CN103555728A - Porcine transcription factor coup-tf1, its recombinant vector and application - Google Patents
Porcine transcription factor coup-tf1, its recombinant vector and application Download PDFInfo
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Abstract
The invention relates to a porcine transcription factor coup-tf1, its recombinant vector and application. The nucleotide sequence of the porcine transcription factor coup-tf1 is shown as SEQ ID NO.19. Overexpression of the porcine transcription factor coup-tf1 can reduce the expression of CYP11A1, CYP17A1 and StAR genes, lower the androstenone content in pork, inhibit the accumulation of odor substance, and ultimately improve the quality of the pork.
Description
Technical field
The invention belongs to genetically engineered field, particularly a boar transcription factor coup-tf1, also relates to the recombinant vectors and the application that contain pig transcription factor coup-tf1.
Background technology
Pig industry is the autonomous industry of China's livestock industry, and pork is the main animal food of population of China.Compare with castration boar, full boar not the comprehensive productivity effect of castration boar will improve 30%, have significant society and economy and be worth, but the existence of boar smell of mutton makes it be difficult to be accepted by human consumer, eliminating or reducing boar smell of mutton is to promote full boar to raise the problem that must solve.Research shows, boar smell of mutton is mainly derived from two kinds of materials that are deposited in pork adipose tissue: skatole and androsterone (rare-3 α-one of 5 α-steroid-16-), wherein androsterone is the synthetic steroidal compounds of testis tissue mesenchymal cell, there is piss smell, androsterone is discharged in blood and laggard people's sialisterium and fatty tissue by spermatic vein, and metabolism in liver, through urine excretion; Skatole is that daily ration and endogenous protein decompose that the tryptophane producing is degraded under the effect of large intestine anaerobion and the aromatic essence that produces has excrement stink; Skatole is through intestines wall absorbed into serum, mainly through liver metabolism by urinating discharge, another part is stored in fatty tissue.Therefore, the concentration of reduction androsterone and skatole can reach the object of eliminating smell of mutton.
Summary of the invention
One of object of the present invention is to provide a boar transcription factor coup-tf1; Two of object of the present invention is to provide the recombinant vectors that contains pig transcription factor coup-tf1; Three of object of the present invention is to provide the application of pig transcription factor coup-tf1 in reducing pork smell of mutton; Four of object of the present invention is to provide the application of pig transcription factor coup-tf1 in suppressing CYP11A1 genetic expression; Five of object of the present invention is to provide the application of pig transcription factor coup-tf1 in suppressing CYP17A1 genetic expression; Six of object of the present invention is to provide the application of pig transcription factor coup-tf1 in suppressing StAR genetic expression; Seven of object of the present invention is to provide the application of pig transcription factor coup-tf1 in reducing androsterone content.
1. pig transcription factor coup-tf1, is characterized in that, the nucleotide sequence of described pig transcription factor coup-tf1 is as shown in SEQ ID NO.19.
2. the recombinant vectors that contains pig transcription factor coup-tf1 described in claim 1.
Preferably, described recombinant vectors contains cell screening gene neo.
3. the application of pig transcription factor coup-tf1 in reducing pork smell of mutton described in.
4. the application of pig transcription factor coup-tf1 in suppressing CYP11A1 genetic expression described in.
5. the application of pig transcription factor coup-tf1 in suppressing CYP17A1 genetic expression described in.
6. the application of pig transcription factor coup-tf1 in suppressing StAR genetic expression described in.
7. the application of pig transcription factor coup-tf1 in suppressing androsterone content described in.
Beneficial effect of the present invention is: the invention discloses pig transcription factor coup-tf1, and by overexpression pig transcription factor coup-tf1, regulation and control CYP17 and STAR genetic expression, thus directly or indirectly the interior androsterone of negative regulation pig body is synthetic; Can also regulate skatole main metabolic enzyme CYP450, the degraded of regulation and control skatole simultaneously; Because the expression of COUP-TF is played negative regulation effect to the metabolism of androsterone, further weakened the restraining effect of androsterone to skatole metabolism simultaneously, and the collaborative concentration of skatole in pig body that reduces; Utilize pig transcription factor coup-tf1 disclosed by the invention can reduce pork smell of mutton, can or regulate COUP-TF I to obtain the pig genetic strain of low smell of mutton for seed selection, there is good application prospect.
Accompanying drawing explanation
Fig. 1 is for the total RNA(1 of the fragrant porcine tissue of bar horse, 2 is for clinging to horse testis RNA; 3,4 is liver rna).
Fig. 2 is the expression of main smell of mutton gene in different pig kind livers.
Fig. 3 is the expression of main smell of mutton gene in different pig kind testis.
Fig. 4 is the color atlas of 100ng/mL skatole.
Fig. 5 is skatole typical curve.
Fig. 6 is that (the A:4 monthly age is clung to horse pig muscle skatole color atlas for 4 monthly ages bar horse pig muscles and blood plasma skatole color atlas; The B:4 monthly age is clung to horse porcine blood plasma skatole color atlas).
Fig. 7 is the typical curve that androsterone is measured.
Fig. 8 be do not sieve at 1 monthly age and 6 monthly ages the state of culture method institute cultured cells after 3 β-HSD dyeing (magnification: 400 *, the A:6 monthly age culture method that do not sieve; The B:1 monthly age culture method that do not sieve)
Fig. 9 is the total RNA agarose gel electrophoresis of pig liver figure.
Figure 10 is COUP-TF I PCR product agarose gel electrophoresis figure.
Figure 11 is pEGFP-C1 restriction enzyme digestion and electrophoresis figure (M2: λ-Hind III digest 1:Vector DNA).
Figure 12 is for take the pcr amplification result figure (M1:DL2000 DNA Marker 1:PCR amplified production) that COUP-TF I-pMD19-T carries out as template.
Figure 13 is that (M:DL1,000DNA Marker are identified in the pig testis mesenchymal cell plasmid cracking of 1 monthly age of transient transfection; 1: blank; 2: transfection pEGFP-C1 plasmid; 3: transfection COUP-TF I-neo recombinant plasmid).
Figure 14 is that (M:DL1000 DNA Marker is identified in the pig testis mesenchymal cell plasmid cracking of 6 monthly age of transient transfection; 1: blank; 2: transfection pEGFP-C1 plasmid; 3: transfection COUP-TF I-neo recombinant plasmid).
Figure 15 is cell total rna electrophorogram cell total rna electrophorogram (the total RNA electrophorogram of A:1 monthly age interstitial glands; The total RNA electrophorogram of B:6 monthly age interstitial glands; 1: transfection COUP-TF I-neo recombinant plasmid; 2: transfection pEGFP-C1 plasmid; 3: blank).
Figure 16 is 1 monthly age of transient transfection pig testis mesenchymal cell gene relative expression quantity analytical results.
Figure 17 is the gene relative expression quantity analytical results of 6 monthly age of transient transfection pig testis mesenchymal cell.
Figure 18 is gene relative expression quantity analytical results after 1 monthly age and 6 monthly ages transfection COUP-TF I-neo.
Figure 19 is 1 monthly age of transfection COUP-TF I-neo recombinant plasmid and the gene relative expression quantity analytical results of 6 monthly age interstitial glandses.
Embodiment
Hereinafter with reference to embodiment, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition or the condition of advising according to manufacturer.
The present invention's experiment material used is commercially available prod if no special instructions.
At present, the research that improves boar smell of mutton is mainly to carry out marker assisted selection research for the fatty deposits level of androsterone and skatole, the one, and quantitative trait locus (quantitative trait locus, QTL) Position Research; The 2nd, candidate gene research.Androsterone and skatole are the compounds that causes pork smell of mutton, and both depositions in fatty tissue depend on its synthetic and catabolism level in pig body.CYP11A1 has been found in research, CYB5A, CYP17, the oligogene of the synthetic and metabolism of StAR and 3 β-HSD, SULT2A1 philosophy androsterone; The synthetic of skatole only depends on trophic factor, and metabolism is mainly completed by CYP2E1, CYP2A6, SULT1A1 etc.Androsterone has suppressed the metabolism of skatole simultaneously, makes both produce synergistic effect.
COUP-TF I, has another name called EAR-3 or NR2F1, is a hypotype of COUP-TFs gene, and chicken ovalbumin gene upstream promoter transcription factor, is the lonely nuclear receptor of nuclear receptor superfamily.At present, the research of COUP-TF is mainly concentrated on people and mouse, in domestic animal, the research of this gene is relatively less.
One. the relation of COUP-TF I and smell of mutton genes involved and smell of mutton material in different pig kinds
StAR, CYP17A1, CYP11A1 are mainly present in the testis of people and animal, participate in the synthetic of androsterone; CYP2C34, CYP2A19, CYP2E1 are mainly present in the liver of people and animal, are the main metabolic enzymes of skatole metabolism.It is significant to control boar smell of mutton that in research landrace, yorker, Taihu Lake pig, Rongchang Pig and Ba Ma pig liver and testis, COUP-TF1 affect expression level and the characteristic of the oligogene that smell of mutton expresses.
1. take β-actin as reference gene, application fluorescent quantitation method is carried out quantitatively smell of mutton genes involved and COUP-TF1 gene expression dose in landrace, yorker, Taihu Lake pig, Rongchang Pig, bar horse pig liver, analyze its expression characteristic, the primer that fluorescent quantitation is used is as shown in table 1.
The primer sequence of table 1 smell of mutton genes involved real-time quantitative
Concrete grammar is: extract landrace, yorker, Taihu Lake pig, Rongchang Pig and Ba Ma pig liver and the total RNA of testis, and detect RNA and extract quality, the total RNA of its mini-bus horse pig as shown in Figure 1.Then total RNA is reversed to cDNA and as template, the sequence shown in table 1 of take is carried out fluorescent quantitation detection as primer, and fluorescent quantitation is used iQ
tMsYBR
green BIO-RAD test kit, reaction system is as follows: the iQ of 5 μ L
tMsYBR
green, the Forward Primer of 0.5 μ L (10 μ M), the cDNA template of the Reverse Primer of 0.5 μ L (10 μ M) and 4 μ L, and detect by following condition: 95 ℃ of denaturation 5min; 35cycles(95 ℃ of sex change 15s, 56 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate 35 times, last 4 ℃ of preservations.Detected result is analyzed, processed data and image with Bio-Rad CFX Manager software.Adopt relatively Cq value method to compare analysis to fluorescent quantitative PCR result, obtain result as shown in Figure 2.As shown in Figure 2, each pig kind COUP-TF1 in liver, all has expression in CYP2E1, CYP2A19, CYP2C34.What wherein the content of COUP-TF1 showed in bar horse pig kind is the highest, is secondly York and landrace, and Rongchang County and Taihu Lake pig take second place.CYP2E1 gene is high at the expression amount of landrace, yorker and Ba Ma pig, and Rongchang Pig and Taihu Lake pig expression amount are low; And landrace CYP2E1 gene expression amount is 12 times of Rongchang Pig and Taihu Lake pig CYP2E1 gene expression amount, yorker CYP2E1 gene expression amount is Rongchang Pig and Taihu Lake pig CYP2E1 gene expression amount 13 times, bar horse pig is Rongchang Pig and Taihu Lake pig CYP2E1 gene expression amount 20 times, difference is (P < 0.01) extremely significantly, the landrace high with respect to expression amount, yorker and Ba Ma pig, the expression amount of bar horse pig is the highest, significant difference (P < 0.05); CYP2A19 gene expression amount in landrace, yorker and Ba Ma pig is high, and expression amount is low in Rongchang Pig and Taihu Lake pig; Wherein the expression amount of landrace CYP2A19 gene is Rongchang Pig and Taihu Lake pig 51 times, the expression amount of yorker CYP2A19 gene is Rongchang Pig and Taihu Lake pig 23 times, the expression amount of bar horse pig CYP2A19 gene is Rongchang Pig and Taihu Lake pig 4 times, significant difference (P < 0.05); In CYP2A19 gene, what expression amount was the highest is landrace.CYP2C34 gene expression amount in landrace, yorker and Ba Ma pig is high, and expression amount is relatively low in Rongchang Pig and Taihu Lake pig, and difference is not obvious each other.
2. according to above-mentioned identical method, smell of mutton essential substance androsterone synthetic gene expression level in landrace, yorker, Taihu Lake pig, Rongchang Pig, bar horse pig testis is carried out quantitatively, analyzing its expression characteristic, result as shown in Figure 3.As can be seen from Figure 3, each pig kind COUP-TF1 in testis, all have expression, and CYP2E1, CYP2A19 and CYP2C34 expression amount in testis is extremely low in CYP11A1, CYP17A1, star.In Rongchang Pig and Taihu Lake pig, the expression amount of CYP11A1 gene is higher than bar horse pig, landrace and yorker, and wherein landrace and yorker are minimum; Rongchang Pig and Taihu Lake pig expression amount are respectively its 8 times and 6 times, and difference is (P < 0.01) extremely significantly.CYP17A1 gene is high at bar horse pig expression amount, and the expression amount of Rongchang Pig and Taihu Lake pig is higher than landrace and yorker.StAR gene expression amount in Rongchang Pig, Taihu Lake pig and Ba Ma pig is higher, and wherein the expression amount of Rongchang Pig is up to other 11 times, significant difference (P < 0.01).But the content of COUP-TF1 in testis does not have too big-difference in various different pig kinds.
3, utilize HPLC method to measure the skatole content in different pig kinds
Skatole (3-methylindole) standard substance are purchased from Sigma company, and the reference liquid that skatole is mixed with to different concns detects (LC-2OAT pump, RF-10A in high-efficient liquid chromatographic instrument system
xLfluorimetric detector, CTO-20AC column oven, LC solution chromatographic working station, Shim-pack VP-ODS C18 post (5 μ m, 4.6 * 150nm) is all purchased from Japanese Shimadzu).Moving phase is second eyeball: water is 60:40(v/v), post case temperature is 30 ℃, and flow velocity is 1.0mL/min, and sample size is 20 μ L; Fluorimetric detector condition: excitation wavelength 285nm, emission wavelength 345nm; UV-detector condition is 254nm, obtains the color atlas of different concns reference liquid, and wherein the color atlas of 100ng/mL skatole as shown in Figure 4.Then according to chromatogram result, drawing standard curve, result is as shown in Figure 5.As shown in Figure 5, skatole typical curve is y=55304x-42085, R
2=0.9999.
Muscle samples is prepared: accurately take different pig kind muscle samples 2.0 ± 0.59g, add respectively homogenate after 5mL methyl alcohol, in ice bath after freezing homogenate, with high speed freezing centrifuge with the centrifugal 8min of rotating speed 6500r/min, through 0.45 μ m membrane filtration, filtrate is detection sample.Then according to above-mentioned condition, detect smelly excretin concentration in sample, result is as shown in table 2, and its mini-bus horse pig muscle skatole color atlas as shown in Figure 6A.
Plasma sample is prepared: get plasma sample in the centrifuge tube of 1.5mL with acetonitrile by volume for 1:1 mixes, with protein precipitation, then by test tube vortex 30s, and write and remain on 15min-20 ℃ of conditions, then the centrifugal 20min of 4800g at 4 ℃, collects supernatant, and supernatant liquor is detected to skatole concentration according to above-mentioned condition, result is as shown in table 2, and its mini-bus horse porcine blood plasma skatole color atlas as shown in Figure 6B.
The mensuration of skatole concentration in table 2 different pig kind muscle tissue and blood
Note: a represents P < 0.05; A represents P < 0.01
As can be known from Table 2, in Rongchang Pig muscle tissue and blood plasma, skatole concentration is respectively 29.7182 ± 1.1461ng/mL, 37.4997 ± 1.8394ng/mL, in Taihu Lake pig muscle tissue and blood plasma, skatole concentration is respectively 39.8411 ± 2.3543ng/mL, 21.0334 ± 1.6803ng/mL, therefore in Rongchang Pig and Taihu Lake pig, all contain the skatole of higher concentration, significant difference (P < 0.05).And the muscle tissue of landrace and yorker and the skatole concentration in blood plasma is all in about 18.50ng/mL, difference is not remarkable.
4, use Elisa method to measure the content of the androsterone in different pigs
Use double-antibody sandwich Elisa method to measure the content of pig androsterone, pig androsterone (ADT) Elisa test kit is purchased from Shanghai Bo Gu biotech company (production code member: PEA087), measure pig androsterone content according to test kit specification sheets.The androsterone of preparation different concns is reference liquid drawing standard curve, as shown in Figure 7, i.e. and y=0.2012+0.1472, R2=0.9478.
Blood plasma detects sample preparation: get porcine blood plasma, then using heparin as antithrombotics, standing 10-20min after mixing, then under 1000g condition centrifugal 15min, collect supernatant, obtain detection sample.
Muscle detects sample preparation: cut muscle specimen, take weight 0.5mg, add the PBS solution of 500 μ L, then by muscle specimen homogenate, then under 2000-3000rmp condition centrifugal 20min, collect supernatant and detect.
The blood plasma of preparation is detected to sample and muscle and detect pig androsterone (ADT) Elisa test kit for sample and detect its content, then according to typical curve, calculate the content of androsterone in different pig kind muscle tissue and blood plasma, result is as shown in table 3.
The mensuration of androsterone concentration in table 3 different pig kind muscle tissue and blood
Note: a represents P < 0.05; A represents P < 0.01
As shown in Table 3, in different pig kinds (landrace, Rongchang Pig, Taihu Lake pig and yorker), carry out Elisa muscle and the concentration determination of blood plasma androsterone, Rongchang Pig and Taihu Lake pig are higher than landrace, yorker and Ba Ma pig, especially the highest with the content of Taihu Lake pig, significant difference (P < 0.05).
From the above results, CYP2E1, CYP2A19 and CYP2C34 gene mainly concentrate on the gene of expressing in liver, and do not express in testis.According to the expression of five pig kinds (landrace, Rongchang Pig, Taihu Lake pig, yorker, bar horse pig) main smell of mutton gene in pig body, can find out, because CYP2E1, CYP2A19 gene are phase metabolic enzymes of skatole catabolism, the expression of Rongchang Pig and Taihu Lake pig is lower, illustrate that its vigor is lower, so the skatole content in pork fat is more, skatole content is apparently higher than landrace, yorker and Ba Ma pig.COUP-TF1 is transcription factor with having affected the activation of CYP2E1, CYP2A19 promotor and having transcribed, so COUP-TF1 gene and CYP2E1, CYP2A19 show positively related relation.Meanwhile, in testis tissue, affect androsterone synthetic oligogene CYP11A1, CYP17A1, the expression amount of star in Rongchang Pig and Taihu Lake pig is higher, and landrace and yorker expression amount are relatively low.The androsterone synthetic gene of high expression level increases the androsterone content in Rongchang Pig and Taihu Lake pig, and then stop the specific binding of COUP-TF and CYP2E1 or CYP2A6 promotor to cause the increase of skatole content by androsterone, and then increased the smell of mutton content of Rongchang Pig and Taihu Lake pig.
The molecular mechanism research of two .COUP-TF I on the impact of smell of mutton material
It is the negative regulation albumen of CYP17 genetic transcription that COUP-TF I has been proved in mouse, ox and human adrenal gland cortex cell, and meanwhile, it can suppress the expression of star and suppress the formation of carrier compound.But do not have relevant research to show direct relation between COUP-TF I and CYP11A1, CYP17A1 that in pig testis, androsterone synthetic molecules mechanism is relevant, StAR gene.The present embodiment is used cell cultures, the method such as construction of eukaryotic expression vector and transient transfection, utilize the analysis means such as qRT-PCR, ELISA, probe in COUP-TF I and pig testis the relation between CYP11A1, CYP17A1 and StAR in androsterone route of synthesis, thereby illustrate the molecule mechanism of COUP-TF I on the synthetic impact of androsterone, for improving boar smell of mutton, the good boar of seed selection provides theoretical foundation.
The pig testis in different varieties and Different Month stage and the physiological function of interstitial glands thereof are different.There are some researches show, low stage at monthly age boar androsterone level lower than the androsterone level in high stage at monthly age.The fragrant pig testis of bar horse at 1 monthly age of young stage and 6 monthly ages of Adulthood is chosen in this experiment, mainly from two aspects, be studied: (1) usings β-actin as reference gene, utilize qRT-PCR to analyze the relative expression quantity of Different Month COUP-TF I, CYP11A1, CYP17A1 and StAR gene in the stage, utilize ELISA method to measure the level of androsterone in testis tissue; (2) utilize the culture method that do not sieve to carry out vitro culture to the fragrant pig testis mesenchymal cell of the bar horse at 1 monthly age and 6 monthly ages, COUP-TF I carrier for expression of eukaryon COUP-TF I-neo that structure contains screening-gene neo, and this recombinant plasmid proceeding to the pig testis mesenchymal cell at 1 monthly age and 6 monthly ages by the method for transient transfection, the cell with transfection pEGFP-C1 plasmid and untransfected compares simultaneously.Utilize qRT-PCR to analyze the variation of the relative expression quantity of COUP-TF I, CYP11A1, CYP17A1 and StAR in cell after transfection, utilize ELISA to detect the variation of androsterone level in cell after transfection.
1, the cultivation of interstitial glands
Remove testis surface reticular tissue and peel off after tunicle, by tissue shear to 1mm
2size, according to 10cm culture dish 1cm between left and right every, be inoculated in culture dish, by piece in a organized way one facing up, standing 5 hours, then, gently culture dish is turned, add primary cell perfect medium, with tissue block, do not fall down and be advisable, 37 ℃, 5%CO
2, standing cultivation under saturated humidity, result is as shown in Figure 8.
2, the amplification of COUP-TF I in pig liver
Extract the total RNA of pig liver, through agarose gel electrophoresis, detect, result as shown in Figure 9.As seen from the figure, total RNA integrity of extracting is better.Then design primer and increase, upstream primer is: COUP-TF I F:5 '-cgtcagatcc
gctagcggtcgccaccatggcaatggtagttagcag-3 ' (SEQ ID NO.17), underscore is Nhe I restriction enzyme site; Downstream primer is: COUP-TF I R:5 '-cttgagctcg
agatctctaggagcactgg-3 ' (SEQ ID NO.18), underscore is Bgl II restriction enzyme site, the test kit of use is
hS DNA Polymerase with GC Buffer(Code No.DR044A), reaction conditions: 98 ℃ of sex change 10s, step back 10s for 55 ℃, 72 ℃ are extended 60s totally 30 circulations, extend 5min after last 72 ℃.Amplified production is carried out to agarose gel electrophoresis, and result as shown in figure 10.As shown in Figure 10, amplification obtains the band of about 1200bp left and right, amplified production is connected to pMD19-T carrier, obtain COUP-TF I-pMD19-T recombinant vectors, then recombinant vectors is transformed to DH5 α competent cell, through order-checking, obtain and contain the COUP-TF I gene ORF that the length as shown in SEQ ID NO.19 is 1269bp.
3, the structure of COUP-TF I-neo carrier for expression of eukaryon
PEGFP-C1 carrier is carried out to double digestion (Figure 11) by Nhe I and Bgl II, reclaim the DNA fragmentation of about 4Kbp, called after Vector DNA.Then COUP-TF I-pMD19-T carrier of take is template, and sequence shown in SEQ ID NO.18 and SEQ ID NO.19 is that primer carries out pcr amplification (Figure 12), by the COUP-TF I of amplification with using In-Fusion after Nhe I and Bgl II double digestion
hD Cloning Kit(Clontech Code No.639633) be connected into Vector DNA, obtain COUP-TF I-neo carrier.
4, transfectional cell cracking is identified
1) preparation of interstitial glands and experiment grouping
The trysinization that is 0.25% with massfraction by the interstitial glands that is cultured to logarithmic phase in 60mm culture dish, 3.0 * 10
5/ hole is inoculated in 6 porocyte culture plates, and every hole adds perfect medium 2mL, in 37 ℃, and 5%CO
2in incubator, be cultured to cell density 80%～90%, then carry out transfection.
Experiment is divided into three groups: transfection COUP-TF I-neo recombinant plasmid group, and transfection pEGFP-C1 plasmid group and without transfection blank group, each group is established 3 repetitions, and experiment is in triplicate.
2) transfection step
1. before transfection, 2h changes liquid with perfect medium to interstitial glands in 6 orifice plates.
2. add 200 μ L transfection composites.Transfection composite is: 8 μ L X-tremeGENE HP DNA Transfection Reagent transfection reagents, the plasmid after 2 μ g purifying, supplies 200 μ L systems with DMEM.Order of addition is DMEM, plasmid, transfection reagent.Standing 15～20min at room temperature after mixing.Blank group is only changed liquid, not transfection.
3. in conjunction with after complete, with rifle, draw 200 μ L and carry out transfection in 6 orifice plates, 200 μ L/ holes.
4. after transfection, temperature is 37 ℃ again, 5%CO
2incubator is hatched, and changes substratum after 12h.
5. cultivate after 48h, collect the cell conditioned medium liquid in every hole, carry out mark.Adherent cell is carried out to cracking evaluation and RT-PCR experiment.
3) lysis is identified
1. by the cell after transfection with after 0.25% trysinization, with the neutralization of appropriate perfect medium, at rotating speed, be then centrifugal 5min under 1500rpm condition, abandon supernatant.
2. in precipitation, add 75 μ L lysates, piping and druming mixes, cracking condition: first at 56 ℃ of insulation 1h, then process 10min at 94 ℃.
3. split product is carried out to PCR checking, reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of sex change 15s, 56 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations; After again at 72 ℃, extend 8min; Last 4 ℃ of preservations.
Checking primer is as shown in table 4:
Table 4, COUP-TF I and pEGFP-C1 checking primer
Qualification result as shown in Figure 14 and Figure 15.As seen from the figure, after the pig testis mesenchymal cell plasmid cracking of 1 monthly age of transient transfection, COUP-TF I-neo plasmid length is 396bp, and pEGFP-C1 plasmid length is 316bp; After the pig testis mesenchymal cell plasmid cracking of 6 monthly age of transient transfection, COUP-TF I-neo length is 396bp, pEGFP-C1 length is 316bp, contrast with corresponding positive plasmid, the band length of two plasmids is identical with the band length of positive plasmid, therefore the plasmid that, cracking is identified is correct plasmid.
5, extract the total RNA of transfectional cell
Extract the cell total rna after 1 monthly age and 6 monthly age untransfecteds and transfection plasmid, and detect the RNA OD that carries
260and OD
280value, then calculates OD
260/ OD
280, result is as shown in table 4 and table 5.From table 4 and table 5, total RNAOD
260/ OD
280all, between 1.7～2.1, show that extracted RNA purity is higher.Through electrophoresis detection result as shown in figure 16, result shows: the 28S of the untransfected at 1 monthly age and 6 monthly ages and total RNA of the cell after transfection plasmid, and 18S, the electrophoretic band of 5S is easy to observe, and integrity is better.To sum up, the requirement that total RNA integrity of extracting and purity meet reverse transcription.
The purity of the total RNA of 1 monthly age of table 4 transient transfection pig testis mesenchymal cell and concentration (N=9)
The purity of the total RNA of 6 monthly age of table 5 transient transfection pig testis mesenchymal cell and concentration (N=9)
6, qRT-PCR analyzes
Total RNA by extracting, carries out qRT-PCR analysis with primer in table 1, and the relative expression quantity of 1 monthly age of transient transfection pig testis mesenchymal cell gene as shown in figure 17, analyze shown in Figure 18 by the relative expression quantity of 6 monthly age of transient transfection pig testis mesenchymal cell gene.As shown in Figure 17,1 monthly age interstitial glands transcription factor COUP-TF I and CYP11A1, CYP17A1, StAR all have expression.Wherein, difference extremely significantly (P<0.01) between the relative expression quantity of COUP-TF I, CYP11A1, CYP17A1 and StAR in transfection COUP-TF I-neo recombinant plasmid group and corresponding blank group and the relative expression quantity of transfection pEGFP-C1 plasmid group, between blank group and the relative expression quantity of transfection pEGFP-C1 plasmid group without significant difference.And in transfection COUP-TF I-neo recombinant plasmid group, the relative expression quantity of COUP-TF I is higher than blank group and transfection pEGFP-C1 plasmid group, and the relative expression quantity of CYP11A1, CYP17A1 and StAR is lower than blank group and transfection pEGFP-C1 plasmid group.By result, can be found out, COUP-TF I-neo recombinant plasmid successfully proceeds to 1 monthly age pig testis mesenchymal cell, and after this recombinant plasmid of transfection, the relative expression quantity of CYP11A1, CYP17A1 and StAR obviously reduces.As shown in Figure 18,6 monthly age interstitial glands transcription factor COUP-TF I and CYP11A1, CYP17A1, StAR all have expression.Wherein, difference extremely significantly (P<0.01) between the relative expression quantity of COUP-TF I, CYP11A1, CYP17A1 and the StAR in transfection COUP-TF I-neo recombinant plasmid group and corresponding blank group and the relative expression quantity of transfection pEGFP-C1 plasmid group; Between the relative expression quantity of blank group and transfection pEGFP-C1 plasmid group without significant difference.And in transfection COUP-TF I-neo recombinant plasmid group, the relative expression quantity of COUP-TF I is significantly higher than corresponding blank group and transfection pEGFP-C1 plasmid group, and the relative expression quantity of CYP11A1, CYP17A1 and StAR is significantly lower than corresponding blank group and transfection pEGFP-C1 plasmid group, wherein the reduction degree of CYP11A1 and CYP17A1 relative expression quantity clearly.By result, can be found out, COUP-TF I-neo recombinant plasmid successfully proceeds to 6 monthly age pig testis mesenchymal cells, and after this recombinant plasmid of transfection, the relative expression quantity of CYP11A1, CYP17A1 and StAR obviously reduces, and CYP11A1 and CYP17A1 relative expression quantity extremely reduce.
Extract respectively 1 monthly age of untransfected and total RNA of 6 monthly age interstitial glandses, with primer shown in table 1, analyze related gene expression amount, result as shown in figure 19.As shown in Figure 19, the relative expression quantity of COUP-TF I and CYP11A1, CYP17A1 and comparing in 1 monthly age cell in 6 monthly age cells, difference is (P<0.01) extremely significantly; And the relative expression quantity significant difference (P<0.05) of StAR.
1 monthly age of transfection COUP-TF I-neo recombinant plasmid and the gene relative expression component analysis of 6 monthly age interstitial glandses: extract respectively 1 monthly age of transfection COUP-TF I-neo recombinant plasmid and total RNA of 6 monthly age interstitial glandses, then with primer shown in table 1, analyze related gene expression amount, result as shown in figure 20.As shown in Figure 20, after transfection COUP-TF I-neo recombinant plasmid, the relative expression quantity of COUP-TF I, CYP11A1 and CYP17A1 and comparing in 1 monthly age cell in 6 monthly age cells, difference is (P<0.01) extremely significantly; And the relative expression quantity significant difference (P<0.05) of StAR.Also can find out by scheming, in 6 monthly age cells, the relative expression quantity of COUP-TF I is higher than the cell at 1 monthly age; And the relative expression quantity of CYP11A1, CYP17A1 and StAR is starkly lower than the cell at 1 monthly age.This explanation, after transfection COUP-TF I-neo recombinant plasmid, in 6 monthly age cells, the degree of the expression by inhibitation system of CYP11A1, CYP17A1 and StAR is greater than the cell at 1 monthly age.Wherein, the difference of CYP17A1 relative expression quantity is very obvious, is secondly CYP11A1, is finally StAR.
7, ELISA measures androsterone level
The level that detects androsterone in 1 monthly age of transient transfection and 6 monthly age interstitial glandses according to method as hereinbefore, result is as shown in table 6.
Table 6,1 monthly age of transient transfection and 6 monthly age at monthly age interstitial glands androsterone levels (N=9)
Note:
*significant difference (P<0.05) has been compared in expression with blank group,
*indicate utmost point significant difference (P<0.01)
As shown in Table 6, the androsterone level of the interstitial glands of 6 monthly ages transfection COUP-TF I-neo recombinant plasmid is compared with the negative control group of transfection pEGFP-C1 with its blank group, and difference is (P<0.01) extremely significantly; The androsterone level of the interstitial glands of 1 monthly age transfection COUP-TF I-neo recombinant plasmid is compared with the negative control group of transfection pEGFP-C1 with its blank group, significant difference (P<0.05).
At present, in the analytical procedure of research transcriptional control, transient transfection assays method is the most conventional.The method is, by certain transfection program, the plasmid containing object control region is imported to recipient cell, and this plasmid is present on free carrier, and unconformability, to karyomit(e), just can obtain a kind of method of the expression product of gene within a short period of time.In a typical case, control region regulation and control " reporter gene " transcribes.And reporter gene is in the level of mRNA and protein, to be all easy to the gene that correctly detected.Although stable gene expression technique is the gold standard of biological study, its operating process is more numerous and diverse, is unfavorable for large flux, finds new medical protein expeditiously.On the other hand, transient transfection assays method is more convenient, quick, and its result is easily quantitative, therefore becomes the prefered method that promoter function is analyzed.The conventional host cell of transient transfection assays method has 293 cells, COS-7 cell.According to requirement of experiment, this experiment utilizes pig testis mesenchymal cell as the host cell of transient transfection, has successfully built pig testis mesenchymal cell transient transfection model.
In the present invention, by the technology of transient transfection, COUP-TF I-neo recombinant plasmid is proceeded to after interstitial glands, from the relative expression quantity of its gene, COUP-TF I has successfully been proceeded to.And after transfection 1 monthly age and CYP11A1, the CYP17A1 at 6 monthly ages and the relative expression quantity of StAR all few than corresponding blank.Show, when cell proceeds to after COUP-TF I-neo, the expression of CYP11A1, CYP17A1 and StAR has been subject to inhibition, and this has also illustrated that COUP-TF I can significantly suppress the expression of CYP11A1, CYP17A1 and StAR.And COUP-TF I is obviously better than 1 monthly age cell to the restraining effect of 6 monthly age cell CYP11A1, CYP17A1 and StAR, this also may be relevant with the monthly age of interstitial glands, and COUP-TF I is better than the mesenchymal cell at low monthly age to the restraining effect of high monthly age mesenchymal cell.And ELISA result shows that in 6 monthly age interstitial glandses, androsterone level is higher than 1 monthly age cell, this detected result is also consistent with androsterone level detection result in 1 monthly age and 6 monthly age testis tissues.After proceeding to COUP-TF I-neo recombinant plasmid, in 1 monthly age and 6 monthly age interstitial glandses all there is decline in androsterone level, and the level at 6 monthly ages reduces more obviously, illustrates that the rise of COUP-TF I can suppress the generation of androsterone level.This result is also consistent with the result of study of Lehmann etc.The rise that is COUP-TF I can suppress CYP11A1, the expression of CYP17A1 and StAR, thus cause the level of androsterone to reduce, and COUP-TF I is better than the low stage at monthly age to the restraining effect of the androsterone level in high stage at monthly age.
By above experiment, obtained following result: built COUP-TF I-neo recombinant plasmid, and proceeded to the fragrant pig testis mesenchymal cell of bar horse at 1 monthly age and 6 monthly ages.QRT-PCR result shows: after proceeding to COUP-TF I-neo recombinant plasmid, reduction has all appearred in CYP11A1, the CYP17A1 in 1 monthly age and 6 monthly age cells and the relative expression quantity of StAR.And the relative expression quantity of 1 monthly age and CYP11A1, CYP17A1 in 6 monthly age cells and StAR is compared with the negative control group of transfection pEGFP-C1 with corresponding blank, difference is all extremely significantly (P<0.01).1 monthly age and the 6 monthly age cells after transfection and transfection COUP-TF I-neo recombinant plasmid are not carried out in contrast, COUP-TF I, the relative expression quantity of CYP11A1, CYP17A1 and comparing of 1 monthly age in 6 monthly age cells, difference is extremely significantly (P<0.01) all, and the relative expression quantity difference of StAR all significantly (P<0.05); And do not carry out the relative expression quantity of CYP11A1, CYP17A1 and StAR in 6 monthly age cells of transfection higher than 1 monthly age cell, in 6 monthly age cells after transfection COUP-TF I-neo recombinant plasmid, the relative expression quantity of CYP11A1, CYP17A1 and StAR is lower than 1 monthly age cell.By ELISA, detect discovery, the androsterone level of 6 monthly age cells of transfection COUP-TF I-neo recombinant plasmid is compared with the negative control group of transfection pEGFP-C1 with blank, and difference is (P<0.01) extremely significantly; The androsterone level of 1 monthly age cell of transfection COUP-TF I-neo recombinant plasmid is compared with the negative control group of transfection pEGFP-C1 with blank, significant difference (P<0.05).
According to above result, can show that the expression of rise COUP-TF I can significantly suppress the generation of the expression of CYP11A1, CYP17A1 and StAR and the level of androsterone.Therefore, pig transcription factor coup-tf1 can be used as the gene that regulates smell of mutton in pork.
Claims (8)
1. pig transcription factor coup-tf1, is characterized in that, the nucleotide sequence of described pig transcription factor coup-tf1 is as shown in SEQ ID NO.19.
2. the recombinant vectors that contains pig transcription factor coup-tf1 described in claim 1.
3. recombinant vectors according to claim 2, is characterized in that: described recombinant vectors contains cell screening gene neo.
4. the application of pig transcription factor coup-tf1 in reducing pork smell of mutton described in claim 1.
5. the application of pig transcription factor coup-tf1 in suppressing CYP11A1 genetic expression described in claim 1.
6. the application of pig transcription factor coup-tf1 in suppressing CYP17A1 genetic expression described in claim 1.
7. the application of pig transcription factor coup-tf1 in suppressing StAR genetic expression described in claim 1.
8. the application of pig transcription factor coup-tf1 in reducing androsterone content described in claim 1.
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| CN111110868A (en) * | 2020-01-21 | 2020-05-08 | 深圳市人民医院 | Application of NR2F6 gene and encoding protein thereof in preparation of medicines |
| CN111110868B (en) * | 2020-01-21 | 2023-04-21 | 深圳市人民医院 | NR2F6 gene and application of coded protein thereof in preparation of medicines |
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