CN103091498A

CN103091498A - Plant in-vitro ubiquitin protein degradation system and application thereof

Info

Publication number: CN103091498A
Application number: CN2013100070419A
Authority: CN
Inventors: 谢旗; 赵庆臻
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2013-01-08
Filing date: 2013-01-08
Publication date: 2013-05-08
Anticipated expiration: 2033-01-08
Also published as: CN103091498B

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

The outer ubiquitin protein degeneration system of plant and application thereof

Technical field

The present invention relates to the outer ubiquitin protein degeneration system of a kind of plant and application thereof, particularly a kind of whole active component all comes from the outer ubiquitin protein degeneration system of plant and the application thereof of arabidopsis.

Background technology

Ubiquitin in most eukaryotes/26S proteasome system (ubiquitin/26S proteasome system UPS) abnormal protein of can not only degrading, and can remove the overwhelming majority very short adjusting protein (as transcription factor, cyclin etc.) of half life period, therefore play important regulating and controlling effect in keeping the cell normal activities.Existing studies confirm that, 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.With ubiquitin as the covalently bound effect successively that needs ubiquitin kinase (E1), ubiquitin binding enzyme (E2) and ubiquitin ligase (E3) to the protein target molecule of albumen label.Ubiquitination on substrate has polytype, the poly ubiquitin that connects as single ubiquitin, K48-, the poly ubiquitin that K63-connects etc., the type decided of ubiquitination the destiny of substrate protein.Have 2 E1,37 E2 in model plant arabidopsis gene group and surpass 1400 E3 genes.The substrate specificity of ubiquitination is mainly determined by E3, and E2 is the crucial factor of determination of poly ubiquitin chain structure type on substrate.37 E2 belong to 12 subfamilies, studies show that existing in vivo a large amount of E2-E3 acts synergistically specifically.

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

Summary of the invention

The purpose of this invention is to provide the outer ubiquitin protein degeneration system of a kind of plant and application thereof.

The outer ubiquitin protein degeneration system of plant provided by the present invention be following (A) or (B) shown in the composition that is used for vitro detection testing protein ubiquitination activity.

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

(1) ubiquitin kinase UBA2 and wild type monomer ubiquitin protein;

The amino acid sequence of described ubiquitin kinase UBA2 is as shown in sequence in sequence table 1; The amino acid sequence of described wild type monomer 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 amino acid sequence as shown in sequence in sequence table 5; The ubiquitin binding enzyme UBC1 of amino acid sequence as shown in sequence in sequence table 7; The ubiquitin binding enzyme UBC2 of amino acid sequence as shown in sequence in sequence table 9; The ubiquitin binding enzyme UBC3 of amino acid sequence as shown in sequence in sequence table 11; The ubiquitin binding enzyme UBC10 of amino acid sequence as shown in sequence in sequence table 13; The ubiquitin binding enzyme UBC32 of amino acid sequence as shown in sequence in sequence table 15; The ubiquitin binding enzyme UBC13 of amino acid sequence as shown in sequence in sequence table 17; The ubiquitin binding enzyme UBC4 of amino acid sequence as shown in sequence in sequence table 19; The ubiquitin binding enzyme UBC5 of amino acid sequence as shown in sequence in sequence table 21; The ubiquitin binding enzyme UBC6 of amino acid sequence as shown in sequence in sequence table 23; The ubiquitin binding enzyme UBC35 of amino acid sequence as shown in sequence in sequence table 25; The ubiquitin binding enzyme UBC19 of amino acid sequence as shown in sequence in sequence table 27; The ubiquitin binding enzyme UBC16 of amino acid sequence as shown in sequence in sequence table 29; The ubiquitin binding enzyme UBC22 of amino acid sequence as shown in sequence in sequence table 31;

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

The amino acid sequence of described point mutation ubiquitin protein UbK48R is for replacing with the lysine of the 48th of sequence in sequence table 3 sequence of gained after arginine; The amino acid sequence of described point mutation ubiquitin protein UbK63R is for replacing with the lysine of the 63rd of sequence in sequence table 3 sequence of gained after arginine;

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

A further 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 following (I) or (II):

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

1) express the plasmid of ubiquitin kinase UBA2, and the plasmid of expressing wild type monomer ubiquitin protein;

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

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

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

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

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

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

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

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

The recombinant bacterium that contains any 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 Escherichia coli, as e. coli bl21 (DE3).

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

(a) detect testing protein and whether have the ubiquitin ligase activity;

(b) detecting the testing protein with ubiquitin ligase activity is combined with any or several ubiquitin binding enzymes.

A further object of the present invention is to provide a kind of method whether testing protein has the ubiquitin ligase activity that detects.

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 kinase UBA2, described wild type monomer ubiquitin protein, and any in described 14 kinds of ubiquitin binding enzymes;

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

The 3rd group: described testing protein, described ubiquitin kinase UBA2, and described wild type monomer ubiquitin protein;

The 4th group: described testing protein, described ubiquitin kinase UBA2, described wild type monomer ubiquitin protein, and with first group in identical described ubiquitin binding enzyme;

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

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

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

In said method, first to three group is control group, is equivalent to quality control standard; The 4th group is experimental group.

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

Any in described composition of the testing protein that detection provided by the present invention has a ubiquitin ligase activity or the method that several ubiquitin binding enzymes are combined specifically can comprise the steps:

(b1) with in described 14 kinds of ubiquitin binding enzymes all or part of each respectively with testing protein, described ubiquitin kinase UBA2, and described wild type monomer ubiquitin protein mixes, and forms several reaction systems;

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

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

In one embodiment of the invention, the ubiquitin binding enzyme of selecting 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.

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

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

Also purpose of the present invention is to provide a kind of testing protein that detects ubiquitin ligase and the described composition formed poly ubiquitin chain that interacts and whether contains the method for the poly ubiquitin chain of poly ubiquitin chain that K48-is connected or K63-connection.

Whether detection testing protein provided by the present invention and the described composition formed poly ubiquitin chain that interacts contains the method for the poly ubiquitin chain of poly ubiquitin chain that K48-is connected or K63-connection, specifically can comprise the steps:

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

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

Second group: testing protein, described wild type monomer ubiquitin protein, and described ubiquitin kinase UBA2;

The 3rd group: testing protein, described wild type monomer ubiquitin protein, described ubiquitin kinase UBA2, and with first group in identical described ubiquitin binding enzyme;

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

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

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

(c3) according to the result of immuning hybridization in step (c2), reaction product at first group, second group sample does not have poly ubiquitin chain signal, and the reaction product of the 3rd group of sample has under the prerequisite of poly ubiquitin chain signal, determines as follows whether described testing protein and the described composition formed poly ubiquitin chain that interacts contains poly ubiquitin chain that K48-is connected or the poly ubiquitin chain of K63-connection:

If satisfy the condition of (d1), do not satisfy simultaneously the condition of (d2), described testing protein contains with the formed poly ubiquitin chain of described composition interaction the poly ubiquitin chain that K48-is connected, and does not contain the poly ubiquitin chain that K63-connects;

If do not satisfy the condition of (d1), satisfy simultaneously the condition of (d2), described testing protein contains with the formed poly ubiquitin chain of described composition interaction the poly ubiquitin chain that K63-is connected, and does not contain the poly ubiquitin chain that K48-connects;

If satisfy simultaneously (d1) and condition (d2), described testing protein had both contained with the formed poly ubiquitin chain of described composition interaction the poly ubiquitin chain that K48-is connected, and also contained the poly ubiquitin chain that K63-connects;

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

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

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

In described method, when testing protein described in practical application is known as ubiquitin binding enzyme (as UBC35), all do not add any in described 14 kinds of ubiquitin binding enzymes in the reaction system of above-mentioned first group to the 5th group, do not add described testing protein in second group simultaneously; Certainly active according to the ubiquitin binding enzyme of described testing protein, can selectivity add the ubiquitin binding enzyme (as UBC35) as testing protein is had its auxilin of being combined with ubiquitin (UEV1D) of promotion.

In described method, first group and second group is control group, and this reaction system of two groups can be carried out suitable change in practical operation, and principle is that the component in this two group reactions system of assurance is compared with the 3rd group, lacks a component.

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

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

In the present invention, each albumen in described composition obtains by prokaryotic expression.In order to facilitate purifying and subsequent applications, each albumen has all been introduced 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 in the label that described testing protein is introduced and described composition, each albumen is introduced is different.

In an embodiment of the present invention, the encoding gene of each albumen in described composition all is building up to prokaryotic expression carrier pET8a(with 6 * His and T7 albumen label), obtain corresponding recombinant plasmid; The gained recombinant plasmid transforms e. coli bl21 (DE3) by the heat shock method, through the IPTG induction expression protein, and gained total bacterial protein supernatant Ni-NTA(Qiagen) purifying and super filter tube (Millipore) desalination and concentration.The encoding gene of described testing protein is building up on prokaryotic expression carrier with the albumen label except 6 * His and T7, as pGEX serial carrier (recombinant protein is with glutathione-S-transferase GST label) or pMalC2(recombinant protein with maltose-binding protein (MBP) label) etc.; The gained recombinant plasmid transforms e. coli bl21 (DE3) by the heat shock method, through the IPTG induction expression protein, the packing-80 of gained total bacterial protein supernatant is ℃ frozen standby, or with corresponding method purifying and desalination and concentration by ultrafiltration, as MBP fusion amylase resin(NEB) purifying, gst fusion protein glutathione agarose Glutathione-sepharose4B (GE healthcare) purifying.

In above all described methods, the reaction that relates to is all 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 are water;

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

The present invention has following advantage: than external ubiquitin reacted constituent commonly used at present, the whole components in composition provided by the present invention all derive from the model plant arabidopsis, and are more genuine and believable for the testing protein analysis result of plant origin; Comprised in addition the component that can represent arabidopsis ubiquitin binding enzyme overwhelming majority subfamily feature in composition provided by the present invention, broad covered area, utilize these components can more fully detect the active of ubiquitin ligase and analyze the specific binding situation of E2-E3, the research that can be gene function provides more clue.

Description of drawings

Fig. 1 is the active testing result of arabidopsis ubiquitin kinase UBA2 and arabidopsis wild type monomer ubiquitin protein.(a) be the arabidopsis ubiquitin kinase UBA2(His-AtE1 with the His label) and wheat ubiquitin kinase (His-wE1) carries out with people's ubiquitin binding enzyme UBCH5b and arabidopsis wild type monomer ubiquitin protein respectively, and ubiquitinization is active to be detected; (b) be (a) middle reaction His-AtE1 used and the protein content of His-wE1.

Fig. 2 detects the result of a plurality of ubiquitin binding enzyme activity of arabidopsis with the formation experiment of the thioester bond of DTT-sensitivity.In figure, contain this material in the corresponding reaction system of "+" expression; Do not contain this material in the corresponding reaction system of "-" expression; Arrow represents the ubiquitin binding enzyme that generates-ubiquitin protein compound in reaction (connecting by the thioester bond to the DTT sensitivity between them); Triangle represents to have neither part nor lot in the ubiquitin binding enzyme of reaction; Asterisk represents to have neither part nor lot in the arabidopsis wild type monomer ubiquitin protein of reaction.

Fig. 3 is that the active result that detects of ubiquitinization is carried out in different E2-E3 combinations.(a) may make up respectively the result of carrying out the ubiquitin reaction for testing protein CBL-like and a plurality of ubiquitin binding enzyme of ubiquitin ligase for inferring; (b) make up respectively the result of carrying out the ubiquitin reaction for ubiquitin ligase RHA2a and a plurality of ubiquitin binding enzyme; (c) be the result that the ubiquitin reaction is carried out in the combination of ubiquitin binding enzyme UBC35 and a plurality of ubiquitin ligase; (d) be the result that the ubiquitin reaction is carried out in the combination of ubiquitin binding enzyme UBC6 and a plurality of ubiquitin ligase.In figure, the swimming lane of mark "+" represents to have occured the poly ubiquitination, and the swimming lane of mark "+" does not represent not occured the poly ubiquitination.

Fig. 4 utilizes wild type and point mutation ubiquitin protein to detect the testing result of poly ubiquitin chain type.In figure, contain this material in the corresponding reaction system of "+" expression; Do not contain this material in the corresponding reaction system of "-" expression.

Embodiment

The experimental technique that uses in following embodiment is conventional method if no special instructions.

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

Experiment material used in following embodiment:

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

His antibody is available from Santa-Cruz company (catalog number (Cat.No.): sc-0836); GST antibody available from the large genome company of China (article No. AbM59001-2H5-PU) ubiquitin (Ub) antibody available from sigma company (catalog number (Cat.No.): U0508); Two is anti-available from the proteintech(catalog number (Cat.No.): 00001-1); ATP is available from sigma company (catalog number (Cat.No.): A9187); The NC film is available from Amersham company (catalog number (Cat.No.): RPN303C); The point mutation kit is available from Qiagen company (catalog number (Cat.No.): 200518); Western detects the chemiluminescence HRP substrate reagent box of use available from Millipore company (article No. WBKLS0500).

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

The structure of embodiment 1, recombinant expression carrier

One, the preparation of recombinant plasmid pET28a-UBA2

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

2, the primer that adopts step 1 carries out enzyme with restriction enzyme EcoRI and SalI and cuts by the CDS complete sequence that the method clone of PCR obtains the UBA2 gene.

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

4, the enzyme of step 2 is cut product and be connected with the carrier framework of step 3, obtain recombinant plasmid pET28a-UBA2.Show through order-checking, recombinant plasmid pET28a-UBA2 is for having inserted GenBank:U40566.1 between the EcoRI of skeleton carrier pET28a and SalI site, from 414 to 3647 nucleotide sequences of 5 ' end, the recombinant plasmid that namely obtains after DNA shown in sequence 2 in sequence table.Albumen shown in sequence 1 (ubiquitin kinase UBA2) in sequence 2 code sequence lists.

Two, the preparation of recombinant plasmid pET28a-Ub

1, a ubiquitin protein sequence monomer (GenBank:NM_001125450.1 in the CDS of design amplification arabidopsis wild type ubiquitin protein UBQ14 gene, from the 71st to 298 nucleotide sequences of 5 ' end, and connect terminator codon TAG, sequence 4 at its 3 ' end) primer and add respectively BamHI and EcoRI restriction enzyme site at its two ends.(annotate: UBQ14 is the PolyUb gene that comprises 4 ubiquitin monomeric proteins, and the coded sequence that the present invention has cloned one of them ubiquitin monomer is DNA sequence dna shown in sequence 4, is used for the ubiquitin protein shown in expressed sequence 3)

2, the primer that adopts step 1 carries out enzyme with restriction enzyme BamHI and EcoRI and cuts by the CDS sequence that the method clone of PCR obtains wild type monomer ubiquitin protein gene.

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

4, the enzyme of step 2 is cut product and be connected with the carrier framework of step 3, obtain recombinant plasmid pET28a-Ub.Show through order-checking, recombinant plasmid pET28a-Ub is for having inserted GenBank:NM_001125450.1 between the BamHI of skeleton carrier pET28a and EcoRI site, from the 71st to 298 nucleotide sequence+TAG of 5 ' end, the recombinant plasmid that namely obtains after DNA shown in sequence 4 in sequence table.Albumen shown in sequence 3 (wild type monomer ubiquitin protein) in sequence 4 code sequence lists.

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

The example that is prepared as with recombinant plasmid pET28a-UBC10:

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

2, the primer that adopts step 1 carries out enzyme with restriction enzyme EcoRI and SalI and cuts by the CDS complete sequence that the method clone of PCR obtains the Ub gene.

4, the enzyme of step 2 is cut product and be connected with the carrier framework of step 3, obtain recombinant plasmid pET28a-UBC10.Show through order-checking, recombinant plasmid pET28a-UBC10 is for having inserted GenBank:Z14991.1 between the EcoRI of skeleton carrier pET28a and SalI site, from 133 to 579 nucleotide sequences of 5 ' end, the recombinant plasmid that namely obtains after DNA shown in sequence 14 in sequence table.Albumen (ubiquitin binding enzyme UBC10) in sequence 14 code sequence lists shown in sequence 13.

The multiple ubiquitin binding enzyme that arrives involved in the present invention is all used with the similar method of UBC10 to be cloned on the pET28a carrier, 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 the 118th to 936 nucleotide sequence of 5 ' end, and connect terminator codon TAG at its 3 ' end, it is sequence 16, albumen shown in sequence 15 in the code sequence list), UBC4(GenBank:NM_123499.4, from the 175th to 738 nucleotide sequence of 5 ' end, sequence 20, albumen shown in sequence 19 in the code sequence list), UBC5(GenBank:NM_105055.2, from the 78th to 635 nucleotide sequence of 5 ' end, sequence 22, albumen shown in sequence 21 in the code sequence list), UBC6(GenBank:NM_130166.3, from the 87th to 638 nucleotide sequence of 5 ' end, sequence 24, albumen shown in sequence 23 in the code sequence list) be the recombinant vector that the effect by restriction enzyme BamH I and Sac I obtains.UBC1(GenBank:NM_101307.4 is from the 254th to 712 nucleotide sequence of 5 ' end, sequence 8, albumen shown in sequence 7 in the code sequence list) and UBC2(GenBank:NM_126331.3, from the 254th to 712 nucleotide sequence of 5 ' end, sequence 10, the albumen shown in sequence 19 in the code sequence list) be that effect by restriction enzyme SacI and HindIII obtains recombinant vector.remaining comprises UBC27(GenBank:NM_124465.5, from the 109th to 687 nucleotide sequence of 5 ' end, sequence 6, albumen shown in sequence 5 in the code sequence list), UBC3(GenBank:NM_125648.3, from the 271st to 723 nucleotide sequence of 5 ' end, sequence 12, albumen shown in sequence 11 in the code sequence list), UBC13(GenBank:NM_114513.5, from the 138th to 638 nucleotide sequence of 5 ' end, sequence 18, albumen shown in sequence 17 in the code sequence list), UBC19(GenBank:NM_112897.3, from the 168th to 713 nucleotide sequence of 5 ' end, sequence 28, albumen shown in sequence 27 in the code sequence list), UBC35(GenBank:NM_106535.3, from the 136th to 597 nucleotide sequence of 5 ' end, sequence 26, albumen shown in sequence 25 in the code sequence list), UBC16(GenBank:NM_106198.4, from the 137th to 622 nucleotide sequence of 5 ' end, sequence 30, albumen shown in sequence 29 in the code sequence list), UBC22(GenBank:NM_120590.2, from the 300th to 1055 nucleotide sequence of 5 ' end, sequence 32, albumen shown in sequence 31 in the code sequence list) and ubiquitin-like desmoenzyme UEV1D(GenBank:NM_180353.2, from the 131st to 574 nucleotide sequence of 5 ' end) be all to utilize restriction enzyme EcoRI and SalI to carry out the structure of recombinant vector.

Above each sequence is the CDS complete sequence of each ubiquitin binding enzyme gene, and the gained recombinant plasmid is all to insert the CDS complete sequence of ubiquitin binding enzyme gene separately between the corresponding restriction enzyme site of skeleton carrier pET28a.

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

Design is with the primer in point mutation site, the recombinant plasmid pET28a-Ub that obtains in the step 2 is as template, utilize Qiagen company point mutation kit, carry out according to operational manual, 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, wherein the base of capitalization is the mutational site, corresponding to the wild type monomer ubiquitin protein gene shown in sequence in sequence table 4 the 143rd and 144 sports GG with former AA.

(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, wherein the base of capitalization is the mutational site,, corresponding to the wild type monomer ubiquitin protein gene shown in sequence in sequence table 4 the 188th sports G with former A.

Order-checking shows, recombinant plasmid pET28a-Ub (K48R) for the A of the 143rd of sequence 4 in having inserted sequence table between the BamHI of skeleton carrier pET28a and EcoRI site and the 144th 's A all replace with G after gained point mutation ubiquitin gene.This point mutation ubiquitin gene Protein sequence replaces with arginine for the lysine of the 48th with sequence in sequence table 3.Recombinant plasmid pET28a-Ub (K63R) for the A of the 188th of sequence 4 in having inserted sequence table between the BamHI of skeleton carrier pET28a and EcoRI site replace with G after gained point mutation ubiquitin gene.This point mutation ubiquitin gene Protein sequence replaces with arginine for the lysine of the 63rd with sequence in sequence table 3.

Five, the preparation of recombinant plasmid pET28a-wE1

List of references: 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 has been for having inserted GenBank:M55604.1 between the EcoRI of skeleton carrier pET28a and XhoI site, the recombinant plasmid that obtains after the wheat ubiquitin kinase gene order shown in 5 ' end 183-3338 position.

Six, the preparation of recombinant plasmid pET15b-UBCH5b.

List of references: 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 has been for having inserted GenBank:U39317.1 between the NcoI of skeleton carrier pET15b and BamHI site, the recombinant plasmid that obtains after the people's ubiquitin binding enzyme UBCH5b gene order shown in 23 to 466,5 ' end.

Annotate: in this recombinant plasmid, (6 * His) sequences excisions (being located between NcoI and BamHI site at 6 * His sequence label on the pET15b carrier) are therefore the UBCH5b albumen that goes out with this plasmid expression and without His and other albumen labels with the albumen label on carrier.

Seven, the preparation of recombinant plasmid pMalC2-SDIR1

List of references: 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 has been for having inserted GenBank:NM_115410.3 between the EcoRI of skeleton carrier pMalC2 and SalI site, the recombinant plasmid that obtains after arabidopsis ubiquitin ligase SDIR1 gene (At3g55530) sequence shown in 5 ' end 169-990 position.

Eight, the preparation of recombinant plasmid pMalC2-Rma1

List of references: 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 has been for having inserted GenBank:NM_116589.3 between the XbaI of skeleton carrier pMalC2 and PstI site, the recombinant plasmid that obtains after arabidopsis ubiquitin ligase Rma1 gene (At4g03510) sequence shown in 5 ' end 191-940 position.

Nine, the preparation of recombinant plasmid pMalC2-CIP8

List of references: 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 piece list of references (that build in document is GST-CIP8, is MBP-CIP8 in the present invention).

Recombinant plasmid pMalC2-CIP8 has been for having inserted GenBank:NM_125891.3 between the XbaI of skeleton carrier pMalC2 and PstI site, the recombinant plasmid that obtains after arabidopsis ubiquitin ligase CIP8 gene (At5g64920) sequence shown in 5 ' end 101-1105 position.

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 has been for having inserted GenBank:NM_128855.3 between the EcoRI of skeleton carrier pMalC2 and SalI site, the recombinant plasmid that obtains after arabidopsis ubiquitin ligase COP1 gene (At2g32950) sequence shown in 5 ' end 68-2095 position.

11, the preparation of recombinant plasmid pMalC2-HOS1

List of references: 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 has been for having inserted GenBank:NM_129540.4 between the BamHI of skeleton carrier pMalC2 and PstI site, the recombinant plasmid that obtains after arabidopsis ubiquitin ligase HOS1 gene (At2g39810) sequence shown in 5 ' end 142-2925 position.

12, the preparation of recombinant plasmid pMalC2-RHA2a

List of references: 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 has been for having inserted GenBank:NM_101378.2 between the EcoRI of skeleton carrier pMalC2 and BamHI site, the recombinant plasmid that obtains after arabidopsis ubiquitin ligase RHA2a gene (At1g15100) sequence shown in 5 ' end 34-501 position.

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

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

2, the primer that adopts step 1 carries out enzyme with restriction enzyme EcoRI and SalI and cuts by the CDS complete sequence that the method clone of PCR obtains the CBL-like gene.

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

4, the enzyme of step 2 is cut product and be connected with the carrier framework of step 3, obtain recombinant plasmid pMalC2-SDIR1.Show through order-checking, recombinant plasmid pMalC2-SDIR1 is for having inserted the recombinant plasmid that obtains after the DNA shown in the 1st to 1278 nucleotide sequence of GenBank:5 ' end between the EcoRI of skeleton carrier pMalC2 and SalI site.

The preparation of embodiment 2, albumen

One, 6 * His fusion and UBCH5b protein expression and purifying

1, preparation Escherichia coli Bl21 (DE3) competent cell.

2, the recombinant plasmid difference heat shock method take pET28a as carrier framework for preparing in embodiment 1 step 1 step 6 is transformed Bl21 (DE3) competent cell.

3, picking 2-3 monoclonal is added with in corresponding antibiotic LB fluid nutrient medium in 3-10mL, and 37 ℃, 200 rev/mins of overnight incubation.

4, the cell culture after spending the night joins 200-500mL and contains corresponding microbiotic and 0.2%(0.2g/100mL) in the LB fluid nutrient medium of glucose, making OD600 is 0.1 left and right.

When 5,18 ℃ of concussions were cultured to OD600 and are 0.5-0.6, adding derivant isopropyl-β-D-sulfo-galactopyranoside (IPTG) to final concentration was 0.2mM; Induce 12-16h for 18 ℃.

6,4 ℃, 4000 rev/mins, centrifugal 10 minutes, collect thalline.

7, cell precipitation is filled a prescription with the lysis buffer A(of precooling: 50mMNaH ₂PO ₄300mMNaCl; Transfer to pH8.0 with HCl, with before adding 1mM PMSF) suspend.

8, carry out clasmatosis with Ultrasonic Cell Disruptor, each 10 seconds, totally 10 times, every minor tick 30 seconds.

9, the clasmatosis lysate is at 4 ℃, and 15000 rev/mins, centrifugal 30 minutes.

10, get supernatant, protein purification is preserved or directly carried out to packing for-70 ℃.

(annotate: people's ubiquitin binding enzyme UBCH5b is because His sequence label excision when construction recombination plasmid, so arriving this step, namely finishes protein expression, the packing of albumen supernatant is frozen, be during form with the albumen supernatant adds reaction system during the ubiquitin reaction outside carrying out dependent body, all the other protein are all to proceed the 11-12 step to carry out purifying standby.）

11, the purifying of 6 * His fusion: the Ni-NTA agarose purifying 6 * His fusion with QIAGEN company, carry out according to the product operation instructions.

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

by above operation, the arabidopsis ubiquitin kinase UBA2(with the His label that obtains after purifying is denoted as albumen His-AtE1), arabidopsis wild type monomer ubiquitin protein (being denoted as albumen His-Ub) with the His label, 14 kinds of ubiquitin binding enzymes with the His label, arabidopsis point mutation ubiquitin protein UbK48R(with the His label is denoted as albumen His-UbK48R), arabidopsis point mutation ubiquitin protein UbK63R(with the His label is denoted as albumen His-UbK63R), wheat ubiquitin kinase (being denoted as albumen His-wE1) with the His label, and people's ubiquitin binding enzyme UBCH5b(protein crude extract.

Two, the expression and purification of MBP fusion

1, preparation Escherichia coli Bl21 (DE3) competent cell.

2, the recombinant plasmid take pMalC2 as carrier framework that embodiment 1 step 7 is prepared in step 13 heat shock method respectively transforms Bl21 (DE3) competent cell.

3-6, with the 3-6 in step 1.

7, cell precipitation is filled a prescription with the Columnbuffer(of precooling: 200mMNaCl, and 20mM Tris-HCl, pH7.4,1mM EDTA is with before adding 1mM PMSF, 1mM DTT) suspend.

8-10, with the 8-10 in step 1.

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

12, with 12 in step 1.

by above operation, the arabidopsis ubiquitin ligase SDIR1(with the MBP label that obtains after purifying is denoted as albumen MBP-SDIR1), arabidopsis ubiquitin ligase Rma1(with the MBP label is denoted as albumen MBP-Rma1), arabidopsis ubiquitin ligase CIP8(with the MBP label is denoted as albumen MBP-CIP8), arabidopsis ubiquitin ligase COP1(with the MBP label is denoted as albumen MBP-COP1), arabidopsis ubiquitin ligase HOS1(with the MBP label is denoted as albumen MBP-HOS1), arabidopsis ubiquitin ligase RHA2a(with the MBP label is denoted as albumen MBP-RHA2a), and be denoted as albumen MBP-CBL-like with the CBL-like(that is predicted to be ubiquitin ligase that comes from arabidopsis of MBP label).

The resulting following albumen of the present embodiment purifying has consisted of the outer ubiquitin protein degeneration system of plant of the present invention, specifically is comprised of following (1)-(3):

(1) ubiquitin kinase UBA2 and wild type monomer ubiquitin protein (partial sequence in UBQ14);

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

The amino acid sequence of described point mutation ubiquitin protein UbK48R is for replacing with the lysine of the 48th of sequence in sequence table 3 sequence of gained after arginine; The amino acid sequence of described point mutation ubiquitin protein UbK63R is for replacing with the lysine of the 63rd of sequence in sequence table 3 sequence of gained after arginine.

The arabidopsis ubiquitin kinase of embodiment 3, embodiment 2 purifying and the activity of arabidopsis ubiquitin protein detect

The arabidopsis ubiquitin kinase that the present embodiment obtains embodiment 2 purifying as follows and the activity of arabidopsis ubiquitin protein detect:

(1) get the Eppendorf pipe of 2 1.5mL, be denoted as respectively sample 1, sample 2.All add people's ubiquitin binding enzyme (UBCH5b), 3-5 μ g arabidopsis wild type monomer ubiquitin protein (His-Ub) and the 1 μ L20 * reaction buffer (formula: 1M Tris pH7.5 that obtains in 0.2 μ g embodiment 2 in 2 sample hoses, 100mMATP, 50mM MgCl ₂, 2mM DTT, solvent are water).Add in addition 0.05 μ g arabidopsis ubiquitin kinase UBA2(His-AtE1 in sample 1), add the wheat ubiquitin kinase (His-wE1) of equivalent in sample 2.All add ddH in two samples ₂O to cumulative volume be 20 μ L.

(2) after the mixing sample, take out the reactant liquor of 1/2 volume, add immediately the 4 * SDS sample-loading buffer that contains 0.1mol/L DTT, boiled 5 minutes, this is 0 minute sample of reaction; Residual reaction liquid adds the 4 * SDS sample-loading buffer that contains 0.1mol/LDTT at 37 ℃ after hatching 15 minutes again, boils 5 minutes, and this is 15 minutes samples of reaction.

(3) sample is carried out the SDS-PAGE electrophoresis; The rear electricity consumption of electrophoresis end turns method protein transduction is moved on on nitrocellulose filter (NC film); After transferring film finishes, carry out immuning hybridization with anti-His antibody and detect.Detect principle: the reactivity band that detects due to this research institute is approximately 27kD of UBCH5b-His-Ub(molecular weight), all the other can be gone out by the anti-His antibody test albumen such as His-AtE1 and the His-wE1 of band with the His label, molecular weight all (is seen (b) in Fig. 1) more than 130kD, therefore can not affect testing result.UBCH5b albumen itself there is no the His label, and as not being combined with His-Ub, the band that does not have 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) the position on signal all detected.As seen arabidopsis ubiquitin kinase UBA2 and arabidopsis wild type monomer ubiquitin protein (His-Ub) can make people's ubiquitin binding enzyme UBCH5b that ubiquitination occurs, and response intensity and wheat ubiquitin kinase (wE1) roughly the same.Wheat ubiquitin kinase (wE1) and people's ubiquitin binding enzyme UBCH5b is E1 and E2 commonly used in now widely used external ubiquitin reaction system, and the ubiquitin ligase that major part has been delivered (E3) activity detects with wE1 and UBCH5b.This result shows that arabidopsis ubiquitin kinase UBA2 provided by the invention and arabidopsis wild type monomer ubiquitin protein (His-Ub) are activated, can be used for the detection of external ubiquitin reaction.

The activity of the arabidopsis ubiquitin binding enzyme (E2) of embodiment 4, embodiment 2 purifying detects

The purpose of the present embodiment is to detect the activity of the multiple ubiquitin binding enzyme albumen of embodiment 2 purifying, all can be used for the detection of the active detection of ubiquitin ligase and E2-E3 specific binding to prove them.Because embodiment 3 has proved arabidopsis ubiquitin kinase UBA2(His-AtE1) with wheat ubiquitin kinase (His-wE1), identical activity is arranged, so these two kinds of ubiquitin kinases all can be used for the present embodiment reaction.What this experiment was used is active detection of ubiquitin binding enzyme that wheat ubiquitin kinase (His-wE1) carries out.

The present embodiment utilizes the formation experiment of the thioester bond of DTT-sensitivity, and whether the following 9 kinds of arabidopsis ubiquitin binding enzymes that detect embodiment 2 purifying have activity: UBC3, UBC19, UBC6, UBC35, UBC5, UBC22, UBC32, UBC13 and UBC27.Its principle is: a conservative Cys is arranged in all ubiquitin binding enzyme albumen, the avtive spot of being combined with ubiquitin protein, both in conjunction with the time the 76th of sulfydryl on the Cys side chain and ubiquitin protein C end Gly(G76) the carboxyl condensation form thioester bond (immuning hybridization shows as both when detecting, and there is band the position of molecular weight sum.Attention: owing to being not that all ubiquitin binding enzyme albumen and ubiquitin proteins all can form this heterodimer, so when both with identical albumen label, when detecting with corresponding tag antibody immuning hybridization, also can detect band in the molecular weight position of free ubiquitin binding enzyme and ubiquitin protein); This thioester bond can be reduced agent DTT and open, if therefore add DTT in system after reaction is completed, established ubiquitin binding enzyme-ubiquitin dimer molecule is free ubiquitin binding enzyme and the ubiquitin molecule (immuning hybridization shows as the position of molecular weight sums both without band when detecting, and in the molecular weight position of independent ubiquitin binding enzyme and ubiquitin protein, band is arranged) of again forming separated from one another again.Concrete operations are as follows:

(1) get two 1.5mL Eppendorf pipes, be denoted as respectively sample 1, sample 2.All add the wheat ubiquitin kinase (His-wE1) that obtains in 50ng embodiment 2 in 2 sample hoses, 200ng ubiquitin binding enzyme (in 9 kinds a kind), 1 μ L20 * reaction buffer (formula is with embodiment 3), 2 μ L concentration are the arabidopsis wild type monomer ubiquitin protein (His-Ub) of 2 μ g/ μ L, add ddH ₂O to cumulative volume be 20 μ L.

Hatch 5min for (2) 37 ℃; Add in the sample 1 and contain 4 * SDS-PAGE sample-loading buffer, the 6.7 μ L that final concentration is the DTT of 0.1mol/L, add 4 * SDS-PAGE sample-loading buffer, the 6.7 μ L that do not contain DTT in sample 2, two samples all boil 5min in 100 ℃.

(3) two samples are carried out the SDS-PAGE electrophoresis; The rear electricity consumption of electrophoresis end turns method protein transduction is moved on on nitrocellulose filter (NC film); After transferring film finishes, carry out immuning hybridization with anti-His antibody and detect.

Result 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 the DTT-sensitivity except UBC27, illustrate that they are all activated ubiquitin binding enzymes.

(annotate: although UBC27 self can not form the thioester bond to the DTT-sensitivity, but still can make the reaction of part ubiquitin ligase generation ubiquitin, see step 1 in following examples 5 for details, result such as Fig. 3 (a) be the second swimming lane from left to right, and the band between 34kD and 55kD is UBC27-Ub dimer band; This explanation UBC27 also has ubiquitin binding enzyme active.）

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

The purpose of the present embodiment is to detect multiple to be measured or known albumen whether to have the ubiquitin ligase activity, and the situation of E2-E3 specific binding.Because embodiment 3 has proved arabidopsis ubiquitin kinase UBA2(His-AtE1) with wheat ubiquitin kinase (His-wE1), identical activity is arranged, so these two kinds of ubiquitin kinases all can be used for the present embodiment reaction.What this experiment was used is that wheat ubiquitin kinase (His-wE1) carries out coherent detection.

One, there is ubiquitin reaction in situation in testing protein (supposition may be ubiquitin ligase) CBL-like at different ubiquitin binding enzymes

(1) get 9 1.5mL Eppendorf pipes, all add the wheat ubiquitin kinase (His-wE1), 4 μ g arabidopsis wild type monomer ubiquitin proteins (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 are water), and the testing protein CBL-like of 0.2 μ gMBP mark.

(2) add the ubiquitin binding enzyme 200ng that obtains in a kind of embodiment 2 respectively in each sample hose, relate to altogether following 9 kinds of ubiquitin binding enzymes: UBCH5b(people source), UBC27, UBC3, UBC10, UBC32, UBC13, UBC6, UBC19 and UBC35(arabidopsis source).Add ddH ₂O to cumulative volume be 30 μ L.

(3) sample hose is placed on EppendorfThermomixer comfort instrument, 30 ℃, 900 rev/mins, hatched 1.5 hours.

(4) add respectively 10 μ L to contain 4 * SDS sample-loading buffer cessation reaction of 0.1mol/L DTT in sample hose, 100 ℃ are boiled 5min; Get 15 μ L samples and carry out the SDS-PAGE electrophoresis; The rear electricity consumption of electrophoresis end turns method protein transduction is moved on on nitrocellulose filter (NC film); After transferring film finishes, carry out immuning hybridization with anti-His antibody or anti-Ub antibody and detect.When the detection principle that adopts anti-His antibody as follows: because the molecular weight of ubiquitin binding enzyme is (most of in the 20kD up and down, UBC16 and UBC22 molecular weight are slightly large), (molecular weight generally can be greater than 30kD and the ubiquitin ligase molecular weight is generally greater than ubiquitin binding enzyme, some meetings are greater than 100kD, as COP1 etc.annotate: the RHA2a molecular weight, about 17kD), and ubiquitin binding enzyme merges His label (albumen that the His label gives expression to is the 3kD left and right approximately), ubiquitin ligase merges MBP label (albumen that the MBP label gives expression to is the 42kD left and right approximately), so two kinds of fusion protein molecule amounts differ greatly, and because reaction result is generally to add poly ubiquitin chain on the ubiquitin ligase molecule, the immuning hybridization detected representation is the smear that makes progress of the larger POS INT of molecular weight, and the activity of ubiquitin binding enzyme shows as and adds single or a few ubiquitin molecule, the immuning hybridization detected representation is the obvious band of single or minority of molecular weight position, therefore the band of ubiquitin binding enzyme and the band of ubiquitin ligase are easy to distinguish in position.Below be same case in the reaction example.

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

Two, there is ubiquitin reaction in situation in ubiquitin ligase RHA2a at different ubiquitin binding enzymes

(1) get 9 1.5mL Eppendorf pipes, all add the wheat ubiquitin kinase (His-wE1), 4 μ g arabidopsis wild type monomer ubiquitin proteins (His-Ub), the 1.5 μ L20 * reaction buffers (the same step 1 of filling a prescription) that obtain in 50ng embodiment 2, and the arabidopsis ubiquitin ligase RHA2a of 0.2 μ gMBP mark.

(2)-(4) are with (2) in step 1-(4).

Result shown in (anti-His antibody), at UBCH5b, when UBC10 exists, has all detected protein signal as arabidopsis ubiquitin ligase RHA2a on the position greater than MBP-RHA2a molecular weight (approximately 70KD) as shown in (b) in Fig. 3; And when with other seven kinds of ubiquitin binding enzymes combinations, this protein signal not.As seen arabidopsis ubiquitin ligase RHA2a be respectively at UBCH5b, can produce the poly ubiquitination when UBC10 exists, but can not carry out the ubiquitin reaction when respectively with other seven kinds of ubiquitin binding enzymes combinations.

Three, there is ubiquitin reaction in situation in ubiquitin binding enzyme UBC35 at different ubiquitin ligases

(1) get 8 1.5mL Eppendorf pipes, all add the wheat ubiquitin kinase (His-wE1), 4 μ g arabidopsis wild type monomer ubiquitin proteins (His-Ub), the 1.5 μ L20 * reaction buffers (the same step 1 of filling a prescription) that obtain in 50ng embodiment 2, and 0.2 μ g arabidopsis ubiquitin binding enzyme UBC35.

(2) add the ubiquitin ligase 200ng that obtains in a kind of embodiment 2 respectively in each sample hose, relate to altogether following 6 kinds of ubiquitin ligase: Rma1, CIP8, SDIR1, CBL-like, COP1, HOS1, residue two pipes are 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 1-(4).

Result is as shown in (c) in Fig. 3, shown in (anti-His antibody), as ubiquitin binding enzyme UBC35 at SDIR1, when COP1 exists, all greater than corresponding ubiquitin ligase molecular weight (the MBP-SDIR1 molecular weight is about 72KD), the MBP-COP1 molecular weight is about 120KD) the position on stronger protein signal detected; When Rma1 exists, greater than weak protein signal having been detected on the Rma1 molecular weight position of (being about 70KD); And when with other three kinds of ubiquitin ligase combinations, there is no corresponding protein signal.As seen arabidopsis ubiquitin binding enzyme UBC35 can make SDIR1, COP1 produce the modification of poly ubiquitin chain, makes Rma1 produce weak ubiquitin signal, but can not make other three kinds of ubiquitin ligases carry out the ubiquitin reaction.

Four, there is ubiquitin reaction in situation in ubiquitin binding enzyme UBC6 at different ubiquitin ligases

(1) get 8 1.5mL Eppendorf pipes, all add the wheat ubiquitin kinase (His-wE1), 4 μ g arabidopsis wild type monomer ubiquitin proteins (His-Ub), the 1.5 μ L20 * reaction buffers (the same step 1 of filling a prescription) that obtain in 50ng embodiment 2, and 0.2 μ g arabidopsis ubiquitin binding enzyme UBC6.

(2)-(4) are with (2) in step 1-(4).

Result shown in (anti-His antibody),, (is being about on the position of 140KD and stronger protein signal detected greater than the MBP-HOS1 molecular weight when HOS1 exists as ubiquitin binding enzyme UBC6 as shown in (d) in Fig. 3; When CBL-like and COP1 exist, all weak protein signal detected greater than on the corresponding ubiquitin ligase molecular weight position of (the MBP-CBL-like molecular weight is about 90KD, and the MBP-COP1 molecular weight is about 120KD); And when with other three kinds of ubiquitin ligase combinations, there is no corresponding protein signal.As seen arabidopsis ubiquitin binding enzyme UBC6 can make HOS1 produce the modification of poly ubiquitin chain, makes CBL-like and COP1 produce weak ubiquitin signal, but can not make other three kinds of ubiquitin ligases carry out the ubiquitin reaction.

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

The present embodiment will utilize the ubiquitin protein of wild type that embodiment 2 prepares and point mutation to detect the type of the upper formed poly ubiquitin chain of arabidopsis ubiquitin binding enzyme UBC35.Specific as follows:

(1) get 5 1.5mL Eppendorf pipes, be denoted as respectively sample 1, sample 2, sample 3, sample 4 and sample 5.All add the wheat ubiquitin kinase (His-wE1) that obtains in 50ng embodiment 2 in 5 sample hoses, and 1 μ L20 * reaction buffer (formula is with embodiment 3).

(2) add 0.2 μ g arabidopsis ubiquitin binding enzyme UBC35 and 4 μ g arabidopsis wild type monomer ubiquitin proteins (His-Ub) in sample 1; Add the 0.2 μ g ubiquitin-like desmoenzyme UEV1D(UEV1D and the ubiquitin binding enzyme UBC35 that obtain in embodiment 2 one to form heterodimer in sample 2, promote specifically the generation of the poly ubiquitin chain that K63-connects.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 the 1st phases of volume in 2008: 213-227.) He 4 μ g arabidopsis arabidopsis wild type monomer ubiquitin proteins (His-Ub); Add 0.2 μ g arabidopsis ubiquitin binding enzyme UBC35,0.2 μ g ubiquitin-like desmoenzyme UEV1D and 4 μ g arabidopsis wild type monomer ubiquitin proteins (His-Ub) in sample 3; Add 0.2 μ g arabidopsis ubiquitin binding enzyme UBC35,0.2 μ g ubiquitin-like desmoenzyme UEV1D and 4 μ g arabidopsis point mutation ubiquitin protein UbK48R in sample 4; Add 0.2 μ g arabidopsis ubiquitin binding enzyme UBC35,0.2 μ g ubiquitin-like desmoenzyme UEV1D and 4 μ g arabidopsis point mutation ubiquitin protein UbK63R in sample 5.All use at last ddH ₂O mend to cumulative volume be 20 μ L.

Hatch 2h for (3) 37 ℃; Add 4 * SDS-PAGE sample-loading buffer, the 6.7 μ L that do not contain DTT, 100 ℃ are boiled 5min.

(4) sample is carried out the SDS-PAGE electrophoresis; The rear electricity consumption of electrophoresis end turns method protein transduction is moved on on nitrocellulose filter (NC film); After transferring film finishes, carry out immuning hybridization with anti-Ub antibody and detect.

Result as shown in Figure 4, sample 1 is not in the situation that there is no UEV1D, UBC35 is combined with the wild type ubiquitin protein, produces two bands, is respectively UBC35-Ub1 and UBC35-Ub2, but can not forms poly ubiquitin chain; In sample 2 UEV1D separately with the reaction of wild type ubiquitin protein without the band generation, illustrate that UEV1D self can not be by ubiquitination; Sample 3-5 is respectively in the situation that UBC35 and UEV1D all exist, and the ubiquitin protein with wild type, K48R and K63R point mutation reacts respectively.As seen there is stronger poly ubiquitin chain to form in sample 3, the signal of poly ubiquitin chain does not become substantially when using the ubiquitin protein UbK48R of point mutation, but poly ubiquitin chain disappears substantially when using the ubiquitin protein UbK63R of point mutation, and what illustrate that UBC35 is connected with UEV1D that when existing, catalysis produces is the poly ubiquitin chain of K63-connection.

Claims

1. the composition that is used for vitro detection testing protein ubiquitination activity, for following (A) or (B):

(A) formed by following (1)-(3):

(1) ubiquitin kinase UBA2 and wild type monomer ubiquitin protein;

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

(B) formed by described (1) in (A) and described (2).

2. the plasmid group that is used for vitro detection testing protein ubiquitination activity, for following (I) or (II):

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

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

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

4. contain the recombinant bacterium of having the right to want any or several plasmids in 2 or 3 described plasmid groups.

In claim 1-4 arbitrary described composition or plasmid group or recombinant bacterium at following (a) or the application (b):

(a) detect testing protein and whether have the ubiquitin ligase activity;

6. detect the method whether testing protein has the ubiquitin ligase activity, comprise the steps:

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

First group: the ubiquitin kinase UBA2 described in claim 1 or 3, described wild type monomer ubiquitin protein, and any in described 14 kinds of ubiquitin binding enzymes;

Second group: the wild type monomer ubiquitin protein described in testing protein, claim 1 or 3, and with first group in identical described ubiquitin binding enzyme;

The 3rd group: the ubiquitin kinase UBA2 described in testing protein, claim 1 or 3, and described wild type monomer ubiquitin protein;

The 4th group: the ubiquitin kinase UBA2 described in testing protein, claim 1 or 3, described wild type monomer ubiquitin protein, and with first group in identical described ubiquitin binding enzyme;

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

7. detect any in the described composition of claim 1 or 3 of testing protein with ubiquitin ligase activity or the method that several ubiquitin binding enzymes are combined, comprise the steps:

(b1) with in 14 kinds of ubiquitin binding enzymes described in claim 1 or 3 all or part of each respectively with the ubiquitin kinase UBA2 described in testing protein, claim 1 or 3, and described wild type monomer ubiquitin protein mixing, form several reaction systems;

8. arbitrary described composition or plasmid group or the recombinant bacterium application in detecting poly ubiquitin chain type in claim 1-4.

9. detect testing protein and the described composition of the claim 1 or 3 formed poly ubiquitin chain that interacts and whether contain the method for the poly ubiquitin chain of poly ubiquitin chain that K48-is connected or K63-connection, comprise the steps:

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

First group: the wild type monomer ubiquitin protein described in testing protein, claim 1 or 3, and any in described 14 kinds of ubiquitin binding enzymes;

Second group: the wild type monomer ubiquitin protein described in testing protein, claim 1 or 3, and described ubiquitin kinase UBA2;

The 3rd group: the wild type monomer ubiquitin protein described in testing protein, claim 1 or 3, described ubiquitin kinase UBA2, and with first group in identical described ubiquitin binding enzyme;

The 4th group: the point mutation ubiquitin protein UbK48R described in testing protein, claim 1 or 3, described ubiquitin kinase UBA2 and with first group in identical described ubiquitin binding enzyme;

The 5th group: the point mutation ubiquitin protein UbK63R described in testing protein, claim 1 or 3, described ubiquitin kinase UBA2 and with first group in identical described ubiquitin binding enzyme;

(c3) according to the result of immuning hybridization in step (c2), reaction product at first group, second group sample does not have poly ubiquitin chain signal, and the reaction product of the 3rd group of sample has under the prerequisite of poly ubiquitin chain signal, determines as follows whether the described composition of described testing protein and the claim 1 or 3 formed poly ubiquitin chain that interacts contains poly ubiquitin chain that K48-is connected or the poly ubiquitin chain of K63-connection:

If satisfy the condition of (d1), do not satisfy simultaneously the condition of (d2), described testing protein contains with the formed poly ubiquitin chain of the described composition interaction of claim 1 or 3 the poly ubiquitin chain that K48-is connected, and does not contain the poly ubiquitin chain that K63-connects;

If do not satisfy the condition of (d1), satisfy simultaneously the condition of (d2), described testing protein contains with the formed poly ubiquitin chain of the described composition interaction of claim 1 or 3 the poly ubiquitin chain that K63-is connected, and does not contain the poly ubiquitin chain that K48-connects;

If satisfy simultaneously (d1) and condition (d2), described testing protein had both contained with the formed poly ubiquitin chain of the described composition interaction of claim 1 or 3 the poly ubiquitin chain that K48-is connected, and also contained the poly ubiquitin chain that K63-connects;

If do not satisfy simultaneously (d1) and condition (d2), described testing protein neither contains with the formed poly ubiquitin chain of the described composition interaction of claim 1 or 3 the poly ubiquitin chain that K48-is connected, and does not also contain the poly ubiquitin chain that K63-connects;

10. arbitrary described composition, plasmid group, recombinant bacterium, application or method according to claim 1-7, it is characterized in that: described testing protein is vegetable protein.