Eriocheir sinensis ELOVL7 gene and application thereof

Abstract

The invention discloses an Eriocheir sinensis ELOVL7 gene and application thereof, the nucleotide sequence of the Eriocheir sinensis ELOVL7 gene is as shown in SEQ ID NO: 1, and the amino acid sequence of protein encoded by the Eriocheir sinensis ELOVL7 gene is as shown in SEQ ID NO: 2. The Eriocheir sinensis ELOVL7 gene sequence is successfully obtained through a PCR technology for the first time, the function of the ELOVL7 gene is verified through an in-vitro experiment, it is proved that the gene serves as an elongase gene to play a role in the Eriocheir sinensis fatty acid synthesis process, the function of promoting LC-PUFA synthesis is achieved, a foundation is laid for studying the LC-PUFA anabolism mechanism of the Eriocheir sinensis, and the Eriocheir sinensis ELOVL7 gene has a good application prospect. And a reference is provided for the functional research of other crustacean ELOVL7.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an ELOVL7 gene of Chinese mitten crab and an application thereof. Background Art

[0002] Long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3), and arachidonic acid (ARA; 20:4n-6), play an important role in the growth and reproduction of aquatic organisms. They are also important components of nervous tissue and cell membranes, maintaining the integrity of cellular structure, energy storage, and signal transduction. DHA and EPA possess significant anti-inflammatory properties, enhancing the immune function of aquatic animals, thereby improving disease resistance and overall health. The synthesis of LC-PUFAs requires the catalysis of multiple enzymes, one key enzyme being fatty acid elongase (ELOVL). ELOVL elongates fatty acids through carbon chain elongation reactions, a crucial process in the biosynthesis of LC-PUFAs, converting short-chain precursors (linoleic acid and α-linolenic acid) into functional LC-PUFAs. Studies have shown that the ELOVL family has seven main members (ELOVL1-ELOVL7), and ELOVL8 has also been found in some fish. These ELOVLs have different substrate preferences and tissue expression patterns.

[0003] The Chinese mitten crab (Eriocheir sinensis) is an important aquatic species with high economic value in China's Yangtze River Basin. In recent years, with increasing market demand, improving its growth performance has become a research focus in the aquaculture industry. Studies have shown that long-chain polyunsaturated fatty acids (LC-PUFA) are key nutrients essential for the growth of Chinese mitten crabs. Studies have found that the appropriate addition of LC-PUFA (such as DHA and EPA) can significantly promote the growth of juvenile crabs and improve their physiological metabolic capacity. ELOVL fatty acid elongase plays a vital role in the synthesis of LC-PUFA. In-depth research on its regulatory mechanism in LC-PUFA synthesis will help optimize the proportion of fish oil replacement in aquatic feeds and promote the development of sustainable aquaculture.

[0004] Currently, little research has been conducted on ELOVL7 within the ELOVL family. Some studies have shown that ELOVL7 plays a key role in the metabolism of long-chain and very-long-chain fatty acids, catalyzing the elongation of long-chain fatty acids such as C18:0 (stearic acid) and C18:3 (linolenic acid). Therefore, cloning the ELOVL7 gene from the Chinese mitten crab (Eriocheir sinensis) is of great significance for studying LC-PUFA biosynthesis in crustaceans and, consequently, reducing reliance on fish oil in aquaculture. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the main purpose of the present invention is to provide an ELOVL7 gene of the Chinese mitten crab. For the first time, the full-length cDNA sequence of the ELOVL7 gene of the Chinese mitten crab was successfully obtained by PCR technology, and its expression in multiple tissues and different developmental stages was verified. The complete sequence of the ELOVL7 gene in the Chinese mitten crab was obtained, providing a reference for the functional study of the ELOVL7 gene in crustaceans.

[0006] Another object of the present invention is to provide an application of the Chinese mitten crab ELOVL7 gene.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a Chinese mitten crab ELOVL7 gene is provided, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] The second aspect of the present invention provides a method for cloning the ELOVL7 gene of the Chinese mitten crab, comprising the following steps:

[0010] (1) Primers were designed based on the known ELOVL7 gene sequence, and PCR reaction was performed using the first strand of cDNA reverse transcribed from the intestinal RNA of Chinese mitten crab as a template to obtain the core fragment of the ELOVL7 gene of Chinese mitten crab. The core fragment was cloned into a vector and sequenced;

[0011] (2) Based on the ELOVL7 gene core fragment obtained above, 3'-ELOVL7 RACE downstream primers and 5'-ELOVL7 RACE upstream primers were designed, and the extracted Chinese mitten crab intestinal RNA was reverse transcribed into 3'-cDNA and 5'-cDNA first strands. Using these as templates, 3' and 5' fragment RACE were performed, and the obtained 3' and 5' fragments were ligated with vectors, cloned, and sequenced;

[0012] (3) The obtained 3' and 5' fragments were spliced with the core fragment to obtain the full-length sequence of the ELOVL7 gene of Chinese mitten crab.

[0013] Preferably, in steps (1) and (2), the Chinese mitten crab intestinal RNA is extracted from the Chinese mitten crab intestinal tract by TRIzol method.

[0014] Preferably, in steps (1) and (2), the vector is pMD-19T plasmid.

[0015] In a third aspect, the present invention provides a protein encoded by the ELOVL7 gene of Eriocheir sinensis, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0016] In a fourth aspect, the present invention provides an expression vector comprising the Chinese mitten crab ELOVL7 gene.

[0017] The fifth aspect of the present invention provides a transfected cell, which includes the protein encoded by the ELOVL7 gene of the Chinese mitten crab or the expression vector.

[0018] Preferably, the transfected cells are Escherichia coli TOP10 competent cells.

[0019] The sixth aspect of the present invention provides the use of the Chinese mitten crab ELOVL7 gene or the protein encoded by the Chinese mitten crab ELOVL7 gene in promoting the biosynthesis of long-chain polyunsaturated fatty acids in Chinese mitten crab.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention reports for the first time the isolation and cloning of the ELOVL7 gene from Eriocheir sinensis, and verifies the function of the ELOVL7 gene through in vitro experiments, indicating that the gene plays a role as an elongase gene in the fatty acid synthesis process of Eriocheir sinensis, promoting the synthesis of LC-PUFA.

[0022] (2) The ELOVL7 cDNA cloned by the present invention is 1,600 base pairs (bp) in length (accession number, PQ867554), which includes a 1,200 bp open reading frame (ORF) that can encode 399 amino acids (AA). Its 5' untranslated region (UTR) is 176 bp in length, and its 3' non-coding region (3'-UTR) is 224 bp. Protein sequence analysis showed that the protein encoded by the cDNA sequence has a typical ELOVL highly conserved motif, including a typical histidine box HXXHH and multiple conserved regions (KXXEXXDT, HXXXLHXXHH, NXXXHXXNYXYY, TXXQXXQ). Structural prediction analysis found that the amino acid sequence of ELOVL7 of the ELOVL of the Chinese mitten crab contains 8 transmembrane regions.

[0023] (3) The present invention compared the amino acid sequences of the proteins encoded by ELOVL7 of zebrafish (Danio rerio), olive mud crab (Scylla olivacea), three-spotted swimming crab (Portunus trituberculatus), Indian shrimp (Penaeus indicus), whiteleg shrimp (Penaeus vannamei) and Chinese mitten crab. The results showed that the similarities were 44.69%, 73.82%, 73.76%, 56.27% and 52.29%, respectively. The results showed that ELOVL7 of Chinese mitten crab clustered in the same branch with ELOVL7 of other crustaceans, and was located in a different branch with ELOVL7 of mammals, fish and molluscs. Among them, the ELOVL7 of Chinese mitten crab was most closely related to olive mud crab and three-spotted swimming crab.

[0024] (4) The fatty acid detection results showed that INVSc1 brewer's yeast itself contains C16:0, C16:1n-7, C18:0, and C18:1n-9 fatty acids. Without the addition of exogenous fatty acids, INVSc1 brewer's yeast carrying the pYES2-ELOVL7 expression vector produces one more new fatty acid than INVSc1 brewer's yeast carrying the pYES2 empty vector. After comparison, it was found that the fatty acid formed was C20:1n-9, which is an elongation product of C18:1n-9, with a conversion rate of 2.4%. This proves that the ELOVL7 gene of Chinese mitten crab obtained by the present invention has the function of promoting LC-PUFA synthesis, laying the foundation for in-depth research on the LC-PUFA synthesis and metabolism mechanism of Chinese mitten crab, and also provides a reference for the functional research of ELOVL7 in other crustaceans. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The cDNA sequence and predicted amino acid sequence of the Chinese mitten crab intestinal tissue elongase 7 (ELOVL7) in the examples are shown; each two lines are grouped together, the first line is the nucleotide sequence (the numbers on the left represent the nucleotide sequence number), the second line is the corresponding predicted amino acid, and the start codon and stop codon are highlighted in black with white letters.

[0026] Figure 2 This is the prediction result of the transmembrane region of Eriocheir sinensis in the examples.

[0027] Figure 3 The hydrophilicity and hydrophobicity analysis results of the ELOVL7 protein in the examples are shown.

[0028] Figure 4 The secondary and tertiary structures of the ELOVL7 protein in the examples are predicted; A: secondary structure; B: tertiary structure.

[0029] Figure 5The following are the homology comparison results of the ELOVL7 amino acid sequences of various species in the examples; the compared species sequences are, in order: zebrafish (Danio rerio) NP 955826.1; Chinese mitten crab (Eriocheir sinensis) PQ867554; three-spotted swimming crab (Portunus trituberculatus) XP 045111474.1; olive mud crab (Scylla olivacea) AWM30548.1; whiteleg shrimp (Penaeus vannamei) XP 027227008.1; and Indian shrimp (Penaeus indicus) XP 063584662.1.

[0030] Figure 6 : This is the phylogenetic tree of the ELOVL7 gene of Chinese mitten crab in the examples.

[0031] Figure 7 : The relative expression levels of ELOVL7 in different tissues of Chinese mitten crab in the examples.

[0032] Figure 8 This is the in vitro functional verification of the ELOVL7 gene in the example; A: fatty acid peak diagram of INVSc1 Saccharomyces cerevisiae with empty vector; B: fatty acid peak diagram of INVSc1 Saccharomyces cerevisiae with pYES2-ELOVL7 expression vector. DETAILED DESCRIPTION

[0033] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the following is a further detailed and complete description of the technical effects produced by the present invention in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, and not all of them. It should be pointed out that for those skilled in the art, other embodiments obtained without departing from the concept of the present invention are all within the scope of protection of the present invention.

[0034] Example 1

[0035] Cloning of the ELOVL7 gene from Chinese mitten crab

[0036] (1) Based on the known partial sequence of ELOVL7, primers were designed using Primer Premier 6.0 software (Table 1). RNA from the intestine of Eriocheir sinensis was extracted using Trizol and reverse transcribed into first-strand cDNA, which was used as a template for PCR to amplify the ELOVL7 core fragment.

[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), and 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 ELOVL7 gene of Chinese mitten crab was cloned into the pMD-19T vector and sequenced.

[0040] (2) Based on the ELOVL7 gene core fragment obtained above, 3' and 5' RACE primers were designed (Table 1). 3' and 5' cDNA first strands were synthesized as templates for rapid gene amplification. The ELOVL7 gene of Chinese mitten crab was amplified according to the reaction system and reaction conditions specified in the Gene Racer™ Kit instructions.

[0041] The reaction system was: 0.5 μL of 3 / 5 RACE template, 1 μL of 3 / 5 Gene Racer outer primer (10 μM), 1 μL of ELOVL7-F1 / R1 (10 μM), and 22.5 μL of Platinum PCR Supermix High Fidelity (Invitrogen).

[0042] Table 1: Summary of primers used

[0043] Primer name Sequence (5′ to 3′) use ELOVL7-F ATGGCTCCGAACGAAACC CDS Area ELOVL7-R TCACGAAGCCTTGTTGGG CDS Area 3'ELOVL7-F GCTTCCTGAACGGCTTCATCCACATCGT 3' RACE primer 3'ELOVL7-F2 CACCACCCTCCAGATGGTGCAGTTC 3' RACE primer 5'ELOVL7-F GCAGTAGGCAACCTGCAGGGCGT 5' RACE primer 5'ELOVL7-F2 TACATCCAGGAATCGACTCGCGGGTCC 5' RACE primer qRT-ELOVL7-F CTCTGGTATGGCATCCGACA Gene expression qRT-ELOVL7-R AACAAACATGGCAACGAACTG Gene expression β-actin-F ACCTCGGTTCTATTTTGTCGG Internal reference primer β-actin-R ATGCTTTCGCAGTAGTTCGTC Internal reference primer ELOVL7-F2 CCCAAGCTTATGGCTCCGAACGAAACCG Open reading frame cloning ELOVL7-R2 CCGGAATTCTCACGAAGCCTTGTTGGGGA Open reading frame cloning T7 TAATACGACTCACTATAGGG Bacterial liquid RCR CYC1 Terminator GTGACATAACTAATTACATGATG Bacterial liquid RCR

[0044] 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 for 30 s, 72°C for 2.5 min; 5 cycles of 94°C for 30 s, 70°C for 2.5 min; and 25 cycles of 94°C for 30 s, 66°C for 2.5 min.

[0045] The second-round PCR reaction system was: 0.5 μL of the first-round product of 3 / 5-RACE PCR, 1 μL of 3 / 5Gene Racer outer primer (10 μM), 1 μL of ELOVL7-F1 / R1 (10 μM), and 22.5 μL of Platinum PCR Supermix High Fidelity (Invitrogen).

[0046] The reaction conditions for the second round of PCR were as follows: pre-denaturation at 94°C for 2 min; 30 cycles of 94°C for 30 s and 66°C for 2.5 min.

[0047] The reaction conditions for the first round of 5'RACE PCR were as follows: pre-denaturation at 94°C for 2 min; 5 cycles of 94°C for 30 s, 72°C for 1 min; 5 cycles of 94°C for 30 s, 70°C for 1 min; and 25 cycles of 94°C for 30 s, 66°C for 1 min.

[0048] The reaction conditions for the second round of 5'RACE PCR were as follows: pre-denaturation at 94°C for 2 min; 30 cycles of 94°C for 30 s and 66°C for 1 min.

[0049] The obtained 3' and 5' fragments were ligated with pMD-19T, cloned and sequenced.

[0050] (3) The obtained 3' and 5' fragments were spliced with the core fragment to obtain the full-length cDNA sequence of ELOVL7 of Chinese mitten crab.

[0051] The ELOVL7 cDNA of the Chinese mitten crab obtained by cloning is 1,600 base pairs (bp) in length (accession number, PQ867554), as follows:

[0052] ATGGGGAGTGTGTGTGCGCTAGTGTTACACGGAAGGCAGCTACGCTGGAAAGTGTAGACGCTGTGTGGGTGTCTGGGAGGGAGTGAAGTGCTCGTCGTGAAAGGTGGTAAAATAGCATCTCATTGCACGCAACATTTGATATTAGCTACCTGTGTCCAGCAGCTGACGAAGACAGG ATG TGA AAAACTTAAATAATCGCTGGACAATCCAGCAAAACGTGATAAATATATATATATATATATATATATATATATATATATATATATATATATATGTGTATATATGTATATATATATATATATATATATATATATATATATATATCATTATCATTACACATTCTACGCTGCATGCTTAAAAAAAATCCCATTTAAATCAGTACACACCGAAATGCTATTGCTCAAAAAAAAAAAAAAAA (SEQ ID NO: 1), which includes a 1,200 bp open reading frame (ORF) encoding 399 amino acids (AA), as follows:

[0053] MAPNETAENMTHLRRTYDPKSLEHDTPSHQLADAEVRLQPIDRQQLDPRGRSHDHTIETKTHHSKDTGDPTQPTSVERKLLSSGIKAAAITLFLYFYGNHMVSSQMKKDPRVDSWMYGLYSSPTPTLLACLAYIAGITYIGPRLMRGRQPPKWLKTVMVMYNALQVAYCSWMFYEAGMAGWFGSYSFICQPCDFSNSPSAL RMLRVAIAYHLSKFLDFFDTIFFVLNHKYSHVSLLHVTHHALMPMGLWYGIRHEPGGQTTFFGFLNGFIHIVMYLYYLLAALGPRVRPYLWWKRYLTTLQM VQFVAMFVHALQSLILGCPAGLPLMKIIMVMAAIFQVLFTDFYIKAYRKKATQTSPKPIKLPMMCSSINQQEDVTGSEAPSEGENQNLRNRVPNKAS(SEQ ID NO:2), of which the 5' untranslated region (UTR) is 176 bp long and the 3' non-coding region (3'-UTR) is 224 bp ( Figure 1 Protein sequence analysis showed that the protein encoded by the cDNA sequence has a typical ELOVL highly conserved motif, including a typical histidine box HXXHH and multiple conserved regions (KXXEXXDT, HXXXLHXXHH, NXXXHXXNYXYY, TXXQXXQ). Structural prediction analysis found that the amino acid sequence of ELOVL7 of Chinese mitten crab contains 8 transmembrane regions ( Figure 2 ).

[0054] The theoretical isoelectric point of the protein is 9.326, the molecular weight is about 45.61kD, and the total average hydrophilicity is -0.123, which is speculated to be a hydrophilic protein. Figure 3 ), it is speculated that it is a hydrophilic protein. The secondary structure of ELOVL7 protein mainly consists of 45.86% random coil, 41.10% α-helix, 13.03% extended chain ( Figure 4 A). The results of the tertiary structure construction of ELOVL7 protein showed that the sequence similarity between ELOVL7 protein and the green crab (AWM30548.1) was 73.82%, and the global model estimation value (GMQE) was 0.73, indicating that the generated three-dimensional model has a certain degree of reliability ( Figure 4 B).

[0055] The amino acid sequences of ELOVL7 from zebrafish (Danio rerio), olive mud crab (Scylla olivacea), swimming crab (Portunus trituberculatus), Indian shrimp (Penaeus indicus), whiteleg shrimp (Penaeus vannamei) and Chinese mitten crab were compared. The results showed that the similarities were 44.69%, 73.82%, 73.76%, 56.27% and 52.29%, respectively, indicating high homology ( Figure 5 ). MEGA11.0 software was used to perform maximum likelihood phylogenetic analysis on ELOVL7 sequences from different groups ( Figure 6 ), the analysis results showed that the ELOVL7 of Chinese mitten crab was clustered in the same branch as the ELOVL7 of other crustaceans, and was located in a different branch from the ELOVL7 of mammals, fish and mollusks, among which the ELOVL7 of Chinese mitten crab was most closely related to the olive green mud crab and swimming crab.

[0056] Example 2

[0057] Verification of the function of the ELOVL7 gene in Chinese mitten crab

[0058] (1) The full-length cDNA sequence of ELOVL7 from Chinese mitten crab was experimentally analyzed and resolved using Snapgene software. Based on the ELOVL7 ORF and pYES2 vector, specific primers ELOVL7-F2 and ELOVL7-R2 with HindIII and EcoRI restriction sites were designed, and PCR was performed using the cDNA as a template. The specific steps are as follows:

[0059] 1) ORF amplification was performed using Vazyme 2×Taq PCR Master Mix.

[0060] 2) PCR reaction program: 94°C for 10 min; 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, for a total of 30 cycles; 72°C for 5 min; end at 16°C.

[0061] 3) PCR product recovery and purification: The specific operation is the same as above. The recovered product is sent for testing and verification. After the sequence is confirmed to be correct, it is stored at -20℃ for future use.

[0062] (2) Recovered products of ELOVL7 ORF amplification and pYES2 vector

[0063] The ELOVL7 ORF amplified product and the pYES2 vector were double digested with restriction endonucleases HindIII and EcoRI. The specific reaction system is as follows:

[0064] Table 2: Enzyme digestion system

[0065] Reagents Volume (μL) 10×FastDigest-Buffer (Green) 2 plasmids 3 (μg) Recycled products 3 (μg) HindIII 1 EcoR I 1 sterile water Make up to 20

[0066] Incubate in a constant temperature water bath at 37°C for 0.5 to 1 hour, then inactivate in a water bath at 80°C for 5 minutes. The recovered products of ELOVL7 ORF amplification and pYES2 vector were digested and recovered by electrophoresis. After recovery, the quality and concentration were detected using a Q5000 ultra-micro spectrophotometer.

[0067] (3) Enzyme digestion products were connected to form recombinant pYES2-ELOVL7

[0068] The ligase-digested products were ligated in the following reaction system:

[0069] Table 3: Connection system

[0070] Reagents Volume (μL) <![CDATA[10×T4 DNALigase Buffer]]> 2 Linearized pYES2 vector (100 ng) 100(ng) ORF amplification and recovery products after enzyme digestion × <![CDATA[T4 DNALigase]]> 1 sterile water Make up to 20

[0071] Note: The molar ratio of linearized vector to PCR product after enzyme digestion is 3:1.

[0072] After the system is prepared, place it in a PCR instrument, perform enzyme coupling at 16°C overnight, transform and screen positive clones, randomly select and sequence, obtain monoclonal strains with the correct sequence, expand culture, preserve in glycerol, and store at -80°C.

[0073] (4) Extraction and testing of pYES2-ELOVL7 plasmid

[0074] The preserved bacterial suspension was expanded and the pYES2-ELOVL7 plasmid was extracted from the bacterial suspension using the QuickPure Plasmid Mini Kit. The specific steps are as follows:

[0075] 1) Centrifuge 10 mL of bacterial suspension at 13,000 rpm for 1 min, discard the supernatant, and resuspend in 600 μL of the original bacterial suspension.

[0076] 2) Add 100 μL of Buffer L2 and gently invert to mix until the solution turns clear purple.

[0077] 3) Immediately add 350 μL of Buffer N3 (containing premixed RNase A), mix by inversion 8-10 times until the solution turns yellow and a precipitate forms. Centrifuge at 13,000 rpm for 3 min.

[0078] 4) Add the supernatant to the adsorption column, centrifuge at 13,000 rpm for 15 seconds, and discard the waste liquid;

[0079] 5) Add 150 μL of Buffer PB, centrifuge at 13,000 rpm for 15 seconds, and discard the waste liquid;

[0080] 6) Add 400 μL of Buffer PW (containing anhydrous ethanol), centrifuge at 13,000 rpm for 1 min, and discard the waste liquid; centrifuge the empty column for 2 min;

[0081] 7) Transfer the adsorption column to a new EP tube, add 80 μL of Buffer EB, let it stand for 2 minutes, and then centrifuge at 13,000 rpm for 1 minute to collect the plasmid;

[0082] 8) The recovered plasmid was verified by PCR and double enzyme digestion, and finally sent for testing.

[0083] (5) Preparation of competent Saccharomyces cerevisiae

[0084] 1) Inoculate 40 μL of Saccharomyces cerevisiae into 40 mL of YPD medium and culture overnight at 30°C and 200 rpm.

[0085] 2) Determine the OD of the bacterial solution 600 The value was inoculated into 500 mL YPD medium at a ratio of 1:100 and cultured until OD 600 ≈0.4, culture for about 4-5h;

[0086] 3) Place the culture flask on ice and pre-cool for 15 minutes;

[0087] 4) Centrifuge at 5000 rpm for 5 min at 4°C to remove the supernatant and collect the cell pellet;

[0088] 5) Add 25 mL of pre-cooled sterile water to the collected cell pellet, mix gently, centrifuge at 5000 rpm at 4°C for 5 minutes, and discard the supernatant. Repeat this step to ensure that the cells are completely washed.

[0089] 6) Add 20 mL of 1 M sorbitol solution, mix gently, and centrifuge at 5000 rpm for 5 min at 4°C; discard the supernatant.

[0090] 7) Repeat step (6);

[0091] 8) Resuspend the cells in 0.5 mL of 1 M sorbitol, then add 0.5 mL of fresh sorbitol, mix gently, and place on ice until ready to use.

[0092] (6) Expression plasmid transformation into competent yeast

[0093] 1) Take 10 μL each of plasmid pYES2-ELOVL7 and vector PYES2, add them to 40 μL of prepared competent Saccharomyces cerevisiae, and mix them by gently pipetting;

[0094] 2) Transfer the cells to a pre-chilled 0.2 cm electroporation cuvette and place on ice for 5 minutes;

[0095] 3) Use the electric shock program Sc2 (voltage 1.5 kV, pulse time 5 ms) to perform one electric shock treatment;

[0096] 4) Immediately add 1 mL of pre-chilled YPD medium to the cuvette, gently pipette, and transfer to a centrifuge tube.

[0097] 5) Incubate the transformed mixture at 30°C with low-speed shaking for 2-5 hours;

[0098] 6) Centrifuge at 5000 rpm for 5 min, discard most of the supernatant, resuspend the yeast cells to achieve a concentrated effect, then spread on Sc-Ura solid medium and culture at 30°C for 48-72 h.

[0099] (7) Screening of positive brewer's yeast

[0100] Pick a single yeast colony into Sc-Ura liquid medium and culture overnight at 30°C and 200 rpm. Take 1 mL of yeast culture and use the Kangwei Century Yeast Plasmid Mini Kit to extract the plasmid for PCR verification. The specific steps are as follows:

[0101] 1) Take 1 mL of yeast culture and centrifuge at 12,000 rpm for 30 seconds. Remove and discard the supernatant as much as possible to collect the yeast cells.

[0102] 2) Add 250 μL of Buffer P1 (containing premixed RNase A) to the collected yeast cells and gently pipette to resuspend the yeast cells.

[0103] 3) Add 40 mg of glass beads and vortex for 10 min to disrupt the cell walls;

[0104] 4) Add 250 μL of Buffer P2, mix thoroughly by inversion, and let stand at room temperature for 5 minutes;

[0105] 5) Add 350 μL of Buffer N3, mix by inversion until a white precipitate appears, and centrifuge at 12,000 rpm for 20 min;

[0106] 6) Add 200 μL of Buffer PS to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0107] 7) Transfer the supernatant from step (5) to an adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0108] 8) Add 150 μL Buffer PB, centrifuge for 1 min, and discard the waste liquid;

[0109] 9) Add 750 μL of Buffer PW (containing anhydrous ethanol), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and centrifuge the empty column for 2 min to remove the ethanol;

[0110] 10) Place the adsorption column in a new centrifuge tube, add 50 μL of Buffer EB, let it stand for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. Repeat this step to improve the recovery rate.

[0111] 11) Perform PCR verification using pYES2 vector universal primers T7 and CYC1Terminator to verify the correct recombinant plasmid for sequencing;

[0112] 12) The Saccharomyces cerevisiae strain into which the plasmid has been successfully introduced is cultured and then stored in glycerol at -80°C.

[0113] (8) Protein induction expression

[0114] 1) The recombinant yeast was inoculated into Sc-Ura liquid medium and cultured at 30°C and 200 rpm overnight until OD 600 The value is 2 to 5;

[0115] 2) Each culture medium was inoculated into fresh Sc-Ura liquid medium at a ratio of 1:10 and cultured until OD 600 =1.6;

[0116] 3) Centrifuge at 5000 rpm for 10 min at room temperature and remove the supernatant;

[0117] 4) Resuspend the yeast cells in 1–2 mL of induction medium containing 2% galactose, then inoculate into 100 mL of induction medium containing fatty acid standards (C18:2n-6, C18:3n-6, C18:3n-3, C20:4n-6, C20:5n-3) and incubate at 30°C, 200 rpm for 48–72 h.

[0118] 5) Recover yeast cells by multiple centrifugation of the yeast culture at 4°C, 5000 rpm for 5 min, discard the supernatant, resuspend the cells in sterile water, centrifuge at 12,000 rpm for 30 s, remove the supernatant, and freeze-dry for 48 h before use.

[0119] (9) Fatty acid determination

[0120] 1) Accurately weigh 200 mg of each freeze-dried yeast sample using a balance and add them to the methyl esterification bottle with a cap;

[0121] 2) Add 3 mL of 1N KOH-methanol solution, incubate at 80°C for 20 min, and cool to room temperature;

[0122] 3) Add 3 mL of 2N HCl-methanol solution, incubate at 80°C for 20 min, and cool again to complete the methyl esterification;

[0123] 4) Add 1 mL of chromatography-grade n-hexane, shake thoroughly for extraction, centrifuge at 3500 rpm for 5 min, and allow to stand for separation;

[0124] 5) Take 600 μL of supernatant and transfer to a 1.5 mL sterile centrifuge tube;

[0125] 6) Aspirate the entire supernatant with a sterile 1 mL syringe, filter through a 0.22 μm filter, and inject into a sample vial for gas chromatography analysis. The column temperature program is as follows: increase to 70°C and hold for 1 min, then increase to 210°C at 10°C / min, then increase to 220°C, and finally increase to 235°C at 10°C / min and hold for 8 min. The total analysis time is 25.5 min.

[0126] 7) Determine the various fatty acids by comparing with fatty acid standard samples.

[0127] (10) Tissue expression determination

[0128] Fluorescence quantitative PCR was used to analyze the expression of ELOVL7 mRNA in different tissues of Chinese mitten crab. The results showed that the expression level of ELOVL7 gene was highest in the intestine, followed by gills. The expression levels of these two tissues were significantly higher than those in other tissues (P<0.05) ( Figure 7 RNA was extracted from different tissues, and cDNA templates of each tissue were reverse synthesized. Primer5 software was used to design 18-20 bp long forward and reverse primers for real-time fluorescence quantitative PCR (qPCR). The primers were designed in the cloned ELOVL7 core coding region (see primers (Table 1)). β-actin was selected as the internal reference gene, and qPCR was performed using the cDNA of each tissue as a template. The PCR system was as follows:

[0129] Table 4: PCR reaction system

[0130] Components Volume (μL) SYBR Premix Ex Taq 2 Forward primer 0.4 Reverse primer 0.4 cDNA template 2 <![CDATA[ddH2O]]> 7.2 Total 20

[0131] The PCR procedure is as follows:

[0132] Table 5: PCR reaction program

[0133]

[0134]

[0135] Using intestinal cDNA from the Chinese mitten crab (Eriocheir sinensis) as a template, PCR amplification was performed using primers ELOVL7-F2 and ELOVL7-R2, which contain HindIII and EcoRI restriction sites. PCR products were then analyzed by agarose gel electrophoresis. Specifically, the target fragment and the pYES2 vector were digested with Fast Digest HindIII and Fast Digest EcoRI (Thermo Scientific). The digested target fragment and pYES2 vector were recovered and ligated using T4 DNA ligase. The resulting fragments were then transformed into TOP10 competent cells. Positive clones were verified by bacterial culture PCR, double restriction enzyme digestion, and sequencing. The results showed that bacterial PCR yielded a band of the expected size at 1,200 bp. Double enzyme digestion of the recombinant plasmid produced two specific bands, approximately 5,800 bp and 1,200 bp in length, respectively. The 5,800 bp band was consistent in size with the double enzyme digestion product of the pYES2 vector, while the 1,200 bp band was consistent in length with the ELOVL7 ORF sequence of the Chinese mitten crab (Eriocheir sinensis), confirming that the plasmid was a positive clone. Sequencing comparison confirmed the complete sequence identity, further validating the results and confirming the successful construction of the eukaryotic expression vector pYES2-ELOVL7.

[0136] The pYES2-ELOVL7 recombinant plasmid was transformed into INVSc1 yeast cells by electroporation. Single colonies were selected by plate screening and expanded. The plasmid was extracted from the expanded yeast cells, and PCR was performed using universal primers for the vector to confirm plasmid integration. Gel electrophoresis of the PCR product revealed a band at 1,200 bp, consistent with the open reading frame of ELOVL7 in Chinese mitten crab (Eriocheir sinensis). Sequencing results from plasmids that successfully validated the PCR product were identical to the pYES2-ELOVL7 recombinant plasmid sequence. PCR and sequencing confirmed that the recombinant plasmid had been successfully introduced into INVSc1 yeast, demonstrating the successful establishment of the INVSc1 yeast eukaryotic expression system for Chinese mitten crab (Eriocheir sinensis).

[0137] The fatty acid detection results showed that INVSc1 Saccharomyces cerevisiae itself contains C16:0, C16:1n-7, C18:0, and C18:1n-9 fatty acids. Without the addition of exogenous fatty acids, INVSc1 Saccharomyces cerevisiae carrying the pYES2-ELOVL7 expression vector produced one more new fatty acid ( Figure 8 ), after comparison, it was found that the fatty acid formed was C20:1n-9, which was the elongation product of C18:1n-9, and the conversion rate was calculated to be 2.4%.

[0138] In summary, the present invention obtained the full-length ELOVL7 gene of Chinese mitten crab and verified its function, laying the foundation for in-depth research on its LC-PUFA synthesis and metabolism mechanism and providing a reference for the functional research of ELOVL7 in other crustaceans, which is of great significance.

[0139] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A Chinese mitten crab ELOVL7 gene, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The method for cloning the ELOVL7 gene of Eriocheir sinensis according to claim 1, characterized in that: The steps include: (1) Primers were designed based on the known ELOVL7 gene sequence, and PCR reaction was performed using the first strand of cDNA reverse transcribed from the intestinal RNA of Chinese mitten crab as a template to obtain the core fragment of the ELOVL7 gene of Chinese mitten crab. The core fragment was cloned into a vector and sequenced; (2) Based on the ELOVL7 gene core fragment obtained above, 3'-ELOVL7 RACE downstream primers and 5'-ELOVL7 RACE upstream primers were designed, and the extracted Chinese mitten crab intestinal RNA was reverse transcribed into 3'-cDNA and 5'-cDNA first strands. Using these as templates, 3' and 5' fragment RACE were performed, and the obtained 3' and 5' fragments were ligated with vectors, cloned, and sequenced; (3) The obtained 3' and 5' fragments were spliced with the core fragment to obtain the full-length sequence of the ELOVL7 gene of Chinese mitten crab.

3. The method for cloning the ELOVL7 gene of Chinese mitten crab according to claim 2, characterized in that: In steps (1) and (2), the Chinese mitten crab intestinal RNA is extracted from the Chinese mitten crab intestinal tract by TRIzol method.

4. The method for cloning the ELOVL7 gene of Chinese mitten crab according to claim 2, characterized in that: In steps (1) and (2), the vector is pMD-19T plasmid.

5. A protein encoded by the ELOVL7 gene of Chinese mitten crab, the amino acid sequence of which is shown in SEQ ID NO:

2. An expression vector comprising the ELOVL7 gene of Chinese mitten crab according to claim 1 .

7. A transfected cell comprising the protein encoded by the ELOVL7 gene of Chinese mitten crab according to claim 5 or the expression vector according to claim 6.

8. The transfected cell according to claim 7, characterized in that The transfected cells are Escherichia coli TOP10 competent cells.

9. Use of the Chinese mitten crab ELOVL7 gene according to claim 1 or the protein encoded by the Chinese mitten crab ELOVL7 gene according to claim 5 in promoting the biosynthesis of long-chain polyunsaturated fatty acids in Chinese mitten crab.

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