CN105543275B - Double gene expression vector and its application - Google Patents
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Abstract
The invention discloses three dual-gene plant expression vectors, number pDT1, pDT7 and pDT7G, the serial carrier is from pCAMBIA3301-ACTIN2:: 3xHA-LUC transformation, energy is simple, reliable by a Plant Transformation, two genes are fast and efficiently expressed in plant, the relationship of interaction and multiprotein complex between research albumen and albumen provides new Research approach, and can be by simple restoration and reuse in other species.
Description
Technical field
The present invention relates to the three double gene expression plant vectors-being transformed from pCAMBIA3301-ACTIN2::3xHA-LUC
PDT1, pDT7 and pDT7G.
Background technique
The building of transgenic plant is the very important prerequisite of modern plants biology.And carrier is as foreign gene
Into the transporter of the genome of target plant, very important role is play in terms of the generation of transgenic plant.However
As researcher is to the needs of relationship between multiple albumen and polygenes co- controlling plant growth and development research, especially
The interaction of polyprotein, pervious single-gene vectors are no longer satisfied present needs.So researcher needs one kind
Efficient method enters in plant to convert polygenes to obtain while express the transgenic plant of multiple genes.
Currently, the method for traditional acquisition polygenes transgenic plant includes cotransformation, then converts and hybridize.But this
The shortcomings that a little conventional methods be the transformation efficiency of two or more genes of cotransformation be it is uncontrollable, each gene is in target base
Because the insertion position of group is also different, and Gene Isolation will lead to unstable hereditary offspring.In addition it converts again and miscellaneous
Handing over all is time-consuming process.
And in order to overcome these disadvantages, the multigene carriers with single or multiple transcription boxes come into being and successfully
Applied to multiple researchs, but the application of these polygene expression vectors is not promoted and applied widely.Because most of
Method need be subcloned so that clone spent time elongate；There is no label or reporter gene to be connected with target gene, this
Sample results in the identification of albumen or gene relatively difficult；In addition, certain methods are as used isocaudarner to construct multigene carrier
Method is limited to few this problem of quantity of available enzyme.So building multigene carrier needs are set according to research purpose
Meter.
Summary of the invention
Dual-gene plant expression vector can be expressed technical problem to be solved by the invention is to provide three, which carries
Body is adapted from pCAMBIA3301-ACTIN2::3xHA-LUC, and (carrier is given by Chinese Academy of Agricultural Sciences Liu Bin professor laboratory
Send, the present inventor laboratory saves), it can structures simple and quick, reliable, efficient for dual-gene expression and its genetically modified plants
It builds.
Present invention provide the technical scheme that three can be used for the plant expression vector of double gene expression, the carrier is
PCAMBIA3301-ACTIN2::3xHA-LUC transformation, number pDT1, pDT7 and pDT7G, specific as follows: one kind is biradical
Because of plant expression vector, it is adapted from pCAMBIA3301-ACTIN2::3xHA-LUC, independent transcript unit that there are three tools,
In one be in the bar genetic transcription unit controlled by 35S promoter, one is the label protein controlled by ACTIN2 promoter
The transcript unit of myc gene and one are the transcript units of the label protein HA gene controlled by UBQ10 promoter.
The dual-gene plant expression vector, the linear structure of the carrier are as shown in Figure 1A.
Above-mentioned dual-gene plant expression vector, the vector construction process are as follows:
(1) it is ready to carry required for skeleton carrier pCAMBIA3301-ACTIN2::3xHA-LUC and clone's target fragment
Body and DNA；
(2) primer containing connector for being suitble to in-fusion clone is designed；
(3) 3xHA segment is cloned from skeleton pCAMBIA3301-ACTIN2::3xHA-LUC, is cloned on pGARD-Myc
4xmyc segment clones 6xHis-cGR segment on pKYLX-CRY2-GR, and wherein 6xHis has been synthesized on primer, is cloned on PER8
TNOS；
(4) SpeI&BamHI digestion pCAMBIA3301-ACTIN2::3xHA-LUC is used, removes 3xHA-LUC, and use In-
On carrier after fusion enzyme connection 4xmyc segment to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::
4xmyc；
(5) connected ASC I restriction enzyme site that UBQ10 promoter sequence is added with 3xHA segment by design of primers and with T4
It connects enzyme bridging and is connected into UBQ10::3xHA segment, two sections of the segment are all by design of primers the 5 ' of UBQ10 promoter sequence
It joined AvrII, joined MfeI, Aat II, Pac I and Nco I the 3 ' of 3xHA；
(6) Pst I digestion intermediate vector pCAMBIA3301-ACTIN2::4xmyc is used, is connected with the method for In-fusion
On carrier after UBQ10::3xHA to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::Myc-T35S-UBQ10::
HA；
(7) with the intermediate vector in Pac I and Nco I digestion (6), T is seamlessly connected with In-fusion enzymeNOSSegment is to enzyme
On carrier after cutting, as final carrier pDT1 (pCAMBIA3301-ACTIN2::Myc-T35S-UBQ10::HA-TNOS)。
Another dual-gene plant expression vector, is adapted from pCAMBIA3301-ACTIN2::3xHA-LUC, there are three tools
Independent transcript unit, one of them bar genetic transcription unit controlled by 35S promoter, one by ACTIN2 promoter control
The series connection label H is-cGR gene that the transcript unit of the label protein myc gene of system and one are controlled by UBQ10 promoter
Transcript unit, wherein cGR is the amino acid residue of mouse glucocorticoid albumen 508 to 795, the caryoplasm relied on dexamethasone
Forwarding function.
The dual-gene plant expression vector, the linear structure of the carrier are as shown in Figure 1B.
The dual-gene plant expression vector, the vector construction process are as follows:
(1) it is ready to carry required for skeleton carrier pCAMBIA3301-ACTIN2::3xHA-LUC and clone's target fragment
Body and DNA；
(2) primer containing connector for being suitble to in-fusion clone is designed；
(3) 3xHA segment is cloned from skeleton pCAMBIA3301-ACTIN2::3xHA-LUC, is cloned on pGARD-Myc
4xmyc segment clones 6xHis-cGR segment (6xHis has been synthesized on primer) on pKYLX-CRY2-GR, clones on PER8
TNOS；
(4) SpeI&BamHI digestion pCAMBIA3301-ACTIN2::3xHA-LUC is used, removes 3xHA-LUC, and use In-
On carrier after fusion enzyme connection 4xmyc segment to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::
4xmyc；
(5) the ASC I restriction enzyme site that UBQ10 promoter sequence and 6xHis-cGR segment are added by design of primers is simultaneously
It is connected into UBQ10::6xHis-cGR segment with T4 ligase bridging, two sections of the segment are all opened by design of primers in UBQ10
The 5 ' of promoter sequences joined AvrII, joined MfeI, Aat II, Pac I and Nco I the 3 ' of 3xHA and 6xHis-cGR；
(6) Pst I digestion intermediate vector pCAMBIA3301-ACTIN2::4xmyc is used, is connected with the method for In-fusion
On carrier after UBQ10::6xHis-cGR to the digestion, as intermediate vector pCABIA3301-ACTIN2::Myc-T35S-
UBQ10::His-cGR；
(7) with the intermediate vector in Pac I and Nco I digestion (6), T is seamlessly connected with In-fusion enzymeNOSSegment is to enzyme
On carrier after cutting, as final carrier pDT7 (pCABIA3301-ACTIN2::Myc-T35S-UBQ10::His-cGR–
TNOS)。
Another dual-gene plant expression vector, is adapted from pCAMBIA3301-ACTIN2::3xHA-LUC, is upper
On the basis of the double gene expression vector stated, the Gypsy insulator sequence from drosophila, insulator Gypsy sequence are introduced
Third is transcribed in the reversed opposite left side in second transcript unit of double gene expression vector as claimed in claim 4
The specific transcriptional control that position effect guarantees promoter is eliminated with this in the right side of unit；And 6xHis sequence is replaced with
3xFlag sequence.
The dual-gene plant expression vector, the linear structure of the carrier are as shown in Figure 1 C.
The dual-gene plant expression vector, the vector construction process are as follows:
(1) segment between above-mentioned dual-gene plant expression vector 6614nt and 8178nt is cloned down, and in addition will
The segment with Kpn I, remove by the digestion from double gene expression vector described in claim 6, and purification and recovery large fragment；
(2) forward and reverse Gypsy insulator sequence is cloned from carrier；
(3) with the reverse primer of Gypsy segment in the forward primer of the segment cloned in (1) and (2), with the two pieces
The mixture of section is cloned as template；
(4) bridge-clip being cloned into (3) is connected to by T4 ligase and has used the described of Kpn I digestion in (1)
Carrier；
(5) reversed Gypsy sequence is connected in (4) and has been connected to positive Gypsy sequence and with the load of Nco I digestion
On body；
(6) sequence between pDT7Asc I and Aat II improved in (5) is cloned and is building up to pGWC (T load
Body) on, then by " T " clonal mutation of the position 11095nt remove at " C " a Nsi I restriction enzyme site (ATGCAT, 11090
~11095bp), finally with Nsi I and Nde I, by 6xHis sequence fragment, the digestion from carrier T is removed；
(7) 3xFlag segment is by being annealed into double-stranded DNA, being connected to Nsi I and Nde I digestion after synthesizing single-stranded ssDNA
In carrier T afterwards；
(8) sequence digestion improved in carrier T is got off with Asc I and Aat II, and be connected to the two enzyme enzymes
On pDT7 after cutting, final as pDT7G.
Meanwhile dual-gene application is expressed in plant the present invention also provides dual-gene carrier: by the CDS of TCP2 and CRY1
It is cloned into the Spe I and Mef I restriction enzyme site building TCP2-CRY1-pDT1 carrier of pDT1 respectively, is transfected by mediated by agriculture bacillus
Method is transferred in arabidopsis and tobacco.Additionally construct TCP2-Myc-pDT1 and HA-CRY1-pDT1 individual gene carrier conduct
Control has been transferred in tobacco.Pass through the expression of immune-blotting method TCP2 and CRY1 in arabidopsis and tobacco.
Construct CRY1-TCP2-pDT7 and CIB1-B7-pDT7 (B7:AT2G02320) respectively in the same way.And by this
Two carriers are transferred in arabidopsis, obtain the arabidopsis transgenic plant of glyphosate resistance, then to glyphosate resistance plant into
Row immunoblotting and real-time quantitative PCR detect dual-gene expression.
One pair of genes A-B and LUC-GFP are building up on pDT7G carrier, obtain following recombinant vector: A-B-pDT7G,
LUC-GFP-pDT7G and GFP-LUC-pDT7G.A-B-pDT7G carries out immune-blotting method after being transferred in arabidopsis.By LUC-
GFP-pDT7G and GFP-LUC-pDT7G is transferred in tobacco leaf cell, transient expression LUC and GFP albumen.Carry out fluorescence mycin
Test experience (luciferase assay) detects the expression of LUC albumen and the expression position of fluorescence microscope experimental observation GFP.
Pass through the mutual of co-immunoprecipitation verification experimental verification CRY1 and TCP2 in TCP2-CRY1-pDT1/WT transgenic line
Effect；In addition analysis CIB1/CIB2-CRY2-pDT7G/cry2 transgenic seedlings are under blue light and dark to DEX evoked response
Hypocotyl phenotype.
Pass through the coexpression rate and CIBs of SPA1 and mCRY1 in analysis SPA1-mCRY1-pDT1/WT transgenic line
The coexpression efficiency of CIBs and CRY2 in (1-5)-CRY2-pDT7G/cry2 transgenic line.
The invention has the following advantages:
Serial double gene expression plant expression vector through the invention can construct double gene expression transgenosis using it and plant
Then object studies correlation or interaction between the two genes.One, this method can make compared to traditional method
The building of dual-gene transgenic plant is simpler quickly.Two, the systemic vectors have multiple transcription boxes, and each transcription
Box has the expression of different promoter and terminator control target gene, reduces the probability of two foreign gene co-suppressions.
Three, each target gene is connect with the label protein with commercial antibodies, to identify that the expression of destination protein is provided convenience.
Four, it in the way of In-fusion on cloned target gene to carrier, can avoid caused by digestion connection or other clonal fashions
The shortcomings that can not being cloned with restriction enzyme site, can clone most target gene using the system.Five, in the system pDT7 and
PDT7G can also by expression of the DEX Induction Control target gene in nucleus and cytoplasm, the use of the inducible system,
Determination for research purpose gene function position provides evidence, can successfully construct lethal gene genetically modified plants.Six, exist
Gypsy is introduced in pDT7G, and the specific transcriptional control that position effect guarantees promoter is eliminated with this.Therefore, double gene expression carries
Body correlation between research protein-protein interaction and albumen or gene has very important use value.
The present invention is provides studying correlation between protein-protein interaction and albumen or gene in plant
New approach.Three double gene expression vectors of the present invention can express simultaneously two genes in tobacco and arabidopsis,
It will not influence gene mutual biochemical functions while expressing dual-gene, double gene coexpression is high-efficient.Therefore, of the invention
There is provided three double gene expression vectors research double gene expression and albumen, protein-interacting and albumen/gene it
Between correlation have very big Exploitative potential and value.
Detailed description of the invention
Fig. 1: the rough schematic of three double gene expression vectors of the invention；
Fig. 2: the cyclic annular schematic diagram for the double gene expression vector that present invention number is pDT1；
Fig. 3: present invention number is pDT7 and the double gene expression vector ring-type schematic diagram of pDT7G；
Fig. 4: present invention number is pDT1 and the dual-gene carrier of pDT7 expresses dual-gene knot in tobacco and arabidopsis
Fruit figure；
Fig. 5: the double gene expression vector that number is pDT7G in the present invention expresses dual-gene and cGR fusion protein to DEX
The result figure of evoked response；
Fig. 6: it is analyzed by the dual-gene biochemical functions that dual-gene carrier is expressed；
Fig. 7: the efficiency analysis pie chart of double gene expression vector double gene coexpression in transgenic plants.
Specific embodiment:
Embodiment 1: the building of double gene expression vector
With high copy number, stabilization and the derivative vector pCAMBIA3301- of complete binary vector pCAMBIA is sequenced
ACTIN2::3xHA-LUC (carrier is given by Chinese Academy of Agricultural Sciences Liu Bin professor laboratory, this laboratory saves) is bone
Frame.Three can be used for the plant expression vector of double gene expression, and the carrier is pCAMBIA3301-ACTIN2::3xHA-LUC
Transformation, number pDT1, pDT7 and pDT7G.PCAMBIA3301-ACTIIN2::3xHA-LUC is by Scientia Agricultura Sinica
Institute Liu Bin professor laboratory is adapted from pCAMBIA3300 (www.cambia.org), and specific transformation process are simply as follows: using Sac
The ACTIN2 promoter sequence (C-terminal joined Spe I restriction enzyme site) of I and Bam HI digestion pCAMBIA3300 and clone, purifying
The carrier of digestion is connected with segment with T4 ligase after recycling；Then using Spe I and Bam HI by intermediate vector and gram
Grand 3xHA-LUC segment carries out digestion connection；Last Bam HI and PstI is by the intermediate vector and 35S of previous step
Terminator segment carries out digestion connection；The carrier name finally obtained are as follows: pCAMBIA3301-ACTIN2::3xHA-LUC.
And the construction of dual-gene carrier is carried out as skeleton.
The building process of pDT1, pDT7 are as follows: (1) be ready to skeleton carrier pCAMBIA3301-ACTIN2::3xHA-LUC and
Clone carrier and DNA required for target fragment；(2) primer containing connector for being suitble to in-fusion clone is designed；(3) from
Skeleton pCAMBIA3301-ACTIN2::3xHA-LUC clones 3xHA segment, clones 4xmyc segment, pKYLX- on pGARD-Myc
6xHis-cGR segment (6xHis has been synthesized on primer) is cloned on CRY2-GR, clones T on PER8NOS；(4) SpeI&BamHI is used
Digestion pCAMBIA3301-ACTIN2::3xHA-LUC removes 3xHA-LUC, and extremely with In-fusion enzyme connection 4xmyc segment
On carrier after the digestion, as intermediate vector pCAMBIA3301-ACTIN2::4xmyc；(5) by UBQ10 promoter sequence point
It the ASC I restriction enzyme site that is added by design of primers of 3xHA, 6xHis-cGR segment and is connected into not and with T4 ligase bridging
(two sections of the two segments are all by design of primers in UBQ10 promoter for UBQ10::3xHA and UBQ10::6xHis-cGR segment
The 5 ' of sequence joined AvrII, joined MfeI, Aat II, Pac I and Nco I the 3 ' of 3xHA and 6xHis-cGR；(6) it uses
Pst I digestion intermediate vector pCAMBIA3301-ACTIN2::4xmyc, is separately connected UBQ10: with the method for In-fusion:
On carrier after 3xHA and UBQ10::6xHis-cGR to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::Myc-
T35S- UBQ10::HA and pCABIA3301-ACTIN2::Myc-T35S-UBQ10::His-cGR；(7) Pac I and Nco I enzyme are used
Two intermediate vectors in (6) are cut, are seamlessly connected T with In-fusion enzymeNOSIt is as final on carrier after segment to digestion
Carrier pDT1 (pCAMBIA3301-ACTIN2::Myc-T35S-UBQ10::HA-TNOS) and pDT7 (pCABIA3301-ACTIN2::
Myc-T35S-UBQ10::His-cGR–TNOS)。
The building process of pDT7G are as follows: (1) pDT7G is the transformation and upgrade carried out on the basis of pDT7, in pDT76614nt
Segment between 8178nt is cloned down, and is removed with the Kpn I digestion at the two positions；(2) it is cloned just from carrier
To with reversed Gypsy insulator sequence；(3) in the forward primer of the segment cloned in (1) and (2) Gypsy segment it is anti-
To primer, cloned using the mixture of the two segments as template；(4) bridge-clip being cloned into (3) is passed through into T4
Ligase is connected on the pDT7 for having used Kpn I digestion in (1)；(5) reversed Gypsy sequence is connected in (4) and has been connected
Positive Gypsy sequence and on the pDT7 carrier of Nco I digestion；(6) by pDT7Asc I and Aat II improved in (5)
Between sequence clone and be building up in pGWC (carrier T), then by " T " clonal mutation of the position 11095nt at " C " with
Remove a Nsi I restriction enzyme site (ATGCAT, 11090~11095bp), finally uses Nsi I and Nde I by 6xHis tract
Section digestion from carrier T is removed；(7) 3xFlag segment is by being annealed into double-stranded DNA, being connected to Nsi after synthesizing single-stranded ssDNA
In carrier T after I and Nde I digestion；(8) sequence digestion improved in carrier T is got off with Asc I and Aat II, and even
It is connected to on the pDT7 after the two enzyme digestions, finally as pDT7G.
There are three independent transcript unit (Fig. 1-Fig. 3) for serial carrier tool.Three transcript units are all by different startings
Son and terminator control.There is something in common also to have difference, there are two identical for pDT1 and pDT7 tool between pDT1 and pDT7
Transcript unit, one is in the bar genetic transcription unit controlled by 35S promoter, another is by ACTIN2 promoter
The transcript unit of the label protein myc gene of control.The difference is that third transcript unit, the third transcript unit of pDT1 are
The transcript unit of the label protein HA gene controlled by UBQ10 promoter and the third transcript unit of pDT7 is opened by UBQ10
The transcript unit of the series connection label H is-cGR gene of mover control.CGR is the amino acid of mouse glucocorticoid albumen 508 to 795
Residue, the caryoplasm forwarding function relied on dexamethasone (dexamethasone, DEX).
The Gypsy insulator sequence from drosophila is introduced on the basis of pDT7, which can guarantee foreign gene
It is effective and accurately express.Third is a in the Gypsy sequence reversed opposite left side in second transcript unit of pDT7
The specific transcriptional control that position effect guarantees promoter is eliminated with this in the right side of transcript unit；And 6xHis sequence is replaced with
3xFlag sequence (Fig. 1 C, Fig. 3 B).The improved dual-gene carrier of pDT7 is pDT7G.Be shown in table 1 dual-gene carrier can
Restriction enzyme site；Table 2 is primer sequence table used in building double gene expression vector.
The last restriction enzyme site that can be used for In-Fusion clone of 1 pDT carrier of table
Cloning primer used in 2 pDTs vector construction process of table
Embodiment 2: double gene expression vector is expressed dual-gene in tobacco and arabidopsis in the present invention
1.pDT1 and pDT7 expresses dual-gene in tobacco and arabidopsis
Dual-gene expression of the analysis building on double gene expression vector.TCP2-CRY1-pDT1 carrier passes through agriculture bar
Bacterium mediated transfection method is transferred in arabidopsis and tobacco.Additionally construct the single base of TCP2-Myc-pDT1 and HA-CRY1-pDT1
Because carrier has been transferred in tobacco as control.By the expression of immune-blotting method TCP2 and CRY1 in arabidopsis and tobacco,
It was found that TCP2 and CRY1 can co-express (Fig. 4 A, B) in both plants.
Two carriers of CRY1-TCP2-pDT7 and CIB1-B7-pDT7 (B7:AT2G02320) are transferred in arabidopsis respectively,
The arabidopsis transgenic plant of glyphosate resistance is obtained, immunoblotting and real-time quantitative then are carried out to glyphosate resistance plant
PCR detects dual-gene expression.The result shows that CIB1-Myc and CRY1-Myc are overexpressed (Fig. 4 C, E) in arabidopsis, and
TCP2, which is much higher than to compare with the mRNA expression of B7, shows that the two genes also realize overexpression (Fig. 4 D, F) in plant.
2.pDT7G expresses response of the target gene of dual-gene and GR fusion to DEX in tobacco and arabidopsis
PDT7G expresses dual-gene and cGR fusion protein in plant and analyzes the response results that DEX is induced.By A-B-
PDT7G passes through immune-blotting method after being transferred in arabidopsis, discovery A-Myc and Flag-cGR-B co-expresses (figure in arabidopsis
5A).LUC-GFP-pDT7G and GFP-LUC-pDT7G are transferred in tobacco leaf cell, transient expression LUC and GFP albumen.It is glimmering
LUC fluorescence in light mycin test experience (luciferase assay) detection discovery LUC-GFP-pDT7G and GFP-LUC-pDT7G
Signal can capture (Fig. 5 B) by CCD camera in tobacco leaf cell.Discovery GFP-L is detected by fluorescence microscopy
The GFP signal of UC-pDT7 has (Fig. 5 C) in cytoplasm and nucleus.And the GFP of LUC-GFP-pDT7 due to 3xFlag-
CGR fusion, at DEX or the not processing of DEX, the expression position of GFP is different, Fig. 5 D be under the disposition of not DEX,
GFP protein signal is only present in cytoplasm；And be under 30mM DEX processing in Fig. 5 E, GFP protein signal is only present in carefully
In karyon.The above results show improved pDT7 carrier-pDT7G, have and express dual-gene function in plant, and have
The cGR caryoplasm forwarding function for thering is DEX induction to rely on.
Embodiment 3: it is analyzed by the dual-gene biochemical functions that double gene expression vector is expressed
Whether the function that albumen is co-expressed to understand pDTs works orderly in plant cell, and the present invention is using immune
Co-precipitation inspection protein-interacting and known albumen assess plant growth and development influence the reality of dual-gene carrier pDTs
The property used.TCP2 and CRY1 has been proved to interact in yeast and plant, so passing through the interaction of the two albumen
Whether the biochemical functions that two albumen of pDT carrier expression can be verified normally play.Co-immunoprecipitation the results show that
CRY1 albumen can detected in the sediment composite of myc-TCP2 albumen, it was demonstrated that CRY1 and TCP2 is by pDT1 in plant
Expression still has normal biochemical functions (Fig. 6 A).In addition CRY2 has been proved under blue light in the research of forefathers
Inhibit the elongation of hypocotyl, and this function is realized in nucleus.CIB1/CIB5-CRY2-pDT7G/cry2 transgenosis
CGR-CRY2 obviously inhibits the elongation of hypocotyl under 30mM DEX processing (Fig. 6 B, C) under blue light in plant.These result tables
Bright pDTs carrier can convert in two genes to plant simultaneously and guarantee the normal function of gene, control the position of protein expression.
Embodiment 4: the analysis of double gene coexpression efficiency
Determine that the dual-gene efficiency co-expressed in plant is to assess an important index of dual-gene carrier.The present invention
PDTs is assessed by analyzing the efficiency of double gene coexpression in the transgenic plant with glyphosate resistance.Co-express efficiency point
The positive rate that analysis is expressed in glyphosate resistant transgenic plant by each albumen of immune-blotting method.Totally 133 plants of tables
Up to SPA1-mCRY1s-pDT1 there is glyphosate resistance plant to be used for immune-blotting method.SPA1-Myc and HA-m CRY1s is
The ratio for the plant that can be detected is 35%；The ratio for being only able to detect the plant of SPA1-Myc is 39%, only 5% ratio
Plant is only able to detect the expression of HA-mCRY 1s (mutation gene that mCRY1 is CRY1) albumen, can neither detect
Ratio be 21% (Fig. 7 A).In addition, it is dual-gene also to analyze pDT7G expression by CIBs-CRY2-pDT7G transgenic plant
Efficiency.189 plants of transgenic plants with glyphosate resistance are applied to immune-blotting method.CIBs-Myc and Flag-
The ratio that cGR-CRY2 detected is 42%, and the ratio that only detected CIBs-Myc is 6%, however only detects Flag-
The probability of cGR-CRY2 is 21% (Fig. 7 B).
Claims (2)
1. a kind of dual-gene plant expression vector, wherein being characterized in that: it is with pCAMBIA3301-ACTIN2::3xHA-LUC-
T35SIt is improved for skeleton carrier, the dual-gene plant expression vector construction process is as follows:
(1) skeleton carrier pCAMBIA3301-ACTIN2::3xHA-LUC-T is got out35SIt is carried with required for clone's target fragment
Body and DNA；The skeleton carrier pCAMBIA3301-ACTIIN2::3xHA-LUC-T35SIt is to be adapted from pCAMBIA3300, tool
Body transformation process is simply as follows: with the ACTIN2 promoter sequence of Sac I and Bam HI digestion pCAMBIA3300 and clone,
In, C-terminal joined Spe I restriction enzyme site, be connected the carrier of digestion with segment with T4 ligase after purification and recovery；Then it uses
Intermediate vector and the 3xHA-LUC segment of clone are carried out digestion with Bam HI and connected by Spe I；Last Bam HI and PstI will
The intermediate vector and 35S terminator segment of previous step carry out digestion connection；The carrier finally obtained is i.e. are as follows:
pCAMBIA3301-ACTIN2::3xHA-LUC-T35S；
(2) primer containing connector for being suitble to in-fusion clone is designed；
(3) from skeleton pCAMBIA3301-ACTIN2::3xHA-LUC-T35S3xHA segment is cloned, clones 4xmyc piece on carrier
Section, 6xHis-cGR segment is cloned on pKYLX-CRY2-GR, wherein 6xHis has been synthesized on primer, and T is cloned on PER8NOS；
(4) SpeI&BamHI digestion pCAMBIA3301-ACTIN2::3xHA-LUC-T is used35S, remove 3xHA-LUC, and use In-
On carrier after fusion enzyme connection 4xmyc segment to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::
4xmyc-T35S；
(5) the ASC I restriction enzyme site that UBQ10 promoter sequence and 3xHA segment is added by design of primers and with T4 ligase
Bridging is connected into UBQ10::3xHA segment, and the both ends of the UBQ10::3xHA segment all pass through design of primers in UBQ10 promoter
The 5 ' of sequence joined AvrII, joined MfeI, Aat II, Pac I and Nco I the 3 ' of 3xHA；
(6) Pst I digestion intermediate vector pCAMBIA3301-ACTIN2::4xmyc is used, is connected with the method for In-fusion
On carrier after UBQ10::3xHA to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::4x Myc-T35S-
UBQ10::3xHA；
(7) with the intermediate vector pCABIA3301-ACTIN2::4xMyc-T in Pac I and Nco I digestion (6)35S-UBQ10::
3xHA is seamlessly connected T with In-fusion enzymeNOSOn carrier after segment to digestion, as final carrier pCAMBIA3301-
ACTIN2::4xMyc-T35S-UBQ10::3xHA-TNOS, referred to as pDT1.
2. a kind of dual-gene plant expression vector, wherein being characterized in that: it is with pCAMBIA3301-ACTIN2::3xHA-LUC-
T35SIt is improved for skeleton carrier, the dual-gene plant expression vector construction process is as follows:
(1) skeleton carrier pCAMBIA3301-ACTIN2::3xHA-LUC-T is got out35SIt is carried with required for clone's target fragment
Body and DNA；The skeleton carrier pCAMBIA3301-ACTIIN2::3xHA-LUC-T35SIt is to be adapted from pCAMBIA3300, tool
Body transformation process is simply as follows: with the ACTIN2 promoter sequence of Sac I and Bam HI digestion pCAMBIA3300 and clone,
In, C-terminal joined Spe I restriction enzyme site, be connected the carrier of digestion with segment with T4 ligase after purification and recovery；Then it uses
Intermediate vector and the 3xHA-LUC segment of clone are carried out digestion with Bam HI and connected by Spe I；Last Bam HI and PstI will
The intermediate vector and 35S terminator segment of previous step carry out digestion connection；The carrier finally obtained is i.e. are as follows:
pCAMBIA3301-ACTIN2::3xHA-LUC-T35S；
(2) primer containing connector for being suitble to in-fusion clone is designed；
(3) from skeleton pCAMBIA3301-ACTIN2::3xHA-LUC-T35S3xHA segment is cloned, clones 4xmyc piece on carrier
Section, 6xHis-cGR segment is cloned on pKYLX-CRY2-GR, clones T on PER8NOS；
(4) SpeI&BamHI digestion pCAMBIA3301-ACTIN2::3xHA-LUC-T is used35S, remove 3xHA-LUC, and use In-
On carrier after fusion enzyme connection 4xmyc segment to the digestion, as intermediate vector pCAMBIA3301-ACTIN2::
4xmyc-T35S；
(5) by ASC I restriction enzyme site that UBQ10 promoter sequence and 6xHis-cGR segment are added by design of primers and T4 is used
Ligase bridging is connected into UBQ10::6xHis-cGR segment, and the both ends of the UBQ10::6xHis-cGR segment are all set by primer
Meter joined AvrII the 5 ' of UBQ10 promoter sequence, and 3 ' in 3xHA and 6xHis-cGR joined MfeI, AatII, Pac
I and Nco I；
(6) Pst I digestion intermediate vector pCAMBIA3301-ACTIN2::4xmyc is used, is connected with the method for In-fusion
On carrier after UBQ10::6xHis-cGR to the digestion, as intermediate vector pCABIA3301-ACTIN2::4x Myc-T35S-
UBQ10::6xHis-cGR；
(7) with the intermediate vector pCABIA3301-ACTIN2::4xMyc-T in PacI and Nco I digestion (6)35S-UBQ10::
6xHis-cGR is seamlessly connected T with In-fusion enzymeNOSOn carrier after segment to digestion, as final carrier
pCABIA3301-ACTIN2::4x Myc-T35S-UBQ10::6xHis-cGR–TNOS, referred to as pDT7.
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