CN113186211A

CN113186211A - Pig ASMT gene and specific primer pair and detection kit thereof

Info

Publication number: CN113186211A
Application number: CN202110575590.0A
Authority: CN
Inventors: 刘国世; 李广栋; 张鲁; 朱天奇; 王立凯; 关盛宇; 于海颖; 马骁; 阎来庆
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2021-07-30

Abstract

The invention relates to an isolated porcine ASMT gene and corresponding expression vectors and host cells. The invention also relates to a specific primer pair and a detection kit aiming at the pig ASMT gene. The invention defines the sequence of the pig ASMT gene and provides a new reference for the biosynthesis pathway related to the gene and the research on the physiological and pathological aspects.

Description

Pig ASMT gene and specific primer pair and detection kit thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a separated pig ASMT gene, a corresponding expression vector and a host cell, and also relates to a specific primer pair and a detection kit aiming at the pig ASMT gene.

Background

Melatonin (Melatonin, MT, N-acetyl-5-methoxytryptamine) is also called "brain platinum", and is an important indole-like active small molecule substance synthesized and secreted by organs such as pineal bodies, ovaries and the like of mammals, and widely distributed in a plurality of tissues of animals. Melatonin is a very important endocrine hormone whose synthesis is inhibited by light and exhibits rhythmic oscillations of low day and high night, and is therefore also called "dark hormone". Melatonin has a variety of physiological and pharmacological functions, such as: improving sleep, regulating biological rhythm, relieving stress, regulating immune system, enhancing immunity, resisting tumor, and resisting aging. Melatonin and metabolites thereof are important broad-spectrum antioxidants, can remove oxygen Radical (ROS) in and out of an animal body, high-activity nitrogen Radical (RNS) and the like, and can activate the expression of intracellular antioxidant enzyme genes to further play an antioxidant function. In addition, melatonin can protect the stability of cellular DNA, reduce lipid damage, maintain mitochondrial function, promote expression of anti-apoptotic genes, and the like. Melatonin is also a key reproductive hormone, directly or indirectly regulates the secretion of the reproductive hormone, follicular development, embryonic development, implantation and the like in animal reproduction activities, and also participates in the regulation of the oestrus cycle of seasonal oestrous animals.

In mammals, the primary source of melatonin synthesis is L-trpophan (tryptophan). L-tryptophan undergoes a hydroxylation reaction catalyzed by TPH (tryptophane hydroxylase) to generate 5-Hydroxytryptophan (5-Hydroxytryptophan, 5-HTP), and then undergoes an AAAD (L-aromatic amino acid decarboxylase) decarboxylation reaction to be changed into 5-Hydroxytryptamine (5-Hydroxytryptamine, 5-HT), also called Serotonin (Serotonin). The 5-hydroxytryptamine generates N-Acetyl-5-hydroxytryptamine (NAS) under the action of AANAT (Arylalkylamine-N-acetylamine, Arylalkylamine N-Acetyltransferase). Finally, methylation is carried out under the catalytic action of ASMT (acetyl serotonin O-methyltransferase, acetyl-5-hydroxytryptamine-oxygen-methyltransferase) so as to generate the final melatonin.

China is the country with the largest stock of live pigs in the world, the healthy development of the pig raising industry and the stable supply of pork are closely related to the national civilian life, and the continuous development of experimental model pigs greatly promotes the research progress of biomedicine and medicines in China. However, for pigs with important edible and medical values, the inventor finds that the NCBI database does not have a pig ASMT gene, and supposes that the pig ASMT gene may be similar to mouse ASMT, and the high mutation rate of a complex region may cause that the pig ASMT gene is not successfully mined so far, which is also a main reason for the relevant research on the pig melatonin biosynthesis pathway to be stopped.

Disclosure of Invention

The invention aims to solve the problem that the ASMT gene information of pigs is disordered and unclear in the prior art, relocates and defines the ASMT gene of the pigs by using a bioinformatics method, and provides a new reference for further biological research and application of the gene.

The specific technical scheme is as follows:

in a first aspect, the present invention provides an isolated porcine ASMT gene having a nucleotide sequence as shown in SEQ ID No. 1.

In a second aspect, the invention provides a protein encoded by the porcine ASMT gene, which has the amino acid sequence shown in SEQ ID No. 2.

In a third aspect, the invention provides an expression vector for expressing a pig ASMT gene, which comprises a nucleotide sequence shown as SEQ ID NO. 1.

In a fourth aspect, the invention provides a host cell for expressing a pig ASMT gene, wherein the expression vector of the third aspect is exogenously transferred into the host cell. The host cell may be Escherichia coli or eukaryotic cell.

In a fifth aspect, the invention provides the isolated porcine ASMT gene, a protein encoded by the porcine ASMT gene, the expression vector or the host cell for use in physiological or pathological studies related to melatonin and/or metabolites thereof.

In a sixth aspect, the invention provides an application of the isolated pig ASMT gene, the protein coded by the pig ASMT gene, the expression vector or the host cell in preparing a medicament for preventing or treating melatonin and/or diseases related to metabolites thereof.

In a seventh aspect, the invention provides a primer pair specific to the pig ASMT gene of the first aspect.

As a specific embodiment, the sense strand of the primer has the nucleotide sequence shown by SEQ ID NO.3, and the antisense strand of the primer has the nucleotide sequence shown by SEQ ID NO. 4.

As a specific embodiment, the sense strand of the primer has the nucleotide sequence shown by SEQ ID NO.3, and the antisense strand of the primer has the nucleotide sequence shown by SEQ ID NO. 5.

A large number of experiments show that the primer pair has strong specificity on the pig ASMT gene, the amplification efficiency is high, and the working concentration of the primer is reasonable.

In an eighth aspect, the invention provides a kit for detecting pig ASMT gene, which comprises the specific primer pair described in the seventh aspect.

In a ninth aspect, the invention provides an application of the specific primer pair or the kit in pig ASMT gene detection.

The primer pair and the kit containing the specific primer pair provided by the invention can be used for quickly and accurately detecting the ASMT gene of the pig, and provide a convenient way for researching the relevant biosynthesis, biorhythm, reproductive biology and neuroendocrine of the ASMT gene of the pig.

In a tenth aspect, the present invention provides a method for detecting a porcine ASMT gene, the method comprising: and carrying out PCR amplification on the target gene by using the specific primer pair of the seventh aspect or the kit of the eighth aspect.

In the invention, when the target gene is preferentially amplified by PCR, a Slow-down PCR method aiming at cloning the gene with high GC content is adopted, and the working procedure of the PCR method is specifically as follows:

pre-denaturation at 98 ℃ for 2 min;

10s at 98 ℃, 30s at 72 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 71 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 70 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 69 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 68 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 67 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 66 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 65 ℃ and 45s at 72 ℃ for 4 cycles;

after the circulation is finished, the temperature is 72 ℃ for 10 min.

Compared with the prior art, the invention successfully locates the ASMT gene of the pig for the first time by utilizing a bioinformatics method, proves that the NCBI database machine annotation error is proved, and discovers a new transcript lacking the third last exon and the fourth last exon by molecular cloning, thereby having important scientific significance for the research of the functions, biological rhythms, reproductive biology and neuroendocrine of the ASMT gene of the pig and the large animal, and providing a new reference for the research related to the mammalian melatonin biosynthesis pathway.

Drawings

FIG. 1 shows the sequence analysis and molecular cloning of pig ASMT gene;

FIG. 2 shows the sequence alignment of pig ASMT gene clone.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Examples

Ovarian tissue from 3 adult large white pigs was harvested, and the harvested tissue was quickly placed into a 1.5ml EP tube (DEPC water treated) and placed into a liquid nitrogen tank, which was then stored in a freezer at-80 ℃.

1. Extraction of Total RNA

(1) Baking all glassware at high temperature for 6h before test, sterilizing, soaking all EP tubes and gun heads required for extracting RNA in 0.1% DEPC water for 12-18h, autoclaving at 121 deg.C for 15min, and oven drying at 60 deg.C in vacuum drying oven. Of particular note are: all steps of RNA extraction operation are carried out in a special RNA working chamber and a special RNA superclean working platform, all liquid-transferring guns, gun heads and the like used in the experiment are special for RNA, and 0.1% DEPC water is required for solution preparation; (2) opening the low-temperature high-speed centrifuge in advance to pre-cool the centrifuge at 4 ℃; preparing 75% ethanol for washing RNA by DEPC water, and pre-cooling in a refrigerator at 4 ℃; (3) samples were removed from the-80 ℃ freezer and weighed quickly (approximately 0.05-0.1g) and subjected to tissue grinding using a liquid nitrogen pre-cooled mortar. Liquid nitrogen was added continuously and intermittently during the milling process until all the clumps were ground to a powder. Then using a medicine spoon to grind the tissue; transferring the powder to a 1.5ml centrifuge tube (the process requires rapidness and accuracy), adding 1ml Ttrizol, and shaking for 2-3 min to fully crack; (4) respectively adding 200 mu L of chloroform into each sample, shaking violently and uniformly, standing at room temperature for 10min, centrifuging at 12000r/min at low temperature for 15min, and sucking supernatant at 4 ℃; (5) adding isopropanol with the same volume, repeatedly reversing and mixing uniformly, standing at room temperature for 10min, centrifuging at 12000r/min for 15min, and removing supernatant, wherein precipitate is visible at the bottom of the tube; (6) adding 500 μ L75% precooled alcohol, carefully cleaning the precipitate, centrifuging at 7500r/min for 5min, and removing the ethanol; (7) repeating the step (6) once; (8) placing the precipitate in a ventilated place, volatilizing excessive alcohol for 12min until the precipitate changes from white to near transparent; the precipitate was dissolved in 30. mu.L of DEPC water and then the total RNA purity was checked by NanoDrop.

Reverse transcription of RNA

Removing genome from RNA, taking 2 μ l of 5 XgDNA Eraser, 1 μ l of gDNA Eraser Buffer, 1 μ l of DEPC water and 6 μ l of RNA, putting the system into a PCR instrument, and reacting for 2min at 42 ℃; then add 4. mu.l

Buffer 2、1μl

RT Enzyme Mix I, 1. mu.l RT Primer Mix, 4. mu.l DEPC water, reacted in PCR instrument at 37 ℃ for 20min, 85 ℃ for 5s to obtain cDNA, and stored at-20 ℃. Kit PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) was purchased from Takara.

3. Design and Synthesis of primers

Primers were designed using Primer Premier 6.0 and Oligo 7.0 software, with the parameters set for screening primers between 20-30bp in length, between 60-75% GC content, Tm >60 ℃ and Tm Difference <1 ℃. The upstream and downstream primers span the CDS region. The primers were synthesized by Beijing Biotechnology Ltd.

The specific information is as follows:

the pig ASMT gene (SEQ ID NO.1) has the following specific sequence:

GGTGATCCCCGAGGCCAGGACCGCTGGGGTCCCCCGAGCTGGGCCAGCCCACAGGGAAAATGGGTTCCCCGCATGATCAGGCCTACCGTCTCCTGAAGGAATATTCCAACGGCTTCATGGTCTCCCAGGTTCTCTTCGCTGCCTGTGAGCTGGGCGTGTTTGACCTTCTGGCCGAGGCCCCGGGGCCCCTGGGCTCGGCCGCAGTGGCCGCACATCTGGGCATCAGCTGCCGGGGGACGGAGCAACTGCTGGATGCCTGTGTGCTCCTGAAGCTGCTTCATGTGGAAATGAGGAGAGGAGAAGCTGTGTACGCCAACACCGAGCTGGCCAGCGCCTACCTGGCCGGGACAAGCCCCACGTCCCAGCAGCACATGCTGCTCTACGTGGCCAGGACCACCTACCTGTGCTGGGCCCACCTGGCCGAGGCCGTGAGGGAAGGGAAGAACCAGTATCTGAAGACGTTCGGGGTTCCCTCTGAGGAGCTCTTCAGCGCCATCTACAGGAGATTTCTTTAAAGACCCCCTCCCGGAGGCGGACCTGTACCTGCTGGCCAGGGTCCTGCACGACTGGACGGACGACAAGTGCTCCCGCCTGCTGGCCAGAATCCACGGCGCCTGCAGGACAGGCGGTGGCATCCTGGTCATTGAGAGCCTCCTGGATGCCGATGGGCGGGGCCCCCTGACCACACAGCTCTACTCGCTCAACATGCTCGTGCAGACCGAGGGCCGCGAGCGGACCCCCGCCCAGTACCGCGCACTCCTGGCCCCCGCTGGCTTCCACGACATCCAGTGCCGCAGAACCGGGGGCACCTACGACGCCATCCTGGCCAGGAAGTGACCCCCAGCGTCATCTGAGACCCCTCACGGACTGCCCTCCAGAGGCCGTGTCTGGCACATTTCCTGGTTTTGTGCCCGGAGA

the amino acid sequence (SEQ ID NO.2) coded by the pig ASMT gene has the following specific sequence:

MGSPHDQAYRLLKEYSNGFMVSQVLFAACELGVFDLLAEAPGPLGSAAVAAHLGISCRGTEQLLDACVLLKLLHVEMRRGEAVYANTELASAYLAGTSPTSQQHMLLYVARTTYLCWAHLAEAVREGKNQYLKTFGVPSEELFSAIYRRFL

the sense strand (SEQ ID NO.3) of the primer pair 1 has the following specific sequence:

5'-CCCCAGTTCCCGCACAC-3'；

the antisense strand (SEQ ID NO.4) of primer pair 1 has the following specific sequence:

5'-CAGAAGCTCAGCATCGCTCT-3'。

the amplification product of primer pair 1 was 1214 bp.

The sense strand (SEQ ID NO.3) of primer pair 2 has the following specific sequence:

5'-CCCCAGTTCCCGCACAC-3'；

the antisense strand (SEQ ID NO.5) of primer pair 2 has the following specific sequence:

5'-GACCCCTCACTTCATCACATGCAA-3'。

the amplification product of primer pair 2 was 1351 bp.

According to the invention, a plurality of groups of primer pairs are designed, and a large number of experiments show that the two primer pairs have strong specificity on pig ASMT genes, high amplification efficiency and reasonable working concentration of the primers, and particularly have excellent amplification effect on a Slow-down PCR method (see Chinese patent ZL201811496751.1) for cloning genes with high GC content.

4.RT-PCR

The amplification system is as follows: 3% DMSO, high fidelity DNA Polymerase PrimeSTAR MAX DNA Polymerase (R045A, Takara) 0.5. mu.l, dNTPmix (2.5mM each) 25. mu.l, GC Buffer II (RR02AG, Takara) 8. mu.l, Forward primer 1. mu.l, Reverse primer 1. mu.l, cDNA 2. mu.l, 11.5. mu. l H₂And O. The PCR products were electrophoretically detected using a 1.5% agarose gel.

Pig LOC110258194(ASMT) gene sequence information is shown in figure 1A. The present invention designed multiple sets of primers spanning the CDS region, and failed amplification by ordinary PCR (FIG. 1C, with two failed sets of first and second lanes as examples). The structure of the pig ASMT gene is analyzed by Editseq software, and the GC content of the pig ASMT gene is higher (64.74%, shown in figure 1B). Then, we used the existing Slow-down PCR method (see chinese patent ZL201811496751.1) for cloning high GC content genes to screen out two pairs of primer pairs with strong specificity and high efficiency (i.e. primer pair 1 and primer pair 2 above) and successfully amplify the target genes (fig. 1C, third and fourth lanes, PCR product size is about 1250 bp).

The working procedure of the Slow-down PCR method is specifically as follows:

pre-denaturation at 98 ℃ for 2 min;

10s at 98 ℃, 30s at 72 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 71 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 70 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 69 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 68 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 67 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 66 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 65 ℃ and 45s at 72 ℃ for 4 cycles;

after the circulation is finished, the temperature is 72 ℃ for 10 min.

The cloned sequence is subjected to Sanger sequencing (a peak diagram is shown in figure 2B), then is compared with a target sequence, the comparison result of DNAman software is 98.80% (figure 2A), a segment is found to be deleted in the middle of the sequence, and then the SnapGene software is used for comparison, and the result shows that the cloned sequence is deleted for the third last and fourth exons (figure 2C), namely a new transcript.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> university of agriculture in China

<120> pig ASMT gene and specific primer pair and detection kit thereof

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ggtgatcccc gaggccagga ccgctggggt cccccgagct gggccagccc acagggaaaa 60

tgggttcccc gcatgatcag gcctaccgtc tcctgaagga atattccaac ggcttcatgg 120

tctcccaggt tctcttcgct gcctgtgagc tgggcgtgtt tgaccttctg gccgaggccc 180

cggggcccct gggctcggcc gcagtggccg cacatctggg catcagctgc cgggggacgg 240

agcaactgct ggatgcctgt gtgctcctga agctgcttca tgtggaaatg aggagaggag 300

aagctgtgta cgccaacacc gagctggcca gcgcctacct ggccgggaca agccccacgt 360

cccagcagca catgctgctc tacgtggcca ggaccaccta cctgtgctgg gcccacctgg 420

ccgaggccgt gagggaaggg aagaaccagt atctgaagac gttcggggtt ccctctgagg 480

agctcttcag cgccatctac aggagatttc tttaaagacc ccctcccgga ggcggacctg 540

tacctgctgg ccagggtcct gcacgactgg acggacgaca agtgctcccg cctgctggcc 600

agaatccacg gcgcctgcag gacaggcggt ggcatcctgg tcattgagag cctcctggat 660

gccgatgggc ggggccccct gaccacacag ctctactcgc tcaacatgct cgtgcagacc 720

gagggccgcg agcggacccc cgcccagtac cgcgcactcc tggcccccgc tggcttccac 780

gacatccagt gccgcagaac cgggggcacc tacgacgcca tcctggccag gaagtgaccc 840

ccagcgtcat ctgagacccc tcacggactg ccctccagag gccgtgtctg gcacatttcc 900

tggttttgtg cccggaga 918

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Met Gly Ser Pro His Asp Gln Ala Tyr Arg Leu Leu Lys Glu Tyr Ser

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Asn Gly Phe Met Val Ser Gln Val Leu Phe Ala Ala Cys Glu Leu Gly

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Val Phe Asp Leu Leu Ala Glu Ala Pro Gly Pro Leu Gly Ser Ala Ala

35 40 45

Val Ala Ala His Leu Gly Ile Ser Cys Arg Gly Thr Glu Gln Leu Leu

50 55 60

Asp Ala Cys Val Leu Leu Lys Leu Leu His Val Glu Met Arg Arg Gly

65 70 75 80

Glu Ala Val Tyr Ala Asn Thr Glu Leu Ala Ser Ala Tyr Leu Ala Gly

85 90 95

Thr Ser Pro Thr Ser Gln Gln His Met Leu Leu Tyr Val Ala Arg Thr

100 105 110

Thr Tyr Leu Cys Trp Ala His Leu Ala Glu Ala Val Arg Glu Gly Lys

115 120 125

Asn Gln Tyr Leu Lys Thr Phe Gly Val Pro Ser Glu Glu Leu Phe Ser

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Ala Ile Tyr Arg Arg Phe Leu

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Claims

1. The separated pig ASMT gene is characterized by having a nucleotide sequence shown as SEQ ID NO. 1.

2. The protein coded by the pig ASMT gene is characterized by having an amino acid sequence shown as SEQ ID NO. 2.

3. An expression vector for expressing a pig ASMT gene is characterized by comprising a nucleotide sequence shown as SEQ ID NO. 1.

4. A host cell expressing a porcine ASMT gene, wherein the expression vector of claim 3 is exogenously introduced into the host cell;

preferably, the host cell is escherichia coli or a eukaryotic cell.

5. Use of the isolated porcine ASMT gene according to claim 1, the protein according to claim 2, the expression vector according to claim 3 or the host cell according to claim 4 for physiological or pathological studies related to melatonin and/or its metabolites.

6. Use of the isolated porcine ASMT gene according to claim 1, the protein according to claim 2, the expression vector according to claim 3, or the host cell according to claim 4 for the preparation of a medicament for the prevention or treatment of a disease associated with melatonin and/or a metabolite thereof.

7. The specific primer pair of the pig ASMT gene is characterized in that a sense strand of the primer has a nucleotide sequence shown in SEQ ID NO.3, and an antisense strand of the primer has a nucleotide sequence shown in SEQ ID NO. 4;

or, the sense strand of the primer has the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand of the primer has the nucleotide sequence shown in SEQ ID NO. 5.

8. A kit for detecting a pig ASMT gene, which comprises the specific primer pair of claim 7.

9. Use of the specific primer pair of claim 7 or the kit of claim 8 for the detection of pig ASMT gene.

10. A method of detecting a porcine ASMT gene, comprising: performing PCR amplification on a target gene by using the specific primer pair of claim 7 or the kit of claim 8;

preferably, the working procedure of the PCR amplification is specifically:

pre-denaturation at 98 ℃ for 2 min;

10s at 98 ℃, 30s at 72 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 71 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 70 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 69 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 68 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 67 ℃, 45s at 72 ℃ and 4 cycles;

10s at 98 ℃, 30s at 66 ℃ and 45s at 72 ℃ for 4 cycles;

10s at 98 ℃, 30s at 65 ℃ and 45s at 72 ℃ for 4 cycles;

after the circulation is finished, the temperature is 72 ℃ for 10 min.