CN113388019A - Application of sepiella maindroni neuropeptide - Google Patents
Application of sepiella maindroni neuropeptide Download PDFInfo
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- CN113388019A CN113388019A CN202110647717.5A CN202110647717A CN113388019A CN 113388019 A CN113388019 A CN 113388019A CN 202110647717 A CN202110647717 A CN 202110647717A CN 113388019 A CN113388019 A CN 113388019A
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- gnrh
- sepiella maindroni
- neuropeptide
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Abstract
The invention discloses an application of Sepiella maindroni neuropeptide, the Sepiella maindroni neuropeptide GnRH gene cDNA complete sequence is 432bp, the open reading frame is 273bp, 90 amino acids are coded, 5 '-UTR 86bp, 3' -UTR 73bp, the precursor protein MW is 10.1kDa, and the isoelectric point pI is 7.73. The sepiella maindroni neuropeptide provided by the invention can effectively carry out reproduction regulation and control by utilizing neuropeptide and an antagonist thereof in the artificial breeding process of sepiella maindroni.
Description
The application is a divisional application of Chinese patent application with the application date of 2018, 1 month and 25 days, the application number of 201810075238.9 and the invented name of 'Sepiella maindroni neuropeptide and application thereof'.
Technical Field
The invention relates to the technical field of neuropeptides, in particular to application of sepiella maindroni neuropeptide.
Technical Field
Sepiella maindroni (Sepiella japonica) genus mollusk (Mollusca), Cephalopoda (Cepalopoda), Decales (Decapoda), Sepiella (Sepiidae), and Sepiella (Sepiella). The Sepiella maindroni is an annual medium-sized cuttlefish commonly known as cuttlefish, has quick growth and delicious meat quality, and is a seafood food popular in the market. Cephalopoda, sepiaceae. Shield shape of the trunk. The length of the carcass is 2 times of the width of the carcass. The carcass back has a plurality of nearly elliptical white spots. The meat fin is narrow in front and wide in back, is positioned at the whole edges of two sides of the body part, and is separated only at the tail end. The length of the wrist without the handle is generally 4 to 3 to 1 to 2, and the suction cup has 4 rows. The fourth left carpal stalk of the male. The ear-shaped narrow handle is in the shape of a small and dense sucking disc with about 20 rows. The medical department is oval. Record the maximum carcass length 0.19m and maximum body weight 0.7 kg. Distributed in the coastal waters of the northwest pacific ocean and the north indian ocean. The main operation fishing ground is in the coastal waters of Zhejiang and Fujian in China. The annual production is more than 8 ten thousand tons, and the method is an important economic species in the world. The fresh food is mainly used as an economic cephalopod with high medicinal and edible values, the number of natural populations is rapidly reduced from the 20 th century and 80 th century, and numerous scholars research the resource protection and reproductive development of the cephalopod.
Neuropeptides are bioactive polypeptides which are distributed in the body mainly in nervous tissues and have a neurohormone-like effect or a neuron information transmission effect on the body, and play an important role in the aspects of functions of connecting the whole nervous system and other systems of the body, such as growth and development, respiratory regulation, thermoregulation and the like.
Disclosure of Invention
The invention aims to provide sepiella maindroni neuropeptide and application of the sepiella maindroni neuropeptide in reproduction control by using neuropeptide and an antagonist thereof in a fish culture process, analyzes a gene sequence of the sepiella maindroni neuropeptide and tissue expression specificity of the gene by using a molecular biology means, performs cell positioning on a specific expression site of mRNA of the sepiella maindroni neuropeptide in a brain tissue of a sepiella maindroni, provides a certain theoretical basis for germplasm resource protection and artificial breeding of sepiella maindroni by using a whole sepiella maindroni reproduction control mechanism, and provides a certain theoretical basis for germplasm resource protection and artificial breeding of sepiella maindroni.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the application of the Sepiella maindroni neuropeptide is provided, in the aquaculture process, the GnRH analogue LHRH-A or sGnRH-A and the dopamine antagonist DOM can be used as an oxytocic to induce fish to carry out artificial breeding, and in the artificial breeding process of the Sepiella maindroni, the neuropeptide and the antagonist thereof can be used for carrying out reproductive regulation and control more effectively;
the gene cDNA complete sequence of the Sepiella maindroni neuropeptide has the length of 432bp, the Open Reading Frame (ORF) is 273bp, 90 amino acids are coded, the 5 '-UTR is 86bp, and the 3' -UTR is 73 bp; the ORF includes a segment of a signal peptide of 31 amino acids and a helper sequence of 44 amino acids, as well as a mature peptide of 12 amino acids: pQNYHFSN GWPPG; the MW of the precursor protein is 10.1kDa, and the isoelectric point pI is 7.73; the Sepiella maindroni neuralgia GnRH can participate in regulating and controlling the reproduction activity of Sepiella maindroni.
The complete sequence of cDNA of the gene of the sepiella maindroni neuropeptide is as follows: ACTTCCTTTACACTCGTCCCTCGCCTAAGACAAAAGAACACTTCAAATCTCCATCATCAGCTAAACATCTACCAGACAGTGACATCATGTCAACCTCCACAGCCTCGTCCAGCCTGAGAAGAATCCAATTTTTCACCTGTGCTATTCTTCCCCTCTCTTTCTGCATGCATATCCAGGCACAGAATTACCATTTTAGCAATGGATGGCACCCCGGTGGTAAACGAAGTGGACTTCCAGACATGCAGTGTCATTTCAGACCACAAACAAAAGCTACAATCGAGAAACTCTTAGACGAGGAAATCACACGTATAATTACTACATGTACCAATACAGTCAATGACATCGCAGACTTGCAGTAATTTTTCACAGGTCAAGCAAATTCACTGGATATACAACTACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA are provided.
The molecular biology analysis step that the Sepiella maindroni neuropeptide GnRH can participate in the regulation of the Sepiella maindroni reproductive activity comprises the following steps: the method comprises the following steps of neuropeptide gene cloning and sequence analysis, neuropeptide tissue specificity expression analysis and neuropeptide brain tissue expression positioning analysis, and specifically comprises the following steps:
neuropeptide gene cloning and sequence analysis operations were:
selecting mature and healthy Sepiella maindroni, extracting total RNA from the brain tissue of the Sepiella maindroni by using a Trizol reagent, and determining that the absorbance R value of the extracted total RNA is between 1.8 and 2.0, thereby indicating that the total RNA is effective; spotting 3 μ L of RNA solution on 1% agarose gel, running gel for 15-16 min under 200V and 150mA electrophoresis apparatus, collecting gel, analyzing RNA strip condition in UVP ultraviolet gel imaging apparatus to obtain clear 28S and 18S at a ratio of 1.9-2.1: 1; synthesizing a first cDNA chain by using total RNA extracted from the sepiella maindroni brain tissue by using a reverse transcription kit; taking Sepiella maindroni brain tissue cDNA as a template, carrying out PCR amplification on GnRH gene core fragments by degenerate primers GnRH-F and GnRH-R, detecting a PCR product by using 1% agarose electrophoresis, finding a band with an expected size, carrying out tapping recovery and purification by using a gel recovery kit, and carrying out clone connection on the purified target fragments; respectively taking total RNA of sepiella maindroni brain tissue as templates to obtain template cDNA required by amplifying 3 ' RACE fragments and 5 ' RACE fragments, amplifying 3 ' RACE unknown fragments by utilizing nested PCR, and performing gel cutting recovery and cloning connection on PCR products; splicing the 3 'end, the 5' end and the core fragment sequence of the GnRH gene by using Lasergene software to obtain the cDNA full-length sequence of the Sepiella maindroni GnRH gene; the online tool of Predict Protein and Scratch Protein Predictor can be used for predicting an Open Reading Frame (ORF) of a GnRH gene and presuming an amino acid sequence coded by the ORF, an online analysis site SignalP v3.0 is used for predicting the signal peptide of the amino acid sequence, NCBI is used for performing Blast analysis on the amino acid sequence, ClustalW2 is used for performing comparison analysis on the amino acid sequence of the GnRH and a homologous gene thereof, ExpasyProtParam online analysis software is used for estimating the relative molecular mass and isoelectric point pI of a precursor Protein, MEGAv5.0 software is used for constructing an evolutionary tree based on an NJ method, and 1000 cycles are repeated; the primer sequences used during neuropeptide gene cloning and sequence analysis are shown in table 1.
TABLE 1 GnRH Gene cloning and primers for sequence analysis
| Primer Name | Primer sequence(5′-3′) |
| GnRH-F | CAGACNCAAGCACARAAYTA |
| GnRH-R | TYTCTATCAAAGCYTTTGT |
| 5′-GnRH-outter | TTACCACCAGGGTGCCATCC |
| 5′-GnRH-inner | TTGCTAAAATGGTAATTCTG |
| 3′-GnRH-outter | CACCTGTGCTATTCTTCCCCTCTCTTTC |
| 3′-GnRH-inner | TGCATATCCAGGCACAGAATTACCA |
| M13-F | TGTAAAACGACGGCCAGT |
| M13-R | CAGGAAACAGCTATGACC |
| 5′RACE Adapter | GGCCAGGCGTCGACTAGTACGGGGGGGGGG |
| 5′RACE Outer Primer | ACUACUACUAGGGGCGTCGACGTA |
| 5′RACE Inner Primer | ACUACUACUAGGGGCGTCGA |
| 3′RACE Adapter | GCGAGCACAGAATTAATATTTTTTTTTTTT |
| 3′RACE Outer Primer | GCGGATCCGAATTAATACGACT |
| 3′RACE Inner Primer | GCGAGCACAGAATTAATACGACT |
The gene cDNA complete sequence of Sepiella maindroni neuropeptide GnRH is 432bp, ORF273bp, 90 coded amino acids, 86bp 5 '-UTR and 73bp 3' -UTR; the ORF includes a segment of a signal peptide of 31 amino acids and a helper sequence of 44 amino acids, as well as a mature peptide of 12 amino acids: pQNYHFSN GWPPG; the precursor protein MW is 10.1kDa, and the pI is 7.73; the prediction analysis of the signal peptide of the precursor protein amino acid sequence shows that the N end of the GnRH precursor protein contains a signal peptide with 31 amino acids in length, and the C end of the GnRH mature peptide sequence contains a shearing site and an auxiliary sequence with 44 amino acids in length, and the auxiliary sequence can be combined with the mature peptide to play a stabilizing role. The results of the alignment of the GnRH amino acid sequences show that only the mature polypeptide portions are conserved and cluster as one branch with invertebrate GnRH in the evolutionary tree analysis.
And acquiring GnRH gene sequences of other species from an NCBI database, and comparing the GnRH amino acid sequence of Sepiella maindroni with the homologous sequences of other species by using a ClustalW2 online analysis tool. And constructing an evolutionary tree of the selected homologous sequences by MEGA5.0 software. The results of homologous alignment show that the Sepiella maindroni GnRH is 90% similar to the Sepiella tigerina, 86% and 71% similar to the Sepiella jiangensis and the Sepiella maindroni GnRH, 47%, 41% and 31% similar to the pacific oyster, the Japanese scallop and the sea snail, and the mature peptide part in the sequence is very conservative.
The neuropeptide tissue specific expression analysis was performed as follows:
selecting 3 different development stages of Sepiella maindroni with healthy weight and strong vitality in stages III, IV and V, quickly killing the Sepiella maindroni, taking out each tissue, putting the tissue into RNA preservation solution, wherein the male tissue is as follows: brain, visual lobe, liver, muscle, testis, heart, gill, female tissues are: storing the brain, the visual leaves, the liver, the muscles, the ovary, the heart, the branchia, the periwinal gland and the accessory periwinal gland at a temperature of between 80 ℃ below zero and 100 ℃ below zero for later use; total RNA was extracted from each tissue of Sepiella maindroni with Trizol reagent, cDNA of different tissues was synthesized using a kit, specific expression of GnRH gene in different development stages and different tissues was measured using a fluorescence quantitative PCR method using Sepiella maindroni beta-actin gene (JN564496.1) as an internal reference gene and the gene expression level of the heart as a reference standard, and the expression level of GnRH gene in different tissues was measured using a 2-. DELTA.Ct method using primers shown in Table 2.
TABLE 2 primers for fluorescent quantitative PCR of GnRH genes
| Primer name | Sequence of |
| RT-GnRH-F | ACTTCCTTTACACTCGTCCCT |
| RT-GnRH-R | AGGGTGCCATCCATTGCTAA |
| RT-actin-F | TGAGAGGGAGATTGTGCGTG |
| RT-actin-R | GAACATAGATTCTGGAGCACGG |
The result of detecting the mRNA expression level of the Sepiella maindroni GnRH gene by fluorescence quantitative PCR shows that the GnRH gene is obviously expressed in the brain tissues of the Sepiella maindroni in three stages. In contrast, GnRH is expressed in minute amounts in the optic leaves in phase iii and in minute amounts in the female cuttlefish ovary, the perigonal gland and the paragonal gland in phase v. However, in adult squid, semi-quantitative PCR was used to detect the GnRH expression level in different tissues, but the results showed that the PCR product was amplified only in brain tissue, and the ovary of female squid, like other tissues, did not express GnRH. Adult sepiella maindroni and octopus ocellatus are also expressed only in the brain and ovary of female sepiella maindroni.
The neuropeptide brain tissue expression localization analysis operation is as follows:
carrying out PCR amplification by taking cuttlefish brain tissue cDNA as a template, wherein the PCR amplification reaction program is as follows: 94 ℃ for 2 min; 94 ℃ for 20s, 64 ℃ for 40s, 10 cycles; annealing at 94 ℃ for 20s and 62 ℃ for 40s for 15 cycles; 10 cycles of 94 ℃ for 20s, 60 ℃ for 30s, 72 ℃ for 30 s; 8min at 72 ℃; detecting the product by electrophoresis; performing gel cutting purification, enzyme digestion, connection, transformation and screening on the PCR product obtained by amplification, and preparing an antisense probe and a sense probe by using a DIG RNAlabelling kit SP6/T7 kit; carrying out in-situ hybridization treatment on the complete brain tissue section of the sepiella maindroni, and sealing and storing after the section is photographed. The primer capable of specifically amplifying the Sepiella maindroni GnRH gene comprises the following components: ISHGnRH-F: CCGTCACGTCCATCATCAGGTAAACATC, ISH-GnRH-R: CCGGAAGGGAGTGATTTCCTCGTCTAAG are provided.
Preferably, the preparation method of the paraffin section of the complete brain tissue of the sepiella maindroni comprises the following steps:
1) fixing: soaking brain tissue of cuttlefish in 4% paraformaldehyde, and fixing at 4 deg.C for 16 hr; rinsing in PBS for 5 min;
2) and (3) dehydrating: dehydrating brain tissue in gradient staining jar containing 70%, 80%, 90% and 100% ethanol for 1 hr respectively, wherein the reagent can be replaced once;
3) and (3) transparency: brain tissue was transferred to xylene: soaking in 1:1 ethanol for 30min, and soaking in organic solvent xylene for 30min to make tissue transparent;
4) wax dipping: brain tissue was first transferred to paraffin: 1h in a ratio of 1:1, and then transferring the mixture into paraffin for 1 h;
5) embedding: preheating a metal box in advance, and putting brain tissues which are fully waxed into the metal box for paraffin embedding;
6) block repairing: repairing the embedded wax block;
7) slicing: placing the repaired wax block on a paraffin slicer to slice, and longitudinally cutting, wherein the slice thickness is 7 mu m;
8) unfolding and baking: spreading for 5min on a 42 ℃ film spreading machine; baking the slices on a 50 ℃ slice baking machine for 30 min; the dried paraffin slices can be stored in a refrigerator at the temperature of-20 ℃ for later use;
9) dewaxing: placing the cut brain tissue slices in xylene for dewaxing for 30min, and replacing the reagent once;
10) rehydration: rehydrating brain tissue in gradient staining jars filled with 100%, 90%, 80% and 70% ethanol for 10min respectively; rinsing in PBS for 10min, and replacing the reagent once;
preferably, the in situ hybridization step is:
1) pretreatment: immersing the paraffin section of the brain tissue into 4% paraformaldehyde for fixing for 10 min; rinsing with PBS for 10 min; 0.1 MPBS/glycine for 5 min; 0.3% Triton X-100/PBS for 15 min; rinsing with PBS for 10 min;
2) and (3) permeation treatment: treating in protease K solution at 37 deg.C for 20 min; washing with 0.1M amino acid/PBS for 1 min; rinsing with PBS for 10 min; 0.1M triethanolamine (containing 0.25% ammonium acetate) for 10 min; finally rinsing for 10min by using a salt solution 2 XSSC;
3) prehybridization and hybridization: incubating the prehybridization solution for 1h at 45 ℃; incubating the hybridization solution for 6h at 46 ℃;
4) and (3) post-treatment: after incubation of the hybridization solution, rinsing with 4 XSSC for 1 min; rinsing with 2 XSSC at 37 ℃ for 30 min; rinsing with 1 XSSC for 30 min;
5) antibody hybridization: blocking buffer solution is sealed for 1h at room temperature; AP-labeled anti-DIG antibody (1:500), and incubating for 1h at 37 ℃; then washing with PBS for 1 min;
6) color development: adding a chromogenic substrate NBT/BCIP, and developing for 30min-24h in a dark place; washing with DEPC water for 3min, sealing with glycerol, taking pictures after two days, and storing.
Cellular localization of the expression site of sepiella maindroni GnRH gene mRNA in brain tissue was performed by the in situ hybridization method, and as shown in fig. 6, clear GnRH gene mRNA positive hybridization signals were observed in a plurality of regions of sepiella maindroni brain tissue, whereas no signals were observed in fig. 6A where hybridization was performed with sense probes. The positive hybridization signals were most intense in the supraesophageal nerve plexus, the subcrural lobe and the subvertical lobe (6B), and significant hybridization signals were observed in the medullary region of the entire subvertical lobe margin. Positive signals were also observed in the vertical leaf above the sub-vertical leaf (6B), but the signals were weaker. Clear positive signals were also observed in the anterior basal lobe (6C) and posterior basal lobe (6D). Hybridization signals obtained by hybridization staining with the antisense probe were observed in the lower frontal lobe (6C), but not in the upper frontal lobe (6C). The functional leaflets of the subgsophageal nerve group: the hybridization signals in the giant cell leaf, coat visceral leaf, pre-pigmented cell, foot leaf and wrist leaf (6E) are very dark colored and very strong. Clear hybridization positive signals were also observed in the medullary region of the optic leaf (6F), while no positive signals were observed in the cortical region.
Through in-situ hybridization of GnRH gene mRNA of Sepiella maindroni brain tissue, the GnRH gene is found to be expressed in 3 main components of Sepiella maindroni brain tissue. Positive hybridization signals of different intensities were observed in the subungual, perpendicular, sub-perpendicular, antero-basal, postero-basal and inferior frontal lobes of the brain of the supraesophageal nerve plexus. Effective positive hybridization signals were also observed in the giant cell leaf, the mantle visceral leaf, the anterior pigmented cell leaf, the foot leaf, the carpal nerve leaf, and the optic leaf of the subgsophageal nerve group.
Compared with the prior art, the invention has the advantages that:
1) the gene sequence and the tissue expression specificity of the gene of the Sepiella maindroni neuropeptide are analyzed by using a molecular biology method, the cell positioning is carried out on the specific expression site of the mRNA in the brain tissue of the Sepiella maindroni, and the research data can provide a certain theoretical basis for further researching the cephalopodium reproduction regulation mechanism;
2) the result of GnRH amino acid sequence comparison shows that only mature polypeptide part is conservative, and is clustered into one with invertebrate GnRH in the evolutionary tree analysis, the neuropeptide GnRH gene is detected to have significant expression in the brain tissues of cuttlefish in stages III, IV and V by a fluorescence quantitative PCR method, the result of in situ hybridization shows that the GnRH gene has expression in SVL, SPL, ABL, PBL and lower frontal lobe (IFL) in the nerve group on the esophagus of cuttlefish brain, MAG, PVL, ACL, foot lobe (PL) and wrist nerve lobe BL and visual lobe of the nerve group under the esophagus, and the GnRH is presumed to be possibly involved in regulating and controlling the Sepiella maindroni reproductive activity according to the data.
Drawings
FIG. 1 is a schematic diagram showing the result of PCR electrophoresis of the GnRH gene of the present invention;
FIG. 2 is a schematic representation of the 5' RACE amplification electrophoresis band of the present invention;
FIG. 3 is a schematic representation of the 3' RACE amplification electrophoresis band of the present invention;
FIG. 4 is a schematic diagram showing the full length of cDNA of the GnRH gene according to the present invention;
FIG. 5 is a schematic representation of the specificity of GnRH differential tissue expression in the present invention;
FIG. 6 is a schematic diagram of brain tissue in situ hybridization of GnRH gene mRNA in accordance with the present invention.
Reference numerals: in FIG. 4, the predicted amino acid sequence is shown below the base sequence, the predicted signal peptide sequence is indicated by black underlining, the predicted GnRH mature peptide is indicated by black double underlining, the primary cleavage site is indicated by black double-dashed line, the C-terminally amidated glycine is indicated by a circle, the helper sequence is indicated by black dashed line, the predicted start codon is ATG and the stop codon is TAA; in fig. 6, a: a sense probe control; b: a sub-vertical leaf; c: lower frontal lobe; d: posterior basal leaves; e: the sub-esophageal nerve ball; f: a visual leaf; ABL: a pre-basal leaf; ASEM: the lower anterior esophageal nerve plexus; MSEM: the lower middle esophageal nerve plexus; PBL: posterior basal leaves; PSEM: the posterior sub-esophageal nerve ball; SVL: a sub-vertical leaf; black spikes indicate positive areas and black bars indicate a length of 1000 μm.
Detailed Description
The scheme of the invention is further illustrated by the following examples:
example 1:
the sepiella maindroni neuropeptide, has gene cDNA complete sequence length of 432bp, ORF273bp, encoding 90 amino acids, 5 '-UTR 86bp and 3' -UTR 73 bp. The ORF includes a segment of a signal peptide of 31 amino acids and a helper sequence of 44 amino acids, as well as a mature peptide of 12 amino acids: pQNYHFSN GWPPG; the MW of the precursor protein is 10.1kDa, and the isoelectric point pI is 7.73; the results of the alignment of the GnRH amino acid sequences show that only the mature polypeptide portions are conserved and cluster as one branch with invertebrate GnRH in the evolutionary tree analysis. The fluorescent quantitative PCR method is used for detecting that the neuropeptide GnRH gene has obvious expression in the brain tissues of the cuttlefish in stages III, IV and V. In contrast, GnRH is expressed in trace amounts in the optic leaves at the stage III, and in the female cuttlefish ovary, the perigonal gland and the paraperigonal gland at the stage V. The results of in situ hybridization showed that the GnRH gene was expressed in SVL, SPL, ABL, PBL and lower frontal lobe (IFL) in the supraesophageal nerve group of the brain of cuttlefish, MAG, PVL, ACL, Plantar Lobe (PL) and carpal lobe BL in the infraesophageal nerve group, and in the optic lobe. According to the data, GnRH can participate in the regulation of the sepiella maindroni reproductive activity.
The method for analyzing the participation of the sepiella maindroni neuropeptide GnRH in the regulation of the reproductive activity mechanism of the sepiella maindroni comprises the following steps:
A. preparing materials:
(1) DEPC water: adding 500mL double distilled water and 500 mu LDEPC into a blue-mouth reagent bottle, and shaking overnight at 37 ℃ for later use;
(2) DEPC treated Water: treating DEPC water in autoclave at 121 deg.C for 30min, cooling, and storing at 4 deg.C. 75% of ethanol: adding 25mL of EPC treated water into 75mL of 100% ethanol, and storing at-20 ℃ for later use;
(3) LB liquid medium: adding yeast extract with concentration of 0.5% into deionized water, adding 1% NaCl and 1% peptone, adjusting pH to neutral, sterilizing at high temperature and high pressure for 30min, and storing in 4 deg.C refrigerator;
(4) LB solid medium: adding yeast extract with final concentration of 0.5%, 1% NaCl, 1% peptone and 2% agar powder into deionized water, adjusting pH to neutral, sterilizing at high temperature and high pressure for 30min, taking out, shaking, pouring into sterilized culture dish, and storing in refrigerator at 4 deg.C.
B. Extracting total RNA:
(1) selecting a mature and strong-activity Sepiella maindroni individual, placing the Sepiella maindroni individual on ice, anesthetizing and killing the Sepiella maindroni individual under a low-temperature condition, taking a Sepiella maindroni brain tissue, carefully peeling a nodule tissue around a nerve tissue, placing the Sepiella maindroni brain tissue in a preservation solution, and performing cryopreservation at an ultralow temperature of-80 ℃;
(2) a clean 1.5mL centrifuge tube (RNAase free) was added to 500. mu.L Trizol (4 ℃ C.) with a RNAase-free tip, 30mg of brain tissue sample was clamped with DEPC-treated forceps and immersed in Trizol, homogenized for 30s using an electric homogenizer, and 500. mu.L of Trizol was added;
(3) slightly shaking the centrifuge tube to fully dissolve the tissues into Trizol, and standing for 5 minutes at room temperature;
(4) adding 200 mu L of chloroform into a centrifuge tube, violently shaking for 15s, and standing for 5 minutes at room temperature;
(5) centrifuging at 12000rpm for 15min at 4 deg.C;
(6) after centrifugation, dividing the centrifugal tube into 3 layers, and taking the supernatant on the uppermost layer in a new centrifugal tube;
(7) adding 500 mu L of isopropanol into a centrifuge tube, shaking up, and standing for 10 minutes at room temperature;
(8) centrifuging at 12000rpm for 10min at 4 deg.C in a centrifuge;
(9) after centrifugation, the supernatant is removed and the precipitate is retained;
(10) adding 1mL of 75% ethanol, and blowing and sucking the sediment by using a gun head;
(11) centrifuging at 7500rpm for 5min at 4 deg.C in a centrifuge, sucking off the liquid part with a gun head, and retaining the precipitate;
(12) standing at room temperature for about 20 minutes until RNA is in a semi-dry transparent state, and dissolving with DEPC water for later use.
The substances in the preservation solution in the step (1) and the contents thereof are as follows: 2g/L of dimethyl sulfoxide, 1.6g/L of acetamide, 1g/L, PEG-8000.5 g/L of 1, 2-propylene glycol, 0.15g/L of cromolyn sodium, 0.02g/L of reducing glutathione, 6.0g/L of lecithin, 5g/L of benzoate, 12.5g/L of sodium nitrate, 16g/L of glycerol, 150IU/mL of penicillin, 110IU/mL of streptomycin, 120mg/L of erythrose and 23mg/L of (S) -ethyl lactate, the pH is adjusted to be 7.3 by phosphate buffer solution, and the balance is double distilled water; the sepiella maindroni brain tissue is frozen and preserved by the preservation solution, so that the efficient freezing and preservation of the brain tissue can be realized, the liquefaction and the brain cell autolysis of the sepiella maindroni brain tissue can be effectively prevented, the biological characteristics can be efficiently preserved, and the frozen and preserved brain tissue can be directly used for tissue analysis and is beneficial to the efficient and complete extraction of RNA; erythrose and (S) -ethyl lactate in a specific proportion have a synergistic gain effect, the gain effect can enable the (S) -ethyl lactate to be in cross-linked association with saccharides in brain cell membranes, and hydroxyl groups of the (S) -ethyl lactate can replace hydroxyl groups of water on the surfaces of proteins to form a layer of water layer on the surfaces of the proteins, so that the cross-linked association positions of the (S) -ethyl lactate and the saccharides can be protected from being directly exposed to the surrounding environment, the cross-linked association can change the microenvironment on the surfaces of the cell membranes, and water molecules can be reduced from diffusing to the outside of the cells, so that brain tissue dehydration is prevented, and brain tissue cell shrinkage is reduced; meanwhile, erythrose can generate steric hindrance among proteins, can better inhibit the formation of ice crystals during frozen storage, and control and buffer the change of the pH value of brain tissues in the freezing process, so that the structural integrity and the functional integrity of the brain tissues and the stability of the brain tissues during freezing are ensured, the activity of the brain tissues is prolonged, the reproductive regulation activity of the cuttlefish neuropeptide is maintained, and the reproductive regulation feed additive can effectively play the reproductive regulation roles of promoting cell growth, differentiation, proliferation and the like; under the condition that requirements of tissue analysis and DNA extraction can be met, the preservation period of the preservation solution added with erythrose and ethyl (S) -lactate can be prolonged by more than 40% compared with the preservation solution without erythrose and ethyl (S) -lactate, which shows that the erythrose and the ethyl (S) -lactate have important effects on preservation and quality guarantee of the cuttlefish brain tissue.
The absorbance R value of the extracted total RNA must be measured, the general R value between 1.8 and 2.0 indicates that the extracted RNA is effective total RNA and can be continuously used for subsequent use, and if the value is less than 1.8, the extracted RNA contains more impurities, the accuracy is influenced, and the extracted RNA cannot be used; if the R value is more than 2.0, the extracted RNA is degraded and cannot be used; and after the absorbance value is determined, the absorbance value is between 1.8 and 2.0, which indicates that the extracted RNA is possibly effective, the electrophoresis detection is continuously carried out, 3 mu L of RNA liquid is sampled on 1 percent agarose gel, the gel is run for about 15 minutes under an electrophoresis apparatus of 200v and 150mA, the gel is taken out to analyze the RNA band condition in a UVP ultraviolet gel imaging instrument, and if a clear 28S and 18S band can be seen and the proportion is about 2:1, the extracted RNA has good effect and can meet the requirement of further operation.
C. First strand cDNA Synthesis:
the first strand synthesis of cDNA is carried out by using M-MLV (RNase H-) reverse transcription kit produced by Takara company and total RNA extracted from sepiella maindroni brain tissue, and the specific operation steps are as follows:
(1) the following reaction system was added to a 0.2mL centrifuge tube:
| total RNA | 5.0μL |
| Oligo DT | 1.0μL |
(2) Mixing the reaction system uniformly, centrifuging, placing the centrifugal tube in a PCR instrument, incubating at 70 ℃ for 10min, and immediately standing on ice for 2 min;
(3) the following reaction systems were added to the centrifuge tubes:
| 5×M-MLV Buffer | 2.0μL |
| dNTP Mixture(10mM) | 0.5μL |
| RNase Inhibitior(40U/μL) | 0.25μL |
| M-MLV RTase(200U/μL) | 0.25μL |
| DEPC treated Water | 1.0μL |
(4) Mixing the reaction system, centrifuging, placing the centrifuge tube in a PCR instrument, incubating at 42 deg.C for 1h, incubating at 70 deg.C for 15min, and rapidly standing on ice for 15 min;
(5) the synthesized cDNA was stored in a refrigerator at-20 ℃ for further use.
D. Neuropeptide gene cloning and sequence analysis:
(1) designing a primer:
according to the reported mollusk species GnRH gene sequence obtained from NCBI database, a plurality of pairs of degenerate primers for cloning the core sequence segment of the Sepiella maindroni GnRH gene are synthesized by analyzing the conserved region of the amino acid sequence. The 5 'RACE and 3' RACE primers are designed by searching the obtained core fragment sequences by using primer 5 software; the primers required for cloning are as follows:
(2) obtaining a core sequence:
1) and (3) PCR amplification: taking Sepiella maindroni brain tissue cDNA as a template, carrying out PCR amplification on GnRH gene core segments by degenerate primers GnRH-F and GnRH-R, wherein the reaction system is as follows:
| sterilized water | 15.5μL |
| 10×PCR buffer | 2.5μL |
| MgCl2(25mM) | 2.5μL |
| dNTP(10mM) | 1.0μL |
| GnRH-F | 1.0μL |
| GnRH-R | 1.0μL |
| Brain tissue cDNA | 1.0μL |
| Taq enzyme | 0.5μL |
After the sample loading was completed, the PCR amplification procedure was as follows: 5min at 95 ℃; 30s at 94 ℃, 30s at 52 ℃, 30s at 72 ℃ and 35 cycles; 12min at 72 ℃.
2) Purification and connection of PCR products: detecting the PCR product by using 1% agarose electrophoresis, finding a band which is in line with the expected size, and performing tapping recovery and purification by using a gel recovery kit, wherein the specific operation steps are as follows:
firstly, running glue, observing a target strip by using a gel imaging system, cutting the strip by using a clean blade after finding the strip, putting the cut strip into a 1.5mL centrifugal tube of RNAase free, weighing the weight of the cut strip, adding 1mL Solution I into the centrifugal tube filled with a glue block according to the calculated amount of adding 1mL Solution I into each gram of glue, carrying out water bath at 56 ℃ for 10min, and oscillating once every 2-3min to help to accelerate dissolution;
secondly, transferring the Solution I mixed Solution containing the agarose gel into a centrifugal column, centrifuging at 10000rpm for 2min, and if the mixed Solution is excessive, performing the centrifugation for multiple times;
collecting the solution in the step II, transferring the solution into an adsorption column AC, centrifuging for 1min under the same eccentricity condition, discarding waste liquid, and then putting the centrifugal column back into a collecting pipe;
adding 700 mu L of rinsing liquid into the adsorption column, centrifuging at 10000rpm for 1min, discarding the waste liquid, and returning the centrifugal column into the collecting pipe;
fifthly, removing the rinsing liquid as much as possible, adding 500 mu L of the rinsing liquid into the adsorption column again, centrifuging for 1min under the same eccentricity condition, pouring the waste liquid, putting the centrifugal column back into the tube, and centrifuging for 1 min;
sixthly, replacing a clean collecting pipe, adding 30 mu L of LDEPC treatment water in the middle part of the adsorption film, centrifuging for 2min at 12000rpm, and collecting the solution;
seventhly, taking 4 mu L of recovered DNA for detection, using 5 mu L of recovered DNA for connection, and storing the DNA at the temperature of minus 20 ℃.
3) Cloning of the fragment of interest:
connecting a PCR product with a pUcm-T vector at 16 ℃ overnight, then mixing the PCR product with DH5 alpha competence uniformly, standing the mixture on ice for 30min, treating the mixture at 42 ℃ for 90s, and standing the mixture in ice bath for 5min quickly;
adding 1mLLB liquid culture medium, placing the centrifuge tube containing the bacterial liquid in a constant temperature shaking table, and shaking the shaking table at 37 ℃ and 200rpm for 1 h;
taking out the centrifugal tube from the constant-temperature shaking table, placing the centrifugal tube in a centrifugal machine, centrifuging for 5min at the room temperature of 4000rpm, removing 1mL of supernatant, and blowing and sucking the heavy suspension thalli by a gun head;
fourthly, coating a plate, and culturing for 16 hours in an incubator at 37 ℃;
screening blue and white spots, re-inoculating colonies, and carrying out positive detection on clones by using a vector primer M13-F/M13-R. The PCR system for screening clones was as follows:
| bacterial liquid | 16.5μL |
| 10×PCR buffer | 2.5μL |
| MgCl2(25mM) | 2.5μL |
| dNTP(10mM) | 1.0μL |
| M13-F | 1.0μL |
| M13-R | 1.0μL |
| Taq enzyme | 0.5μL |
And (5) sending the bacterial liquid to Shanghai biological engineering for sequencing.
As the length of the core fragment is only about 100bp and is not suitable for gel cutting purification, the PCR product is directly connected with pUcm-T vector and transformed into DH5 alpha competent bacteria. And (5) sequencing after culturing and screening.
(3) Obtaining a 3' end cDNA sequence:
1) 3' end RACE template cDNA preparation: specific primers 3 ' -GnRH-outter and 3 ' -GnRH-inner (see Table 1) for amplification of the RACE at the 3 ' end of the GnRH gene were designed using the known sequence of the gene of interest. The primers used were 3 ' RACE Outer Primer and 3 ' -GnRH-inner carried by SMARTTM RACE cDNAamplification kit manufactured by 3 ' -GnRH-outter and Clontech.
And (3) taking total RNA of sepiella maindroni brain tissue as a template to obtain template cDNA required by amplifying 3 'RACE fragments, and amplifying 3' RACE unknown fragments by utilizing nested PCR. The procedure was performed according to the kit provided instructions.
Adding the following reaction system into a 0.2mL centrifuge tube:
mixing the reaction system, centrifuging a little, placing the centrifugal tube in a PCR instrument, incubating for 2min at 70 ℃, and quickly moving to ice for standing for 2 min.
② adding the following reaction system into the reaction liquid in the first step 1):
| 5 Xfirst Strand buffer | 2.0μL |
| dNTP(10mM) | 1.0μL |
| DTT | 1.0μL |
| PowerScript Reverse Transcriptase | 1.0μL |
Mixing the reaction system, centrifuging a little, placing the centrifuge tube in a PCR instrument, incubating for 1.5h at 42 ℃, incubating for 7min at 70 ℃, and adding 100 mu L of sterilized water to dilute the product. The diluted template cDNA was stored at-20 ℃.
2) PCR amplification of 3' end RACE
And performing two rounds of PCR reactions by using a nested PCR method to amplify a sequence of a RACE fragment at the 3' end of the GnRH gene. The reaction system is as follows:
first reaction of nested PCR. The following reaction system was added to a 0.2mL centrifuge tube:
| sterilized water | 29.1μL |
| 10 × Advantage 2PCR buffer | 4.0μL |
| dNTP(10mM) | 0.8μL |
| 50×Advantage 2Polymerase Mix | 0.8μL |
| 3′RACE cDNA | 2.5μL |
| 3′-GnRH-outter | 5.0μL |
| 3′RACE Outer primer | 1.0μL |
Mixing the reaction system evenly, centrifuging a little, placing the centrifugal tube into a PCR instrument, and carrying out the reaction procedures of: 94 ℃ for 2 min; 5s at 94 ℃, 10s at 66 ℃ and 3min at 72 ℃ for 20 cycles; extension at 72 ℃ for 5 min.
② the amplification product of the first round 1) is used as template for this round. The second round of reaction system is as follows:
| sterilized water | 29.1μL |
| 10 × Advantage 2PCR buffer | 4.0μL |
| dNTP(10mM) | 2.5μL |
| 50×Advantage 2Polymerase Mix | 0.8μL |
| First round PCR product | 0.8μL |
| 3′-GnRH-inner | 5.0μL |
| 3′RACE Inner primer | 1.0μL |
Mixing the reaction system evenly, centrifuging a little, placing the centrifugal tube into a PCR instrument, and carrying out the reaction procedures of: 94 ℃ for 2 min; 5s at 94 ℃, 12s at 65 ℃, 3min at 72 ℃ and 20 cycles; 6min at 72 ℃. And (4) detecting the PCR product by electrophoresis.
Thirdly, the PCR product is subjected to gel cutting recovery and cloning connection, and the positive clone is sent to the Shanghai worker for sequencing.
(4) Obtaining a 5' end cDNA sequence:
1) preparation of 5' end RACE template cDNA: specific primers 5 ' -GnRH-outter and 5 ' -GnRH-inner (Table 1) for amplification of the 5 ' RACE of GnRH gene were designed using the known sequence of the gene of interest. The amplification of the RACE fragment at the 5 ' end of the GnRH gene was carried out using 5 ' -GnRH-OuterPrimer and 5 ' RACE Inner Primer carried by the 5 ' -GnRH-outter/5 ' -GnRH-Inner and SMARTTM RACE cDNAamplification kit manufactured by Clontech as primers.
After obtaining the template cDNA required for amplifying the 5 'RACE fragment by using the total RNA of the sepiella maindroni brain tissue as a template, the 5' RACE fragment is amplified by utilizing nested PCR. The procedure was performed according to the kit provided instructions.
Adding the following reaction system into a 0.2mL centrifuge tube:
| total RNA of | 1μg | |
| 5′RACE Adapter | 1μL | |
| DEPC treated Water | Make up to 15.5 mu L |
Mixing the reaction system, centrifuging a little, placing the centrifugal tube in a PCR instrument, incubating for 10min at 70 ℃, and quickly moving to ice for standing for 2 min.
Adding the following reaction system into the reaction solution obtained in the previous step 1):
| 10×PCR buffer | 2.5μL |
| MgCl2(25mM) | 2.5μL |
| dNTP(10mM) | 1.0μL |
| 0.1M DTT | 2.5μL |
mixing the reaction solution, slightly centrifuging, placing the centrifugal tube in PCR, and incubating for 1min at 42 ℃; adding 1 μ L SperScriptTM IIRT, incubating at 42 deg.C for 50min, and incubating at 70 deg.C for 15 min; mu.L of RNase mix was added and incubated at 37 ℃ for 30 min.
③ purifying the cDNA: adding 120 mu L of Solution I into the cDNA, transferring the mixed Solution into a centrifugal column, centrifuging for 30s under the condition of 10000rpm eccentricity, taking the centrifugal column, and sucking out liquid; adding 350 μ L Solution II, centrifuging at 12000rpm for 20s, removing supernatant, and repeating for 2 times; washing with 70% ethanol for 2 times; centrifuging at 12000rpm for 60s, and removing ethanol; changing the centrifuge tube, adding 50 mu L of sterilized water into the centrifugal column, and centrifuging for 60s under the same eccentricity condition to obtain a purified product.
Fourthly, tailing reaction, wherein the reaction system is as follows:
| DEPC treated Water | 6.5 |
| 5×Tailing Buffer | 5.0μL |
| 2mM dCTP | 2.5μL |
| Purified cDNA | 10.0μL |
Mixing the reaction system, centrifuging a little, placing the centrifugal tube in a PCR instrument, incubating for 3min at 94 ℃, and rapidly standing for 1min on ice; adding 1 mu L of TdT; incubating at 37 ℃ for 10min, and performing heat shock at 65 ℃ for TdT 10 min; the product was diluted by the addition of 100. mu.L of sterile water. The diluted template cDNA was stored at-20 ℃.
2) PCR amplification of 5' end RACE
First reaction of nested PCR. The following reaction system was added to a 0.2mL centrifuge tube:
| sterilized water | 31.5μL |
| 10×PCR buffer | 5.0μL |
| MgCl2(25mM) | 3.0μL |
| dNTP(10mM) | 1.0 |
| 5′RACE Outer primer | 2.0μL |
| 5′-GnRH-outter | 2.0μL |
| Tailed cDNA | 5.0μL |
| Taq enzyme | 0.5μL |
Mixing the reaction system evenly, centrifuging a little, placing the centrifugal tube into a PCR instrument, and carrying out the reaction procedures of: 94 ℃ for 2 min; 30s at 94 ℃, 35s at 62 ℃, 45s at 72 ℃ and 32 cycles; and 8min at 72 ℃.
② the amplification product of the first round 1) is used as template for this round. The second round of reaction system is as follows:
| sterilized water | 31.5μL |
| 10×PCR buffer | 5.0μL |
| MgCl2(25mM) | 3.0μL |
| dNTP(10mM) | 1.0 |
| 5′RACE Inner primer | 1.0μL |
| 5′-GnRH-inner | 1.0μL |
| First round PCR product | 5.0μL |
| Taq enzyme | 0.5μL |
Mixing the reaction system evenly, centrifuging a little, placing the centrifugal tube into a PCR instrument, and carrying out the reaction procedures of: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles; extension at 72 ℃ for 8 min. And (4) detecting the PCR product by electrophoresis.
Thirdly, the PCR product is subjected to gel cutting recovery and cloning connection, and the positive clone is sent to the Shanghai worker for sequencing.
(5) The sequence information analysis method comprises the following steps:
splicing the 3 'end, the 5' end and the core fragment sequence of the GnRH by using Lasergene software to obtain the cDNA full-length sequence of the Sepiella maindroni GnRH gene. On-line tools such as Predict Protein, Scratch Protein predictor, etc. can be used to Predict the Open Reading Frame (ORF) of GnRH gene and to Predict the amino acid sequence encoded by the ORF. And (3) performing signal peptide prediction on the amino acid sequence by using an online analysis site SignalP v 3.0. The amino acid sequence was subjected to blast (blastx and blastn) analysis using NCBI, and the amino acid sequence of GnRH and its homologous gene was subjected to alignment analysis using ClustalW 2. The estimation of the relative molecular mass isoisoelectric point of the GnRH precursor protein uses the online analysis software of Expasy-ProtParam. Construction of an NJ-based evolutionary tree was performed using MEGA v5.0 software, and repeated 1000 cycles.
And (3) finding a published homologous sequence comparison in NCBI, designing a degenerate primer for amplifying a core sequence by using a relative conserved region, and carrying out PCR amplification by using sepiella maindroni brain tissue cDNA as a template to obtain a fragment.
Specific primers are designed by using the detected cDNA core sequence of the Sepiella maindroni FMRF to amplify the 5 'end and 3' end non-coding region sequences, and bands of the amplified products in an imaging system after agar gel electrophoresis are shown in figures 2 and 3, wherein 5 'RACE products are 289bp bands, and 3' RACE products are 267bp bands.
The total length of the cDNA sequence of the Sepiella maindroni GnRH gene is 432bp, the open reading frame is 273bp, 90 coded amino acids are obtained, the 5 'non-coding region is 86bp, and the 3' non-coding region is 73 bp. The precursor protein has a predicted relative molecular mass of 10.1kDa and an isoelectric point pI of 7.73. According to the GnRH sequence of other 3 known cephalopods, the mature peptide product of Sepiella maindroni is a dodecapeptide like other 2 known cephalopods: pQNYHFSSNGWPPG (shown in FIG. 4). The prediction analysis of the signal peptide of the precursor protein amino acid sequence shows that the N end of the GnRH precursor protein contains a signal peptide with 31 amino acids in length, and the C end of the GnRH mature peptide sequence contains a shearing site and an auxiliary sequence with 44 amino acids in length, and the auxiliary sequence can be combined with the mature peptide to play a stabilizing role.
And acquiring GnRH gene sequences of other species from an NCBI database, and comparing the GnRH amino acid sequence of Sepiella maindroni with the homologous sequences of other species by using a ClustalW2 online analysis tool. And constructing an evolutionary tree of the selected homologous sequences by MEGA5.0 software. The results of homologous alignment show that the Sepiella maindroni GnRH is 90% similar to the Sepiella tigerina, 86% and 71% similar to the Sepiella jiangensis and the Sepiella maindroni GnRH, 47%, 41% and 31% similar to the pacific oyster, the Japanese scallop and the sea snail, and the mature peptide part in the sequence is very conservative.
E. Tissue-specific expression analysis of neuropeptides:
according to the division of the ovum generation stage and the ovary development stage, selecting Sepiella maindroni with healthy weight and strong activity in 3 different development stages of stage III, stage IV and stage V, wherein the weight is 500-1000g, quickly killing the Sepiella maindroni, immediately taking out various tissues and placing the tissues into RNA preservation solution for preservation at-80 ℃ for later use; tissues include, male squid: brain, optic lobe, liver, muscle, testis, heart, gill; female cuttlefish: brain, folium Camelliae sinensis, liver, muscle, ovary, heart, branchia, periwiny gland, and parawiny gland.
(1) Designing a primer: based on the full length of the cDNA sequence of the Sepiella maindroni GnRH gene, primers required by the fluorescent quantitative PCR operation of the GnRH gene are designed by using Primer 5.0 software and a Primer 3.0 online Primer searching tool. And (3) carrying out gradient PCR screening and sequencing on the primer, and finally determining that the primer can specifically amplify the Sepiella maindroni GnRH gene. The primers used in the fluorescent quantitative PCR operation of the GnRH gene are as follows:
| RT-GnRH-F | ACTTCCTTTACACTCGTCCCT |
| RT-GnRH-R | AGGGTGCCATCCATTGCTAA |
| RT-actin-F | TGAGAGGGAGATTGTGCGTG |
| RT-actin-R | GAACATAGATTCTGGAGCACGG |
(2) fluorescent quantitative PCR: the expression specificity of different tissues of GnRH gene of female and male Sepiella maindroni at three different development stages (stage III, stage IV and stage V, which are divided according to the stage of ovum generation and ovary development) is respectively detected by using a relative quantitative method. Sepiella maindroni beta-actin gene (JN564496.1) was used as an internal reference gene, and the gene expression level in the heart was used as a reference standard. Fluorescent quantitative PCR was performed according to the SYBR Premix Ex TaqTM II kit (Tli RNaseH plus) kit instructions.
Place the eight tubes on ice and add the following reaction:
| 2×SYBR Premix Ex TaqTMⅡ | 10.0μL |
| cDNA template | 0.8μL |
| Sterilized water | 7.2μL |
| RT-FMRF-F | 0.8μL |
| RT-FMRF-R | 0.8μL |
| ROX Reference DyeⅡ | 0.4μL |
The reaction system was mixed well, centrifuged slightly, and the eight tubes placed in an ABI 7500Real Time PCR instrument, set up the procedure as follows: 30s at 94 ℃; 5s at 94 ℃, 30s at 60 ℃ and 40 cycles; gradually heating to 55-95 deg.C for 2 min. 3 replicates were performed.
(3) Data processing: the standard curve, Ct value, etc. were automatically completed by ABI 7500Real Time PCR System Detection software of the ABI 7500Real Time PCR apparatus itself. The expression level of LFRFamide gene of different tissues is measured by using a 2-delta-Ct method. Analysis of variance was performed using the one way ANOVA function of the SPSS 18.0 statistical software, with multiple comparisons testing the significance of differences between groups using the Duncan method, P < 0.05 check for significance. Statistics and analysis of the data were performed using Excel 2010 software, and the software Sigma Plot 12.5 was used to make the relevant bar graph.
Taking tissues of female and male Sepiella maindroni, wherein the tissues of male Sepiella maindroni: brain, optic lobe, liver, muscle, testis, heart, gill; female cuttlefish: the GnRH gene expression was detected in brain, retinal, liver, muscle, ovary, heart, gill, perigonal, and paragonal tissues as shown in FIG. 5. The result of detecting the mRNA expression level of the Sepiella maindroni GnRH gene by fluorescence quantitative PCR shows that the GnRH gene is obviously expressed in the brain tissues of the Sepiella maindroni in three stages. In contrast, GnRH is expressed in minute amounts in the optic leaves in phase iii and in minute amounts in the female cuttlefish ovary, the perigonal gland and the paragonal gland in phase v. However, in adult squid, semi-quantitative PCR was used to detect the GnRH expression level in different tissues, but the results showed that the PCR product was amplified only in brain tissue, and the ovary of female squid, like other tissues, did not express GnRH. Adult sepiella maindroni and octopus ocellatus are also expressed only in the brain and ovary of female sepiella maindroni.
F. Neuropeptide brain tissue expression localization analysis:
(1) preparing a reagent:
1) 4% paraformaldehyde: 4g of paraformaldehyde is added into 100mL of deionized water and is placed at room temperature for 2 days, and the mixture is stored at 4 ℃ after the paraformaldehyde is completely dissolved and is used within 2 weeks;
2)0.2M EDTA: EDTA is added into the sterilized water according to the final concentration of 0.2M, and the pH is adjusted to 8.0;
3) PBS buffer: adding 0.2g of potassium chloride, 0.27g of monopotassium phosphate, 1.42g of sodium dihydrogen phosphate and 8g of sodium chloride into sterilized water, adjusting the pH value to 7.4, and fixing the volume to 1000 mL;
4) preparation of 0.1M Glycine/PBS solution: adding 7.5g of glycine into the 1LPBS solution, and shaking up;
5) preparation of 20 XSSC buffer: adding 175.2g of sodium chloride and 88.2g of sodium citrate into 800mL of deionized water, adjusting the pH value to be neutral, and metering the volume to 1000 mL;
(2) designing a primer:
based on the full length of the cDNA sequence of the Sepiella maindroni GnRH gene, primers required by GnRH in-situ hybridization are designed by using Primer 5.0 software and a Primer 3.0 online Primer searching tool. When designing a primer, a restriction enzyme cleavage site is screened in a GnRH gene cDNA sequence, and a cleavage site base is added to the 5' ends of the front and rear primers F/R. Performing gradient PCR screening and sequencing on the primers, and finally determining the primers capable of specifically amplifying the Sepiella maindroni GnRH gene: ISH-GnRH-F: CCGTCACGTCCATCATCAGGTAAACATC and ISH-GnRH-R: CCGGAAGGGAGTGATTTCCTCGTCTAAG are provided.
(3) Preparing a section:
preparing a paraffin section of the complete brain tissue of the sepiella maindroni:
1) fixing: soaking brain tissue of cuttlefish in 4% paraformaldehyde, and fixing at 4 deg.C for 16 hr; rinsing in PBS for 5 min;
2) and (3) dehydrating: dehydrating brain tissue in gradient staining jar containing 70%, 80%, 90% and 100% ethanol for 1 hr respectively, wherein the reagent can be replaced once;
3) and (3) transparency: brain tissue was transferred to xylene: soaking in 1:1 ethanol for 30min, and soaking in organic solvent xylene for 30min to make tissue transparent;
4) wax dipping: brain tissue was first transferred to paraffin: 1h in a ratio of 1:1, and then transferring the mixture into paraffin for 1 h;
5) embedding: preheating a metal box in advance, and putting brain tissues which are fully waxed into the metal box for paraffin embedding;
6) block repairing: repairing the embedded wax block;
7) slicing: placing the repaired wax block on a paraffin slicer to slice, and longitudinally cutting, wherein the slice thickness is 7 mu m;
8) unfolding and baking: spreading for 5min on a 42 ℃ film spreading machine; baking the slices on a 50 ℃ slice baking machine for 30 min; the dried paraffin slices can be stored in a refrigerator at the temperature of-20 ℃ for later use;
9) dewaxing: placing the cut brain tissue slices in xylene for dewaxing for 30min, and replacing the reagent once;
10) rehydration: rehydrating brain tissue in gradient staining jars filled with 100%, 90%, 80% and 70% ethanol for 10min respectively; rinse in PBS for 10min, during which time the reagents were changed once.
(4) In situ hybridization:
1) preparation of hybridization probe template: designing a primer ISH-GnRH-F, ISH-GnRH-R according to the cDNA sequence of GnRH, carrying out PCR amplification by taking cuttlefish brain tissue cDNA as a template, and preparing a PCR product used as an in situ hybridization probe template. Restriction enzymes AccI and Hind III were used in the preparation of the probes.
And (3) carrying out PCR amplification on the probe template, wherein the reaction system is as follows:
mixing the reaction system evenly, centrifuging a little, placing the centrifugal tube into a PCR instrument, and carrying out the reaction procedures of: 94 ℃ for 2 min; 94 ℃ for 20s, 64 ℃ for 40s, 10 cycles; annealing at 94 ℃ for 20s and 62 ℃ for 40s for 15 cycles; 10 cycles of 94 ℃ for 20s, 60 ℃ for 30s, 72 ℃ for 30 s; and 8min at 72 ℃. And detecting the product by electrophoresis.
2) Enzyme digestion, connection, transformation and screening of target fragments and vectors:
PCR products obtained by amplification are subjected to gel cutting and purification;
secondly, performing double enzyme digestion in a 0.2mL centrifuge tube by using restriction enzymes AccI and Hind III after purification, wherein the enzyme digestion system is as follows:
| PCR purified product | 5.0μL |
| 10×M Buffer | 1.0μL |
| AccⅠ | 1.0μL |
| HindⅢ | 1.0μL |
| Sterilized water | 12.0μL |
Uniformly mixing the reaction system, centrifuging a little, placing the centrifugal tube in a PCR instrument, and incubating for 8h at 37 ℃;
③ pSPT18 also adopts EcoRI and Hind III to carry out double enzyme digestion, the enzyme digestion system is the same as above;
the PCR product and the enzyme-cleaved pSPT18 were subjected to Gel cutting and purification again using E.Z.N.A.TM.gel Extraction Kit (50) Kit, and then ligated using Solution I manufactured by Takara, in the following manner:
GnRH sequence fragment 2.0 μ L
pSPT18 3.0μL
Sterilized water 5.0 μ L
Fifthly, transforming to DH5 alpha competence, coating a plate, selecting proper bacterial colony individually, and enlarging and culturing the thallus. Plasmids were extracted using a Plasmid extraction Kit D6942-01 type Plasmid Mini Kit I (100). Storing the extracted plasmid in a refrigerator at the temperature of 20 ℃ below zero, sending the plasmid to Shanghai workers for sequencing, and detecting the correctness of the insertion sequence;
sixthly, carrying out EcoRI single enzyme digestion on the correct recombinant plasmid. The single enzyme digestion reaction system is as follows:
| plasmids | 5.0μL |
| 10×H Buffer | 5.0μL |
| EcoRⅠ | 5.0μL |
| Sterilized water | 35.0μL |
Mixing the reaction system, centrifuging a little, placing the centrifugal tube in a PCR instrument, incubating for 5h at 37 ℃, re-extracting the product with phenol/chloroform, precipitating with ethanol, re-dissolving the precipitate in 15 mu L of DEPC treated water, and taking 2 mu L of electrophoresis for detection.
3) Preparing a probe: antisense probes were prepared using the DIG RNA Labeling kit SP6/T7 kit: adding the following reaction system into a 0.2mL centrifuge tube:
| treated plasmid | 2.0μL |
| 10×Transcription Buffer | 2.0μL |
| 10×DIG Labeling mixture | 2.0μL |
| RNA inhibitor(20U/μL) | 2.0μL |
| T7 RNA polymerase(20U/μL) | 2.0μL |
| DEPC treated Water | 10.0μL |
② uniformly mixing the reaction system, centrifuging a little, placing the centrifuge tube in a PCR instrument, incubating for 2h at 37 ℃, adding 2.0 μ L EDTA to terminate the reaction;
③ adding 75 mu L of precooled absolute ethyl alcohol into the centrifuge tube, uniformly mixing the reaction liquid, and standing for 3 hours at the temperature of minus 20 ℃;
fourthly, centrifuging for 15min at 4 ℃ and at 12,000rpm/min, and removing supernatant fluid;
adding 100 μ L of 70% ethanol, blowing and sucking with a gun head, centrifuging at 7500rpm/min at 4 deg.C for 5min, removing supernatant, adding 50 μ L of DEPC treated water, collecting 2 μ L for electrophoresis detection, and storing in a refrigerator at-20 deg.C;
the preparation system of the sense probe comprises the following steps:
| treated plasmid | 2.0μL |
| 10×Transcription Buffer | 2.0μL |
| 10×DIG Labeling mixture | 2.0μL |
| RNA inhibitor(20U/μL) | 2.0μL |
| SP6 RNA polymerase(20U/μL) | 2.0μL |
| DEPC treated Water | 10.0μL |
The preparation steps of the antisense probe are the same as those of the sense probe.
4) In situ hybridization:
pretreatment: immersing the paraffin section of the brain tissue into 4% paraformaldehyde for fixing for 10 min; rinsing with PBS for 10 min; 0.1M PBS/glycine for 5 min; 0.3% Triton X-100/PBS for 15 min; rinsing with PBS for 10 min;
permeation treatment: treating in protease K solution at 37 deg.C for 20 min; washing with 0.1M amino acid/PBS for 1 min; rinsing with PBS for 10 min; 0.1M triethanolamine (containing 0.25% ammonium acetate) for 10 min; finally rinsing for 10min by using a salt solution 2 XSSC;
③ prehybridization and hybridization: incubating the prehybridization solution for 1h at 45 ℃; incubating the hybridization solution for 6h at 46 ℃;
fourthly, post-treatment: after incubation of the hybridization solution, rinsing with 4 XSSC for 1 min; rinsing with 2 XSSC at 37 ℃ for 30 min; rinsing with 1 XSSC for 30 min;
antibody hybridization: blocking buffer solution is sealed for 1h at room temperature; AP-labeled anti-DIG antibody (1:500), and incubating for 1h at 37 ℃; then washing with PBS for 1 min;
sixthly, color development: adding a chromogenic substrate NBT/BCIP, and developing for 30min-24h in a dark place; washing with DEPC water for 3min, sealing with glycerol, taking pictures after two days, and storing.
The cellular localization of the expression site of Sepiella maindroni GnRH gene mRNA in brain tissue is carried out by the in situ hybridization method. As shown in FIG. 6, a clear signal for positive hybridization of GnRH gene mRNA was observed in a plurality of regions of the cuttlefish brain tissue, whereas no signal was observed in FIG. 6A in which hybridization was carried out with the sense probe. The positive hybridization signals were most intense in the supraesophageal nerve plexus, the subcrural lobe and the subvertical lobe (6B), and significant hybridization signals were observed in the medullary region of the entire subvertical lobe margin. Positive signals were also observed in the vertical leaf above the sub-vertical leaf (6B), but the signals were weaker. Clear positive signals were also observed in the anterior basal lobe (6C) and posterior basal lobe (6D). Hybridization signals obtained by hybridization staining with the antisense probe were observed in the lower frontal lobe (6C), but not in the upper frontal lobe (6C). The functional leaflets of the subgsophageal nerve group: the hybridization signals in the giant cell leaf, coat visceral leaf, pre-pigmented cell, foot leaf and wrist leaf (6E) are very dark colored and very strong. Clear hybridization positive signals were also observed in the medullary region of the optic leaf (6F), while no positive signals were observed in the cortical region.
Through in-situ hybridization of GnRH gene mRNA of Sepiella maindroni brain tissue, the GnRH gene is found to be expressed in 3 main components of Sepiella maindroni brain tissue. Positive hybridization signals of different intensities were observed in the subungual, perpendicular, sub-perpendicular, antero-basal, postero-basal and inferior frontal lobes of the brain of the supraesophageal nerve plexus. Effective positive hybridization signals were also observed in the giant cell leaf, the mantle visceral leaf, the anterior pigmented cell leaf, the foot leaf, the carpal nerve leaf, and the optic leaf of the subgsophageal nerve group.
The conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Zhejiang ocean university
<120> application of sepiella maindroni neuropeptide
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 432
<212> DNA
<213> Sepiella maindroni de Rochebrune)
<400> 1
acttccttta cactcgtccc tcgcctaaga caaaagaaca cttcaaatct ccatcatcag 60
ctaaacatct accagacagt gacatcatgt caacctccac agcctcgtcc agcctgagaa 120
gaatccaatt tttcacctgt gctattcttc ccctctcttt ctgcatgcat atccaggcac 180
agaattacca ttttagcaat ggatggcacc ccggtggtaa acgaagtgga cttccagaca 240
tgcagtgtca tttcagacca caaacaaaag ctacaatcga gaaactctta gacgaggaaa 300
tcacacgtat aattactaca tgtaccaata cagtcaatga catcgcagac ttgcagtaat 360
ttttcacagg tcaagcaaat tcactggata tacaactacc aaaaaaaaaa aaaaaaaaaa 420
aaaaaaaaaa aa 432
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cagacncaag cacaraayta 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tytctatcaa agcytttgt 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttaccaccag ggtgccatcc 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttgctaaaat ggtaattctg 20
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cacctgtgct attcttcccc tctctttc 28
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgcatatcca ggcacagaat tacca 25
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tgtaaaacga cggccagt 18
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caggaaacag ctatgacc 18
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggccaggcgt cgactagtac gggggggggg 30
<210> 11
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acuacuacua ggggcgtcga cgta 24
<210> 12
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
acuacuacua ggggcgtcga 20
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gcgagcacag aattaatatt tttttttttt 30
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gcggatccga attaatacga ct 22
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcgagcacag aattaatacg act 23
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acttccttta cactcgtccc t 21
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
agggtgccat ccattgctaa 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tgagagggag attgtgcgtg 20
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gaacatagat tctggagcac gg 22
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ccgtcacgtc catcatcagg taaacatc 28
<210> 21
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ccggaaggga gtgatttcct cgtctaag 28
<210> 22
<211> 12
<212> PRT
<213> Sepiella maindroni de Rochebrune)
<400> 22
Gln Asn Tyr His Phe Ser Asn Gly Trp His Pro Gly
1 5 10
Claims (1)
1. The application of sepiella maindroni neuropeptide is characterized in that: the sepiella maindroni neuropeptide is used for carrying out reproduction regulation by using neuropeptides and antagonists thereof in the fish culture process;
the gene cDNA complete sequence of the Sepiella maindroni neuropeptide GnRH is 432bp in length, an Open Reading Frame (ORF) is 273bp, 90 amino acids are coded, 5 '-UTR 86bp and 3' -UTR 73 bp; the ORF includes a segment of a signal peptide of 31 amino acids and a helper sequence of 44 amino acids, as well as a mature peptide of 12 amino acids: pQNYHFSN GWPPG; the MW of the precursor protein is 10.1kDa, and the isoelectric point pI is 7.73; the complete sequence of the cDNA of the gene of the Sepiella maindroni neuropeptide GnRH is shown in SEQ ID NO. 1;
the primer capable of specifically amplifying the Sepiella maindroni GnRH gene comprises the following components:
ISH-GnRH-F CCGTCACGTCCATCATCAGGTAAACATC；
ISH-GnRH-R CCGGAAGGGAGTGATTTCCTCGTCTAAG。
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| CN110438130B (en) * | 2019-08-15 | 2023-01-03 | 浙江海洋大学 | Sepiella maindroni TNF gene and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050143567A1 (en) * | 2000-06-30 | 2005-06-30 | Suntory Limited | Novel gonadotropin-releasing hormone, precursor peptides thereof and genes encoding the same |
| CN102627687A (en) * | 2012-03-23 | 2012-08-08 | 上海海洋大学 | Eriocheir sinensis GnRH analogue, and preparation method and application thereof |
| CN107586327A (en) * | 2017-11-02 | 2018-01-16 | 浙江海洋大学 | Tiger spot cuttlefish neuropeptide FMRFamide genital regulating agent |
| CN107586326A (en) * | 2017-11-02 | 2018-01-16 | 浙江海洋大学 | Tiger spot cuttlefish neuropeptide FMRFamide and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050143567A1 (en) * | 2000-06-30 | 2005-06-30 | Suntory Limited | Novel gonadotropin-releasing hormone, precursor peptides thereof and genes encoding the same |
| CN102627687A (en) * | 2012-03-23 | 2012-08-08 | 上海海洋大学 | Eriocheir sinensis GnRH analogue, and preparation method and application thereof |
| CN107586327A (en) * | 2017-11-02 | 2018-01-16 | 浙江海洋大学 | Tiger spot cuttlefish neuropeptide FMRFamide genital regulating agent |
| CN107586326A (en) * | 2017-11-02 | 2018-01-16 | 浙江海洋大学 | Tiger spot cuttlefish neuropeptide FMRFamide and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486825A (en) * | 2018-04-28 | 2019-03-19 | 浙江海洋大学 | Sepiella maindroni Spef1 and application thereof |
| CN109486825B (en) * | 2018-04-28 | 2022-07-29 | 浙江海洋大学 | Sepiella maindroni Spef1 and application thereof |
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Application publication date: 20210914 |