CN103091498B - Plant in-vitro ubiquitin protein degradation system and application thereof - Google Patents

Abstract

The invention discloses a plant in-vitro ubiquitin protein degradation system and an application thereof. The plant in-vitro ubiquitin protein degradation system provided by the invention is a composition consisting of one ubiquitin activating enzyme from arabidopsis thaliana, 14 ubiquitin-conjugating enzymes and wild-type K48R and K63R point mutation ubiquitin protein. Compared with the currently common used in-vitro ubiquitination reaction component, all components of the composition provided by the invention come from the model plant arabidopsis thaliana; when the composition is used for detecting whether the to-be-detected protein of plant origin has ubiquitination activity, the analysis result is more real and credible; the composition provided by the invention contains the component capable of representing most of the subfamily characteristics of the ubiquitin-conjugating enzyme of arabidopsis thaliana, and the coverage is wide; the activity of ubiquitin ligase can be detected and the specific binding condition of E2-E3 can be analyzed more comprehensively by use of the components, and more clues can be provided for studying the gene functions; and moreover, the type of polyubiquitin chain formed by the ubiquitination reaction can be analyzed by use of the point mutation ubiquitin protein.

Description

Plant in-vitro ubiquitin protein degradation system and application thereof

Technical field

The present invention relates to a kind of plant in-vitro ubiquitin protein degradation system and application thereof, particularly a kind of whole active ingredient all comes from plant in-vitro ubiquitin protein degradation system and the application thereof of Arabidopis thaliana.

Background technology

Ubiquitin in most eukaryotes/26S proteasomal system (ubiquitin/26S proteasome system UPS) abnormal protein of can not only degrading, and overwhelming majority transformation period very short Function protein matter (as transcription factor, cyclin etc.) can be removed, therefore in maintenance cell normal activities, play important regulating and controlling effect.Existing research confirms, the impact of this system in plant is even more important, and its function almost relates to the control of all physiological pathway committed steps in plant, is one of adjustment and control system the meticulousst in plant.Ubiquitin is covalently attached to as protein tag the effect successively protein target molecule needing ubiquitin activating enzyme (E1), ubiquitin binding enzyme (E2) and ubiquitin ligase (E3).Ubiquitination on substrate has broad variety, the poly ubiquitination etc. that the poly ubiquitination connected as single ubiquitination, K48-, K63-connect, the type decided of the ubiquitination destiny of substrate protein.2 E1,37 E2 are had and more than 1400 E3 genes in model plant arabidopsis gene group.The substrate specificity of ubiquitination determines primarily of E3, and E2 is the key determinant of poly ubiquitin chain structure type on substrate.37 E2 belong to 12 subfamilies, and research shows that there is a large amount of E2-E3 in vivo acts synergistically specifically.

Ubiquitin ligase is predicted as certain or by the agnoprotein of the substrate protein of ubiquitination, utilize external ubiquitination system to carry out the analysis of its ubiquitination activity, be indispensable research contents, experimental result can provide important clue for follow-up study.But for the testing protein of plant gene coding, there is following shortcoming in external ubiquitination reaction system conventional at present: the ubiquitin activating enzyme that (1) is used, ubiquitin binding enzyme (being directed in the detection of ubiquitin ligase self ubiquitination activity) and ubiquitin protein are not that the albumen of plant code is (as conventional yeast ubiquitin activating enzyme mostly, ubiquitin binding enzyme UBCH5b or UBCH5c of people, and the ubiquitin protein etc. of people), because the homogenic function of different plant species is likely incomplete same, therefore in such reaction system, detect the activity of plant gene products, can not truth completely in On behalf of plant system, so false positive or false-negative result often can be produced, (2) ubiquitin binding enzyme kind used is single, but reality also exists the specificity combination of a large amount of E2-E3 in vivo, utilizes single E2 can not detect the activity of all E3 albumen, therefore occurs that the false-negative result of part is inevitable.

Summary of the invention

The object of this invention is to provide a kind of plant in-vitro ubiquitin protein degradation system and application thereof.

Plant in-vitro ubiquitin protein degradation system provided by the present invention is the composition for vitro detection testing protein ubiquitination activity shown in following (A) or (B).

(A) the described composition for vitro detection testing protein ubiquitination activity is made up of following (1)-(3):

(1) ubiquitin activating enzyme UBA2 and wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

(2) following 14 kinds of ubiquitin binding enzymes is all or part of: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31;

(3) point mutation ubiquitin protein UbK48R and point mutation ubiquitin protein UbK63R;

The aminoacid sequence of described point mutation ubiquitin protein UbK48R is the sequence Methionin of the 48th of sequence in sequence table 3 the being replaced with gained after arginine; The aminoacid sequence of described point mutation ubiquitin protein UbK63R is the sequence Methionin of the 63rd of sequence in sequence table 3 the being replaced with gained after arginine;

(B) the described composition for vitro detection testing protein ubiquitination activity is made up of (1) described in (A) and described (2).

Another object of the present invention is to provide a kind of plasmid group for vitro detection testing protein ubiquitination activity.

Plasmid group provided by the present invention can be (I) or (II) as follows:

(I) by following 1)-3) form:

1) express the plasmid of ubiquitin activating enzyme UBA2, and express the plasmid of wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

2) all or part of in 14 kinds of plasmids, is expressed as follows in 14 kinds of ubiquitin binding enzymes a kind of: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5 respectively; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31;

3) express the plasmid of point mutation ubiquitin protein UbK48R, and express the plasmid of point mutation ubiquitin protein UbK63R;

The aminoacid sequence of described point mutation ubiquitin protein UbK48R is the sequence Methionin of the 48th of sequence in sequence table 3 the being replaced with gained after arginine; The aminoacid sequence of described point mutation ubiquitin protein UbK63R is the sequence Methionin of the 63rd of sequence in sequence table 3 the being replaced with gained after arginine;

(II) by described 1 in (I)) and 2) form.

In above-mentioned (A) and (I), described detection testing protein ubiquitination activity be all specially following one or its two: one, detect testing protein whether there is ubiquitin ligase activity; Its two, detect any or several ubiquitin binding enzyme of testing protein in described composition and be combined.

In above-mentioned (B) and (II), described detection testing protein ubiquitination activity be all specially one in described (A) and (I), its two or as follows its three: its three, detect the interact poly ubiquitin chain that formed of testing protein and described composition and whether contain poly ubiquitin chain that K48-is connected or the poly ubiquitin chain that K63-connects.

Each albumen in above-mentioned (1) and (2) all derives from Arabidopis thaliana.(2) the described 14 kinds of ubiquitin binding enzymes in belong to the different subfamily of Arabidopis thaliana ubiquitin binding enzyme, can cover arabidopsis gene group major part ubiquitin binding enzyme feature.

In an embodiment of the present invention, in described plasmid group, each plasmid is the prokaryotic expression carrier carrying corresponding protein encoding gene, and its skeleton plasmid is pET28a.

In the present invention, the encoding gene (At5g06460) of described ubiquitin activating enzyme UBA2 is specially the DNA molecular shown in sequence 2 in sequence table; The encoding gene (At4g02890) of described wild type monomeric ubiquitin protein is specially the DNA molecular shown in sequence 4 in sequence table; The encoding gene (At5g50870) of described ubiquitin binding enzyme UBC27 is specially the DNA molecular shown in sequence 6 in sequence table; The encoding gene (At1g14400) of described ubiquitin binding enzyme UBC1 is specially the DNA molecular shown in sequence 8 in sequence table; The encoding gene (At2g02760) of described ubiquitin binding enzyme UBC2 is specially the DNA molecular shown in sequence 10 in sequence table; The encoding gene (At5g62540) of described ubiquitin binding enzyme UBC3 is specially the DNA molecular shown in sequence 12 in sequence table; The encoding gene (At5g53300) of described ubiquitin binding enzyme UBC10 is specially the DNA molecular shown in sequence 14 in sequence table; The encoding gene (At3g17000, disappearance membrane spaning domain) of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene (At3g46460) of described ubiquitin binding enzyme UBC13 is specially the DNA molecular shown in sequence 18 in sequence table; The encoding gene (At5g41340) of described ubiquitin binding enzyme UBC4 is specially the DNA molecular shown in sequence 20 in sequence table; The encoding gene (At1g63800) of described ubiquitin binding enzyme UBC5 is specially the DNA molecular shown in sequence 22 in sequence table; The encoding gene (At2g46030) of described ubiquitin binding enzyme UBC6 is specially the DNA molecular in sequence table shown in sequence 24; The encoding gene (At1g78870) of described ubiquitin binding enzyme UBC35 is specially the DNA molecular shown in sequence 26 in sequence table; The encoding gene (At3g20060) of described ubiquitin binding enzyme UBC19 is specially the DNA molecular shown in sequence 28 in sequence table; The encoding gene (At1g75440) of described ubiquitin binding enzyme UBC16 is specially the DNA molecular shown in sequence 30 in sequence table; The encoding gene (At5g05080) of described ubiquitin binding enzyme UBC22 is specially the DNA molecular shown in sequence 32 in sequence table; The encoding gene of described point mutation ubiquitin protein UbK48R is specially the DNA molecular A of the A and the 144th of the 143rd of sequence in sequence table 4 all being replaced with gained after G; The encoding gene of described point mutation ubiquitin protein UbK63R is specially the DNA molecular A of the 188th of sequence in sequence table 4 the being replaced with gained after G.

Recombinant bacterium containing any one or several plasmids in described plasmid group also belongs to protection scope of the present invention.

In the present invention, described recombinant bacterium is specially intestinal bacteria, as e. coli bl21 (DE3).

Described composition or described plasmid group or the application of described recombinant bacterium in following (a) or (b) also belong to protection scope of the present invention:

A () is detected testing protein and whether is had ubiquitin ligase activity;

B () is detected the testing protein with ubiquitin ligase activity and is combined with any or several ubiquitin binding enzyme.

Another object of the present invention is to provide and a kind ofly detects the method whether testing protein has ubiquitin ligase activity.

Whether detection testing protein provided by the present invention has the method for ubiquitin ligase activity, specifically can comprise the steps:

(a1) following 4 groups of samples are reacted respectively:

First group: described ubiquitin activating enzyme UBA2, described wild type monomeric ubiquitin protein, and any one in described 14 kinds of ubiquitin binding enzymes;

Second group: described testing protein, described wild type monomeric ubiquitin protein, and the described ubiquitin binding enzyme identical with first group;

3rd group: described testing protein, described ubiquitin activating enzyme UBA2, and described wild type monomeric ubiquitin protein;

4th group: described testing protein, described ubiquitin activating enzyme UBA2, described wild type monomeric ubiquitin protein, and the described ubiquitin binding enzyme identical with first group;

(a2) reaction product of 4 groups of samples in step (a1) is carried out SDS-PAGE electrophoresis respectively, after transferring film, carry out immuning hybridization with the antibody of anti-described wild type monomeric ubiquitin protein;

(a3) according to the result of immuning hybridization in step (a2), determine whether described testing protein has ubiquitin ligase activity as follows: if the reaction product of the 4th group of sample has the antibody signal of anti-described wild type monomeric ubiquitin protein in the position that molecular weight is greater than described testing protein, first group simultaneously, the reaction product of second group and the 3rd group sample is greater than the antibody signal of all not anti-described wild type monomeric ubiquitin protein in position of described testing protein at molecular weight, then described testing protein has ubiquitin ligase activity or candidate has ubiquitin ligase activity, otherwise then described testing protein does not have ubiquitin ligase activity or candidate does not have ubiquitin ligase activity.Described antibody signal is mainly the smear being greater than the position appearance of described testing protein at molecular weight.

In the process, described testing protein, described ubiquitin activating enzyme UBA2, described wild type monomeric ubiquitin protein, and be that 200-500ng(is as 200ng with the quality proportioning of described ubiquitin binding enzyme): 50ng:3-5 μ g(is as 4 μ g): 200ng.

In the above-mentioned methods, the first to three group is control group, is equivalent to quality control standard; 4th group is experimental group.

Another object of the present invention is to provide the method that any or several ubiquitin binding enzyme of testing protein in described composition that a kind of detection has ubiquitin ligase activity is combined.

The method that any or several ubiquitin binding enzyme of testing protein in described composition that detection provided by the present invention has ubiquitin ligase activity is combined, specifically can comprise the steps:

(b1) by described 14 kinds of ubiquitin binding enzymes all or part of in each respectively with testing protein, described ubiquitin activating enzyme UBA2, and described wild type monomeric ubiquitin protein mixing, form several reaction systems;

(b2) after reaction terminates, the product of several reaction systems in step (b1) is carried out SDS-PAGE electrophoresis respectively, after transferring film, carry out immuning hybridization with the antibody of anti-described wild type monomeric ubiquitin protein;

(b3) according to the result of immuning hybridization in step (b2), determine that any or several ubiquitin binding enzyme of described testing protein in described composition is combined as follows: the ubiquitin binding enzyme met in the reaction system of following condition is the ubiquitin binding enzyme that described testing protein combines with it; The ubiquitin binding enzyme do not met in the reaction system of following condition is described testing protein uncombined ubiquitin binding enzyme with it: the antibody signal having anti-described wild type monomeric ubiquitin protein in the position that molecular weight is greater than described testing protein.

In the process, described testing protein, described ubiquitin activating enzyme UBA2, described wild type monomeric ubiquitin protein, and be that 200-500ng(is as 200ng with the quality proportioning of described ubiquitin binding enzyme): 50ng:3-5 μ g(is as 4 μ g): 200ng.

In one embodiment of the invention, the ubiquitin binding enzyme selected in step (b1) is described ubiquitin binding enzyme UBC27, described ubiquitin binding enzyme UBC3, described ubiquitin binding enzyme UBC10, described ubiquitin binding enzyme UBC32, described ubiquitin binding enzyme UBC13, described ubiquitin binding enzyme UBC6, described ubiquitin binding enzyme UBC19 and described ubiquitin binding enzyme UBC35.

Described composition or described plasmid group or described recombinant bacterium also belong to protection scope of the present invention detecting the application in poly ubiquitin chain type.

In above-mentioned application, described detection poly ubiquitin chain type is specially and detects the interact poly ubiquitin chain that formed of testing protein and described composition and whether contain poly ubiquitin chain that K48-is connected or the poly ubiquitin chain that K63-connects.

Also object of the present invention is to provide the interact poly ubiquitin chain that formed of a kind of testing protein detecting ubiquitin ligase and described composition and whether contains the method for poly ubiquitin chain that K48-is connected or the poly ubiquitin chain that K63-connects.

Detection testing protein provided by the present invention and described composition interact the method for poly ubiquitin chain that whether the poly ubiquitin chain that formed be connected containing K48-or the poly ubiquitin chain that K63-connects, and specifically can comprise the steps:

(c1) following 5 groups of samples are reacted respectively:

First group: testing protein, described wild type monomeric ubiquitin protein, and any one in described 14 kinds of ubiquitin binding enzymes;

Second group: testing protein, described wild type monomeric ubiquitin protein, and described ubiquitin activating enzyme UBA2;

3rd group: testing protein, described wild type monomeric ubiquitin protein, described ubiquitin activating enzyme UBA2, and the described ubiquitin binding enzyme identical with first group;

4th group: testing protein, described point mutation ubiquitin protein UbK48R, described ubiquitin activating enzyme UBA2 and the described ubiquitin binding enzyme identical with first group;

5th group: testing protein, described point mutation ubiquitin protein UbK63R, described ubiquitin activating enzyme UBA2 and the described ubiquitin binding enzyme identical with first group;

(c2) reaction product of 5 groups of samples in step (c1) is carried out SDS-PAGE electrophoresis respectively, after transferring film, carry out immuning hybridization with the antibody of anti-described wild type monomeric ubiquitin protein;

(c3) according to the result of immuning hybridization in step (c2), poly ubiquitin chain signal is not had in the reaction product of first group, second group sample, and under the reaction product of the 3rd group of sample has the prerequisite of poly ubiquitin chain signal, the poly ubiquitin chain that the poly ubiquitin chain determined to interact described testing protein and described composition as follows whether the poly ubiquitin chain that formed be connected containing K48-or K63-connect:

If meet the condition of (d1), do not meet the condition of (d2), then described testing protein and described composition interact the poly ubiquitin chain that the poly ubiquitin chain that formed is connected containing K48-, and do not contain the poly ubiquitin chain of K63-connection simultaneously;

If do not meet the condition of (d1), meet the condition of (d2), then described testing protein and described composition interact the poly ubiquitin chain that the poly ubiquitin chain that formed is connected containing K63-, and do not contain the poly ubiquitin chain of K48-connection simultaneously;

If meet the condition of (d1) and (d2) simultaneously, then the interacts poly ubiquitin chain that formed of described testing protein and described composition had both contained the poly ubiquitin chain that K48-is connected, and also contained the poly ubiquitin chain of K63-connection;

If do not meet the condition of (d1) and (d2), then the interacts poly ubiquitin chain that formed of described testing protein and described composition neither contains the poly ubiquitin chain that K48-is connected, and does not also contain the poly ubiquitin chain of K63-connection simultaneously;

(d1) reaction product of the 4th group of sample is without poly ubiquitin chain signal, or has the poly ubiquitin chain signal that strength of signal is less than the reaction product of the 3rd group of sample;

(d2) reaction product of the 5th group of sample is without poly ubiquitin chain signal, or has the poly ubiquitin chain signal that strength of signal is less than the reaction product of the 3rd group of sample;

Described poly ubiquitin chain signal is the antibody signal that there is anti-described wild type monomeric ubiquitin protein the position being greater than described testing protein at molecular weight.

In the process, when testing protein described in practical application is known as ubiquitin binding enzyme (as UBC35), then all do not add any one in described 14 kinds of ubiquitin binding enzymes in the reaction system of above-mentioned first group to the 5th group, simultaneously then do not add described testing protein in second group; Certainly active according to the ubiquitin binding enzyme of described testing protein, selectivity can add to have the ubiquitin binding enzyme (as UBC35) as testing protein and promote its accessory protein be combined with ubiquitin (UEV1D).

In described method, first group and second group is control group, and this reaction system of two groups can carry out suitable variation in actually operating, and principle ensures that component in this two group reactions system is compared with the 3rd group, lacks a component.

In the process, described testing protein, described ubiquitin activating enzyme UBA2, described ubiquitin protein (wild type monomeric ubiquitin protein, or point mutation ubiquitin protein UbK48R, or point mutation ubiquitin protein UbK63R), and can be 200-500ng(as 200ng with the quality proportioning of described ubiquitin binding enzyme): 50ng:3-5 μ g(is as 4 μ g): 200ng.

In the present invention, above-mentioned all described testing proteins are preferably vegetable-protein.

In the present invention, each albumen in described composition is obtained by prokaryotic expression.Conveniently purifying and subsequent applications, each albumen all introduces label (coming from the marker gene on skeleton carrier) in described prokaryotic expression process.In above-mentioned all described application and method, the label that described testing protein is introduced is different from the label that each albumen in described composition is introduced.

In an embodiment of the present invention, the encoding gene of each albumen in described composition is all building up to prokaryotic expression carrier pET8a(with 6 × His and T7 protein tag), obtain corresponding recombinant plasmid; Gained recombinant plasmid is by heat shock method transformation of E. coli BL21(DE3), through IPTG induction expression protein, gained total bacterial protein supernatant Ni-NTA(Qiagen) purifying super filter tube (Millipore) desalination and concentration.The encoding gene of described testing protein is building up on the prokaryotic expression carrier with the protein tag except 6 × His and T7, if pGEX serial carrier (recombinant protein is with glutathione-S-transferase GST label) or pMalC2(recombinant protein are with maltose binding protein (MBP) label) etc.; Gained recombinant plasmid is by heat shock method transformation of E. coli BL21(DE3), through IPTG induction expression protein, gained total bacterial protein supernatant packing-80 DEG C is frozen for subsequent use, or purify and desalination and concentration by ultrafiltration by corresponding method, as MBP fusion rotein amylase resin(NEB) purifying, gst fusion protein glutathione agarose Glutathione-sepharose4B (GE healthcare) purifying.

In described methods all above, the reaction related to all is carried out in following reaction buffer: 20 × reaction buffer.Formula is following (a) or (b):

(a) 1M Tris pH7.5,40mM ATP, 100mM MgCl ₂, 40mM DTT, solvent is water;

(b) 1M Tris pH7.5,100mM ATP, 50mM MgCl ₂, 2mM DTT, solvent is water.

Tool of the present invention has the following advantages: compared to external ubiquitination reacted constituent conventional at present, the whole components in composition provided by the present invention all derive from model plant Arabidopis thaliana, and the testing protein analytical results for plant origin is more genuine and believable; The component that can represent Arabidopis thaliana ubiquitin binding enzyme overwhelming majority subfamily feature is contained in addition in composition provided by the present invention, broad covered area, utilize these components more fully can detect the activity of ubiquitin ligase and analyze the specific binding situation of E2-E3, the research that can be gene function provides more clue.

Accompanying drawing explanation

Fig. 1 is the Activity determination result of Arabidopis thaliana ubiquitin activating enzyme UBA2 and Arabidopis thaliana wild type monomeric ubiquitin protein.A () is the Arabidopis thaliana ubiquitin activating enzyme UBA2(His-AtE1 of band His label) and wheat ubiquitin activating enzyme (His-wE1) carry out ubiquitination Activity determination respectively with people's ubiquitin binding enzyme UBCH5b and Arabidopis thaliana wild type monomeric ubiquitin protein; B () is the protein content of His-AtE1 and His-wE1 that in (a), reaction is used.

Fig. 2 is the result detecting the multiple ubiquitin binding enzyme activity of Arabidopis thaliana with the formation experiment of the thioester bond of DTT-sensitivity.In figure, "+" represents in corresponding reaction system containing this material; "-" represents in corresponding reaction system not containing this material; Arrow to represent in reaction generate ubiquitin binding enzyme-ubiquitin protein mixture (by connecting the thioester bond of DTT sensitivity between them); Triangle represents the ubiquitin binding enzyme having neither part nor lot in reaction; Asterisk represents the Arabidopis thaliana wild type monomeric ubiquitin protein having neither part nor lot in reaction.

Fig. 3 is the result that ubiquitination Activity determination is carried out in different E2-E3 combinations.A () may combine the result of carrying out ubiquitination reaction for the testing protein CBL-like of ubiquitin ligase and multiple ubiquitin binding enzyme respectively for inferring; B () combines the result of carrying out ubiquitination reaction respectively for ubiquitin ligase RHA2a and multiple ubiquitin binding enzyme; C () combines the result of carrying out ubiquitination reaction for ubiquitin binding enzyme UBC35 and multiple ubiquitin ligase; D () combines the result of carrying out ubiquitination reaction for ubiquitin binding enzyme UBC6 and multiple ubiquitin ligase.In figure, the swimming lane of mark "+" represents and there occurs poly ubiquitination, and the swimming lane not marking "+" represents and not there occurs poly ubiquitination.

Fig. 4 is the detected result utilizing wild-type and point mutation ubiquitin protein to detect poly ubiquitin chain type.In figure, "+" represents in corresponding reaction system containing this material; "-" represents in corresponding reaction system not containing this material.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Experiment material used in following embodiment:

The amylose resin that the Glutathione Sepharose4B beads that the albumen of purified fusion GST label uses uses purchased from QIAGEN company (article No. R10-22-4042/43), MBP fusion protein purification purchased from the Ni-NTA agarose of GE Healthcare company (article No. 17-0756-01), purifying 6 × His fusion rotein is purchased from NEB company (article No. E8201L);

His antibody is purchased from Santa-Cruz company (catalog number (Cat.No.): sc-0836); GST antibody purchased from Hua Da genome company (article No. AbM59001-2H5-PU) ubiquitin (Ub) antibody purchased from sigma company (catalog number (Cat.No.): U0508); Two is anti-purchased from proteintech(catalog number (Cat.No.): 00001-1); ATP is purchased from sigma company (catalog number (Cat.No.): A9187); NC film is purchased from Amersham company (catalog number (Cat.No.): RPN303C); Point mutation test kit is purchased from Qiagen company (catalog number (Cat.No.): 200518); The chemiluminescent HRP substrate test kit that Western detects is purchased from Millipore company (article No. WBKLS0500).

PET28a carrier and pET15b carrier: Novagen company; PMalC2 carrier: NEB company.

The structure of embodiment 1, recombinant expression vector

One, the preparation of recombinant plasmid pET28a-UBA2

1, design the primer of the CDS complete sequence (GenBank:U40566.1 holds 414 to 3647 nucleotide sequences from 5 ', sequence 2) of amplification Arabidopis thaliana ubiquitin activating enzyme UBA2 gene and add EcoRI and SalI restriction enzyme site respectively at its two ends.

2, adopt the primer of step 1 to obtain the CDS complete sequence of UBA2 gene by the method clone of PCR, carry out enzyme with restriction enzyme EcoRI and SalI and cut.

3, cut pET28a carrier with restriction enzyme EcoRI and SalI enzyme, obtain carrier framework.

4, the digestion products of step 2 is connected with the carrier framework of step 3, obtains recombinant plasmid pET28a-UBA2.Show through order-checking, recombinant plasmid pET28a-UBA2 inserts GenBank:U40566.1 between EcoRI and the SalI site of skeleton carrier pET28a, from 5 ' end, 414 to 3647 nucleotide sequences, the recombinant plasmid namely obtained after DNA shown in sequence 2 in sequence table.Albumen (ubiquitin activating enzyme UBA2) in sequence 2 polynucleotide shown in sequence 1.

Two, the preparation of recombinant plasmid pET28a-Ub

1, a ubiquitin protein sequence monomer (GenBank:NM_001125450.1 in the CDS of amplification Arabidopis thaliana wild-type ubiquitin protein UBQ14 gene is designed, from 5 ' end the 71st to 298 nucleotide sequences, and at its 3 ' end connection termination codon TAG, sequence 4) primer and add BamHI and EcoRI restriction enzyme site respectively at its two ends.(note: UBQ14 is the PolyUb gene comprising 4 ubiquitin monomeric proteins, and the present invention has cloned DNA sequence dna shown in the encoding sequence of one of them ubiquitin monomer and sequence 4, the ubiquitin protein for shown in expressed sequence 3)

2, adopt the primer of step 1 to obtain the CDS sequence of wild type monomeric ubiquitin protein gene by the method clone of PCR, carry out enzyme with restriction enzyme BamHI and EcoRI and cut.

3, cut pET28a carrier with restriction enzyme BamHI and EcoRI enzyme, obtain carrier framework.

4, the digestion products of step 2 is connected with the carrier framework of step 3, obtains recombinant plasmid pET28a-Ub.Show through order-checking, recombinant plasmid pET28a-Ub inserts GenBank:NM_001125450.1 between BamHI and the EcoRI site of skeleton carrier pET28a, from 5 ' end the 71st to 298 nucleotide sequence+TAG, the recombinant plasmid namely obtained after DNA shown in sequence 4 in sequence table.Albumen (wild type monomeric ubiquitin protein) in sequence 4 polynucleotide shown in sequence 3.

Three, multiple take pET28a as the ubiquitin binding enzyme of vector background and the preparation of ubiquitin-like desmoenzyme UEV1D recombinant plasmid

Example is prepared as with recombinant plasmid pET28a-UBC10:

1, design the primer of the CDS complete sequence (GenBank:Z14991.1 holds 133 to 579 nucleotide sequences from 5 ', sequence 14) of amplification ubiquitin binding enzyme UBC10 gene and add EcoRI and SalI restriction enzyme site respectively at its two ends.

2, adopt the primer of step 1 to obtain the CDS complete sequence of Ub gene by the method clone of PCR, carry out enzyme with restriction enzyme EcoRI and SalI and cut.

3, cut pET28a carrier with restriction enzyme EcoRI and SalI enzyme, obtain carrier framework.

4, the digestion products of step 2 is connected with the carrier framework of step 3, obtains recombinant plasmid pET28a-UBC10.Show through order-checking, recombinant plasmid pET28a-UBC10 inserts GenBank:Z14991.1 between EcoRI and the SalI site of skeleton carrier pET28a, from 5 ' end, 133 to 579 nucleotide sequences, the recombinant plasmid namely obtained after DNA shown in sequence 14 in sequence table.Albumen (ubiquitin binding enzyme UBC10) in sequence 14 polynucleotide shown in sequence 13.

The multiple ubiquitin binding enzyme arrived involved in the present invention, is all cloned on pET28a carrier by the method that same UBC10 is similar, obtains corresponding recombinant plasmid.According to the difference of sequence signature, restriction enzyme used can be slightly different.Be specially: UBC32(lacks membrane spaning domain, GenBank:NM_112576.2, from 5 ' end the 118 to 936 nucleotide sequence, and at its 3 ' end connection termination codon TAG, i.e. sequence 16, albumen shown in sequence 15 in polynucleotide), UBC4(GenBank:NM_123499.4, from 5 ' end the 175 to 738 nucleotide sequence, sequence 20, albumen shown in sequence 19 in polynucleotide), UBC5(GenBank:NM_105055.2, from 5 ' end the 78 to 635 nucleotide sequence, sequence 22, albumen shown in sequence 21 in polynucleotide), UBC6(GenBank:NM_130166.3, from 5 ' end the 87 to 638 nucleotide sequence, sequence 24, albumen shown in sequence 23 in polynucleotide) be the recombinant vectors obtained by the effect of restriction enzyme BamH I and Sac I.UBC1(GenBank:NM_101307.4 is from 5 ' end the 254 to 712 nucleotide sequence, sequence 8, albumen shown in sequence 7 in polynucleotide) and UBC2(GenBank:NM_126331.3, from 5 ' end the 254 to 712 nucleotide sequence, sequence 10, the albumen shown in sequence 19 in polynucleotide) be obtain recombinant vectors by the effect of restriction enzyme SacI and HindIII.Remaining comprises UBC27(GenBank:NM_124465.5, from 5 ' end the 109 to 687 nucleotide sequence, sequence 6, albumen shown in sequence 5 in polynucleotide), UBC3(GenBank:NM_125648.3, from 5 ' end the 271 to 723 nucleotide sequence, sequence 12, albumen shown in sequence 11 in polynucleotide), UBC13(GenBank:NM_114513.5, from 5 ' end the 138 to 638 nucleotide sequence, sequence 18, albumen shown in sequence 17 in polynucleotide), UBC19(GenBank:NM_112897.3, from 5 ' end the 168 to 713 nucleotide sequence, sequence 28, albumen shown in sequence 27 in polynucleotide), UBC35(GenBank:NM_106535.3, from 5 ' end the 136 to 597 nucleotide sequence, sequence 26, albumen shown in sequence 25 in polynucleotide), UBC16(GenBank:NM_106198.4, from 5 ' end the 137 to 622 nucleotide sequence, sequence 30, albumen shown in sequence 29 in polynucleotide), UBC22(GenBank:NM_120590.2, from 5 ' end the 300 to 1055 nucleotide sequence, sequence 32, albumen shown in sequence 31 in polynucleotide) and ubiquitin-like desmoenzyme UEV1D(GenBank:NM_180353.2, from 5 ' end the 131 to 574 nucleotide sequence) are all the structures utilizing restriction enzyme EcoRI and SalI to carry out recombinant vectors.

Each sequence is the CDS complete sequence of each ubiquitin binding enzyme gene above, and gained recombinant plasmid is all the CDS complete sequences inserting respective ubiquitin binding enzyme gene between the corresponding restriction enzyme site of skeleton carrier pET28a.

Four, with the recombinant plasmid pET28a-Ub (K48R) of point mutation ubiquitin gene and the preparation of pET28a-Ub (K63R)

Design the primer with point mutation site, with the recombinant plasmid pET28a-Ub obtained in step 2 for template, utilize Qiagen company point mutation test kit, carry out according to process specifications, obtain pET28a-Ub (K48R) and pET28a-Ub (K63R) recombinant plasmid.The primer sequence is respectively:

(1) the upstream and downstream primer of construction recombination plasmid pET28a-Ub (K48R):

Upstream primer: 5 '-gcttattttcgccggaaGGcagctagaggatggccg-3 ';

Downstream primer: 5 '-cggccatcctctagctgCCttccggcgaaaataagc-3 '.

Upstream and downstream primer reverse complemental, the base wherein capitalized is mutational site, corresponding to the wild type monomeric ubiquitin protein gene shown in sequence in sequence table 4 the 143rd and 144, former AA is sported GG.

(2) the upstream and downstream primer of construction recombination plasmid pET28a-Ub (K63R):

Upstream primer: 5 '-ctgattacaatatccagaGggaatccaccctccacttg-3 ';

Downstream primer: 5 '-caagtggagggtggattccCtctggatattgtaatcag-3 '.

Upstream and downstream primer reverse complemental, the base wherein capitalized is mutational site, corresponding to the 188th of the wild type monomeric ubiquitin protein gene shown in sequence in sequence table 4, former A is sported G.

Order-checking shows, recombinant plasmid pET28a-Ub (K48R) for insert between BamHI and the EcoRI site of skeleton carrier pET28a the A of the A and the 144th of the 143rd of sequence in sequence table 4 the is all replaced with G after gained point mutation ubiquitin gene.The protein sequence of this point mutation ubiquitin gene coding is that the Methionin of the 48th of sequence in sequence table 3 the is replaced with arginine.Recombinant plasmid pET28a-Ub (K63R) for insert between BamHI and the EcoRI site of skeleton carrier pET28a the A of the 188th of sequence in sequence table 4 the is replaced with G after gained point mutation ubiquitin gene.The protein sequence of this point mutation ubiquitin gene coding is that the Methionin of the 63rd of sequence in sequence table 3 the is replaced with arginine.

Five, the preparation of recombinant plasmid pET28a-wE1

Reference: Peggy M.Hatfield, Judy Callis and Richard D.Vierstra.Cloning of Ubiquitin Activating Enzyme from Wheat and Expression of a Functional Protein in Escherichia coli. " THE JOURNAL OF BIOLOGICAL CHEMISTRY ", nineteen ninety, 265 volumes, 26 phases, 15813-15817.(removing this document)

Recombinant plasmid pET28a-wE1 inserts GenBank:M55604.1 between EcoRI and the XhoI site of skeleton carrier pET28a, the recombinant plasmid obtained after holding the wheat ubiquitin activating enzyme gene order shown in 183-3338 position from 5 '.

Six, the preparation of recombinant plasmid pET15b-UBCH5b.

Reference: Jane P.Jensen, Paul W. Bates, Mei Yang, Richard D.Vierstra, and Allan M.Weissman.Identification of a Family of Closely Related Human Ubiquitin Conjugating Enzymes. " THE JOURNAL OF BIOLOGICAL CHEMISTRY " nineteen ninety-five, 270 volumes, 51 phases, 30408-30414.

Recombinant plasmid pET15b-UBCH5b inserts GenBank:U39317.1 between NcoI and the BamHI site of skeleton carrier pET15b, the recombinant plasmid obtained after the people's ubiquitin binding enzyme UBCH5b gene order shown in 5 ' 23 to 466, end.

Note: by protein tag (6 × His) sequence excision (6 × His sequence label is located between NcoI and BamHI site on pET15b carrier) on carrier in this recombinant plasmid, therefore the UBCH5b albumen gone out with this plasmid expression without His and other protein tag.

Seven, the preparation of recombinant plasmid pMalC2-SDIR1

Reference: Yiyue Zhang, ChengweiYang, Yin Li, Nuoyan Zheng, Hao Chen, Qingzhen Zhao, Ting Gao, Huishan Guo, and Qi Xie.SDIR1Is a RING Finger E3Ligase That Positively Regulates Stress-Responsive Abscisic Acid Signaling in Arabidopsis. " The Plant Cell ", 2007,19 volumes, 1912-1929.

Recombinant plasmid pMalC2-SDIR1 inserts GenBank:NM_115410.3 between EcoRI and the SalI site of skeleton carrier pMalC2, the recombinant plasmid obtained after holding Arabidopis thaliana ubiquitin ligase SDIR1 gene (At3g55530) sequence shown in 169-990 position from 5 '.

Eight, the preparation of recombinant plasmid pMalC2-Rma1

Reference: Noriyuki Matsuda, Toshiaki Suzuki, Keiji Tanaka and Akihiko Nakano.Rma1, a novel type of RING finger protein conservedfrom Arabidopsis to human, is a membrane-boundubiquitin ligase. " Journal of Cell Science ", 2007,4 volumes, 1949-1957.

Recombinant plasmid pMalC2-Rma1 inserts GenBank:NM_116589.3 between XbaI and the PstI site of skeleton carrier pMalC2, the recombinant plasmid obtained after holding Arabidopis thaliana ubiquitin ligase Rma1 gene (At4g03510) sequence shown in 191-940 position from 5 '.

Nine, the preparation of recombinant plasmid pMalC2-CIP8

Reference: Christian S.Hardtke Haruko Okamoto Chatanika Stoop-Myer Xing Wang Deng.Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1interacting protein8 (CIP8). " the Plant Journal ", 2002,30 volumes, 4 phases, 385-394.Remove this section of reference (that build in document is GST-CIP8, is MBP-CIP8 in the present invention).

Recombinant plasmid pMalC2-CIP8 inserts GenBank:NM_125891.3 between XbaI and the PstI site of skeleton carrier pMalC2, the recombinant plasmid obtained after holding Arabidopis thaliana ubiquitin ligase CIP8 gene (At5g64920) sequence shown in 101-1105 position from 5 '.

Ten, the preparation of recombinant plasmid pMalC2-COP1

Christian S.Hardtke Haruko Okamoto Chatanika Stoop-Myer Xing Wang Deng.Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1interacting protein8 (CIP8). " the Plant Journal ", 2002,30 volumes, 4 phases, 385-394.

Recombinant plasmid pMalC2-COP1 inserts GenBank:NM_128855.3 between EcoRI and the SalI site of skeleton carrier pMalC2, the recombinant plasmid obtained after holding Arabidopis thaliana ubiquitin ligase COP1 gene (At2g32950) sequence shown in 68-2095 position from 5 '.

11, the preparation of recombinant plasmid pMalC2-HOS1

Reference: Chun-Hai Dong, Manu Agarwal, Yiyue Zhang, Qi Xie, and Jian-Kang Zhu.The negative regulator of plant cold responses, HOS1, is a RING E3ligase that mediates the ubiquitination and degradation of ICE1. " Proceedings of National Academy Sci ence in U S A. ", 2006,103 volumes, 21 phases, 8281-8286.

Recombinant plasmid pMalC2-HOS1 inserts GenBank:NM_129540.4 between BamHI and the PstI site of skeleton carrier pMalC2, the recombinant plasmid obtained after holding Arabidopis thaliana ubiquitin ligase HOS1 gene (At2g39810) sequence shown in 142-2925 position from 5 '.

12, the preparation of recombinant plasmid pMalC2-RHA2a

Reference: Qingyun Bu, Hongmei Li, Qingzhen Zhao, Hongling Jiang, Qingzhe Zhai, Jie Zhang, Xiaoyan Wu, Jiaqiang Sun, Qi Xie, Daowen Wang, and Chuanyou Li.The Arabidopsis RING Finger E3Ligase RHA2a Is a Novel Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development. " Plant Physiology ", 2009,150 volumes, 463-481.

Recombinant plasmid pMalC2-RHA2a inserts GenBank:NM_101378.2 between EcoRI and the BamHI site of skeleton carrier pMalC2, the recombinant plasmid obtained after holding Arabidopis thaliana ubiquitin ligase RHA2a gene (At1g15100) sequence shown in 34-501 position from 5 '.

13, the preparation of recombinant plasmid pMalC2-CBL-like

1, design amplification Arabidopis thaliana agnoprotein (prediction may be ubiquitin ligase) CBL-like(At2g42030) gene CDS complete sequence (GenBank:AY096666.1 holds the 1 to 1278 nucleotide sequence from 5 ') primer and add EcoRI and SalI restriction enzyme site respectively at its two ends.

2, adopt the primer of step 1 to obtain the CDS complete sequence of CBL-like gene by the method clone of PCR, carry out enzyme with restriction enzyme EcoRI and SalI and cut.

3, cut pMalC2 carrier with restriction enzyme EcoRI and SalI enzyme, obtain carrier framework.

4, the digestion products of step 2 is connected with the carrier framework of step 3, obtains recombinant plasmid pMalC2-SDIR1.Show through order-checking, recombinant plasmid pMalC2-SDIR1 for inserting the recombinant plasmid obtained after GenBank:5 ' holds the DNA shown in the 1 to 1278 nucleotide sequence between EcoRI and the SalI site of skeleton carrier pMalC2.

The preparation of embodiment 2, albumen

One, the expression and purification of 6 × His fusion rotein and UBCH5b albumen

1, intestinal bacteria Bl21 (DE3) competent cell is prepared.

2, by prepare in embodiment 1 step one step 6 with pET28a be carrier framework recombinant plasmid respectively heat shock method transform Bl21 (DE3) competent cell.

3, picking 2-3 mono-clonal is added with in corresponding antibiotic LB liquid nutrient medium in 3-10mL, 37 DEG C, 200 revs/min of overnight incubation.

4, the cell culture after spending the night is joined 200-500mL containing corresponding microbiotic and 0.2%(0.2g/100mL) in the LB liquid nutrient medium of glucose, make OD600 be about 0.1.

5,18 DEG C of concussions are cultured to OD600 when being 0.5-0.6, and adding inducer isopropylthio-β-D-Thiogalactopyranoside (IPTG) to final concentration is 0.2mM; 18 DEG C of induction 12-16h.

6,4 DEG C, 4000 revs/min, centrifugal 10 minutes, thalline is collected.

7, the lysis buffer A(of cell precipitation precooling fills a prescription: 50mMNaH ₂pO ₄; 300mMNaCl; PH8.0 is adjusted to, with front adding 1mM PMSF with HCl) suspend.

8, cytoclasis is carried out with Ultrasonic Cell Disruptor, each 10 seconds, totally 10 times, every minor tick 30 seconds.

9, cytoclasis lysate is at 4 DEG C, 15000 revs/min, centrifugal 30 minutes.

10, get supernatant, packing, preserve or directly carry out protein purification for-70 DEG C.

(note: people's ubiquitin binding enzyme UBCH5b is because when construction recombination plasmid by His sequence label excision, namely terminate so the expression of albumen arrives this step, the packing of albumen supernatant is frozen, be add in reaction system with the form of albumen supernatant when carrying out the outer ubiquitination reaction of dependent body, all the other protein are all proceed 11-12 step to carry out purifying for subsequent use.）

11, the purifying of 6 × His fusion rotein: purify 6 × His fusion rotein with the Ni-NTA agarose of QIAGEN company, carry out according to product operation specification sheets.

12, by the Ultra15(Millipore company of the albumen after purifying, concrete model is determined according to molecular weight of albumen) by albumen desalination and concentration, illustrate according to product operation and carry out.

By above operation, obtain the Arabidopis thaliana ubiquitin activating enzyme UBA2(with His label after purifying and be denoted as albumen His-AtE1), with the Arabidopis thaliana wild type monomeric ubiquitin protein (being denoted as albumen His-Ub) of His label, with 14 kinds of ubiquitin binding enzymes of His label, Arabidopis thaliana point mutation ubiquitin protein UbK48R(with His label is denoted as albumen His-UbK48R), Arabidopis thaliana point mutation ubiquitin protein UbK63R(with His label is denoted as albumen His-UbK63R), with the wheat ubiquitin activating enzyme (being denoted as albumen His-wE1) of His label, and people's ubiquitin binding enzyme UBCH5b(protein crude extract.

Two, the expression and purification of MBP fusion rotein

1, intestinal bacteria Bl21 (DE3) competent cell is prepared.

2, by prepare in embodiment 1 step 7 to step 13 with pMalC2 be carrier framework recombinant plasmid respectively heat shock method transform Bl21 (DE3) competent cell.

3-6, with the 3-6 in step one.

7, the Columnbuffer(formula of cell precipitation precooling: 200mMNaCl, 20mM Tris-HCl, pH7.4,1mM EDTA, with front adding 1mM PMSF, 1mM DTT) suspend.

8-10, with the 8-10 in step one.

11, the purifying of MBP fusion rotein: purify MBP fusion rotein with the amylase resin of NEB company, carry out according to product operation specification sheets.

12, with 12 in step one.

By above operation, obtain the Arabidopis thaliana ubiquitin ligase SDIR1(with MBP label after purifying and be denoted as albumen MBP-SDIR1), Arabidopis thaliana ubiquitin ligase Rma1(with MBP label is denoted as albumen MBP-Rma1), Arabidopis thaliana ubiquitin ligase CIP8(with MBP label is denoted as albumen MBP-CIP8), Arabidopis thaliana ubiquitin ligase COP1(with MBP label is denoted as albumen MBP-COP1), Arabidopis thaliana ubiquitin ligase HOS1(with MBP label is denoted as albumen MBP-HOS1), Arabidopis thaliana ubiquitin ligase RHA2a(with MBP label is denoted as albumen MBP-RHA2a), and be denoted as albumen MBP-CBL-like with the CBL-like(being predicted to be ubiquitin ligase coming from Arabidopis thaliana of MBP label).

The following albumen that the present embodiment purifying obtains constitutes plant in-vitro ubiquitin protein degradation system of the present invention, is specifically made up of following (1)-(3):

(1) ubiquitin activating enzyme UBA2 and wild type monomeric ubiquitin protein (in UBQ14 partial sequence);

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

(2) following 14 kinds of ubiquitin binding enzymes: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31;

(3) point mutation ubiquitin protein UbK48R and point mutation ubiquitin protein UbK63R;

The aminoacid sequence of described point mutation ubiquitin protein UbK48R is the sequence Methionin of the 48th of sequence in sequence table 3 the being replaced with gained after arginine; The aminoacid sequence of described point mutation ubiquitin protein UbK63R is the sequence Methionin of the 63rd of sequence in sequence table 3 the being replaced with gained after arginine.

Embodiment 3, the Arabidopis thaliana ubiquitin activating enzyme of embodiment 2 purifying and the Activity determination of Arabidopis thaliana ubiquitin protein

The activity of the Arabidopis thaliana ubiquitin activating enzyme that the present embodiment obtains embodiment 2 purifying as follows and Arabidopis thaliana ubiquitin protein detects:

(1) get the Eppendorf pipe of 2 1.5mL, be denoted as sample 1, sample 2 respectively.The people's ubiquitin binding enzyme (UBCH5b), 3-5 μ g Arabidopis thaliana wild type monomeric ubiquitin protein (His-Ub) and the 1 μ L20 × reaction buffer (formula: 1M Tris pH7.5 that obtain in 0.2 μ g embodiment 2 is all added in 2 sample hoses, 100mMATP, 50mM MgCl ₂, 2mM DTT, solvent is water).0.05 μ g Arabidopis thaliana ubiquitin activating enzyme UBA2(His-AtE1 is added in addition in sample 1), in sample 2, add the wheat ubiquitin activating enzyme (His-wE1) of equivalent.All ddH is added in two samples ₂o is 20 μ L to cumulative volume.

(2), after mixing sample, take out the reaction solution of 1/2 volume, add the 4 × SDS sample-loading buffer containing 0.1mol/L DTT immediately, boil 5 minutes, this is reaction 0 minute sample; Residual reaction liquid adds the 4 × SDS sample-loading buffer containing 0.1mol/LDTT again after hatching 15 minutes at 37 DEG C, boil 5 minutes, this is reaction 15 minutes samples.

(3) sample is carried out SDS-PAGE electrophoresis; Electrophoresis terminates rear electricity consumption robin and is moved on to by protein transduction on nitrocellulose filter (NC film); After transferring film terminates, carry out immuning hybridization detection with anti-His antibody.Cleaning Principle: the reactive behavior band detected due to this institute is that UBCH5b-His-Ub(molecular weight is about 27kD), all the other can be gone out the albumen of band as His-AtE1 and His-wE1 by anti-His antibody test with His label, molecular weight all at more than 130kD (in see Fig. 1 (b)), therefore can not affect detected result.UBCH5b albumen itself there is no His label, and as not being combined with His-Ub, then the band not having 27kD produces.

Result as shown in (a) in Fig. 1, the sample of 15 minutes of sample 1 and sample 2 at about 27KD(sizableness in UBCH5b and His-Ub sum) position on all signal detected.Visible Arabidopis thaliana ubiquitin activating enzyme UBA2 and Arabidopis thaliana wild type monomeric ubiquitin protein (His-Ub) can make the ubiquitin binding enzyme UBCH5b of people that ubiquitination occurs, and response intensity is roughly the same with wheat ubiquitin activating enzyme (wE1).The ubiquitin binding enzyme UBCH5b of wheat ubiquitin activating enzyme (wE1) and people is E1 and E2 conventional in now widely used external ubiquitination reaction system, and ubiquitin ligase (E3) the activity wE1 and UBCH5b that major part has been delivered detects.This result shows that Arabidopis thaliana ubiquitin activating enzyme UBA2 provided by the invention and Arabidopis thaliana wild type monomeric ubiquitin protein (His-Ub) are activated, may be used for the detection of external ubiquitination reaction.

The Activity determination of the Arabidopis thaliana ubiquitin binding enzyme (E2) of embodiment 4, embodiment 2 purifying

The object of the present embodiment is the activity of the multiple ubiquitin binding enzyme albumen detecting embodiment 2 purifying, to prove that they all can be used for the detection of ubiquitin ligase activity detection and E2-E3 specific binding.Because embodiment 3 has proved Arabidopis thaliana ubiquitin activating enzyme UBA2(His-AtE1) there is identical activity with wheat ubiquitin activating enzyme (His-wE1), therefore these two kinds of ubiquitin activating enzymes all can be used for the present embodiment reaction.The ubiquitin binding enzyme Activity determination that this experiment uses wheat ubiquitin activating enzyme (His-wE1) to carry out.

The present embodiment utilizes the formation of the thioester bond of DTT-sensitivity to test, and whether the following 9 kinds of Arabidopis thaliana ubiquitin binding enzymes detecting embodiment 2 purifying have activity: UBC3, UBC19, UBC6, UBC35, UBC5, UBC22, UBC32, UBC13 and UBC27.Its principle is: have the Cys that conservative in all ubiquitin binding enzyme albumen, the avtive spot be combined with ubiquitin protein, the two in conjunction with time Cys side chain on sulfydryl and the Gly(G76 of ubiquitin protein C-terminal the 76th) carboxyl condensation form thioester bond (there is band the position showing as the two molecular weight sum when immuning hybridization detects.Attention: due to and the ubiquitin binding enzyme albumen of not all and ubiquitin protein all can form this heterodimer, so when the two is with identical protein tag, when detecting with corresponding tag antibody immuning hybridization, also band can be detected at the molecular weight of free ubiquitin binding enzyme and ubiquitin protein); This thioester bond can be reduced agent DTT and open, if therefore when adding DTT after reaction completes in system, established ubiquitin binding enzyme-ubiquitin dimer molecule is separated from one another again forms free ubiquitin binding enzyme and ubiquitin molecule (show as the position of the two molecular weight sum when immuning hybridization detects without band, and have band at the molecular weight of independent ubiquitin binding enzyme and ubiquitin protein) again.Concrete operations are as follows:

(1) get two 1.5mL Eppendorf to manage, be denoted as sample 1, sample 2 respectively.The wheat ubiquitin activating enzyme (His-wE1) obtained in 50ng embodiment 2 is all added in 2 sample hoses, 200ng ubiquitin binding enzyme (1 kinds in 9 kinds), 1 μ L20 × reaction buffer (same embodiment 3 of filling a prescription), 2 μ L concentration are the Arabidopis thaliana wild type monomeric ubiquitin protein (His-Ub) of 2 μ g/ μ L, add ddH ₂o is 20 μ L to cumulative volume.

Hatch 5min for (2) 37 DEG C; Adding in sample 1 containing final concentration is 4 × SDS-PAGE sample-loading buffer 6.7 μ L of the DTT of 0.1mol/L, and add in sample 2 not containing 4 × SDS-PAGE sample-loading buffer 6.7 μ L of DTT, two samples all boil 5min in 100 DEG C.

(3) two samples are carried out SDS-PAGE electrophoresis; Electrophoresis terminates rear electricity consumption robin and is moved on to by protein transduction on nitrocellulose filter (NC film); After transferring film terminates, carry out immuning hybridization detection with anti-His antibody.

As shown in Figure 2, as can be seen from the figure above 9 kinds of ubiquitin binding enzymes all can form the thioester bond to DTT-sensitivity to result except UBC27, illustrate that they are all activated ubiquitin binding enzymes.

(note: although UBC27 self can not form the thioester bond to DTT-sensitivity, but still part ubiquitin ligase generation ubiquitination can be made to react, refer to step one in following examples 5, result is as Fig. 3 (a) the second swimming lane from left to right, and the band between 34kD and 55kD is UBC27-Ub dimer band; This illustrates that UBC27 also has ubiquitin binding enzyme active.）

Whether embodiment 5, self ubiquitination reaction detection testing protein external have ubiquitin ligase activity and E2-E3 specificity combined situation

The object of the present embodiment detects multiple to be measured or known albumen whether to have ubiquitin ligase activity, and the situation of E2-E3 specific binding.Because embodiment 3 has proved Arabidopis thaliana ubiquitin activating enzyme UBA2(His-AtE1) there is identical activity with wheat ubiquitin activating enzyme (His-wE1), therefore these two kinds of ubiquitin activating enzymes all can be used for the present embodiment reaction.This experiment uses wheat ubiquitin activating enzyme (His-wE1) to carry out correlation detection.

One, testing protein (inferring may be ubiquitin ligase) the ubiquitination reaction of CBL-like under different ubiquitin binding enzyme exists situation

(1) get 9 1.5mL Eppendorf to manage, all add the wheat ubiquitin activating enzyme (His-wE1), 4 μ g Arabidopis thalianas wild type monomeric ubiquitin protein (His-Ub), the 1.5 μ L20 × reaction buffers (formula: 1M Tris pH7.5 that obtain in 50ng embodiment 2,40mMATP, 100mM MgCl ₂, 40mM DTT, solvent is water), and the testing protein CBL-like that 0.2 μ gMBP marks.

(2) respectively to adding the ubiquitin binding enzyme 200ng obtained in a kind of embodiment 2 in each sample hose, relate to following 9 kinds of ubiquitin binding enzymes altogether: UBCH5b(people source), UBC27, UBC3, UBC10, UBC32, UBC13, UBC6, UBC19 and UBC35(Arabidopis thaliana source).Add ddH ₂o is 30 μ L to cumulative volume.

(3) sample hose is placed on EppendorfThermomixer comfort instrument, 30 DEG C, 900 revs/min, hatches 1.5 hours.

(4) in sample hose, add the 4 × SDS sample-loading buffer termination reaction of 10 μ L containing 0.1mol/L DTT respectively, 100 DEG C are boiled 5min; Get 15 μ L samples and carry out SDS-PAGE electrophoresis; Electrophoresis terminates rear electricity consumption robin and is moved on to by protein transduction on nitrocellulose filter (NC film); After transferring film terminates, carry out immuning hybridization detection with anti-His antibody or anti-Ub antibody.When adopting, the Cleaning Principle of anti-His antibody is as follows: because the molecular weight of ubiquitin binding enzyme is (most of upper and lower at 20kD, UBC16 and UBC22 molecular weight is slightly large), and ubiquitin ligase molecular weight is generally greater than ubiquitin binding enzyme, and (molecular weight generally can be greater than 30kD, some meetings are greater than 100kD, as COP1 etc.Note: RHA2a molecular weight, about 17kD), and ubiquitin binding enzyme merges His label (albumen that His tag expression goes out is about about 3kD), ubiquitin ligase merges MBP label (albumen that MBP tag expression goes out is about about 42kD), so two kinds of fusion protein molecule amounts differ greatly, and due to reaction result be generally on ubiquitin ligase molecule, add poly ubiquitin chain, immuning hybridization detected representation is the smear upwards of the larger POS INT of molecular weight, and the activities present of ubiquitin binding enzyme is for adding single or a few ubiquitin molecule, immuning hybridization detected representation is the single of molecular weight position or the obvious band of minority, therefore the band of the band of ubiquitin binding enzyme and ubiquitin ligase is easy to distinguish in position.Below react in example and be same case.

Result is as shown in (a) in Fig. 3 (anti-His antibody), when Arabidopis thaliana testing protein (may be ubiquitin ligase) CBL-like exists at UBCH5b, UBC10, UBC35, all on the position being greater than MBP-CBL-like molecular weight (about 90KD), detect protein signal; And when combining with other six kinds of ubiquitin binding enzymes, not this protein signal.Visible Arabidopis thaliana testing protein CBL-like is activated ubiquitin ligase, and it all can produce poly ubiquitination when UBCH5b, UBC10, UBC35 exist, but can not carry out ubiquitination reaction when combining with other six kinds of ubiquitin binding enzymes.

Two, the ubiquitination reaction of ubiquitin ligase RHA2a under different ubiquitin binding enzymes exists situation

(1) get 9 1.5mL Eppendorf to manage, all add the wheat ubiquitin activating enzyme (His-wE1), 4 μ g Arabidopis thalianas wild type monomeric ubiquitin protein (His-Ub), the 1.5 μ L20 × reaction buffers (same step one of filling a prescription) that obtain in 50ng embodiment 2, and the Arabidopis thaliana ubiquitin ligase RHA2a that 0.2 μ gMBP marks.

(2)-(4) are with (2) in step one-(4).

Result, as shown in (b) in Fig. 3, shown in (anti-His antibody), when Arabidopis thaliana ubiquitin ligase RHA2a exists at UBCH5b, UBC10, all detects protein signal on the position being greater than MBP-RHA2a molecular weight (about 70KD); And when combining with other seven kinds of ubiquitin binding enzymes, not this protein signal.Visible Arabidopis thaliana ubiquitin ligase RHA2a can produce poly ubiquitination when UBCH5b, UBC10 exist respectively, but can not carry out ubiquitination reaction when combining with other seven kinds of ubiquitin binding enzymes respectively.

Three, the ubiquitination reaction of ubiquitin binding enzyme UBC35 under different ubiquitin ligases exists situation

(1) get 8 1.5mL Eppendorf to manage, all add the wheat ubiquitin activating enzyme (His-wE1), 4 μ g Arabidopis thalianas wild type monomeric ubiquitin protein (His-Ub), the 1.5 μ L20 × reaction buffers (same step one of filling a prescription) that obtain in 50ng embodiment 2, and 0.2 μ g Arabidopis thaliana ubiquitin binding enzyme UBC35.

(2) respectively to adding the ubiquitin ligase 200ng obtained in a kind of embodiment 2 in each sample hose, relate to following 6 kinds of ubiquitin ligases altogether: Rma1, CIP8, SDIR1, CBL-like, COP1, HOS1, remain two pipes as the negative contrast of experiment, one pipe does not separately add albumen, and a pipe adds label protein MBP.

(3)-(4) are with (3) in step one-(4).

Result is as shown in (c) in Fig. 3, shown in (anti-His antibody), when ubiquitin binding enzyme UBC35 exists at SDIR1, COP1, all be greater than corresponding ubiquitin ligase molecular weight (MBP-SDIR1 molecular weight is about 72KD), MBP-COP1 molecular weight is about 120KD) position on detect stronger protein signal; When Rma1 exists, the position being greater than Rma1 molecular weight (being about 70KD) detects more weak protein signal; And when combining with other three kinds of ubiquitin ligases, there is no corresponding protein signal.Visible Arabidopis thaliana ubiquitin binding enzyme UBC35 can make SDIR1, COP1 produce the modification of poly ubiquitin chain, makes Rma1 produce more weak ubiquitination signal, but other three kinds of ubiquitin ligases can not be made to carry out ubiquitination reaction.

Four, the ubiquitination reaction of ubiquitin binding enzyme UBC6 under different ubiquitin ligase exists situation

(1) get 8 1.5mL Eppendorf to manage, all add the wheat ubiquitin activating enzyme (His-wE1), 4 μ g Arabidopis thalianas wild type monomeric ubiquitin protein (His-Ub), the 1.5 μ L20 × reaction buffers (same step one of filling a prescription) that obtain in 50ng embodiment 2, and 0.2 μ g Arabidopis thaliana ubiquitin binding enzyme UBC6.

(2)-(4) are with (2) in step one-(4).

Result, as shown in (d) in Fig. 3, shown in (anti-His antibody), when ubiquitin binding enzyme UBC6 exists at HOS1, (position being about 140KD detects stronger protein signal being greater than MBP-HOS1 molecular weight; When CBL-like and COP1 exists, all on the position being greater than corresponding ubiquitin ligase molecular weight (MBP-CBL-like molecular weight is about 90KD, and MBP-COP1 molecular weight is about 120KD), detect more weak protein signal; And when combining with other three kinds of ubiquitin ligases, there is no corresponding protein signal.Visible Arabidopis thaliana ubiquitin binding enzyme UBC6 can make HOS1 produce the modification of poly ubiquitin chain, makes CBL-like and COP1 produce more weak ubiquitination signal, but other three kinds of ubiquitin ligases can not be made to carry out ubiquitination reaction.

Embodiment 6, the ubiquitin protein of wild-type and point mutation is utilized to detect the type of the poly ubiquitin chain formed

The ubiquitin protein of the wild-type utilizing embodiment 2 to prepare and point mutation is detected the type of the poly ubiquitin chain that Arabidopis thaliana ubiquitin binding enzyme UBC35 is formed by the present embodiment.Specific as follows:

(1) get 5 1.5mL Eppendorf to manage, be denoted as sample 1, sample 2, sample 3, sample 4 and sample 5 respectively.The wheat ubiquitin activating enzyme (His-wE1) obtained in 50ng embodiment 2 is all added in 5 sample hoses, and 1 μ L20 × reaction buffer (same embodiment 3 of filling a prescription).

(2) in sample 1, add 0.2 μ g Arabidopis thaliana ubiquitin binding enzyme UBC35 and 4 μ g Arabidopis thalianas wild type monomeric ubiquitin protein (His-Ub); In sample 2, add the 0.2 μ g ubiquitin-like desmoenzyme UEV1D(UEV1D and ubiquitin binding enzyme UBC35 obtained in embodiment 2 one form heterodimer, promote the generation of the poly ubiquitin chain that K63-connects specifically.See document: Rui Wen, J.Antonio Torres-Acosta, Landon Pastushok, Xiaoqin Lai, Lindsay Pelzer, Hong Wang, and Wei Xiao.Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and is involved in DNA Damage Response. " Plant Cell ", 20 volumes the 1st phase: 213-227. in 2008) and 4 μ g Arabidopis thalianas Arabidopis thaliana wild type monomeric ubiquitin protein (His-Ub); 0.2 μ g Arabidopis thaliana ubiquitin binding enzyme UBC35,0.2 μ g ubiquitin-like desmoenzyme UEV1D and 4 μ g Arabidopis thalianas wild type monomeric ubiquitin protein (His-Ub) are added in sample 3; 0.2 μ g Arabidopis thaliana ubiquitin binding enzyme UBC35,0.2 μ g ubiquitin-like desmoenzyme UEV1D and 4 μ g Arabidopis thaliana point mutation ubiquitin protein UbK48R are added in sample 4; 0.2 μ g Arabidopis thaliana ubiquitin binding enzyme UBC35,0.2 μ g ubiquitin-like desmoenzyme UEV1D and 4 μ g Arabidopis thaliana point mutation ubiquitin protein UbK63R are added in sample 5.Finally all use ddH ₂o benefit is 20 μ L to cumulative volume.

Hatch 2h for (3) 37 DEG C; Add not containing 4 × SDS-PAGE sample-loading buffer 6.7 μ L of DTT, 100 DEG C are boiled 5min.

(4) sample is carried out SDS-PAGE electrophoresis; Electrophoresis terminates rear electricity consumption robin and is moved on to by protein transduction on nitrocellulose filter (NC film); After transferring film terminates, carry out immuning hybridization detection with anti-Ub antibody.

Result as shown in Figure 4, deposit in case not having UEV1D, and UBC35 is combined with wild-type ubiquitin protein, produces two bands, is UBC35-Ub1 and UBC35-Ub2 respectively, but can not form poly ubiquitin chain by sample 1; In sample 2 UEV1D separately and wild-type ubiquitin protein react and produce without band, illustrate that UEV1D self can not by ubiquitination; Sample 3-5 is respectively and all deposits in case at UBC35 and UEV1D, reacts respectively with the ubiquitin protein of wild-type, K48R and K63R point mutation.Visible have stronger poly ubiquitin chain formation in sample 3, when using the ubiquitin protein UbK48R of point mutation, the signal of poly ubiquitin chain does not become substantially, but when using the ubiquitin protein UbK63R of point mutation, poly ubiquitin chain disappears substantially, and what when illustrating that UBC35 with UEV1D exists jointly, catalysis produced is the poly ubiquitin chain that K63-is connected.

Claims (17)

1., for the composition of vitro detection vegetable-protein ubiquitination to be measured activity, be made up of following (1)-(3):

(1) ubiquitin activating enzyme UBA2 and wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

(2) 14 kinds of ubiquitin binding enzymes: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31;

(3) point mutation ubiquitin protein UbK48R and point mutation ubiquitin protein UbK63R;

The aminoacid sequence of described point mutation ubiquitin protein UbK48R is the sequence Methionin of the 48th of sequence in sequence table 3 the being replaced with gained after arginine; The aminoacid sequence of described point mutation ubiquitin protein UbK63R is the sequence Methionin of the 63rd of sequence in sequence table 3 the being replaced with gained after arginine.

2. composition according to claim 1, is characterized in that: the encoding gene of described ubiquitin activating enzyme UBA2 is the DNA molecular shown in sequence in sequence table 2; The encoding gene of described wild type monomeric ubiquitin protein is the DNA molecular shown in sequence in sequence table 4; The encoding gene of described ubiquitin binding enzyme UBC27 is the DNA molecular shown in sequence in sequence table 6; The encoding gene of described ubiquitin binding enzyme UBC1 is the DNA molecular shown in sequence in sequence table 8; The encoding gene of described ubiquitin binding enzyme UBC2 is the DNA molecular shown in sequence in sequence table 10; The encoding gene of described ubiquitin binding enzyme UBC3 is the DNA molecular shown in sequence in sequence table 12; The encoding gene of described ubiquitin binding enzyme UBC10 is the DNA molecular shown in sequence in sequence table 14; The encoding gene of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene of described ubiquitin binding enzyme UBC13 is the DNA molecular shown in sequence in sequence table 18; The encoding gene of described ubiquitin binding enzyme UBC4 is the DNA molecular shown in sequence in sequence table 20; The encoding gene of described ubiquitin binding enzyme UBC5 is the DNA molecular shown in sequence in sequence table 22; The encoding gene of described ubiquitin binding enzyme UBC6 is the DNA molecular in sequence table shown in sequence 24; The encoding gene of described ubiquitin binding enzyme UBC35 is the DNA molecular shown in sequence in sequence table 26; The encoding gene of described ubiquitin binding enzyme UBC19 is the DNA molecular shown in sequence in sequence table 28; The encoding gene of described ubiquitin binding enzyme UBC16 is the DNA molecular shown in sequence in sequence table 30; The encoding gene of described ubiquitin binding enzyme UBC22 is the DNA molecular shown in sequence in sequence table 32; The encoding gene of described point mutation ubiquitin protein UbK48R is the DNA molecular A of the 143rd of sequence in sequence table 4 the and the 144th all being replaced with gained after G; The encoding gene of described point mutation ubiquitin protein UbK63R is the DNA molecular A of the 188th of sequence in sequence table 4 the being replaced with gained after G.

3., for the composition of vitro detection vegetable-protein ubiquitination to be measured activity, be made up of following (1) and (2):

(1) ubiquitin activating enzyme UBA2 and wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

(2) 14 kinds of ubiquitin binding enzymes: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31.

4. composition according to claim 3, is characterized in that: the encoding gene of described ubiquitin activating enzyme UBA2 is the DNA molecular shown in sequence in sequence table 2; The encoding gene of described wild type monomeric ubiquitin protein is the DNA molecular shown in sequence in sequence table 4; The encoding gene of described ubiquitin binding enzyme UBC27 is the DNA molecular shown in sequence in sequence table 6; The encoding gene of described ubiquitin binding enzyme UBC1 is the DNA molecular shown in sequence in sequence table 8; The encoding gene of described ubiquitin binding enzyme UBC2 is the DNA molecular shown in sequence in sequence table 10; The encoding gene of described ubiquitin binding enzyme UBC3 is the DNA molecular shown in sequence in sequence table 12; The encoding gene of described ubiquitin binding enzyme UBC10 is the DNA molecular shown in sequence in sequence table 14; The encoding gene of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene of described ubiquitin binding enzyme UBC13 is the DNA molecular shown in sequence in sequence table 18; The encoding gene of described ubiquitin binding enzyme UBC4 is the DNA molecular shown in sequence in sequence table 20; The encoding gene of described ubiquitin binding enzyme UBC5 is the DNA molecular shown in sequence in sequence table 22; The encoding gene of described ubiquitin binding enzyme UBC6 is the DNA molecular in sequence table shown in sequence 24; The encoding gene of described ubiquitin binding enzyme UBC35 is the DNA molecular shown in sequence in sequence table 26; The encoding gene of described ubiquitin binding enzyme UBC19 is the DNA molecular shown in sequence in sequence table 28; The encoding gene of described ubiquitin binding enzyme UBC16 is the DNA molecular shown in sequence in sequence table 30; The encoding gene of described ubiquitin binding enzyme UBC22 is the DNA molecular shown in sequence in sequence table 32.

5. for the plasmid group of vitro detection vegetable-protein ubiquitination to be measured activity, by following 1)-3) form:

1) express the plasmid of ubiquitin activating enzyme UBA2, and express the plasmid of wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

2) 14 kinds of plasmids, are expressed as follows in 14 kinds of ubiquitin binding enzymes a kind of: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5 respectively; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31;

3) express the plasmid of point mutation ubiquitin protein UbK48R, and express the plasmid of point mutation ubiquitin protein UbK63R;

The aminoacid sequence of described point mutation ubiquitin protein UbK48R is the sequence Methionin of the 48th of sequence in sequence table 3 the being replaced with gained after arginine; The aminoacid sequence of described point mutation ubiquitin protein UbK63R is the sequence Methionin of the 63rd of sequence in sequence table 3 the being replaced with gained after arginine.

6. plasmid group according to claim 5, is characterized in that: the encoding gene of described ubiquitin activating enzyme UBA2 is the DNA molecular shown in sequence in sequence table 2; The encoding gene of described wild type monomeric ubiquitin protein is the DNA molecular shown in sequence in sequence table 4; The encoding gene of described ubiquitin binding enzyme UBC27 is the DNA molecular shown in sequence in sequence table 6; The encoding gene of described ubiquitin binding enzyme UBC1 is the DNA molecular shown in sequence in sequence table 8; The encoding gene of described ubiquitin binding enzyme UBC2 is the DNA molecular shown in sequence in sequence table 10; The encoding gene of described ubiquitin binding enzyme UBC3 is the DNA molecular shown in sequence in sequence table 12; The encoding gene of described ubiquitin binding enzyme UBC10 is the DNA molecular shown in sequence in sequence table 14; The encoding gene of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene of described ubiquitin binding enzyme UBC13 is the DNA molecular shown in sequence in sequence table 18; The encoding gene of described ubiquitin binding enzyme UBC4 is the DNA molecular shown in sequence in sequence table 20; The encoding gene of described ubiquitin binding enzyme UBC5 is the DNA molecular shown in sequence in sequence table 22; The encoding gene of described ubiquitin binding enzyme UBC6 is the DNA molecular in sequence table shown in sequence 24; The encoding gene of described ubiquitin binding enzyme UBC35 is the DNA molecular shown in sequence in sequence table 26; The encoding gene of described ubiquitin binding enzyme UBC19 is the DNA molecular shown in sequence in sequence table 28; The encoding gene of described ubiquitin binding enzyme UBC16 is the DNA molecular shown in sequence in sequence table 30; The encoding gene of described ubiquitin binding enzyme UBC22 is the DNA molecular shown in sequence in sequence table 32; The encoding gene of described point mutation ubiquitin protein UbK48R is the DNA molecular A of the 143rd of sequence in sequence table 4 the and the 144th all being replaced with gained after G; The encoding gene of described point mutation ubiquitin protein UbK63R is the DNA molecular A of the 188th of sequence in sequence table 4 the being replaced with gained after G.

7. for the plasmid group of vitro detection vegetable-protein ubiquitination to be measured activity, by following 1) and 2) form:

1) express the plasmid of ubiquitin activating enzyme UBA2, and express the plasmid of wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

2) 14 kinds of plasmids, are expressed as follows in 14 kinds of ubiquitin binding enzymes a kind of: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5 respectively; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31.

8. plasmid group according to claim 7, is characterized in that: the encoding gene of described ubiquitin activating enzyme UBA2 is the DNA molecular shown in sequence in sequence table 2; The encoding gene of described wild type monomeric ubiquitin protein is the DNA molecular shown in sequence in sequence table 4; The encoding gene of described ubiquitin binding enzyme UBC27 is the DNA molecular shown in sequence in sequence table 6; The encoding gene of described ubiquitin binding enzyme UBC1 is the DNA molecular shown in sequence in sequence table 8; The encoding gene of described ubiquitin binding enzyme UBC2 is the DNA molecular shown in sequence in sequence table 10; The encoding gene of described ubiquitin binding enzyme UBC3 is the DNA molecular shown in sequence in sequence table 12; The encoding gene of described ubiquitin binding enzyme UBC10 is the DNA molecular shown in sequence in sequence table 14; The encoding gene of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene of described ubiquitin binding enzyme UBC13 is the DNA molecular shown in sequence in sequence table 18; The encoding gene of described ubiquitin binding enzyme UBC4 is the DNA molecular shown in sequence in sequence table 20; The encoding gene of described ubiquitin binding enzyme UBC5 is the DNA molecular shown in sequence in sequence table 22; The encoding gene of described ubiquitin binding enzyme UBC6 is the DNA molecular in sequence table shown in sequence 24; The encoding gene of described ubiquitin binding enzyme UBC35 is the DNA molecular shown in sequence in sequence table 26; The encoding gene of described ubiquitin binding enzyme UBC19 is the DNA molecular shown in sequence in sequence table 28; The encoding gene of described ubiquitin binding enzyme UBC16 is the DNA molecular shown in sequence in sequence table 30; The encoding gene of described ubiquitin binding enzyme UBC22 is the DNA molecular shown in sequence in sequence table 32.

9. a recombinant bacterium combination, by following 1)-3) form:

1) recombinant bacterium of the plasmid containing expression ubiquitin activating enzyme UBA2, and contain the recombinant bacterium of the plasmid of expressing wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

2) 14 kinds of recombinant bacteriums, respectively containing the one in 14 kinds of plasmids, described 14 kinds of plasmids are expressed as follows in 14 kinds of ubiquitin binding enzymes a kind of respectively: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31;

3) recombinant bacterium of the plasmid containing expression point mutation ubiquitin protein UbK48R, and contain the recombinant bacterium of the plasmid of expressing point mutation ubiquitin protein UbK63R;

The aminoacid sequence of described point mutation ubiquitin protein UbK48R is the sequence Methionin of the 48th of sequence in sequence table 3 the being replaced with gained after arginine; The aminoacid sequence of described point mutation ubiquitin protein UbK63R is the sequence Methionin of the 63rd of sequence in sequence table 3 the being replaced with gained after arginine.

10. recombinant bacterium combination according to claim 9, is characterized in that: the encoding gene of described ubiquitin activating enzyme UBA2 is the DNA molecular shown in sequence in sequence table 2; The encoding gene of described wild type monomeric ubiquitin protein is the DNA molecular shown in sequence in sequence table 4; The encoding gene of described ubiquitin binding enzyme UBC27 is the DNA molecular shown in sequence in sequence table 6; The encoding gene of described ubiquitin binding enzyme UBC1 is the DNA molecular shown in sequence in sequence table 8; The encoding gene of described ubiquitin binding enzyme UBC2 is the DNA molecular shown in sequence in sequence table 10; The encoding gene of described ubiquitin binding enzyme UBC3 is the DNA molecular shown in sequence in sequence table 12; The encoding gene of described ubiquitin binding enzyme UBC10 is the DNA molecular shown in sequence in sequence table 14; The encoding gene of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene of described ubiquitin binding enzyme UBC13 is the DNA molecular shown in sequence in sequence table 18; The encoding gene of described ubiquitin binding enzyme UBC4 is the DNA molecular shown in sequence in sequence table 20; The encoding gene of described ubiquitin binding enzyme UBC5 is the DNA molecular shown in sequence in sequence table 22; The encoding gene of described ubiquitin binding enzyme UBC6 is the DNA molecular in sequence table shown in sequence 24; The encoding gene of described ubiquitin binding enzyme UBC35 is the DNA molecular shown in sequence in sequence table 26; The encoding gene of described ubiquitin binding enzyme UBC19 is the DNA molecular shown in sequence in sequence table 28; The encoding gene of described ubiquitin binding enzyme UBC16 is the DNA molecular shown in sequence in sequence table 30; The encoding gene of described ubiquitin binding enzyme UBC22 is the DNA molecular shown in sequence in sequence table 32; The encoding gene of described point mutation ubiquitin protein UbK48R is the DNA molecular A of the 143rd of sequence in sequence table 4 the and the 144th all being replaced with gained after G; The encoding gene of described point mutation ubiquitin protein UbK63R is the DNA molecular A of the 188th of sequence in sequence table 4 the being replaced with gained after G.

11. 1 kinds of recombinant bacterium combinations, by following 1) and 2) form:

1) recombinant bacterium of the plasmid containing expression ubiquitin activating enzyme UBA2, and contain the recombinant bacterium of the plasmid of expressing wild type monomeric ubiquitin protein;

The aminoacid sequence of described ubiquitin activating enzyme UBA2 is as shown in sequence in sequence table 1; The aminoacid sequence of described wild type monomeric ubiquitin protein is as shown in sequence in sequence table 3;

2) 14 kinds of recombinant bacteriums, respectively containing the one in 14 kinds of plasmids, described 14 kinds of plasmids are expressed as follows in 14 kinds of ubiquitin binding enzymes a kind of respectively: the ubiquitin binding enzyme UBC27 of aminoacid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of aminoacid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of aminoacid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of aminoacid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of aminoacid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of aminoacid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of aminoacid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of aminoacid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of aminoacid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of aminoacid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of aminoacid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of aminoacid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of aminoacid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of aminoacid sequence as shown in sequence in sequence table 31.

12. recombinant bacterium combination according to claim 11, is characterized in that: the encoding gene of described ubiquitin activating enzyme UBA2 is the DNA molecular shown in sequence in sequence table 2; The encoding gene of described wild type monomeric ubiquitin protein is the DNA molecular shown in sequence in sequence table 4; The encoding gene of described ubiquitin binding enzyme UBC27 is the DNA molecular shown in sequence in sequence table 6; The encoding gene of described ubiquitin binding enzyme UBC1 is the DNA molecular shown in sequence in sequence table 8; The encoding gene of described ubiquitin binding enzyme UBC2 is the DNA molecular shown in sequence in sequence table 10; The encoding gene of described ubiquitin binding enzyme UBC3 is the DNA molecular shown in sequence in sequence table 12; The encoding gene of described ubiquitin binding enzyme UBC10 is the DNA molecular shown in sequence in sequence table 14; The encoding gene of described ubiquitin binding enzyme UBC32 is the DNA molecular shown in sequence in sequence table 16; The encoding gene of described ubiquitin binding enzyme UBC13 is the DNA molecular shown in sequence in sequence table 18; The encoding gene of described ubiquitin binding enzyme UBC4 is the DNA molecular shown in sequence in sequence table 20; The encoding gene of described ubiquitin binding enzyme UBC5 is the DNA molecular shown in sequence in sequence table 22; The encoding gene of described ubiquitin binding enzyme UBC6 is the DNA molecular in sequence table shown in sequence 24; The encoding gene of described ubiquitin binding enzyme UBC35 is the DNA molecular shown in sequence in sequence table 26; The encoding gene of described ubiquitin binding enzyme UBC19 is the DNA molecular shown in sequence in sequence table 28; The encoding gene of described ubiquitin binding enzyme UBC16 is the DNA molecular shown in sequence in sequence table 30; The encoding gene of described ubiquitin binding enzyme UBC22 is the DNA molecular shown in sequence in sequence table 32.

Arbitrary described composition in 13. claim 1-4, or arbitrary described plasmid group in claim 5-8, or arbitrary described recombinant bacterium combination in claim 9-12, the application in following (a) or (b):

A () is detected vegetable-protein to be measured and whether is had ubiquitin ligase activity;

B () is detected the vegetable-protein to be measured with ubiquitin ligase activity and is combined with any or several ubiquitin binding enzyme.

14. detect the method whether vegetable-protein to be measured has ubiquitin ligase activity, comprise the steps:

(a1) following 4 groups of samples are reacted respectively:

First group: claim 1-4 arbitrary described in ubiquitin activating enzyme UBA2, described wild type monomeric ubiquitin protein, and any one in described 14 kinds of ubiquitin binding enzymes;

Second group: the wild type monomeric ubiquitin protein described in vegetable-protein to be measured, claim 1-4 are arbitrary, and the described ubiquitin binding enzyme identical with first group;

3rd group: the ubiquitin activating enzyme UBA2 described in vegetable-protein to be measured, claim 1-4 are arbitrary, and described wild type monomeric ubiquitin protein;

4th group: the ubiquitin activating enzyme UBA2 described in vegetable-protein to be measured, claim 1-4 are arbitrary, described wild type monomeric ubiquitin protein, and the described ubiquitin binding enzyme identical with first group;

(a2) reaction product of 4 groups of samples in step (a1) is carried out SDS-PAGE electrophoresis respectively, after transferring film, carry out immuning hybridization with the antibody of anti-described wild type monomeric ubiquitin protein;

(a3) according to the result of immuning hybridization in step (a2), determine whether described vegetable-protein to be measured has ubiquitin ligase activity as follows: if the reaction product of the 4th group of sample has the antibody signal of anti-described wild type monomeric ubiquitin protein in the position that molecular weight is greater than described vegetable-protein to be measured, first group simultaneously, the reaction product of second group and the 3rd group sample is greater than the antibody signal of all not anti-described wild type monomeric ubiquitin protein in position of described vegetable-protein to be measured at molecular weight, then described vegetable-protein to be measured has ubiquitin ligase activity or candidate has ubiquitin ligase activity, otherwise then described vegetable-protein to be measured does not have ubiquitin ligase activity or candidate does not have ubiquitin ligase activity.

15. detect the method that any or several ubiquitin binding enzyme of vegetable-protein to be measured in claim 1-4 in arbitrary described composition with ubiquitin ligase activity be combined, and comprise the steps:

(b1) the ubiquitin activating enzyme UBA2 described in each in 14 kinds of ubiquitin binding enzymes described in claim 1-4 is arbitrary is arbitrary with vegetable-protein to be measured, claim 1-4 respectively, and described wild type monomeric ubiquitin protein mixing, form several reaction systems;

(b2) after reaction terminates, the product of several reaction systems in step (b1) is carried out SDS-PAGE electrophoresis respectively, after transferring film, carry out immuning hybridization with the antibody of anti-described wild type monomeric ubiquitin protein;

(b3) according to the result of immuning hybridization in step (b2), determine that any or several ubiquitin binding enzyme of described vegetable-protein to be measured in described composition is combined as follows: the ubiquitin binding enzyme met in the reaction system of following condition is the ubiquitin binding enzyme that described vegetable-protein to be measured combines with it; The ubiquitin binding enzyme do not met in the reaction system of following condition is described vegetable-protein to be measured uncombined ubiquitin binding enzyme with it: the antibody signal having anti-described wild type monomeric ubiquitin protein in the position that molecular weight is greater than described vegetable-protein to be measured.

Described in plasmid group described in composition described in 16. claims 1 or 2 or claim 5 or 6 or claim 9 or 10, recombinant bacterium is combined in the application detected in poly ubiquitin chain type.

Whether 17. detect the interact poly ubiquitin chain that formed of vegetable-proteins to be measured and composition described in claim 1 or 2 contains the method for poly ubiquitin chain that K48-is connected or the poly ubiquitin chain that K63-connects, and comprises the steps:

(c1) following 5 groups of samples are reacted respectively:

First group: the wild type monomeric ubiquitin protein described in vegetable-protein to be measured, claim 1 or 2, and any one in described 14 kinds of ubiquitin binding enzymes;

Second group: the wild type monomeric ubiquitin protein described in vegetable-protein to be measured, claim 1 or 2, and described ubiquitin activating enzyme UBA2;

3rd group: the wild type monomeric ubiquitin protein described in vegetable-protein to be measured, claim 1 or 2, described ubiquitin activating enzyme UBA2, and the described ubiquitin binding enzyme identical with first group;

4th group: the point mutation ubiquitin protein UbK48R described in vegetable-protein to be measured, claim 1 or 2, described ubiquitin activating enzyme UBA2 and the described ubiquitin binding enzyme identical with first group;

5th group: the point mutation ubiquitin protein UbK63R described in vegetable-protein to be measured, claim 1 or 2, described ubiquitin activating enzyme UBA2 and the described ubiquitin binding enzyme identical with first group;

(c2) reaction product of 5 groups of samples in step (c1) is carried out SDS-PAGE electrophoresis respectively, after transferring film, carry out immuning hybridization with the antibody of anti-described wild type monomeric ubiquitin protein;

(c3) according to the result of immuning hybridization in step (c2), poly ubiquitin chain signal is not had in the reaction product of first group, second group sample, and under the reaction product of the 3rd group of sample has the prerequisite of poly ubiquitin chain signal, the poly ubiquitin chain that the poly ubiquitin chain determined to interact described vegetable-protein to be measured and composition described in claim 1 or 2 as follows whether the poly ubiquitin chain that formed be connected containing K48-or K63-connect:

If meet the condition of (d1), do not meet the condition of (d2) simultaneously, then described vegetable-protein to be measured and composition described in claim 1 or 2 interact the poly ubiquitin chain that the poly ubiquitin chain that formed is connected containing K48-, and containing the poly ubiquitin chain of K63-connection;

If do not meet the condition of (d1), meet the condition of (d2) simultaneously, then described vegetable-protein to be measured and composition described in claim 1 or 2 interact the poly ubiquitin chain that the poly ubiquitin chain that formed is connected containing K63-, and containing the poly ubiquitin chain of K48-connection;

If meet the condition of (d1) and (d2) simultaneously, then described vegetable-protein to be measured and composition described in claim 1 or 2 interact the poly ubiquitin chain that formed both containing the poly ubiquitin chain that K48-is connected, also containing the poly ubiquitin chain of K63-connection;

If do not meet the condition of (d1) and (d2) simultaneously, then described vegetable-protein to be measured and composition described in claim 1 or 2 interact the poly ubiquitin chain that formed neither containing the poly ubiquitin chain that K48-is connected, also containing the poly ubiquitin chain of K63-connection;

(d1) reaction product of the 4th group of sample is without poly ubiquitin chain signal, or has the poly ubiquitin chain signal that strength of signal is less than the reaction product of the 3rd group of sample;

(d2) reaction product of the 5th group of sample is without poly ubiquitin chain signal, or has the poly ubiquitin chain signal that strength of signal is less than the reaction product of the 3rd group of sample;

Described poly ubiquitin chain signal is the antibody signal that there is anti-described wild type monomeric ubiquitin protein the position being greater than described vegetable-protein to be measured at molecular weight.