CN102977199A - Application of Ubc13 protein serving as plant internal control protein - Google Patents
Application of Ubc13 protein serving as plant internal control protein Download PDFInfo
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- CN102977199A CN102977199A CN2012104991841A CN201210499184A CN102977199A CN 102977199 A CN102977199 A CN 102977199A CN 2012104991841 A CN2012104991841 A CN 2012104991841A CN 201210499184 A CN201210499184 A CN 201210499184A CN 102977199 A CN102977199 A CN 102977199A
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Abstract
The invention relates to the field of molecular biology, and particularly relates to an application of a Ubc13 protein serving as a plant internal control protein. According to the invention, the Ubc13 protein in plant can be identified better in western hybrid detection by use of human Ubc13 protein monoclonal antibody (4E11). Since the Ubc13 protein has the characteristics of house-keeping gene in plant, the human Ubc13 protein monoclonal antibody (4E11) can be used as a general western hybrid internal control identification antibody in different plants. The human Ubc13 protein monoclonal antibody (4E11) is the first monoclonal antibody which can be applied to cross-plant western hybrid internal control protein identification.
Description
Technical field
The present invention relates to biology field, relate to particularly Ubc13 protein as the application of plant confidential reference items albumen.
Background technology
The protein ubiquitin is a kind of important protein post-translational modification process, and this process participates in function and the destiny of regulation protein.Ubiquitin is the little modified protein that a kind of 76 amino acid form, it can be connected on the lysine residue of another ubiquitin or target protein with individual form or with the form of ubiquitin polymkeric substance, and front a kind of modified forms is called then a kind of poly ubiquitin that is called of single ubiquitinization.Thereby several lysine residues that other ubiquitin forms poly ubiquitin chain that can be used for connecting are contained on the ubiquitin surface.The target protein that different poly ubiquitin connection modes is modified is composed with different functions, and it is very wide that these functions relate to, and is changed the biological pathway of their participations by being degraded to of modifying protein from regulation and control.Ubc13(is called again UBE2N) be a kind of ubiquitin ligase (E2), and be that the 63rd Methionin of unique known catalysis ubiquitin connects the ubiquitin ligase of poly ubiquitin chain formation up to now.Ubc13 usually need to form a heterodimer with Mms2 or Uev1A and exercise the function of its poly ubiquitin ligase.In zooblast, thereby two kinds of heterodimers of Ubc13/Mms2 and Ubc13/Uev1A mediate multiple different signal path with the ubiquitin of different pattern catalysis target proteins respectively, these paths comprise the dna damage reparation, p53 and cell cycle check position regulation and control, cell cycle, the inflammatory reaction signal transduction, marrow generates, immunity receptor signal transduction and cell endocytic.
Summary of the invention
The objective of the invention is Ubc13 protein as the application of plant confidential reference items albumen.
And then a further object of the present invention provides human Ubc13 protein monoclonal antibody 4E11 identifies the Ubc13 protein in the plant in the western hybridization check application.
According to technical scheme of the present invention, described plant Ubc13 albumen can be all known plant-sourced Ubc13 protein before the applying date, and for example its aminoacid sequence is shown in SEQ ID No.1.MANSNLPRRI IKETQRLLSE PAPGISASPS EDNMRYFNVM ILGPTQSPYE GRGVFKLELF 60LPEEYPMAAP KVRFLTKIYH PNIDKLGRIC LDILKDKWSP ALQIRTVLLS IQALLSAPNP 120DDPLSENIAK HWKSNEAEAV DTAKEWTRLY ASGA 154
The human Ubc13 protein monoclonal antibody 4E11 that the present invention uses can be available from handsome (invitrogen) company, trade name: Mouse anti-Ubc13, article No.: 37-1100.
According to the specific embodiment of the present invention, utilize human Ubc13 protein monoclonal antibody (4E11) that the Ubc13 protein in Arabidopis thaliana (Colombia's 0 type) tissue whole protein extract and paddy rice (Japan is fine) the tissue whole protein extract is detected.At first extract Arabidopis thaliana (Colombia's 0 type) and paddy rice (Japan fine) and entirely organize total protein, then by the SDS-PAGE electrophoresis two kinds of whole proteins are separated and by the human Ubc13 protein of western hybridization technique utilization monoclonal antibody the Ubc13 protein in two kinds of albumen samples is detected.
Human Ubc13 protein monoclonal antibody 4E11 is a kind of protein that can be used as plant western hybridization confidential reference items.Arabidopis thaliana is a kind ofly to be used as model plant and to be widely applied to small-sized flowering plant in the plant biology research.Arabidopsis is in dicotyledonous cress, and the member of same section also has Caulis et Folium Brassicae capitatae and radish etc.Although Arabidopis thaliana is not farm crop, because the considerable advantage of self provides valuable help for the fundamental research in genetics and molecular biological field.Equally as model plant, although the paddy rice growth cycle will be grown than Arabidopis thaliana, planting environment is more complicated also, the seed of paddy rice---rice, as the main farm crop of our times, the researching value at aspects such as crop yield and qualities is that Arabidopis thaliana can not be compared.Paddy rice belongs to annual unifacial leaf herbaceous plant, and also there is certain difference in the dicotyledons with the Arabidopis thaliana representative from the growth characteristic to the physiological structure etc.Therefore, paddy rice also becomes the important model plant that people study plant physiology and molecular cytobiology.
The present invention retrieves by the expression chip database to Arabidopis thaliana and paddy rice, and discovery Ubc13 gene is stablized high expression level in Arabidopis thaliana and paddy rice.Simultaneously the present invention has also passed through western hybridization, take coomassie brilliant blue staining albumen sample in the situation of reference, utilize human Ubc13 protein monoclonal antibody, Ubc13 expression amount in the paddy rice total protein sample of unlike signal processing is detected, find that the protein level of Ubc13 is keeping well consistence between the sample of different treatment.
The present invention utilizes the well Ubc13 protein in the identification plant in the western hybridization check of human Ubc13 protein monoclonal antibody (4E11).And because Ubc13 protein has house-keeping gene in plant, human Ubc13 protein monoclonal antibody (4E11) can be used as western hybridization confidential reference items identification antibody general in the different plants.This is that first can be used as the monoclonal antibody of striding the identification of plant western hybridization confidential reference items albumen.
Description of drawings
Ubc13 albumen in the human Ubc13 protein of Fig. 1 monoclonal antibody 4E11 identification Arabidopis thaliana (Colombia's 0 type) total protein and paddy rice (Japan the is fine) total protein, among the figure 1 and 2 swimming lanes marks be respectively Arabidopsis thaliana Seedlings full tissue in period and adult plant leaf is organized total protein, 3 and 4 swimming lanes marks be respectively Seed Germination of Rice full tissue in period and adult period leaf tissue total protein, the 17kD position is the Ubc13 albumen of 4E11 identification.
Ubc13 albumen under the human Ubc13 protein of Fig. 2 monoclonal antibody 4E11 identification Whitfield's ointment and the methyl jasmonic acid ester disposition in the Arabidopis thaliana total protein.The total protein applied sample amount is 20 micrograms.
The human Ubc13 protein of Fig. 3 monoclonal antibody 4E11 identification Whitfield's ointment, the Ubc13 albumen under methyl jasmonic acid ester and the dormin disposition in the paddy rice total protein.The total protein applied sample amount is 20 micrograms.
Embodiment
(1) from Arabidopis thaliana (Colombia's 0 type) and paddy rice (Japan is fine) tissue, extract gross protein:
In mortar, with liquid nitrogen is auxiliary plant sample is clayed into power, and forward the plant sample of milled to mixing in the 1.5ml EP pipe that is added with protein cleavage liquid (adding the ground plant sample powder of 0.5mg in every 1ml protein cleavage liquid).The composition of protein cleavage liquid is as follows: 50mM Tris-HCl pH=8.0,0.3M NaCl, 2mM EDTA, 10% glycerine, 0.1%TritonX-100,10mM PMSF, 3mM DDT and proteinase inhibitor (Luo Shi).
The protein sample of mixing is placed 4 degrees centigrade of whizzers, and 14000 rpms centrifugal 15 minutes.Get supernatant and change in the new 1.5ml EP pipe, again 4 degrees centigrade 14000 rpms centrifugal 15 minutes.At last supernatant is forwarded in the new 1.5mlEP pipe, so far the plant sample total protein extracts and finishes.
(2) vegetable-protein sample SDS-PAGE electrophoretic separation:
Get plant total protein sample 50ul, adding 10ul 6X SDS sample-loading buffer (buffer formulation: 0.75MTris-HCl pH=6.5,60%[mass volume ratio] glycerine, the 24%[mass volume ratio] SDS, the 0.03%[mass volume ratio] tetrabromophenol sulfonphthalein) mixing, 100 degrees centigrade were boiled 5 minutes.After the cooling with centrifugal 2 minutes of 14000 rpms in sample (will be evaporated to sample collection that pipe covers to the pipe end).
Concentration is the configuration of 12%SDS-PAGE glue: separation gel: 12% pre-prepared colloid, 0.375M Tris-HCl pH=8.8,0.1%SDS, 0.1% ammonium persulphate and 0.04%TEMED; Concentrated glue: 5.1% pre-prepared colloid, 0.1875MTris-HClpH=6.8,0.1%SDS, 0.1% ammonium persulphate and 0.1%TEMED.
Determination of protein concentration: determination of protein concentration uses Bio-Rad D
CProtein Assay Reagent, typical curve 0mg/ml, 2mg/ml, 4mg/ml, 6mg/ml, 8mg/ml, the 10mg/ml BSA aqueous solution is measured and is drawn as standard substance.
Each sample is got the well-done total protein sample of 20ug loading, constant voltage 100V electrophoresis 90 minutes
(3) Western hybridization detailed step:
Electricity turns the liquid composition: glycine 14.41g/L, and Tris 3.03g/L, 800ml water, 200ml methyl alcohol prepares rear 4 degrees centigrade of precoolings.Soaked 10 minutes with methyl alcohol before pvdf membrane uses, filter paper turns the liquid immersion with the electricity of precooling.Assemble in the following order membrane-transferring device: positive plate, sponge, three filter paper, the glue that pvdf membrane, electrophoresis are finished, three filter paper, sponge, negative plate.The membrane-transferring device that installs is put into electric turn trough, and 4 degrees centigrade of 100V turn 90 minutes.
Sealing: place 5% skim-milk to be dissolved into phosphoric acid buffer (PBS) pvdf membrane that takes a turn for the better, and place on the rolling shaking table room temperature sealing 1 hour.PBS prescription: KCl 0.2g, KH
2PO
40.2g, NaCl 0.8g, Na
2HPO
412H
2O 3g.
Primary antibodie hybridization: human Ubc13 protein monoclonal antibody (4E11) is dissolved in the PBS solution that contains 5% skimmed milk with 1:5000.The pvdf membrane that sealing is finished changes in the primary antibodie solution, places on the rolling shaking table incubated at room one hour.
Two anti-hybridization: primary antibodie is hatched complete pvdf membrane put into PBST (the PBS damping fluid adds 0.1% polysorbas20), cleaned 10 minutes at shaking table, repeat 3 times.With sheep anti-mouse igg-HRP(Santa Cruz, sc2005) be dissolved in the PBS solution of 5% skimmed milk with 1:5000.The pvdf membrane of rinsing well is put into two anti-solution, incubated at room 1 hour.
Develop: after two anti-hybridization finish, pvdf membrane is cleaned 3 times according to the method among the 3d.With SuperSignal(Thermo, 34080) two kinds of solution 1:1 in mix, and are sprinkled upon uniformly on the pvdf membrane, reacts 2 minutes, place afterwards LAS-4000(FUJIFILM) development.
Claims (2)
1.Ubc13 albumen is as the application of plant confidential reference items albumen.
2. human Ubc13 protein monoclonal antibody 4E11 identifies the application of the Ubc13 protein in the plant in the western hybridization check.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2012104991841A CN102977199A (en) | 2012-08-14 | 2012-11-29 | Application of Ubc13 protein serving as plant internal control protein |
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| CN201210288253 | 2012-08-14 | ||
| CN2012104991841A CN102977199A (en) | 2012-08-14 | 2012-11-29 | Application of Ubc13 protein serving as plant internal control protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507678A (en) * | 2022-02-22 | 2022-05-17 | 中国科学院东北地理与农业生态研究所 | Rice ABA signal negative regulatory factor OsUBC12 gene and encoding protein and application thereof |
-
2012
- 2012-11-29 CN CN2012104991841A patent/CN102977199A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| ÉRICA DUARTE SILVEIRA等: "Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha", 《BMC PLANT BIOLOGY》 * |
| RUI WEN: "《A Thesis Submitted to the College of Graduate Studies and Research》", 30 September 2010 * |
| RUI WEN等: "Arabidopsis thaliana UBC13: implication of error-free DNA damage tolerance and Lys63-linked polyubiquitylation in plants", 《PLANT MOLECULAR BIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507678A (en) * | 2022-02-22 | 2022-05-17 | 中国科学院东北地理与农业生态研究所 | Rice ABA signal negative regulatory factor OsUBC12 gene and encoding protein and application thereof |
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Application publication date: 20130320 |