CN106244624B - Plasmid system and its application for the building of plant polygene expression vector - Google Patents
Plasmid system and its application for the building of plant polygene expression vector Download PDFInfo
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Abstract
The invention discloses a kind of plasmid systems and its application for the building of plant polygene expression vector.By being transformed to e. coli plasmid vector pUC18 and double-core Agrobacterium Ti plasmid pBI121, recombinant plasmid pKAFCR0 and pKAFCR100 are formd.PKAFCR0 has the most common 35S promoter of gene expression in plants and NOS terminator, and at its upstream, intermediate and downstream introduce multiple clone's restriction enzyme sites, can be connected in many ways among the 35S and NOS of pKAFCR0 using the target gene fragment that the methods of PCR clone obtains；PKAFCR100 has kanamycins NPT II tolerance gene and sGFP Green fluorescent protein fusion vector and in-between polyclone enzyme enzyme site.Two plasmids of pKAFCR0 and pKAFCR100 match, and construct the single or multiple expression vectors of plant with can be convenient.
Description
Technical field
The present invention relates to biological fields, and in particular to it is a kind of for plant polygene expression vector building plasmid system and
It is applied.
Background technique
The rapid development and extensive use of plant gene engineering technology are that the genetic improvement of plant has opened up wide prospect.
The technology overcomes the limitation of plant sexual hybridization, can will derive from the even artificial synthesized gene transfered plant of different plant species,
To improve plant trait, good quality and high output New Crop Varieties are cultivated.In gene cloning, gene function analysis and genetic transformation etc.
In the research and application process of molecular biology, vector construction is common technological means, using suitable expression vector and structure
Construction method can make experiment effect get twice the result with half the effort.But it is usually used in the plasmid of plant gene expression vector building, such as pBIN19 at present
(Bevan, 1984), pBI121 (Jefferson, 1987), pIG121-Hm (Ohta et al., 1999), pMSIsGFP
Plasmid vectors such as (Kawasaki et al., 1999), since restriction endonuclease sites are limited in its field T-DNA, purpose base
Because segment is difficult to be inserted into and connects, also need to construct multiple intermediate vectors sometimes, operation is more troublesome, and these classical matter
Grain carrier is only limited to not be commercialized in researcher in the ranks transferring the possession of mutually at present, not fully up to expectations in use.For this purpose, one
A little international bio major companies, such as Invitrogen founding of the company TOPO clone (Patel, 2009), Gateway recombinant technique
(Chung et al., 2005), ClonTech founding of the company Infusion round pcr (Berrow et al., 2007) etc..
Although TOPO clone is simple, purpose carrier is limited in scope, Gateway and infusion PCR can not consider restriction enzyme site,
But the technology and carrier of these enterprise developments, are primarily directed to the building of the expression vector of animal, microorganism, lack plant
The function element such as promoter necessary to object gene expression, terminator, selection markers, and all more expensive (3000- of expense
10000 yuan or so), in addition can be only applied to research purpose since patent limits, above it is restricted in application.In addition, at present
The technology of these common plasmid vectors and exploitation can only largely construct individual gene expression vector, however in plant gene
In improvement, many characters are to require the coordinate expression of several related genes, it is often necessary to structure by multiple gene co- controllings
Build multiple expression vectors.
Summary of the invention
To solve the above problems, the present invention passes through to most common e. coli plasmid vector pUC18 and double-core Agrobacterium
Ti-plasmids pBI121 carries out comprehensive transformation, and is inserted into the function such as the common promoter of gene expression in plants, terminator, selection markers
Energy element, while multiple clone's restriction enzyme sites are introduced, it establishes a set of for the single or multiple expression vectors buildings of plant
Plasmid system, and provide the application of the plasmid system.
To achieve the above object, the technical scheme adopted by the invention is as follows:
For the plasmid system of plant polygene expression vector building, including pKAFCR0 plasmid and pKAFCR100 plasmid；
The pKAFCR0 plasmid is constructed by following steps:
S11, using restriction enzyme Hind III and EcoR I, to e. coli plasmid vector pUC18 (Yanisch-
Perron et al., 1985) DNA carries out double digestion processing；Main purpose is excision its multiple cloning sites MCS (multiple
Cloning site) the part Hind III to EcoR I；
S12, also with restriction enzyme Hind III and EcoR I, to double-core Agrobacterium Ti plasmid pBI121 DNA
Carry out double digestion processing；Main purpose is to cut that it includes 35S promoters most-often used when plant transgene
(cauliflower mosaic virus 35S promoter), GUS (β-glucuronidase) reporter gene and NOS are terminated
The segment of sub (nopaline synthase terminator).
S13, by the pUC18 after double digestion, and the pBI121 after same double digestion, through 1% agarose gel electrophoresis
After separation, purpose band is cut, passes through gel recovery method or gel reclaims kit (such as QIAGEN QIAquick Gel
Extraction Kit) obtain the 35S-GUS- for having cut off the pUC18 of the part Hind III-EcoR I and having cut from pBI121
NOS segment；
S14, using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit), will
The 35S-GUS-NOS segment for having cut off the pUC18 of the part Hind III-EcoR I and having cut from pBI121 is attached；Connection
Recombinant plasmid afterwards, is transformed into the competent cell of e.colistraindh5α using thermal excitation；
S15, flat be applied on the LB solid medium containing 25mg/L penicillin of the Escherichia coli after conversion is cultivated, picking
Independent bacterium colony carries out bacterium colony PCR identification, and containing recombinant plasmid, (35S-GUS-NOS segment has been inserted into the Hind of pUC18 for screening
III and EcoR I site) positive bacterium colony；
S16, by positive bacterium colony through the LB liquid medium of 25mg/L penicillin expand it is numerous after, utilize conventional plasmid extraction side
Method or plasmid extraction kit (such as QIAGEN Qiaprep Spin Miniprep Kit) extract recombinant plasmid dna, the recombination
Plasmid is named as pKAFCR1, i.e., on Hind III and the EcoR I site of pUC18, is inserted into 35S-GUS-NOS segment；
S17, using restriction endonuclease sma I and Sac I, double digestion processing is carried out to recombinant plasmid pKAFCR1 DNA；
Main purpose is excision gus reporter gene segment.
S18, by the pKAFCR1 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through
Gel recovery method or gel reclaims kit obtain the pKAFCR1 for having cut off gus reporter gene segment；
S19, artificial synthesized section of DNA, the segment contain restriction endonuclease sma I, Kpn I, Xho I and Sac I
Point；Using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit) by the DNA fragmentation
It is attached with the pKAFCR1 of excision gus reporter gene；Recombinant plasmid after connection is transformed into Escherichia coli using thermal excitation
In the competent cell of DH5 α bacterial strain；Escherichia coli after conversion are put down and are applied to the LB solid medium containing 25mg/L penicillin
Upper culture, the independent bacterium colony of picking carry out bacterium colony PCR identification, screen the positive bacterium colony containing recombinant plasmid；Positive bacterium colony is passed through
After the LB liquid medium expansion of 25mg/L penicillin is numerous, (such as using conventional plasmid extracting method or plasmid extraction kit
QIAGEN Qiaprep Spin Miniprep Kit) extract recombinant plasmid dna；The recombinant plasmid is named as pKAFCR2, that is, goes
In addition to the gus reporter gene of pKAFCR1, and Kpn I and Xho I restriction enzyme site is increased on this position；
S120, using restriction enzyme Hind III and Pst I, double digestion is carried out to recombinant plasmid pKAFCR2 DNA
Processing；
S121, by the pKAFCR2 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through
Gel recovery method or gel reclaims kit obtain the pKAFCR2 of digestion.
S122, artificial synthesized section of DNA, the segment contain restriction enzyme Hind III, Hpa I, Stu I, Sph
I, Pst I and Sbf I site；Utilize T4-DNA ligase or plasmid connection kit (such as TOYOBO Ligation High
Kit) DNA fragmentation and the pKAFCR2 of digestion are attached；Recombinant plasmid after connection is transformed into greatly using thermal excitation
In the competent cell of enterobacteria DH5 α bacterial strain；Escherichia coli after conversion are put down and are applied to the LB solid containing 25mg/L penicillin
It is cultivated on culture medium, the independent bacterium colony of picking carries out bacterium colony PCR identification, screens the positive bacterium colony containing recombinant plasmid；By positive bacteria
It falls after the expansion of the LB liquid medium of 25mg/L penicillin is numerous, utilizes conventional plasmid extracting method or plasmid extraction kit
(such as QIAGEN Qiaprep Spin Miniprep Kit) extracts recombinant plasmid dna；The recombinant plasmid is named as pKAFCR0,
Hpa I, Stu I and Sph I restriction enzyme site are increased in the upstream 35S；PKAFCR0 in the upstream 35S, among 35S and NOS and
Polyclone enzyme enzyme site is all had in the downstream NOS；
The pKAFCR100 plasmid is constructed by following steps:
S21, using restriction enzyme Hind III and EcoR I, double-core Agrobacterium Ti plasmid pBI121 DNA is carried out
Double digestion processing.Main purpose is to cut off its 35S-GUS-NOS segment.
S22, using restriction enzyme Hind III and EcoR I, to double-core Agrobacterium Ti plasmid pBsGFP
(Kajiyama et al., 2007) DNA carries out double digestion processing；Main purpose is to cut its 35S Ω promoter (35S
promoter with an additional omega element translational enhancer)、sGFP
(synthetic green-fluorescent protein with S65T mutation) reporter gene and NOS terminator
Segment.
S23, by the pBI121 after double digestion, and the pBsGFP after same double digestion, through 1% agarose gel electrophoresis
After separation, purpose band is cut, passes through gel recovery method or gel reclaims kit (such as QIAGEN QIAquick Gel
Extraction Kit), obtain the pBI121 for having cut off 35S-GUS-NOS segment, and the 35S Ω-cut from pBsGFP
SGFP-NOS segment；
S24, using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit), will
35S Ω-sGFP-NOS the segment for having cut off the pBI121 of 35S-GUS-NOS segment and having cut from pBsGFP is attached；Connection
Recombinant plasmid afterwards, is transformed into the competent cell of e.colistraindh5α using thermal excitation；By the large intestine bar after conversion
Flat be applied on the LB solid medium containing 50mg/L kanamycins of bacterium is cultivated, and the independent bacterium colony of picking carries out bacterium colony PCR identification, sieve
Select the positive bacterium colony containing recombinant plasmid；By positive bacterium colony after the expansion of the LB liquid medium of 50mg/L kanamycins is numerous, utilize
Conventional plasmid extracting method or plasmid extraction kit (such as QIAGEN Qiaprep Spin Miniprep Kit) extracts weight
Group Plasmid DNA, the recombinant plasmid are named as pKAFCR31, i.e., the 35S-GUS of pBI121 are replaced as 35S Ω-sGFP；
S25, using restriction enzyme Hind III and Pst I, double digestion is carried out to recombinant plasmid pKAFCR31 DNA
Processing；
S26, the plasmid pKAFCR31 after double digestion is cut into purpose item after 1% agarose gel electrophoresis separation
Band is obtained by gel recovery method or gel reclaims kit (such as QIAGEN QIAquick Gel Extraction Kit)
Obtain the pKAFCR31 of digestion；
S27, artificial synthesized section of DNA, the segment contain restriction enzyme Hind III, Hpa I, Stu I, Sph I
With Sbf I site；It, will using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit)
The DNA fragmentation and the pKAFCR31 of digestion are attached；Recombinant plasmid after connection is transformed into Escherichia coli using thermal excitation
In the competent cell of DH5 α bacterial strain；Escherichia coli after conversion are put down and are applied to the LB solid culture containing 50mg/L kanamycins
It is cultivated on base, the independent bacterium colony of picking carries out bacterium colony PCR identification, screens the positive bacterium colony containing recombinant plasmid；Positive bacterium colony is passed through
After the LB liquid medium expansion of 50mg/L kanamycins is numerous, (such as using conventional plasmid extracting method or plasmid extraction kit
QIAGEN Qiaprep Spin Miniprep Kit) recombinant plasmid dna is extracted, which is named as pKAFCR100, i.e.,
Hpa I, Stu I and Sph I restriction enzyme site are increased in 35S Ω promoter upstream；PKAFCR100 have the card independently expressed that
Mycin NPT II (neomycin phosphotransferase II) tolerance gene and sGFP Green fluorescent protein fusion vector,
And in-between polyclone enzyme enzyme site.
The above-mentioned plasmid system for the building of plant polygene expression vector can be used for the single or multiple gene tables of plant
Up to the building of carrier.
The invention has the following advantages:
1, the 35S promoter and NOS that the recombinant plasmid pKAFCR0 that transformation is formed has gene expression in plants most-often used are whole
It is only sub, it can easily be connected to pKAFCR0's in many ways using the target gene fragment that the methods of PCR clone obtains
Among 35S and NOS.
2, the recombinant plasmid pKAFCR100 that transformation is formed have the kanamycins NPT II tolerance gene independently expressed and
SGFP Green fluorescent protein fusion vector and in-between polyclone enzyme enzyme site.
3, matching using pKAFCR0 and pKAFCR100 plasmid constructs the single or multiple bases of plant in which can be convenient
Because of expression vector.Thus the expression vector of method building, the upstream and downstream of target gene are connected separately in plant
Promoter and terminator necessary to gene expression enable the target gene of insertion to stablize expression in plant；Have simultaneously
There are the selection markers such as the kanamycins NPT II tolerance gene independently expressed and sGFP Green fluorescent protein fusion vector, can be used for
The choice and observation of transgenic protocol cell, effectively improve transgene efficiency.
4, the 35S promoter of target gene upstream and downstream and NOS terminator, NPT II antibiotic resistance gene and sGFP
There is restriction enzyme site before and after Green fluorescent protein fusion vector, can be needed to select more suitable Genetic elements to it according to experiment
It is replaced.
Detailed description of the invention
Fig. 1 is pKAFCR0 plasmid multiple cloning sites and pKAFCR100 plasmid T-DNA schematic diagram in the embodiment of the present invention.
Fig. 2 is SrUGT76G1, SrUGT85C2 and SrUGT91D2m full length gene PCR amplification in the embodiment of the present invention.Figure
In: M:DNA Marker；1:SrUGT76G1；2:SrUGT85C2；3:SrUGT91D2m.
Fig. 3 is that SrUGT76G1, SrUGT85C2 and SrUGT91D2m target gene are connected individually in the embodiment of the present invention
Bacterium colony PCR identification after pKAFCR0 plasmid.In figure: M:DNA Marker；1-4:SrUGT76G1 (4 be feminine gender)；5-8:
SrUGT85C2 (6 be feminine gender)；9-12:SrUGT91D2m.
Fig. 4 is SrUGT76G1, SrUGT85C2 and SrUGT91D2m assortment of genes PCR amplification in the embodiment of the present invention.Figure
In: M:DNA Marker；1-3:SrUGT76G1；4-6:SrUGT91D2m；7-9:SrUGT85C2.
Fig. 5 is that the combination of SrUGT76G1, SrUGT85C2 and SrUGT91D2m target gene individually connects in the embodiment of the present invention
Bacterium colony PCR identification after being connected to pKAFCR100 plasmid.In figure: M:DNA Marker；1-3:SrUGT76G1；4-6:
SrUGT85C2；7-9:SrUGT91D2m (7 be feminine gender).
Fig. 6 is that SrUGT76G1, SrUGT85C2 and SrUGT91D2m target gene combine independent structure in the embodiment of the present invention
Build the recombinant plasmid schematic diagram to pKAFCR100.In figure: upper: pNULPGE01101 (pKAFCR100+SrUGT76G1 gene
Combination)；In: pNULPGE01102 (the pKAFCR100+SrUGT85C2 assortment of genes)；Under: pNULPGE01103 (pKAFCR100+
The SrUGT91D2m assortment of genes).
Fig. 7 is that 3 target gene combinations of SrUGT76G1, SrUGT85C2 and SrUGT91D2m are complete in the embodiment of the present invention
Portion is connected to the bacterium colony PCR identification after pKAFCR100 plasmid.In figure: M:DNA Marker；1-5:SrUGT76G1；6-10:
SrUGT85C2；11-15:SrUGT91D2m.
Fig. 8 is that 3 target gene combinations of SrUGT76G1, SrUGT85C2 and SrUGT91D2m are complete in the embodiment of the present invention
Portion is connected to the recombinant plasmid pNULPGE01105 schematic diagram after pKAFCR100.
Specific embodiment
In order to which objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
It is described in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
For the plasmid system of plant polygene expression vector building, including pKAFCR0 plasmid and pKAFCR100 plasmid；
The pKAFCR0 plasmid is constructed by following steps:
S11, using restriction enzyme Hind III and EcoR I, to e. coli plasmid vector pUC18 (Yanisch-
Perron et al., 1985) DNA carries out double digestion processing；Main purpose is excision its multiple cloning sites MCS (multiple
Cloning site) the part Hind III to EcoR I；
S12, also with restriction enzyme Hind III and EcoR I, to double-core Agrobacterium Ti plasmid pBI121 DNA
Carry out double digestion processing；Main purpose is to cut that it includes 35S promoters most-often used when plant transgene
(cauliflower mosaic virus 35S promoter), GUS (β-glucuronidase) reporter gene and NOS are terminated
The segment of sub (nopaline synthase terminator).
S13, by the pUC18 after double digestion, and the pBI121 after same double digestion, through 1% agarose gel electrophoresis
After separation, purpose band is cut, passes through gel recovery method or gel reclaims kit (such as QIAGEN QIAquick Gel
Extraction Kit) obtain the 35S-GUS- for having cut off the pUC18 of the part Hind III-EcoR I and having cut from pBI121
NOS segment；
S14, using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit), will
The 35S-GUS-NOS segment for having cut off the pUC18 of the part Hind III-EcoR I and having cut from pBI121 is attached；Connection
Recombinant plasmid afterwards, is transformed into the competent cell of e.colistraindh5α using thermal excitation；
S15, flat be applied on the LB solid medium containing 25mg/L penicillin of the Escherichia coli after conversion is cultivated, picking
Independent bacterium colony carries out bacterium colony PCR identification, and containing recombinant plasmid, (35S-GUS-NOS segment has been inserted into the Hind of pUC18 for screening
III and EcoR I site) positive bacterium colony；
S16, by positive bacterium colony through the LB liquid medium of 25mg/L penicillin expand it is numerous after, utilize conventional plasmid extraction side
Method or plasmid extraction kit (such as QIAGEN Qiaprep Spin Miniprep Kit) extract recombinant plasmid dna, the recombination
Plasmid is named as pKAFCR1, i.e., on Hind III and the EcoR I site of pUC18, is inserted into 35S-GUS-NOS segment；
S17, using restriction endonuclease sma I and Sac I, double digestion processing is carried out to recombinant plasmid pKAFCR1 DNA；
Main purpose is excision gus reporter gene segment.
S18, by the pKAFCR1 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through
Gel recovery method or gel reclaims kit obtain the pKAFCR1 for having cut off gus reporter gene segment；
S19, artificial synthesized section of DNA, the segment contain restriction endonuclease sma I, Kpn I, Xho I and Sac I
Point；Using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit) by the DNA fragmentation
It is attached with the pKAFCR1 of excision gus reporter gene；Recombinant plasmid after connection is transformed into Escherichia coli using thermal excitation
In the competent cell of DH5 α bacterial strain；Escherichia coli after conversion are put down and are applied to the LB solid medium containing 25mg/L penicillin
Upper culture, the independent bacterium colony of picking carry out bacterium colony PCR identification, screen the positive bacterium colony containing recombinant plasmid；Positive bacterium colony is passed through
After the LB liquid medium expansion of 25mg/L penicillin is numerous, (such as using conventional plasmid extracting method or plasmid extraction kit
QIAGEN Qiaprep Spin Miniprep Kit) extract recombinant plasmid dna；The recombinant plasmid is named as pKAFCR2, that is, goes
In addition to the gus reporter gene of pKAFCR1, and Kpn I and Xho I restriction enzyme site is increased on this position；
S120, using restriction enzyme Hind III and Pst I, double digestion is carried out to recombinant plasmid pKAFCR2 DNA
Processing；
S121, by the pKAFCR2 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through
Gel recovery method or gel reclaims kit obtain the pKAFCR2 of digestion.
S122, artificial synthesized section of DNA, the segment contain restriction enzyme Hind III, Hpa I, Stu I, Sph
I, Pst I and Sbf I site；Utilize T4-DNA ligase or plasmid connection kit (such as TOYOBO Ligation High
Kit) DNA fragmentation and the pKAFCR2 of digestion are attached；Recombinant plasmid after connection is transformed into greatly using thermal excitation
In the competent cell of enterobacteria DH5 α bacterial strain；Escherichia coli after conversion are put down and are applied to the LB solid containing 25mg/L penicillin
It is cultivated on culture medium, the independent bacterium colony of picking carries out bacterium colony PCR identification, screens the positive bacterium colony containing recombinant plasmid；By positive bacteria
It falls after the expansion of the LB liquid medium of 25mg/L penicillin is numerous, utilizes conventional plasmid extracting method or plasmid extraction kit
(such as QIAGEN Qiaprep Spin Miniprep Kit) extracts recombinant plasmid dna；The recombinant plasmid is named as pKAFCR0,
Hpa I, Stu I and Sph I restriction enzyme site are increased in the upstream 35S；PKAFCR0 in the upstream 35S, among 35S and NOS and
Polyclone enzyme enzyme site is all had in the downstream NOS；As shown in Figure 1.
The pKAFCR100 plasmid is constructed by following steps:
S21, using restriction enzyme Hind III and EcoR I, double-core Agrobacterium Ti plasmid pBI121DNA is carried out
Double digestion processing.Main purpose is to cut off its 35S-GUS-NOS segment.
S22, using restriction enzyme Hind III and EcoR I, to double-core Agrobacterium Ti plasmid pBsGFP
(Kajiyama et al., 2007) DNA carries out double digestion processing；Main purpose is to cut its 35S Ω promoter (35S
promoter with an additional omega element translational enhancer)、sGFP
(synthetic green-fluorescent protein with S65T mutation) reporter gene and NOS terminator
Segment.
S23, by the pBI121 after double digestion, and the pBsGFP after same double digestion, through 1% agarose gel electrophoresis
After separation, purpose band is cut, passes through gel recovery method or gel reclaims kit (such as QIAGEN QIAquick Gel
Extraction Kit), obtain the pBI121 for having cut off 35S-GUS-NOS segment, and the 35S Ω-cut from pBsGFP
SGFP-NOS segment；
S24, using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit), will
35S Ω-sGFP-NOS the segment for having cut off the pBI121 of 35S-GUS-NOS segment and having cut from pBsGFP is attached；Connection
Recombinant plasmid afterwards is transformed into the competent cell of escherichia coli DH5a bacterial strain using thermal excitation；By the large intestine bar after conversion
Flat be applied on the LB solid medium containing 50mg/L kanamycins of bacterium is cultivated, and the independent bacterium colony of picking carries out bacterium colony PCR identification, sieve
Select the positive bacterium colony containing recombinant plasmid；By positive bacterium colony after the expansion of the LB liquid medium of 50mg/L kanamycins is numerous, utilize
Conventional plasmid extracting method or plasmid extraction kit (such as QIAGEN Qiaprep Spin Miniprep Kit) extracts weight
Group Plasmid DNA, the recombinant plasmid are named as pKAFCR31, i.e., the 35S-GUS of pBI121 are replaced as 35S Ω-sGFP；
S25, using restriction enzyme Hind III and Pst I, double digestion is carried out to recombinant plasmid pKAFCR31 DNA
Processing；
S26, the plasmid pKAFCR31 after double digestion is cut into purpose item after 1% agarose gel electrophoresis separation
Band is obtained by gel recovery method or gel reclaims kit (such as QIAGEN QIAquick Gel Extraction Kit)
Obtain the pKAFCR31 of digestion；
S27, artificial synthesized section of DNA, the segment contain restriction enzyme Hind III, Hpa I, Stu I, Sph I
With Sbf I site；It, will using T4-DNA ligase or plasmid connection with kit (such as TOYOBO Ligation High Kit)
The DNA fragmentation and the pKAFCR31 of digestion are attached；Recombinant plasmid after connection is transformed into Escherichia coli using thermal excitation
In the competent cell of DH5 α bacterial strain；Escherichia coli after conversion are put down and are applied to the LB solid culture containing 50mg/L kanamycins
It is cultivated on base, the independent bacterium colony of picking carries out bacterium colony PCR identification, screens the positive bacterium colony containing recombinant plasmid；Positive bacterium colony is passed through
After the LB liquid medium expansion of 50mg/L kanamycins is numerous, (such as using conventional plasmid extracting method or plasmid extraction kit
QIAGEN Qiaprep Spin Miniprep Kit) recombinant plasmid dna is extracted, which is named as pKAFCR100, i.e.,
Hpa I, Stu I and Sph I restriction enzyme site are increased in 35S Ω promoter upstream；PKAFCR100 have the card independently expressed that
Mycin NPT II (neomycin phosphotransferase II) tolerance gene and sGFP Green fluorescent protein fusion vector,
And in-between polyclone enzyme enzyme site, as shown in Figure 1.
The above-mentioned plasmid system for the building of plant polygene expression vector can be used for the single or multiple gene tables of plant
Up to the building of carrier, specifically there are following methods:
1, the upstream and downstream of target gene adds promoter and terminator (target gene and pKAFCR0 plasmid respectively
Connection)
Because pKAFCR0 has polyclone enzyme enzyme site among 35S and NOS, obtained mesh is cloned using the methods of PCR
Genetic fragment can easily be connected among the 35S and NOS of pKAFCR0, target gene upstream and downstream distinguish
In addition promoter necessary to plant gene expression in vivo and terminator:
It (1) is the connection of cohesive end after 5 ', 3 ' both ends digestions of target gene pcr amplification product
Target gene is introduced by 5 ' ends of forward and reverse primer in PCR amplification and pKAFCR0 is in 35S-NOS interphase
Same cohesive end restriction enzyme site (one of which of Xba I, BamH I, Kpn I, Xho I and Sac I), after expanding in this way
PCR product 5 ', 3 ' both ends after digestion are cohesive end, can be attached with the pKAFCR0 after same digestion.If
Two ends are identical restriction enzyme sites, also need to carry out the walking direction of Insert Fragment after connection.
(2) after 5 ', 3 ' both ends digestions of target gene pcr amplification product wherein one section be cohesive end, the other end is flat
The connection of sliding end
Target gene is introduced by 5 ' ends of forward primer or reverse primer in PCR amplification and pKAFCR0 exists
(forward primer introduces the one of which of Xba I and BamH I to identical cohesive end restriction enzyme site, or anti-among 35S-NOS
The one of which of Kpn I, Xho I and Sac I are introduced to primer), and another any smooth end of 5 ' end introducings to primer
Restriction enzyme site is held, wherein one end is cohesive end to the PCR product after expanding in this way after digestion, and the other end is smooth end, can
With with one end through same cohesive end digestion, after the smoothed end digestion of the other end (one of which of Xma I or Sma I)
PKAFCR0 is attached.
(3) be after 5 ', 3 ' both ends digestions of target gene pcr amplification product smooth end connection
Target gene introduces any smooth end in PCR amplification, through 5 ' ends of forward primer and reverse primer
Restriction enzyme site, two ends after digestion of the PCR product after amplification in this way are smooth end, can be with smoothed end digestion
PKAFCR0 after (one of which of Xma I or Sma I) is attached.Need to carry out the walking direction of Insert Fragment after connection.
(4) 5 ', 3 ' both ends of target gene pcr amplification product are the connection of smooth end
Using some archaeal dna polymerases (such as PHUSION high-fidelity DNA polymerase), smooth end can be generated after PCR amplification
The characteristics of holding product can directly carry out with the pKAFCR0 after smoothed end digestion (one of which of Xma I or Sma I)
Connection.Need to carry out the walking direction of Insert Fragment after connection.
Target gene in PCR amplification, can also be introduced by 5 ' ends of forward primer or reverse primer with
(forward primer introduces wherein the one of Xba I and BamH I to pKAFCR0 for identical cohesive end restriction enzyme site among 35S-NOS
Kind or reverse primer introduce the one of which of Kpn I, Xho I and Sac I), and another hold to the 5 ' of primer does not introduce digestion
Site is wherein cohesive end after the digestion of one end although generating the product that both ends are smooth end after such PCR amplification,
And the other end not digestion still maintains as smooth end, it can be with one end through same cohesive end digestion, the smoothed end of the other end
PKAFCR0 behind end digestion (one of which of Xma I or Sma I) is attached.
(5) sequencing after target gene is connect with pKAFCR0 plasmid is examined
After the connection of target gene and pKAFCR0 plasmid, the special primer of pKAFCR0 can use, such as CR0 MCS
Insert 35S-S (5 '-CAC AAT CCC ACT ATC CTT CGC A-3 ') and CR0MCS insert NOS-A (5 '-TCC
ACT CTA ATC ATA AAA ACC CAT CTC-3 '), or using the common primer of pUC18, such as M13-F (5 '-GTA
AAA CGA CGG CCA GT-3 and M13-R (5 '-GGA AAC AGC TAT GAC CAT G-3 ') are sequenced, and are surveyed
Whether sequence examines the target gene of insertion correct.
2, the building (connection of single target gene combination and pKAFCR100 plasmid) of plant individual gene expression vector
The upstream and downstream of target gene adds promoter respectively and the segment of terminator is known as the assortment of genes
(Cassette).After above-mentioned purpose gene is connect with pKAFCR0 plasmid, have using pKAFCR0 in the upstream 35S and the downstream NOS
Polyclone enzyme enzyme site (upstream 35S be Pvu II, Hind III, Hpa I, Stu I, Sph I and Pst I (Sbf I), NOS
Downstream is EcoR I and Pvu II), target gene, which is obtained, after digestion combines segment.After the assortment of genes segment and same digestion
PKAFCR100 plasmid be attached, the carrier of gene expression can be carried out in plant by finally obtaining.
Thus the expression vector of method building, the upstream and downstream of target gene are connected separately with gene in plant
Promoter necessary to expressing and terminator, while there is the kanamycins NPT II tolerance gene independently expressed and sGFP green
Fluorescent protein report gene, the former can use the culture medium added with kanamycins and selects to transgenic plant cells,
The latter can use fluorescence microscope and transgenic plant cells judged and observed.
In addition, the 35S promoter and NOS terminator of target gene upstream and downstream, NPT II antibiotic resistance gene and
There is restriction enzyme site before and after sGFP Green fluorescent protein fusion vector, can also be needed to select more suitable gene member according to experiment
Part is replaced (Fig. 1) to it.
3, the building (connections of multiple target gene combinations and pKAFCR100 plasmid) of the multiple expression vectors of plant
Cohesive end digestion after first target gene is connect with pKAFCR0 plasmid, using pKAFCR0 in the upstream 35S
The smooth end restriction enzyme site (Pvu II) in site (Hind III) and the downstream NOS carries out digestion；Or utilize the downstream NOS
After cohesive end restriction enzyme site (EcoR I) digestion, then smoothing techniques are carried out, obtaining one 5 ' and holding is cohesive end, 3 ' ends are
The target gene of smooth end combines segment.And pKAFCR100 plasmid then carries out same cohesive end digestion (Hind III),
With different smooth end digestions (Hpa I or Stu II), after the target gene combination segment and digestion after such digestion
After pKAFCR100 plasmid is attached, the novel plasmid of formation still retains original cohesive end digestion at 5 ' ends of junction
Site (Hind III), and 3 ' hold because being different smooth end digestion, smooth end restriction enzyme site disappears after connection.But
Since the target gene combination of insertion has same cohesive end restriction enzyme site (Hind III) in the upstream of 35S, and it is smooth
End restriction enzyme site (Hpa I or Stu II), second target gene after being connect with pKAFCR0 plasmid, equally using above-mentioned
Enzymatic cleavage methods obtain the target gene that one 5 ' end be cohesive end, 3 ' ends are smooth end and combine segment, are connected to existing the
On the plasmid of one target gene combination.
It is ingenious in this way using pKAFCR0 and pKAFCR100 plasmid with the identical restriction enzyme site in part, two plasmids are matched
Set uses, and multiple target gene can be combined segment and be connected to pKAFCR100 plasmid, be built into plant multi-gene expression load
Body.
If target gene combination segment and pKAFCR100 plasmid do not have matched restriction enzyme site, can also be again to mesh
Assortment of genes segment carry out PCR amplification.Using above-mentioned in PCR amplification, by forward primer 5 ' end introduce with
PKAFCR100 plasmid cohesive end restriction enzyme site having the same (Cla I or Ase I), and utilization can generate smooth end
The archaeal dna polymerase (such as PHUSION high-fidelity DNA polymerase) of product forms smooth end at 3 ' ends, in this way can also be constantly
Multiple target gene combination segment is connected to pKAFCR100 plasmid.
Embodiment
The clone of STEVIA REBAUDIANA glucosyltransferase SrUGT76G1, SrUGT85C2 and SrUGT91D2m gene and carrier structure
It builds:
1, the extraction and the synthesis of first chain of cDNA of STEVIA REBAUDIANA blade total serum IgE
Using the blade on mountain two in STEVIA REBAUDIANA strain as experimental material, dosage 80-100mg.Utilize RNAprep Pure
Polysaccharide polyphenol plant total RNA extraction reagent box (Tiangeng) extracts total serum IgE.The total serum IgE of extraction is through NanoDrop 2000 (Thermo)
After measuring concentration, with RNase-free ddH2O is diluted to 100ng/ μ L solution, utilizes TransScript Fly First-
The synthesis of Strand cDNA Synthesis SuperMix kit (Quan Shijin) progress first chain of cDNA.Concrete operations step
The rapid operation instruction for pressing kit.
2, target gene overall length PCR amplification and purification of products
(1) design of primers: STEVIA REBAUDIANA SrUGT76G1 (AY345974), SrUGT85C2 referring to disclosed in the databases such as NCBI
(AY345978) and the sequence class of SrUGT91D2m (EU751291) gene each gene PCR amplimer, is designed, 5 ' ends of primer are drawn
Enter and pKAFCR0 identical cohesive end restriction enzyme site among 35S-NOS:
SrUGT76G1, SrUGT85C2 and SrUGT91D2m target gene overall length PCR amplification primer
(2) PCR amplification: with the cDNA (50ng/ μ L) of above-mentioned synthesis for template, high-fidelity Phusion High- is used
Fidelity DNA Polymerase PCR (Thermo) kit is expanded (Fig. 2) to the overall length of said gene, and PCR is anti-
Answering condition is 98 DEG C of 30s, 94 DEG C of 60 DEG C of 10s, 72 DEG C of 30s 45s 30cycles, 72 DEG C of 7min, 4 DEG C.Concrete operations step
The rapid operation instruction for pressing kit.
(3) PCR product purifies: after above-mentioned 2 μ L electrophoresis detection of PCR product, utilizing QIAquick PCR
Purification Kit kit (QIAGEN) purifies PCR product, and concrete operation step presses the operation instruction of kit.
3, the extraction of pKAFCR0 and pKAFCR100 Plasmid DNA
Using Qiaprep Spin Miniprep Kit kit (QIAGEN), pKAFCR0 is extracted from bacillus coli DH 5 alpha
With pKAFCR100 Plasmid DNA, concrete operation step presses the operation instruction of kit.It is dense that its is measured using NaroDrop 2000
Degree.
4, the connection of single target gene and pKAFCR0 plasmid
(1) double digestion is handled: using restriction enzyme Kpn I and Sac I respectively to through obtained by PCR amplification, purification
SrUGT85C2 and SrUGT91D2m target gene overall length and pKAFCR0 plasmid carry out double digestion processing, and use it is restricted
Restriction endonuclease BamH I and Xho I carries out the SrUGT76G1 target gene overall length and pKAFCR0 plasmid obtained through PCR amplification double
Digestion processing.The reaction condition of double digestion processing is 37 DEG C of 4h, inactivates 60 DEG C of 5min.
(2) target fragment recycles: will carry out electrophoresis through double digestion treated reaction product, testing goal genetic fragment with
And the digestion effect of plasmid, then with QIAquick Gel Extraction Kit kit (QIAGEN) to target fragment into
The recycling of row glue, concrete operation step press the operation instruction of kit.
(3) connection reaction: the target gene fragment recycled after identical digestion through glue is connected with pKAFCR0 respectively
It connects, respectively in the ratio of target gene fragment 125fmol, plasmid 25fmol, by 65 DEG C of 5min pre-treatments to eliminate DNA shape
After secondary structure, the half Ligation High ligase (TOYOBO) for measuring volume is added, is connected under the conditions of 16 DEG C of 12h
It is reversed to answer.
(4) bacillus coli gene converts: the reaction product for taking appropriate (10 μ L of <) to have connected is transformed into using thermal excitation
In the competent cell of e.colistraindh5α.
(5) bacterium colony PCR is identified: the Escherichia coli liquid (100 μ L) after plasmid is converted is coated in solid LB Antibiotic medium
One evening of culture on (penicillin 25mg/L), picking single bacterium colony carry out bacterium colony PCR identification (Fig. 3) using above-mentioned primer.By PCR
Positive bacterium colony is placed in one evening of liquid LB Antibiotic medium (penicillin 25mg/L) culture, again after PCR mirror, saves positive bacteria
Liquid.
(6) target gene insertion sequencing: by the positive bacterium solution kept expand it is numerous after, utilize Qiaprep Spin
Miniprep Kit kit, the Plasmid DNA after extracting building are sent to invitrogen company and carry out sequence verification.Sequencing is with drawing
Object is CR0MCS insert 35S-S and CR0MCS insert NOS-A.
Target gene is successfully constructed to the recombinant plasmid of pKAFCR0 and is respectively designated as:
PNULPGE01001:pKAFCR0+SrUGT76G1
PNULPGE01002:pKAFCR0+SrUGT85C2
PNULPGE01003:pKAFCR0+SrUGT91D2m
5, single-gene expression vector is constructed
Since (target gene is constructed to pKAFCR0, and upstream and downstream adds starting respectively for 3 target gene combinations
The segment of son and terminator) all without Ase I restriction enzyme site, it is constructed to the recombinant plasmid dna of pKAFCR0 using target gene as mould
Plate introduces Ase I cohesive end restriction enzyme site at 5 ' ends by forward primer using PCR amplification method, and utilization can generate
The archaeal dna polymerase (such as PHUSION high-fidelity DNA polymerase) of smooth end product forms smooth end, such PCR at 3 ' ends
Amplified production, after Ase I digestion processing, can with through Ase I and Stu I digestion, treated that pKAFCR100 is attached,
Construct single-gene expression vector.Junction Ase I cohesive end restriction enzyme site still retains, and Stu I smooth end digestion position
Point disappears.
(1) recombinant plasmid dna extracts: extracting each target gene respectively from bacillus coli DH 5 alpha and constructs to pKAFCR0's
Recombinant plasmid pNULPGE01001, pNULPGE01002 and pNULPGE01003.Its concentration is measured using NaroDrop 2000.
(2) PCR amplification and purification of products: the PCR amplification design of primers of target gene combination is as follows, and 5 ' ends of primer introduce
Ase I cohesive end restriction enzyme site:
The PCR amplification primer of target gene combination
After recombinant plasmid pNULPGE01001, pNULPGE01002, pNULPGE01003 DNA are diluted to 10ng/ μ L, point
Not as template, PCR is carried out using high-fidelity Phusion High-Fidelity DNA Polymerase PCR kit
It expands (Fig. 4), PCR reaction system and condition are same as above.After above-mentioned 2 μ L electrophoresis detection of PCR product, QIAquick PCR is utilized
Purification Kit kit purifies PCR product.
(3) it target gene combination pcr amplification product and the processing of pKAFCR100 double digestion: combines the target gene of purification
Pcr amplification product carry out digestion with Ase I, pKAFCR100 carries out double digestion with Ase I and Stu I.Endonuclease reaction condition
Are as follows: 37 DEG C of 4h, 90 DEG C of 5min.
(4) target fragment recycles: carrying out electrophoresis detection through digestion treated reaction product for above-mentioned, then uses
QIAquick Gel Extraction Kit kit carries out glue recycling to target fragment.
(5) connection reaction: the pKAFCR100 that the target gene combination that glue recycles is recycled with glue, by above-mentioned
After method is attached, Escherichia coli convert (antibiotic of culture medium is changed to kanamycins 50mg/L) and bacterium colony PCR identification
(Fig. 5) saves positive bacterium colony.Target gene, which is combined into function and constructs to the recombinant plasmid of pKAFCR100 (Fig. 6), to be respectively designated as:
The pNULPGE01101:pKAFCR100+SrUGT76G1 assortment of genes；
The pNULPGE01102:pKAFCR100+SrUGT85C2 assortment of genes；
The pNULPGE01103:pKAFCR100+SrUGT91D2m assortment of genes.
6, two expression vectors are constructed
When constructing single-gene expression vector, the junction Ase I cohesive end digestion of target gene combination and pKAFCR100
Site still retains, and Stu I smooth end restriction enzyme site disappears.But since the target gene of insertion is combined in the upper of 35S
Trip has same cohesive end restriction enzyme site Ase I and Stu I, and second target gene combination may be coupled to existing first
On the plasmid of a target gene combination.
(1) recombinant plasmid dna extract: from bacillus coli DH 5 alpha extract SrUGT85C2 target gene combination building to
The recombinant plasmid pNULPGE01102 of pKAFCR100.Its concentration is measured using NaroDrop 2000.
(2) pNULPGE01102 double digestion is handled: carrying out double digestion with Ase I and Stu I.Endonuclease reaction condition are as follows: 37
DEG C 4h, 90 DEG C of 5min.
(3) target fragment recycles: carrying out electrophoresis detection through digestion treated reaction product for above-mentioned, then uses
QIAquick Gel Extraction Kit kit carries out glue recycling to target fragment.
(4) connection reaction: the SrUGT91D2m target gene combination that 5 (4) glue recycle is recycled with glue
PNULPGE01102 is attached according to the above method, (antibiotic of culture medium is changed to kanamycins 50mg/ for Escherichia coli conversion
L) and after bacterium colony PCR identification, positive bacterium colony is saved.Two target gene are combined into function and construct to the recombination matter of pKAFCR100
Grain name are as follows:
The pNULPGE01104:pKAFCR100+SrUGT91D2m assortment of genes+SrUGT85C2 the assortment of genes.
7, three expression vectors are constructed
(1) recombinant plasmid dna extracts: recombinant plasmid pNULPGE01104 is extracted from bacillus coli DH 5 alpha.It utilizes
NaroDrop 2000 measures its concentration.
(2) pNULPGE01104 double digestion is handled: carrying out double digestion with Ase I and Stu I.Endonuclease reaction condition are as follows: 37
DEG C 4h, 90 DEG C of 5min.
(3) target fragment recycles: carrying out electrophoresis detection through digestion treated reaction product for above-mentioned, then uses
QIAquick Gel Extraction Kit kit carries out glue recycling to target fragment.
(4) connection reaction: the SrUGT76G1 target gene combination that 5 (4) glue recycle is recycled with glue
PNULPGE01104 is attached according to the above method, (antibiotic of culture medium is changed to kanamycins 50mg/ for Escherichia coli conversion
L) and after bacterium colony PCR identification (Fig. 7), positive bacterium colony is saved.Three target gene are combined into function and construct to the weight of pKAFCR100
Group plasmid (Fig. 8) name are as follows:
Combination+SrUGT91D2m the assortment of genes+the SrUGT85C2 of pNULPGE01105:pKAFCR100+SrUGT76G1 mesh
The assortment of genes.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (1)
1. the plasmid system for the building of plant polygene expression vector, which is characterized in that including pKAFCR0 plasmid and
PKAFCR100 plasmid；
The pKAFCR0 plasmid is constructed by following steps:
S11, using restriction enzyme Hind III and EcoR I, double enzymes are carried out to e. coli plasmid vector pUC18 DNA
Cut processing；
S12, also with restriction enzyme Hind III and EcoR I, double-core Agrobacterium Ti plasmid pBI121 DNA is carried out
Double digestion processing；
S13, by the pUC18 after double digestion, and the pBI121 after same double digestion is separated through 1% agarose gel electrophoresis
Afterwards, purpose band is cut, the part Hind III-EcoR I has been cut off by gel recovery method or gel reclaims kit acquisition
PUC18 and the 35S-GUS-NOS segment that is cut from pBI121；
S14, using T4-DNA ligase or plasmid connection kit, the pUC18 of the part Hind III-EcoR I will have been cut off
It is attached with the 35S-GUS-NOS segment cut from pBI121；Recombinant plasmid after connection is transformed into large intestine using thermal excitation
In the competent cell of bacillus DH5 α bacterial strain；
S15, flat be applied on the LB solid medium containing 25mg/L penicillin of the Escherichia coli after conversion is cultivated, picking is independent
Bacterium colony carries out bacterium colony PCR identification, screens the positive bacterium colony containing recombinant plasmid；
S16, by positive bacterium colony through the LB liquid medium of 25mg/L penicillin expand it is numerous after, using conventional plasmid extracting method or
Plasmid extraction kit extracts recombinant plasmid dna, which is named as pKAFCR1, i.e., in the Hind III of pUC18 and
On EcoR I site, it is inserted into 35S-GUS-NOS segment；
S17, using restriction endonuclease sma I and Sac I, double digestion processing is carried out to recombinant plasmid pKAFCR1 DNA；
S18, by the pKAFCR1 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through gel
Recovery method or gel reclaims kit obtain the pKAFCR1 for having cut off gus reporter gene segment；
S19, artificial synthesized section of DNA, the segment contain restriction endonuclease sma I, Kpn I, Xho I and Sac I site；Benefit
The pKAFCR1 of the DNA fragmentation and excision gus reporter gene is connected with T4-DNA ligase or plasmid connection with kit
It connects；Recombinant plasmid after connection, is transformed into the competent cell of e.colistraindh5α using thermal excitation；After conversion
Flat be applied on the LB solid medium containing 25mg/L penicillin of Escherichia coli is cultivated, and the independent bacterium colony of picking carries out bacterium colony PCR mirror
It is fixed, screen the positive bacterium colony containing recombinant plasmid；By positive bacterium colony after the expansion of the LB liquid medium of 25mg/L penicillin is numerous, benefit
Recombinant plasmid dna is extracted with conventional plasmid extracting method or plasmid extraction kit；The recombinant plasmid is named as pKAFCR2,
The gus reporter gene of pKAFCR1 is eliminated, and increases Kpn I and Xho I restriction enzyme site on this position；
S120, using restriction enzyme Hind III and Pst I, double digestion processing is carried out to recombinant plasmid pKAFCR2 DNA；
S121, by the pKAFCR2 after double digestion through 1% agarose gel electrophoresis separation after, cut purpose band, pass through gel
Recovery method or gel reclaims kit obtain the pKAFCR2 of digestion.
S122, artificial synthesized section of DNA, the segment contain restriction enzyme Hind III, Hpa I, Stu I, Sph I, Pst
I and Sbf I site；Using T4-DNA ligase or plasmid connection kit by the DNA fragmentation and the pKAFCR2 of digestion into
Row connection；Recombinant plasmid after connection, is transformed into the competent cell of e.colistraindh5α using thermal excitation；It will conversion
Flat be applied on the LB solid medium containing 25mg/L penicillin of Escherichia coli afterwards is cultivated, and the independent bacterium colony of picking carries out bacterium colony
PCR identification, screens the positive bacterium colony containing recombinant plasmid；Positive bacterium colony is numerous through the LB liquid medium expansion of 25mg/L penicillin
Afterwards, recombinant plasmid dna is extracted using conventional plasmid extracting method or plasmid extraction kit；The recombinant plasmid is named as
PKAFCR0 increases Hpa I, Stu I and Sph I restriction enzyme site in the upstream 35S；PKAFCR0 is in the upstream 35S, 35S and NOS
It is intermediate and all have polyclone enzyme enzyme site in the downstream NOS；
The pKAFCR100 plasmid is constructed by following steps:
S21, using restriction enzyme Hind III and EcoR I, double enzymes are carried out to double-core Agrobacterium Ti plasmid pBI121 DNA
Cut processing；
S22, using restriction enzyme Hind III and EcoR I, double enzymes are carried out to double-core Agrobacterium Ti plasmid pBsGFPDNA
Cut processing；
S23, by the pBI121 after double digestion, and the pBsGFP after same double digestion is separated through 1% agarose gel electrophoresis
Afterwards, purpose band is cut, by gel recovery method or gel reclaims kit, acquisition has cut off 35S-GUS-NOS segment
PBI121, and the 35S Ω-sGFP-NOS segment cut from pBsGFP；
S24, using T4-DNA ligase or plasmid connection kit, by cut off 35S-GUS-NOS segment pBI121 and
35S Ω-sGFP-NOS the segment cut from pBsGFP is attached；Recombinant plasmid after connection is transformed into greatly using thermal excitation
In the competent cell of enterobacteria DH5 α bacterial strain；It is applied to the Escherichia coli after conversion are flat the LB containing 50mg/L kanamycins and consolidates
It is cultivated on body culture medium, the independent bacterium colony of picking carries out bacterium colony PCR identification, screens the positive bacterium colony containing recombinant plasmid；It will be positive
Bacterium colony utilizes conventional plasmid extracting method or plasmid to extract reagent after the expansion of the LB liquid medium of 50mg/L kanamycins is numerous
Box extracts recombinant plasmid dna, which is named as pKAFCR31, i.e., the 35S-GUS of pBI121 is replaced as 35S Ω-
sGFP；
S25, using restriction enzyme Hind III and Pst I, double digestion processing is carried out to recombinant plasmid pKAFCR31 DNA；
S26, the plasmid pKAFCR31 after double digestion is cut into purpose band after 1% agarose gel electrophoresis separation, led to
Gel recovery method or gel reclaims kit are crossed, the pKAFCR31 of digestion is obtained；
S27, artificial synthesized section of DNA, the segment contain restriction enzyme Hind III, Hpa I, Stu I, Sph I and Sbf
I site；Using T4-DNA ligase or plasmid connection kit, the DNA fragmentation and the pKAFCR31 of digestion are connected
It connects；Recombinant plasmid after connection, is transformed into the competent cell of e.colistraindh5α using thermal excitation；After conversion
Flat be applied on the LB solid medium containing 50mg/L kanamycins of Escherichia coli is cultivated, and the independent bacterium colony of picking carries out bacterium colony PCR
The positive bacterium colony containing recombinant plasmid is screened in identification；Positive bacterium colony is numerous through the LB liquid medium expansion of 50mg/L kanamycins
Afterwards, recombinant plasmid dna is extracted using conventional plasmid extracting method or plasmid extraction kit, which is named as
PKAFCR100 increases Hpa I, Stu I and Sph I restriction enzyme site in 35S Ω promoter upstream；PKAFCR100 has only
The kanamycins NPT II tolerance gene and sGFP Green fluorescent protein fusion vector of vertical expression and in-between polyclone enzyme
Enzyme site；
The application of the plasmid system for plant gene expression vector building, is used for the single or multiple gene expressions of plant
The building of carrier, and transgenic plant cells or plant using expression vector progress genetic transformation.
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