CN102206675A - Novel vector for raising content of polyunsaturated fatty acids in animal body - Google Patents
Novel vector for raising content of polyunsaturated fatty acids in animal body Download PDFInfo
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Abstract
The invention discloses a novel vector for raising the content of polyunsaturated fatty acids in an animal body. The expression vector provided by the invention can simultaneously transcribe and express omega-3 fatty acid dehydrogenase and delta12 fatty acid dehydrogenase, and the vector sequence contains gene sequences of Sequence NO.1 and Sequence NO.2. In addition, the sequences of the enhancer SP163 and IRES are added between the sFat and Fat2 genes, and the vector provided by the invention can highly express two types of dehydrogenase at the same time. The vector provided by the invention is also transfected into the CHO cells, followed by the screening of antibiotics to obtain a stable cell line for highly expressing the sFat and Fat2 genes with the gene expression amount being 105 times that of untransfected cells. The invention also discloses a concrete method for raising offspring animals having high content of polyunsaturated fatty acids by using the vector. The vector provided by the invention lays a foundation for producing animal products having high content of omega-3 polyunsaturated fatty acids and improving the quality of meat products or dairy products by producing transgenic livestock.
Description
Technical field
The present invention relates to a kind of structure of new expression vector.Particularly, cloned gene sFat-1 and Fat-2 sequence from nematode Caenorhabditis elegans, and carried out codon optimized.The present invention has made up high-expression vector pcDNA3.1-sFat-SP163-IRES-Fat2, can be applied to improve the content of omega-3 polyunsaturated fatty acids in the animal body.
Background technology
Polyunsaturated fatty acid (PUFAs) is meant more than 16 carbon and contains the lipid acid of two keys more than two that ω-3PUFAs and ω-6PUFAs are wherein most important two types.Alpha-linolenic acid (α-linolenic acid, α LA), eicosapentaenic acid (eicosapentaenoic acid, EPA), docosapentanoic acid (docosapentaenoic acid, DPA), (docosahexaenoic acid DHA) all belongs to ω-3PUFAs to docosahexenoic acid; And linolic acid (linoleic acid, LA), (γ-lenolenic acid, (arochidonic acid AA) waits and belongs to ω-6PUFAs gamma-linolenic acid for γ-LA), arachidonic acid.More and more studies have shown that [Chen W X, Chen D G, Cheng X F, etc.Effects of ω-3Polyunsaturated Fatty Acids And Taurine on Rat Brain Development.Acta Nutrimenta Sinica, 1998,20 (4): 25-30.], PUFAs has biological function widely, they participate in the formation of cytolemma and as the signaling molecule in many cell response processes, with being closely related of the mankind's multiple disease, its suitable content is for human and other mammiferous normal developments and keep good condition of health of crucial importance.
Omega-3 polyunsaturated fatty acids is the important component of cellularity, keeps the effect that a certain amount of ω-3PUFAs can play preventing cardiovascular disease, nerve degenerative diseases even cancer in the mammalian body.Along with the increase of people, the food demand that is rich in this composition is also increased greatly n-3 polyunsaturated fatty acid understanding.But Mammals self can not be synthesized ω-3PUFAs owing to lack the omega-fatty acid desaturase, can only replenish from food.The Fat-1 gene extensively is present in fungi, plant and some lower animals, and it can be converted into ω-3 form with polyunsaturated fatty acid from ω-6 and bring into play function by producing the omega-fatty acid desaturase.The researchist is transgenic animal, as mouse, pig etc., expressed the fat-1 gene, for research ω-3PUFAs function provides efficiently, medical science, trophology animal model accurately.SFat-1 gene and fat-1 gene action are similar, also can be transcribed into the omega-fatty acid desaturase.
Discover that arachidonic acid, docosapentanoic acid and docosahexenoic acid exist on structural fat (structural lipids) the middle and high concentration ground of central nervous system (CNS), so it is necessary that these PUFAs are considered to brain, retina and nervous system development, indispensable in infantile nutrition, the deficiency of ω-3PUFAs can cause visual sensitivity and learning capacity to reduce.Physiological effects such as (prothombotic) was reacted in blood coagulation before ω-3 and ω-6PUFAs had tangible differentiation: ω-6PUFAs such as arachidonic excessive meeting to cause in metabolism and on the function, and caused inflammation and disease such as cardiovascular; On the contrary, ω-3PUFAs such as eicosapentaenic acid, docosahexenoic acid have been proved and can have prevented and treat multiple diseases such as cardiovascular disorder, sacroiliitis, cancer, and therefore, the importance of ω-3PUFAs is more outstanding.
The omega-3-aliphatic acid desaturase gene is ω-3PUFAs synthetic key gene, and they utilize ω-6PUFAs to be substrate, generates a unsaturated link(age) (ethylene linkage) in the 3rd carbon catalysis of fatty acid carbon chain methyl end, thus synthetic corresponding ω-3PUFAs.1997, people such as Spychalla have cloned the ω-3 delta 8 desaturase genes Fat-1[Spychalla J P that derives from nematode (Caenorhabditis elegans), Kinney A J, Browse J.Identification of an animal ω-3 fatty acid desaturase by heterologous expression in Arabidopsis.Proc Natl Acad Sci USA, 1997,94 (4): 1142～1147.], find that in mammalian cell Fat-1 albumen can catalysis be that substrate produces ω-3PUFAs with ω-6PUFAs.2004, the Mammals of the first fat-1 of commentaries on classics gene was born, and these change, and corresponding n-3PUFAs content increases in each tissue of fat-1 dna murine, and n-6/n-3 ratio also obviously descends.This transgenic mice [the Kang J X that is rich in n-3PUFAs, Wang J, Wu L, et al.Transgenic mice:fat-1mice convert n-6 to n-3 fatty acids.Nature, 2004,427:504], for effect, mechanism and the many associated disease researches of further studying n-3PUFAs provides good animal model, also provide a good thinking for improving human dietary.The lipid acid of transgenic mice detects and shows that the decapacitation of sFat-1 gene is brought into play outside the effect of its ω-3 desaturase fully, can also produce more long-chain eicosapentaenic acid and docosahexenoic acid.
The long-chain PUFAs of higher animal is positioned the desaturase on the film and is prolonged enzyme catalysis linoleic acid plus linolenic acid synthetic by a series of.Yet they but lack the synthetic necessary desaturase of long-chain PUFAs precursor linoleic acid plus linolenic acid; And ω-3 and ω-6 lipid acid can't transform in the higher animal body mutually.Therefore, higher animal must obtain these lipid acid PUFAs from food: specific spermatophyte (LA, GLA, ALA) and marine fishes (AA, EPA and DHA) and some Mammalss.Occurring in nature ω-6PUFAs content is abundant and ω-3PUFAs content is few, cause ω in the animal body-6PUFAs Excessive Intake and ω-3PUFAs takes in wretched insufficiency, the ratio that makes ω-6/ ω-3 is up to more than 16, according to relevant research, the ratio of ω-6 and ω-3PUFAs should be at 1: 1 for suitable, so human body is in the under-supply state of ω-3PUFAs usually.The omega-fatty acid dehydrogenase gene can be converted into omega-fatty acid by catalysis ω-6 lipid acid, thereby the expression study of omega-fatty acid dehydrogenase gene has become the focus that utilizes genetically engineered to produce PUFAs.Yet the omega-fatty acid desaturase is present in most of plants and the minority lower animal body, does not exist in Mammals and the human body.Though the omega-fatty acid desaturase is of a great variety but also be not utilized in the zooscopy in the plant, because embrane-associated protein separates and the difficulty of purifying, fixed animal omega-fatty acid desaturase is also very limited on the animal.At present, though the research of omega-fatty acid dehydrogenase gene is obtaining remarkable progress aspect catalysis and the regulation mechanism, for organism how to experience, transmission information, how to induce with regulate gene expression etc. also to be not very clear.
The lipid metabolism of organism can utilize the required lipid acid (lipid acid is synthetic) of the synthetic body of basic material; Or lipid acid decomposed obtain some little compositions (lipid acid decomposition); Can also become another form (lipid acid conversion) to lipid acid from a kind of form, body is regulated the ratio of lipid acid just in this way, to satisfy self needs, studies show that the Fat-2 gene can produce Δ 12 fatty acid dehydrogenases in vivo, the dehydrogenation on 12 of Δs of this enzyme catalysis oleic acid (18:1n-9) generates linolic acid (18:2n-6).The omega-fatty acid desaturase has the lipid acid transition function, the carbon-carbon single bond of specific position on the fatty acid carbon chain can be oxidized to two keys, forms unsaturated fatty acids or high-grade polyunsaturated fatty acid more.Therefore, want to improve ω-6 and ω-3PUFAs ratio in the mammalian body, improve the content of ω-3PUFAs in the mammalian body, the food that has higher ω-3PUFAs content except absorption, it is also conceivable that the omega-fatty acid dehydrogenase gene is imported in the animal body by transgenic method, make and himself can be converted into ω-3PUFAs to too much ω-6PUFAs, tackle the problem at its root.
Linolic acid, linolenic acid are the intravital indispensable fatty acids of Mammals, and self can not synthesize but Mammals is owing to lacking Δ 12 and omega-fatty acid desaturase, can only absorb from food.Δ 12 and omega-fatty acid desaturase are present in fungi, plant and some lower animals.ω-3PUFAs content is few because occurring in nature ω-6PUFAs content is abundant, causes ω in the animal body-6PUFAs Excessive Intake and ω-3PUFAs takes in wretched insufficiency.Solve ω-6PUFAs and ω-3PUFAs imbalance problem in animal body for this reason, have great importance.The present invention has designed a kind of carrier for expression of eukaryon, omega-fatty acid desaturase and Δ 12 fatty acid dehydrogenases can be transcribed and express to this expression vector simultaneously, Δ 12 fatty acid dehydrogenases can increase linoleic content in the cell, the omega-fatty acid desaturase can be converted into ω-3PUFAs with linolic acid and other ω-6PUFAs, thereby increase the content of ω-3PUFAs in the animal body better, improve the ratio of ω-6/ ω-3, and then generate the animal product that is rich in ω-3PUFAs the human body beneficial.The present invention produces breeding transgenic livestock and lays the foundation for improving meat product or milk preparation quality, produces the animal product (meat, milk) that is rich in ω-3PUFAs and provides a good approach for improving human dietary.
Summary of the invention
The present invention has made up a kind of novel carriers that improves content of polyunsaturated fatty acid in the animal body, and described carrier comprises omega-fatty acid dehydrogenase gene sFat, Δ 12 fatty acid dehydrogenase gene Fat2, enhanser SP163 and IRES sequence.Their gene order is respectively Sequnence NO.1, Sequnence NO.2, Sequnence NO.3 and Sequnence NO.4.
The contained Sequnence NO.1 sequence of carrier that the present invention makes up is to carry out codon optimized obtaining by the sFat-1 gene (omega-3-aliphatic acid desaturase gene) to nematode Caenorhabditis elegans.Can better in eukaryote, express through the codon optimized sFat gene that obtains.
Carrier of the present invention is transfected to Chinese hamster ovary celI, through the screening of microbiotic G418, has obtained the stable cell line of while high expression level sFat and Fat2 gene.Its sFat of the stable cell line that obtains and Fat2 expression of gene amount are 10 of non-transfected cells expression amounts
5Doubly.
Carrier of the present invention is expressed omega-fatty acid desaturase and Δ 12 fatty acid dehydrogenases simultaneously in eukaryotic cell, improved the content of cell and animal omega-3 polyunsaturated fatty acids.Because omega-fatty acid desaturase and Δ 12 fatty acid dehydrogenases are transcribed and expressed to described carrier simultaneously, Δ 12 fatty acid dehydrogenases can increase linoleic content in the cell, the omega-fatty acid desaturase can be converted into ω-3PUFAs with linolic acid and other ω-6PUFAs, thereby has increased the content of ω-3PUFAs in cell and the animal body better.
Utilize carrier of the present invention to set up omega-3 polyunsaturated fatty acids content method in a kind of raising filial generation animal body, comprising:
1) preparation carrier for expression of eukaryon pcDNA3.1-sFat-SP163-IRES-Fat2;
2) carrier for expression of eukaryon pcDNA3.1-sFat-SP163-IRES-Fat2, liposome and platform phenol indigo plant are mixed with transfection liquid;
3) adopt Minimally Invasive Surgery, above-mentioned transfection liquid is gone into minute multi-point injection in the testis of animal both sides again and the jenny mating;
4),, detect filial generation animal sFat and Fat2 expression of gene with the Auele Specific Primer of sFat and Fat2 gene by round pcr.Detect the eliminating false positive by the Southern trace.Utilize the method for real-time quantitative PCR to detect the expression of positive animal goal gene.
The method of the invention can be applied to cultivate the animal new variety that are rich in omega-3 polyunsaturated fatty acids.
Description of drawings
The KpnI/NotI double digestion electrophorogram of Fig. 1 pEASY-Blunt-Simple-sFat-1 and pcDNA3.1.
1:pcDNA3.1 KpnI, Not I enzyme are cut; 2:1Kb marker; 3:DL2000marker; 4:pEASY-Blunt-Simple-sFat-1KpnI, NotI enzyme are cut.
Fig. 2
And the NotI/XbaI double digestion electrophorogram of pcDNA3.1 (+)-sFat-SP163.
NotI, XbaI enzyme cutting; 2:1Kb marker; (3:pcDNA3.1+)-sFat-SP163, NotI, XbaI enzyme cutting.
Fig. 3 pcDNA3.1 (+)-sFat-SP163 collection of illustrative plates.
Fig. 4 pcDNA3.1-sFat-SP163-IRES-Fat2 carrier structure figure.
Three kinds of plasmid destination gene expressions of Fig. 5 transient transfection level.Solid column is represented the sFat-1 expression level, and grid posts is represented the Fat-2 expression level.A group transfection pcDNA3.1 plasmid, B group transfection pcDNA3.1-sFat1-SP163 plasmid, C group transfection pcDNA3.1-sFat-SP163-IRES-Fat2 plasmid.
Embodiment one Construction of eukaryotic and functional evaluation
One. research method
1. vector construction
1) the gene complete sequence of sFat-1 and Fat-2 sequence is analyzed, checked whether gene inside contains complicated secondary structure and tumor-necrosis factor glycoproteins.According to the particular case design synthetic schemes of sequence, the design of carrying out strand oligo earlier reaches synthesizes, and utilizes PCR that synthetic oligo is spliced into complete gene.
2) will synthesize pack into pEASY-Blunt Simple carrier and be converted into competent cell Trans1-T1 Phage Resistant Cell of good sFat-1 gene, in the sequence verification recombinant clone gene order whether with require to conform to.
3) contain the purpose fragment that the segmental carrier of purpose obtains 1398bp with the full gene synthetic of KpnI/NotI double digestion, the pcDNA3.1 carrier obtains the linearized vector sequence of 5.4Kbp by the KpnI/NotI double digestion.
4) cut the dna fragmentation that glue reclaims the purpose size, use T4DNA ligase room temperature and connect 2h.
5) will connect product and be converted into competent cell, in the sequence verification recombinant clone gene order whether with require to conform to, obtain carrier pcDNA3.1 (+)-sFat-SP163.
6) connect bacterium at 1: 1000, spend the night and shake bacterium.Man-PureLink HiPure Plasmid Filter Purification Kits extracts plasmid DNA ,-20 ℃ of preservations.
7) will synthesize good Fat-2 gene packs into
Carrier, and be converted into competent cell Top10 competent cell, in the sequence verification recombinant clone gene order whether with require to conform to.
8) obtain the fragment of about 6740bp with NotI/XbaI double digestion pcDNA3.1 (+)-sFat-SP163, contain the fragment that the segmental carrier of purpose obtains about 1750bp with the full gene synthetic of NotI/XbaI double digestion.
9) cut the dna fragmentation that glue reclaims the purpose size, use T4DNA ligase room temperature and connect 2h.
10) will connect product and be converted into competent cell, in the sequence verification recombinant clone gene order whether with require to conform to.
11) connect bacterium at 1: 1000, spend the night and shake bacterium.Man-PureLink HiPure Plasmid Filter Purification Kits extracts plasmid DNA, the plasmid of gained-20 ℃ preservation.
2.CHO cell wink Pignus pignoris grain and detection of expression
1) cultivates Chinese hamster ovary celI to logarithmic phase;
2) with the Invitrogen lipofectamine2000 of company transfection pcDNA3.1-sFat1-SP163 plasmid, qPCR testing goal gene sFat1 expresses; The pcDNA3.1-sFat-SP163-IRES-Fat2 of transfection simultaneously plasmid, qPCR detects sFat and Fat2.
3. cell microbiotic the best is screened dosage experiments (Kill Curves)
1) growth conditions is good, be in the cell of logarithmic phase, be inoculated in 24 orifice plates than 1: 10 according to density.Behind the cell bed board 24 hours, can add microbiotic and screen.
2) set suitable screening dose gradient according to the filter information of antibiotic dosage range and relevant target cell, each gradient is provided with two multiple holes.
3) the lasting growing state of observing and writing down cell changed a subculture in per three days, added microbiotic and continued to screen.
4) target cell microbiotic the best is screened dosage for can cause a large amount of necrocytosiss in 3 days, and kills the concentration of all cells in two weeks.
4. three mixed stability cell line selections (pcDNA3.1-sFat1-SP163, pcDNA3.1-sFat-SP163-IRES-Fat2 and negative control pcDNA3.1 empty carrier)
1) uses the high purity plasmid kit, the plasmid that the preparation fs makes up;
2) cultivate Chinese hamster ovary celI to growing the vigorous stage;
3) plasmid transfection changes liquid;
4) add G418 and screened for 3 weeks, the mixed zone resistant cell is expanded to q.s and is used for detecting, simultaneously frozen part cell;
5) qPCR detects cell mixing goal gene mRNA.
Two. the result
1. sequence verification result:
With complete synthesis sFat-1 gene sequencing, its sequence is seen Sequnence NO.1, and the sequence after the sequencing result demonstration is optimized with the Fat-1 gene codon of nematode Caenorhabditis elegans is in full accord.
With synthetic IRES-Fat2 gene sequencing, its sequence is seen Sequnence NO.2, and sequencing result shows with the sequence that is provided in full accord.
2. enzyme is cut the result:
The synthetic good right-on sFat-1 gene of sequence is cut with KpnI/NotI is two from pEASY-Blunt Simple-sFat-1 carrier, simultaneously with the two pcDNA3.1 carriers of cutting of KpnI/NotI, obtain the purpose fragment of 1398bp and the linearized vector of 5.4Kbp respectively, electrophoresis result is seen Fig. 1.
With the synthetic good right-on Fat-2 gene of sequence from
Cut with NotI/XbaI is two on the carrier, cut pcDNA3.1 (+)-sFat-SP163 with NotI/XbaI is two simultaneously, obtain the purpose fragment of 1750bp and the linearized vector of 6740bp respectively, electrophoresis result is seen Fig. 2.
3.pcDNA3.1 (+)-sFat-SP163 collection of illustrative plates, pcDNA3.1-sFat-SP163-IRES-Fat2 carrier structure figure see Fig. 3 and Fig. 4.
4. destination gene expression effect assessment
1) transient transfection destination gene expression effect assessment
PcDNA3.1, pcDNA3.1-sFat1-SP163 plasmid and pcDNA3.1-sFat-SP163-IRES-Fat2 plasmid transient transfection Chinese hamster ovary celI, sample qPCR testing goal expression of gene situation is collected in transfection after 48 hours, destination gene expression content all has significantly lifting, wherein:
Transfection pcDNA3.1-sFat1-SP163 plasmid cell sFat1 genetic expression promotes 31000.4 times;
Transfection pcDNA3.1-sFat-SP163-IRES-Fat2 plasmid cell sFat1 genetic expression promotes 21668.8 times;
Transfection pcDNA3.1-sFat-SP163-IRES-Fat2 plasmid cell Fat2 genetic expression promotes 25064.1 times.
The results are shown in Figure 5
2) situation after the CHO-K1 dosing:
After adding pharmaceutical culture medium, death all appears in each gradient cell, the cell basic all dead (2 multiple hole situations are identical) that 4 days drug levels are 600ug/ml after the dosing; Use the dosing culture medium culturing about 2 weeks, the G418 final concentration is that the cell of 400ug/ml is all dead, and the medicine final concentration is that the cell of 200ug/ml still has survival, so select 400ug/ml to be screening concentration.
3) mixed stability clone destination gene expression effect assessment
In pcDNA3.1, pcDNA3.1-sFat1-SP163 plasmid and the pcDNA3.1-sFat-SP163-IRES-Fat2 plasmid stable transfection Chinese hamster ovary celI, the destination gene expression level all is significantly improved, and improves multiple all 10
5More than.
The research that implementation column two testis injection methods are set up high expression level omega-3 polyunsaturated fatty acids Transgenic Sheep
At first preparation can be expressed the carrier for expression of eukaryon pcDNA3.1-sFat-SP163-IRES-Fat2 of omega-fatty acid desaturase and Δ 12 fatty acid dehydrogenases simultaneously, then this carrier for expression of eukaryon is mixed with liposome and platform phenol indigo plant, is prepared into transfection liquid.Then adopt Minimally Invasive Surgery, with above-mentioned transfection liquid with in the testis that divides multi-point injection to go into the sheep both sides again with female mating.At last by round pcr, with the Auele Specific Primer detection filial generation animal sFat and the Fat2 expression of gene of sFat and Fat2 gene.Detect the eliminating false positive by the Southern trace.Utilize the method for real-time quantitative PCR to detect the expression of positive animal goal gene.
The expression amount that its omega-fatty acid desaturase of positive offspring and Δ 12 fatty acid dehydrogenases are organized in preliminary experimental result demonstration injection is significantly higher than the animal of not injection group.
Claims (4)
1. a novel carriers that improves content of polyunsaturated fatty acid in the animal body is characterized in that described carrier comprises gene order Sequnence NO.1, Sequnence NO.2, Sequnence NO.3 and Sequnence NO.4.
2. a kind of novel carriers that improves content of polyunsaturated fatty acid in the animal body according to claim 1 is characterized in that described carrier is expressed omega-fatty acid desaturase and Δ 12 fatty acid dehydrogenases simultaneously in eukaryotic cell.
3. method that improves omega-3 polyunsaturated fatty acids content in the filial generation animal body comprises:
1) preparation carrier for expression of eukaryon pcDNA3.1-sFat-SP163-IRES-Fat2;
2) carrier for expression of eukaryon pcDNA3.1-sFat-SP163-IRES-Fat2, liposome and platform phenol indigo plant are mixed with transfection liquid;
3) adopt Minimally Invasive Surgery, above-mentioned transfection liquid is gone into minute multi-point injection in the testis of animal both sides again and the jenny mating;
4),, detect filial generation animal sFat and Fat2 expression of gene with the Auele Specific Primer of sFat and Fat2 gene by round pcr;
5) detect to get rid of false positive by the Southern trace, utilize the method for real-time quantitative PCR to detect the expression of positive animal goal gene.
4. a kind of method that improves omega-3 polyunsaturated fatty acids content in the filial generation animal body according to claim 3 is characterized in that described method can be applied to cultivate the animal new variety that are rich in omega-3 polyunsaturated fatty acids.
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Cited By (5)
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| CN102660576A (en) * | 2012-04-12 | 2012-09-12 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing DHA in mammal cells |
| CN104059881A (en) * | 2013-03-21 | 2014-09-24 | 中国农业大学 | Method for producing polyunsaturated fatty acid-containing transgenic animal |
| CN104711271A (en) * | 2013-12-11 | 2015-06-17 | 中国农业科学院北京畜牧兽医研究所 | Artificially-synthesized fatty acid desaturase coding gene and applications thereof |
| CN105219853A (en) * | 2015-09-25 | 2016-01-06 | 中国农业科学院北京畜牧兽医研究所 | One turns sFat-1 genetic animal loop-mediated isothermal amplification detection method |
| CN106047926A (en) * | 2016-05-25 | 2016-10-26 | 华南农业大学 | Transgenic pig with transgenic delta 12 and delta 15 fatty acid desaturase genes as well as preparation method and applications of transgenic pig |
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| CN101886061A (en) * | 2009-05-11 | 2010-11-17 | 北京济普霖生物技术有限公司 | Method for breeding transgenic livestock rich in polyunsaturated fatty acid |
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| CN102660576A (en) * | 2012-04-12 | 2012-09-12 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing DHA in mammal cells |
| CN104059881A (en) * | 2013-03-21 | 2014-09-24 | 中国农业大学 | Method for producing polyunsaturated fatty acid-containing transgenic animal |
| CN104711271A (en) * | 2013-12-11 | 2015-06-17 | 中国农业科学院北京畜牧兽医研究所 | Artificially-synthesized fatty acid desaturase coding gene and applications thereof |
| CN104711271B (en) * | 2013-12-11 | 2018-05-25 | 中国农业科学院北京畜牧兽医研究所 | A kind of artificial synthesized fatty acid desaturase encoding gene and its application |
| CN105219853A (en) * | 2015-09-25 | 2016-01-06 | 中国农业科学院北京畜牧兽医研究所 | One turns sFat-1 genetic animal loop-mediated isothermal amplification detection method |
| CN105219853B (en) * | 2015-09-25 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | One kind turning sFat-1 genetic animal loop-mediated isothermal amplification detection method |
| CN106047926A (en) * | 2016-05-25 | 2016-10-26 | 华南农业大学 | Transgenic pig with transgenic delta 12 and delta 15 fatty acid desaturase genes as well as preparation method and applications of transgenic pig |
| CN106047926B (en) * | 2016-05-25 | 2019-09-03 | 华南农业大学 | One kind turning 15 fatty acid desaturase gene pig of Δ 12 and Δ and its preparation method and application |
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