CN103952405B - A kind of gene site-directed modification system of goat MSTN and application thereof - Google Patents
A kind of gene site-directed modification system of goat MSTN and application thereof Download PDFInfo
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Abstract
The invention discloses the gene site-directed modification system of a kind of goat MSTN and application method thereof, the gene site-directed modification system of described goat MSTN, namely can effectively identify, modify the transcriptional activation sample effector nuclease TALENs of goat MSTN gene target sequence; Transcriptional activation sample effector nuclease TALENs provided by the invention is by forming by nuclease TALEN-R and TALEN-L of identification module TALMEX-1F and TALMEX-1R described in specific recognition respectively; Does is described nuclease TALEN-L SEQ? ID? NO.2 or SEQ? ID? protein nucleotide sequence coded shown in NO.3, does is described nuclease TALEN-R SEQ? ID? NO.4 or SEQ? ID? protein nucleotide sequence coded shown in NO.5.The invention also discloses and use the above-mentioned system that knocks out to carry out targeting modification to goat cells MSTN gene, obtain the method for mutational cell line.The present invention has major application be worth for knocking out or transform to obtain the new variety of premium muscle rate and MSTN gene regulating study mechanism to MSTN gene on goat.
Description
Technical field
The present invention relates to gene engineering technology field, particularly relate to a pair transcriptional activation increment effector nuclease and encoding gene thereof and application, utilize a pair transcriptional activation effector nuclease pointed decoration goat MSTN gene specifically.
Background technology
Flesh generates statin (Myostatin, MSTN) and is extensively present in animal skeleton intramuscular, is the negative regulatory factor of Skeletal Muscle Growth, and MSTN gene has expression in the skeletal muscle of Embryonic Development in Animal phase and Adulthood.The cDNA of the MSTN gene of mouse, rat, people, ox, pig, sheep, chicken, turkey, zebra fish is cloned, checks order.Result of study shows, MSTN gene contains 3 exons, and between different plant species MSTN gene order high conservative.Knock out MSTN gene and can cause animal myofiber popularity hyperplasia and hypertrophy, skeletal muscle amount significantly increases.The MSTN gene coded sequence origination point sudden change of the species such as mouse, rat, people, pig, chicken, ox, sheep, baboon and zebra fish or phase shift mutation, all present " two flesh symptom " (doublemuscling), show the proterties that muscle mass significantly increases.In the species such as people, ox, sheep, dog, find the sudden change of abiogenous MSTN gene at present, ox as blue in Belgium (Belgianblue), Piemonte ox (Piedmontese) etc. are undergone mutation in exon3 sequence, and body shows double-muscling shape.
According to mankind's wish, the dream that directed modification is many scientists is always carried out to genome.By genetic modifications such as suddenling change to MSTN gene, lack, knock out, obtain gene mutant animals, the animal model with " two flesh symptom " can be constructed on the one hand and be used for biological study and pathogenic mechanism research, the object improving muscle quality and weight can be reached on the other hand, produce the improved seeds domestic animal with commercialization character.
People are finding simple method efficiently always and are carrying out targeting modification to genome.Traditional gene targeting depends on abiogenous homologous chromosomes in cell and to recombinate at random exchange, and the ratio that hits is extremely low, usually only has 10
-6-10
-8, be not widely used.The genome targeting modification technical development of nuclease guidance is in recent years rapid.This kind of nuclease is made up of a DNA recognition structure territory and a non-specific nucleic acid restriction endonuclease structural domain usually, the identification target site special by DNA recognition structure territory, nuclease is navigated to the genome area needing to carry out editing, then DNA double chain is cut off by non-specific nucleic acid restriction endonuclease, cause DNA self-regeneration mechanism, thus cause the sudden change of gene order and promote the generation of homologous recombination.
Artificial Zinc finger nuclease (ZincFingerNucleases, ZFN) technology is the genome pointed decoration technology that development in recent years is got up.ZFN is a kind of albumen of structural domain and FokI restriction endonuclease domain fusion by identifying special DNA sequence dna, by identifying that specific DNA sequence dna makes FokI endonuclease form dimer at target site and produces endonuclease activity, cause target site DNA double chain break, thus cause non-homologous end joining and homologous recombination repair mechanism.The phase shift mutation produced in repair process can be used to carry out gene knockout to goal gene, and homologous recombination can be used to the insertion of foreign gene the gene knock-in performing specific site.ZFN technology be once considered to gene function or produce transgenic animal significant, but be full of hope and disappointment, because ZFN is by combining instability between Context-dependent effects and target spot DNA sequence dna, can not target all sequences, need a large amount of screening and identification work before using, and the transgenation possibility that missing the target property of generation cutting introducing cannot be expected is high.In addition, it is expensive that business buys efficiently special ZFN, and investigator is difficult to designed, designed and goes out special and efficient ZFN.
Studied discovery in recent years, transcriptional activation sample effector (transcriptionactivator-likeeffector, TALE) there is DNA binding specificity, and its recognition code has modularization and simplification feature, TALE-DNA binding domains is made up of tandem repeat unit, and most of unit contains 34 amino acid, the 12nd and 13 amino acids alterable heights of unit, become repetition variable region (repeatvariablediresidues, RVDs).The RVDs of TALE identifies that 4 bases of DNA sequence dna have high specificity, and the 13rd amino acids directly and the base specific combination of DNA.Investigator according to DNA sequence dna, can build special TALE-DNA and identifies binding domain in any site, can be widely used in gene order sudden change modification and gene targeting etc.
Setting DNA target sequence, assembling TALE-DNA binding domain, the non-specific DNA merging FokI restriction endonuclease cuts territory, is assembled into TALE nuclease (tanscriptionactivator-likeeffectornucleases, TALENs).TALENs target, in conjunction with DNA, FokI Non-specific cleavage DNA sequence dna, produces DNA double splitting of chain (DNAdouble-srandbreaks, DSBs).In eukaryotic cell, DSBs activates two conservative DNA repair pathways: non-homologous end joining reparation (non-homologousendjoining, NHEJ) karyomit(e) of fracture can be reconnected, likely introduce loss or the insertion of small segment base in connection procedure Fracture site, thus affect gene function or produce gene knockout effect.If now introduce homologous recombination vector; Homology instructs reparation (homology-directedrepair, HDR) that similar DNA profiling can be utilized to instruct and repairs, and substitutes the DNA sequence dna around breaking point, realizes specific sudden change or fixed point importing foreign DNA.
Summary of the invention
The object of the invention is to provide the gene site-directed modification system of a kind of goat MSTN and the application method in goat MSTN genetic modification thereof.
In the present invention, term goat MSTN gene refers to goat myogenesis arrestin (Myostatin, MSTN) gene.
In order to solve the problem, this invention takes following technical scheme:
The gene site-directed modification system of a kind of goat MSTN, described pointed decoration system is the transcriptional activation sample effector nuclease TALENs of a pair goat MSTN gene target sequence, effectively can shear goat MSTN gene target sequence;
Described goat MSTN gene target sequence nucleotide sequence as shown in SEQIDNO.1,23-40 position Nucleotide is the reverse complementary sequence of identification module TALMEX-1F, 57-74 position Nucleotide be identification module TALMEX-1R, 41-56 position Nucleotide is intervening sequence;
Described transcriptional activation sample effector nuclease TALENs is by identifying the nuclease TALEN-L of described identification module TALMEX-1F and identifying that the nuclease TALEN-R of described identification module TALMEX-1R forms;
Described nuclease TALEN-L, is characterized in that, is formed with merging through engineered DNA scinderin Fok-L by TALMEX-1F recognition structure territory TALE-L; Described nuclease TALEN-R, is characterized in that, is formed with merging through engineered DNA scinderin Fok-R by TALMEX-1R recognition structure territory TALE-R;
The protein that described recognition structure territory TALE-L is the position of 229-2022 shown in SEQIDNO.2 or SEQIDNO.3 nucleotide coding; The protein that described DNA scinderin Fok-L is the position of 2023-2814 shown in SEQIDNO.2 or SEQIDNO.3 nucleotide coding; The protein that described recognition structure territory TALE-R is the position of 229-2022 shown in SEQIDNO.4 or SEQIDNO.5 nucleotide coding; The protein that described DNA scinderin Fok-R is the position of 2023-2808 shown in SEQIDNO.4 or SEQIDNO.5 nucleotide coding.
The present invention also provides the above-mentioned goat MSTN preparation method of gene site-directed modification system, comprises the following steps:
(1) according to goat MSTN gene Selection SEQIDNO.1, nucleotides sequence is classified as target sequence, in nucleotide sequence shown in SEQIDNO.1,23-40 position Nucleotide is identification module TALMEX-1F, be identification module TALMEX-1R, 41-56 position Nucleotide with the sequence of 57-74 position Nucleotide reverse complemental be intervening sequence;
(2) the transcriptional activation sample effector nuclease TALENs shearing described target sequence is synthesized according to step (1) gained identification module TALMEX-1F and identification module TALMEX-1R; Described transcriptional activation sample effector nuclease TALENs is by identifying the nuclease TALEN-L of described identification module TALMEX-1F and identifying that the nuclease TALEN-R of described identification module TALMEX-1R forms;
The nucleotide coding sequence of described nuclease TALEN-L is as shown in SEQIDNO.2 or SEQIDNO.3, and the nucleotide coding sequence of described nuclease TALEN-R is as shown in SEQIDNO.4 or SEQIDNO.5;
The invention also discloses containing gene site-directed recombinant expression vector, recombinant bacterium or the recombinant cell lines knocking out system coding gene of above-mentioned goat MSTN;
Disclose that described goat MSTN is gene site-directed knocks out the application that system modifies goal gene nucleotide sequence in target cell.Described MSTN is gene site-directed knocks out the application of system in other mammalian cells MSTN genetic modification.
Fixed point knocks out a method for goat MSTN gene, is included in goat cells and expresses the step that above-mentioned fixed point knocks out system.
Further provide the mRNA that can express described TALENs, it is characterized in that, described mRNA sequence arranges as transcription templates with the nucleotides sequence shown in TALEN-L and TALEN-R.And described mRNA deletes the application of MSTN gene in goat cells or zygote.
Compared with prior art, beneficial effect of the present invention is:
1, have been found that the natural mutation of MSTN gene appears in the species such as ox, sheep and people, and can cause double-muscling shape, but be limited to technical factor, application homology Knockout technology or ZFN method are difficult to the efficient sudden change realizing goat MSTN gene.Therefore, the present invention carries out TALENs by TALEN technology to goat MSTN gene and edits, and obtains the clone that MSTN gene target sequence is undergone mutation, for verifying that the regulatory mechanism of MSTN gene pairs goat muscle growth provides feasible materials and methods further.
2, the clone that the MSTN gene target sequence that the present invention obtains is undergone mutation, the nuclear donor cell of MSTN transgenation goat can be prepared as body-cell neucleus transplanting, there is provided new technological approaches for improving goat meat and improving goat dressing percentage, for the Regulation Mechanism knocking out or transform the new variety and research MSTN gene pairs goat muscle growth that obtain premium muscle rate on goat to MSTN gene, there is major application and be worth.
3, TALENs structure of the present invention is the recombinant nucleic acid enzyme for goat MSTN gene particular target sequence, cuts off gene DNA, cause non-homologous end joining reparation in special site, and then produces MSTN gene Knock-out sudden change.He overcomes conventional ZFN method can not identify arbitrary target gene order, and miss rate is high, and recognition sequence is often by problems such as upstream and downstream sequence affect; Also overcome homologous recombination workload large, mutation rate is low, needs to introduce the problem such as foreign gene or marker gene; And there is the equal or higher activity of ZFN, do not introducing in foreign gene or marker gene situation, make MSTN site-directed point mutation become more simple, convenient, and use this type of mutant cell can obtain the higher gene mutant animals strain of biological safety in subsequent applications.
Accompanying drawing explanation
Fig. 1 is the action principle schematic diagram of TALENs pointed decoration goat MSTN gene;
Fig. 2 is pTALEN-L and pTALEN-R plasmid schematic diagram;
Fig. 3 is cell sample amplified production electrophorogram;
Fig. 4 is cell sample amplified production AluI restriction enzyme digestion and electrophoresis figure;
Fig. 5 is TALENs Activity determination order-checking peak figure;
Fig. 6 is mutant clone L4 order-checking peak figure
Fig. 7 is L4, L6, L7, L10, L11, L13 sample TA cloning and sequencing result; Note: WT represents wildtype gene sequence; △ represents and occurs deletion mutant; Fragmesshift represents that base occurs replaces;
Fig. 8 is L9, L16 sample order-checking peak value figure; Note: peak value is single without cover peak, shows that the catastrophe that diallele generation occurs is completely the same;
Embodiment
Be described in further detail the present invention below in conjunction with accompanying drawing and concrete case study on implementation, but not as a limitation of the invention, specific operation process is carried out according to step below:
The first step, to design according to the gene order that need knock out there is specific sequence TALEN target spot;
The TALEN target spot that second step, basis are designed constructs TALEN plasmid;
3rd step, by TALEN plasmid transfection recipient cell;
4th step, to turning, cell then screens, cultivates, picking mono-clonal, and mono-clonal enlarged culturing, qualification monoclonal cell genome mutation situation, picks out the positive cell clone meeting sudden change and require, use in order to subsequent experimental.
The experimental technique used in following example is ordinary method if no special instructions.
Material used in following example, reagent etc., if no special instructions, all can buy acquisition from commercial channels, and available class reagent or test kit substitute.
Example caprine fetal fibroblast cell MSTN genetic modification
MSTN gene regulates and controls relevant with muscle growth.The present invention's application TALENs technology, codon mutation operation is carried out in the AGCT site for goat MSTN gene exon1, thus realizes the modification of MSTN gene.
1, this example material requested and reagent
FastTALETMTALENAssemblykit test kit is provided by SidansaiBiotechnologyCO., LTD (http://www.sidansai.com/).This test kit is by 8 skeleton carriers, and 172 RVD monomer identification modules and 5 kinds of model calling operate the compositions such as liquid.
Competent cell is purchased from raw emerging biotechnology (Nanjing) company limited, ClaI, AluI restriction endonuclease, DNAMarker etc. are purchased from raw biotechnology (Dalian) company limited of treasured, order-checking and primer synthesis are completed by Hua Da gene (Beijing) Science and Technology Ltd., other biochemical reagents are purchased from Sangon Biotech (Shanghai) Co., Ltd., Multiporator, HypotonicElectroporationBuffer and electroporationcuvettes are purchased from Eppendorf company, and caprine fetal fibroblast cell takes from conceived 35 ~ 40 days Sa energy milk goats.
PL15, pR11 skeleton carrier all derives from FastTALETMTALENAssemblykit.
2, the selection of goat MSTN gene specific TALENs target spot
This example for goat MSTN gene exon1 sequence, adopt TALeffectorNucleotideTarger2.0 (https: //tale-nt.cac.cornell.edu/node/add/talen) to design identification module, 5' end retains T base.Choosing TALMEX-1F and TALMEX-1R is that the AGCT site of identification module to goat MSTN gene exon1 sequence is modified.
TALMEX-1FCCTCAGTAAACTTCGCCT
TALMEX-1RTATAGCATCTTTGCTGAT
The RVD sequence that identification module TALMEX-1F is corresponding:
HD-HD-NG-HD-NI-NN-NG-NI-NI-NI-HD-NG-NG-HD-NN-HD-HD-NG or
HD-HD-NG-HD-NI-NH-NG-NI-NI-NI-HD-NG-NG-HD-NH-HD-HD-NG
The RVD sequence that identification module TALMEX-1F is corresponding:
NG-NI-NG-NI-NN-HD-NI-NG-HD-NG-NG-NG-NN-HD-NG-NN-NI-NG or
NG-NI-NG-NI-NH-HD-NI-NG-HD-NG-NG-NG-NH-HD-NG-NH-NI-NG
Wherein HD identifies that C, NI identify that A, NG identify that T, NN or NH identify G.
The nucleotide sequence of this example code TALEN-L cog region protein is as shown in the Nucleotide of SEQIDNO.2 or SEQIDNO.3 sequence 229-2022 position, the nucleotide sequence of coding TALEN-R cog region protein is as shown in the Nucleotide of SEQIDNO.4 or SEQIDNO.4 sequence 229-2022 position, and (TALEN-L identifies CCTCAGTAAACTTCGCCT to 18 Nucleotide in identification MSTN gene that respectively can be special; TALEN-R identifies TATAGCATCTTTGCTGAT), jointly form TALENs abruptly-changing system by TALEN-L and TALEN-R, its action principle is as shown in Figure 1.
3, the structure of TALENs expression vector
With reference to FastTALETMTALENAssemblykit operation instruction, corresponding link block is selected according to TALMEX-1F and TALMEX-1R identification module, TALMEX-1F selects 9 identification form modules (CC1-T2-CA3-CT4-AA5-AC6-TT7-CG8-CC9) to be connected with pL15 skeleton carrier, builds pTALEN-L plasmid; TALMEX-1R selects 9 identification form modules (TA1-T2-AG3-CA4-TC5-TT6-TG7-CT8-GA9) to be connected with pR11 skeleton carrier, builds pTALEN-R plasmid.
Transformed competence colibacillus cell is gone forward side by side row filter, and ClaI enzyme is cut qualification and order-checking and obtained and connect correct pTALEN-L and pTALEN-R plasmid, pTALEN-L and pTALEN-R schematic diagram as shown in Figure 2.
4, the acquisition of TALENs Activity determination and mono-clonal mutant clone
PTALEN-L and pTALEN-R plasmid amplification, extraction and purification carry out with reference to the method for " Molecular Cloning: A Laboratory guide " second edition.
Operation method gets 35 age in days Sa energy milk goat fetuses, three times are washed with D-Hank ' s liquid in Bechtop, fetal head is cut off with eye scissors, four limbs and internal organ, residue tissue scissors is cut into the little block organization of 1mm3, to move in 10ml centrifuge tube and add 0.25%Trypsin Digestive system and hold piping and druming digestion 15min, after leaving standstill 2min, draw the larger tissue block in bottom to new centrifuge tube, repeat digestion operation, by supernatant 1500r/min, 5min is centrifugal, supernatant discarded, add fresh DMEM nutrient solution (containing 10%FBS+1%PS) re-suspended cell, be diluted to 3 × 10
5individual/ml, bed board cultivates (37 DEG C, 5%CO
2, saturated humidity CO
2).Growth of Cells to degree of converging 80% time, with 0.25% tryptic digestion process, washing collect after obtain caprine fetal fibroblast cell for subsequent use.
1mlElectroporationBuffer suspension cell is added in the cell collected, the centrifugal 5min of 1500r/min, abandon supernatant liquor, cell is resuspended in the transfection liquid (pTALEN-L and the pTALEN-R plasmid of HypotonicElectroporationBuffer respectively containing 20 μ g/ml) of 400 μ L, makes cell density be 1 ~ 3 × 10
6individual/ml, the cell suspension of 400 μ l is transferred in electroporationcuvettes (the wide 2mm in footpath), puts into Multiporator electrotransfection groove and carry out electrotransfection (transfection pattern (Mode) is: Eukaryotes " ⊙ ", Voltage (V) 400V, Timeconstant (τ) 300us, No.ofpulse (n) 1 time).After electricimpulse, cell suspension is made to leave standstill 5min under room temperature condition in cuvette (transfection cup); Cell suspension is added normal cell nutrient solution, be laid on 6 orifice plates and be placed on 37 DEG C, 5%CO
2, saturated humidity cultivate.
Screening and culturing liquid (DMEM+10%FBS+1%PS+3 μ g/mlPuro) is changed after bed board 24h, be replaced by normal cell nutrient solution after screening 72h to continue to cultivate, digestion collection 3 porocyte carries out Activity determination, be numbered L1 ~ L3, all the other cells continue enlarged culturing 5 ~ 10 days, when picking cell clone 96 to 48 orifice plates continue to be cultured to degree of converging 70%, collect part cell and carry out abrupt climatic change, be numbered L4 ~ L45, collect part normal cell as a control group simultaneously, be numbered W1 ~ W3.
Activity determination and abrupt climatic change is carried out by singe-cell PCR method.The cell sample collected is numbered: L1 ~ L3 is the cell collected after screening 72h; L4 ~ L45 is monoclonal cell strain; W1 ~ W3 is non-transfected cells.Be placed in by cell sample in 200 μ l centrifuge tubes, centrifugal segregation nutrient solution, add 10 μ L cell pyrolysis liquids (see table 1), hatch 15min for 45 DEG C, hatch directly as the DNA profiling that regular-PCR detects after 20min for 96 DEG C ,-20 DEG C save backup.
Table 1 cell pyrolysis liquid
| Moiety | Volume (μ l) |
| 10×PCR buffer | 1 |
| 1％tween20 | 1 |
| 0.1％triton | 1 |
| Protin K(25mg/ml) | 0.2 |
| Distilled water | 6.8 |
Get 2 μ l split products and carry out PCR reaction as template, PCR primer designs in goat MSTN genome, and it is good that goat cells genome extracts quality, and all can amplify object band, primer sequence is in table 2, and reaction system is in table 3.
Table 2PCR primer
Table 3PCR reaction system
| Moiety | Volume (μ l) | Final concentration |
| 10×PCR buffer | 4.8 | 1× |
| dNTP(10Mm) | 2 | 200μM |
| TAL-2se(10μM) | 0.3 | 0.1μM |
| TAL-2an(10μM) | 0.3 | 0.1μM |
| Taq enzyme (5U/ μ l) | 0.3 | 2.5U/reaction |
| Template | 2 | |
| ddH 2O | 40.3 |
PCR reaction conditions is: 95 DEG C of 5min; 95 DEG C of 50sec, 52 DEG C of 30sec, 72 DEG C of 45sec, totally 35 circulations; 72 DEG C of 10min; 4 DEG C of preservations.Get 5 μ lPCR products and carry out agarose gel electrophoresis detection, as shown in Figure 3, L1, L4 ~ L17, L19, W1 cell sample all amplifies 439bp specificity object band to partial detection.Get 10 μ lL1, L4 ~ L17, W1 sample P CR product AluI restriction enzyme to carry out enzyme and cut qualification, electrophoresis detection enzyme cuts situation, detected result as shown in Figure 4, result shows: L1 sample P CR product only can be partly cut across, illustrate that PCR primer is mixed with multiple fragment, there is the sudden change of AGCT restriction enzyme site in Partial Fragment, can not be cut open; L4, L5, L6, L8, L11, L12, L14 sample P CR product only can be partly cut across, illustrate PCR primer for mixing fragment, obtain cell clone be monoallelic sudden change; L7, L9, L10, L13, L16 sample P CR product can not be cut open, and illustrates that the diallele that sports existed in obtained cell suddenlys change; L15, L17, W1 sample P CR product can be cut completely, illustrates that L15, L17, W1 cell sample is not undergone mutation.
By L1, L4, L6, L11, L7, L9, L10, L13, L16 cellular genome amplified production, and after amplified production connection carrier T, send Hua Da gene (Beijing) Science and Technology Ltd. to check order, sequencing primer is CX-se:TTGGCTTGGCGTTACTCA.
Sequencing result shows:
(1) L1 cellular genome amplified production sequencing result as shown in Figure 5, occur in goat MSTN gene exon1 sequence A GCT site obviously overlapping peak, illustrating has multiple sequence signal to occur in this site, there is special sequence to change, prove that the TALENs of this example design has MSTN transgenation active;
(2), there is overlapping peaks (as L4 sample order-checking peak figure is shown in Fig. 6) in L4, L6, L7, L9, L10, L11, L13, L16 sample sequencing result display: L4, L6, L7, L9, L10, L11, L13 amplified production direct Sequencing is presented at MSTN gene exon1 sequence A GCT location proximate and occurs base deletion and replacement simultaneously; Amplified production connects the display of TA cloning and sequencing result L4, L6, L11 sample monosome gene and undergos mutation, and L7, L10, L13 diplochromosome allelotrope is all undergone mutation, and the catastrophe that every sample contains as shown in Figure 7; The catastrophe of L9, L16 sample allelotrope target site is completely the same, and be homozygous mutation, catastrophe as shown in Figure 8.
(3) when detected result illustrates and uses TALENs process caprine fetal fibroblast cell of the present invention, can efficiently fix a point to goat myostatin (Myostatin, MSTN) gene is modified, thus realizes the transformation of MSTN gene coded sequence or knock out.
Above example is only exemplary embodiment of the present invention, and be not used in restriction the present invention, protection scope of the present invention is defined by the claims.Those skilled in the art can in essence of the present invention and protection domain, and make various amendment or equivalent replacement to the present invention, this amendment or equivalent replacement also should be considered as dropping in protection scope of the present invention.
Claims (5)
1. the gene site-directed modification system of goat MSTN, is characterized in that efficient gene pointed decoration system is shear the transcriptional activation sample effector nuclease TALENs of goat MSTN gene target sequence;
Described goat MSTN gene target sequence nucleotide sequence as shown in SEQIDNO.1,23-40 position Nucleotide is identification module TALMEX-1F, be identification module TALMEX-1R, 41-56 position Nucleotide is intervening sequence with the complementary sequence of 57-74 position Nucleotide;
Described transcriptional activation sample effector nuclease TALENs is by identifying the nuclease TALEN-L of described identification module TALMEX-1F and identifying that the nuclease TALEN-R of described identification module TALMEX-1R forms;
Described nuclease TALEN-L, is formed with merging through engineered DNA scinderin Fok-L by TALMEX-1F recognition structure territory TALE-L; Described nuclease TALEN-R, is formed with merging through engineered DNA scinderin Fok-R by TALMEX-1R recognition structure territory TALE-R;
The protein that described recognition structure territory TALE-L is the position of 229-2022 shown in SEQIDNO.2 or SEQIDNO.3 nucleotide coding; The protein that described DNA scinderin Fok-L is the position of 2023-2814 shown in SEQIDNO.2 or SEQIDNO.3 nucleotide coding; The protein that described recognition structure territory TALE-R is the position of 229-2022 shown in SEQIDNO.4 or SEQIDNO.5 nucleotide coding; The protein that described DNA scinderin Fok-R is the position of 2023-2808 shown in SEQIDNO.4 or SEQIDNO.5 nucleotide coding.
2. the preparation method of the gene site-directed modification system of goat MSTN described in claim 1, is characterized in that, comprise the following steps:
(1) according to goat MSTN gene Selection SEQIDNO.1, nucleotides sequence is classified as target sequence, and the 23-40 of nucleotide sequence shown in SEQIDNO.1 position Nucleotide is identification module TALMEX-1F, be identification module TALMEX-1R, 41-56 position Nucleotide with the sequence of 57-74 position nucleotide complementary be that intervening sequence, 47-50 position Nucleotide are for shear target spot and to can be used as AluI digestion with restriction enzyme abrupt climatic change site;
(2) the transcriptional activation sample effector nuclease TALENs shearing described target sequence is synthesized according to step (1) gained identification module TALMEX-1F and identification module TALMEX-1R; Described transcriptional activation sample effector nuclease TALENs is by identifying the nuclease TALEN-L of described identification module TALMEX-1F and identifying that the nuclease TALEN-R of described identification module TALMEX-1R forms;
Described nuclease TALEN-L, is formed with merging through engineered DNA scinderin Fok-L by TALMEX-1F recognition structure territory TALE-L; Described nuclease TALEN-R, is formed with merging through engineered DNA scinderin Fok-R by TALMEX-1R recognition structure territory TALE-R;
The protein that described recognition structure territory TALE-L is the position of 229-2022 shown in SEQIDNO.2 or SEQIDNO.3 nucleotide coding; The protein that described DNA scinderin Fok-L is the position of 2023-2814 shown in SEQIDNO.2 or SEQIDNO.3 nucleotide coding; The protein that described recognition structure territory TALE-R is the position of 229-2022 shown in SEQIDNO.4 or SEQIDNO.5 nucleotide coding; The protein that described DNA scinderin Fok-R is the position of 2023-2808 shown in SEQIDNO.4 or SEQIDNO.5 nucleotide coding.
3. the recombinant expression vector containing the gene site-directed modification system of goat MSTN described in claim 1, recombinant bacterium or recombinant cell lines.
4. the application of the gene site-directed modification system of goat MSTN described in claim 1 in goat cells MSTN gene recognition, modification.
5. the application of the gene site-directed modification system of MSTN in goat cells MSTN genetic modification according to claim 1.
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| CN105985943B (en) * | 2015-03-16 | 2021-07-06 | 中国科学院遗传与发育生物学研究所 | Method for site-specific modification of plant genome by using non-genetic material |
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