CN103834643A - Genetic marker of peptide tyrosine-tyrosine gene PYY3' UTR for regulating and controlling feed intake of pigs and application thereof - Google Patents

Abstract

The invention discloses a genetic marker of a peptide tyrosine-tyrosine gene PYY3' UTR for regulating and controlling the feed intake of pigs and application thereof. The nucleotide sequence of the genetic marker of the PYY3' UTR is shown as SEQ ID NO. 1, wherein three SNP (Single Nucleotide Polymorphism) loci, namely, bases positioned at the 137th bit, the 146th bit and the 169th bit of the sequence respectively, are included. PCR (Polymerase Chain Reaction) amplification is performed by taking pig genome DNA (Deoxyribonucleic Acid) containing the peptide tyrosine-tyrosine gene as a template and adopting a primer pair consisting of a forward primer, namely, 5'-CTGAGCCGCTACTACGC-3' and a reverse primer, namely, 5'-CTCCACAACCCCATCTT-3' to obtain the genetic marker shown as SEQ ID NO. 1. The functions of the PYY on the aspects of pig nutrition and breeding are compared and analyzed through the genetic marker of the PYY3' UTR in combination with the physiological features and economic traits of two pig breeds.

Description

Genetic marker and the application thereof of pig feed intake regulation and control junket junket peptide gene PYY3 ' UTR

Technical field

The present invention relates to belong to nutrition regulation and the Animal Genetics field of pig, refer to particularly genetic marker and the application thereof of a kind of pig feed intake regulation and control junket junket peptide gene PYY.

Background technology

(Peptide tyrosine-tyrosine is Peptide YY to junket junket peptide, be called for short PYY) be to be separated from pig ileal mucous membrane by Tatemoto etc. for 1981, the amino of this peptide and the amino acid of C-terminal are all tyrosine (Tyr), then must be called junket junket peptide, from then on start afterwards a burst of research boom.Junket junket peptide is the derivative hormone of enteron aisle, belong to pancreatic polypeptide family member, it is a kind of peptide matters with amidation C-terminal, for straight-chain polypeptide, within 1993, confirm that by Taylor its molecular structure is by being to have colonic secretory cell to be produced, also can also have existence at the neurone of central nervous system (hypothalamus, brain stem, medullary substance, pons and spinal cord) and peripheral nervous system by the secreted while of the endocrine cell of stomach and pancreas.

Early stage research finds that the rising of blood plasma level PYY has restraining effect to the secretion of gastrointestinal motility and digestion pancreatin after animal body is on the feed, thereby and then to a certain extent regulate intake control animal body body weight.Just find at Adrian TE in 1985 etc., PYY can suppress by the caused gastric acid secretion of pentagastrin, cholinergic agonist, vagus nerve stimulation and histamine and reduce pepsic content, can also increase the PYY concentration in blood pressure simultaneously.Nieuwwnhui zen in 1994 etc. find again, and in the experiment inside and outside body, PYY can directly act on together and reduce the secretion that cyclic monophosphate suppresses Regular Insulin.In mouse, research is found, PYY major part is from blood plasma, other synthesize in tongue epithelium gustatory cell, also appear in mouse and people's saliva, and PYY in the recycle system is a kind of apocleisis hormone being come by the secretion of enteric epithelium L-endocrine cell after searching for food, in order to reply a large amount of energy intake of body.PYY has important regulating and controlling effect in appetite with above body obesity thus, in mouse test, reducing after searching for food the release of PYY shows and whets the appetite and be negative correlation with obesity, and mouse performance is voracious to fat after disappearance PYY gene, the release of contrary slowly adjusting PYY can reduce obesity, and the transgenic mice that increases PYY has certain resistance to the obesity that has food consumption induction.In some structural researches, compared with other two kinds of pancreas polypeptide, their primary structure is extremely similar, but tertiary structure variant be the reason that causes biological activity special.In the recycle system, PYY is with PYY(1-36) and PYY(3-36) two kinds of forms exist, body is on the feed under state, PYY(3-36) be the principal mode in circulation, be about PYY(1-36) three times, and under fasting state PYY(3-36) be only 37%.

The adjusting of PYY to feeding behavior finally summed up in the point that in these effects, and the PYY content increase in saliva causes animal apocleisis, regulates slowly saliva PYY(3-36) can control that animal blindly searches for food and the sharp increase of body weight, obesity is had to good prophylactic effect.Whether PYY has other effects requires study.Recent research shows PYY and also have other more physiological function except gi tract being had to effect.Wherein vital effect is played in the non-coding region of PYY gene in post-transcriptional control.Nearest research is found, the intragentic expression of polymorphism regulation and control active somatic cell, the secretory volume of PYY and the generation of some metabolic syndromes of PYY gene 3 ' UTR district and nearly promoter region.

The acceptor of PYY refers to by G albumen coupling through 7 times across a film formed receptoroid.Up to the present the main species of finding has hypotype in 6 (Y1, Y2, Y3, Y4, Y5 and Y6).Wherein PYY(1-36) to the avidity of Y5 higher than PYY(3-36), and for Y2 PYY(3-36) it is highly had a preference for, the biological significance of Y6 is not illustrated yet, Y1 and Y3 are very little to the bonding force of PYY.In people and mice study, Y2 great expression is in the precursor cell substrate of tongue epithelium and Weir Von Ebner's glands body, and the effect of PYY is to realize in the expression of tongue epithelium by specificity Y2 acceptor.

Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to the sequence polymorphism being caused by single nucleotide diversity in genomic level, comprises insertion or the disappearance etc. of conversion, transversion and single base of single base.Be distributed widely in Animal genome, can occur in gene and compile district and non-coding region.SNP is the main existence form of life genetic material genovariation, and these SNPs are carried out to deep research important directive significance in the breeding process of pig.

MicroRNA (miRNA) is the small-sized non-coding RNA of a class, is made up of about 22 Nucleotide, and it is encoded by chromosomal DNA, in the function of post-transcriptional level performance regulate gene expression.Increasing research is found, miRNA has the biological functions such as regulation of embryonic development, cell proliferation, differentiation, apoptosis and tumour generation, miRNA is mainly combined by or incomplete base complementrity complete with 3 ' UTR of said target mrna, regulate the function of said target mrna to realize its negativity, and then the expression of regulatory gene.The specific combination site of miRNA on gene has the SNP of function, therefore miRNA calmodulin binding domain CaM 3 ' UTR single nucleotide polymorphism may have influence on the adjusting function of miRNA, due to the variation of base, affect the specific binding of miRNA and said target mrna, thereby affect target gene and protein expression.

The contribution of the appetite regulation and control of PYY to animal and body body-mass index is great for the Research Significance of the animal of economic type, verify the effect of pig PYY gene different characteristics to gene expression regulation in animal body by research, for molecule mechanism and the transcriptional control network analysis of pig appetite and obesity and metabolic syndrome.Also may find the new SNP site about pig economic characters, as effective new molecule marker.For some metabolic syndrome shapes, provide certain prevention theoretical basis simultaneously.In a word, PYY has important effect to zoologizeing the adjusting of control, energy metabolism and the body quality of body to food consumption.It is research object that pig is chosen in this experiment, and the single nucleotide polymorphism (SNP) that has function from the binding site of pig PYY gene 3 ' UTR region miRNA on gene is studied its regulating and controlling effect in body and the dependency of special construction and its biological action.

Eukaryotic gene expression regulation has important biological significance at transcriptional level, mainly to be subject to the location of mRNA and the regulation and control of selectivity translation, the regulating and controlling sequence that relates to this adjusting is clustered in non-translational region (the untranslated regions of transcript mostly, UTR) in, comprise 5 ' UTR and 3 ' UTR, wherein 3 ' UTR is even more important.3 ' UTR of eukaryote mRNA is the critical function element of its mRNA, play a part very important at aspects such as gene expression regulation and mRNA location, and sudden change in 3 ' UTR can also affect the expression of one or more gene and changes, thereby cause some pathologies of body.In forefathers' research, also find that the miRNA that compares 5 ' UTRHe CDS district about 53% in human genome has binding site and plays a role at 3 ' UTR, therefore, the research of PYY3 ' UTR is significant.

Summary of the invention

Technical problem to be solved by this invention is just to provide genetic marker and the application thereof of a kind of pig feed intake regulation and control junket junket peptide gene PYY, the present invention is by 3 ' UTR region of clone pig PYY gene, acquisition may be that nature is present in interracial SNP, thereby analyzes the effect of PYY aspect Animal nutrition and breeding.

For solving the problems of the technologies described above, its nucleotide sequence of genetic marker of a kind of pig feed intake regulation and control junket junket peptide gene PYY3 ' UTR provided by the invention is as shown in SEQ IDNO.1, wherein contain 3 SNP sites, lay respectively at the 137th, 146 and the base of 169 of this sequence.

The present invention also provides the primer pair for obtaining genetic marker, and described primer pair is:

Forward primer: 5'-CTGAGCCGCTACTACGC-3';

Reverse primer: 5'-CTCCACAACCCCATCTT-3'.

The present invention also provides the preparation method of genetic marker, taking the pig genomic dna with junket junket peptide gene as template, adopts following primer pair:

Forward primer: 5'-CTGAGCCGCTACTACGC-3';

Reverse primer: 5'-CTCCACAACCCCATCTT-3'.

Carry out pcr amplification, its genetic marker obtaining is SEQ IDNO.1.

Be reference sequences according to pig genome sequence PYY complete genome sequence in NCBI, design primer with Primerpremier5.0, complete after amplification, the purified rear clone order-checking of PCR product, and carry out sequential analysis, obtain the genome base sequence of 3 ' UTR district 222bp of PYY gene.The base sequence of acquisition is carried out to BLAST comparative analysis, obtain single nucleotide polymorphism (SNP) site between pig kind.

The present invention also provides the application of genetic marker in pig feed intake regulation and control.

The present invention also provide genetic marker at miRNA to the application in expression of target gene study on regulation.

The invention provides 3 ' the UTR district prediction of the miRNA of combination with it of pig PYY gene, also have the verification method of miRNA to expression of target gene regulation and control aspect effect, in accordance with the following steps:

(1) prediction of target gene miRNA.Adopt the method for information biology to carry out the prediction of miRNA binding site to base sequence;

(2) predict the outcome and SNP site that second step comparative analysis obtains according to above-mentioned, study emphatically the binding of SNP to miRNA and the regulating and controlling effect to target gene in the Seed Sequences of miRNA on its target gene.

Taking pig genomic dna as template, carry out respectively pcr amplification with Auele Specific Primer, PCR product reclaims and purifying, upper to T carrier (pmd-18T) through molecular cloning, and enzyme is cut and is run glue and reclaim.

(3) described to (2) product is connected with carrier (PmirGLO), By Transfecting Porcine nephrocyte after qualification is correct, measures its activity.

The principle of the invention

The present invention obtains the genetic marker sequence SEQ IDNO.1 of junket junket peptide gene sequence SEQ IDNO.2 and junket junket peptide gene PYY3 ' UTR by NCBI, genetic marker sequence comprises three SNP sites, wherein, the binding of the 146th SNP site and miRNA, thus research miRNA regulates and controls expression of target gene.

Beneficial effect of the present invention is:

The object of the invention is to 3 ' UTR region of clone pig PYY gene, find the difference of 3 ' UTR amplifying nucleic acid base between two kinds of different physiological characteristic pig kinds (Mei Shan and great Bai) by order-checking, acquisition may be that nature is present in interracial SNP.Further gather Mei Shan and Large White portion of tissue sample, do relative quantitative assay taking β-actin as internal reference, obtain respectively the express spectra of Mei Shan and Large White PYY gene, contact physiological characteristic and economic characters between two pig kinds, the effect of comparative analysis PYY aspect pig nutrition and breeding.

Brief description of the drawings

Fig. 1 is sequence comparison diagram;

Wherein Query1 is 3 ' UTR nucleotide sequence of plum mountain pig PYY gene, and Sbject1 is 3 ' UTR nucleotide sequence of Large White PYY gene.

Fig. 2 is the portion of tissue expression pattern analysis figure of pig PYY gene.

Fig. 3 is for organizing sample RNA electrophoresis detection figure.

Fig. 4 becomes cDNA detection figure for organizing sample to extract reverse transcription after RNA.M is DL2000maker, and 1,2,3,4,5,6,7 are and organize sample.

Fig. 5 is the measurement result figure of recombinant plasmid and miRNA mimic cotransfection luciferase relative reactivity.

Fig. 6 is pmirGLO carrier figure.

Embodiment

In order to explain better the present invention, further illustrate main contents of the present invention below in conjunction with specific embodiment, but content of the present invention is not only confined to following examples.

Embodiment 1: the preparation of gene downstream 3 ' UTR regional sequence

Select totally 57 of Large White and Mei Shan pigs, the PYY whole genome sequence providing according to NCBI is reference sequences, designs following primer, and product size is 892bp, and primer sequence is as follows:

Forward primer: 5'-CTGAGCCGCTACTACGC-3';

Reverse primer: 5'-CTCCACAACCCCATCTT-3'.

In Large White and Mei Shan pig genomic dna, carry out pcr amplification with above-mentioned primer, PCR reaction system is 20 μ l, wherein template DNA is 50ng, regular-PCR mix10 μ l(NovoStar Green Pcr Mix2x, NovoGene), every primer concentration is 10 μ mol, adds deionized water to cumulative volume 20 μ l; PCR response procedures: 95 DEG C of denaturation 3min; Then 95 DEG C of sex change 30s, 61 DEG C of annealing 30s, 72 DEG C of extensions 1min, totally 35 circulations; Last 72 DEG C are extended 8min, preserve 30min for 12 DEG C.

The PCR product of different pig kinds reclaims (reclaiming test kit specification sheets referring to Shanghai raw work SanPreP pillar DNA glue) purifying (being century Quick DNAPurification Kit specification sheets referring to health), Ke Longhou through gel glue, carry out sequencing, sequencing is completed by the prosperous biotech company of Beijing AudioCodes.Amplified production, after cloning and sequencing, adopts NCBI-BLAST to analyze and finds, in Mei Shan and great Bai, PYY gene 3 ' UTR region total length is all 222bp.Its sequence as shown in Figure 1.

Embodiment 2: Mei Shan and Large White PYY gene 3 ' UTR sequence SNP detect

Open information biology website NCBI(http: //www.ncbi.nlm.nih.gov/), click BLAST option, after entering BLAST, select nucleotide blast, eject the Standard Nucleotide BLAST page (http://blast.ncbi.nlm.nih.gov/Blast.cgi PROGRAM=blastn & PAGE_TY PE=BlastSearch & LINK_LOC=blasthome), select Align two or more sequences, respectively by two comparison frames of the PYY gene of two pig kinds that obtain in embodiment 13 ' UTR sequence input, in option, select Optimize for Highly similar sequences (megablast) and database to select Others (nr etc.), other parameter constants, selection result appears at new dialog box after setting completed, then click BLAST button, obtain the comparison diagram of two sequences, sectional drawing saving result after analyzing relatively.

Analytical results detects 8 variant sites altogether, is started to calculate by first of PYY gene 3 ' UTR, and making a variation more stable is the 137th (C → T), the 146th (C → T) and the 169th (C → T).Because plum mountain pig belongs to local variety, three places are VITAMIN B4 C; Large White is for cultivating pig kind, and under comparing, C/T gene frequency is respectively: 79.41%, 20.59%; 52.94%, 47.06%; 44.12%, 45.88%.Can obtain thus the 137th of 3 ' the UTR region sequence on mountain and Large White PYY gene after the rainy season and be partial to VITAMIN B4 C, other two position C/T are without base skewed popularity.Sequence alignment concrete outcome is shown in Fig. 1, and red frame shows SNP site.

Embodiment 3: Mei Shan and great Bai portion of tissue expression pattern analysis

1. design of primers

According to pig PYY gene order in NCBI, design quantitative primer according to the principle of quantitative analysis design of primers, ripe β-actin has been in reference gene choice experiment chamber, and primer sequence is specific as follows:

The quantitative primer of goal gene PYY

Forward primer: 5'-GGCAAGTCGTGGTAAAAGCG-3';

Reverse primer: 5'-AGAGTCTGGGGGGTGGTCA-3';

Reference gene β-actin primer

Forward primer 2:5'-CCAGGTCATCACCATCGG-3';

Reverse primer 2:5'-CCGTGTTGGCGTAGAGGT-3';

2. gather tissue sample, grind away

Instrument and reagent are prepared: scalpel, and operating scissors, tweezers, without RNA enzyme centrifuge tube, old lab-gown, mouth mask, gloves label paper, scotch tape, physiological saline, 75% alcohol, liquid nitrogen, ice, disk, mortar, tinfoil etc. are pressed consumption and are prepared.

Sampling: it is lethal first live pig to be butchered fast to bloodletting (can blood-sample withdrawal), rapidly whole pig is placed on ice as far as possible, going organizing of each several part first to put into physiological saline cleans, then frozen by organizing sample to shred to put into rapidly the centrifuge tube of mark to be positioned over rapidly liquid nitrogen container, after finishing, sample is taken out to be placed in-80 DEG C of refrigerators for subsequent use before liquid nitrogen is evaporated completely.Notice that the necessary processing rapidly of operating process is in case RNA degraded.

Grind away: take out from-80 DEG C of refrigerators the sample of adopting back and be placed in liquid nitrogen, the environment of a lower temperature poured liquid nitrogen into and makes it to form therein by the mortar of taking out precooling, under the prerequisite that has liquid nitrogen to exist, get 50～100mg homogeneous microstructure and grind evil spirit 1～2min, ground tissue juice is transferred to 15ml centrifuge tube, add the TRIZOL of 1ml when complete Deng liquid nitrogen volatilization, melt and mix with oscillator, then static 5min, is positioned over-80 DEG C of refrigerators for subsequent use after finishing.

3. extracting is organized RNA and is run glue and detect

From-80 DEG C of refrigerators get milled 2 organize sample, the TRIZOL of every use 1ml adds 0.2ml chloroform, concuss 15s, room temperature is placed 3min or ice bath 10min; 2～8 DEG C, the centrifugal 15min of 12000rpm, sample are divided into three layers: bottom is yellow organic phase, and middle layer is white, and upper strata is colourless water, RNA mainly in water volume be about 600 μ l; Water is transferred in new 1.5ml centrifuge tube and adds 500 μ l Virahols (precipitated rna), turn upside down 7～8 times, ice bath 10min; , now there is precipitation in 2～8 DEG C, the centrifugal 10min of 12000rpm, abandons supernatant; Add 1ml75% ethanol that RNA is suspended wherein, 2～8 DEG C, the centrifugal 5min of 8000rpm, abandon supernatant, and hyposynchronization can repeat twice; Room temperature is placed the dry RNA of 5～10min, adds 80 μ lDEPC water, inhales to beat RNA is dissolved with rifle head, and sealing mark, finally puts into RNA sample-80 DEG C of refrigerators temporary for subsequent use.

The sepharose of preparation 0.8%, gets 2 μ lRNA stostes, adds the loadingbuffer of 1 μ l, and after mixing, in point sample hole point sample electrophoresis detection, 28S and the clear explanation of 18S band RNA can use.Run glue and the results are shown in Fig. 3.

4.RNA surveys concentration, and reverse transcription becomes cDNA and detection

Measure RNA concentration: use Thermo NVNO Drop2000Spectrophotometer to measure RNA concentration and absorbancy 260/280=1.8～2.0 to determine RNA quality, after measured the RNA that carries all qualified, can do follow-up test.

CDNA is synthetic: test kit used is TaKaRa company rT reagent Kit with gDNA Eraser, operates in strict accordance with test kit specification sheets.Specifically be divided into: remove genomic dna reaction, reverse transcription reaction, concrete operations system and condition are in table 1 and table 2.

Table 1 is removed genomic dna reaction system

Note: reaction conditions is: 42 DEG C of 2min, remove gene DNA; 4 DEG C.

Table 2 reverse transcription reaction system

Note: reverse transcription reaction condition is: 37 DEG C, reverse 15min; 85 DEG C, 5sec carries out enzyme-deactivating;-20 DEG C of preservations.

CDNA detects: after cDNA is synthetic, utilize reference gene β-actin to carry out reverse transcription effect detection, whether the quantitative primer of reference gene is also overstated an intron according to quantitative design of primers principle, be also can detect to exist genomic dna to pollute like this at detection cDNA simultaneously.Reverse transcription reaction system and condition are in table 3.Configuration PCR reaction system, increases with BIO-RAD T100hermal cycler PCR instrument, is positioned over 4 DEG C after finishing, and system 2.0% sepharose runs electrophoresis detection, and the cDNA after the reverse transcription of result demonstration RNA sample can be used in follow-up test.Run glue and the results are shown in Fig. 4, M is 2000bp marker, the 1,2,3,4,5,6, the 7th, and the goal gene fragment of a cDNA sample amplification.

5.QRT-PCR relative quantification expression pattern analysis

According to the reaction system shown in table 3, configuration reaction solution, notices that quantitative MIX must pad pasting after wanting lucifuge, packing to complete while adding system, and ensures once to paste successfully.

Table 3PCR reaction system and reaction conditions

Note: reaction conditions is: 95 DEG C of denaturation 2min; 95 DEG C of sex change 10s, 62 DEG C of annealing 10s, 72 DEG C are extended 10s, 40 circulations; 72 DEG C are extended 10min; Preserve 5min for 25 DEG C.

As shown in Figure 2, PYY gene is obviously high than its hetero-organization at the expression amount of great Bai and Mei Shan tissue digestion road, heart and liver for result, and each organizes the expression amount of PYY all higher than Large White with regard to tissue expression plum mountain pig.

Embodiment 4: the structure that contains goal gene downstream 3 ' UTR sequence expression vector

1. design of primers

(be purchased from Promega according to pmirGLO carrier; as Fig. 6) multiple clone site figure and the feature of pig PYY gene 3 ' UTR sequence and consider that the technical qualification of order-checking company design primer; upstream and downstream primer 5 ' end has respectively SacI and XhoI restriction enzyme site; and adding respectively 1 or 2 protection bases in restriction enzyme site recognition sequence upstream, guarantee restriction enzyme site is not lost.Carry out PCR reaction taking Mei Shan and Large White genomic dna as template, the annealing temperature of amplification is 61 DEG C.Primer sequence is as follows:

Forward primer 1:5'-C gAGCTCgCAGAAGGCGCCTACCTATGG

Reverse primer 1:5'-CC cTCGAGgGCAGCCCTGGGGTTTGGTAAT

Forward primer 2:5'-C gAGCTCgCATCCCCCTCATCTCGATTCC

Reverse primer 2:5'-CC cTCGAGgGCTTCACCCCAACCCTGTCTT note: underscore place base represents restriction enzyme site.

2. double digestion

After will PCR product reclaiming purifying, carry out double digestion, it is as follows that enzyme is cut system: PCR purified product 30 μ L, FD buffer(Thermo Scientific) 4 μ L, 10U XhoI and SacI restriction endonuclease, add water to cumulative volume 40 μ L.37 DEG C of enzymes are cut the above rear electrophoresis of 2h and are reclaimed.Simultaneously, pmirGLO carrier is also carried out to enzyme to be cut, double digestion system cumulative volume is 20 μ L, wherein contain through plasmid extraction obtain pmirGLO carrier DNA 7 μ L(approximately 5 μ g), FD buffer(Thermo Scientific) 2 μ L, 5U Xho I and SacI restriction endonuclease, add water to cumulative volume 15 μ L.Put into constant incubator case, 37 DEG C of enzymes are cut the above rear electrophoresis of 2h and are reclaimed.For preventing self connecting of carrier, after recovery, build immediately recon, other is in-20 DEG C of preservations, and be generally no more than 2 months storage period.

3. connect

In ligation system, inserting DNA and carrier DNA is undertaken by the mole number of 5 ︰ 1, reaction cumulative volume is 10 μ L, wherein, 1 μ L10xLigase buffer, T4DNA ligase enzyme (Promega company) 1 μ L, the PCR double digestion purified product 5 μ L of each cloning vector plasmids, the enzyme of pmirGLO carrier is cut purified product 1 μ L, distilled water 2 μ L.Spend the night 4 DEG C of connections.Then be transformed into bacillus coli DH 5 alpha.

4. the prokaryotic expression of recombinant plasmid qualification

Picking transforms single bacterium colony of having grown, and is inoculated in the liquid LB substratum of Amp resistance (5～8mL), more than 37 DEG C on shaking table, 220r/min cultivates 12h.Draw 1 μ l bacterium liquid, carry out PCR qualification with original primer.

5. the extracting of plasmid and enzyme are cut qualification

Plasmid extraction is extracted test kit with the raw work SanPreP in Shanghai without intracellular toxin plasmid DNA sales volume, and main operational steps is as follows:

(1) while using for the first time this test kit, take out RNaseA (test kit is worn) from-20 DEG C and all join Buffer P1, after mixing, carry out mark and be placed in 4 DEG C of preservations.Other reagent of test kit is stored in room temperature;

(2) getting PCR is accredited as the positive about 5mL of bacterium liquid and is placed in 10mL centrifuge tube, the centrifugal 1min of 4000rpm under room temperature; Abandon supernatant.

(3) add 1ml Bacterial Endotoxin Erasol, gentleness mixes, and under room temperature, the centrifugal 1min of 12000rpm, abandons supernatant.

(4) repeating the 3rd step just can obtain without endotoxic thalline for 2～3 times.

(5) add 250 μ l Buffer P1, on vortex vibrator, vibration is until break up bacterial aggregate, add Buffer P2250 μ l, mix up and down gently and make the liquid shape that is translucent for 5-10 time, to under centrifuge tube room temperature, place 2～4min, add 350 μ l Buffer P3, upper and lower gentleness is put upside down centrifuge tube and is mixed for 5-10 time immediately, visible adularescent floss occurs, the centrifugal 5～10min of 12000xG under room temperature;

(6) from test kit, take out adsorption column post, by all careful adsorption column, centrifugal 30sec of 9000xGrpm under room temperature of moving into of supernatant liquor.Outwell the liquid in collection tube, adsorption column is put into same collection tube.

(7) in adsorption column, add 500 μ l Wash Solution, the centrifugal 30sec of 9000xG.Outwell the liquid in collection tube, adsorption column is put into same collection tube.

(8) repeating step (7) once.

(9) suction attached column and collection tube are put into whizzer, the centrifugal 1min of 9000xG.

(10) abandon collection tube, adsorption column is put into new 1.5ml centrifuge tube, add 45 μ l Elution Buffer to adsorption film central authorities, room temperature leaves standstill 1～2min, the centrifugal 1min of 9000xG.By obtained plasmid DNA solution be placed in-20 DEG C preserve or for follow-up test.(Elution Buffer is preheated to 60 DEG C can further improve yield)

After plasmid extraction, carry out concentration and purity testing and carry out double digestion qualification.Enzyme is cut result as shown in Figure 4, and enzyme is cut rear agarose electrophoresis and detected, in conjunction with PCR qualification result, and the exactness of sending bacterium liquid to check order to identify insertion sequence in recon, sequencing result shows that insertion sequence is aim sequence, and does not undergo mutation.

The foundation of embodiment 5:miRNA to expression of target gene study on regulation method

1. cell cultures

By porcine kidney cell (Porcine Kidney Cell, PK-15) 10% new-born calf serum containing dual anti-DMEM(Dulbecco ' s Modified Eagle ' s Medium) in substratum, its culture condition is 37 DEG C, 5% CO2.When cell is during in exponential phase of growth, the cultivation of going down to posterity, operate as follows: with twice of the quick washed cell of PBS, add 300 μ l pancreatin, 37 DEG C of about 3min of digestion, examine under a microscope cell and after glomeration, discard immediately pancreatin, in substratum, add cell growth medium, and it is existed with suction pipe piping and druming cell as far as possible with the form of individual cells.A cell suspension part for acquisition is stayed to the cultivation of going down to posterity in Tissue Culture Flask, and another part equal-volume divides and installs in 24 orifice plates, is placed in incubator and cultivates for transfection.

2. transient transfection

When cell in 24 orifice plates reach 40～50% converge rate time prepare transfection.Test adopts the method for expression vector and miRNA stand-in (mimic) cotransfection in cell, with NC(Negativecontrol) as internal reference, blank plasmid is in contrast.Every porocyte expression vector plasmid 0.2 μ g, miRNA mimic and NC mimic are 2 μ l, the every hole 2 μ l of LipofectamineTM2000 transfection reagent.Recombinant plasmid and pmirGLO carrier are carried out to concentration determination, by the different add-ons of calculating every porocyte of concentration.First prepare expression vector plasmid and add mimic liquid, two pig kinds totally four pipes, add a blank plasmid control group in addition, and every pipe adds 150 μ l opti-MEM substratum.(being referred to as above A liquid) adds every LipofectamineTM2000 transfection reagent hole 2 μ l in 150 μ L opti-MEM substratum, by hole summary preparation mixed solution.(being referred to as B liquid) above two kinds of liquid mix gently, add each A liquid to mix B liquid 150 μ l, then room temperature leave standstill 20min under room temperature after standing 5min.Get transfection liquid 100 μ L and add in one of them hole of 24 orifice plates, each like this recombinant plasmid can ensure to repeat for 3 times.After evenly mixing, put into incubator incubation, abandon liquid in clean 24 orifice plates after 6h, what add 10% new-born calf serum continues cultivation without dual anti-DMEM substratum.Collecting cell after 24h.

3. luciferase relative reactivity is measured and is analyzed

Use Dual-Luciferase report analysis system (Dual-Luciferase Reporter AssaySystem) to measure active, before mensuration, prepare following reagent: (1) 1 × PLB, 5 × PLB that test kit is provided mixes in 1 ︰ 4 ratios with aqua sterilisa, configuration before each use; (2) LAR II (Luciferase Assay Reagent II), the freeze-drying fluorometric analysis substrate that test kit is provided is resuspended in 10mL Luciferase Assay Buffer II, mixes, marks after packing and be placed in-70 DEG C and save backup; (3)

reagent, configuration before each use, by 50 × Stop &

substrate and Stop &

buffer is by the volume mixture of 1 ︰ 49.The complete laggard row uciferase activity of reagent configuration is measured, and operation steps is as follows:

(1) there is the attached cell 1 × PBS of target DNA to wash once transfection in above-mentioned 24 orifice plates, discard raffinate, add the freshly prepared 1 × PLB of 100 μ L, under room temperature, be placed on shaking table and vibrate 20min with lysing cell;

(2) lysate is collected to 200 μ l centrifuge tubes, carried out mark;

(3) every pipe is got 10 μ l and is added enzyme plate, all adds 50 μ L LAR II solution (70 DEG C of taking-ups, melt under room temperature), for subsequent use; (noting operation lucifuge)

(4) open Perkin Elmer2030Workstation power switch, open two fluorometric assay programs, select the hole count of measuring for the first time and preserve.Operation, fluorescence A value measured in record.Add again the freshly prepared Stop & of 50 μ L

reagent, after mixing, puts back in sample detection pond, operation, and fluorescence B value measured in record.Instrument just can calculate renilla luciferase vigor (internal reference) again, and calculates two kinds of luciferase vigor ratios, i.e. luciferase relative activities.Record result, then detect next sample with same method.Treat that whole mensuration finishes that result is inputted to Excel form and carries out data analysis, draw histogram.

Result as shown in Figure 5.At two expression vectors of 3 ' UTR sequence of constructed pig PYY gene, wherein the uciferase activity of ssc-miR-652mimic and negative control all reach significantly (P<0.05) or utmost point conspicuous level (P<0.01) in Large White, result shows that it has regulating and controlling effect to target gene, and with respect to plum mountain pig, it is expressed and significantly lowers (P<0.05).Ssc-miR-4339mimic uciferase activity and equal indifference of negative control in two groups in addition, result shows, it does not have large difference to the expression of PYY gene in two pig kinds.

Other unspecified part is prior art.Although above-described embodiment has been made detailed description to the present invention; but it is only the present invention's part embodiment; instead of whole embodiment, people can also obtain other embodiment according to the present embodiment under without creative prerequisite, and these embodiment belong to protection domain of the present invention.

Sequence table

 

Claims (5)

1. a genetic marker of pig feed intake regulation and control junket junket peptide gene PYY3 ' UTR, is characterized in that: its nucleotide sequence, as shown in SEQ IDNO.1, wherein contains 3 SNP sites, lays respectively at the 137th, 146 and the base of 169 of this sequence.

2. for obtaining the primer pair of genetic marker described in claim 1, it is characterized in that: described primer pair is:

Forward primer: 5'-CTGAGCCGCTACTACGC-3';

Reverse primer: 5'-CTCCACAACCCCATCTT-3'.

3. by the preparation method of the genetic marker described in claim 1 or 2, it is characterized in that: taking the pig genomic dna with junket junket peptide gene as template, adopt following primer pair:

Forward primer: 5'-CTGAGCCGCTACTACGC-3';

Reverse primer: 5'-CTCCACAACCCCATCTT-3'.

Carry out pcr amplification, its genetic marker obtaining is SEQ IDNO.1.

4. the application of genetic marker claimed in claim 1 in pig feed intake regulation and control.

Genetic marker described in claim 1 or 4 at miRNA to the application in expression of target gene study on regulation.

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