NPY of babylonia areolata and structural domain, coding gene and application thereof

Abstract

The invention relates to NPY of Babylonia areolata and a structural domain, a coding gene and application thereof, and belongs to the technical field of molecular biology, the nucleotide sequence of the gene is shown as SEQ ID NO.1, the amino acid sequence of the gene is shown as SEQ ID NO.2, the structural domain gene of the gene is shown as SEQ ID NO.2, and the structural domain protein of the gene is shown as SEQ ID NO.4. The NPY of the babylonia areolata and the structural domain protein of the NPY can promote the growth of the babylonia areolata.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to NPY of Babylonia areolata and its structural domain, encoding gene and application. Background Art

[0002] Babylonia square Babylonia areolata ) is an important aquaculture species in the southeast coast of my country. The factory farming of "high density + constant flowing water + fresh ice bait" is currently the main farming model of Babylonia square spot. At present, artificial compound feed is generally used in the farming of Babylonia square spot and chemical attractants (such as amino acids, betaine, etc.) are added to promote feeding. However, such traditional attractants have problems such as unclear target of action and unstable feeding induction effect. Excessive addition of attractants may lead to risks such as increased feed costs, eutrophication of water bodies and drug residues, which restricts the sustainable development of large-scale farming. Summary of the invention

[0003] The purpose of the present invention is to provide a Babylonia areolata neuropeptide Y (neuropeptide Y, NPY for short) that can be used as a growth-promoting additive for snail seedlings, based on the problem that there are no effective growth-promoting additives in the existing artificial breeding of Babylonia areolata. The present invention analyzes the expression pattern and signal pathway of the Babylonia areolata NPY gene, which can not only provide a reference for revealing the neuroendocrine mechanism of mollusks' feeding regulation, but also provide theoretical support for designing efficient and environmentally compatible attractants, which has important application value in promoting the breeding industry to upgrade to precision and green.

[0004] The present invention is achieved through the following technical solutions: One of the purposes of the present invention is to provide a gene of NPY of Babylonia areolata, the nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0005] The second object of the present invention is to provide a recombinant expression vector, which contains the nucleotide sequence described in SEQ ID NO.1.

[0006] The third object of the present invention is to provide a recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the nucleotide sequence described in SEQ ID NO.1.

[0007] A fourth object of the present invention is to provide a domain of NPY of Babylonia areolata, the nucleotide sequence of the domain is shown in SEQ ID NO.3, and the amino acid sequence of the domain is shown in SEQ ID NO.4.

[0008] The fifth object of the present invention is to provide a recombinant expression vector of a structural domain, wherein the recombinant expression vector contains the nucleotide sequence described in SEQ ID NO.3.

[0009] The sixth object of the present invention is to provide a recombinant engineered bacterium of a structural domain, wherein the recombinant engineered bacterium contains the nucleotide sequence described in SEQ ID NO.3.

[0010] The seventh object of the present invention is to provide a growth-promoting preparation for Babylonia areolata, wherein the growth-promoting preparation contains a protein with an amino acid sequence as shown in SEQ ID NO.2 or SEQ ID NO.4.

[0011] The beneficial effects of the present invention compared with the prior art are as follows: the present invention discovers neuropeptides and their structural domains in Babylonia areolata, and the neuropeptides and their structural domains have the function of promoting the growth of Babylonia areolata. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure is a gel electrophoresis analysis diagram of PCR amplification of NPY products with restriction endonucleases using cDNA of Babylonia square areolata as template, wherein M: molecular weight standard; 1, 2: NPY gene PCR amplification products with restriction endonucleases introduced by primers; Figure 2 It is a gel electrophoresis diagram of PCR identification of pEASY®-T1-NPY, wherein M: molecular weight standard; 1: PCR amplification product using SEQ ID NO.5 and SEQ ID NO.6 as primers, 2: PCR amplification product using SEQ ID NO.5 and SEQ ID NO.8 as primers, 3: PCR amplification product using SEQ ID NO.6 and SEQ ID NO.7 as primers, 4: PCR amplification product using SEQ ID NO.6 and SEQ ID NO.7 as primers; Figure 3 is a map of the expression vector pET32a; Figure 4 It is the gel electrophoresis analysis diagram of the enzyme digestion identification of the recombinant plasmid pET32a-NPY, wherein 1, 2: pET32a-NPY double digestion, the upper band is the cut plasmid band, the lower band is the target band, M: molecular weight standard; Figure 5 This is a diagram of the immunological activity identification of the recombinant neuropeptide Y protein of Babylonia areolata using the immunoblotting method. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further explained below by examples, but the technical solution of the present invention is not limited in any form by the examples.

[0014] Example 1 The gene sequence of neuropeptide Y gene of Babylonia square areolata was obtained by molecular cloning, including the following steps: PCR amplification, purification, ligation to pEASY®-T1 vector, transformation of Escherichia coli DH5α, screening of positive clones, identification and sequencing analysis, and finally confirmed that the nucleotide sequence of neuropeptide Y gene of Babylonia square areolata is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The required primer sequences are as follows: NPY-F; 5'-CTCGAGATGCTGGCGCCCCCT-3' (SEQ ID NO.5), NPY-R;5'-GAATTCCGGTCAGGGGGGCGC-3' (SEQ ID NO.6), M13-F; 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO.7), M13-R;5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO.8), In this example, a prokaryotic expression vector containing the neuropeptide Y gene of Babylonia square areolata was constructed. The specific method is as follows: NPY gene fragments with XhoI and EcoRI restriction sites introduced at both ends were obtained by PCR amplification (primer sequences such as SEQ ID NO: 5-6), and after double restriction digestion and gel purification, they were connected to the pET32a vector digested with the same restriction enzymes to obtain the recombinant plasmid pET32a-NPY. The required primer sequences are as follows: NPY-F; 5'-CTCGAGATGCTGGCGCCCCCT-3' (SEQ ID NO.5), NPY-R;5'-GAATTCCGGTCAGGGGGGCGC-3' (SEQ ID NO.6), T7;5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO.9), T7ter-R; 5'-GCTAGTTATTGCTCAGCGG-3' (SEQ ID NO. 10).

[0015] Example 2: Synthesis of the Babylonia areolata neuropeptide Y domain gene with an enzyme-cut linker According to the sequenced amino acid sequence of neuropeptide Y (NPY) of Babylonia square areolata (SEQ ID NO.2), the position of its mature peptide (domain) was determined (SEQ ID NO.4). According to the newly designed nucleotide sequence of neuropeptide Y mature peptide (domain) of Babylonia square areolata (SEQ ID NO.3), 4 pairs of primers were designed, named P1, P2, P3, and P4, respectively, and the sequences were shown in SEQ ID NO: 5-8, wherein P1 contained the recognition site sequence of restriction endonuclease XhoI, P2 contained the recognition site sequence of EcoRI enzyme cutting, P3 was M13F sequence, and P4 was M13R sequence. According to the instruction manual, the PCR reaction system was prepared, and the first round of PCR amplification was carried out. The PCR reaction conditions were: 95℃ pre-denaturation for 3 minutes; the following was 35 cycles, 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 30 seconds; and finally 72℃ extension for 5 minutes. The PCR product was recovered using FastPure® Gel DNA Extraction Mini Kit (Novozymes) and subjected to agarose gel electrophoresis to separate and purify the neuropeptide Y domain gene fragment of Babylonia square areolata. The NPY domain fragment was connected to the pEASY®-T1 vector by heat shock method and then transformed into Escherichia coli DH5α. The recombinant plasmid was named pEASY®-T1-NPY.

[0016] Example 3: Analysis and comparison of the nucleotide sequence and amino acid sequence of the NPY domain gene cloned by TA with the original sequence The recombinant plasmid pEASY®-T1-NPY was used as a template and P1, P2, P3, and P4 were used as primers for cross-validation. Figure 2 The constructed recombinant plasmid pEASY®-T1-NPY was sent to BGI for DNA sequencing to obtain the nucleotide sequence of the NPY domain, which was then compared with the original sequence of the NPY gene using snapgene software.

[0017] Amino acid sequence of NPY domain: MLAPPDRPQEFRSPGELRRYLKALNEYYAIVGRPRF (SEQ ID NO. 3); Nucleotide of the NPY domain: ATGCTGGCGCCCCTGACCGGCCCCAGGAGTTCA GGAGTCCCGGGGAGCTCCGGCGCTACCTCAAGGCCCTCAACGAGTACTACGCCATTGTCGGCAGGCCCAGG (SEQ ID NO. 4).

[0018] Example 3: Construction of the prokaryotic expression vector pET32a-NPY containing the gene of the neuropeptide Y domain of Babylonia areolata The vector plasmid pET32a was treated with restriction endonucleases XhoI and EcoRI to separate and purify the 5.8kb large fragment, which was mixed with the neuropeptide Y gene fragment of Babylonia quadrata at a ratio of 1:5, and then ligated with T4 ligase at 16°C for 16 hours before being transferred into Escherichia coli DE3. Bacteria with Amp resistance were screened on LB plates, and plasmids were extracted using standard methods. Recombinant plasmids with a size of about 6.0 kb were screened, and the recombinant plasmids were double-digested with restriction endonucleases XhoI and EcoRI to obtain two fragments of 119bp and 5.8kb, which were the same size as the neuropeptide Y gene of Babylonia quadrata and the expression vector pET32a, respectively, proving that the neuropeptide Y gene of Babylonia quadrata had been cloned into the Escherichia coli expression vector pET32a, and the recombinant plasmid was named pET32a-NPY. See the plasmid map for details. Figure 3 , PCR identification diagram and enzyme digestion analysis diagram are shown in Figure 4 .

[0019] Example 5: Production of recombinant Babylonia areolata neuropeptide Y domain protein using pET32a-NPY-DE3 A single colony of pET32a-NPY was picked and inoculated into 100 ml LB liquid medium (100 μg / mL Amp) at 37°C and 180 rpm until the bacterial solution OD600≈0.4-0.6; after the bacteria returned to room temperature, the confirmed optimal induction concentration of IPTG was added, and after induction, the supernatant was discarded, the precipitate was collected, and 6 mL of 0 mM imidazole buffer (pH=8.0) was added to thoroughly resuspend the bacteria; the bacterial solution was quickly frozen in liquid nitrogen (about 10 min), and then taken out for low-temperature thawing (4°C refrigerator); then the ultrasonic mode was set to work for 2 s and stop for 4 s, the power was 130 W, and the protein was subjected to low-temperature ultrasonic fragmentation for 10 min; 12000 g low-temperature (4°C) centrifugation was performed for 30 min, and the soluble protein in the supernatant was collected at low temperature, and the supernatant was the expressed recombinant neuropeptide Y domain protein of Babylonia square-spotted. After mixing the supernatant protein sample with 5× protein loading buffer, boil it for 15 min and run SDS-PAGE gel electrophoresis according to the standard method. At the same time, take the negative control and treat it in the same way and then run electrophoresis. The negative control is the supernatant protein of the monoclonal culture grown after the empty plasmid pET32a is transformed into DE3. The pET32a-NPY expression supernatant is purified and desalted using N Ni-NTA-Sepharose column. Finally, the recombinant neuropeptide Y protein of Babylonia square isolata was identified by immunoblotting (Western blot) method (see Figure 5 ).

[0020] Example 6 The concentrated and desalted NPY protein obtained in Example 5 was added to the feed at 1 μg / g for 30 days, and the snails fed with normal feed were used as the control. During the breeding process, the seawater salinity was 31.20±0.84, the temperature was 27.56±0.64℃, the pH was 7.56±0.36, and the dissolved oxygen (DO) concentration was above 5.0 mg / L. After 30 days, the feeding and body weight were analyzed. The results showed that the body weight of the snails fed with NPY protein feed increased by 13% compared with the control group.

[0021] Babylonia square areolata neuropeptide Y gene (SEQ ID NO.1): atgcagaaagcagtgctgtgcgccttgctcgtgatctgcctggtggtggcggaagtgacgtgtcaggaagcg atgctggcgccccctgaccggccccaggagttcaggagtcccggggagctccggcgctacctcaaggccctc aacgagtactacgccattgtcggcaggcccaggttc ggacgcagtgtcaacaccaacaagagatcagtggaagagctgggagacctcaagtccgatgactggggatacggccctgagtacccgtgggaagatgaagaggaattcctacgccgctga; Babylonia square areolata neuropeptide Y gene (SEQ ID NO.2): MQKAVLCALLVICLVVAEVTCQEA MLAPPDRPQEFRSPGELRRYLKALNEYYAIVGRPRF GRSVNTNKRSVEELGDLKSDDWGYGPEYPWEDEEEFLRR.

Claims

1. A neuropeptide Y of Babylonia areolata, characterized in that: The amino acid sequence of the neuropeptide Y is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the neuropeptide Y is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleotide sequence described in SEQ ID NO.

1.

3. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contains the nucleotide sequence described in SEQ ID NO.

1.

4. A mature peptide of neuropeptide Y of Babylonia areolata, characterized in that: The nucleotide sequence of the gene encoding the mature peptide is shown in SEQ ID NO.3, and the amino acid sequence of the mature peptide is shown in SEQ ID NO.

4.

5. A recombinant expression vector of a mature peptide, characterized in that: The recombinant expression vector contains the nucleotide sequence described in SEQ ID NO.

3.

6. A recombinant engineering bacterium of a mature peptide, characterized in that: The recombinant engineered bacteria contains the nucleotide sequence described in SEQ ID NO.

3.

7. A growth-promoting preparation for Babylonia areolata, characterized in that: The growth-promoting preparation contains a protein with an amino acid sequence as shown in SEQ ID NO.2 or SEQ ID NO.4.

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