CN101962645A - Method for promoting growth and lactation of mammary epithelial cells of milk cow - Google Patents
Method for promoting growth and lactation of mammary epithelial cells of milk cow Download PDFInfo
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- CN101962645A CN101962645A CN 201010263328 CN201010263328A CN101962645A CN 101962645 A CN101962645 A CN 101962645A CN 201010263328 CN201010263328 CN 201010263328 CN 201010263328 A CN201010263328 A CN 201010263328A CN 101962645 A CN101962645 A CN 101962645A
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Abstract
The invention provides a method for promoting growth and lactation of mammary epithelial cells of a milk cow, and relates to a method for promoting the growth and lactation of the mammary gland. The method comprises the following steps: (1) culturing the mammary epithelial cells in vitro; (2) designing primers, carrying out PCR augmentation to generate a complete sequence of Kcnmal gene, then connecting the sequence with pGEM-t vectors, then converting escherichia coli DH5alpha and obtaining cloning vector plasmids; and (3) carrying double digestion on pSV-beta-Galactosidase Control Vector plasmids and the cloning vector plasmids, connecting and building expression vectors, then carrying out transfection on the mammary epithelial cells cultured in vitro, leading the Kcnmal gene to be excessively expressed in the mammary epithelial cells cultured in vitro, and completing. In the invention, the Kcnmal gene is highly expressed in the mammary gland of the milk cow during a lactation period, and the experiment verifies that the Kcnmal gene is surely an important functional gene capable of promoting the growth and lactation of mammary epithelial cells of the milk cow.
Description
Technical field
The present invention relates to a kind of method that promotes mammogenesis and lactation.
Background technology
Milk cow is one of economic animal important in the livestock breeding industry, and milk yield of milk cow and milk quality are the major criterions of weighing breed level and economic benefit.China is milk cattle cultivating big country, and the livestock on hand milk cow reaches 1,216 ten thousand, but the average year unit yield of milk cow has only 3884kg, and the milk cow per unit area yield of the U.S. then can reach 7767kg, and Israel is 8615kg, and material milk is than reaching 1: 3.Milk yield is low, butterfat relative with milk protein content not high, ratio for input and output is lower is the principal element that restriction China milk cattle cultivating already develops.
According to National Program for Medium-to Long-term Scientific and Technological Development, " national Eleventh Five-Year Plan scientific and technical development program " and " 863 Program Eleventh Five-Year Plan development outline ", by using various information biology means and gene manipulation techniques, filtering out influences the critical function of milk character gene, has important in theory meaning and actual application value to promoting China's milk cattle cultivating already to develop.Mammary gland is important lactation function organ, and gene expression dose is subjected to the dual regulation and control of h and E factor in the mammary gland.Lactation critical function expression of gene level changes can regulate and control the growing of mammary gland, differentiation and tissue reconstruction, and important physical processes such as milk is synthetic, secretion and transportation.Mammary epithelial cell is the basic structure and the functional unit of lactation, by studying lactation Expression of Related Genes in it, can how the mammary specific expression gene be activated in order to disclose, how initial sum is kept lactation to lactation critical function gene, regulation and control milk component provides theoretical foundation.Simultaneously, help to set up a kind of mammary gland biological modulated control techniques of regulating and control milk bovine lactation critical function gene expression dose and significantly improving milk yield.
Milk cow functional genomics research at present is in the starting stage, is following research emphasis to the functional study that influences Niu Jiankang, disease, meat milk quality genes involved.Domestic research work to the relevant milk cow functional gene of lactation is in space state substantially, also has only less research report, the lactation functional gene comparatively small amt of acquisition abroad.Adopt modern functions genomics research method, study the relevant gene of the important productive capacity of this milk cow of lactation, and milk cow has great importance with the interaction between the important gene of these control lactations from the various nutritive substances that feed obtained.Can be the raising of cow producing milk performance and the fair supply research of nutritive substance important techniques theoretical foundation and technique means are provided.
Summary of the invention
The invention provides a kind of method that promotes cow mammary gland epithelial cells growth and lactation.
Promoting that the cow mammary gland epithelium is fine educates and the method for lactation is carried out according to the following steps: one, get the He Sitan bovine lactation phase and organize sample and adopt collagenase-trypsin digestion to separate epithelial cell, place cell grown cultures liquid to cultivate for 3 generations then, get the mammary epithelial cell of external cultivation; Two, according to the mRNA sequences Design primer of Kcnma1 gene, pcr amplification goes out the complete sequence of Kcnma1 gene, is connected with the pGEM-T carrier then, connects product transformed into escherichia coli DH5 α, obtains cloning vector plasmids; Three, pSV-β-Galactosidase Control Vector plasmid and cloning vector plasmids use AgeI and XbaI to carry out double digestion respectively, reclaim required purpose fragment, connect and construction of expression vector, use the mammary epithelial cell of expression vector transfection vitro culture again, make Kcnma1 gene overexpression in the mammary epithelial cell of vitro culture, finish promptly that the cow mammary gland epithelium is fine educates and the promotion of lactation; Wherein cell grown cultures liquid is made up of the DMEM high glucose medium of 90ml, FBS (foetal calf serum) and the Regular Insulin that 10ml concentration is 50mg/ml, the penicillin of 100 μ l, 100,000 units/ml and the Streptomycin sulphate of 100 μ l, 100,000 units/ml of 10ml in the step 1; In the step 2 PCR increase used upstream primer be 5 '-GCACCGGTTGCTAGCTATGGCAAATGGTGGC-3 ', downstream primer is 5 '-GCTCTAGAGGCAGTGGATACACACATCAGAG-3 ', and added the restriction enzyme site of restriction enzyme A geI and XbaI respectively at 5 of upstream and downstream primer ' end.
The big electricity of potassium is led calcium active channel subfamily M, α member 1 (potassium large conductance calcium-activated channel, subfamily M, alpha member 1, Kcnma1) a kind of BK potassium-channel proteins of coding; It is made up of two subunits, is responsible for passage α subunit that forms and the β subunit that plays regulating effect respectively; Intracellular calcium is regulated the interaction between α and the β subunit; Potassium channel is by the depolarize or the interior Ca of raising cell of film
2+Concentration is activated, and regulates K
+Transportation; It also can be by Mg in the cell
2+Activate; Its activation will cushion Ca in the cell
2+Therefore concentration and/or membrane potential depolarize can make the membrane potential repolarization.
Kcnma1 gene high expression level in milk cow in lactation period mammary gland among the present invention has verified that at cell levels the Kcnma1 gene is the critical function gene that can promote that cow mammary gland epithelial cells is grown and breast produces really by gene overexpression experiment and RNA interference experiment.
The Kcnma1 gene is to influence cell proliferation and milk-protein synthetic critical function gene in the cow mammary gland among the present invention, plays important key effect in cow mammary gland growth and lactation function; By regulation and control to the Kcnma1 gene, can change the lactation function of cow mammary gland, reach the change milk character, increase the purpose of economic benefit; The present invention also is the function that has proved that first the Kcnma1 gene has stimulates mammogenesis and lactation in mammary gland, and can be used for gene regulating and change related application such as breeding performonce fo animals.
Description of drawings
Fig. 1 is Kcnma1 gene overexpression cell casein and pSTAT5 detection figure in the mammary epithelial cell of vitro culture in the embodiment one; Fig. 2 is the cell cycle detection figure of Kcnma1 gene overexpression cell in the mammary epithelial cell of vitro culture in the embodiment one, and wherein A is Kcnma1 gene overexpression curve in the mammary epithelial cell of vitro culture, and B is contrast; Fig. 3 is that the complete sequence that pcr amplification goes out the Kcnma1 gene in the embodiment one carries out RNA gradient curve interference time figure; Fig. 4 is Weatern Blot detection figure behind the Kcnma1 gene silencing in the embodiment one; Fig. 5 is the cell cycle detection figure of Kcnma1 gene silencing cell in the embodiment one, and wherein A is the cell cycle detection curve of Kcnma1 gene silencing cell, and B is contrast.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment promotes that the cow mammary gland epithelium is fine and educates and the method for lactation is carried out according to the following steps: one, get the He Sitan bovine lactation phase and organize sample and adopt collagenase-trypsin digestion to separate epithelial cell, place cell grown cultures liquid to cultivate for 3 generations then, get the mammary epithelial cell of external cultivation; Two, according to the mRNA sequences Design primer of Kcnma1 gene, pcr amplification goes out the complete sequence of Kcnma1 gene, is connected with the pGEM-T carrier then, connects product transformed into escherichia coli DH5 α, obtains cloning vector plasmids; Three, pSV-β-Galactosidase Control Vector plasmid and cloning vector plasmids use AgeI and XbaI to carry out double digestion respectively, reclaim required purpose fragment, connect and construction of expression vector, use the mammary epithelial cell of expression vector transfection vitro culture again, make Kcnma1 gene overexpression in the mammary epithelial cell of vitro culture, finish promptly that the cow mammary gland epithelium is fine educates and the promotion of lactation; Wherein cell grown cultures liquid is made up of the DMEM high glucose medium of 90ml, FBS (foetal calf serum) and the Regular Insulin that 10ml concentration is 50mg/ml, the penicillin of 100 μ l, 100,000 units/ml and the Streptomycin sulphate of 100 μ l, 100,000 units/ml of 10ml in the step 1; In the step 2 the used upstream primer of pcr amplification be 5 '-GCACCGGTTGCTAGCTATGGCAAATGGTGGC-3 ', downstream primer is 5 '-GCTCTAGAGGCAGTGGATACACACATCAGAG-3 ', and added the restriction enzyme site of restriction enzyme A geI and XbaI respectively at 5 of upstream and downstream primer ' end.
Cell grown cultures liquid in the present embodiment step 1, after the filtering with microporous membrane degerming of 0.22 μ m, regulating the pH value is 7.2.
The process that transforms in the present embodiment step 2: a, melt 50 μ l bacillus coli DH 5 alphas on ice, add 5 μ l and connect product, place 30min in-20 ℃ behind the mixing, place 42 ℃ of water-baths 45 seconds then, keep EP pipe transfixion therebetween; B, the rapid taking-up are positioned over 2min in the ice bath, add 950 μ l LB liquid nutrient mediums, and 1.5h are cultivated in 37 ℃ of concussions; C, the centrifugal 10min of cultivation back 3000r/min, abandoning supernatant is with the resuspended solid precipitation of 200 μ l LB liquid nutrient mediums; D, get 100 μ l coating and contain penicillin and scribble X-gal and the LB flat board of IPTG, after drying up, cultivate the 12h observationss for 37 ℃; Behind e, the cultivation 12h, the bacterium colony of picking white on the LB flat board is inoculated in 20ml and contains in the LB liquid nutrient medium of penicillin, and 12h are cultivated in 37 ℃ of concussions.
Get positive recombinant 20 μ l, send the order-checking of the big gene company limited of Beijing China, the gained sequencing result is compared with the Kcnma1 gene order, and homology is 100%.
Transfection process in the present embodiment step 3: a, with 2 * 10
5The mammary epithelial cell of individual external cultivation adds 6 porocyte culture plates, cultivates 24h and makes the cell attachment growth; The substratum 400ul, the recombinant vectors 30ul that mix 37 ℃ of preheatings in b, the sterilization centrifuge tube, after the short term oscillation, add lipofectamine 16ul, vibration mixing, incubated at room mixture 10min, cell culture medium is removed in suction, with mixture short term oscillation mixing, add in the cell cultures hole, cultivate lh for 37 ℃, the adding of every hole is preheating to 37 ℃ serum-free and microbiotic substratum 1.5ml, places 5%CO
2, cultivate 24h in 37 ℃ of cell culture incubators; C, change cell grown cultures liquid in the step 1 into, continue to cultivate 24h.
The Kcnma1 gene is in the mammary epithelial cell of vitro culture behind the overexpression in the present embodiment, detects the variation of milk-protein synthesis capability in the mammary epithelial cell of vitro culture and the variation of mammary epithelial cell multiplication capacity; Weatern Blot detected result as shown in Figure 1, the cell casein ability to express of visible Kcnma1 gene overexpression obviously strengthens, the pSTAT5 expression amount increases; Flow cytometer detects the cell cycle result as shown in Figure 2, and the cell fission propagation of visible Kcnma1 gene overexpression is more active, and more cell is in division stage; Experimental result shows that the Kcnma1 gene overexpression makes cell casein synthesis capability strengthen (P＜0.05), and the cell-proliferation activity of Kcnma1 gene overexpression increases (P＜0.05).
The complete sequence that pcr amplification goes out the Kcnma1 gene in the present embodiment step 2 carries out the RNA interference experiment: little RNA sequences are disturbed in synthetic 3 pairs of design, by screening Optimal Jamming sequence, Optimal Jamming dosage and Optimal Jamming time behind the liposome transfection mammary epithelial cell; The result uses and disturbs 24 hours effects of sheet effect better as shown in Figure 3, and the proliferation and differentiation, casein output and the stat5 signal path that detect mammary epithelial cell after goal gene is by silence start situation; As can be seen from the results, the Kcnma1 gene silencing descends (P＜0.05) cell casein synthesis capability, and the disturbed cell-proliferation activity of Kcnma1 gene reduces (P＜0.05); Weatern Blot detected result as shown in Figure 4, the mammary epithelial cell casein resultant quantity of visible Kcnma1 gene silencing descends, the pSTAT5 expression amount reduces, this milk-protein synthesis capability that all shows mammary epithelial cell this moment descends; Flow cytometer detects the cell cycle result as shown in Figure 5, and the mammary epithelial cell division growth of visible Kcnma1 gene silencing becomes inactive, and the cell that is in division stage reduces.
Experiment shows: the Kcnma1 gene is to influence cell proliferation and milk-protein synthetic critical function gene in the cow mammary gland, plays important key effect in cow mammary gland growth and lactation function; By regulation and control to the Kcnma1 gene, can change the lactation function of cow mammary gland, reach the change milk character, increase the purpose of economic benefit; Proved that the Kcnma1 gene has specific triumph function in mammary gland, and can be used for gene regulating and change related application such as breeding performonce fo animals.
Embodiment two: present embodiment and embodiment one are different is that the reaction system of pcr amplification in the step 2 is 25 μ L reaction systems, is made up of following ingredients:
| Composition | Consumption |
| TaKaRa?Ex?Taq(5U/μl) | 0.25μl |
| 10×PCR?Buffer(Mg 2+Plus) | 5μl |
| DNTP Mixture (each 2.5mmol/L) | 4μl |
| The cDNA masterplate | 1μl |
| Upstream primer (20 μ mol/L) | 1μl |
| Downstream primer (20 μ mol/L) | 1μl |
| The sterilization tri-distilled water | 12.75μl |
The pcr amplification condition is: 94 ℃ of sex change 3min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 5min, totally 38 circulations, 72 ℃ are extended 10min, 4 ℃ of insulations again.Other step and parameter are identical with embodiment one.
Embodiment three: what present embodiment was different with one of embodiment one to two is the concrete use step operational manual of pSV-β in the step 2-Galactosidase Control Vector.Other step and parameter are identical with one of embodiment one to two.
PSV-β in the present embodiment-Galactosidase Control Vector (pSV-beta-galactosidase enzymes control vector) plasmid is for buying Bai Puluomaige (Beijing) Bioisystech Co., Ltd.
Embodiment four: present embodiment is different with one of embodiment one to three is that the system that connects in the step 2 is as follows:
| Composition | Consumption |
| The complete sequence of Kcnma1 gene | 3.0 μ L |
| The pGEM-T carrier | 1.0 μ L |
| 2 * Buffer | 5.0 μ L |
| T 4Ligase enzyme | 1.0 μ L. |
Other step and parameter are identical with one of embodiment one to three.
Embodiment five: present embodiment is different with one of embodiment one to four is that the total order that pcr amplification goes out the Kcnma1 gene in the step 2 is classified as
gttgctagct?atggcaaatg?gtggcggcgg?cggcggcggc?ggcggcggag?gcagcagtct 60
tagaatgagc?agcaatatcc?acgcgaacca?tctcagccta?gacgcgtcct?cctcctcctc 120
ttcttcctcc?tcctcctctt?cctcgtcgtc?ctcggtccac?gagcccaaga?tggatgcgct 180
catcatcccg?gtgaccatgg?aggtgccgtg?cgacagccgg?ggccaacgga?tgtggtgggc 240
tttcctggcc?tcctccatgg?tgactttctt?cggcggcctc?ttcatcatct?tgctctggag 300
gacgctcaag?tacctgtgga?ccgtttgctg?ccactgcggg?ggcaaaacga?aggaggccca 360
gaagattaac?aatggctcaa?gccaggcaga?tggcactctc?aagccagtgg?atgaaaaaga 420
ggagacggtg?gcagccgagg?tcggctggat?gacctccgtg?aaagactggg?cgggggtgat 480
gatatctgcc?cagacgctga?ctggcagagt?cctggttgtc?ttagtctttg?ctctcagcat 540
tggtgcactt?gtaatatact?tcatagattc?gtcaaaccca?atagaatcct?gccagaattt 600
ctacaaagat?ttcacattac?agatcgacat?ggcgttcaac?gtattcttcc?ttctctactt 660
tggcttgcgg?tttattgcag?ccaacgataa?gctatggttc?tggcttgaag?tgaactctgt 720
ggtagatttc?ttcacggtcc?cccctgtgtt?tgtgtccgtg?tacttaaaca?gaagttggct 780
tggtttgaga?tttttaagag?ctctcagact?gatccagttt?tcagaaattt?tacagtttct 840
gaatatcctg?aaaacaagta?attccatcaa?gctggtgaat?ctgctctcca?tttttatcag 900
cacgtggctc?acagcagccg?ggttcatcca?tttggtggag?aattcagggg?acccatggga 960
aaatttccaa?aacaaccagg?ctcttaccta?ctgggaatgt?gtctatctgc?tcatggtgac 1020
catgtccact?gttggttacg?gggatgttta?tgcaaaaacc?acgctcgggc?gtctcttcat 1080
ggtcttcttc?atcctcgggg?gactggccat?gtttgccagc?tacgtccctg?aaatcataga 1140
gttaatagga?aaccgcaaga?aatacggggg?ctcctatagt?gcggttagtg?gaagaaagca 1200
catagtggtc?tgtggacaca?tcacactgga?gagcgtttcc?aacttcctga?aggactttct 1260
gcacaaggac?cgggacgatg?tcaatgtgga?gatcgtcttt?cttcacaaca?tctcccctaa 1320
cctggagctg?gaagccttgt?tcaaacgaca?ttttactcag?gtggagtttt?atcagggttc 1380
agtcctcaat?ccacatgatc?ttgcaagagt?caagatagag?tcagcagatg?cgtgcctgat 1440
ccttgccaat?aagtactgcg?cagaccccga?tgctgaagat?gcctccaaca?tcatgagagt 1500
catctccata?aagaactacc?acccgaagat?aagaatcatc?actcagatgc?tgcagtatca 1560
caataaggcc?catctgctaa?acatcccgag?ctggaattgg?aaagaggggg?atgatgcaat 1620
ctgcctcgca?gaactgaagc?tggggttcat?cgcccagagc?tgcctggctc?aagggctctc 1680
caccatgctc?gccaacctct?tctccatgag?gtcattcata?aagattgagg?aagacacgtg 1740
gcagaaatac?tacttggaag?gagtctcaaa?tgaaatgtac?acagaatatc?tctccagtgc 1800
cttcgtgggt?ctgtccttcc?cgactgtttg?tgagctgtgt?tttgtgaagc?tcaagcttct 1860
catgatagcc?attgagtaca?agtccgccaa?tcgagagagc?cgtatattaa?ttaatcctgg 1920
aaaccatctt?aagatccaag?aaggtacttt?aggatttttc?atcgcaagtg?atgccaaaga 1980
agttaaaagg?gcattttttt?actgcaaggc?ctgtcatgat?gacatcacgg?atcccaaaag 2040
gataaaaaaa?tgcggctgca?aacggcttga?agatgaacag?ccgtcgacac?tgtcacccaa 2100
aaaaaagcag?cgcaacggag?gcatgcggaa?ctcacccagc?tcgtcgccca?agctgatgag 2160
gcatgacccc?ttgttaattc?ctggcaatga?tcagattgac?aacatggact?ccaatgtgaa 2220
gaaatatgac?tctactggga?tgtttcactg?gtgtgcgccc?aaggagatag?agaaagtcat 2280
cctgacccga?agtgaagctg?ccatgaccgt?cctgagcggc?cacgtggtgg?tctgcatctt 2340
tggcgacgtc?agctctgccc?tgattggcct?tcggaacctg?gtgatgccgc?tccgtgccag 2400
caactttcac?taccacgagc?tcaagcacat?cgtgtttgtg?ggctccatcg?agtacctcaa 2460
gcgggaatgg?gagacactgc?ataacttccc?caaggtttcc?atcttgcctg?gtacgccatt 2520
aagtcgggct?gatttaaggg?ccgtcaacat?caacctctgt?gacatgtgcg?ttatcctgtc 2580
agccaatcag?aataatattg?atgatacttc?gctgcaggac?aaggaatgca?tcttggcgtc 2640
actcaacatc?aaatctatgc?agtttgatga?cagcatcggg?gtcttgcagg?ctaattccca 2700
agggttcaca?cctccaggaa?tggaccgatc?ctcaccagat?aacagcccgg?tacatgggat 2760
gttacgccag?ccctccatca?ctactggggt?caacatcccc?atcatcactg?aactagtgaa 2820
cgatactaat?gttcagtttt?tggaccaaga?cgatgacgat?gaccccgaca?cagaactgta 2880
cctcacgcag?ccgtttgcat?gtgggacagc?gttcgccgtc?agtgtcctgg?attcactcat 2940
gagcgcaaca?tacttcaatg?acaacatcct?taccctgata?cggaccctgg?tgactggagg 3000
agccacacca?gagctggagg?ctttgatcgc?tgaggagaat?gcactccgcg?ggggctacag 3060
caccccccag?acgctggcca?acagggaccg?ctgccgtgtg?gcccagttag?cgctgctgga 3120
cgggcccttt?gcggacctgg?gggatggtgg?ttgttatggt?gatctgttct?gcaaagctct 3180
gaaaacatac?aatatgcttt?gttttggaat?ttatcggctg?agagatgctc?acctcagcac 3240
ccccagccag?tgcacgaaga?ggtatgtcat?caccaacccc?ccgtacgagt?ttgagctcgt 3300
gccgacggac?ctgatcttct?gcttaatgca?gtttgaccac?aacgccggcc?agtcccgggc 3360
cagcctctcc?cattcctccc?actcgtccca?gtcctctagc?aagaagagct?cctccgtcca 3420
ctccatccca?tccacggcaa?accgacagaa?ccggcccaag?tcccgggagt?cccgagacaa 3480
acagaagtac?gtgcaggaag?agcggctctg?atgtgtgtat?ccactgcctc 3530。
Other step and parameter are identical with one of embodiment one to four.
Embodiment six: present embodiment promotes that the cow mammary gland epithelium is fine and educates and the method for lactation is carried out according to the following steps: one, get the He Sitan bovine lactation phase and organize sample and adopt collagenase-trypsin digestion to separate epithelial cell, place cell grown cultures liquid to cultivate for 3 generations then, get the mammary epithelial cell of external cultivation; Two, according to the mRNA sequences Design primer of Kcnma1 gene, pcr amplification goes out the complete sequence of Kcnma1 gene, is connected with the pGEM-T carrier then, connects product transformed into escherichia coli DH5 α, obtains cloning vector plasmids; Three, pSV-β-Galactosidase Control Vector plasmid and cloning vector plasmids use AgeI and XbaI to carry out double digestion respectively, reclaim required purpose fragment, connect and construction of expression vector, use the mammary epithelial cell of expression vector transfection vitro culture again, make Kcnma1 gene overexpression in the mammary epithelial cell of vitro culture, finish promptly that the cow mammary gland epithelium is fine educates and the promotion of lactation; Wherein cell grown cultures liquid is made up of the DMEM high glucose medium of 90ml, FBS (foetal calf serum) and the Regular Insulin that 10ml concentration is 50mg/ml, the penicillin of 100 μ l, 100,000 units/ml and the Streptomycin sulphate of 100 μ l, 100,000 units/ml of 10ml in the step 1; In the step 2 the used upstream primer of pcr amplification be 5 '-GCACCGGTTGCTAGCTATGGCAAATGGTGGC-3 ', downstream primer is 5 '-GCTCTAGAGGCAGTGGATACACACATCAGAG-3 ', and added the restriction enzyme site of restriction enzyme A geI and XbaI respectively at 5 of upstream and downstream primer ' end.
The reaction system of pcr amplification is 25 μ L reaction systems in the present embodiment step 2, is made up of following ingredients:
| Composition | Consumption |
| TaKaRa?Ex?Taq(5U/μl) | 0.25μl |
| 10×PCR?Buffer(Mg 2+Plus) | 5μl |
| DNTP Mixture (each 2.5mmol/L) | 4μl |
| The cDNA masterplate | 1μl |
| Upstream primer (20 μ mol/L) | 1μl |
| Downstream primer (20 μ mol/L) | 1μl |
| The sterilization tri-distilled water | 12.75μl |
The pcr amplification condition is: 94 ℃ of sex change 3min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 5min, totally 38 circulations, 72 ℃ are extended 10min, 4 ℃ of insulations again.
The system that connects in the present embodiment step 2 is as follows:
| Composition | Consumption |
| The complete sequence of Kcnma1 gene | 3.0 μ L |
| The pGEM-T carrier | 1.0 μ L |
| 2 * Buffer | 5.0 μ L |
| T 4Ligase enzyme | 1.0 μ L. |
The experiment proved that the Kcnma1 gene plays important key effect in the present embodiment in cow mammary gland growth and lactation function, can change the lactation function of cow mammary gland, reach the change milk character, can promote cow mammary gland epithelial cells to grow and lactation.
Claims (4)
1. one kind promotes cow mammary gland epithelial cells to grow and the method for lactation, it is characterized in that promoting that the cow mammary gland epithelium is fine educates and the method for lactation is carried out according to the following steps: one, get the He Sitan bovine lactation phase and organize sample and adopt collagenase-trypsin digestion to separate epithelial cell, place cell grown cultures liquid to cultivate for 3 generations then, get the mammary epithelial cell of external cultivation; Two, according to the mRNA sequences Design primer of Kcnma1 gene, pcr amplification goes out the complete sequence of Kcnma1 gene, is connected with the pGEM-T carrier then, connects product transformed into escherichia coli DH5 α, obtains cloning vector plasmids; Three, pSV-β-GalactosidaseControl Vector plasmid and cloning vector plasmids use AgeI and XbaI to carry out double digestion respectively, reclaim required purpose fragment, connect and construction of expression vector, use the mammary epithelial cell of expression vector transfection vitro culture again, make Kcnma1 gene overexpression in the mammary epithelial cell of vitro culture, finish promptly that the cow mammary gland epithelium is fine educates and the promotion of lactation; Wherein cell grown cultures liquid is made up of the DMEM high glucose medium of 90ml, FBS and the Regular Insulin that 10ml concentration is 50mg/ml, the penicillin of 100 μ l, 100,000 units/ml and the Streptomycin sulphate of 100 μ l, 100,000 units/ml of 10ml in the step 1; In the step 2 the used upstream primer of pcr amplification be 5 '-GCACCGGTTGCTAGCTATGGCAAATGGTGGC-3 ', downstream primer is 5 '-GCTCTAGAGGCAGTGGATACACACATCAGAG-3 ', and added the restriction enzyme site of restriction enzyme A geI and XbaI respectively at 5 of upstream and downstream primer ' end.
2. a kind of method that promotes cow mammary gland epithelial cells growth and lactation according to claim 1, the reaction system that it is characterized in that pcr amplification in the step 2 is 25 μ L reaction systems, is made up of following ingredients:
The composition consumption
TaKaRa?Ex?Taq(5U/μl) 0.25μl
10×PCR?Buffer(Mg
2+Plus) 5μl
DNTP Mixture (each 2.5mmol/L) 4 μ l
CDNA masterplate 1 μ l
Upstream primer (20 μ mol/L) 1 μ l
Downstream primer (20 μ mol/L) 1 μ l
Sterilization tri-distilled water 12.75 μ l
The pcr amplification condition is: 94 ℃ of sex change 3min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 5min, totally 38 circulations, 72 ℃ are extended 10min, 4 ℃ of insulations again.
3. a kind of method that promotes cow mammary gland epithelial cells growth and lactation according to claim 1 and 2 is characterized in that the system that connects in the step 2 is as follows:
The composition consumption
The complete sequence 3.0 μ L of Kcnma1 gene
PGEM-T carrier 1.0 μ L
2×Buffer 5.0μL
T
4Ligase enzyme 1.0 μ L.
4. a kind of cow mammary gland epithelial cells that promotes according to claim 3 is grown and the method for lactation, it is characterized in that the total order that pcr amplification in the step 2 goes out the Kcnma1 gene classifies as
gttgctagct?atggcaaatg?gtggcggcgg?cggcggcggc?ggcggcggag?gcagcagtct 60
tagaatgagc?agcaatatcc?acgcgaacca?tctcagccta?gacgcgtcct?cctcctcctc 120
ttcttcctcc?tcctcctctt?cctcgtcgtc?ctcggtccac?gagcccaaga?tggatgcgct 180
catcatcccg?gtgaccatgg?aggtgccgtg?cgacagccgg?ggccaacgga?tgtggtgggc 240
tttcctggcc?tcctccatgg?tgactttctt?cggcggcctc?ttcatcatct?tgctctggag 300
gacgctcaag?tacctgtgga?ccgtttgctg?ccactgcggg?ggcaaaacga?aggaggccca 360
gaagattaac?aatggctcaa?gccaggcaga?tggcactctc?aagccagtgg?atgaaaaaga 420
ggagacggtg?gcagccgagg?tcggctggat?gacctccgtg?aaagactggg?cgggggtgat 480
gatatctgcc?cagacgctga?ctggcagagt?cctggttgtc?ttagtctttg?ctctcagcat 540
tggtgcactt?gtaatatact?tcatagattc?gtcaaaccca?atagaatcct?gccagaattt 600
ctacaaagat?ttcacattac?agatcgacat?ggcgttcaac?gtattcttcc?ttctctactt 660
tggcttgcgg?tttattgcag?ccaacgataa?gctatggttc?tggcttgaag?tgaactctgt 720
ggtagatttc?ttcacggtcc?cccctgtgtt?tgtgtccgtg?tacttaaaca?gaagttggct 780
tggtttgaga?tttttaagag?ctctcagact?gatccagttt?tcagaaattt?tacagtttct 840
gaatatcctg?aaaacaagta?attccatcaa?gctggtgaat?ctgctctcca?tttttatcag 900
cacgtggctc?acagcagccg?ggttcatcca?tttggtggag?aattcagggg?acccatggga 960
aaatttccaa?aacaaccagg?ctcttaccta?ctgggaatgt?gtctatctgc?tcatggtgac 1020
catgtccact?gttggttacg?gggatgttta?tgcaaaaacc?acgctcgggc?gtctcttcat 1080
ggtcttcttc?atcctcgggg?gactggccat?gtttgccagc?tacgtccctg?aaatcataga 1140
gttaatagga?aaccgcaaga?aatacggggg?ctcctatagt?gcggttagtg?gaagaaagca 1200
catagtggtc?tgtggacaca?tcacactgga?gagcgtttcc?aacttcctga?aggactttct 1260
gcacaaggac?cgggacgatg?tcaatgtgga?gatcgtcttt?cttcacaaca?tctcccctaa 1320
cctggagctg?gaagccttgt?tcaaacgaca?ttttactcag?gtggagtttt?atcagggttc 1380
agtcctcaat?ccacatgatc?ttgcaagagt?caagatagag?tcagcagatg?cgtgcctgat 1440
ccttgccaat?aagtactgcg?cagaccccga?tgctgaagat?gcctccaaca?tcatgagagt 1500
catctccata?aagaactacc?acccgaagat?aagaatcatc?actcagatgc?tgcagtatca 1560
caataaggcc?catctgctaa?acatcccgag?ctggaattgg?aaagaggggg?atgatgcaat 1620
ctgcctcgca?gaactgaagc?tggggttcat?cgcccagagc?tgcctggctc?aagggctctc 1680
caccatgctc?gccaacctct?tctccatgag?gtcattcata?aagattgagg?aagacacgtg 1740
gcagaaatac?tacttggaag?gagtctcaaa?tgaaatgtac?acagaatatc?tctccagtgc 1800
cttcgtgggt?ctgtccttcc?cgactgtttg?tgagctgtgt?tttgtgaagc?tcaagcttct 1860
catgatagcc?attgagtaca?agtccgccaa?tcgagagagc?cgtatattaa?ttaatcctgg 1920
aaaccatctt?aagatccaag?aaggtacttt?aggatttttc?atcgcaagtg?atgccaaaga 1980
agttaaaagg?gcattttttt?actgcaaggc?ctgtcatgat?gacatcacgg?atcccaaaag 2040
gataaaaaaa?tgcggctgca?aacggcttga?agatgaacag?ccgtcgacac?tgtcacccaa 2100
aaaaaagcag?cgcaacggag?gcatgcggaa?ctcacccagc?tcgtcgccca?agctgatgag 2160
gcatgacccc?ttgttaattc?ctggcaatga?tcagattgac?aacatggact?ccaatgtgaa 2220
gaaatatgac?tctactggga?tgtttcactg?gtgtgcgccc?aaggagatag?agaaagtcat 2280
cctgacccga?agtgaagctg?ccatgaccgt?cctgagcggc?cacgtggtgg?tctgcatctt 2340
tggcgacgtc?agctctgccc?tgattggcct?tcggaacctg?gtgatgccgc?tccgtgccag 2400
caactttcac?taccacgagc?tcaagcacat?cgtgtttgtg?ggctccatcg?agtacctcaa 2460
gcgggaatgg?gagacactgc?ataacttccc?caaggtttcc?atcttgcctg?gtacgccatt 2520
aagtcgggct?gatttaaggg?ccgtcaacat?caacctctgt?gacatgtgcg?ttatcctgtc 2580
agccaatcag?aataatattg?atgatacttc?gctgcaggac?aaggaatgca?tcttggcgtc 2640
actcaacatc?aaatctatgc?agtttgatga?cagcatcggg?gtcttgcagg?ctaattccca 2700
agggttcaca?cctccaggaa?tggaccgatc?ctcaccagat?aacagcccgg?tacatgggat 2760
gttacgccag?ccctccatca?ctactggggt?caacatcccc?atcatcactg?aactagtgaa 2820
cgatactaat?gttcagtttt?tggaccaaga?cgatgacgat?gaccccgaca?cagaactgta 2880
cctcacgcag?ccgtttgcat?gtgggacagc?gttcgccgtc?agtgtcctgg?attcactcat 2940
gagcgcaaca?tacttcaatg?acaacatcct?taccctgata?cggaccctgg?tgactggagg 3000
agccacacca?gagctggagg?ctttgatcgc?tgaggagaat?gcactccgcg?ggggctacag 3060
caccccccag?acgctggcca?acagggaccg?ctgccgtgtg?gcccagttag?cgctgctgga 3120
cgggcccttt?gcggacctgg?gggatggtgg?ttgttatggt?gatctgttct?gcaaagctct 3180
gaaaacatac?aatatgcttt?gttttggaat?ttatcggctg?agagatgctc?acctcagcac 3240
ccccagccag?tgcacgaaga?ggtatgtcat?caccaacccc?ccgtacgagt?ttgagctcgt 3300
gccgacggac?ctgatcttct?gcttaatgca?gtttgaccac?aacgccggcc?agtcccgggc 3360
cagcctctcc?cattcctccc?actcgtccca?gtcctctagc?aagaagagct?cctccgtcca 3420
ctccatccca?tccacggcaa?accgacagaa?ccggcccaag?tcccgggagt?cccgagacaa 3480
acagaagtac?gtgcaggaag?agcggctctg?atgtgtgtat?ccactgcctc 3530。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424008A (en) * | 2020-03-31 | 2020-07-17 | 吉林大学 | Experimental method for promoting animal mammary gland development by nicotinic acid |
| CN112626236A (en) * | 2021-01-14 | 2021-04-09 | 华南农业大学 | CircRNA marker related to cow mammary epithelial cell apoptosis and application thereof |
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| WO2009052567A1 (en) * | 2007-10-23 | 2009-04-30 | Clinical Genomics Pty. Ltd. | A method of diagnosing neoplasms - ii |
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| WO2009052567A1 (en) * | 2007-10-23 | 2009-04-30 | Clinical Genomics Pty. Ltd. | A method of diagnosing neoplasms - ii |
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|---|
| 《GenBank》 20100121 Santi CM et al Bos taurus potassium large conductance calcium-activated channel, subfamily M, alpha member 1 (KCNMA1) 4 , 2 * |
| 《中国博士学位论文全文数据库农业科技辑》 20100315 侯晓明 奶牛乳腺发育及泌乳重要功能基因的筛选 1-3 , 第3期 2 * |
| 《中国博士学位论文全文数据库农业科技辑》 20100315 侯晓明 奶牛乳腺发育及泌乳重要功能基因的筛选 4 , 第3期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424008A (en) * | 2020-03-31 | 2020-07-17 | 吉林大学 | Experimental method for promoting animal mammary gland development by nicotinic acid |
| CN112626236A (en) * | 2021-01-14 | 2021-04-09 | 华南农业大学 | CircRNA marker related to cow mammary epithelial cell apoptosis and application thereof |
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