CN104911199B - Cloned dna molecule method - Google Patents
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Abstract
The present invention relates to a kind of cloned dna molecule method, it includes：The destination carrier containing ccdB genes is obtained, the ccdB genes both ends carry SfiI restriction enzyme sites, the first joint sequence and the second joint sequence, and the joint sequence is 15 30bp, and the joint sequence is in SfiI restriction enzyme sites periphery；Target gene is obtained, the target gene makes its both ends consistent with above-mentioned first joint sequence and the second joint sequence difference again with joint sequence, its sequence through PCR amplification；By the destination carrier containing ccdB genes and the target gene, SfiI enzymes and Gibson cloning reaction liquid hybrid reactions, then inverted Escherichia coli obtain recombinant clone.Can be by DNA by single step reaction Direct Cloning to cricoid destination carrier, from target dna sequence limitation by the method for the present invention, and zero background.
Description
Technical field
The present invention relates to biology field.A kind of in particular it relates to method of cloned dna molecule.
Background technology
Cloned dna molecule technology is routine and basic technology in molecular biology, is had in molecular biology research
Important function.Traditional digestion connection method is earliest and still widely used DNA clone technology so far, it is using restricted
Digestions and DNA ligase are attached.But there is also some obvious limitations, such as crucial enzyme for this conventional method
Enzyme site possibly be present in the sequence for needing to assemble, and more difficult gram that more than 2 DNA fragmentations are carried out using this technology
It is grand.Daniel Gibson have invented a kind of new cloning process-Gibson splicing methods in 2009, for the straight of DNA fragmentation
Connect splicing and clone.Gibson splicings method is a kind of simple isothermal one step splicing method, and independent of the sequence on DNA fragmentation
Row, only need to mix DNA fragmentation or DNA fragmentation with linearized vector, add T5 exonucleases, Phusion polymerases and Taq
Ligase, can complete to splice when 50 DEG C of reactions 1 are small.
But Gibson splicings method can only be by DNA clone to the carrier linearized, therefore need to lead to plasmid vector before reacting
Cross PCR amplification or digestion method is linearized.The prior art fails offer one kind and fast and effectively arrives DNA Direct Clonings
On circular plasmid vectors.
The content of the invention
In view of the problems of the existing technology, first aspect present invention provides a kind of cloned dna molecule method, it is included such as
Lower step：The destination carrier containing ccdB genes is obtained, the ccdB genes both ends have SfiI restriction enzyme sites and first respectively
Joint sequence and the second joint sequence, first joint sequence and the second joint sequence are 15-30bp, the first connector sequence
Row and the second joint sequence are respectively in SfiI restriction enzyme sites both sides；Target gene is obtained, the target gene both ends are respectively provided with
3rd joint sequence and the 4th joint sequence, wherein first joint sequence is as the 3rd joint sequence sequence, described
Two joint sequences and the 4th joint sequence are the same；Preferably, the first joint sequence is ACCTAGGTTCTGGGCCAGGA, and second connects
Header sequence is CCTGAGACCGAAGGCCCAGA.By the destination carrier containing ccdB genes and the target gene, SfiI
Enzyme and Gibson cloning reaction liquid hybrid reactions.In a preferred embodiment of the invention, what the ccdB genes carried
SfiI restriction enzyme sites and the first joint sequence are introduced by PCR amplification.
In a preferred embodiment of the invention, the primer used in the PCR amplification is：Sense primer FP (SEQ ID
NO:1)：GAGACCTAGGTTCTGGGCCAGGATGGCCATTCTCGACTAAGTTGGCAG, anti-sense primer RP (SEQ ID NO:
2)：AACCACCTGAGACCGAAGGCCCAGATGGCCCTGTGTATAAGGGAGCCTG,
In a preferred embodiment of the invention, the pcr amplification product recycles rear clone to weight through Ago-Gel
In group carrier.
In a preferred embodiment of the invention, the second joint sequence that the target gene carries passes through PCR amplification
Introduce.
In a preferred embodiment of the invention, the target gene is arabidopsis VIP1 genes, it is furthermore preferred that described
Sense primer FP-1 (the SEQ ID NO of PCR amplification:4)：ACCTAGGTTCTGGGCCAGGAATGGAAGGAGGAGGAAGAGG,
And anti-sense primer RP-1 (SEQ ID NO:5)：CCTGAGACCGAAGGCCCAGAGCCTCTCTTGGTGAAATCCA.
In a preferred embodiment of the invention, the target gene is arabidopsis ABCG11 genes, it is furthermore preferred that institute
State sense primer FP-2 (the SEQ ID NO of PCR amplification:6)：
ACCTAGGTTCTGGGCCAGGAATGGAGATAGAAGCAAGCAGAC, and anti-sense primer RP-2 (SEQ ID NO:7)：
CCTGAGACCGAAGGCCCAGACCATCTGCGAGCTCCATCTG。
In a preferred embodiment of the invention, by the destination carrier containing ccdB genes and the target base
Cause, SfiI enzymes and Gibson cloning reactions liquid carry out hybrid reaction, and reaction condition reacts 60 minutes for 50 DEG C.
In a preferred embodiment of the invention, by the recombinant vector containing ccdB genes and the target base
After the step of cause, SfiI enzymes and Gibson cloning reaction liquid hybrid reactions, 5 μ L reaction mixtures are taken to convert E. coli
Competent cell, selects 6 bacterium colonies and is identified at random.
Another aspect of the present invention provides a kind of recombinant vector of target gene, and the carrier is by above-mentioned cloned dna molecule
Method and prepare.
The technical solution of the offer of the present invention compared with prior art, has the following advantages that：
First, Gibson of the prior art clone can only be by gene clonings to linearized vector, therefore are needed before cloning
First pass through digestion or PCR amplification linearizes plasmid vector.And this method utilizes ccdB genes and SfiI enzymes, can pass through
Gene Direct Cloning to circular plasmid vector is eliminated the linearisation of carrier this single stepping, when having saved by Gibson reactions
Between and cost.
Second, this method can eliminate the interference of background plasmid carrier using ccdB genes, realize zero background of recombinant vector；
Using SfiI enzymes can from the sequence of target gene limit (SfiI enzyme recognition sites be rare restriction enzyme site), and operating temperature and
Gibson reactions are consistent, and SfiI endonuclease reactions (by vector linearization) while carries out Gibson reactions, and cloning efficiency is high.We
Method, which simplifies cloning process, to boil over.
Brief description of the drawings
Fig. 1 is the step schematic diagram of cloned dna molecule method in the embodiment of the present invention, wherein target cloning vector (ring-type
Plasmid) cloning site at contain SfiI restriction enzyme sites, the structure of ccdB genes.Connecing for 15-30bp is with outside target DNA fragments
Header sequence is identical with two SfiI restriction enzyme site peripheral tabs sequences of destination carrier respectively.Target DNA fragments, SfiI enzymes and
Gibson cloning reactions liquid is mixed with ring target carrier, and conversion is carried out after reaction and obtains the recombinant plasmid containing target dna.
Fig. 2 is the agarose gel electrophoresis figure of the PCR product of ccdB genes, wherein M：(Dalian is precious raw by DL2000Marker
Thing)；1：CcdB gene PCR amplified productions.
The agarose gel electrophoresis figure of Fig. 3 arabidopsis VIP1 gene PCR products, wherein M：(Dalian is precious by DL2000Marker
Biology)；1：VIP1 gene PCR amplified productions.
The agarose gel electrophoresis figure of Fig. 4 arabidopsis ABCG11 gene PCRs, wherein M：(Dalian is precious raw by DL2000Marker
Thing)；1：ABCG11 gene PCR amplified productions.
Embodiment
The embodiment of the present invention is described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or has the function of same or like element.Below with reference to attached
The embodiment of figure description is exemplary, and is only used for explaining the present invention, and is not considered as limiting the invention.In embodiment
The person that is not specified actual conditions, the condition suggested according to normal condition or manufacturer carry out.Production is not specified in agents useful for same or instrument
Manufacturer person, being can be with conventional products that are commercially available.
Present disclosure is first to build a matched destination carrier, contains SfiI digestions position at its cloning site
Point, ccdB genes (lethal gene), the structure of Sfi I restriction enzyme sites；According to target dna and the SfiI digestions of carrier insert division two
The sequence design synthesis PCR amplification primer of site periphery, then PCR amplification, obtain target dna；Directly by destination carrier and target
DNA is mixed, and adds SfiI enzymes and Gibson cloning reactions liquid (NEB companies), 50 degree react 1 it is small when after can convert Escherichia coli
Competent cell, cultivate and after picking colony through identification obtain correct recombinant clone (scheme and formula that embodiment is not described in detail,
Refer to《Molecular Cloning:A Laboratory guide》, the third edition, Science Press).
Embodiment 1：The structure of target recombinant plasmid vector pccdB containing ccdB genes and joint sequence
(1) clone of the ccdB genes containing joint sequence
According to nucleotide sequence (the GenBank database logins number of ccdB genes：JQ085427) and design of primers it is general
Rule, designs two primers, sense primer FP (SEQ ID NO:1)：GAGACCTAGGTTCTGGGCCAGGATGGCC
(sequence for wherein having underscore is joint sequence to ATTCTCGACTAAGTTGGCAG, and shade sequence is SfiI restriction enzyme sites, is not had
The sequence of underscore is ccdB gene specific sequences), and anti-sense primer RP (SEQ ID NO:2)：AACCACCTGAGACCGAAGGCCCAGATGGCC(sequence for wherein having underscore is connector sequence to CTGTGTATAAGGGAGCCTG
Row, shade sequence is SfiI restriction enzyme sites, and the sequence without underscore is ccdB gene specific sequences).
With plasmid pRNAi-GG (the GenBank database logins number of the gene containing ccdB：JQ085427 it is) template, in utilization
State two primers and carry out PCR amplification, PCR amplification program is：94 DEG C 4 minutes；94 DEG C 30 seconds again, 55 DEG C 30 seconds, 72 DEG C 60 seconds, 30
A circulation；Last 72 DEG C extend 5 minutes.PCR amplification obtains the PCR product of ccdB genes.
The PCR product of above-mentioned ccdB genes is detected into row agarose gel electrophoresis, the result is shown in Fig. 2.Electrophoresis result is shown
Amplification obtains a band between 500bp and 750bp, meets with the 666bp of expected size.Therefore this band is carried out
Recycling, recycles Ago-Gel QIAquick Gel Extraction Kit (Shanghai life work), carries out to specifications.
(2) ccdB gene clonings are to pMD-T carriers
The PCR recovery products of above-mentioned ccdB genes and pMD-T carriers (Dalian treasured biotech firm, product identification 6011) is mixed
Close, carry out TA clones.The clone of acquisition send Invitrogen companies (Guangzhou) to be sequenced, containing correct in the results show recombinant vector
CcdB gene orders and with primer introduce SfiI restriction enzyme sites and joint sequence.
Above-mentioned clone there are into ccdB genes and SfiI restriction enzyme sites and the name of the restructuring pMD-T carriers of cloning adapters sequence
For pccdB, its structure diagram is shown in Fig. 1.The sequence of pccdB is shown in SEQ ID NO:3.
SEQ ID NO:3
cggagaccatggagggccttcaaggccattctcgactaagttggcagcatcacccgacgcactttgcgccgaataaa
tacctgtgacggaagatcacttcgcagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaac
ttttggcgaaaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccggg
cgtattttttgagttatcgagattttcaggagctaaggaagctaaacttttgctgacgagaacagggactggtgaaa
tgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgac
acgcctgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttaccc
ggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtatccgttatcgggg
aagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatg
acaggctcccttatacacagggcctagtgggccggtctcttcaggaggtaaatctctagaggatccccgggtaccga
gctcgaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacga
gccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcc
cgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgta
ttgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcac
tcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaa
ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc
gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg
cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctca
tagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttc
agcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggca
gcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta
cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct
cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaa
ggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggatttt
ggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagta
tatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgt
tcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgc
aatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgca
gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgcca
gttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcatt
cagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtc
ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttact
gtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg
accgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattg
gaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgca
cccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaa
aaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagg
gttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccc
cgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcc
ctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtc
tgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaac
tatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaa
ataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctat
tacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgt
tgtaaaacgacggccagtgccaagcttgcatgcctgcaggtcgacgatt
Embodiment 2：Arabidopsis VIP1 gene clonings are to pccdB
(1) arabidopsis RNA extractions and cDNA synthesis
Arabidopsis leaf is chosen, after liquid nitrogen grinding, uses the Trizol reagent (article No.s of Invitrogen companies：
15596-026) extract plant total serum IgE, its concrete operation step reference reagent box specification.
Using the arabidopsis total serum IgE of said extracted as template, Takara RNA PCR Kit (AMV) Ver.3.0 reagents are used
Box (TaKaRa companies, article No.：RR019A cDNA, its concrete operation step reference reagent box specification) are synthesized.
(2) amplification of arabidopsis VIP1 genes
According to the sequence of arabidopsis VIP1 genes (VIRE2-interacting protein 1) in Genbank
(Genbank accession number：AY117284), two primers of design amplification VIP1 genes, sense primer FP-1 (SEQ ID NO:
4)：ACCTAGGTTCTGGGCCAGGA(sequence for wherein having underscore is joint sequence to ATGGAAGGAGGAGGAAGAGG, is not had
The sequence for having underscore is VIP1 gene specific sequences), and anti-sense primer RP-1 (SEQ ID NO:5)：CCTGAGACCGAAGGCCCAGAGCCTCTCTTGGTGAAATCCA (sequence for wherein having underscore is joint sequence, not under
The sequence of line is VIP1 gene specific sequences).Using above-mentioned arabidopsis cDNA as template, PCR is carried out using above-mentioned two primers
Amplification, PCR amplification program are：94 DEG C 4 minutes；94 DEG C 30 seconds again, 54 DEG C 30 seconds, 72 DEG C 60 seconds, 30 circulation；Last 72 DEG C are prolonged
Stretch 5 minutes.PCR amplification obtains the PCR product of arabidopsis VIP1 genes, is then detected into row agarose gel electrophoresis, the result is shown in
Fig. 3.Electrophoresis result display amplification obtains one and is located at the bands of 1000bp slightly above, meets with the 1063bp of expected size,
Therefore this product is recycled.
(3) arabidopsis VIP1 genes Direct Cloning is to ring-type pccdB
VIP1 genes after above-mentioned recycling are directly mixed with ring-type pccdB, add SfiI enzymes and Gibson cloning reactions
Liquid, reaction system are：VIP1 gene recovery product 3.2ul, pccdB plasmid 6ul, SfiI enzyme 0.8ul (1-2U/ul), Gibson
Reaction solution 10ul, common 20ul reaction solutions.Reaction solution mixing is placed on 50 DEG C and reacts 60 minutes, takes 5 μ L to convert Escherichia coli
E.coli competent cells, transformed bacteria solution are coated on the LB culture medium flat plates of the antibiotic of benzyl containing ammonia, and 16h is cultivated in 37 DEG C.Wherein
Gibson reaction solutions form：T5 exonucleases 0.006-0.009U/ul, Phusion archaeal dna polymerase 0.04-0.06U/
Ul, Taq DNA ligase 5-10U/ul (or being not added with this enzyme), NAD 1-3mM, DTT 10-30mM, dNTP 0.3-0.5mM,
Mgcl 10-30mM, Tris-Hcl 50-150mM (pH 7.5).Gibson reaction solutions are purchased from NEB companies, and product identification is
E2611S。
The colonies more than 100 are obtained after conversion, select 6 bacterium colonies at random through PCR and sequencing identification, the results showed that
6 all correct recons of clone.
Embodiment 3：Arabidopsis ABCG11 gene clonings are to pccdB
(1) amplification of arabidopsis ABCG11 genes
According to the sequence of arabidopsis ABCG11 genes in Genbank (ABC transporter G family member 11)
Arrange (Genbank accession number：NM101647), two primers of design amplification ABCG11 genes, sense primer FP-2 (SEQ ID
NO:6)：ACCTAGGTTCTGGGCCAGGA(sequence for wherein having underscore is connector sequence to atggagatagaagcaagcagac
Row, the sequence without underscore is ABCG11 gene specific sequences), and anti-sense primer RP-2 (SEQ ID NO:7)：CCTGAGACCGAAGGCCCAGA(sequence for wherein having underscore is connector to ccatctgcgagctccatctg
Sequence, the sequence without underscore are ABCG11 gene specific sequences).Using the arabidopsis cDNA in embodiment 2 as
Template, carries out PCR amplification, PCR amplification program is using above-mentioned two primers：94 DEG C 4 minutes；94 DEG C 30 seconds again, 55 DEG C 30 seconds,
72 DEG C 120 seconds, 30 circulation；Last 72 DEG C extend 8 minutes.PCR amplification obtains the PCR product of arabidopsis ABCG11 genes, with
Laggard row agarose gel electrophoresis detection, the result is shown in Fig. 3.Electrophoresis result shows that amplification obtains one and is located at 2000bp slightly above
Band, meet with the 2149bp of expected size, therefore this product is recycled.
(2) arabidopsis ABCG11 genes Direct Cloning is to ring-type pccdB
ABCG11 genes after above-mentioned recycling are directly mixed with ring-type pccdB, SfiI enzymes is added and Gibson clones is anti-
Liquid is answered, reaction system is：ABCG11 gene recovery product 4ul, pccdB plasmid 5.4ul, SfiI enzyme 0.6ul, Gibson reaction solutions
10ul, common 20ul reaction solutions.Reaction solution mixing is placed on 50 DEG C and reacts 60 minutes, takes 5 μ L conversion E. coli impressions
State cell, transformed bacteria solution are coated on the LB culture medium flat plates of the antibiotic of benzyl containing ammonia, and 16h is cultivated in 37 DEG C.
Obtained after conversion more than the colonies of 100, select 6 bacterium colonies at random and identified through PCR, and send wherein 4 into
Row sequencing, sequencing result show 4 all correct recons of clone.
These results suggest that can be by the various genes of different sequences and length (with VIP1 genes and ABCG11 using the present invention
The embodiment of gene is representative) fast and efficiently it is cloned into circular plasmid vector.The present invention provides newly for genetic engineering research
Technological means.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (8)
1. a kind of cloned dna molecule method, described method includes following steps：
The destination carrier containing ccdB genes is obtained, the ccdB genes both ends have SfiI restriction enzyme sites and the first connector respectively
Sequence and the second joint sequence, first joint sequence and the second joint sequence are 15-30bp, first joint sequence and
Second joint sequence is respectively in SfiI restriction enzyme sites both sides；
Target gene is obtained, the target gene both ends are respectively provided with the 3rd joint sequence and the 4th joint sequence, wherein described
For first joint sequence as the 3rd joint sequence sequence, second joint sequence and the 4th joint sequence are the same；First connects
Header sequence is ACCTAGGTTCTGGGCCAGGA, and the second joint sequence is CCTGAGACCGAAGGCCCAGA；
By the destination carrier containing ccdB genes and the target gene, SfiI enzymes and the mixing of Gibson cloning reactions liquid are anti-
Should.
2. the cloned dna molecule method described in claim 1, it is characterised in that：The SfiI digestions position that the ccdB genes carry
Point and joint sequence are introduced by PCR amplification, and the primer used in the PCR amplification is：Sense primer FP (SEQ ID NO:1)：
GAGACCTAGGTTCTGGGCCAGGATGGCCATTCTCGACTAAGTTGGCAG, anti-sense primer RP (SEQ ID NO:2)：
AACCACCTGAGACCGAAGGCCCAGATGGCCCTGTGTATAAGGGAGCCTG, the pcr amplification product is through Ago-Gel
Rear clone is recycled into recombinant vector.
3. the cloned dna molecule method described in claim 1, it is characterised in that：The joint sequence that the target gene carries leads to
PCR amplification introducing is crossed, the target gene is arabidopsis VIP1 genes, sense primer FP-1 (the SEQ ID of the PCR amplification
NO:4)：ACCTAGGTTCTGGGCCAGGAATGGAAGGAGGAGGAAGAGG, and anti-sense primer RP-1 (SEQ ID NO:5)：
CCTGAGACCGAAGGCCCAGAGCCTCTCTTGGTGAAATCCA。
4. the cloned dna molecule method described in claim 1, it is characterised in that：The joint sequence that the target gene carries leads to
PCR amplification introducing is crossed, the target gene is arabidopsis ABCG11 genes, sense primer FP-1 (the SEQ ID of the PCR amplification
NO:6)：ACCTAGGTTCTGGGCCAGGAATGGAGATAGAAGCAAGCAGAC, and anti-sense primer RP-2 (SEQ ID NO:
7)：CCTGAGACCGAAGGCCCAGACCATCTGCGAGCTCCATCTG.
5. the cloned dna molecule method described in claim 1, it is characterised in that：By the destination carrier containing ccdB genes
With the target gene, SfiI enzymes and Gibson cloning reactions liquid carry out hybrid reaction, and reaction condition is 50 DEG C of reactions 15 to 60
Minute.
6. the cloned dna molecule method described in claim 1, it is characterised in that：Carried by the target containing ccdB genes
After the step of body and the target gene, SfiI enzymes and Gibson cloning reaction liquid hybrid reactions, reaction mixtures conversion is big
Enterobacteria E.coli competent cells, random choosing colony are identified.
7. the cloned dna molecule recombinant vector prepared according to any one of foregoing claim.
8. a kind of kit of cloned dna molecule, it includes：Destination carrier containing ccdB genes, SfiI enzymes and Gibson grams
Grand reaction solution.
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| CN105177041B (en) * | 2015-11-04 | 2018-07-17 | 中国热带农业科学院热带生物技术研究所 | A kind of expression vector and its application for the research of bimolecular fluorescence complementary |
| CN105400809A (en) * | 2015-12-21 | 2016-03-16 | 生工生物工程（上海）股份有限公司 | Cloning vector and preparation and application thereof |
| CN106497925A (en) * | 2016-06-22 | 2017-03-15 | 浙江省农业科学院 | A kind of connection non-dependent cloning adapters sequence and application |
| CN106906232B (en) * | 2017-04-07 | 2023-10-13 | 中国热带农业科学院热带生物技术研究所 | DNA molecular cloning method and application thereof |
| CN110863004A (en) * | 2019-12-06 | 2020-03-06 | 中国热带农业科学院热带生物技术研究所 | Yeast two-hybrid vector, construction method and application thereof in protein interaction |
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