CN109929874A - The construction method of Agrobacterium Ti plasmid carrier PCHF1302 and application - Google Patents
The construction method of Agrobacterium Ti plasmid carrier PCHF1302 and application Download PDFInfo
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Abstract
The construction method of Agrobacterium Ti plasmid carrier PCHF1302 and application belong to plant gene technical field, the present invention is to carrying out double digestion simultaneously at the restriction enzyme site ECORI and HindIII on Agrobacterium Ti plasmid Pcambia1302, the complete gene expression frame that PCHF3300 includes 35S promoter, NOS terminator and MCS is added into homology arm by PCR, the expression cassette that 35S promoter starts successfully is connected on plasmid Pcambia1302, PCHF1302 is built into；The target gene obtained through the methods of PCR clone can be conveniently attached to multiple clone's restriction enzyme sites (MCS), and target gene is enable to stablize expression in plant；Contain hygromycin gene and mGFP5 Green fluorescent protein fusion vector in the region TDNA, the screening effect of genetically modified plants can be significantly improved.
Description
Technical field
The invention belongs to plant gene technical fields, and in particular to a kind of building of Agrobacterium Ti plasmid carrier PCHF1302
Method and application.
Background technique
Plant gene engineering technology is quickly grown in recent years, and especially transgenic technology plays pass in gene functional research
The effect of key, by transgenic technology, the gene that we can will study is transferred to the originating species or other modes of gene
Go to study the function of the gene in plant.Need to complete the building of expression vector before transgenosis.But it is common at present
Plant expression vector all there is various defects.As Pcambia1301, Pcambia1302 lack the 35S of destination gene expression
Promoter and terminator cannot be used directly for building expression vector.
Summary of the invention
A kind of construction method for being designed to provide Agrobacterium Ti plasmid carrier PCHF1302 of invention.Through the methods of PCR
Clone obtained target gene, the 35S promoter and E9 that the plasmid vector can be connected in many ways terminate between it is more
A clone's restriction enzyme site (MCS) enables target gene to stablize expression in plant；The plasmid vector is other than the region TDNA
Contain hygromycin resistance in the region TDNA convenient for screening monoclonal positive bacterium colony in prokaryotic cell containing that resistant gene of card
Gene and mGFP5 Green fluorescent protein fusion vector, can significantly improve the screening effect of genetically modified plants.
The construction method of Agrobacterium Ti plasmid carrier PCHF1302 of the invention, including the following steps:
It S1. include the acquisition of the expression cassette of 35S promoter, E9 terminator and multiple cloning sites MCS
S11. Agrobacterium Ti plasmid Pcambia1302 is made into recombinant plasmid compared with the region MCS of PCHF3300
There are most restriction enzyme sites in the region PCHF1302MCS.Finally selected in the region MCS (Fig. 3) of Agrobacterium Ti plasmid Pcambia1302
The two restriction enzyme sites of EcoRI and HindIII.
S12. 18bp sequence is selected as the homology arm of upstream primer in EcoRI restriction enzyme site, be denoted as B-F in HindIII
Homology arm of the downstream 18bp sequence of restriction enzyme site as downstream primer is denoted as B-R and selects in the upstream expression cassette 35S PCHF3300
Bp selects bp as downstream primer E9-R as upstream primer 35S-F, in PCHF3300 expression cassette E9 terminator downstream.
Upstream primer F (B-F+35S-F): TGACCATGATTACGAATTAAAGGGCAATTG is obtained from above
Downstream primer R (B-R+E9-R): GACGGCCAGTGCCAAGCTAGCTTCGATTGA
S13. using PCHF3300 plasmid as template, with KOD-FX high fidelity enzyme will include 35S promoter, E9 terminator with
And the complete gene expression frame of MCS adds homology arm
The acquisition of S2.Pcambia1302 carrier framework
Pcambia1302 (Fig. 2) is subjected to double digestion with restriction enzyme EcoRI and HindIII, excision EcoRI and
MCS among HindIII cuts purpose by the Pcambia1302 after double digestion after 1% agarose gel electrophoresis separation
Band is recycled by conventional gel recovery method.
The building of S3.PCHF1302 plasmid
S31. utilize seamless Cloning Kit, by the method for homologous recombination by gel extraction include 35S promoter,
The expression cassette of E9 terminator and multiple cloning sites MCS are connected on Pcambia1302 carrier framework, and the recombinant plasmid after connection utilizes
Heat shock method is transformed into the competent cell of escherichia coli DH5a bacterial strain.
S32. the Escherichia coli after conversion are put down and is applied to the LB culture medium culture containing 50mg/L kanamycins, picking is independent
Bacterium colony carries out bacterium colony PCR identification, screens the positive bacterium colony containing 35S-MCS-E9 recombinant plasmid.
S33. positive bacterium colony is sent and is sequenced in sequencing company after the expansion of the LB liquid medium of 50mg/L kanamycins is numerous,
To correct bacterium solution is sequenced, recombinant plasmid vector DNA is extracted using plasmid extraction kit, which is named as
PCHF1302 (Fig. 4).
Provided by the present invention for the plasmid vector PCHF1302 of plant gene expression vector building, because starting in 35S
Son and E9 have polyclone enzyme enzyme site MCS between terminating at, can be very square using the target gene that the methods of PCR clone obtains
Just it is connected to 35S promoter and between E9 terminates at, the upstream and downstream of target gene is made to add promoter and end respectively
It is only sub, it is independently expressed in plant.Target gene in PCR amplification, can by the end 5' of forward and reverse primer introduce with
The corresponding restriction enzyme site of the MCS of PCHF1302 (Eco53K1, Sac 1, BanII, Acc65I, Kpn 1, BamH1, XbaI,
SaL1, PstI), it is attached, can also incite somebody to action with the PCHF1302 carrier after same restriction enzymes double zyme cutting
After PCHF1302 carrier is by above-mentioned restriction enzyme single endonuclease digestion, double digestion, passed through using seamless Cloning Kit homologous heavy
The method of group, genetic fragment is connected on PCHF1302 carrier.
The plasmid vector also has the hygromycin gene independently expressed and mGFP5 Green fluorescent protein fusion vector,
The former can use the culture medium added with hygromycin and screens to transgenic plant cells, tissue, even transgenic plant,
The latter can use inverted fluorescence microscope and observe Transgenic plant tissue, further screening transgenic positive tissue
And plant, the two combine the selection effect that can significantly improve genetically modified plants.It overcomes and is usually used in plant gene at present
Since restriction enzyme site is limited, target gene fragment is difficult to be inserted into and connect the plasmid of expression vector establishment, and lacks in plant
The defect of the function element such as promoter, terminator, the selection markers that expression needs in vivo.
Detailed description of the invention
Fig. 1 is the electrophoretogram of OLEOSIN2 gene, and 1-4 is the PCR electrophoresis result of OLEOSIN2 gene in figure
Fig. 2 is the schematic diagram of Agrobacterium Ti plasmid Pcambia1302
Fig. 3 is PCHF3300 plasmid schematic diagram
Fig. 4 is the schematic diagram of PCHF1302 plasmid
Fig. 5 is the electrophoretogram that OLEOSIN2 gene connects bacterium solution PCR after PCHF1302 carrier.1-5 is bacterium solution PCR sun in figure
Property result
Specific embodiment
In order to show the object of the invention and advantage clearly, with reference to embodiments to the present invention carry out into
Description is described in detail in one step.The purposes of some aspects described herein that specific examples are only used to explain the present invention, not
For limiting the present invention.
The embodiment of the invention provides the application methods of plasmid vector PCHF1302:
Embodiment
The building of soybean OLEOSIN2 gene cloning and expression carrier
The acquisition of one, target gene
1. the sequence of OLEOSIN2 gene is obtained at the website Soybase (https: //www.soybase.org/), according to
Transcript profile sequence utilizes the website NCBI primer blast (https: //www.ncbi.nlm.nih.gov) design primer OLE-
F and OLE-R.
OLE-F:ACACACCCCACTAACAATTCC
OLE-R:AGACACGAACGAACGTCCCTAC
2. cloning OLEOSIN2 gene (Fig. 1) using RT-PCR technology, PCR is produced using the cDNA of WILLIAMS-DARLING Ton 82 as template
Object is recycled after 1% agarose gel electrophoresis with Tiangeng TIANgel Midi Purification Kit kit.Tool
Gymnastics is carried out according to kit specification.
3., according to the range of segment and carrier molar concentration ratio 1-10, being closed using the concentration of apparatus measures recovery product
The volume of segment and TAKARA PMD-18T carrier is added in reason adjustment.16 DEG C of connections overnight.
4. converting DH5 α, uniformly it is applied on the LB solid medium of 50mg/l ampicillin, 37 degree are incubated overnight.
5. picking single bacterium colony carries out bacterium colony PCR identification, PCR positive bacterium colony is placed in liquid LB Antibiotic medium (ammonia
Parasiticin 50mg/L) it is incubated overnight, after bacterium solution PCR identification, bacterium is protected to positive bacterium solution, 200 μ l are sucked out and send sequencing.
6. bacterium solution of the sequencing after correct is continued to use Tiangeng TIANprep Mini Plasmid Kit reagent after expanding culture
Box extracts plasmid, and operation detailed process carries out to specifications.
97., with primer OLE-F2, OLE-R2 with restriction enzyme site, OLEOSIN2 gene is added enzyme using plasmid as template
Enzyme site is again coupled to PMD-18T conversion DH5 α, subclone sequencing is carried out, after the continuous expansion culture by bacterium solution of sequencing successful subsequent
Plasmid concrete operations are extracted with above-mentioned steps 4-6.
The extraction of two .PCH1302 Plasmid DNA
Using Tiangeng TIANprep Mini Plasmid Kit kit, PCH1302 matter is extracted from bacillus coli DH 5 alpha
Grain DNA, concrete operation step press the operation instruction of kit.Its concentration is measured using NanoPhotometer.
The connection of three, target gene and PCH1302 plasmid
(1) double digestion handle: using restriction enzyme Sam 1 and Xba1 respectively to OLEOSIN2 gene plasmid with
PCH1302 plasmid carries out double digestion processing.The reaction condition of double digestion processing is 37 degree of reaction 2h, is added in every 50 μ l digestion system
Enter 5 μ l 10Xlodingbuffer and terminates endonuclease reaction.
(2) target fragment recycles: will carry out electrophoresis, testing goal gene and matter through double digestion treated reaction product
Then the digestion effect of grain carries out glue recycling to target fragment with kit, concrete operation step presses the operation instruction of kit.
(3) connection reaction: the target gene OLEOSIN2 recycled after identical digestion through glue and PCHF1302 is carried out
Connection adjusts the amount of target fragment and plasmid respectively in the ratio of target gene fragment 125fmol, plasmid 25fmol, and 2 μ are added
LT4 connection buffer, 1 μ lT4 ligase, rest part supply 20 μ l, 16 DEG C of connections overnight with water.
(4) Escherichia coli convert: the total overall reaction product that oneself connects is transformed into escherichia coli DH5a bacterium using heat shock method
In the competent cell of strain.
(5) bacterium colony PCR is identified: the Escherichia coli liquid (100 μ L) after plasmid is converted is coated in solid LB Antibiotic medium
It is inverted and is incubated overnight on (kanamycins 50mg/L), picking single bacterium colony carries out bacterium colony PCR identification (Fig. 5), and PCR primer is as follows.
PCR positive bacterium colony is placed in liquid LB Antibiotic medium (kanamycins 50mg/L) to be incubated overnight, again after PCR identification, is protected
Deposit positive bacterium solution.The plasmid that target gene OLEOSIN2 is successfully connected to PCHF1302 is named as PCHF1302-OLESIN2 (figure
5)。
OLE-F2:5'-TCCCCCGGG ATGACCACACAAGTACCAC-3'
OLE-R2:5'-GCTCTAGATCATGCGGTTGCGGTTGT-3'
Four, Agrobacterium-mediated Transformations and utilization
PCHF1302 Plasmid DNA is extracted from escherichia coli DH5a in same way as described above, takes appropriate (1ng or so) plasmid
DNA is transformed into the competent cell of Agrobacterium K599 bacterial strain using thermal excitation, carries out bacterium colony PCR identification again, is saved positive
Bacterium solution.Positive bacterium solution after preservation is put in -80 DEG C of long-term preservations, can be used for plant rooting after taking out the activation of 5 μ l plate streakings
Conversion.
Sequence table
<120>construction method of Agrobacterium Ti plasmid carrier PCHF1302 and application
<110>Jilin University
The sequence of PCHF1302
(i) sequence signature: (A) length: 11550bp；(B) type: plasmid；(C) chain: closed loop double-strand
(ii) molecule type: nucleotide
(iii) sequence description: plasmid vector PCHF1302
1 CATGGTAGAT CTGACTAGTA AAGGAGAAGA ACTTTTCACT GGAGTTGTCC CAATTCTTGT
61 TGAATTAGAT GGTGATGTTA ATGGGCACAA ATTTTCTGTC AGTGGAGAGG GTGAAGGTGA
121 TGCAACATAC GGAAAACTTA CCCTTAAATT TATTTGCACT ACTGGAAAAC TACCTGTTCC
181 GTGGCCAACA CTTGTCACTA CTTTCTCTTA TGGTGTTCAA TGCTTTTCAA GATACCCAGA
241 TCATATGAAG CGGCACGACT TCTTCAAGAG CGCCATGCCT GAGGGATACG TGCAGGAGAG
301 GACCATCTTC TTCAAGGACG ACGGGAACTA CAAGACACGT GCTGAAGTCA AGTTTGAGGG
361 AGACACCCTC GTCAACAGGA TCGAGCTTAA GGGAATCGAT TTCAAGGAGG ACGGAAACAT
421 CCTCGGCCAC AAGTTGGAAT ACAACTACAA CTCCCACAAC GTATACATCA TGGCCGACAA
481 GCAAAAGAAC GGCATCAAAG CCAACTTCAA GACCCGCCAC AACATCGAAG ACGGCGGCGT
541 GCAACTCGCT GATCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC
601 AGACAACCAT TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA
661 CCACATGGTC CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT
721 ATACAAAGCT AGCCACCACC ACCACCACCA CGTGTGAATT GGTGACCAGC TCGAATTTCC
781 CCGATCGTTC AAACATTTGG CAATAAAGTT TCTTAAGATT GAATCCTGTT GCCGGTCTTG
841 CGATGATTAT CATATAATTT CTGTTGAATT ACGTTAAGCA TGTAATAATT AACATGTAAT
901 GCATGACGTT ATTTATGAGA TGGGTTTTTA TGATTAGAGT CCCGCAATTA TACATTTAAT
961 ACGCGATAGA AAACAAAATA TAGCGCGCAA ACTAGGATAA ATTATCGCGC GCGGTGTCAT
1021 CTATGTTACT AGATCGGGAA TTAAACTATC AGTGTTTGAC AGGATATATT GGCGGGTAAA
1081 CCTAAGAGAA AAGAGCGTTT ATTAGAATAA CGGATATTTA AAAGGGCGTG AAAAGGTTTA
1141 TCCGTTCGTC CATTTGTATG TGCATGCCAA CCACAGGGTT CCCCTCGGGA TCAAAGTACT
1201 TTGATCCAAC CCCTCCGCTG CTATAGTGCA GTCGGCTTCT GACGTTCAGT GCAGCCGTCT
1261 TCTGAAAACG ACATGTCGCA CAAGTCCTAA GTTACGCGAC AGGCTGCCGC CCTGCCCTTT
1321 TCCTGGCGTT TTCTTGTCGC GTGTTTTAGT CGCATAAAGT AGAATACTTG CGACTAGAAC
1381 CGGAGACATT ACGCCATGAA CAAGAGCGCC GCCGCTGGCC TGCTGGGCTA TGCCCGCGTC
1441 AGCACCGACG ACCAGGACTT GACCAACCAA CGGGCCGAAC TGCACGCGGC CGGCTGCACC
1501 AAGCTGTTTT CCGAGAAGAT CACCGGCACC AGGCGCGACC GCCCGGAGCT GGCCAGGATG
1561 CTTGACCACC TACGCCCTGG CGACGTTGTG ACAGTGACCA GGCTAGACCG CCTGGCCCGC
1621 AGCACCCGCG ACCTACTGGA CATTGCCGAG CGCATCCAGG AGGCCGGCGC GGGCCTGCGT
1681 AGCCTGGCAG AGCCGTGGGC CGACACCACC ACGCCGGCCG GCCGCATGGT GTTGACCGTG
1741 TTCGCCGGCA TTGCCGAGTT CGAGCGTTCC CTAATCATCG ACCGCACCCG GAGCGGGCGC
1801 GAGGCCGCCA AGGCCCGAGG CGTGAAGTTT GGCCCCCGCC CTACCCTCAC CCCGGCACAG
1861 ATCGCGCACG CCCGCGAGCT GATCGACCAG GAAGGCCGCA CCGTGAAAGA GGCGGCTGCA
1921 CTGCTTGGCG TGCATCGCTC GACCCTGTAC CGCGCACTTG AGCGCAGCGA GGAAGTGACG
1981 CCCACCGAGG CCAGGCGGCG CGGTGCCTTC CGTGAGGACG CATTGACCGA GGCCGACGCC
2041 CTGGCGGCCG CCGAGAATGA ACGCCAAGAG GAACAAGCAT GAAACCGCAC CAGGACGGCC
2101 AGGACGAACC GTTTTTCATT ACCGAAGAGA TCGAGGCGGA GATGATCGCG GCCGGGTACG
2161 TGTTCGAGCC GCCCGCGCAC GTCTCAACCG TGCGGCTGCA TGAAATCCTG GCCGGTTTGT
2221 CTGATGCCAA GCTGGCGGCC TGGCCGGCCA GCTTGGCCGC TGAAGAAACC GAGCGCCGCC
2281 GTCTAAAAAG GTGATGTGTA TTTGAGTAAA ACAGCTTGCG TCATGCGGTC GCTGCGTATA
2341 TGATGCGATG AGTAAATAAA CAAATACGCA AGGGGAACGC ATGAAGGTTA TCGCTGTACT
2401 TAACCAGAAA GGCGGGTCAG GCAAGACGAC CATCGCAACC CATCTAGCCC GCGCCCTGCA
2461 ACTCGCCGGG GCCGATGTTC TGTTAGTCGA TTCCGATCCC CAGGGCAGTG CCCGCGATTG
2521 GGCGGCCGTG CGGGAAGATC AACCGCTAAC CGTTGTCGGC ATCGACCGCC CGACGATTGA
2581 CCGCGACGTG AAGGCCATCG GCCGGCGCGA CTTCGTAGTG ATCGACGGAG CGCCCCAGGC
2641 GGCGGACTTG GCTGTGTCCG CGATCAAGGC AGCCGACTTC GTGCTGATTC CGGTGCAGCC
2701 AAGCCCTTAC GACATATGGG CCACCGCCGA CCTGGTGGAG CTGGTTAAGC AGCGCATTGA
2761 GGTCACGGAT GGAAGGCTAC AAGCGGCCTT TGTCGTGTCG CGGGCGATCA AAGGCACGCG
2821 CATCGGCGGT GAGGTTGCCG AGGCGCTGGC CGGGTACGAG CTGCCCATTC TTGAGTCCCG
2881 TATCACGCAG CGCGTGAGCT ACCCAGGCAC TGCCGCCGCC GGCACAACCG TTCTTGAATC
2941 AGAACCCGAG GGCGACGCTG CCCGCGAGGT CCAGGCGCTG GCCGCTGAAA TTAAATCAAA
3001 ACTCATTTGA GTTAATGAGG TAAAGAGAAA ATGAGCAAAA GCACAAACAC GCTAAGTGCC
3061 GGCCGTCCGA GCGCACGCAG CAGCAAGGCT GCAACGTTGG CCAGCCTGGC AGACACGCCA
3121 GCCATGAAGC GGGTCAACTT TCAGTTGCCG GCGGAGGATC ACACCAAGCT GAAGATGTAC
3181 GCGGTACGCC AAGGCAAGAC CATTACCGAG CTGCTATCTG AATACATCGC GCAGCTACCA
3241 GAGTAAATGA GCAAATGAAT AAATGAGTAG ATGAATTTTA GCGGCTAAAG GAGGCGGCAT
3301 GGAAAATCAA GAACAACCAG GCACCGACGC CGTGGAATGC CCCATGTGTG GAGGAACGGG
3361 CGGTTGGCCA GGCGTAAGCG GCTGGGTTGT CTGCCGGCCC TGCAATGGCA CTGGAACCCC
3421 CAAGCCCGAG GAATCGGCGT GACGGTCGCA AACCATCCGG CCCGGTACAA ATCGGCGCGG
3481 CGCTGGGTGA TGACCTGGTG GAGAAGTTGA AGGCCGCGCA GGCCGCCCAG CGGCAACGCA
3541 TCGAGGCAGA AGCACGCCCC GGTGAATCGT GGCAAGCGGC CGCTGATCGA ATCCGCAAAG
3601 AATCCCGGCA ACCGCCGGCA GCCGGTGCGC CGTCGATTAG GAAGCCGCCC AAGGGCGACG
3661 AGCAACCAGA TTTTTTCGTT CCGATGCTCT ATGACGTGGG CACCCGCGAT AGTCGCAGCA
3721 TCATGGACGT GGCCGTTTTC CGTCTGTCGA AGCGTGACCG ACGAGCTGGC GAGGTGATCC
3781 GCTACGAGCT TCCAGACGGG CACGTAGAGG TTTCCGCAGG GCCGGCCGGC ATGGCCAGTG
3841 TGTGGGATTA CGACCTGGTA CTGATGGCGG TTTCCCATCT AACCGAATCC ATGAACCGAT
3901 ACCGGGAAGG GAAGGGAGAC AAGCCCGGCC GCGTGTTCCG TCCACACGTT GCGGACGTAC
3961 TCAAGTTCTG CCGGCGAGCC GATGGCGGAA AGCAGAAAGA CGACCTGGTA GAAACCTGCA
4021 TTCGGTTAAA CACCACGCAC GTTGCCATGC AGCGTACGAA GAAGGCCAAG AACGGCCGCC
4081 TGGTGACGGT ATCCGAGGGT GAAGCCTTGA TTAGCCGCTA CAAGATCGTA AAGAGCGAAA
4141 CCGGGCGGCC GGAGTACATC GAGATCGAGC TAGCTGATTG GATGTACCGC GAGATCACAG
4201 AAGGCAAGAA CCCGGACGTG CTGACGGTTC ACCCCGATTA CTTTTTGATC GATCCCGGCA
4261 TCGGCCGTTT TCTCTACCGC CTGGCACGCC GCGCCGCAGG CAAGGCAGAA GCCAGATGGT
4321 TGTTCAAGAC GATCTACGAA CGCAGTGGCA GCGCCGGAGA GTTCAAGAAG TTCTGTTTCA
4381 CCGTGCGCAA GCTGATCGGG TCAAATGACC TGCCGGAGTA CGATTTGAAG GAGGAGGCGG
4441 GGCAGGCTGG CCCGATCCTA GTCATGCGCT ACCGCAACCT GATCGAGGGC GAAGCATCCG
4501 CCGGTTCCTA ATGTACGGAG CAGATGCTAG GGCAAATTGC CCTAGCAGGG GAAAAAGGTC
4561 GAAAAGGTCT CTTTCCTGTG GATAGCACGT ACATTGGGAA CCCAAAGCCG TACATTGGGA
4621 ACCGGAACCC GTACATTGGG AACCCAAAGC CGTACATTGG GAACCGGTCA CACATGTAAG
4681 TGACTGATAT AAAAGAGAAA AAAGGCGATT TTTCCGCCTA AAACTCTTTA AAACTTATTA
4741 AAACTCTTAA AACCCGCCTG GCCTGTGCAT AACTGTCTGG CCAGCGCACA GCCGAAGAGC
4801 TGCAAAAAGC GCCTACCCTT CGGTCGCTGC GCTCCCTACG CCCCGCCGCT TCGCGTCGGC
4861 CTATCGCGGC CGCTGGCCGC TCAAAAATGG CTGGCCTACG GCCAGGCAAT CTACCAGGGC
4921 GCGGACAAGC CGCGCCGTCG CCACTCGACC GCCGGCGCCC ACATCAAGGC ACCCTGCCTC
4981 GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC AGCTCCCGGA GACGGTCACA
5041 GCTTGTCTGT AAGCGGATGC CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT
5101 GGCGGGTGTC GGGGCGCAGC CATGACCCAG TCACGTAGCG ATAGCGGAGT GTATACTGGC
5161 TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC
5221 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCTC TTCCGCTTCC TCGCTCACTG
5281 ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA
5341 TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC
5401 AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC
5461 CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT
5521 AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT CCGACCCTGC
5581 CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT TCTCATAGCT
5641 CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG
5701 AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC
5761 CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA
5821 GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC TACACTAGAA
5881 GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA
5941 GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC
6001 AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT ACGGGGTCTG
6061 ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGCATTCT AGGTACTAAA
6121 ACAATTCATC CAGTAAAATA TAATATTTTA TTTTCTCCCA ATCAGGCTTG ATCCCCAGTA
6181 AGTCAAAAAA TAGCTCGACA TACTGTTCTT CCCCGATATC CTCCCTGATC GACCGGACGC
6241 AGAAGGCAAT GTCATACCAC TTGTCCGCCC TGCCGCTTCT CCCAAGATCA ATAAAGCCAC
6301 TTACTTTGCC ATCTTTCACA AAGATGTTGC TGTCTCCCAG GTCGCCGTGG GAAAAGACAA
6361 GTTCCTCTTC GGGCTTTTCC GTCTTTAAAA AATCATACAG CTCGCGCGGA TCTTTAAATG
6421 GAGTGTCTTC TTCCCAGTTT TCGCAATCCA CATCGGCCAG ATCGTTATTC AGTAAGTAAT
6481 CCAATTCGGC TAAGCGGCTG TCTAAGCTAT TCGTATAGGG ACAATCCGAT ATGTCGATGG
6541 AGTGAAAGAG CCTGATGCAC TCCGCATACA GCTCGATAAT CTTTTCAGGG CTTTGTTCAT
6601 CTTCATACTC TTCCGAGCAA AGGACGCCAT CGGCCTCACT CATGAGCAGA TTGCTCCAGC
6661 CATCATGCCG TTCAAAGTGC AGGACCTTTG GAACAGGCAG CTTTCCTTCC AGCCATAGCA
6721 TCATGTCCTT TTCCCGTTCC ACATCATAGG TGGTCCCTTT ATACCGGCTG TCCGTCATTT
6781 TTAAATATAG GTTTTCATTT TCTCCCACCA GCTTATATAC CTTAGCAGGA GACATTCCTT
6841 CCGTATCTTT TACGCAGCGG TATTTTTCGA TCAGTTTTTT CAATTCCGGT GATATTCTCA
6901 TTTTAGCCAT TTATTATTTC CTTCCTCTTT TCTACAGTAT TTAAAGATAC CCCAAGAAGC
6961 TAATTATAAC AAGACGAACT CCAATTCACT GTTCCTTGCA TTCTAAAACC TTAAATACCA
7021 GAAAACAGCT TTTTCAAAGT TGTTTTCAAA GTTGGCGTAT AACATAGTAT CGACGGAGCC
7081 GATTTTGAAA CCGCGGTGAT CACAGGCAGC AACGCTCTGT CATCGTTACA ATCAACATGC
7141 TACCCTCCGC GAGATCATCC GTGTTTCAAA CCCGGCAGCT TAGTTGCCGT TCTTCCGAAT
7201 AGCATCGGTA ACATGAGCAA AGTCTGCCGC CTTACAACGG CTCTCCCGCT GACGCCGTCC
7261 CGGACTGATG GGCTGCCTGT ATCGAGTGGT GATTTTGTGC CGAGCTGCCG GTCGGGGAGC
7321 TGTTGGCTGG CTGGTGGCAG GATATATTGT GGTGTAAACA AATTGACGCT TAGACAACTT
7381 AATAACACAT TGCGGACGTT TTTAATGTAC TGAATTAACG CCGAATTAAT TCGGGGGATC
7441 TGGATTTTAG TACTGGATTT TGGTTTTAGG AATTAGAAAT TTTATTGATA GAAGTATTTT
7501 ACAAATACAA ATACATACTA AGGGTTTCTT ATATGCTCAA CACATGAGCG AAACCCTATA
7561 GGAACCCTAA TTCCCTTATC TGGGAACTAC TCACACATTA TTATGGAGAA ACTCGAGCTT
7621 GTCGATCGAC AGATCCGGTC GGCATCTACT CTATTTCTTT GCCCTCGGAC GAGTGCTGGG
7681 GCGTCGGTTT CCACTATCGG CGAGTACTTC TACACAGCCA TCGGTCCAGA CGGCCGCGCT
7741 TCTGCGGGCG ATTTGTGTAC GCCCGACAGT CCCGGCTCCG GATCGGACGA TTGCGTCGCA
7801 TCGACCCTGC GCCCAAGCTG CATCATCGAA ATTGCCGTCA ACCAAGCTCT GATAGAGTTG
7861 GTCAAGACCA ATGCGGAGCA TATACGCCCG GAGTCGTGGC GATCCTGCAA GCTCCGGATG
7921 CCTCCGCTCG AAGTAGCGCG TCTGCTGCTC CATACAAGCC AACCACGGCC TCCAGAAGAA
7981 GATGTTGGCG ACCTCGTATT GGGAATCCCC GAACATCGCC TCGCTCCAGT CAATGACCGC
8041 TGTTATGCGG CCATTGTCCG TCAGGACATT GTTGGAGCCG AAATCCGCGT GCACGAGGTG
8101 CCGGACTTCG GGGCAGTCCT CGGCCCAAAG CATCAGCTCA TCGAGAGCCT GCGCGACGGA
8161 CGCACTGACG GTGTCGTCCA TCACAGTTTG CCAGTGATAC ACATGGGGAT CAGCAATCGC
8221 GCATATGAAA TCACGCCATG TAGTGTATTG ACCGATTCCT TGCGGTCCGA ATGGGCCGAA
8281 CCCGCTCGTC TGGCTAAGAT CGGCCGCAGC GATCGCATCC ATAGCCTCCG CGACCGGTTG
8341 TAGAACAGCG GGCAGTTCGG TTTCAGGCAG GTCTTGCAAC GTGACACCCT GTGCACGGCG
8401 GGAGATGCAA TAGGTCAGGC TCTCGCTAAA CTCCCCAATG TCAAGCACTT CCGGAATCGG
8461 GAGCGCGGCC GATGCAAAGT GCCGATAAAC ATAACGATCT TTGTAGAAAC CATCGGCGCA
8521 GCTATTTACC CGCAGGACAT ATCCACGCCC TCCTACATCG AAGCTGAAAG CACGAGATTC
8581 TTCGCCCTCC GAGAGCTGCA TCAGGTCGGA GACGCTGTCG AACTTTTCGA TCAGAAACTT
8641 CTCGACAGAC GTCGCGGTGA GTTCAGGCTT TTTCATATCT CATTGCCCCC CGGGATCTGC
8701 GAAAGCTCGA GAGAGATAGA TTTGTAGAGA GAGACTGGTG ATTTCAGCGT GTCCTCTCCA
8761 AATGAAATGA ACTTCCTTAT ATAGAGGAAG GTCTTGCGAA GGATAGTGGG ATTGTGCGTC
8821 ATCCCTTACG TCAGTGGAGA TATCACATCA ATCCACTTGC TTTGAAGACG TGGTTGGAAC
8881 GTCTTCTTTT TCCACGATGC TCCTCGTGGG TGGGGGTCCA TCTTTGGGAC CACTGTCGGC
8941 AGAGGCATCT TGAACGATAG CCTTTCCTTT ATCGCAATGA TGGCATTTGT AGGTGCCACC
9001 TTCCTTTTCT ACTGTCCTTT TGATGAAGTG ACAGATAGCT GGGCAATGGA ATCCGAGGAG
9061 GTTTCCCGAT ATTACCCTTT GTTGAAAAGT CTCAATAGCC CTTTGGTCTT CTGAGACTGT
9121 ATCTTTGATA TTCTTGGAGT AGACGAGAGT GTCGTGCTCC ACCATGTTAT CACATCAATC
9181 CACTTGCTTT GAAGACGTGG TTGGAACGTC TTCTTTTTCC ACGATGCTCC TCGTGGGTGG
9241 GGGTCCATCT TTGGGACCAC TGTCGGCAGA GGCATCTTGA ACGATAGCCT TTCCTTTATC
9301 GCAATGATGG CATTTGTAGG TGCCACCTTC CTTTTCTACT GTCCTTTTGA TGAAGTGACA
9361 GATAGCTGGG CAATGGAATC CGAGGAGGTT TCCCGATATT ACCCTTTGTT GAAAAGTCTC
9421 AATAGCCCTT TGGTCTTCTG AGACTGTATC TTTGATATTC TTGGAGTAGA CGAGAGTGTC
9481 GTGCTCCACC ATGTTGGCAA GCTGCTCTAG CCAATACGCA AACCGCCTCT CCCCGCGCGT
9541 TGGCCGATTC ATTAATGCAG CTGGCACGAC AGGTTTCCCG ACTGGAAAGC GGGCAGTGAG
9601 CGCAACGCAA TTAATGTGAG TTAGCTCACT CATTAGGCAC CCCAGGCTTT ACACTTTATG
9661 CTTCCGGCTC GTATGTTGTG TGGAATTGTG AGCGGATAAC AATTTCACAC AGGAAACAGC
9721 TATGACCATG ATTACGAATT CGCAATTGAG ACTTTTCAAC AAAGGGTAAT ATCCGGAAAC
9781 CTCCTCGGAT TCCATTGCCC AGCTATCTGT CACTTTATTG TGAAGATAGT GGAAAAGGAA
9841 GGTGGCTCCT ACAAATGCCA TCATTGCGAT AAAGGAAAGG CCATCGTTGA AGATGCCTCT
9901 GCCGACAGTG GTCCCAAAGA TGGACCCCCA CCCACGAGGA GCATCGTGGA AAAAGAAGAC
9961 GTTCCAACCA CGTCTTCAAA GCAAGTGGAT TGATGTGATA TCTCCACTGA CGTAAGGGAT
10021 GACGCACAAT CCCACTATCC TTCGCAAGAC CCTTCCTCTA TATAAGGAAG TTCATTTCAT
10081 TTGGAGAGAA CACGGGGGAC GAGCTCGGTA CCCGGGGATC CTCTAGAGTC GACCTGCAGA
10141 GCTTTCGTTC GTATCATCGG TTTCGACAAC GTTCGTCAAG TTCAATGCAT CAGTTTCATT
10201 GCGCACACAC CAGAATCCTA CTGAGTTCGA GTATTATGGC ATTGGGAAAC ATGTTTTTCT
10261 TGTACCATTT GTTGTGCTTG TAATTTACTG TGTTTTTTAT TCGGTTTTCG CTATCGAACT
10321 GTGAAATGGA AATGGATGGA GAAGAGTTAA TGAATGATAT GGTCCTTTTG TTCATTCTCA
10381 AATTAATATT ATTTGTTTTT TCTCTTATTT GTTGTGTGTT GAATTTGAAA ATATAAGAGA
10441 TATGCAAACA TTTTGTTTTG AGTAAAAATG TGTCAAATCG TGGCCTCTAA TGACCGAAGT
10501 TAATATGAGG AGTAAAACAC TTGTAGTTGT ACCATTATGC TTATTCACTA GGCAACAAAT
10561 ATATTTTCAG ACCTAGAAAA GCTGCAAATG TTACTGAATA CAAGTATGTC CTCTTGTGTT
10621 TTAGACATTT ATGAACTTTC CTTTATGTAA TTTTCCAGAA TCCTTGTCAG ATTCTAATCA
10681 TTGCTTTATA ATTATAGTTA TACTCATGGA TTTGTAGTTG AGTATGAAAA TATTTTTTAA
10741 TGCATTTTAT GACTTGCCAA TTGATTGACA ACATGCATCA ATCGAAGAAG CTTGGCACTG
10801 GCCGTCGTTT TACAACGTCG TGACTGGGAA AACCCTGGCG TTACCCAACT TAATCGCCTT
10861 GCAGCACATC CCCCTTTCGC CAGCTGGCGT AATAGCGAAG AGGCCCGCAC CGATCGCCCT
10921 TCCCAACAGT TGCGCAGCCT GAATGGCGAA TGCTAGAGCA GCTTGAGCTT GGATCAGATT
10981 GTCGTTTCCC GCCTTCAGTT TAGCTTCATG GAGTCAAAGA TTCAAATAGA GGACCTAACA
11041 GAACTCGCCG TAAAGACTGG CGAACAGTTC ATACAGAGTC TCTTACGACT CAATGACAAG
11101 AAGAAAATCT TCGTCAACAT GGTGGAGCAC GACACACTTG TCTACTCCAA AAATATCAAA
11161 GATACAGTCT CAGAAGACCA AAGGGCAATT GAGACTTTTC AACAAAGGGT AATATCCGGA
11221 AACCTCCTCG GATTCCATTG CCCAGCTATC TGTCACTTTA TTGTGAAGAT AGTGGAAAAG
11281 GAAGGTGGCT CCTACAAATG CCATCATTGC GATAAAGGAA AGGCCATCGT TGAAGATGCC
11341 TCTGCCGACA GTGGTCCCAA AGATGGACCC CCACCCACGA GGAGCATCGT GGAAAAAGAA
11401 GACGTTCCAA CCACGTCTTC AAAGCAAGTG GATTGATGTG ATATCTCCAC TGACGTAAGG
11461 GATGACGCAC AATCCCACTA TCCTTCGCAA GACCCTTCCT CTATATAAGG AAGTTCATTT
11521 CATTTGGAGA GAACACGGGG GACTCTTGAC
Claims (2)
1. a kind of Agrobacterium Ti plasmid carrier PCHF1302 construction method, it is characterised in that include the following steps:
It S1. include the acquisition of the expression cassette of 35S promoter, E9 terminator and multiple cloning sites MCS
S11. Agrobacterium Ti plasmid Pcambia1302 is made into recombinant plasmid compared with the region MCS of PCHF3300
There are most restriction enzyme sites in the region PCHF1302MCS；Finally in the MCS regional choice EcoRI of Agrobacterium Ti plasmid Pcambia1302
With the two restriction enzyme sites of HindIII；
S12. 18bp sequence is selected to be denoted as B-F as the homology arm of upstream primer in EcoRI restriction enzyme site, in HindIII digestion
The downstream 18bp sequence in site is denoted as B-R as the homology arm of downstream primer, and in the upstream expression cassette 35S PCHF3300, selection bp makees
For upstream primer 35S-F, select bp as downstream primer E9-R in PCHF3300 expression cassette E9 terminator downstream；
Upstream primer F (B-F+35S-F): TGACCATGATTACGAATTAAAGGGCAATTG is obtained from above
Downstream primer R (B-R+E9-R): GACGGCCAGTGCCAAGCTAGCTTCGATTGA
S13. using PCHF3300 plasmid as template, with KOD-FX high fidelity enzyme will include 35S promoter, E9 terminator and
The complete gene expression frame of MCS adds homology arm
The acquisition of S2.Pcambia1302 carrier framework
Pcambia1302 is subjected to double digestion with restriction enzyme EcoRI and HindIII, is cut off in EcoRI and HindIII
Between MCS, by the Pcambia1302 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through
Conventional gel recovery method recycling；
The building of S3.PCHF1302 plasmid
S31. seamless Cloning Kit is utilized, by gel extraction includes 35S promoter, E9 whole by the method for homologous recombination
Only the expression cassette of son and multiple cloning sites MCS are connected on Pcambia1302 carrier framework, and the recombinant plasmid after connection utilizes heat shock
Method is transformed into the competent cell of escherichia coli DH5a bacterial strain；
S32. the Escherichia coli after conversion are put down and is applied to the LB culture medium culture containing 50mg/L kanamycins, the independent bacterium colony of picking
Bacterium colony PCR identification is carried out, the positive bacterium colony containing 35S-MCS-E9 recombinant plasmid is screened；
S33. positive bacterium colony is sent and is sequenced in sequencing company, to survey after the expansion of the LB liquid medium of 50mg/L kanamycins is numerous
The correct bacterium solution of sequence extracts recombinant plasmid vector DNA using plasmid extraction kit, which is named as
PCHF1302。
2. Agrobacterium Ti plasmid carrier PCHF1302 as described in claim 1 is in the application of plant genetic engineering field.
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