time fluorescence quantitative detection on blood cDNA of Epinephelus spheniscus, the real-time fluorescence quantitative amplification system used is 20 μL, including: 5 ng / μL cDNA 3.0 μL, Hieff ® qPCR SYBR Green MasterMix 10.0 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, sterile ultrapure water 6.2 μL.

[0016] The real-time fluorescence quantitative qRT-PCR amplification procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60°C for 30 s for 40 cycles, then melting curves were obtained after 15 s at 95°C, 60 s at 60°C, and 15 s at 95°C, and then stored at 4°C. –∆ ∆CT The relative expression of HIF-1α was calculated.

[0017] The present invention has at least the following beneficial effects: (1) The primers, design methods and applications for detecting HIF-1α, a hypoxia marker of Epinephelus spp. provided by the present invention, are designed based on conservative domain design and comparative verification, using hypoxia acclimation and scientific design methods to design qRT-PCR primers suitable for HIF-1α gene detection in Epinephelus spp., filling the gap in the field of hypoxia inducible factor detection technology for this species, and can be further applied to variety selection and disease prevention and control. They can provide key molecular markers for the progressive hypoxia tolerance acclimation and variety selection of Epinephelus spp., and breed fish with improved hypoxia tolerance. Actual experimental results show that the system can accurately detect the expression level of HIF-1α mRNA in blood tissue samples, has extremely strong significance for hypoxia stress, and has the advantages of rapidity, strong specificity and high sensitivity. The entire detection process only takes 70 minutes to complete, and can be applied to low-cost large-scale sample analysis, as well as large-scale application in the acclimation and breeding of Epinephelus spp.

[0018] (2) The primers, design methods and applications provided by the present invention have extremely strong significance for the hypoxia stress of the fine-spot grouper, and have the advantages of rapidity, strong specificity and high sensitivity. They can be applied to aquaculture risk warning. By monitoring the dynamic changes in the expression of HIF-1α, the hypoxia stress risk of the fine-spot grouper can be predicted in advance, and a dissolved oxygen early warning model can be provided for intensive aquaculture and deep-sea cage aquaculture, thereby optimizing aquaculture density and environmental management and improving aquaculture efficiency. At the same time, by evaluating the expression of immune-related genes, it can assist in the formulation of hypoxia emergency plans and disease prevention and control strategies to reduce economic losses in the aquaculture industry.

[0019] (3) The primers, design methods and applications provided by the present invention solve the technical bottleneck of hypoxia detection in fine-spot grouper through a new targeted domestication and primer design method, and have the advantages of high efficiency, accuracy and speed, providing key technical support for the scientific management of aquaculture and improvement of economic benefits.

[0020] (4) The primers, design methods and applications provided by the present invention have certain potential for technology promotion. The primers and their design methods provided by the present invention are more applicable, less time-consuming, more efficient and more accurate. They can also be extended to the study of hypoxic stress in other aquatic species, promote the advancement of molecular detection technology in the field of aquaculture, and promote the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of the domain structure of HIF-1α predicted by the embodiment of the present invention; Figure 2 This is an electrophoresis diagram of the PCR product detected by agarose gel electrophoresis in an embodiment of the present invention; Figure 3This is a schematic diagram of the real-time fluorescence quantitative results of an embodiment of the present invention. DETAILED DESCRIPTION

[0022] The following is combined with Figure 1-3 The technical solution of the present invention is further described in detail with specific implementation cases so that those skilled in the art can fully understand the technical solution of the present invention. All reagents or materials used in the implementation cases of the present invention, unless otherwise specified, are from commercial channels.

[0023] Example 1 The method for designing primers for detecting the hypoxia marker HIF-1α of Epinephelus spicata provided in an embodiment of the present invention comprises the following steps: S1: Hypoxia acclimation of Epinephelus gracilis Multiple groups of healthy groupers were raised in multiple tanks, fed once in the morning and once in the evening every day, and the water temperature was controlled at 22±0.5℃. The fish were cultured in natural anoxic circulating water without artificially adding dissolved oxygen to the water in the tanks. The fish were acclimated for 21 consecutive days. S2: Hypoxia treatment and sample collection After acclimation, nitrogen was added to the water tank to maintain the dissolved oxygen concentration at 0.5 ± 0.1 mg / L, and the dissolved oxygen concentration was tested. Three fish were randomly selected from each group at 0 h, 1 h, 3 h, 6 h, and 9 h, anesthetized, and venous blood was obtained using a syringe; S3: Primer Design Based on the translation of the nucleotide sequence of HIF-1α into the amino acid sequence, the domain structure of HIF-1α was predicted using the domain online website; Based on the NCBI database, 4 pairs of primers were designed in the structural domain of HIF-1α, specifically: The first pair: qHIF-1αF1 5'-TGGGAAGGAGTCTGAGGTGT -3', qHIF-1αR1 5'-TCCGCATGAGCAGTTTCCT; The product length is 138 bp, and the binding position is HLH; The second pair: qHIF-1αF2 5'-GTGTGGGTGGAAACACAAG -3', qHIF-1αR2 5'-CCTGGATGCCACTTAGCACA-3'; The product length is 97 bp, and the binding position is PAC; The third pair: qHIF-1αF3 5'-TGAGGACACTTCCAATGGGC -3', qHIF-1αR3 5' -CAACAGGTCGTCTGGGTCAT -3'; The product length is 199 bp, and the binding site is PAS; The fourth pair: qHIF-1αF4 5'-AGGCCAAGGAACCAAACACA -3', qHIF-1αR4 5'-GCCCATTGGAAGTGTCCTCA-3'; The product length is 165 bp, and the binding site is a non-structured domain position between two PASs; S4: Primer Validation S4-1 extracts mRNA from blood of Epinephelus gracilis; S4-2 reverse transcribes blood mRNA of Epinephelus spicata into cDNA; S4-3 respectively uses the four pairs of primers to perform qRT-PCR amplification on the blood cDNA of Epinephelus gracilis; S4-4 After qRT-PCR, the performance of each primer pair was observed through melting curve analysis. A single peak indicated that the amplification effect of the primer was good.

[0024] S5: Agarose gel electrophoresis S5-1 Take agarose and add it to 1×TAE, then heat it in a microwave oven to dissolve it into 1% agarose gel, add nucleic acid dye; seal both ends of the electrophoresis template, pour in the agarose gel solution, and insert the comb; after condensation, pull out the comb and put the electrophoresis gel into the electrophoresis tank; S5-2 Take 10 μL of the amplified product, add the loading buffer, mix well, and add to the sample well. At the same time, add 5 μL of Maker to the first lane; maintain the power at 130V, and stop the electrophoresis until the bromophenol blue gun reaches 2 / 3 of the bottom of the gel; After S5-3 electrophoresis, the agarose gel was taken out and then placed in a gel imager for observation and photography to screen out the best pair of primers.

[0025] The primers for detecting the hypoxia marker HIF-1α of Epinephelus spicata provided in this embodiment are designed by the aforementioned method, and are qRT-PCR primer pairs for detecting the HIF-1α gene of Epinephelus spicata based on real-time fluorescence quantitative PCR, including upstream primers and downstream primers, and the DNA sequences are respectively: Upstream primer: F 5'-TGGGAAGGAGTCTGAGGTGT-3', Downstream primer: R 5′-TCCGCATTGAGCAGTTTCCT-3′.

[0026] Application of a primer for detecting HIF-1α, a hypoxia marker of Epinephelus gracilis, in breeding of Epinephelus gracilis, comprising the following steps: The primers were used to perform real-time fluorescence quantification on blood cDNA of multiple groups of Epinephelus spp. samples after hypoxia acclimation treatment for 0 h, 1 h, 3 h, 6 h, and 9 h, and the relative expression levels of HIF-1α after different hypoxia stress times were detected. After analysis and comparison, the fish with no change or small change in relative expression level were selected as breeding fish of Epinephelus spp.

[0027] When the primers are used to perform real-time fluorescence quantitative detection on cDNA of blood of grouper, the real-time fluorescence quantitative amplification system is 20 μL, including: 3.0 μL of 5 ng / μL cDNA, 10.0 μL of Hieff® qPCR SYBR Green Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, and 6.2 μL of sterile ultrapure water.

[0028] The real-time fluorescence quantitative qRT-PCR amplification procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60°C for 30 s for 40 cycles, then melting curves were obtained after 15 s at 95°C, 60 s at 60°C, and 15 s at 95°C, and then stored at 4°C. –∆ ∆CT The relative expression of HIF-1α was calculated.

[0029] Example 2 The primers, design method and application for detecting the hypoxia marker HIF-1α of Epinephelus spiculus provided in the embodiment of the present invention are specific implementations based on the embodiment 1, and the difference is that: The experimental animals used in this example were healthy grouper weighing 35±0.5 g, provided by Guangdong Marine Fisheries Center.

[0030] Experimental materials: Trizol (Novozyme, R411-01), reverse transcription reagent (Yisheng, 11149ES10), fluorescent quantitative PCR reagent (Yisheng, 11202ES03).

[0031] The method for designing primers for detecting the hypoxia marker HIF-1α of Epinephelus spicata comprises the following steps: S1: Acclimation of Epinephelus gracilis for anoxic culture Several groups of healthy groupers were raised in five 200L plastic tanks (50 fish in each tank), and were fed once a day at 9:00 and 17:00 respectively. The water temperature was controlled at 22±0.5℃, and the fish were cultured in natural anoxic circulating water without artificially adding dissolved oxygen to the water body through aerators. The fish were acclimated for 21 consecutive days under natural anoxic conditions. S2: Hypoxia treatment and sample collection After acclimation, nitrogen was added to the water tank to maintain the dissolved oxygen concentration at 0.5 ± 0.1 mg / L, and the dissolved oxygen concentration was tested using a Lei-Magnetic portable dissolved oxygen meter (JPBJ, China). Three fish were randomly selected at 0 h, 1 h, 3 h, 6 h, and 9 h, anesthetized with MS-222, and venous blood was obtained using a 1 mL syringe; S3: Primer Design Based on the translation of the nucleotide sequence of HIF-1α into the amino acid sequence, the domain structure of HIF-1α was predicted using the domain online website Simple Modular Architecture Research Tool (SMART) ( http: / / smart.emblheidelberg.de / ); Then, based on the primer design website Primer designing tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome) of the NCBI database, four pairs of primers were designed in the structural domain of HIF-1α, specifically: The first pair: qHIF-1αF1 5'-TGGGAAGGAGTCTGAGGTGT -3', qHIF-1αR1 5'-TCCGCATGAGCAGTTTCCT; The product length is 138 bp, and the binding position is HLH; The second pair: qHIF-1αF2 5'-GTGTGGGTGGAAACACAAG -3', qHIF-1αR2 5'-CCTGGATGCCACTTAGCACA-3'; The product length is 97 bp, and the binding position is PAC; The third pair: qHIF-1αF3 5'-TGAGGACACTTCCAATGGGC -3', qHIF-1αR3 5' -CAACAGGTCGTCTGGGTCAT -3'; The product length is 199 bp, and the binding site is PAS; The fourth pair: qHIF-1αF4 5'-AGGCCAAGGAACCAAACACA -3', qHIF-1αR4 5'-GCCCATTGGAAGTGTCCTCA-3'; The product length is 165 bp, and the binding site is a non-structured domain position between two PASs; S4: Primer Validation S4-1 extracts mRNA from blood of Epinephelus gracilis; S4-2 reverse transcribes blood mRNA of Epinephelus spicata into cDNA; S4-3 qRT-PCR amplification of blood cDNA of Epinephelus spicata was performed using the above four pairs of primers; The qRT-PCR amplification system is 20 μL, including: 5 ng / μL cDNA 3.0 μL, Hieff ® qPCR SYBRGreen Master Mix 10.0 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, sterile ultrapure water 6.2 μL. The procedure of qRT-PCR amplification in the present invention is: pre-denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, then, obtain the melting curve after 15 s at 95°C, 60 s at 60°C and 15 s at 95°C, and store at 4°C; the fluorescence quantitative instrument is LightCycler ® 480Instrument II.

[0032] S4-4 After qRT-PCR, melting curve analysis was performed. If the primer showed a single peak, it indicated that the amplification effect was good. After qRT-PCR, melting curve analysis showed that all four primer pairs had single peaks, indicating good amplification effects, and agarose gel electrophoresis was used for further detection.

[0033] S5: Agarose gel electrophoresis S5-1 Take 0.3g agarose and add 30mL 1×TAE, then heat in a microwave to dissolve and prepare 1% agarose gel, add 3μL nucleic acid dye; seal both ends of the electrophoresis template, pour in the agarose gel solution, and insert the comb. After condensation, pull out the comb and put the electrophoresis gel into the electrophoresis tank; S5-2 Take 10 μL of the amplified product, add 1 / 6 volume of loading buffer, mix well, add to the sample well, and add 5 μL of Maker to the first lane; maintain the power at 130V, and stop electrophoresis when the bromophenol blue gun reaches 2 / 3 of the bottom of the gel; After S5-3 electrophoresis, take out the agarose gel and then put it into the gel imager for observation and photography. Figure 2 shown. Figure 2In the figure, M: DNA Marker, 1: product of the first pair of primers, 2: product of the second pair of primers, 3: product of the third pair of primers, 4: product of the fourth pair of primers.

[0034] From the attached Figure 2 It can be seen that the bands of the first pair of primers and the second pair of primers in this embodiment are single and have no tailing phenomenon, which has high specificity, but the bands of the third and fourth pairs of primers have tailing phenomenon, which is considered to be not specific, so they are discarded. Through the calculation of the melting curve analysis of the first and second pairs of primers, the amplification efficiency is 96.06% (first pair) and 180.82% (second pair), respectively. It is generally believed that the amplification efficiency of primers should be between 90% and 110%, so the first pair of primers is determined to be the best primer sequence and used as the primer for detecting the hypoxia marker HIF-1α of fine-spotted grouper.

[0035] The invention discloses an application of a primer for detecting HIF-1α, a hypoxia marker of Epinephelus spicata, in the breeding of Epinephelus spicata, which detects and compares the relative expression of HIF-1α in the blood of Epinephelus spicata at different hypoxia stress times, evaluates the domestication effect, and performs breeding, etc., and comprises the following steps: (1) Using the first pair of primers mentioned above, real-time fluorescence quantification of blood cDNA of multiple groups and multiple fine-spotted grouper after hypoxia treatment for 0 h, 1 h, 3 h, 6 h, and 9 h was performed, and then the relative expression levels of HIF-1α in each fish were compared.

[0036] The quantitative results of this example are as follows Figure 3 Observation Figure 3 The real-time fluorescence quantitative results in show that the primers provided in this example can quickly and accurately detect the relative expression level and changes of HIF-1α after different hypoxia stress time.

[0037] The experimental results of the above embodiments of the present invention show that the primers for detecting HIF-1α in the blood of Epinephelus spp. based on PCR provided by the present invention have the advantages of rapidity, strong specificity and high sensitivity, and can provide basic data for the domestication, breeding and prevention of hypoxia and disease in intensive aquaculture and deep-sea aquaculture of Epinephelus spp.

[0038] After actual testing, the primers and design method for detecting the hypoxia marker HIF-1α of fine-spot grouper provided in the above embodiments of the present invention and their application in fine-spot grouper farming can meet the needs of large-scale breeding and production of multiple populations of fine-spot grouper distributed in different regions, and have low overall cost, fast speed and easy implementation.

[0039] The above implementation cases are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above implementation cases. The technicians under the implementation cases all belong to the protection scope of the present invention. Ordinary technicians in the relevant technical field can achieve the purpose of the present invention based on the above disclosure of the present invention. Any improvements and modifications made based on the concept of the present invention fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A method for designing primers for detecting HIF-1α, a hypoxia marker in Epinephelus spicata, characterized in that: It includes the following steps: S1: Hypoxia acclimation of Epinephelus gracilis Multiple groups of healthy groupers were raised in multiple tanks, fed once in the morning and once in the evening every day, the water temperature was controlled at 22±0.5℃, and cultured in anoxic circulating water for 21 consecutive days; S2: Hypoxia treatment and sample collection After acclimation, nitrogen was added to the water tank to maintain the dissolved oxygen concentration at 0.5 ± 0.1 mg / L, and the dissolved oxygen concentration was tested. Three fish were randomly selected from each group at 0 h, 1 h, 3 h, 6 h, and 9 h, anesthetized, and venous blood was obtained using a syringe; S3: Primer Design First, the nucleotide sequence of HIF-1α was translated into an amino acid sequence to predict the structural domain of HIF-1α. Then, based on the NCBI database, 4 pairs of primers were designed within the structural domain of HIF-1α, specifically: The first pair: qHIF-1αF1 5'-TGGGAAGGAGTCTGAGGTGT -3', qHIF-1αR1 5'-TCCGCATGAGCAGTTTCCT; The product length is 138 bp, and the binding position is HLH; The second pair: qHIF-1αF2 5'-GTGTGGGTGGAAACACAAG -3', qHIF-1αR2 5'-CCTGGATGCCACTTAGCACA-3'; The product length is 97 bp, and the binding position is PAC; The third pair: qHIF-1αF3 5'-TGAGGACACTTCCAATGGGC -3', qHIF-1αR3 5' -CAACAGGTCGTCTGGGTCAT -3'; The product length is 199 bp, and the binding site is PAS; The fourth pair: qHIF-1αF4 5'-AGGCCAAGGAACCAAACACA -3', qHIF-1αR4 5'-GCCCATTGGAAGTGTCCTCA-3'; The product is 165 bp in length and the binding site is a non-structural position between two PAS.

2. The method for designing primers for detecting the hypoxia marker HIF-1α of Epinephelus spicata according to claim 1, characterized in that: It also includes step S4 primer verification, specifically: S4-1 extracts mRNA from blood of Epinephelus gracilis; S4-2 reverse transcribes blood mRNA of Epinephelus spicata into cDNA; S4-3 respectively uses the four pairs of primers described in claim 2 to perform qRT-PCR amplification on the blood cDNA of Epinephelus spicata; S4-4 After qRT-PCR, the performance of each primer pair was observed through melting curve analysis. A single peak indicated that the amplification effect of the primer was good.

3. The method for designing primers for detecting the hypoxia marker HIF-1α of Epinephelus spicata according to claim 2, characterized in that: The method further comprises step S5 of agarose gel electrophoresis, specifically comprising: S5-1 Take agarose and add it to 1×TAE, then heat it in a microwave oven to dissolve it into 1% agarose gel, add nucleic acid dye; seal both ends of the electrophoresis template, pour in the agarose gel solution, and insert the comb; after condensation, pull out the comb and put the electrophoresis gel into the electrophoresis tank; S5-2 Take 10 μL of the amplified product, add the loading buffer, mix well, and add to the sample well. At the same time, add 5 μL of Maker to the first lane; maintain the power at 130V, and stop the electrophoresis until the bromophenol blue gun reaches 2 / 3 of the bottom of the gel; After S5-3 electrophoresis, the agarose gel was taken out and then placed in a gel imager for observation and photography to screen out the best pair of primers.

4. A primer for detecting HIF-1α, a hypoxia marker in Epinephelus spicata, characterized in that: It is a qRT-PCR primer pair for detecting the HIF-1α gene of Epinephelus spicata based on real-time fluorescence quantitative PCR, including an upstream primer and a downstream primer, and the DNA sequences are: Upstream primer: F 5'-TGGGAAGGAGTCTGAGGTGT-3', Downstream primer: R 5′-TCCGCATTGAGCAGTTTCCT-3′.

5. Use of the primers for detecting the hypoxia marker HIF-1α of Epinephelus gracilis according to claim 4 in breeding of Epinephelus gracilis.

6. The use according to claim 5, characterized in that: It includes the following steps: The primers are used to perform real-time fluorescence quantification on blood cDNA of multiple groups of Epinephelus samples after hypoxia acclimation treatment for 0h, 1h, 3h, 6h and 9h, and the relative expression of HIF-1α after different hypoxia stress times is detected. After analyzing and comparing the change trends, the relative expression at 0h is selected as the standard for detecting whether the Epinephelus is subjected to hypoxia stress. A value higher than this value indicates that it is subjected to hypoxia stress, and a value close to this value indicates that it is not subjected to hypoxia stress. Fish whose relative expression value does not change significantly after hypoxia stress are used as breeding fish.

7. The use according to claim 6, characterized in that: It includes the following steps: When the primers are used to perform real-time fluorescence quantitative detection on blood cDNA of grouper, the real-time fluorescence quantitative amplification system is 20 μL, including: 3.0 μL of 5 ng / μL cDNA, 10.0 μL of qPCR SYBR Green Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, and 6.2 μL of sterile ultrapure water.

8. The use according to claim 7, characterized in that: It includes the following steps: The real-time fluorescence quantitative qRT-PCR amplification procedure was as follows: pre-denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, then obtaining the melting curve after 15 s at 95°C, 60 s at 60°C, and 15 s at 95°C, and storing at 4°C; then using calculate The relative expression level.

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