Eriocheir sinensis HNF4 gene and cloning method and application thereof

Abstract

The invention discloses an Eriocheir sinensis HNF4 gene and a cloning method and application thereof, the nucleotide sequence of the HNF4 gene is as shown in SEQ ID NO: 1, the amino acid sequence of protein coded by the HNF4 gene is as shown in SEQ ID NO: 2, and the HNF4 gene is separated and cloned from Eriocheir sinensis for the first time to obtain a full-length complete sequence. After the expression of the HNF4 gene is improved, the expression quantities of FAS, SCD, FAD6, FAD9 and ELOVL4 genes in hepatopancreas of the Eriocheir sinensis are remarkably increased, the expression of the HSL gene is remarkably reduced, and the results are opposite after the expression of the HNF4 gene is inhibited, so that the HNF4 gene can promote the expression of fatty acid synthesis related genes, plays a role in the synthesis process of fatty acid of the Eriocheir sinensis as a transcription factor, and can be used for preparing the fatty acid of the Eriocheir sinensis. The synthesis of LC-PUFA is promoted, and a reference is provided for the functional research of the HNF4 gene of the crustacean.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to a Chinese mitten crab HNF4 gene and a cloning method and application thereof. Background Art

[0002] Long-chain polyunsaturated fatty acids (LC-PUFA) refer to polyunsaturated fatty acids with ≥3 double bonds and ≥20 carbon atoms. LC-PUFA has important physiological effects. Among them, EPA and DHA are essential for maintaining body homeostasis. EPA can promote the degradation of cholesterol and TG in the blood and improve human blood microcirculation, while DHA can improve brain cell development. In addition, cell membrane phospholipids also need LC-PUFA composition. LC-PUFA can maintain cell membrane fluidity, regulate gene transcription and intracellular signal transduction, and regulate cell physiological functions.

[0003] Hepatocyte nuclear factor (HNF) is a transcription factor specifically expressed in liver genes, and plays an important role in animal development, hepatocyte proliferation and differentiation, and maintaining the biological function of hepatocytes and the physiological function of the liver. HNF4 is a member of the HNF gene family, mainly present in animal livers, and is an important transcription factor involved in regulating lipid and cholesterol metabolism. It mainly regulates the synthesis of PUFA at the transcriptional level and regulates the activity of genes such as ELOVL and FAD. In addition, HNF4 can also interact with transcription factors to regulate the anabolism of LC-PUFA.

[0004] Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species with extremely high economic value. Studying its lipid accumulation and metabolic mechanism not only helps to improve the anti-stress ability of Chinese mitten crab itself, but also provides basic data for improving its nutritional value. In recent years, the regulatory mechanism and interaction of HNF4 on key enzyme genes and transcription factors of LC-PUFA biosynthesis have been studied, but there is no report that HNF4 gene plays a role as LC-PUFA biosynthesis transcription factor in crustaceans. Therefore, studying and cloning the HNF4 gene of Chinese mitten crab is of great significance for studying LC-PUFA biosynthesis in crustaceans and reducing the dependence of aquaculture on fish oil. Summary of the invention

[0005] The main purpose of the present invention is to provide the Chinese mitten crab HNF4 gene, and to obtain the complete sequence of the Chinese mitten crab HNF4 gene by cloning through RACE technology, so as to provide a reference for the functional study of the crustacean HNF4 gene.

[0006] Another object of the present invention is to provide a method for cloning the Chinese mitten crab HNF4 gene, which is cloned using the RACE technology.

[0007] Another object of the present invention is to provide the application of the Chinese mitten crab HNF4 gene in regulating the biosynthesis of LC-PUFA in crustaceans.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a Chinese mitten crab HNF4 gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0010] The present invention also provides a protein encoded by the Eriocheir sinensis HNF4 gene, the amino acid sequence of which is shown in SEQ ID NO:2.

[0011] The present invention also provides a method for cloning the HNF4 gene of Chinese mitten crab, comprising the following steps:

[0012] Step a: designing primers based on a part of the known HNF4 gene sequence, performing PCR reaction using the first strand of cDNA reverse transcribed from the total RNA of Chinese mitten crab as a template, obtaining the core fragment of the HNF4 gene of Chinese mitten crab, cloning the core fragment of the HNF4 gene into a vector and sequencing;

[0013] Step b: design 3'-HNF4 RACE downstream primers and 5'-HNF4 RACE upstream primers respectively according to the obtained HNF4 gene core fragment, reverse transcribe the total RNA of Chinese mitten crab into 3'-cDNA and 5'-cDNA first chain, use them as templates to perform 3' and 5' fragment RACE, connect the obtained 3' and 5' fragments to the vector respectively, clone and sequence;

[0014] Step c: splicing the obtained 3' and 5' fragments with the obtained HNF4 gene core fragment respectively to obtain the full-length sequence of the Chinese mitten crab HNF4 gene.

[0015] Preferably, in steps a and b, the total RNA of Eriocheir sinensis is extracted from the hepatopancreas of Eriocheir sinensis by using the Trizol method.

[0016] Preferably, in steps a and b, the vector is a pMD-19T plasmid.

[0017] The present invention also provides a vector, which comprises the Chinese mitten crab HNF4 gene.

[0018] The present invention also provides a transfected cell, which comprises the vector.

[0019] Preferably, the transfected cells are Escherichia coli TOP10 competent cells.

[0020] The present invention also provides the use of the Chinese mitten crab HNF4 gene, protein, vector or transfected cell in regulating the biosynthesis of LC-PUFA in crustaceans.

[0021] Preferably, the crustacean comprises Eriocheir sinensis, Procambarus clarkii, Scylla paramamosain, Portunus trituberculatus, Cherax quadricarinatus and Homarus americanus.

[0022] Preferably, the Chinese mitten crab HNF4 gene serves as a transcription factor for LC-PUFA biosynthesis in Chinese mitten crab, promoting the synthesis of LC-PUFA.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention is the first to isolate and clone the HNF4 gene from Chinese mitten crab, and obtain the complete sequence of the full length of the HNF4 gene. After increasing the expression of the HNF4 gene, the expression levels of FAS, SCD, FAD6, FAD9, and ELOVL4 genes in the hepatopancreas of Chinese mitten crab are significantly increased, and the expression of the HSL gene is significantly downregulated, while the results are the opposite after inhibiting the expression of the HNF4 gene. This indicates that the HNF4 gene can promote the expression of genes related to fatty acid synthesis, and indirectly play a role in the fatty acid synthesis process of Chinese mitten crab as a transcription factor, thereby promoting the synthesis of LC-PUFA.

[0025] (2) The cDNA of the HNF4 gene of Chinese mitten crab cloned by the present invention encodes 601 amino acids, the predicted protein molecular weight is 55.85 kDa, and the theoretical isoelectric point is 8.46. Homology analysis found that the HNF4 gene of Chinese mitten crab has high homology with shrimps and crabs such as Procambarus clarkii, Scylla paramamosain, Portunus trituberculatus, Cherax quadricarinatus and Homarus americanus, among which the highest homology with Procambarus clarkii is 79.46%. Phylogenetic tree analysis showed that the amino acid sequence of HNF4 of Chinese mitten crab is clustered with HNF4 of Scylla paramamosain and Homarus americanus, and is closely related to crustaceans such as Procambarus clarkii, Scylla paramamosain, Portunus trituberculatus, Cherax quadricarinatus and Homarus americanus, providing important reference value for the functional study of HNF4 in other crustaceans. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The cDNA sequence of hepatocyte nuclear factor 4 (HNF4) of Chinese mitten crab and its predicted amino acid sequence in the example are grouped into two lines. The first line is the nucleotide sequence (the numbers on the left represent the nucleotide sequence number), and the second line is the corresponding predicted amino acid. The start codon and the stop codon are highlighted in black with white letters.

[0027] Figure 2 For the homology comparison of the amino acid sequences of HNF4 of various species in the examples, the sequences of the compared species are Eriocheir sinensis, Scylla sphaeroides (XP 063885300.1), Portunus trituberculatus (XP 045123926.1), Procambarus clarkii (XP045622671.2), Cherax rubripes (XP 069961229.1), Homarus americanus (XP 042234157.1), Danio rerio (AAI24294.1) and Homo sapiens (AAF00110.1).

[0028] Figure 3 This is the phylogenetic tree of the HNF4 gene of Eriocheir sinensis in the examples.

[0029] Figure 4 The effects of interference in the examples and exposure to Benfluorex and BI-6015 on genes related to lipid metabolism in Chinese mitten crab; A: mRNA expression of hormone-sensitive lipase (HSL); B: mRNA expression of fatty acid synthase (FAS); C: mRNA expression of stearoyl-CoA desaturase (SCD); D: mRNA expression of fatty acid desaturase 6 (FAD6); E: mRNA expression of fatty acid desaturase 9 (FAD9); F: mRNA expression of fatty acid elongase 4 (ELOVL4). The differences among different letter groups were statistically significant (P<0.05).

[0030] Figure 5 The effects of different treatment times on TG, LPS and NEFA of Chinese mitten crab in the examples; A: triglyceride (TG) content; B: lipase (LPS) activity; C: free fatty acid (NEFA); the differences between different letter groups were statistically significant (P<0.05). DETAILED DESCRIPTION

[0031] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that for ordinary technicians in this field, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0032] Example 1 Cloning of the Eriocheir sinensis HNF4 gene

[0033] (1) Based on the known partial HNF4 gene sequence, primers were designed using Primer Premier 6.0 software (Table 1). Total RNA from the hepatopancreas of Eriocheir sinensis was extracted using the Trizol method and reverse transcribed into the first-strand cDNA, which was used as a template for PCR reaction to amplify the HNF4 core fragment.

[0034] Table 1: Summary of primers used

[0035]

[0036]

[0037] PCR sample loading system: 1 μL cDNA template, 10 μL 2× Taq Master Mix, 0.5 μL each of forward and reverse primers (10 μM), add sterile deionized water to a total volume of 15 μL.

[0038] PCR reaction conditions: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 60 s / kb, 34 cycles; extension at 72°C for 7 min, and storage at 4°C.

[0039] The obtained core fragment of the Chinese mitten crab HNF4 gene was cloned into the pMD-19T vector and sequenced.

[0040] (2) Based on the obtained HNF4 gene core fragment, 3' and 5' RACE primers were designed (Table 1). 3' and 5' cDNA first strands were synthesized as templates for rapid gene amplification, and the Chinese mitten crab HNF4 gene was amplified according to the reaction system and reaction conditions in the GeneRacerTM Kit instruction manual.

[0041] The reaction system was: 0.5 μL of 3 / 5 RACE template, 1 μL of 3 / 5 GeneRacer outer primer (10 μM), 1 μL of HNF4-F1 / R1 (10 μM), and 22.5 μL of Platinum PCR Supermix High Fidelity (Invitrogen).

[0042] The reaction conditions for the first round of PCR were as follows: pre-denaturation at 94°C for 2 min; 5 cycles of 94°C, 30 s, 72°C, 2.5 min; 5 cycles of 94°C, 30 s, 70°C, 2.5 min; and 25 cycles of 94°C, 30 s, 66°C, 2.5 min.

[0043] The second-round PCR reaction system was: 0.5 μL of the first-round product of 3 / 5-RACE PCR, 1 μL of 3 / 5GeneRacer outer primer (10 μM), 1 μL of HNF4-F1 / R1 (10 μM), and 22.5 μL of Platinum PCR Supermix High Fidelity (Invitrogen).

[0044] The reaction conditions for the second round of PCR were as follows: pre-denaturation at 94°C for 2 min; 94°C, 30 s, 66°C, 2.5 min, 30 cycles.

[0045] The reaction conditions of the first round of 5'RACE PCR were as follows: pre-denaturation at 94°C for 2 min; 5 cycles of 94°C, 30 s, 72°C, 1 min; 5 cycles of 94°C, 30 s, 70°C, 1 min; and 25 cycles of 94°C, 30 s, 66°C, 1 min.

[0046] The reaction conditions of the second round of 5'RACE PCR were as follows: pre-denaturation at 94°C for 2 min; 94°C, 30 s, 66°C, 1 min, 30 cycles.

[0047] The obtained 3' and 5' fragments were ligated with pMD-19T, cloned and sequenced.

[0048] (3) The obtained 3' and 5' fragments were spliced ​​with the core fragment to obtain the full-length cDNA sequence of Eriocheir sinensis HNF4.

[0049] The cloned Chinese mitten crab HNF4 gene includes a 5' non-coding region of 569 bp and a 3' non-coding region of 1179 bp, and an open reading frame of 1806 bp ( Figure 1

[0050] The protein encoded by the cDNA contains 601 amino acids ( Figure 1 ), the amino acid sequence is shown in SEQ ID NO:2, specifically:.

[0051] Analysis of the gene sequence revealed that the molecular weight of the HNF4 gene of Chinese mitten crab is 55.85 kDa, and the theoretical isoelectric point is 8.46. Homology analysis revealed that the HNF4 gene of Chinese mitten crab has a high degree of homology with shrimps and crabs such as Procambarus clarkii, Scylla pseudoburrows, Portunus trituberculatus, Red Crawfish and Homarus americanus. The highest homology with Procambarus clarkii is 79.46%, with Scylla pseudoburrows being 78.04% and with Portunus trituberculatus being 73.54% ( Figure 2Phylogenetic tree analysis showed that the amino acid sequence of HNF4 in Chinese mitten crab was clustered with HNF4 in mud crab and swimming crab, and was closely related to crustaceans such as Procambarus clarkii, red claw crayfish and American lobster, but was distantly related to bony fish such as zebrafish and mammals such as humans and mice (Mus musculus). Figure 3 ).

[0052] Example 2 Verification of the function of the Chinese mitten crab HNF4 gene

[0053] Benfluorex is an anorectic and hypolipidemic drug that can stimulate HNF4 expression. BI-6015 is an HNF4 antagonist that inhibits the expression of known HNF4 target genes. Benfluorex and BI-6015 and dimethyl sulfoxide (DMSO) were purchased from MedChemExpress (China); NaCl (purity 99%), glacial acetic acid (CHCOOH), and DEPC-treated water were purchased from Sinopharm Shanghai Chemical Reagent Co., Ltd. (China); Ex Purchased from TaKaRa (Japan); MiniBEST DNA Fragment Purification Kit Ver.4.0 purchased from TaKaRa (Japan); T7 RNAiTranscription Kit (TR102, Novozyme, China). According to the instructions, Benfluorex and BI-6015 were prepared into a 10 mM stock solution using dimethyl sulfoxide (DMSO), and the stock solution was diluted with physiological saline to a 5 μg / μL solution, which was stored at -20°C for injection experiments, and the remaining stock solution was stored in a -80°C refrigerator for later use.

[0054] According to the cDNA sequence of the HNF4 gene of Chinese mitten crab, the effective interference interval was predicted using the RNAi target site online design software siDirectversion 2.0, and specific primers for the target sequence were designed using Primer5 software. The forward primer and reverse primer of dsRNA were both added with T7 promoter sequence. Hepatopancreas cDNA was used as the amplification template, and dsRNA-HNF4-F and dsRNA-HNF4-R were used as primers to amplify the target fragment to synthesize dsRNA.

[0055] The PCR reaction system was: 1 μL of cDNA template, 25 μL of 2×Taq Master Mix, 2 μL of each forward and reverse primer (10 μM), and sterile deionized water was added to a total volume of 20 μL.

[0056] The PCR reaction program 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, extension at 72°C for 60 s / kb, extension at 72°C for 2 min, 34 cycles; extension at 72°C for 7 min, and storage at 4°C.

[0057] Purification of linearized template (using purification kit Takara, MiNi Best 9761) to avoid the influence of RNase, protein, RNA and salt residues on the system:

[0058] (1) Add an appropriate amount of Buffer-DC to the template and mix well;

[0059] (2) Transfer the mixed liquid to a new centrifuge column, centrifuge at 13400r at room temperature for 1 min, and discard the supernatant;

[0060] (3) Add 700 μL Buffer WB solution and centrifuge at 13400 r at room temperature for 1 min;

[0061] (4) Discard the supernatant and repeat step (3);

[0062] (5) Centrifugation at 13400 r / min at room temperature for 2 min;

[0063] (6) Leave to air dry on a clean bench for 2 minutes;

[0064] (7) Add 30-40 μL of Elution Buffer solution to the centrifuge column membrane and let it stand for 2 min.

[0065] (8) Centrifuge at 12000 r / min at room temperature for 1 min and save the filtrate;

[0066] Check the concentration and quality of purified DNA.

[0067] dsHNF4 was synthesized according to the instructions of T7 RNAi Transcription Kit.

[0068] (1) The reaction system is: NTP Mix 8 μL, 10× Transcription Buffer 2 μL, T7 Enzyme Mix 2 μL, DNA template (Invitrogen) 6 μL, and sterile deionized water to a total volume of 20 μL;

[0069] (2) Incubate at 37°C in a PCR instrument for 4 h;

[0070] (3) Dilute 100U / μL RNase T1 to 10U / μL according to the instructions;

[0071] (4) Dual enzyme nitrification system: Transcription Product 20 μL, DNase I 17 μL, RNase T1 (10 U / μL) 1 μL, R plus sterile deionized water to a total volume of 40 μL;

[0072] (5) Mix by blowing and sucking;

[0073] (6) Incubate at 37°C for 30 min;

[0074] (7) Detect the size of the transcription product bands by agarose gel electrophoresis.

[0075] Purification of dsRNA transcripts:

[0076] (1) Add 115 μL, 230 μL of DEPC-treated water and 15 μL, 30 μL (40 μL system) of sodium ester to the product, and mix by pipetting;

[0077] (2) Add an equal volume of chloroform and water-saturated phenol (chloroform: RNA water-saturated phenol (RNA extraction phenol supernatant) 1:1), shake well, and centrifuge at 12000r at room temperature for 15min;

[0078] (3) Add an appropriate amount of chloroform to the supernatant, mix well, and centrifuge at 12000r at room temperature for 15min;

[0079] (4) Repeat step (3);

[0080] (5) Take the supernatant, add 2 volumes of anhydrous ethanol, mix well, and let stand at -80°C for precipitation;

[0081] (6) Centrifuge at 12000 r, 4°C for 30 min and discard the supernatant;

[0082] (7) Add 500 μL of 70% pre-cooled ethanol and centrifuge at 12,000 r at 4°C for 15 min;

[0083] (8) Discard the supernatant and let it stand to dry for 2-3 minutes;

[0084] (9) Add an appropriate amount of DEPC-treated water to dissolve the precipitate;

[0085] (10) Detect the concentration and quality of purified DNA;

[0086] (11) The dsRNA that met the expectations was stored in a -80°C refrigerator until use. The preparation method of dsEGFP was the same as that of dsHNF4.

[0087] The female juvenile crabs (5±0.5 g) used in this example were purchased from the Shanghai Chongming breeding base. Before the experiment, the juvenile crabs were placed in a breeding tank and temporarily raised with dechlorinated tap water for 7 days. Benfluorex and BI-6015 were injected at a volume of 5 μg / g (the Chinese mitten crabs were wiped dry and weighed). After thawing, the dsRNA was diluted to a concentration of 1 μg / μL and injected at a volume of 1 μg / g. The microsyringe was injected from the base of the tripod. The control group for Benfluorex and BI-6015 was injected with physiological saline, and the dsHNF4 control group was injected with dsEGFP, which was injected at 0h, 48h, 96h and 6d of the experiment. After 24 h, 48 h, and 7 d of treatment, hepatopancreas samples were collected from 6 crabs randomly selected from each group. RNA in the hepatopancreas samples was extracted and synthesized into cDNA as a PCR template. Specific primers were designed using Primer5 software based on the sequence of Eriocheir sinensis. β-actin was used as an internal reference gene. The quantitative real-time PCR step was performed in a 10 μL reaction mixture containing 5 μL of 2×SybrGreen qPCR Master Mix, 1 μL of cDNA, 0.4 μL of forward and reverse primers, and 3.6 μL of RNase-free H2O (TaKaRa, Japan). PCR amplification was performed under the following conditions: 95°C for 30 s (pre-denaturation); 40 cycles of 95°C for 5 s, 60°C for 34 s, then 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s, using 2 -△△Ct The relative expression levels of mRNA of HNF4, ACC, FAS, SCD, HSL, CPT1, FAD6, FAD9 and ELOVL4 were analyzed by the method.

[0088] The results showed that compared with the control group, dsHNF4 treatment significantly reduced the expression of HNF4, FAS, SCD, FAD6, FAD9 and ELOVL4, and HSL and CPT-1 genes were significantly upregulated. After Benfluorex treatment, the expression of HNF4, FAS, SCD, FAD6, FAD9 and ELOVL4 was significantly increased, while the expression of HSL and CPT-1 was significantly decreased. Similar to the dsHNF4 group, after BI-6015 treatment, the expression of HSL and CPT-1 was significantly increased, the expression of HNF4, FAS, FAD6, FAD9 and ELOVL4 was significantly decreased, and the expression of SCD showed a downward trend. Compared with the dsHNF4 treatment group, the expression of FAS and SCD in the BI-6015 treatment group was relatively high, but still lower than that in the control group ( Figure 4 ).

[0089] The triglyceride, free fatty acid content and lipase activity in the collected hepatopancreas samples were detected using a kit (Nanjing Jiancheng, China). The results showed that after Benfluorex treatment, the TG content in the hepatopancreas samples at 24h, 48h and 7d were significantly higher than those in the control group. The TG content detected at 48h was the highest, and LPS and NEFA in the samples at 48h and 7d were significantly downregulated compared with the control group. There was no significant difference in LPS and NEFA in the hepatopancreas after Benfluorex treatment for 24h compared with the control group. The TG, NEFA content and LPS in the hepatopancreas samples treated with BI-6015 for 48h and 7d were significantly different from those in the Benfluorex treatment group; compared with the control group, the TG content was significantly reduced after BI-6015 treatment, and the activities of LPS and NEFA were significantly upregulated; NEFA reached its maximum value at 7d after BI-6015 treatment ( Figure 5 ).

[0090] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Chinese mitten crab HNF4 gene, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. A protein encoded by the HNF4 gene of Chinese mitten crab, the amino acid sequence of which is shown in SEQ ID NO:

2.

3. A method for cloning the HNF4 gene of Eriocheir sinensis, characterized in that: The following steps are involved: Step a: designing primers, performing PCR reaction using the first strand of cDNA reverse transcribed from the total RNA of Chinese mitten crab as a template, obtaining the core fragment of the HNF4 gene of Chinese mitten crab, cloning the core fragment of the HNF4 gene into a vector and sequencing; Step b: design 3'-HNF4 RACE downstream primers and 5'-HNF4 RACE upstream primers respectively according to the obtained HNF4 gene core fragment, reverse transcribe the total RNA of Chinese mitten crab into 3'-cDNA and 5'-cDNA first chain, use them as templates to perform 3' and 5' fragment RACE, connect the obtained 3' and 5' fragments to the vector respectively, clone and sequence; Step c: splicing the obtained 3' and 5' fragments with the HNF4 gene core fragment respectively to obtain the full-length sequence of the Chinese mitten crab HNF4 gene.

4. The method for cloning the HNF4 gene of Chinese mitten crab according to claim 3, characterized in that: In steps a and b, the total RNA of Eriocheir sinensis is extracted from the hepatopancreas of Eriocheir sinensis by using the Trizol method.

5. The method for cloning the HNF4 gene of Chinese mitten crab according to claim 3, characterized in that: In steps a and b, the vector is pMD-19T plasmid. A vector comprising the Chinese mitten crab HNF4 gene according to claim 1.

7. A transfected cell comprising the vector according to claim 6.

8. Use of the Chinese mitten crab HNF4 gene of claim 1, the protein of claim 2, the vector of claim 6, or the transfected cell of claim 7 in regulating the biosynthesis of LC-PUFA in crustaceans.

9. The use according to claim 8, characterized in that: The crustaceans include Eriocheir sinensis, Procambarus clarkii, Scylla paramamosain, Portunus trituberculatus, Cherax quadricarinatus and Homarus americanus.

10. The use according to claim 8, characterized in that: The Chinese mitten crab HNF4 gene serves as a transcription factor for the biosynthesis of LC-PUFA in the Chinese mitten crab, and promotes the synthesis of LC-PUFA.

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