CN103163009A - Identification method for glycosylation modification sites of proteins - Google Patents
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Abstract
The invention provides an identification method for glycosylation modification sites of proteins. The method comprises the steps of: enriching proteins or polypeptides with glycosylation modification; removing the glycosylation modification of the proteins or polypeptides; using a nucleophilic reagent to mark glycosylation modification sites; and analyzing the proteins or polypeptides marked by the nucleophilic reagent through a mass spectrum. By combining an enrichment technique and a glycosylation modification replacement technique, the enriched proteins with the glycosylation modification sites marked by other marks characterized by smaller molecular weight and stable combination can be obtained. And the proteins are in favor of the change of downstream mass spectrum identification modification sites and quantitative analysis modification levels.
Description
Technical field
The present invention relates to a kind of analytical approach, in particular to a kind of method of decorating site of identification of protein.
Background technology
Along with completing of the international Human Genome Project of in June, 2000, the mankind begin to step into the genome times afterwards comprehensively from the genome epoch, in the genome times afterwards comprehensively, the main study subject of life science is functional genomics, the function of gene is to implement by the executor-protein of its function, thereby the research of protein expression amount, location, decorating state and protein interaction is become the hot research field of genome times afterwards comprehensively.
In life entity, protein needs before function through the transcribing of gene, translation, posttranslational modification process in performance, and specific position performance function in transporte to cells or tissue.Most protein before posttranslational modification occurs be do not have activated, that is to say, the posttranslational modification of protein is extremely important for carrying out the specific function of protein, and it makes the structure of specified protein more complicated, function is more complete, regulatory mechanism is also meticulousr.There is at present the form of more than 20 kind of posttranslational modification in eukaryotic; relatively common are phosphorylation modification, glycosylation modified, acetylation modification, methylate and modify etc., the research object of the technical program is that a kind of special modification of protein glycosylation is that (O-GlcNAc) modified in the amination of O type acetyl glucosamine.
In cell, the albumen of many keys all can occur glycosylation modified, glycosylation modified structure and the function that can affect albumen, participate in many important bioprocess, as immune response, virus copy and infect, Growth of Cells and differentiation etc. (referring to Love, D.C.﹠amp; Hanover, J.A.The hexosamine signaling pathway:deciphering the " O-GlcNAc code " .Sci STKE 2005, re13 (2005) and Love, D.C., Krause, M.W.﹠amp; Hanover, J.A.O-GlcNAc cycling:emerging roles in development and epigenetics.Seminars in cell﹠amp; Developmental biology 21,646-654.).In cell, glycosylation modifiedly comprise that the O type is glycosylation modified, N-type is glycosylation modified and the various ways such as the C type is glycosylation modified.It is glycosylation modified that the amination of O type acetyl glucosamine belongs to the O type, and it is to be single sugar unit that acetylglucosamine (GlcNAc) is connected to specific serine or threonine residues on protein.It is a kind of dynamic modification mode that is present on nucleus and cytoplasm protein that O-GlcNAc modifies, this modification mode and the phosphorylation modification that is widely studied have many similarities, often occur in even identical position close to phosphorylation, the relation that has ' negative and positive adjusting ' with phosphorylation modification, and the role who takes on nutrition receptor and pressoreceptor in cell, be considered to crucial control methods (Wang, the Z. of signal transmission and genetic transcription in cell, Gucek, M.﹠amp; Hart, G.W.Cross-talk between GlcNAcylation and phosphorylation:site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc.Proceedings of the National Academy of Sciences ofthe United States of America 105,13793-13798 (2008)).In addition, constantly there is new evidence to show the substantial connection that has of abnormal O-GlcNAc modification and diabetes, cancer etc.
the amination of O type acetyl glucosamine is modified has important impact to bioprocess numerous in body, yet for the albumen of modifying with O-GlcNAc, the number of identifying at present is very limited, this is mainly because there is the technical barrier of the following aspects: at first, be positioned sugar-modified albumen in extracellular and tube chamber with respect to other of the overwhelming majority, the protein that O-GlcNAc modifies almost all is present in tenuigenin and nucleus, as transcription factor, cytoskeletal protein, nucleoporin etc., content is very low mostly, and this modification is unsettled and height change, at different physiological stimulations such as hormone, growth factor, under the stimulation of mitogen etc., O-GlcNAc modification meeting as phosphorylation modification change and quick must the circulation of specific site generating capacity, these characteristics are all had higher requirement to the sensitivity of analytical technology, secondly, the O-GlcNAc modification group is easy to rupture from the amino acid that connects in Mass Spectrometer Method, and this makes mass spectral:mass spectrographic analysis and identification work also increase difficulty, the 3rd is because O-GlcNAc is simple in structure, usually can not be modified or add to grow up to more complicated structure, and easy d/d enzyme hydrolysis in the process of extracting, and has increased the difficulty of enrichment.
Researchers have proposed many methods and technology with the above-mentioned difficulty of past release in the years of researches process, and wherein more classical have an enzyme mark, the enrichment method of or agglutinin affine based on immunity.Yet all there is defective separately in these methods.
In research in early days, identify that the O-GlcNAc decorating site is the process of a heavy and time-consuming, often will spend near year.In some early stage researchs, the method that is used for the earliest research O type acetylglucosamine is take UDP-3[H at the external galactosyl transferase that utilizes] galactose as substrate with 3[H] galactose transfers to 4 hydroxyls (Torres, C.R.﹠amp of acetylglucosamine; Hart, G.W.Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes.Evidence for O-linked GlcNAc.The Journal of biological chemistry 259,3308-3317 (1984)), with being the order-checking of peptide section by degrading with automatic Edman, then decide on which residue with artificial Edman degraded to contain radioactive label, thereby indirectly obtain the decorating site of O-GlcNAc.
But this Technology Need uses very expensive radioactivity ribotide and corresponding specific installation, and experimental cost significantly rises; Next is need to do repeatedly high performance liquid chromatography and Edman degraded just might identify O-GlcNAc site main on a kind of albumen; The 3rd, the sample requirement is large, and the difficulty of all experimental procedures is all higher, and whole process takes time and effort.
Early stage research O-GlcNAc is based on traditional radioactive galactose it is carried out the detection of enzyme mark, and this method sensitivity is not high, and needs a large amount of purifying proteins thereby expend a large amount of manpowers.Use mass-spectrometric technique can directly observe the O-GlcNAc site, but be difficult to, because in the dissociation process that impact energy is induced, because the very unstable meeting of glycosidic bond is ruptured from the peptide section.
In the method for existing improved evaluation O-GlcNAc decorating site, in order to get rid of the phosphorylation that is positioned on serine or threonine or the interference of other modifications, usually at first to carry out specific enrichment to the O-GlcNAc modified protein.
The simplest O-GlcNAc concentration method is exactly the albumen that utilizes the immune affine or agglutinin chromatogram of antibody to come purifying O-GlcNAc to modify.Antibody for O-GlcNAc mainly contains RL2 and CTD110.6.RL2 is immunoglobulin G (IgG) monoclonal antibody that is produced as antigen induction by nucleoporin complex fragment.CTD110.6 is a kind of immunoglobulin M (IgM) monoclonal antibody, produces as antigen induction with the unique repetitive sequence of chemosynthesis with the peptide section RNAP II carboxyl terminal domain (CTD) of O-GlcNAc.The sugar chain that is GlcNAc to end through the wheat germ agglutinin of succinylation has higher affinity, therefore can be used for the protein that zone of enrichment O-GlcNAc modifies.But, the albumen that uses the method enrichment of antibody or agglutinin to modify with O-GlcNAc, its efficient is lower, and exists and cross reaction that other classes are glycosylation modified.For example, the wheat germ agglutinin of succinylation also can contain other GL-PP of O-GlcNAc in conjunction with end, makes the evaluation of O-GlcNAc modified protein increase false positive.Enrichment means based on antibody often can only be just effective to the albumen that a part of O-GlcNAc modifies, and this is because the binding ability of antibody and O-GlcNAc and near peptide section sequence and the structure glycosylation site have much relations.There is report to point out, cross reaction (Isono also can occur with the specific antibody CTD110.6 of O-GlcNAc in the N-GlcNAc2 that is produced by the glucose deprivation effect, T.O-GlcNAc-specific antibody CTD 110.6 cross-reacts withN-GlcNAc2-modified proteins induced under glucose deprivation.PloS one 6, e18959), can guess that therefore the O-GlcNAc decorating site that utilizes before CTD110.6 to identify also should be identified again.It is pointed out that these two kinds of methods tend to the relatively high or glycoprotein that modify with a plurality of O-GlcNAc of those content of enrichment.By contrast, those content are lower or contain the albumen that single O-GlcNAc modifies and be difficult to be enriched to, therefore use these class methods usually need to use very a large amount of antibody or agglutinin, greatly increased the cost of experiment, and need to carry out a series of control experiment and get rid of nonspecific absorption and false-positive result.
Have two kinds of enrichment methods can overcome defective based on the enrichment method of antibody or agglutinin, a kind of is that the bioengineered enzyme concentration method is (referring to document Khidekel, N., Ficarro, S.B., Peters, E.C.﹠amp; Hsieh-Wilson, L.C.Exploring the O-GlcNAc proteome:direct identification of O-GlcNAc-modified proteins from the brain.Proceedings of the National Academy of Sciences of the United States of America 101,13132-13137 (2004)), another kind is that the interior sugar unit metabolic marker concentration method of living cells is (referring to document Prescher, J.A.﹠amp; Bertozzi, C.R.Chemical technologies for probing glycans.Cell 126,851-854 (2006)).The know-why of these two kinds of methods is very close in fact, all to utilize the O-GlcNAc transferase that the glucose (GlcNAz) of nitrine mark is transferred on the site of being modified by O-GlcNAc, be connected with biotin reaction with chemical tags by nitrine, the albumen that be labeled this moment just can pass through Avidin or the separated purifying of Streptavidin chromatographic column again.Be connected by covalent bond with chemical tags because this kind method makes the O-GlcNAc group, the efficient of this identification and enrichment is far above the non-covalent classic method that is connected based on antibody and agglutinin.Immunoblot experiment shows, and the signal intensity of the O-GlcNAc modified protein that detects by these two class methods is far away higher than common antibody testing method.
Yet, the peptide section with O-GlcNAc that obtains by above-mentioned two kinds of enrichment methods is not suitable for utilizing conventional tandem mass spectrometry (MS/MS) to analyze its decorating site, this mainly contains the reason of the following aspects: be at first because label (for example biotin) quality that is combined on O-GlcNAc is very large, can produce rather serious ion depression effect when mass spectrophotometry, affect normal peptide section sequence analysis; Secondly, in the MS/MS collection of illustrative plates, the fragment of biotin group also can have a strong impact on the interpretation of peptide section sequence; The 3rd is because the GlcNAz-biotin that is connected by glycosidic bond with the peptide section is still unsettled in the process of MS/MS fragmentation, thereby has hindered the evaluation of direct site.Therefore, if the peptide section with O-GlcNAc that directly above-mentioned two kinds of enrichment methods is obtained is carried out mass spectrophotometry, certainly will affect accuracy and the sensitivity of mass spectrometry results.
Summary of the invention
Due in the method in the glycosylation modified site of existing identification of protein, the glycosylation modified protein with label that obtains by bioengineered enzyme concentration method or sugar unit metabolic marker concentration method is unfavorable for mass spectrophotometry, thereby has reduced accuracy and the sensitivity that identifies in the site.Therefore the invention provides a kind of new authentication method, the method is replaced glycosylation modified method phase coupling with one of above-mentioned two kinds of enrichment methods with a kind of, obtain glycosylation modified site less and in conjunction with the protein of the enrichment of stable label institute mark, this protein is conducive to the Mass Spectrometric Identification decorating site in downstream and the variation of quantitative test modification level by other molecular weight.To sum up, the invention provides a kind of enrichment method, replace the method that glycosylation modified method and mass spectrometric analysis method are united use, can be used for the O-GlcNAc decorating site of Direct Identification protein, this authentication method comprises the following steps:
1) enrichment is with glycosylation modified protein or polypeptide
The present invention adopts bioengineered enzyme concentration method or sugar unit metabolic marker concentration method to carry out specific enrichment to the O-GlcNAc modified protein.
The bioengineered enzyme concentration method is in the biological work enzyme of external utilization---β-1, the UDP galactose (UDP-GalNAz) that 4 galactosyl transferase I (Y289LGalT1) will contain nitrine is transferred on four hydroxyls of O-GlcNAc, nitrine can be connected with the alkynyl on the alkynyl biotin (Biotin-alkyne), the purpose (as shown in Figure 1) that then is connected with Avidin by biotin and reaches enrichment O-GlcNAc modified protein.Albumen or the peptide section with the O-GlcNAc modification that are enriched on Avidin medium (Avidin-bead) can use the Avidin of high strength scaling agent or high concentration to elute, and can not be removed in cleaning step with this glycosyl modified protein molecular.
Sugar unit metabolic marker method is close with bioengineered enzyme enrichment ratio juris, and only the method is to introduce the tag molecule (seeing Fig. 2) that contains nitrine on the living cells level.Have and experiment showed, that living cells can absorb and utilize N-Azide acetylglucosamine (GlcNAz) as the substituting substrate of acetylglucosamine.Absorb for the ease of living cells; GlcNAz first processes through acetylation; acetylizad GlcNAz is after by cellular uptake; intracellular esterase can be deacetylated fast with it, and then GlcNAz can enter aminohexose biosynthesis pathway (HBP) and change into UDP-GlcNAz as gucosamine.O-GlcNAc transferase (OGT) is connected to GlcNAz on serine or threonine take UDP-GlcNAz as substrate, and the GlcNAz that is connected on albumen can be combined by the high coupling reaction (as Staudinger ligation or Click chemistry) of specificity and enrichment label such as biotinylated hydrogen phosphide or biotinylated alkynyl external.The method finally also makes protein group that O-GlcNAc modifies all with the label of upper biotin, thereby can use conventional Avidin or Streptavidin medium to carry out purification enrichment.Although the method has successful application report, but it is very low to find that in practice GlcNAz changes into the efficient of UDP-GlcNAz, what therefore the present invention used is through the internal metabolism labelling method after improving---directly feed the galactose (GalNAz) to living cells nitrine mark, through the acetylgalactosamine remedial pathway, UDP-galactose epimerase (GALE) can must change into UDP-GlcNAz with UDP-GalNAz is efficient, thereby as the decorating site of the former UDP-GlcNAc of substrate highly efficient labeling of OGT.Last the present invention utilizes the high affinity between biotin and Avidin to remove more thoroughly the biomolecule of non-specific binding, further improves the efficient of enrichment, reduces the depression effect of foreign ion.
2) replacement is glycosylation modified
Analyze its decorating site because the peptide section with O-GlcNAc that obtains by above-mentioned two kinds of enrichment methods is not suitable for utilizing conventional tandem mass spectrometry (MS/MS), therefore the glycosylation modified of peptide section replaced.
The glycosylation modified embodiment of a preferred replacement of the present invention be removed glycosylation site for can first reacting by β-elimination from serine or threonine, produce unsaturated sulfydryl, with respect to O-α-galactose or O-phosphorylation, O-β-GlcNAc glycosidic bond is more responsive to β-elimination reaction at alkaline environment, therefore the chemical tags and the unsettled glycosidic bond that are marked on GlcNAc can be eliminated in the lump by β-elimination reaction.then use the Michael addition reaction to add stable nucleopilic reagent on original GlcNAc decorating site, thereby nucleopilic reagent and sulfydryl reaction produce the little label of a particular molecule amount on the amino acid of modifying, the very special quality that adds according to these small tenon label removes to identify glycosylation site, namely replace O-GlcNAc modification on serine and threonine in conjunction with β-elimination reaction and Michael addition reaction, wherein being combined with of β-elimination reaction and Michael addition reaction is referred to as β-elimination-Markovnikov addition reaction (beta-Elimination followed by Michael Addtion BEMAD), namely refer to first remove by β-elimination reaction the unsettled acetylglucosamine base that on serine or threonine, O connects under alkali condition, form two keys between α carbon and side chain carbon, add this moment nucleopilic reagent just can open two keys, thereby mark O-GlcNAc decorating site.
The present invention preferably uses dithiothreitol (DTT) (DTT) as nucleopilic reagent, that is, under alkali condition, β-elimination reaction occurs in the modification on serine and threonine, O-GlcNAc group and the chemical tags of carrying thereof are removed in the lump, formed two keys between α carbon and side chain carbon; And then adding dithiothreitol (DTT) (DTT) as nucleopilic reagent, DTT can open two keys, the decorating site at mark O-GlcNAc place.
3) chromatograph-mass spectrometer coupling analysis
Using the chromatograph-mass spectrometer coupling system to carry out the peptide section through the polypeptide sample of above-mentioned enrichment and replacement step separates and Sequence Identification.Preamble is mentioned glycosidic bond and be easy to fracture when cascade mass spectrometry, and after replacing it with DTT, this decorating site just becomes very stable, does not hinder in the mass spectrophotometry peptide section sequence.And DTT modifies the decorating site that the special molecular amount that increases can be used to determine O-GlcNAc, improves the accuracy of location.But also can utilize the relative content of the protein O-GlcNAc level between normal DTT and the cold labeling more different samples of DTT in (deuterium generation), the O-GlcNAc that analyzes protein under different condition colony modifies the quantitative difference of level.
In sum, combine and can high specific obtain the protein group of the O-GlcNAc modification of enrichment replacing one of glycosylation modified method glycoprotein enrichment method different from two kinds, be conducive to the Mass Spectrometric Identification decorating site in downstream and the variation of quantitative test modification level, for the research of the biological process of O-GlcNAc regulation and control provides novel effectively instrument.
The protein group with the O-GlcNAc modification that this new technology flow process that the present invention proposes is used for expressing in enrichment and identification of cell, the method and conventional immune biochemical method phase specific energy significantly improve the sensitivity (3-5 doubly) of purifying, find a large amount of original unknownly with the glycosylation modified albumen of this kind, this provides crucial technology for further investigation with intracellular signal transduction and the Regulation of Gene expression mechanism that O-GlcNAc is modified to the basis.
Description of drawings
Fig. 1 illustrates the schematic diagram that uses bioengineering enzyme process enrichment O-GlcNAc modified protein.
Fig. 2 illustrates sugar unit metabolic marker technology schematic diagram, and wherein (A) substitutes natural O-GlcNAc for Azide monose after by cellular uptake and be expressed on protein substrate; (B) this nitrine functional group and affinity tag molecule or fluorescent dye molecular reaction and connect are used for purifying or the colour developing of O-GlcNAc protein group.
Fig. 3 illustrates the chemical process of β-elimination-Markovnikov addition alternative reaction (BEMAD).
What Fig. 4 (A) showed is after the 293T cell carries out the sugar unit mark, to extract intracellular nucleoprotein, thinks that gel electrophoresis and Western blotting detect; Fig. 4 (B) is the experimental result to mouse embryo stem cell.
Fig. 5 is the tandem mass spectrometry figure with the peptide section of DTT modification.The peptide section derives from respectively (A) actin or (B) myosin.
Embodiment
The following examples can make the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.
What Fig. 4 (A) showed is after the 293T cell carries out the sugar unit mark, to extract intracellular nucleoprotein, carries out SDS-PAGE (separation of one dimension gel) and Western blot (Western blotting) and detects.What (A) upper figure showed is by the glycoproteome of sugar unit mark with upper FLAG label, uses the result that α-FLAG antibody detects.Whether more sensitiveer than traditional antibody detection method in order to investigate chemicobiology method of the present invention, the present invention uses α-O-GlcNAc antibody directly identical sample to be detected (A figure below) simultaneously.The present invention finds that the protein group of using the sugar unit mark to detect the O-GlcNAc modification has higher sensitivity than traditional biochemical immunity method really.Further experiment shows, when cell transfecting enters OGT (O-GlcNAc glucoside transferase), its O-GlcNAc expression rises, the corresponding Western blot signal grow (the upper figure of A) that obtains; Transfection OGA (O-GlcNAc hydrolytic enzyme) can cause the O-GlcNAc expression to descend, so the Western blot signal weaker (A figure below) of glycoprotein.These results illustrate that all what utilize method mark of the present invention and detection is the protein colony of modifying with the amination of O type acetyl glucosamine really.
Fig. 4 (B) is the experimental result to mouse embryo stem cell.The O-GlcNAc modified protein that this embryonic stem cell of the upper figure explanation of B is expressed can by the sugar unit metabolic marker, demonstrate very strong Western blot signal.Compare with the classic method (B figure below) of using O-GlcNAc antibody, the method that the present invention proposes can detect a greater variety of glycoprotein (molecular weight ranges is wider), obtains stronger detection signal.
Above experimental data proves, the sugar unit labelling technique can be used in the glycoproteome with the O-GlcNAc modification of mark, purifying and detection cell inner expression, and the sensitivity of its detection and specificity want obvious must be higher than traditional biochemical immunity method, this Mass Spectrometric Identification for the protein in downstream of the present invention is laid a good foundation.
Embodiment 2
What embodiment 2 showed is the O-GlcNAc decorating site that utilizes on Mass Spectrometric Identification protein.At first the present invention utilizes the peptide section of modifying with O-GlcNAc in the protein digestion sample of sugar unit metabolic marker method enrichment human body 293T cell, then carry out β-elimination-Markovnikov addition alternative reaction and make the O-GlcNAc groups converted become the DTT group, thus the original glycosylation site of mark.As shown in Figure 5, these two peptide sections of carrying DTT in cascade mass spectrometry according to mass number and the distribution of its fracture fragment, the decorating site (being original O-GlcNAc site) of DTT be can accurately must judge, actin (Fig. 5 A) and myosin (Fig. 5 B) come from respectively and its amino acid sequence information can indicate these two peptide sections.B in spectrogram and Y mark be different peptide section fragmention sequences.
Although illustrated and described embodiments of the invention, for the ordinary skill in the art, be appreciated that without departing from the principles and spirit of the present invention and can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claims and equivalent thereof.
Claims (10)
1. the method in the glycosylation modified site of an identification of protein, comprise the following steps: enrichment is with glycosylation modified protein or polypeptide; Remove the glycosylation modified of described protein or polypeptide; With the glycosylation modified site of nucleopilic reagent mark; Utilize mass spectrophotometry by the protein of nucleopilic reagent mark or polypeptide.
2. method according to claim 1 is wherein implemented the glycosylation modified step of the described protein of removal or polypeptide by β-eliminations-Markovnikov addition alternative reaction and with the step in the glycosylation modified site of nucleopilic reagent mark.
3. method according to claim 1, wherein said glycosylation modified be that the amination of O type acetyl glucosamine is modified.
4. method according to claim 1, wherein said enriching step is implemented by the bioengineered enzyme concentration method.
5. method according to claim 1, wherein said enriching step is implemented by sugar unit metabolic marker method.
6. method according to claim 5, wherein said sugar unit metabolic marker method is the internal metabolism labelling method.
7. method according to claim 1, wherein said nucleopilic reagent is dithiothreitol (DTT).
8. method according to claim 1, wherein said nucleopilic reagent is the nucleopilic reagent of cold labeling.
9. method according to claim 8, wherein said nucleopilic reagent is the deuterium dithiothreitol (DTT) in generation.
10. method according to claim 1, wherein implement mass spectrophotometry by method for qualitative analysis or the quantitative analysis method of tandem mass spectrometry.
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CN110609078A (en) * | 2019-09-20 | 2019-12-24 | 南京谱利健生物技术有限公司 | Method for detecting protein phosphorylation and acetylglucosamine saccharification correlation effect |
CN111304240A (en) * | 2020-03-25 | 2020-06-19 | 山东农业大学 | Method for rapidly identifying grape gene function based on tobacco transient expression system |
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CN105467050A (en) * | 2014-09-11 | 2016-04-06 | 中国科学院大连化学物理研究所 | Identification method for O-glycosylation peptide fragment and complete saccharide chain thereof |
CN105467050B (en) * | 2014-09-11 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of identification method for being used for O glycopeptide segments and its complete sugar chain |
CN108761084A (en) * | 2018-05-23 | 2018-11-06 | 同济大学 | A kind of complete N- glycoprotein primary structure comprehensive identification method |
CN110609078A (en) * | 2019-09-20 | 2019-12-24 | 南京谱利健生物技术有限公司 | Method for detecting protein phosphorylation and acetylglucosamine saccharification correlation effect |
CN111304240A (en) * | 2020-03-25 | 2020-06-19 | 山东农业大学 | Method for rapidly identifying grape gene function based on tobacco transient expression system |
CN111304240B (en) * | 2020-03-25 | 2022-02-08 | 山东农业大学 | Method for rapidly identifying grape gene function based on tobacco transient expression system |
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