CN103319574A - Separated polypeptide and application thereof - Google Patents
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- CN103319574A CN103319574A CN2013100512804A CN201310051280A CN103319574A CN 103319574 A CN103319574 A CN 103319574A CN 2013100512804 A CN2013100512804 A CN 2013100512804A CN 201310051280 A CN201310051280 A CN 201310051280A CN 103319574 A CN103319574 A CN 103319574A
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Abstract
The invention relates to a separated polypeptide and an application thereof. The polypeptide comprises: a plurality of repeated amino acid sequence, and the repeated amino acid sequence is: LTPDQVVAIASX1X2GGKQALETVQRLLPVLCQAHG, wherein X1 is H or N, X2 is one selected from I, L, M, W, C, T, P,H, S, N, E, Q, H, K and R. The polypeptide can specifically recognize a base.
Description
Technical field
The present invention relates to biological technical field, more specifically, the present invention relates to isolated polypeptide and application thereof.
Background technology
TALEs(Transcription Activator Like Effectors, transcriptional activation increment effector) mainly is present in plant pathogen (Xanthomonas).When the pathogen infection plant, germ can will comprise that by the III type excretory system of himself the series of effects molecule of TALE is injected in the vegetable cell.These effector molecules are by affecting the signal transmission of host cell, and the modes such as genetic expression assist germ further to increase.TALE then is a class maximum in these effector molecule albumen, the same functionating of the transcription activator of its similar plants.
Yet the at present research about TALE still remains to be improved.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or provides at least a kind of useful commerce to select.For this reason, one object of the present invention is to propose a kind of effectively polypeptide of unity identification DNA base that has.
In a first aspect of the present invention, the present invention proposes a kind of isolated polypeptide, it is characterized in that, described polypeptide comprises:
A plurality of repetition aminoacid sequences, described repetition aminoacid sequence is: LTPDQVVAIASX
1X
2GGKQALETVQRLLPVLCQAHG,
Wherein,
X
1Be H or N,
X
2For be selected from I, L, M, W, C, T, P, H, S, N, E, Q, H, K and R one of.
The contriver finds effectively specific recognition base of this polypeptide.Particularly, by changing X
1X
2The amino-acid residue type, can change the base of identifying.Concrete, be summarized as follows shown in the table 1:
Table 1
Need to prove, adopt in this article single English alphabet to represent amino acid, specifically represent as follows: A, L-Ala; C, halfcystine; D, aspartic acid; E, L-glutamic acid; F, phenylalanine; G, glycine; H, Histidine; I, Isoleucine; K, Methionin; L, leucine; M, methionine(Met); N, l-asparagine; P, proline(Pro); Q, glutamine; R, arginine; S, Serine; T, Threonine; V, L-Ala; W, tryptophane; Y, TYR.
According to embodiments of the invention, aforementioned polypeptides can also have following additional technical feature:
According to one embodiment of present invention, described polypeptide can comprise at least 10 described repetition aminoacid sequences.Preferably, described polypeptide comprises 20 described repetition aminoacid sequences.Thus, can further improve the efficient of described polypeptid specificity identification base.
According to one embodiment of present invention, described polypeptide may further include: dna modification enzyme domain structure territory.Thus, can after polypeptide is identified base, dna sequence dna be modified.
In a second aspect of the present invention, the present invention proposes a kind of oligonucleotide of separation.According to embodiments of the invention, the described oligonucleotide coding foregoing polypeptide of can encoding.Preferably, the sequence of described oligonucleotide is be selected from following sequence at least a:
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTCTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：1），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATACAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：2），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATACAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：3），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：4），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTACGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：5），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTACGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：6），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：7），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：8），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：9），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCTCGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：10），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCTCGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：1），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATATGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：1），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATATGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：12），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATGAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：13），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATGAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：14），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTGTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：15），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTGTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：16），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTGGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：17），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTGGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：18），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAGAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：?19），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAGAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：21），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：22），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：23），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：24），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：25），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATATAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：26），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATATAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：27）。
Thus, can further improve the efficient of utilizing the oligonucleotide coded polypeptide.
In a third aspect of the present invention, the present invention proposes a kind of expression vector.According to embodiments of the invention, this expression vector comprises foregoing oligonucleotide.Thus, utilize this expression vector, can effectively express foregoing polypeptide.
In a fourth aspect of the present invention, the present invention proposes a kind of method that changes cellular genome.According to embodiments of the invention, the method comprises: described cell is comprised or express foregoing polypeptide.Thus, by polypeptid specificity identification base, can further modify the base of specific site in the cellular genome, thus the genome of change cell.According to embodiments of the invention, can be by foregoing expression vector be incorporated in the cell.Thereby further improve the efficient of express polypeptide, the final efficient that changes cellular genome that improves.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 has shown the crystalline structure according to the dHax3 of the embodiment of the invention, and wherein A has represented that at the crystalline structure that does not exist in the DNA situation, B represents the crystalline structure of dHax3 and DNA mixture.
Fig. 2 has shown the molecular basis according to RVD specific recognition DNA among the dHax3 of the embodiment of the invention.Wherein, A has represented that the side chain of RVD points among the dHax3; B has represented that first amino acid is stablized loop zone conformation by hydrogen bond among the RVD; C has represented among the RVD second amino acid and DNA base direct interaction; D has represented among the molecule A to be the conformation of the RVD loop of NG; E has represented among the molecule B to be the conformation of the RVD loop of NG.
Fig. 3 has shown the crystalline structure schematic diagram that contains NI and two kinds of RVD of NN.Wherein, A has represented dHax3 (S505I) and the RVD of dTALE and the dna sequence dna of identification; B has represented dHax3 (S505I) and dTALE structure alignment; C has represented NI, NN and DNA base interaction schematic diagram.
Fig. 4 has shown the crystalline structure of zif268 and DNA mixture.Wherein, A has represented zif268 and DNA mixture one-piece construction schematic diagram; B has represented second zinc fingers schematic diagram in the zif268 albumen.The purple ball represents zinc atom.In conjunction with a zinc atom, zinc atom has played the effect of stabilize proteins conformation at this by coordinate bond for two halfcystines and two Histidines; C has represented amino acid and DNA base interaction schematic diagram in the zif268 albumen; D has represented to be used for the zif268 protein sequence of crystallization, demonstrates 3 Zinc finger domains.Each structural domain is comprised of 25 amino acid; E has represented to be used for the dna sequence dna of crystallization.
Fig. 5 has shown according to an embodiment of the invention crystalline structure schematic diagram.
Embodiment
The below describes embodiments of the invention in detail.These embodiment are exemplary, are intended to for explanation the present invention, and can not be interpreted as limitation of the present invention.
The present invention is based on contriver's following discovery and finishes:
TAL effectors has very high sequence homology.There is a structural domain to be identified by three type excretory systems at the nitrogen end, assists TAL effectors to enter in the vegetable cell by three type excretory systems; Carbon teminal has a nuclear localization signal (NLS) and a transcriptional activation domain (AAD); Tandem repetitive sequence in the middle of being positioned at is DNA binding domains (TAL Repeat Region).This is a brand-new DNA binding domains, does not have sequence homology with all known DNA binding domainss.The series connection repeating unit sequence of different TAL effectors is almost completely consistent.Most tandem repetitive sequence has 34 amino-acid residues.In each tumor-necrosis factor glycoproteins, only have the 12nd and 13 amino-acid residue to change greatly, therefore be named as RVD(repeat variable diresidue).Exactly because but the specific DNA sequence of TALE albumen identification and assembleability flexibly, TALE has been widely used in biological technical field.But people also and do not know that TALE is specific recognition DNA how.Referring to Fig. 1, the contriver has studied the high-resolution crystalline structure of TALE albumen dHax3 under two kinds of different states, has explained how specific recognition DNA of TALE.For follow-up research provides structure theoretical basis.
As depicted in figs. 1 and 2, in the crystalline structure of dHax3 and DNA mixture, first amino acid Histidine (H) or l-asparagine (N) among the RVD do not form direct interaction with the DNA base; On the contrary, it forms interaction of hydrogen bond by the O atom of the L-Ala (A8) in hydrogen bond and the same repeating unit.The effect of stable RVD loop has been played in the interaction of this hydrogen bond, and is extremely important to deputy amino acid specific recognition DNA base among the RVD.The more important thing is that second amino-acid residue and DNA base among the RVD form direct interaction, determine that TAL effector identifies the DNA base specifically.When amino-acid residue was aspartic acid (D), the carboxyl oxygen of aspartic acid can be by the amino interaction of hydrogen bond that directly forms of cytosine(Cyt) among hydrogen bond and the DNA; When amino-acid residue is Serine when being S, the N7 in the Serine in hydroxyl and the VITAMIN B4 forms direct interaction.Owing to same N7 atom being arranged in the guanine, the contriver infers that Serine identification guanine may be to form identical hydrogen bond and interact.Had Van der Waals force between glycine and the thymus pyrimidine methyl and interact, and any other there was the amino acid whose appearance of side chain all can cause conflicting between amino acid side chain and the thymus pyrimidine methyl this moment.By further Crystallographic Study, the contriver has explained that also NI and NN are (CONSTRUCTED SPECIFICATION of RVD and DNA base as shown in Figure 4) of specific recognition DNA base A and G how.N(l-asparagine wherein) form interaction of hydrogen bond by the two key Sauerstoffatoms on amino and 6 C atoms of guanine, and the N(l-asparagine) can identify the DNA bases adenine by similar interaction of hydrogen bond equally; And the I(Isoleucine) more special since with the side chain of Isoleucine be hydrophobic, the interaction of it and VITAMIN B4 can only interact to realize by Van der Waals force.In addition, the contriver analyzes the crystalline structure of TALE and DNA mixture and finds, TALE is the specific identification of the mode DNA base with " base read-out in DNA major groove ".And known zinc finger protein also is to identify in the same way DNA(as shown in Figure 4).And in zinc finger protein, amino-acid residue is particularly important to the identification of DNA bases G.
On the basis of above-mentioned research, the contriver has finished the present invention.
In a first aspect of the present invention, the present invention proposes a kind of isolated polypeptide, it is characterized in that, described polypeptide comprises:
A plurality of repetition aminoacid sequences, described repetition aminoacid sequence is: LTPDQVVAIASX
1X
2GGKQALETVQRLLPVLCQAHG,
Wherein,
X
1Be H or N,
X
2For be selected from I, L, M, W, C, T, P, H, S, N, E, Q, H, K and R one of.
The contriver finds effectively specific recognition base of this polypeptide.Particularly, by changing X
1X
2The amino-acid residue type, can change the base of identifying.Concrete, be summarized as follows shown in the table 1:
Table 1
Need to prove, adopt in this article single English alphabet to represent amino acid, specifically represent as follows: A, L-Ala; C, halfcystine; D, aspartic acid; E, L-glutamic acid; F, phenylalanine; G, glycine; H, Histidine; I, Isoleucine; K, Methionin; L, leucine; M, methionine(Met); N, l-asparagine; P, proline(Pro); Q, glutamine; R, arginine; S, Serine; T, Threonine; V, L-Ala; W, tryptophane; Y, TYR.
According to embodiments of the invention, employed a plurality of finger is more than two in this article, and according to one embodiment of present invention, described polypeptide can comprise at least 10 described repetition aminoacid sequences.Preferably, described polypeptide comprises 20 described repetition aminoacid sequences.Thus, can further improve the efficient of described polypeptid specificity identification base.
According to one embodiment of present invention, described polypeptide may further include: dna modification enzyme domain structure territory.Thus, can after polypeptide is identified base, dna sequence dna be modified.
In a second aspect of the present invention, the present invention proposes a kind of oligonucleotide of separation.According to embodiments of the invention, the described oligonucleotide coding foregoing polypeptide of can encoding.Preferably, the sequence of described oligonucleotide is be selected from following sequence at least a:
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTCTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：1），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATACAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：2），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATACAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：3），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：4），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTACGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：5），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTACGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：6），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：7），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：8），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：9），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCTCGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：?10），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCTCGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：11），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATATGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：12），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATATGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：13），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATGAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：14），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATGAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：15），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTGTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：16），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTGTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：17），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTGGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：18），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTGGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：19），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAGAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：20），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAGAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：21），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：?22），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：23），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：24），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：25），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATATAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：26），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATATAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：27）。Thus, can further improve the efficient of utilizing the oligonucleotide coded polypeptide.
In a third aspect of the present invention, the present invention proposes a kind of expression vector.According to embodiments of the invention, this expression vector comprises foregoing oligonucleotide.Thus, utilize this expression vector, can effectively express foregoing polypeptide.
In a fourth aspect of the present invention, the present invention proposes a kind of method that changes cellular genome.According to embodiments of the invention, the method comprises: described cell is comprised or express foregoing polypeptide.Thus, by polypeptid specificity identification base, can further modify the base of specific site in the cellular genome, thus the genome of change cell.According to embodiments of the invention, can be by foregoing expression vector be incorporated in the cell.Thereby further improve the efficient of express polypeptide, the final efficient that changes cellular genome that improves.
Because but the identification of the specific DNA sequence of TALE albumen and assembleability flexibly are applied to biological technical field increasingly extensively.The present invention has proved the characteristic of TALE protein binding DNA, and the nature that manually designs exists less or non-existent RVD to have equally the recognition capability of DNA base specific, thereby allow the application of TALE become more flexible, the TALE that people are used in TALE uses has had more selectivity.
Below with reference to specific embodiment, the present invention will be described, need to prove, these embodiment only are illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means that adopts among the embodiment is well known to those skilled in the art can carry out with reference to " molecular cloning experiment guide " third edition or related products, and the reagent that adopts and product also are and can commercial obtain.Various processes and the method do not described in detail are ordinary methods as known in the art, the source of agents useful for same, trade(brand)name and be necessary to list its moiety person, all when occurring first, indicate, thereafter used identical reagent if no special instructions, all identical with the content of indicating first.
(1) experiment material
1.DNA in conjunction with albumen dHax3
Hax3 is one of TALE protein family member, and its RVD sequence and the most desirable DNA recognition sequence are as shown in table 2 below:
Table 2
RVD by design and assembly Hax3 has obtained to identify the dHax3 (designed Hax3) of dna sequence dna as shown in table 3 below (in this sequence of code displaying chain DNA only):
Table 3
DHax3 obtains by full gene is synthetic, and sequence is as follows:
ATGGACCCAATACGAAGCAGAACGCCATCACCAGCTAGGGAACTTCTCTCTGGACCACAGCCTGATGGAGTTCAGCCAACTGCAGATCGAGGTGTTTCTCCGCCAGCCGGTGGCCCTTTAGATGGTCTCCCAGCAAGAAGAACAATGTCCCGTACCAGACTCCCAAGTCCCCCTGCCCCGTCGCCAGCCTTTTCAGCTGACTCCTTCTCTGATCTTCTTAGGCAATTTGACCCTTCTCTTTTCAATACATCCCTTTTCGATTCACTTCCTCCTTTCGGCGCACATCATACTGAGGCAGCCACCGGCGAATGGGACGAAGTCCAAAGTGGTTTAAGGGCAGCTGATGCTCCACCACCGACGATGAGAGTCGCTGTTACCGCCGCACGTCCTCCTAGAGCCAAGCCAGCCCCTAGAAGACGAGCTGCGCAACCCTCCGATGCAAGCCCTGCAGCTCAAGTAGACCTTCGAACACTAGGTTACTCCCAGCAACAACAAGAAAAAATAAAGCCAAAGGTTAGATCTACAGTTGCACAACATCACGAAGCCCTAGTCGGACACGGATTTACACATGCTCATATCGTGGCTCTTTCACAACATCCTGCAGCTCTTGGAACAGTCGCTGTCAAATATCAGGATATGATTGCTGCATTGCCAGAAGCTACTCACGAAGCTATCGTCGGAGTTGGGAAACAATGGTCAGGCGCAAGAGCATTAGAGGCGCTTCTCACCGTAGCTGGTGAATTACGAGGTCCTCCACTCCAATTGGATACTGGGCAATTATTAAAAATCGCTAAACGAGGTGGAGTCACTGCTGTCGAAGCCGTTCATGCATGGCGTAACGCTCTCACGGGCGCACCACTAAACCTTACTCCTGAACAGGTTGTCGCAATAGCTTCACATGATGGCGGAAAACAAGCTCTTGAAACAGTGCAACGTCTCCTTCCCGTCCTCTGTCAGGCTCACGGATTGACTCCTCAGCAGGTCGTCGCAATTGCATCACATGATGGAGGCAAACAAGCTTTAGAAACAGTACAAAGACTATTGCCCGTTCTTTGCCAAGCGCATGGGTTAACTCCCGAACAAGTCGTTGCCATTGCAAGTCACGACGGAGGTAAACAAGCTCTCGAAACGGTTCAAGCACTTTTACCCGTTCTCTGTCAAGCACATGGACTCACACCTGAACAAGTAGTTGCTATCGCATCGAATGGAG?GTGGAAAACAAGCACTGGAAACTGTACAAAGACTTTTGCCAGTTTTATGTCAAGCGCACGGTCTTACTCCTCAACAAGTTGTCGCCATTGCCTCTAACGGTGGTGGAAAACAAGCTCTTGAAACTGTCCAGAGACTTCTGCCCGTTCTATGTCAGGCTCATGGGCTAACCCCTCAACAGGTTGTTGCAATCGCATCTAATGGAGGAGGAAAACAAGCTTTAGAAACTGTCCAACGACTACTGCCCGTTCTCTGCCAAGCACACGGACTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTCTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGATTGACCCCACAACAGGTCGTAGCAATCGCATCTAATGGAGGTGGTAAGCAAGCTCTAGAAACGGTACAAAGATTACTTCCCGTGCTTTGTCAAGCTCATGGACTCACTCCTCAACAAGTGGTCGCTATTGCAAGTCATGATGGTGGAAAGCAAGCACTAGAAACCGTCCAACGACTCCTTCCTGTTCTCTGTCAAGCACATGGTCTTACGCCCGAACAAGTTGTTGCTATAGCTTCGAACGGAGGTGGAAAACAAGCTCTCGAAACCGTCCAAAGGCTCCTCCCAGTACTTTGCCAAGCACATGGATTAACCCCTGAGCAAGTAGTTGCAATTGCCTCGCACGACGGAGGAAAGCAAGCATTAGAAACTGTTCAGAGACTTTTGCCTGTCCTGTGTCAAGCCCACGGTCTAACACCACAACAAGTCGTCGCAATCGCTAGTAATGGAGGAGGTAGACCTGCATTGGAGTCGATAGTCGCACAACTATCACGACCTGATCCCGCTCTTGCAGCATTGACAAACGATCATTTAGTCGCACTTGCATGTTTAGGAGGACGACCAGCACTTGATGCCGTTAAGAAAGGACTACCGCACGCCCCTGCATTGATTAAAAGAACAAACAGACGAATCCCGGAGAGAACTTCACATCGTGTAGCCGATCATGCTCAAGTCGTAAGAGTTTTGGGTTTCTTCCAATGTCATTCCCACCCAGCTCAAGCTTTTGACGATGCAATGACTCAATTTGGAATGAGTAGACATGGACTCCTGCAATTATTTCGAAGGGTCGGAGTTACAGAGCTCGAAGCCAGGTCAGGAACGCTGCCCCCCGCATCTCAACGATGGGATAGAATTCTCCAAGCCTCTGGAATGAAAAGAGCTAAACCTTCACCAACGTCCACACAAACACCAGACCAAGCTTCTCTCCACGCTTTTGCCGACTCACTAGAGAGAGATCTAGATGCACCGTCACCTATGCATGAAGGAGACCAAACAAGAGCCTCTTCAAGAAAACGTTCTCGTTCTGATAGAGCTGTCACTGGACCTTCCGCCCAACAATCTTTCGAAGTCCGAGTTCCTGAGCAACGAGATGCCCTACACCTGCCTTTGCTTTCTTGGGGAGTTAAGCGACCACGTACTAGAATTGGTGGACTACTCGATCCAGGTACACCAATGGATGCTGATCTCGTTGCTTCCTCTACCGTAGTATGGGAGCAAGACGCAGACCCCTTCGCTGGAACTGCTGACGATTTCCCAGCCTTTAACGAGGAAGAATTGGCTTGGTTAATGGAACTTCTACCGCAATGA（SEQ?ID?NO：28）。
What be used for protein crystallization experiments is the truncate albumen of dHax3.Truncate albumen (231-720) is cloned into pET-21(Novagen) in the expression vector.The truncate albumen of expressing, sequence is as follows:
MQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP?QQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKLEHHHHHH（SEQ?ID?NO：29）。
The RVD sudden change of all truncate albumen all is that the NS in the 7th tumor-necrosis factor glycoproteins is carried out.The purification process of all mutant proteins is consistent with the purification process of dHax3 wild-type protein.The primer that carries out the point mutation experiment is as shown in table 4 below:
Table 4
| S505P-5 | ATAGCTTCTAATCCAGGTGGTAAACAAGCCCTT（SEQ?ID?NO：30） |
| S505P-3 | TTGTTTACCACCTGGATTAGAAGCTATTGCCAC（SEQ?ID?NO：31） |
| S505T-5 | ATAGCTTCTAATACAGGTGGTAAACAAGCCCTT（SEQ?ID?NO：32） |
| S505T-3 | TTGTTTACCACCTGTATTAGAAGCTATTGCCAC（SEQ?ID?NO：33） |
| S505Y-5 | ATAGCTTCTAATTACGGTGGTAAACAAGCCCTT（SEQ?ID?NO：34） |
| S505Y-3 | TTGTTTACCACCGTAATTAGAAGCTATTGCCAC（SEQ?ID?NO：35） |
| S505H-5 | ATAGCTTCTAATCATGGTGGTAAACAAGCCCTT（SEQ?ID?NO：36） |
| S505H-3 | TTGTTTACCACCATGATTAGAAGCTATTGCCAC（SEQ?ID?NO：37） |
| S505Q-5 | ATAGCTTCTAATCAGGGTGGTAAACAAGCCCTT（SEQ?ID?NO：38） |
| S505Q-3 | TTGTTTACCACCCTGATTAGAAGCTATTGCCAC（SEQ?ID?NO：39） |
| S505L-5 | ATAGCTTCTAATCTCGGTGGTAAACAAGCCCTT（SEQ?ID?NO：40） |
| S505L-3 | TTGTTTACCACCGAGATTAGAAGCTATTGCCAC（SEQ?ID?NO：41） |
| S505M-5 | ATAGCTTCTAATATGGGTGGTAAACAAGCCCTT（SEQ?ID?NO：42） |
| S505M-3 | TTGTTTACCACCCATATTAGAAGCTATTGCCAC（SEQ?ID?NO：43） |
| S505E-5 | ATAGCTTCTAATGAGGGTGGTAAACAAGCCCTT（SEQ?ID?NO：44） |
| S505E-3 | TTGTTTACCACCCTCATTAGAAGCTATTGCCAC（SEQ?ID?NO：45） |
| S505C-5 | ATAGCTTCTAATTGTGGTGGTAAACAAGCCCTT（SEQ?ID?NO：46） |
| S505C-3 | TTGTTTACCACCACAATTAGAAGCTATTGCCAC（SEQ?ID?NO：47） |
| S505W-5 | ATAGCTTCTAATTGGGGTGGTAAACAAGCCCTT（SEQ?ID?NO：48） |
| S505W-3 | TTGTTTACCACCCCAATTAGAAGCTATTGCCAC（SEQ?ID?NO：49） |
| S505R-5 | ATAGCTTCTAATAGAGGTGGTAAACAAGCCCTT（SEQ?ID?NO：50） |
| S505R-3 | TTGTTTACCACCTCTATTAGAAGCTATTGCCAC（SEQ?ID?NO：51） |
| S505K-5 | ATAGCTTCTAATAAGGGTGGTAAACAAGCCCTT（SEQ?ID?NO：52） |
| S505K-3 | TTGTTTACCACCCTTATTAGAAGCTATTGCCAC（SEQ?ID?NO：53） |
| S505N-5 | ATAGCTTCTAATAATGGTGGTAAACAAGCCCTT（SEQ?ID?NO：54） |
| S505N-3 | TTGTTTACCACCATTATTAGAAGCTATTGCCAC（SEQ?ID?NO：55） |
| S505I | ATAGCTTCTAATATAGGTGGTAAACAAGCCCTT（SEQ?ID?NO：56） |
[0121]?
| S505I | TTGTTTACCACCTATATTAGAAGCTATTGCCAC（SEQ?ID?NO：57） |
In the superincumbent table, S505P-5 represents 505 mutant serine is become 5 ' primer of proline(Pro), and S505P-3 represents 505 mutant serine is become 3 ' primer of proline(Pro); The name of the used use of other amino acid mutations all represents with S505X, the amino acid that the X representative is different.
2. the DNA that is used for crystallization experiment
Be to obtain the crystal of protein and dsDNA mixture, the method by chemosynthesis obtains single stranded DNA (17nt): (Invitrogen), see the following form shown in 6:
Table 6
The synthetic single stranded DNA that obtains is dissolved to 1mM, and equimolar ratio is mixed two single stranded DNAs, and 85 ° of C temperature are bathed more than the 3min, are slow cooling to 22 ℃, and this process must not be less than 3 hours.
(2) experimental technique
1. molecular cloning and expression vector establishment
(1) pcr amplification goal gene fragment
50 μ l Standard PC R reaction compositions (if needed proportionally amplification system) as shown in table 7 below:
Table 750 μ lPCR reaction normal system
After the success amplification purpose fragment, directly use common DNA to reclaim the goal gene fragment that test kit reclaims amplification.Note, if the amplification gene fragment of point mutation need to use first agarose gel electrophoresis to remove dna profiling, then use sepharose DNA to reclaim test kit and reclaim goal gene.
(2) restriction enzyme is processed amplified fragments and carrier
Use identical restriction enzyme to process amplified fragments and carrier, thereby produce identical DNA sticky end.50 μ l double digestion reaction system compositions are as shown in table 8 below:
Table 850 μ l double digestion reaction system composition
37 ℃ of temperature are bathed 30-180min, after response estimator is complete, carry out gel electrophoresis, use sepharose DNA recovery test kit to cut glue and reclaim dna fragmentation.
(3) DNA connects
Use the goal gene fragment after the T4DNA ligase enzyme is cut enzyme to be connected into carrier, 16 ℃ or room temperature reaction 30-120min.Linked system is as shown in table 9 below:
Table 9 linked system
(4) transform
To connect product and change over to by the following method in the DH5 α competent cell, prepare screening positive clone: in connecting product, add 50-100 μ lDH5 α competent cell, place 30min on ice; 42 ℃ of thermal shock 90s; Place 2min on ice; All products are added on the ammonia benzyl resistance agar plate, smoothen with spreading rod, be inverted for 37 ℃ and cultivated 14-16 hour.
(5) use bacterium colony PCR method screening positive clone
A mark 4-8 bacterium colony on the flat board that back obtains, use following system check positive colony:
Table 10 bacterium colony PCR system
Use gel electrophoresis to confirm result, picking positive colony, 37 ℃, 220rpm overnight incubation in ammonia benzyl resistance LB substratum.
(6) plasmid extraction
Use the little extraction reagent kit of common plasmid to extract plasmid, send company's order-checking.
2.dHax3 the Expression and purification of recombinant protein
(1) abduction delivering of dHax3 recombinant protein
At BL21(DE3) plasmid that adds the positive colony that 1-2 μ l extracts in the competent cell transforms, and afterwards all bacterium liquid is inoculated in the 100ml ammonia benzyl LB substratum 37 ℃ * 220rpm overnight incubation.According to 1 (bacterium liquid): the ratio of 100 (LB substratum) is inoculated into cultured bacterium liquid in the 1L ammonia benzyl LB substratum, and 37 ℃ * 200rpm enlarged culturing is until OD600 reaches suitable induced concentration.Add 0.2mM IPTG, 22 ℃ * 16h abduction delivering.
(2) collecting cell and cracking
After abduction delivering is finished, use Bradytelic centrifugation of the large capacity machine 4000rpm * 12min collecting cell.Add the ratio re-suspended cell of 10ml lysis buffer according to 1L bacterium liquid, cell suspension is collected in the glass beaker, use the Ultrasonic Cell Disruptor smudge cells.Should be noted in this process, high temperature can cause protein denaturation, so beaker will be in the situation that the ice bath protection be ultrasonic.The ultrasonic apparatus condition setting is as shown in table 11 below:
Table 11 ultrasonic apparatus arranges condition
(3) affinity chromatography
Because histone can be specific in conjunction with nickel, people have developed the post material of chelated nickel ion in order in conjunction with histidine-tagged recombinant protein, reach the purpose of purifying.Concrete steps are as follows:
With the bacterium liquid of ultrasonication, get supernatant behind the high speed centrifugation 14000rpm/min, add affinity column, make it lean on gravity to flow out, repeat where necessary loading 2-3 time; With cleaning buffer solution high salt and that contain a small amount of imidazoles, alternately clean respectively, to remove non-specific binding protein; Use at last the elute soln contain the high density imidazoles, will be with histidine-tagged recombinant protein wash-out from the nickel post.
(4) heparin affinity chromatography
Can be used for purify DNA in conjunction with albumen, will be loaded to again from the albumen that the nickel post elutes heparin sepharose post; Wash after the albumen that does not have hanging column, use again the elutriant wash-out of gradient salt concn, be further purified albumen.
(5) desalination chromatography
The protein that elutes from heparin sepharose post is present in the high level salt solution.Hypersaline environment can affect crystallization and the biochemical test in later stage.So protein is crossed the method for desalination chromatography, the high salt component in the solution at protein place is removed.
3.dHax3 mutant is to the crystallization experiment of albumen and DNA mixture
The dHax3 truncate albumen that purifying is good is adjusted protein concentration at 6-7mg/ml, and the double-stranded DNA after the annealing of adding mol ratio 1.5:1 is hatched 30min for 4 ℃.All truncate albumen and DNA are hatched, directly carry out crystallization experiment.Finally we have found two condition: 10%PEG5000mme (w/v) from Proplex, 12%1-propanol (V/V), 0.1M MES6.5; 15%PEG6000 (w/v); 5%MPD (V/V); 0.1M MES6.5.The crystal that is used for data gathering after optimizing is grown in 8-10%PEG3350 (w/v), 12%ethanol, 0.1M MES pH6.0.
4. data gathering and processing
Use BL17U wire harness station, synchrotron radiation center, Shanghai (SSRF) to carry out data gathering.The diffraction data of all collections carries out integral and calculating with HKL2000 software, and further data processing realizes by CCP4 software.Use the dHax3(PDB code:3V6T of debond DNA) as the pattern of displacement, by the method for molecular replacement, resolve the structure of dHax3 and DNA mixture.Use at last two softwares of Phenix and COOT to finish correcting process to structure.The statistic data of data gathering and structural modifications sees the following form shown in the 12-15:
Table 12
Table 13
Table 14
Table 15
Three, experimental result
Amino acid is to the identification of DNA bases adenine among the RVD.
As shown in Figure 5, in the crystalline structure, 505 amino acid and DNA base interaction Leu/Met/Trp can form Van der Waals force with adenylic acid (AMP) and interact.And halfcystine, Threonine, proline(Pro), Histidine can interact by Van der Waals force and VITAMIN B4.Simultaneously Serine and l-asparagine identification VITAMIN B4 all is by special interaction of hydrogen bond, and same Glu also can interact by interaction of hydrogen bond and VITAMIN B4.As the RVD that determines the DNA identification specificity among the TALE, its deputy amino acid is no matter be that leucine, methionine(Met), tryptophane or L-glutamic acid can specific recognition DNA bases adenine.
And for identification DNA bases G, glutamine, Methionin, Histidine can pass through the special identification DNA base guanine of interaction of hydrogen bond in the positively charged amino acid.As the deputy amino acid among the amino acid RVD that determines the DNA identification specificity among the TALE no matter be that glutamine, Methionin or Histidine can specific recognition DNA bases adenine.
By a series of protein crystal crystallization experiment, the contriver has found except the corresponding relation of the RVD that is in a large number at occurring in nature TALE and DNA base, also can play a role to DNA specific recognition DNA base if amino acid is positioned at the second of RVD.And these are found to be and provide the specificity of TALE identification DNA that new thinking is provided.Offered huge help for the molecular mechanism of further being familiar with TALE identification DNA simultaneously.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or the example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although the above has illustrated and has described embodiments of the invention, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art is not in the situation that break away from principle of the present invention and aim can change above-described embodiment within the scope of the invention, modification, replacement and modification.
Claims (9)
1. an isolated polypeptide is characterized in that, described polypeptide comprises:
A plurality of repetition aminoacid sequences, described repetition aminoacid sequence is: LTPDQVVAIASX
1X
2GGKQALETVQRLLPVLCQAHG,
Wherein,
X
1Be H or N,
X
2For be selected from I, L, M, W, C, T, P, H, S, N, E, Q, H, K and R one of.
2. polypeptide according to claim 1 is characterized in that, described polypeptide comprises at least 10 described repetition aminoacid sequences.
3. polypeptide according to claim 1 is characterized in that, described polypeptide comprises 20 described repetition aminoacid sequences.
4. polypeptide according to claim 1 is characterized in that, further comprises:
Dna modification enzyme domain structure territory.
5. the oligonucleotide of a separation is characterized in that, described each described polypeptide of oligonucleotide coding claim 1-3.
6. oligonucleotide according to claim 5 is characterized in that, the sequence of described oligonucleotide is be selected from following sequence at least a:
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTCTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：1），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATACAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：2），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATACAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：3），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：4），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTACGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：5），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTACGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：6），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：7），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：8），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：9），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATCTCGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：10），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATCTCGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：11），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATATGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：12），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATATGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：13），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATGAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：14），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATGAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：15），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTGTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：16），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTGTGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：17），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATTGGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：18），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATTGGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：19），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAGAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：20），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAGAGGTGGTAAACAAGCCC?TTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：21），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：22），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATAAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：23），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAATGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：24），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATAAGGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：25），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTAATATAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：26），
CTTACCCCACAACAAGTTGTGGCAATAGCTTCTCATATAGGTGGTAAACAAGCCCTTGAGACGGTTCAAAGACTTCTACCAGTTCTTTGTCAGGCACATGGA（SEQ?ID?NO：27）。
7. an expression vector is characterized in that, comprises claim 4 or 5 described oligonucleotide.
8. a method that changes cellular genome is characterized in that, comprising:
Described cell is comprised or express each described polypeptide of claim 1-4.
9. method according to claim 8 is characterized in that, comprising: expression vector claimed in claim 7 is incorporated in the cell.
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| WO2010079430A1 (en) * | 2009-01-12 | 2010-07-15 | Ulla Bonas | Modular dna-binding domains and methods of use |
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| WO2010079430A1 (en) * | 2009-01-12 | 2010-07-15 | Ulla Bonas | Modular dna-binding domains and methods of use |
| US20110145940A1 (en) * | 2009-12-10 | 2011-06-16 | Voytas Daniel F | Tal effector-mediated dna modification |
| CN102558309A (en) * | 2012-02-10 | 2012-07-11 | 浙江大学 | Transcription activator-like effector nucleases, and encoding genes and application thereof |
| CN102702335A (en) * | 2012-05-23 | 2012-10-03 | 上海斯丹赛生物技术有限公司 | Recombinant transcription activator like effector, transcription activator like effector nuclease, as well as coding gene and application thereof |
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