CN105950623A - Two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease) and TALEN construction method - Google Patents
Two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease) and TALEN construction method Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering and relates to a two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease). The TALEN assembly strategy of the two-RVD unite is redesigned aiming to overcome various shortcomings of the existing GG (golden gate)-Vector TALEN assembly method. The two-RVD unite module library comprises individually-packed 144 two-RVD unite modules, 8 one-RVD unite modules and 24 end RVD unites. The two-RVD unite module library based on Golden Gate cloning method can construct TALEN expression vectors targeted against 15-19bp arbitrary DNA sequences through primary reaction.
Description
Technical field
The invention belongs to gene engineering technology field, relate to a kind of for TALEN (transcriptional activation sample effector nuclease) efficiently build double
RVD (repeating double residue that makes a variation) unit module storehouse and TALEN construction method.
Background technology
Genome editing technique (genome editing) refers to carry out rite-directed mutagenesis, site-directed integration, site-directed replacement etc. for the particular sequence of genome
One technology of genetic modification.Genome editing technique is utilized to introduce the change of gene order in situ at genome, to the research gene function side of providing
Just.In application, utilize genome editing technique to formulate animals and plants new varieties, gene radom insertion in existing transgenic technology can be avoided to cause
Express uncertain and to protogene group damage.Appearance along with targeted nuclease technology, it is achieved that genes of interest precisely orients and knocks out
(knock-out), thus obtain the mutant that target gene knocks out.Studying more three technology at present is ZFN (zinc finger nuclease, zinc
Finger nuclease), TALEN (transcription activator-like effectors nuclease, transcriptional activation sample effector nuclease) and
CRISPR/cas9 (The clustered, regularly interspaced, short palindromic repeats-associated protein systems, cluster
The short palindrome repetitive sequence of regular intervals and related protein system).
TALENs albumen includes two ingredients.First ingredient comes from natural TALE (Transcription Activator Like
Effectors, transcriptional activation sample effector) albumen, its N end contains a translocation domain；Centre be one section by 1.5～33.5 TALE not etc.
The repetition aminoacid sequence of unit composition, each unit is made up of 34 aminoacid again, and wherein 32 aminoacid are high conservatives, and the only the 12nd
Position and 13 amino acids can change, it is possible to combining a base sequence specifically, therefore the two aminoacid is otherwise known as and repeats variation pair
Residue (Repeat variant diresidue, RVD), 0.5 last unit comprises only 20 aminoacid above；The C end of natural TALE albumen
Containing a nuclear localization signal and activating transcription factor, TALE albumen can be helped to enter nucleus from Cytoplasm and to play transcriptional activation simultaneously.
As a example by the TALE-AvrXs10 albumen of rice leaf spot bacteria secretion, it combines the DNA sequence of 19bp in host cell needs 17.5 unit,
Finding that in natural TALE albumen first base of its 5' end is the combination that T need not TALE protein unit, last base is by last
About 20 aminoacid of 0.5 unit combine.
In December, 2009, Science the 326th phase has delivered two TALEs albumen cracking plant virus secretion simultaneously can specific recognition base
The mechanism of sequence, wherein Moscou etc. use the method for bioinformatics to obtain the rule of TALEs albumen identification base completely, and Boch etc.
Then decode this " password " by laboratory facilities.They find the 12nd and 13 amino acids combination (asparagine-isoleucine in TALEs albumen
-NI), (Asparagine-Alanine-NA), (histidine-asparate-HD), (Asparagine-Glycine-NG) efficiently can identify base specifically respectively
A, G, C, T, and propose as ZFNs, it to be transformed into the instrument of gene site-directed modification in the future.2010, University of Minnesota
The laboratory of Daniel professor Voytas leader takes the lead in combining the DNA of TALE the nuclease domain fusion of relevant domain and FokI, optimizes
Catenation sequence between the two (linker), it is thus achieved that there is the targeted nuclease TALEN of special cleavage activity for specific dna sequence.Hereafter,
Research worker, with natural TALEs albumen as skeleton, constructs the dTALEs (design for the different biological gene such as people, rat, mice, Brachydanio rerio
TALE), and with FokI Cobra venom endonuclease, transcription factor and epigenetic modification enzyme combine realize the fixed point of different plant species genome is repaiied
Decorations or regulation and control.
Second ingredient of TALENs is the IIS type Cobra venom endonuclease FokI from antibacterial, will play after two FokI occur dimerization
DNA double chain is cut by activity, can start two kinds of repair mechanism processes, a kind of repair machine for the non-homogeneous dependence of end after DNA Damage
System (NHEJ), another kind is the repair mechanism (HDR) relying on homologous recombination.When the homologous sequence not having external source adds fashionable, and cell will start NHEJ
Repair mechanism, the DNA end that will be switched off is reunited, but this repair mechanism is the repair process of a kind of existing defects, often
Introduce new sudden change, the most just reach to carry out the purpose of gene knockout.If the homologous base sequence now adding external source just can start second
Kind of repair mechanism HDR repairs, and utilizes homology repair mechanism, us can be helped to realize gene and drive in and gene repair, but if external source same
Source sequence is existing defects, then equally reach the effect of gene knockout.
Utilize TALEN technology to carry out genome directed modification and generally comprise 1) find TALEN candidate's target site according to target gene；2) for
Target site sequence designs and builds TALE carrier；3) TALEN carrier is obtained with suitable FokI sequence assembling；4) by complete TALEN
Vector introduction purpose cell；5) clone of directed modification is screened.In order to ensure the shearing specificity of TALEN, it is to avoid effect of missing the target, TALEN
The target sequence length of effect is usually chosen in 15～about 20bp.Thus, it is desirable to TALE needs containing at least 15 RVD unit repeated,
The length of nucleotides encoding this TALE will be more than 1.5kb, and the repeatability of sequence is the strongest.The most in actual applications, build by repeating
RVD unit assembles, and identifies that the TALE carrier of specific dna sequence just becomes committed step and the difficult point of this technology.
At present, the TALEN assemble method developed mainly has: Golden Gate cloning (GG-PCR) of PCR-based, based on tradition matter
Golden Gate cloning (GG-Vector) of grain carrier, LIC method based on long sticky end, the continuous cloning connected based on enzyme action, base
High pass in solid phase synthesis is mensuration etc..Wherein, the Golden Gate cloning based on single RVD plasmid vector library of the report such as Cermak
(GG-Vector) it is the most widely used technical system (Cermak T, Starker CG, Voytas DF.2015. in TALEN design and component composition
Efficient design and assembly of custom TALENs using the Golden Gate platform.Methods Molecular Biology,
1239:133-159.Cermak T,Dolye EL,Christian M,et al.,2011.Efficient design and assembly of custom TALEN
and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res,39:e82).The method identifies according to RVD
The rule (NI → A, HD → C, NN → G, NG → T, NK → G) of nucleotide and its all possible positions in TALE albumen, point
Do not construct to comprise and carry in the middle of the plasmid vector of 50 RVD unit modules, 5 last position RVD module plasmid vectors, 13 TALEN assemblings
Body and the library of 4 TALEN expression skeleton plasmid carriers.It is the target according to design that this GG-Vector library assembles the principle of TALEN carrier
Consecutive nucleotides forms, and chooses required different RVD unit carriers from library, produces many by specific II these carriers of type endonuclease digestion
Kind one to one, the sticky end pair of special coupling, be once simultaneously connected with 8-10 RVD unit.
In actual applications, owing to being limited by Golden Gate reaction efficiency, single TALEN assembles the maximum weight of RVD unit in reaction
Plural number mesh not can exceed that 10, therefore to assemble the TALEN carrier identifying 15-20bp target sequence, would have to assemble two length respectively
Less than the TALE sequence of 10 RVD repetitives, then they are coupled together by Golden Gate method.Complete TALEN assembling flow path
Have to be split as 2 independent stages, time-consumingly about 5 working days.This adds experiment undoubtedly and consumes, extends construction schedule, also
Significantly limit the high throughput applications of TALEN technology.
Summary of the invention
The present invention is directed to many deficiencies of existing GG-Vector TALEN assemble method, from the beginning devise " double RVD unit " (two-RVD unite)
TALEN packaging strategy.
The technical scheme is that a kind of double RVD unit module storehouses efficiently built for TALEN, double including 144 independently packed
RVD unit module, 8 single RVD unit modules and 24 last position RVD unit；
The double RVD unit module of described 144 is for identifying adjacent 2 base of combination in any；8 described single RVD unit modules divide
For M08 and M09 two groups, often 4 single RVD unit modules of group, M08 is to identify the base of the 15th during 16bp for target sequence, M09
Group is to identify the base of the 17th during 18bp for target sequence；
24 described last position RVD unit are divided into left group and right group, often group 12, and the last position RVD unit 3 ' end of left group merges Fok
The monomer I of I heterodimer, RVD unit module 3 ' end in position, right group end merges the monomer II of Fok I heterodimer；Left group and right side
Group includes 4 the last position RVD unit identifying target sequence the 15th bit base of 15bp length respectively, identifies the target sequence of 16bp and 17bp length
16th and 4 last position RVD unit of the 17th bit base, identify the target sequence the 18th and the 4 of 19 bit bases of 18bp and 19bp length
Position, individual end RVD unit.
Further, described double RVD unit modules, single RVD unit module two ends according to identifying that base site order in target sequence sets
Put end to end sticky end.
Further, described double RVD unit modules and single RVD unit module are placed on A/T cloning vehicle.
Further, in described last position RVD cell formation to carrier for expression of eukaryon, from 5 ' to 3 ', direction includes CaMV to Expression element successively
35S promoter, nuclear localization signal NLS and 5 ' end TALE, ccdB toxin gene, position, end RVD unit, and 3 ' end TALE and Fok I are different
The dimeric monomer in source I or monomer II.
Concrete, the double RVD unit module of described 144 has the nucleotide sequence as described in SEQ ID No.1～144.
Concrete, 8 described single RVD unit modules have the nucleotide sequence as described in SEQ ID No.143～152.
Concrete, 4 last position RVD unit of target sequence the 15th bit base of described identification 15bp length have such as SEQ ID No.153～156
Shown nucleotide sequence；Identify 4 last position RVD unit of the target sequence the 16th of 16bp and 17bp length and the 17th bit base have as
Nucleotide sequence shown in SEQ ID No.157～160；Identify target sequence the 18th and 4 last positions of 19 bit bases of 18bp and 19bp length
RVD unit has the nucleotide sequence as shown in SEQ ID No.161～164.
Present invention also offers the method using described module library to build TALEN, comprise the steps: to build one section of target sequence for 2
The expression vector of target sequence, the last position RVD unit of 1 expression vector uses left group, and the last position RVD unit of another 1 expression vector uses the right side
Side group；Identify 15,17, the expression vector of 19bp length target sequence choose 7,8,9 double RVD unit modules according to Base sequence in sequence,
With identification the 15th, the 17th, the last position RVD unit module of the 19th bit base；Identification 16, the expression vector root of 18bp length target sequence
Choose 7,8 double RVD unit modules according to Base sequence in sequence, identify the 15th, single RVD unit module of the 17th bit base, and know
Other 16th, the last position RVD unit module of the 18th bit base；Then by Golden Gate cloning one-step synthesis.
By two adjacent single RVD unit modules are connected, form double RVD unit modules of recognizable two nucleotide, exist simultaneously
The two ends design of double RVD unit modules can be finally building up to shape on suitable plasmid vector according to the end to end sticky end of site order
One is become to comprise 144 double RVD unit module libraries (Fig. 1).Double RVD unit modules identify rule and the institute of nucleotide according to RVD
Possible position is designed, and RVD unit modules as double in NI-NI can recognize that AA nucleotide, when AA base is positioned at target sequence the one or two
Time, double RVD unit module carriers of its correspondence are D01-AA:NI-NI (D01-01-AA)；When AA base is positioned at target sequence the three or four
During position, the RVD module carrier of its correspondence is D02-AA:NI-NI (D02-01-AA)；By that analogy, all possible target sequence
With the easy module carrier finding correspondence in 144 double RVD unit libraries.
In addition to 144 double RVD unit modules, this system further comprises 8 single RVD unit modules (Fig. 2).These single RVD unit
Module only one of which RVD repetitive, for target sequence be even length time penultimate RVD build.16bp target is identified as built one
The TALEN carrier of sequence, front 1～14bp selects suitable 7 carriers according to nucleotide combination of two in 144 double RVD unit libraries,
The RVD of the 15th just uses in single RVD unit module M08, then by Golden Gate reaction by these 7 double RVD unit moulds
Block, 1 single RVD unit module couple together with position, corresponding end RVD expression vector, are completed the TALEN of 16 RVD of needs
Expression vector.
Being assembled in TALEN expression vector for the ease of double RVD unit modules, this system also uses 24 left sides and position, end, right side RVD table
Reach carrier (Fig. 3).These position, end RVD expression vectors use plant constitutive promoter CaMV 35S promoter, and the regulation and control of Nos terminator
The expression of gene, its transcript regions contains TALE albumen n end and C terminal sequence, identifies that the last position RVD of last nucleotide of target sequence is heavy
Complex sequences, the sequence of nuclear localization signal NLS, it is easy to the ccdB toxin gene sequence of vector selection and for shearing the nuclease Fok of DNA
I sequence.Different skeleton expression vectors is adapted to identify left side and the assembling of right side TALEN of different length target sequence, completes the TALEN assembled
Protein expression vector can be imported in host cell by agriculture bacillus mediated or additive method easily.Wild type FokI activity shear constitution territory is homology
Dimeric structure, in actual application, in order to reduce non-specific cleavage, the FokI shear constitution territory that often about use, two sequences there are differences, just
Heterodimer is formed in intracellular translation after completing.Therefore, in this application by dimeric 2 monomers carrier construction respectively, containing each
The expression vector of monomer is for the same target sequence in target sequence or different target sequences.
In order to realize this pair of RVD unit module TALEN assembly system, by the way of synthetic, complete synthesis 144 double RVD are mono-
The sequence of modules (SEQ ID No.1:D01-01-AA to SEQ ID No.144:D09-16-TT) of unit, and it is building up to pUC57 load respectively
On body (purchased from Jin Sirui company), form double RVD unit module plasmid vector library.Same, 8 monomer RVD unit of synthetic
Sequence of modules (SEQ ID No.143:M08-01-A to SEQ ID No.152:M09-04-T), be building up to the most respectively on pUC57 carrier,
Form single RVD unit module plasmid vector library.Additionally, the left side that zhang etc. is reported and right side TALEN eukaryotic expression skeleton carrier
PZHY500 (EL), pZHY501 (ER) carry out transforming (Zhang Y, Zhang F, Li X, et al., 2013.Transcription activator-like
effector nucleases enable efficient plant genome engineering.Plant Physiology,161:20-27.).Plant composing type is opened
Mover CaMV 35S promoter (SEQ ID No.165 35S P) and Nos terminator (SEQ ID No.170Nos-T) are by Fusion PCR
Method be connected into respectively in above-mentioned two skeleton carrier, be used for regulating and controlling nuclear localization signal NLS and 5 ' end TALE (SEQ ID No.166
NLS+5 ' TALE), ccdB (SEQ ID No.167ccdB) and 3 ' end TALE and left side Fok I (Seq No.168 3 ' TALE+Fok I-L),
The expression of 3 ' ends TALE and right side Fok I (SEQ ID No.169 3 ' TALE+Fok I-R).Further 24 last position RVD of synthesis are repeated
Unit sequence (Seq NO EL/R07-01-Last A to Seq NO EL/R09-04-Last T) is connected into above-mentioned left side and the right side respectively by fusion DNA vaccine
In side transformation carrier, obtain 12 left side end position RVD-TALEN skeleton expression vectors as shown in Figure 3 and 12 right sides end position RVD-TALEN
Skeleton expression vector.
For target sequence length assembles more than the TALEN of 19bp, be equally useful the vector library of double RVD unit module, simply by
The fragment restriction less than 10 in GG reacts, needs to carry out two steps GG and has reacted assembling structure.But in actual applications, if unilateral
The a length of 19bp of TALEN, the length of left and right sides TALEN has just reached 38bp, and such target sequence long fragment be enough to deal with existing thing
The genome complexity planted, it is achieved specific recognition and the cutting to particular target site, it is to avoid phenomenon of missing the target produces.
Beneficial effects of the present invention: the invention discloses a kind of based on Golden Gate cloning, build TALEN expression vector by primary first-order equation
RVD unit module storehouse, this module library can be with targeting 15～19bp any DNA sequence.Based on this pair of RVD unit module library construction
The method of TALEN expression vector, effectively simplifies construction step, reduces experiment and consume, improve packaging efficiency, will build time-consuming shortening
To 1 working day.The module library utilizing the present invention can significantly optimize TALEN and assembles a process and reduce cost, convenient and swift, spends less,
Apply for the further genralrlization of TALEN technology and provide guarantee, and provide the foundation skeleton carrier for the high throughput applications of TALEN technology.
Accompanying drawing explanation
The permutation matrix of the underlying carrier (16 × 9=144) of Fig. 1 binary RVD module.144 binary RVD modules are in the way of 9 row 16 row
Distribution, is often classified as the position at double RVD place, the sequence of double two adjacent nucleotides corresponding to RVD of every behavior one.In figure, row represent double
2 bases that RVD unit combines position in target sequence, such as D01 represents that 2 bases that this pair of RVD unit combines are positioned at target sequence
1st, 2, D02 represents that 2 bases that this pair of RVD unit combines are positioned at the 3rd of target sequence the, 4, and by that analogy, D09 represents this pair
2 bases that RVD unit combines are positioned at the 17th of target sequence the, 18.In figure, row represents 2 base sequences and the knowledge that double RVD unit combines
The aminoacid of other 2 bases, such as in AA:NI-NI, AA represents that 2 base sequences that double RVD unit combines are that AA, NI-NI represent knot
Close the aminoacid of AA.If the carrier of first row the first row is D01-AA, when representing that being positioned at the one or two bit base of this TALEN is AA, its pair
RVD is NI-NI, and corresponding sequence is SEQ ID No.1:D01-AA；The carrier of the 4th row the third line is D04-AG, represents and is positioned at this TALEN
The seven or eight bit base when being AG, its couple of RVD is NI-NN, and corresponding sequence is SEQ ID No.51:D04-AG.CATG、GGAC、
CCAG, TGTT, TGCA, CGGT, GAAA, TCGA, GCTC: the end to end sticky end in double RVD unit module two ends；
NI-NI、NH-HD、NI-NN、NI-NG、HD-NI、HD-HD、HD-NN、HD-NG、NN-NI、NN-HD、NN-NN、NN-NG、
NG-NI, NG-HD, NG-NN, NG-NG: identify double RVD corresponding DNA sequences of continuous two different IPs thuja acids；Kan: kanamycin
Resistant gene.
The underlying carrier (4 × 2=8) of Fig. 2 monomer RVD module.8 single RVD modules arrange with M08 row and M09 row two, each a line of ACGT
The mode amounting to four row is distributed.M08 row for when target sequence a length of 16 time, the corresponding RVD of penultimate (the 15th) base selects；
M09 row for when target sequence a length of 18 time, the corresponding RVD of penultimate (the 17th) base selects.As built the long 16bp of target sequence,
And during TALEN carrier that the 15th is G, the monomer RVD module selected is M08-G, and corresponding sequence is SEQ ID No.147:M08-G.
GAAA, TCGA, GCTC: the end to end sticky end in single RVD unit module two ends；NI, HD, NN, NG: identify different monokaryon
The RVD corresponding DNA sequence of thuja acid；Kan: kalamycin resistance gene.
TALEN skeleton expression vector (4 × 3 × 2=24) of Fig. 3 end position RVD.24 skeleton expression vectors are divided into left side (EL) and right side
(ER) TALEN two arranges, and is respectively used to when TALEN carrier and right side TALEN carrier on the left of structure use.Each column (side) carrier basis again
Different length target sequence end position RVD identifies that the difference of DNA base is divided into 12 row, when EL/ER07 is used for target sequence a length of 15bp, and EL/ER08
During 16bp and 17bp a length of for target sequence, EL/ER09 is when target sequence a length of 18bp and 19bp.As built the long 16bp of target sequence,
When position, end is carrier on the left of the TALEN of G, the skeleton expression vector selected is EL08-G, and corresponding sequence is SEQ ID No.159:
EL/R08-Last G.35S-P: plant constitutive promoter CaMV 35S promoter；NLS+5 ' TALE: nuclear localization signal+TALE albumen N
Terminal sequence；CTAT, GAAA: cohesive end sequence；CcdB: be easy to the ccdB toxin gene sequence of vector selection；Last-NI: end position RVD
Sequence of modules；3 ' TALE+FokI-L/R:TALE PROTEIN C terminal sequences+for shearing the nuclease Fok I left/right sequence of DNA；Nos-T:
Nos terminator；Amp: ampicillin resistance gene.
Double RVD unit (two-RVD unite) the underlying carrier storehouse of Fig. 4 builds detection.A: the double RVD unit module plasmids as a example by D04 carry
Body PCR testing result, 1～16 represent the double RVD unit module of 16 D04, and M is molecular weight marker；B:8 monomer RVD unit module
Plasmid vector PCR testing result, 1～4 represent 4 M08 mono-RVD modules, and 5～6 represent 4 M09 mono-RVD modules, and M is molecular weight
Labelling；The TALEN skeleton expression vector AatII/AgeI double digestion testing result of C: the last position RVD as a example by 4 EL09,1～4 represent 4
Individual EL09 expresses skeleton carrier, and M is molecular weight marker.
The left side TALEN expression vector schematic diagram of Fig. 5 rice Os DEP1 gene.35S-P: plant constitutive promoter CaMV 35S promoter；
NLS+5 ' TALE: nuclear localization signal+TALE albumen n end sequence；OsDEP1-Left RVD Cluster: Oryza sativa L. DEP1 gene orientation editor's sequence
The left side RVD sequence that row are corresponding；3 ' TALE+FokI-L:TALE PROTEIN C terminal sequences+for shearing sequence on the left of the nuclease Fok I of DNA；
Nos-T:Nos terminator；Amp: ampicillin resistance gene；Ori: replication initiation.
The right side TALEN expression vector schematic diagram of Fig. 6 rice Os DEP1 gene.35S-P: plant constitutive promoter CaMV 35S promoter；
NLS+5 ' TALE: nuclear localization signal+TALE albumen n end sequence；OsDEP1-Left RVD Cluster: Oryza sativa L. DEP1 gene orientation editor's sequence
The left side RVD sequence that row are corresponding；3 ' TALE+FokI-R:TALE PROTEIN C terminal sequences+for shearing sequence on the right side of the nuclease Fok I of DNA
Row；Nos-T:Nos terminator；Amp: ampicillin resistance gene；Ori: replication initiation.
Fig. 7 target gene TALEN expression vector based on double RVD unit (two-RVD unite) library strategy builds and detection.TAL-L01、
TAL-R01 is two TALEN expression vector PCR results of OsDEP1 gene in table 2, the mono-bacterium colony of 2#, 4#, 5#, 6# of TAL-L01
All having amplified the target stripe (19RVD) of about 2000bp, the mono-bacterium colony of 2#, 3#, 4#, 5#, 6# of TAL-R01 has all amplified 1700bp
The target stripe (15RVD) of left and right；TAL-L02, TAL-R02 are two TALEN expression vector PCR of OsBADH2 gene in table 2
As a result, the mono-bacterium colony of 2#, 3#, 4#, 5#, 6# of TAL-L02 has all amplified the target stripe (17RVD) of about 1900bp；TAL-R02
6 single bacterium colonies all amplified the target stripe (16RVD) of about 1800bp；Illustrate that 4 TALEN carriers all successfully construct.
Fig. 8 target gene TALEN expression vector activity rating based on double RVD unit (two-RVD unite) library strategy.Pass through protoplasm
Body transient expression, detects that the shear active of different TALEN carrier, from 25% to 55%, may be used for the endogenous gene directed modification of plant.
Detailed description of the invention
Below by by the concrete embodiment explanation present invention, but these concrete embodiments be understood not to limitation of the present invention, right
Some details is modified within being still within protection domain.
The underlying carrier in 1 pair of RVD unit library of embodiment builds and detection
By the way of synthetic (synthesis of trust money Si Rui bio tech ltd), the module sequence of complete synthesis 144 double RVD unit
Row (SEQ ID No.1D01-AA to SEQ ID No.144D09-TT), and be building up to respectively on pUC57 carrier (purchased from Jin Sirui company),
Form double RVD unit module plasmid vector (Fig. 1).A series of plasmid vectors are directed respectively in escherichia coli DH5a bacterial strain by heat shock method,
Obtain double RVD unit module library, and by double RVD unit areas of the method each plasmid of amplification of bacterium colony PCR, verify the standard in this library
Really property.Now as a example by 16 carriers of D04-AA to D04-TT of D04 row, introduce its verification method.With D04-AA bacterium solution as template, accordingly
Oligonucleotide M13F and M13R be upstream and downstream primer, set up following PCR system: 10 × Taqbuffer5 μ L, dNTP
Mixture(10mM)5μL、M13F(SEQ ID No.171)(10μM)1μL、M13R(SEQ ID No.172)(10μM)1μL、D04-AA1μL、
Taq1μL、ddH2O36μL。
PCR reaction condition is: denaturation 95 DEG C, 3min；Degeneration 94 DEG C, 20s；Anneal 56 DEG C, 20s；Extending 72 DEG C, 15s, 33 are followed
Ring, extends 72 DEG C, 3min.Verify that the PCR system of other 15 carriers is identical with this with reaction condition, only plasmid template and upstream and downstream primer
Different.Gained PCR result electrophoresis is as shown in Figure 4 A: swimming lane 1～16 is respectively the double RVD unit modules of 16 D04, and all amplifications obtain 200bp
The target stripe of left and right, is consistent with expection, and 16 double RVD unit module plamid vector construction successes of D04 are described.
Same, synthetic (synthesis of trust money Si Rui bio tech ltd) sequence of modules (the SEQ ID of 8 monomer RVD unit
No.145M08-A to Seq No.152M09-T), it is building up to the most respectively on pUC57 carrier, forms single RVD unit module plasmid vector library
(Fig. 2).These 8 single RVD module plasmid vectors are directed respectively in escherichia coli DH5a bacterial strain by heat shock method, obtain single RVD module
Library, and single RVD region of each plasmid is expanded by the method for bacterium colony PCR, verify the accuracy in this library.PCR system and reaction condition
Identical with double RVD unit modules, only bacterium solution template is different (primer ibid M13F and M13R).Gained PCR result electrophoresis such as Fig. 4 B
Shown in: swimming lane 1～4 represents 4 M08 mono-RVD modules, and 5～6 represent that 4 M09 mono-RVD modules, all amplification obtain about 100bp
Target stripe, is consistent with expection, and 8 single RVD module plamid vector construction successes of M08 and M09 are described.
In order to build the TALEN skeleton expression vector of 24 last position RVD, the left side that Zhang etc. is reported and right side TALEN eukaryotic expression
Skeleton carrier pZHY500 (EL), pZHY501 (ER) transform.By plant constitutive promoter CaMV 35S promoter (SEQ ID No.165
35S P) and Nos terminator (SEQ ID No.170Nos-T) be connected into respectively in above-mentioned two skeleton carrier by the method for fusion DNA vaccine, be used for
Regulation and control nuclear localization signal NLS and 5 ' end TALE (SEQ ID No.166NLS+5 ' TALE), ccdB (SEQ ID No.167ccdB) and 3 ' ends
TALE and left side Fok I (SEQ ID No.168 3 ' TALE+Fok I-L), 3 ' end TALE and right side Fok I (SEQ ID No.169 3 ' TALE+Fok
I-R) expression.By 12 last position RVD repetitive sequences of synthesis, (the last position RVD of synthesis is 12, in corresponding left side further
In skeleton and right side skeleton carrier, this partial sequence is on all four, and the FokI simply connected is different) (SEQ ID No.153EL/R07-Last A
To SEQ ID No.164EL/R09-Last T) it is connected into respectively in above-mentioned left side and right side transformation carrier by fusion DNA vaccine, obtain as shown in Figure 3
12 left side end position RVD-TALEN skeleton expression vectors and position, end, 12 right sides RVD-TALEN skeleton expression vector.Fusion DNA vaccine anti-
Answer system as follows: 10 × KODbuffer5 μ L, dNTP Mixture (10mM) 5 μ L, Primer F (SEQ ID No.173, SEQ ID No.174
Or SEQ ID No.175) (10 μMs) 1 μ L, Primer R (SEQ ID No.176) (10 μMs) 1 μ L, pZHY500 (EL)/pZHY501 (ER)
1 μ L, KOD1 μ L, ddH2O36μL.PCR reaction condition is: denaturation 95 DEG C, 3min；Degeneration 94 DEG C, 20s；Anneal 56 DEG C, 20s；
Extend 68 DEG C, 15s, 33 circulations, extend 68 DEG C, 3min.
The skeleton expression vector built for checking is the most correct, by AatII/AgeI (being the fast enzyme of Thermo Scientific.Fermentas) double enzymes
Cut into row checking.As a example by 4 carriers of EL09, set up enzyme action system as follows: 10 × Fast Digest Buffer5 μ L, AatII1 μ L, AgeI1 μ L,
Plasmid (plasmid) DNA 20 μ L, ddH2O 23μL.After 37 DEG C of enzyme action 30min, electrophoresis result as shown in Figure 4 C, 4 EL09 bones
Frame expression vector all cuts out the TALEN expression cassette purpose band of treaty an about 4000bp, is consistent with expection, and 24 last positions of EL and ER are described
The success of RVD skeleton expression vector establishment.
Above-mentioned PCR reaction agents useful for same is Japan and spins (Shanghai) bio tech ltd KOD-Plus-Neo test kit.
Embodiment 2 assembles method and the rule of TALEN based on double RVD unit modules
During double RVD unit module tissue T ALEN of application, first select target sequence (length is typically 15～20bp), then design corresponding RVD,
Suitable carrier library is selected, according to Golden again from double RVD unit module storehouses, single RVD module library and position, end RVD skeleton expression vector
Gate reaction carries out building.
According to RVD, double RVD unit modules identify that the rule of nucleotide and all possible position are designed, RVD unit as double in NI-NI
Recognizable AA nucleotide, when AA base is positioned at target sequence the one or two, the RVD module carrier of its correspondence is D01-AA:NI-NI
(D01-01-AA)；When AA base is positioned at target sequence the three or four, the RVD module carrier of its correspondence is D02-AA:NI-NI
(D02-01-AA)；By that analogy, the mould finding correspondence in 144 double RVD unit libraries that all possible target sequence all can be easy
Block carrier.And single RVD unit module only one of which RVD repetitive, for target sequence be even length time penultimate RVD build.
As built a TALEN carrier identifying 16bp target sequence, front 1～14bp according to nucleotide combination of two in 144 double RVD unit libraries
Selecting suitable 7 carriers, the RVD of the 15th just uses in monomer RVD unit module M08, then by GG reaction by this 7
Individual binary carrier, 1 monomeric carrier couple together with position, corresponding end RVD expression vector, are completed the TALEN of 16 RVD of needs
Expression vector.
Table 1 lists different length target sequence corresponding RVD module and selects rule, as assembled the TALEN all identifying 15bp target sequence a pair,
Need according to DNA sequence base composition different, double RVD unit module libraries are selected the corresponding plasmid vector of D01 to D07 row, due to
Skeleton expression vector has contained end position RVD, therefore need not select carrier in single RVD unit module, selects corresponding left side and position, end, right side bone
Frame expression vector, it is possible to assemble complete left side and the TALEN carrier of 15 RVD in right side.
Table 1 TALEN based on double RVD unit modules assembles rule
If same assembles the TALEN identifying 16bp target sequence a pair, according to DNA sequence base composition difference, except selecting double RVD unit
In module library outside the corresponding plasmid vector of D01 to D07 row, in addition it is also necessary to select one according to the 15th nucleotide in single RVD unit module
Corresponding M08 carrier, adds corresponding left side and position, end, right side RVD skeleton expression vector, it is possible to assemble complete left side and right side 16
The TALEN carrier of individual RVD.
Assembling rule for the TALEN of target sequence length 17～19bp is similar with above 15bp, 16bp, is only pair RVD unit modules
Plasmid quantity increase one by one.
For target sequence length assembles more than the TALEN of 19bp, be equally useful the vector library of double RVD unit module, simply by
The fragment restriction less than 10 in GG reacts, needs to carry out two steps GG and has reacted assembling structure.But in actual applications, if unilateral
The a length of 19bp of TALEN, the length of left and right sides TALEN has just reached 38bp, and such target sequence long fragment be enough to deal with existing thing
The genome complexity planted, it is achieved specific recognition and the cutting to particular target site, it is to avoid phenomenon of missing the target produces.
The assembling of embodiment 3 plant endogenous genes based on double RVD unit modules directed modification TALEN carrier and Activity determination
1, the design of plant endogenous genes directed modification TALEN carrier and assembling
Utilize double RVD unit module that plant endogenous genes is oriented for inspection to shear and the efficiency of sudden change, have selected rice varieties Japan fine
OsDEP1(GenBank NO.:FJ039904)、OsBADH2(GenBank NO.:KT993490)、OsCKX2(GenBank NO.:
AB205193) TaMLO (GenBank NO.:KF009556) gene of gene and Semen Tritici aestivi is as target gene, has separately designed 4 couples of TALEN
Its corresponding DNA target sequence is carried out specific cleavage.Target gene title, target site DNA sequence (SEQ ID No.177～184), correspondence
TALEN title, RVD number, RVD sequence, and should select in double RVD unit libraries corresponding binary, monomer and end position
RVD plasmid vector numbering is listed the most in table 2.
Table 2 target gene TALEN expression vector based on double RVD unit module packaging strategies builds situation
2, the TALEN vector construction of endogenous targets gene and Activity determination
With TAL-L01 (Fig. 5), the TAL-R01 (Fig. 6) of OsDEP1 gene, and TAL-L02, TAL-R02 of OsBADH2 gene
As a example by the structure of four TALEN carriers.Based on above-mentioned TALEN design and packaging strategy, with reference to method (Cermak T, the Dolye of Cermak etc.
EL,Christian M,et al.,2011.Efficient design and assembly of custom TALEN and other TAL effector-based
Constructs for DNA targeting.Nucleic Acids Res, 39:e82), by Golden Gate reaction by different RVD and end position RVD
And skeleton expression vector couples together.Connection product, in heat shock method imports escherichia coli DH5a, paves plate incubated overnight.Select 6 lists respectively
Clone carry out bacterium colony PCR detection, result as shown in Figure 5: the mono-bacterium colony of 2#, 4#, 5#, 6# of TAL-L01 expression vector has all amplified 2000bp
The target stripe (19RVD) of left and right；The mono-bacterium colony of 2#, 3#, 4#, 5#, 6# of TAL-R01 expression vector has all amplified about 1700bp
Target stripe (15RVD)；The mono-bacterium colony of 2#, 3#, 4#, 5#, 6# of TAL-L02 expression vector has all amplified the target stripe of about 1900bp
(17RVD)；6 single bacterium colonies of TAL-R02 expression vector have all amplified the target stripe (16RVD) of about 1800bp.Result above table
Bright 4 TALEN expression vector all successfully construct.
The TALEN construction method of other two gene OsCKX2, TaMLO is identical with this, and also passes through bacterium colony PCR detection, it was demonstrated that corresponding
TAL-L03, TAL-R03 and TAL-L04, TAL-R04 all successfully constructs.
In order to verify the TALEN carrier shear active of structure, by every 2 carriers (one be end position RVD be left group, a last position RVD
It is right group) import in protoplasm somatocyte as one group, after 2 days dark culturing, the blue light of wavelength 450～490nm under fluorescence microscope
Excite the GFP fluorescence of rear observation of cell, add up fluorecyte ratio, obtain specific cleavage Activity Results such as Fig. 6 of above-mentioned 8 TALEN carriers
Shown in.8 TALEN carriers all show TALEN shear active, and its activity level, from 25% to 55%, shows above-mentioned TALEN
Carrier may be incorporated in next step plant directed modification research.
Claims (8)
1. the double RVD unit module storehouses efficiently built for TALEN, it is characterised in that: include independently packing
144 double RVD unit modules, 8 single RVD unit modules and 24 last position RVD unit；
The double RVD unit module of described 144 is for identifying adjacent 2 base of combination in any；8 described single RVD
Unit module is divided into M08 and M09 two groups, often 4 single RVD unit modules of group, and M08 is used for knowing when target sequence is 16bp
The base of other 15th, M09 group is to identify the base of the 17th during 18bp for target sequence；
24 described last position RVD unit are divided into left group and right group, often group 12, the last position RVD unit of left group
3 ' ends merge the monomer I of Fok I heterodimer, and RVD unit module 3 ' end in position, right group end merges Fok I heterodimeric
The monomer II of body；Left group and right group include 4 the last position RVD identifying target sequence the 15th bit base of 15bp length respectively
Unit, identifies target sequence the 16th and 4 last position RVD unit of the 17th bit base of 16bp and 17bp length, identifies
The target sequence of 18bp and 19bp length the 18th and 4 last position RVD unit of 19 bit bases.
2. the double RVD unit module storehouses efficiently built for TALEN as claimed in claim 1, it is characterised in that: institute
Double RVD unit modules of stating, single RVD unit module two ends according to identifying that base site order in target sequence arranges head
The sticky end that tail is connected.
3. the double RVD unit module storehouses efficiently built for TALEN as claimed in claim 1 or 2, it is characterised in that:
Described double RVD unit modules and single RVD unit module are respectively placed on A/T cloning vehicle.
4. the double RVD unit module storehouses efficiently built for TALEN as described in any one of claims 1 to 3, its feature
It is: in described last position RVD cell formation to carrier for expression of eukaryon, from 5 ' to 3 ', direction includes CaMV to Expression element successively
35S promoter, nuclear localization signal NLS and 5 ' end TALE, ccdB toxin gene, position, end RVD unit, and 3 ' end TALE
And the monomer I of Fok I heterodimer or monomer II.
5. the double RVD unit module storehouses efficiently built for TALEN as described in any one of Claims 1 to 4, its feature
It is: the double RVD unit module of described 144 has the nucleotide sequence as described in SEQ ID No.1～144.
6. the double RVD unit module storehouses efficiently built for TALEN as described in any one of Claims 1 to 5, its feature
It is: 8 described single RVD unit modules have the nucleotide sequence as described in SEQ ID No.143～152.
7. the double RVD unit module storehouses efficiently built for TALEN as described in any one of claim 1～6, its feature
It is: 4 last position RVD unit of target sequence the 15th bit base of described identification 15bp length have such as SEQ ID
Nucleotide sequence shown in No.153～156；Identify the target sequence the 16th and the 4 of the 17th bit base of 16bp and 17bp length
Position, individual end RVD unit has the nucleotide sequence as shown in SEQ ID No.157～160；Identify the target of 18bp and 19bp length
4 last position RVD unit of sequence the 18th and 19 bit bases have the nucleotide sequence as shown in SEQ ID No.161～164.
8. use the method that module library described in claim 1～7 builds TALEN, it is characterised in that: it is right to comprise the steps:
One section of target sequence builds the expression vector for 2 target sequences, and the last position RVD unit of 1 expression vector uses left group,
The last position RVD unit of another 1 expression vector uses right group；Identify 15,17, the expression vector root of 19bp length target sequence
Choose 7,8,9 double RVD unit modules according to Base sequence in sequence, and identify the 15th, the 17th, the 19th alkali
The last position RVD unit module of base；Identify 16, the expression vector of 18bp length target sequence chooses 7 according to Base sequence in sequence,
8 double RVD unit modules, identify the 15th, single RVD unit module of the 17th bit base, and identification the 16th, the
The last position RVD unit module of 18 bit bases；Then by Golden Gate cloning one-step synthesis.
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