CN102805052A - Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium - Google Patents
Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium Download PDFInfo
- Publication number
- CN102805052A CN102805052A CN2012102700521A CN201210270052A CN102805052A CN 102805052 A CN102805052 A CN 102805052A CN 2012102700521 A CN2012102700521 A CN 2012102700521A CN 201210270052 A CN201210270052 A CN 201210270052A CN 102805052 A CN102805052 A CN 102805052A
- Authority
- CN
- China
- Prior art keywords
- silac
- mark
- fruit bat
- culture media
- special culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 26
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 title abstract description 9
- 150000001413 amino acids Chemical class 0.000 title abstract description 8
- 108010026552 Proteome Proteins 0.000 title abstract description 5
- 238000004113 cell culture Methods 0.000 title abstract description 4
- 238000001948 isotopic labelling Methods 0.000 title abstract description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 93
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 52
- 239000002609 medium Substances 0.000 claims description 38
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 18
- 239000004472 Lysine Substances 0.000 claims description 18
- 108010035533 Drosophila Proteins Proteins 0.000 claims description 14
- 238000002372 labelling Methods 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 claims description 12
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 claims description 12
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 claims description 12
- 229920000936 Agarose Polymers 0.000 claims description 7
- 102000002322 Egg Proteins Human genes 0.000 claims description 7
- 108010000912 Egg Proteins Proteins 0.000 claims description 7
- 210000004681 ovum Anatomy 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 235000013601 eggs Nutrition 0.000 claims description 5
- KDXKERNSBIXSRK-IQUPZBQQSA-N (2s)-2,6-bis(azanyl)hexanoic acid Chemical group N[13CH2][13CH2][13CH2][13CH2][13C@H](N)[13C](O)=O KDXKERNSBIXSRK-IQUPZBQQSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 241000255588 Tephritidae Species 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 9
- 238000002474 experimental method Methods 0.000 abstract description 5
- 241000235342 Saccharomycetes Species 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 18
- 235000018102 proteins Nutrition 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 238000011160 research Methods 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000007222 ypd medium Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 101100455750 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) LYS2 gene Proteins 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 229960005305 adenosine Drugs 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 235000008729 phenylalanine Nutrition 0.000 description 3
- 229960005190 phenylalanine Drugs 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 108010023063 Bacto-peptone Proteins 0.000 description 2
- 241000382353 Pupa Species 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012092 media component Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- WKVZMKDXJFCMMD-UVWUDEKDSA-L (5ar,8ar,9r)-5-[[(2r,4ar,6r,7r,8r,8as)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5h-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one;azanide;n,3-bis(2-chloroethyl)-2-ox Chemical compound [NH2-].[NH2-].Cl[Pt+2]Cl.ClCCNP1(=O)OCCCN1CCCl.COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3C(O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 WKVZMKDXJFCMMD-UVWUDEKDSA-L 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 102100031680 Beta-catenin-interacting protein 1 Human genes 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000993469 Homo sapiens Beta-catenin-interacting protein 1 Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 description 1
- 241001599018 Melanogaster Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000756042 Polygonatum Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 108010066124 Protein S Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108010042388 protease C Proteins 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000007261 sc medium Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and a special culture medium. The special culture medium for marking drosophila by using the SILAC is prepared according to the following method: a saccharomycete body marked by using the SILAC is coated on a drosophila culture medium to obtain the special culture medium for marking the drosophila by using the SILAC. An experiment proves that the culture medium for marking the drosophila by using the SILAC can be specially used for marking the drosophila proteomes by using the SILAC, can achieve rapid and high-efficient marking, and creates conditions for preparation of quantified interior labels and high-precision quantified proteome studying.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special culture media of the SILAC of utilization overall labeling drosophila protein matter group.
Background technology
Fruit bat (Drosophila melanogaster) is individual small insect.Since Morgan began to utilize its research genetics, fruit bat became the model organism of genetics, physiology, growth and evolution biology research gradually.Since fruit bat have growth fast, be prone to raise and breeding, be prone to keep large group, inexpensive, plurality of advantages such as phenotype abundant and most signal pathways are consistent with higher organism, make fruit bat become the golden worm in the modern biology research.
Protein is the direct executor of biological function.Proteomics is that system identifies and quantitative intracellular protein, and studies the new branch of science of its biological function.Along with the completion of drosophila gene group order-checking and the fast development of proteomics research technology, scientist can realize the target that drosophila protein matter group is identified.The quantitative comparison of protein group is the effective means of research gene mutation or biological processes and effect relation thereof, also is the major fields of current proteomics research.So far have the report of fruit bat quantitative proteomics.Like the protein group of fruit bat of having utilized ICAT technology quantitative comparison such as Brunner.Quantitative comparison such as Xun Parkinsonian fruit bat model.Utilizations such as Krijgsveld
15The yeast of the N mark fruit bat of feeding has obtained
15The fruit bat of N mark has begun the research of internal labeling of fruit bat body and quantitative proteomics.But because
15The protein group Isotopic Distribution of N mark is wide; The overlapping quantitative accuracy that causes is low easily between the weight mark; And the weight mark is difficult to distinguish; Be prone to produce technical barriers such as false positive, make quantitative proteomics turn to whole protein group echo technology (stable isotope labeling by amino acid in cell culture, SILAC) goldstandard of this quantitative proteomics research gradually based on the heavy stable isotope labeled amino acid in the cell cultivation process.
Mann research group utilizes the SILAC technology that the SL2 cell-line of fruit bat is studied, and identifies and quantitative more than 6000 protein, and has compared the incidence relation of mRNA and protein abundance.Although SILAC has just begun just to be used for cultured cells, like yeast, mammalian cell etc., the beginning of the heavy stable isotope mark of whole animal has been started in the success of the whole mouse of SILAC mark recently.Sury etc. have utilized SILAC technology mark whole fruit bat, but because of technical limitations, its labeling effciency can only reach about 96%, has influenced the quantitative precision and the sensitivity of detection.
Summary of the invention
An object of the present invention is to provide a kind of SILAC mark fruit bat special culture media.
The present invention provides SILAC mark fruit bat special culture media, prepares according to following method: the yeast thalline of SILAC mark is coated on the fruit bat medium, obtains SILAC mark fruit bat special culture media.
Above-mentioned fruit bat medium is made up of low melting-point agarose, glucose, methyl p-hydroxybenzoate, ethyl acetate and water; The mass ratio of said low melting-point agarose, said glucose, said methyl p-hydroxybenzoate, said ethyl acetate is 0.8:10:0.002:0.8.
The concrete concentration of above-mentioned fruit bat nutrient media components is following: the concentration of said low melting-point agarose in said fruit bat medium is 0.8% (quality percentage composition); The concentration of said glucose in said fruit bat medium is 10% (quality percentage composition); The concentration of said methyl p-hydroxybenzoate in said fruit bat medium is 0.002% (quality percentage composition); The concentration of the said agar of said ethyl acetate in said fruit bat medium is 0.8% (quality percentage composition).
Wherein methyl p-hydroxybenzoate adds in the fruit bat medium with the form of methyl p-hydroxybenzoate ethanolic solution in the fruit bat nutrient media components; The methyl p-hydroxybenzoate ethanolic solution prepares according to following method: methyl p-hydroxybenzoate is dissolved in the mixed liquor that obtains in 95% ethanol water, and the concentration of methyl p-hydroxybenzoate in mixed liquor is 10% (quality percentage composition).
In the above-mentioned special culture media; The yeast thalline of said SILAC mark prepares according to following method: lysine auxotroph yeast strain is cultivated in the SILAC mark yeast special culture media that contains heavy stable isotope mark lysine; Collect thalline, obtain the yeast thalline of SILAC mark.
Lysine auxotroph yeast strain in the embodiments of the invention is knock-out bacterial strain BY4741 △ YBR115C, available from Saccharomyces Genome Deletion Project.
The above-mentioned SILAC mark yeast special culture media that contains heavy stable isotope mark lysine prepares according to following method: lysine, adenosine, uracil, tryptophan, histidine, arginine, methionine, tyrosine, leucine, isoleucine, phenyl alanine, glutamic acid, aspartic acid, valine, threonine, serine and water by yeast nitrogen basis, glucose, heavy stable isotope mark are formed; The concentration of said yeast nitrogen basis in said SILAC mark yeast special culture media is 7g/L; The concentration of said glucose in said SILAC mark yeast special culture media is 20g/L; The lysine L-lysine-of said heavy stable isotope mark
13C
6Concentration in said SILAC mark yeast special culture media is 164.3 μ M; Said adenosine, said uracil, said tryptophan, said histidine, said arginine, the concentration of said methionine in said SILAC mark yeast special culture media are 20mg/L; Said tyrosine, said leucine, the concentration of said isoleucine in said SILAC mark yeast special culture media are 30mg/L; The concentration of said phenyl alanine in said SILAC mark yeast special culture media is 50mg/L; Said glutamic acid and the said aspartic acid concentration in said SILAC mark yeast special culture media is 100mg/L; The concentration of said valine in said SILAC mark yeast special culture media is 150mg/L; The concentration of said threonine in said SILAC mark yeast special culture media is 200mg/L; The concentration of said serine in said SILAC mark yeast special culture media is 400mg/L.The lysine of the heavy stable isotope mark in the above-mentioned SILAC mark yeast special culture media is L-lysine-
13C
6Also can be for the different heavy stable isotope mark lysine of other mass number, like L-lysine-
13C
6 15N
2Deng.
In the saccharomycete preparation of above-mentioned SILAC mark, said best cultivation is 30 ℃ and cultivated for 9 generations, cultivates 18h altogether.
The application of above-mentioned special culture media in SILAC mark drosophila protein matter group also is the scope that the present invention protects.
Another object of the present invention provides a kind of method of SILAC fruit bat medium mark drosophila protein matter group.
Method provided by the invention comprises the steps: that ovum with above-mentioned SILAC mark fruit bat special culture media fruit flies to developing into adult, obtains the fruit bat of SILAC overall labeling drosophila protein matter group, realizes SILAC overall labeling drosophila protein matter group.
In the said method, the ovum of said fruit bat for paired male and female fruit bat on above-mentioned special culture media, cultivate, laying eggs obtains.
Experiment of the present invention proves; A kind of medium that is used for fruit bat SILAC mark of the present invention's exploitation; Can be specifically designed to SILAC mark drosophila protein matter group, and can realize mark fast and efficiently, for condition has been created in target preparation in quantitatively and quantitative protein group research accurately.
Description of drawings
The attach most importance to saccharomycete of the complete mark of stable isotope labeled amino acid of Fig. 1
Fig. 2 is the fruit bat culture apparatus that contains SILAC mark medium
Fig. 3 is the fruit bat of SILAC mark medium breed and the comparison of common fruit bat
Fig. 4 is that SILAC mark medium is cultured the test of fruit bat labeling effciency
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The present invention can more easily understand through following instance, but the present invention is not limited in these instances.
The preparation of embodiment 1, SILAC mark yeast thalline
One, the preparation of SILAC mark yeast special culture media
The component of the SILAC mark yeast special culture media of every 1L (the amino acid whose SC medium that contains heavy stable isotope mark) is following: 7g Difco yeast nitrogen basis (U.S. company BD; 239210), 20g glucose (Chemical Reagent Co., Ltd., Sinopharm Group, article No.: 10010592), the lysine L-lysine-of the heavy stable isotope mark of 164.3 μ mol article No.:
13C
6(U.S. CIL Corp.; Article No.: CNLM-291-0.25), the adenosine of 20mg (U.S. Amresco company, article No.: 0325-50G0325-50G), 20mg uracil (U.S. Amresco company, article No.: 0847-250G0847-250G), 20mg tryptophan (U.S. Amresco company; Article No.: E800-25G), 20mg histidine (U.S. Amresco company; Article No.: E806-100G), 20mg arginine (U.S. Amresco company, article No.: 0877-100G), 20mg methionine (U.S. Amresco company, article No.: E801-100G), 30mg tyrosine (U.S. Amresco company; Article No.: E821-25G), 30mg leucine (U.S. Amresco company; Article No.: E811-100G), 30mg isoleucine (U.S. Amresco company, article No.: E803-25G), 50mg phenyl alanine (U.S. Amresco company, article No.: 0991-100G), 100mg glutamic acid (U.S. Amresco company; Article No.: 0421-1KG), 100mg aspartic acid (U.S. Amresco company; Article No.: 0192-500G), 150mg valine (U.S. Amresco company, article No.: 1B1102-25G), 200mg threonine (U.S. Amresco company, article No.: E808-25G), 400mg serine (U.S. Amresco company; Article No.: 1B1103-25G), water polishing volume.
Two, the acquisition and the checking of SILAC mark yeast thalline
1, SILAC mark yeast
Picking fission yeast lysine auxotrophic strain (knock-out bacterial strain BY4741 △ YBR115C from the YPD solid culture flat board; Available from Saccharomyces Genome Deletion Project) fresh bacterium colony; Be transferred in the YPD liquid nutrient medium (containing 10g bacterium yeast extract, 20g bacto peptone, 20g glucose in every liter); Grow overnight obtains seed culture fluid; Centrifugal seed culture fluid is collected yeast cells under aseptic condition, with aseptic washing twice, and gets a certain amount of above-mentioned SILAC mark yeast special culture media that is inoculated into sterile water after resuspended, makes that the final concentration of initial thalline is 0.0023OD (A
600), cultivated for 9 generations after totally 18 hours for 30 ℃, the final concentration that reaches thalline is 1.2OD (A
600), collected thalline in centrifugal 5 minutes through 10000g again, obtain SILAC mark yeast thalline ,-80 ℃ are frozen subsequent use.
2, the checking of SILAC mark yeast thalline
1) extraction of total protein
With the Tris of 10mM pH8.0, the NaH of 0.1M
2PO
4, 8M urea, 0.02%SDS and 10mM beta-mercaptoethanol be as protein extract, with bead (Biospec Products Inc., Bartlesville) concussion broken wall method cracking SILAC mark yeast thalline.Thalline-bead mixed liquor shook 1 minute the most at a high speed on vortex mixer, cooled on ice 1 minute, obtained pyrolysis product totally for 14 times, with pyrolysis product under the condition of 17000g centrifugal 5 minutes, obtained total protein again.With being used for protease digestion behind the Bradford standard measure commonly used, the result is about 10 μ g/ μ l for the concentration of total protein.
2) digestion
Getting 5 μ g total proteins is that the DTT of 5mM handled 30 minutes through final concentration, and is that the iodoacetamide lucifuge of 20mM was handled 30 minutes down for 25 ℃ with the final concentration, and adding urea to final concentration again is 8M sex change 30 minutes, with 50MmNH
4HCO
3Dilution urea is to 4M concentration, and (article No.: 125-05061) 37 ℃ of digestion is 14 hours, obtains the peptide section for Lys-C, Japanese Wako company with the lysine protease C of 10ng/ μ l.
3) chromatograph-mass spectrometer coupling analysis
With the peptide section of above-mentioned gained with the desalination of C18 post (3M, St.Paul MN), dissolve with 5% acetonitrile of 10 μ l and the chromatograph-mass spectrometer coupling sample-loading buffer of 1% formic acid, get 2 μ l and carry out the chromatograph-mass spectrometer coupling analysis, specifically with reference to Xu, P.; Duong, D.; Peng, J., Systematical optimization of reverse-phase chromatography for shotgun proteomics.J Proteome Res 2009,8 (8), the method for putting down in writing among the 3944-50.Preparing albumen according to the method described above, detect with yeast thalline before the SILAC mark is contrast.
Picked at random ATP combines Protein S SA2 peptide section NQAAMNPANTVFDAK; The result is as shown in Figure 1, can find out, SILAC mark yeast thalline is complete mark yeast thalline; Complete mark after 9 generations, and detect peptide section less than the lysine that contains natural light isotope.
Three, the acquisition of light mark yeast thalline of contrast and contrast yeast thalline
Contrast light mark medium and SILAC mark yeast special culture media component is basic identical, that different is the lysine L-lysine-that will weigh the stable isotope mark
13C
6Replace with common light isotope label lysine (U.S. Amresco company, article No.: 0437-100G)
YPD medium: 1% bacterium yeast extract Bacto-yeast extract (U.S. Amresco company; Article No.: J850-1KG), 2% bacto peptone bacto-peptone (U.S. Amresco company, article No.: J636-500G); 2% glucose (Chemical Reagent Co., Ltd., Sinopharm Group; Article No.: 10010592), 2% bacteria culture media is with agar powder (U.S. Amresco company, article No.: J637-500G).
Adopt above-mentioned two 1 method, yeast BY4741 △ YBR115C is cultivated in the light mark medium of contrast and YPD medium respectively, obtain contrasting light mark yeast thalline and contrast YPD yeast thalline.
The preparation of embodiment 2, SILAC mark fruit bat special culture media
1, SILAC mark fruit bat special culture media preparation
The component of the fruit bat medium of every 1L is following: the quality percentage composition is 0.8% Bio-Rad low melting-point agarose (Bio-Rad Hercules; CA), the quality percentage composition is that 10% glucose, quality percentage composition are that 0.002% methyl p-hydroxybenzoate, final concentration are that 0.8% ethyl acetate and water mix, water constant volume to 1 liter.
SILAC mark fruit bat special culture media prepares according to following method: coating obtains the SILAC mark medium of mark fruit bat by two the 1 SILAC mark yeast thalline yeast thalline of mark (heavy stable isotope) that obtains of embodiment 1 on above-mentioned fruit bat medium.
2, the light mark medium of contrast fruit bat SILAC
The light mark medium of contrast fruit bat SILAC: basic identical with the preparation method of SILAC mark fruit bat special culture media, different is the light mark yeast of the contrast thalline that coating is obtained by three of embodiment 1;
Contrast YPD fruit bat medium: basic identical with the preparation method of SILAC mark fruit bat special culture media, different is the contrast YPD yeast thalline that coating is obtained by three of embodiment 1.
One, SILAC mark
1, material
Select for use wild type fruit bat w118 strain (hereinafter to be referred as fruit bat W118D; Melanogaster w1118; Fruit bat preservation center (Bloomington Drosophila Stock Center at Indiana University) available from U.S. seal ground Anna university makes an experiment, in the fruit bat medium of standard, 25 ℃ and the cultivation down of 70% damp condition.
2, mark
SILAC mark: 100 female fruit bat w118 and 100 male fruit bat w118 are raised together with on above-mentioned SILAC mark fruit bat special culture media lay eggs; Again with ovum subsequently culture transferring to inserting in folding 3 times fruit bat culture tube 3M filter paper and that contain above-mentioned SILAC mark fruit bat special culture media (Fig. 2); About 100 ovum of every pipe; Under 70% damp condition, cultivate in 25 ℃ of incubators, hatch into larva, via the stage of pupa; Develop into ripe fruit bat, obtain SILAC mark fruit bat.
Contrast SILAC light mark fruit bat: adopting uses the same method lays eggs paired fruit bat, cultivates and obtain contrasting the light mark fruit bat of SILAC at the light mark medium of contrast fruit bat SILAC;
Contrast YPD fruit bat: adopting uses the same method lays eggs paired fruit bat, cultivates and obtain the contrasting marking fruit bat at contrast YPD fruit bat medium.
Two, detect
1, profile is observed
SILAC mark fruit bat (heavily marking the SD medium), the contrast light mark fruit bat of SILAC (gently marking the SD medium) and contrasting marking fruit bat (YPD medium) are observed, and the result is as shown in Figure 3, and A is with SILAC mark fruit bat flow chart; B for respectively with the YPD medium, gently mark the SD medium, heavily mark the SD medium culture yeast raising fruit bat pupa shape ratio; C for respectively with the YPD medium, gently mark the SD medium, heavily mark the adult eyes of fruit bat of the yeast raising of SD medium culture; The shape ratio of health and wing; Can find out that with fruit bat behind the mark of above-mentioned medium and method cultivation, the fruit bat that shape and ordinary culture medium are cultivated is in full accord.
2, Mass Spectrometer Method
1) extraction of total protein: with the above-mentioned one SILAC mark fruit bat that obtains according to the second portion 2 of embodiment 1 1) method extract;
2) digestion: with the second portion 2 of embodiment 1 2) identical;
3) chromatograph-mass spectrometer coupling analysis: with the second portion 2 of embodiment 1 3) identical;
The peptide hydrolysis of picked at random protein disulfide bond isomerase (DNEIAIIGFFK), the result is shown in Fig. 4 A, and SILAC mark fruit bat is complete mark fruit bat, after 1 generation, with regard to the complete mark of ability, detects the peptide section less than the lysine that contains natural light isotope.The fruit bat of the complete mark of this heavy stable isotope is suitable for the quantitative proteomics research of high accuracy.
3, the mensuration of the complete mark fruit bat of SILAC labeling effciency
Take a sample zero day (ovum) and 1 phase of larva (L1), 2 phases (L2), 3 phase (L3) and adult stage respectively in the preparation process of above-mentioned SILAC mark fruit bat; Extract total protein, digestion and chromatograph-mass spectrometer coupling analysis, calculating labeling effciency (the amino acid whose ratio of the light mark of mark amino acid replacement of attaching most importance to) according to above-mentioned 2.Quantitative result to each sample carries out gently, the distributional analysis of the logarithm of recalibration value.The experiment triplicate, results averaged.
The result is shown in Fig. 4 B and table 1, and the labeling effciency that is presented at L1, L2, L3 is respectively: 80%, 95% and 96%, and basic detection the in the adult sample less than light target peptide section, system detects and finds that its labeling effciency is more than 98%.Show through a generation and culture, can realize the complete mark of fruit bat.The fruit bat of the complete mark of this heavy stable isotope is suitable for the quantitative proteomics research of high accuracy.
The mark rate of table 1 drosophila protein matter group
#Heavily mark signal/resultant signal behind labeling effciency=mark.
Claims (8)
1. a SILAC mark fruit bat special culture media prepares according to following method: the yeast thalline of SILAC mark is coated on the fruit bat medium, obtains SILAC mark fruit bat special culture media.
2. special culture media according to claim 1 is characterized in that:
Said fruit bat medium is made up of low melting-point agarose, glucose, methyl p-hydroxybenzoate, ethyl acetate and water; The mass ratio of said low melting-point agarose, said glucose, said methyl p-hydroxybenzoate, said ethyl acetate is 0.8:10:0.002:0.8.
3. special culture media according to claim 2 is characterized in that:;
In the said fruit bat medium, the concentration of said low melting-point agarose in said fruit bat medium is 0.8% (quality percentage composition); The concentration of said glucose in said fruit bat medium is 10% (quality percentage composition); The concentration of said methyl p-hydroxybenzoate in said fruit bat medium is 0.002% (quality percentage composition); The concentration of the said agar of said ethyl acetate in said fruit bat medium is 0.8% (quality percentage composition).
4. according to arbitrary described special culture media among the claim 1-3, it is characterized in that:
The yeast thalline of said SILAC mark prepares according to following method: lysine auxotroph yeast strain is cultivated in the SILAC mark yeast special culture media that contains heavy stable isotope mark lysine; Collect thalline, obtain the yeast thalline of SILAC mark.
5. special culture media according to claim 4 is characterized in that:
Said heavy stable isotope mark lysine is L-lysine-
13C
6Said best cultivation is 30 ℃ and cultivated for 9 generations, cultivates 18h altogether.
6. the application of arbitrary described special culture media in SILAC mark drosophila protein matter group among the claim 1-5.
7. the method for a SILAC mark drosophila protein matter group; Comprise the steps: that in claim 1-5 the ovum of fruit flies is to developing into adult on arbitrary described special culture media; Obtain the fruit bat of SILAC overall labeling drosophila protein matter group, realize SILAC overall labeling drosophila protein matter group.
8. method according to claim 7 is characterized in that: the ovum of said fruit bat for will paired male and female fruit bat cultivation on arbitrary described special culture media in claim 1-5, laying eggs obtains.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210270052.1A CN102805052B (en) | 2012-07-31 | 2012-07-31 | Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210270052.1A CN102805052B (en) | 2012-07-31 | 2012-07-31 | Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102805052A true CN102805052A (en) | 2012-12-05 |
CN102805052B CN102805052B (en) | 2014-04-02 |
Family
ID=47229061
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201210270052.1A Expired - Fee Related CN102805052B (en) | 2012-07-31 | 2012-07-31 | Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102805052B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
CN110004074A (en) * | 2019-02-28 | 2019-07-12 | 北京蛋白质组研究中心 | A kind of method and its special culture media using SILAC label mycobacterium smegmatis protein |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1487087A (en) * | 2003-07-31 | 2004-04-07 | 上海新立工业微生物科技有限公司 | Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine |
CN101173926A (en) * | 2006-11-03 | 2008-05-07 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Dual quantification method and reagent kit for stable isotope 18 O marked proteome |
-
2012
- 2012-07-31 CN CN201210270052.1A patent/CN102805052B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1487087A (en) * | 2003-07-31 | 2004-04-07 | 上海新立工业微生物科技有限公司 | Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine |
CN101173926A (en) * | 2006-11-03 | 2008-05-07 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Dual quantification method and reagent kit for stable isotope 18 O marked proteome |
Non-Patent Citations (5)
Title |
---|
JEROEN KRIJGSVELD等: "Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics", 《NATURE BIOTECHNOLOGY》, no. 21, 13 July 2003 (2003-07-13), pages 927 - 931 * |
MATTHIAS D. SURY等: "The SILAC Fly Allows for Accurate Protein Quantification in Vivo", 《MOLECULAR & CELLULAR PROTEOMICS》, no. 9, 5 June 2010 (2010-06-05), pages 2173 - 2183 * |
YVES LAROCHE等: "High-Level Secretion and Very Efficient Isotopic Labeling of Tick Anticoagulant Peptide(TAP) Expressed in the Methylotrophic Yeast,Pichia pastoris", 《NATURE BIOTECHNOLOGY》, no. 12, 4 August 1994 (1994-08-04), pages 1119 - 1124 * |
李树龙等: "稳定同位素标签技术在定量蛋白质组研究的应用", 《生命的化学》, vol. 25, no. 5, 15 October 2005 (2005-10-15), pages 362 - 365 * |
王昕等: "基于质谱技术的蛋白质组定量方法", 《生命的化学》, vol. 31, no. 1, 15 February 2011 (2011-02-15), pages 134 - 140 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
CN110004074A (en) * | 2019-02-28 | 2019-07-12 | 北京蛋白质组研究中心 | A kind of method and its special culture media using SILAC label mycobacterium smegmatis protein |
CN110004074B (en) * | 2019-02-28 | 2021-06-18 | 北京蛋白质组研究中心 | Method for marking mycobacterium smegmatis protein by SILAC and special culture medium thereof |
Also Published As
Publication number | Publication date |
---|---|
CN102805052B (en) | 2014-04-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2015523097A5 (en) | ||
CN106520541B (en) | A kind of kit of zebrafish embryo acute toxicity testing | |
MX2013005058A (en) | Efficient and effective supplement screening for the development of chemically defined media in cell culture. | |
CN103459584A (en) | A functional enviromics method for cell culture media engineering | |
CN104391060B (en) | The sample pre-treatments of GC-MS research the pathogen of Botrytis cinerea metabolism group and detection method | |
Min et al. | In-depth investigation of low-abundance proteins in matured and filling stages seeds of Glycine max employing a combination of protamine sulfate precipitation and TMT-based quantitative proteomic analysis | |
Beckley et al. | A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival*[S] | |
Durand et al. | Combined proteomic and metabolomic profiling of the Arabidopsis thaliana vps29 mutant reveals pleiotropic functions of the retromer in seed development | |
CN102805052B (en) | Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium | |
Cheong et al. | Phenotyping the chilling and freezing responses of young microspore stage wheat spikes using targeted metabolome and lipidome profiling | |
Lucena et al. | Extreme low cytosolic pH is a signal for cell survival in acid stressed yeast | |
Meister et al. | COP9 signalosome interaction with UspA/Usp15 deubiquitinase controls VeA-mediated fungal multicellular development | |
Donzella et al. | Recycling food waste and saving water: optimization of the fermentation processes from cheese whey permeate to yeast oil | |
Olsen et al. | Outdoor cultivation of a novel isolate of the microalgae Scenedesmus sp. and the evaluation of its potential as a novel protein crop | |
Feng et al. | Global metabolic profiling of baculovirus infection in silkworm hemolymph shows the importance of amino-acid metabolism | |
CN103245724B (en) | The detection method of neurocyte discharge performance under Variable Composition drug effect | |
CN102796682B (en) | Method for labeling escherichia coli proteome by using SILAC (Stable Isotope Labeling with Amino Acids in Cell Cultures) and special culture medium | |
CN103937751A (en) | Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody | |
Nakashita et al. | Ecological application of compound-specific stable nitrogen isotope analysis of amino acids: A case study of captive and wild bears | |
CN103163238B (en) | Utilize the method for root system organic acid secretion feature detection Genes For Plant Tolerance Low phosphorus stress ability | |
Ogórek et al. | A culture-based study of micromycetes isolated from the urban nests of grey heron (Ardea cinerea) in SW Poland | |
CN102220257A (en) | Novel yeast two-hybrid method and application thereof | |
CN103278632B (en) | ELISA kit for fast detection of corn phosphoenolpyruvate carboxylase (PEPC) gene transgenic crop and use thereof | |
Yan et al. | Vacuole proteins with optimized microtubule assembly is required for Fum1 protein localization and fumonisin biosynthesis in mycotoxigenic fungus Fusarium verticillioides | |
Wang et al. | Peroxisome proliferator FpPEX11 is involved in the development and pathogenicity in Fusarium pseudograminearum |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140402 |