CN102703498A - Multi-gene plant expression vector and construction method and application thereof - Google Patents
Multi-gene plant expression vector and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a polygene plant expression vector and a construction method and application thereof. The polygenic plant expression vector comprises: plant transformation vector p209, cloning vector p6435x and one or more genes of interest to be transformed. The construction of the polygenic plant expression vector utilizes the characteristic that the original enzyme cutting site disappears after the connection of the isocaudarner, so that the two ends of the vector or the fragment after enzyme cutting are always different sticky segments, the problem of limited endonuclease sites of the traditional vector is solved, meanwhile, the self-connection in the connection process of the vector is avoided, the phosphorylation treatment of the sticky end is not needed, an intermediate vector and a special engineering strain are also not needed, most of restriction endonuclease sites are avoided, only the conventional steps of enzyme cutting, connection, transformation and the like are needed, the operation steps are simplified, the experiment difficulty is reduced, the technology is mature and reliable, the cost is low, the construction can be finished under the common experiment conditions, and the popularization is convenient.
Description
Technical field
The present invention relates to a kind of plant expression vector; Relate in particular to a kind of plant expression vector and construction process thereof that carries single or a plurality of foreign genes; The invention still further relates to the application of this plant expression vector in transgenic plant, belong to the structure field of plant expression vector.
Background technology
Plant transgene research at present mainly is to transform individual gene, and function singleness acts on limited.The common conversion of polygene has become an approach of head it off.
Making up the polygene plant conversion carrier is to realize that polygene transforms basis and the important step of plant simultaneously, and nearly all plant transgene research all is to begin from carrier construction.The structure conditionality restriction enzyme site of polygene expression vector restriction in the past, it is few that carrier carries gene dosage, and some building process transforms also needs dephosphorylation, phosphorylation, sticky end to mend the equality operation, complex steps, difficulty is higher.And some carrier has been introduced " gateway technology " or adopt " the playback restriction endonuclease combines the Cre recombinant technology ", though part addresses the above problem, also has problems such as the intermediate carrier that cost is higher, needs are special, special bacterial strain.
Summary of the invention
One of the object of the invention provides a kind of plant expression vector that carries single or a plurality of foreign genes;
Two of the object of the invention provides a kind of method that makes up said plant expression vector;
Three of the object of the invention is that described plant expression vector is applied to plant transgene.
Above-mentioned purpose of the present invention realizes through following technical scheme:
A kind of plant expression vector that carries single or a plurality of foreign genes comprises: plant conversion carrier p209, cloning vector p6435x and one or more goal gene to be transformed;
Wherein, p209 is a Ti-plasmids, is derived from carrier pBI121, between the HindIII of original pBI121 carrier and two restriction enzyme sites of Eco RI, introduces a fragment that has Xba I restriction enzyme site; Two restriction endonuclease sites of Bsp120 I and Xba I are arranged on the p209 carrier; P209 has foliage filter screening gene npt II.
Said cloning vector p6435x originates from prokaryotic expression carrier pET28a; This carrier has CaMV35S promotor and NOS terminator sequence; 2 Eam1105 I restriction enzyme sites are arranged between the two; Forming the T carrier through single endonuclease digestion links to each other with target gene PCR product; Constitute complete goal gene ORFs (ORF), carry restriction enzyme site Not I and Bsp120I, Spe I and Nhe I restriction enzyme site respectively in the both sides of ORFs successively.
Another object of the present invention provides a kind of said method of carrying the plant expression vector of single or a plurality of foreign genes that makes up, and may further comprise the steps:
(1) makes up plant conversion carrier p209 and cloning vector p6435x respectively;
(2) produce 23 ' overhangs that contain T with restriction endonuclease Eam1105 I enzyme cutting clone carrier p6435x; Form the T carrier; Link to each other with the 1st target gene PCR product that has the 3 ' overhang that contains A; Complete and forward is connected in the cloning vector with the 1st goal gene amplified fragments, is built into the ORFs that comprises CaMV35S promotor+goal gene+NOS terminator sequence; Utilize Not I and Nhe I pair to cut the cloning vector that carries destination gene expression ORFs (ORF), reclaim the 1st destination gene expression ORFs (ORF) fragment, subsequent use;
(3) utilize the two p209 that cut of Bsp120 I and Xba I, reclaim the p209 endonuclease bamhi; The p209 endonuclease bamhi is connected with the 1st destination gene expression ORFs (ORF) fragment that step (2) reclaims; The Not I at last original Bsp120I of p209 and Xba I site and destination gene expression ORFs two ends and Nhe I site disappear; And newly with the plant expression vector that becomes on the Bsp120I and the Spe I site that have; Cut and can have Not I and link to each other through enzyme with the 2nd with destination gene expression ORFs (ORF) fragment in Nhe I site; So constantly repeat, construct the plant expression vector that carries a plurality of goal gene.
Wherein, the construction process of described plant conversion carrier p209 comprises: between the Hind of pBI121 carrier III and two restriction enzyme sites of EcoRI, introduce a fragment that has Xba I restriction enzyme site, promptly get.
P209 is a Ti-plasmids, is derived from carrier pBI121.Self have foliage filter screening gene npt II, be used for Plant Transformation.Between the Hind of original pBI121 carrier III and two restriction enzyme sites of EcoR I, introduce a fragment that has Xba I restriction enzyme site.Two restriction endonuclease sites of Bsp120I and XbaI are arranged on the p209 carrier, behind restriction endonuclease Bsp120 I and Xba I double digestion, can produce two different sticky ends, after corresponding isocaudarner terminal fragment was connected on the cloning vector, the site can disappear.
The construction process of said cloning vector p6435x comprises: cloned the fragment that has CaMV 35S promoter, gfp green fluorescence protein gene and NOS terminator sequence and introduced new restriction enzyme site from carrier pCAMBIA1302 earlier; The fragment that all has Eam1105 I site with two ends replaces the gfp green fluorescence protein gene, forms a new segment, cuts, connects to link new segment through enzyme again to obtain cloning vector p6435x on the pET28a.
Can adopt such as modes such as heat shock methods and transform the Agrobacterium competence to the constructed plant expression vector of the present invention.Utilize agrobacterium-mediated transformation to transform plant,, obtain transfer-gen plant through kantlex (Km) resistance screening.
Plant expression vector of the present invention utilizes has identical sticky end after enzyme is cut between the isocaudarner, and after being connected to each other, the characteristics that original restriction enzyme site disappears make up the plant expression vector that carries a plurality of goal gene.Generally speaking, this carrier portability transforms plant less than the exogenous segment of 30kb.
The present invention through the A-T clone, directly is connected into cloning vector with the target gene PCR fragment, accomplishes the structure of the ORFs (ORF) of goal gene, identifies through simple PCR, promptly can be used for further plant conversion carrier and makes up.In the building process of plant expression vectors containing multiple genes of the present invention; Adopt Not I/Bsp120 I to use simultaneously with two different isocaudarner systems of Spe I/Xba I/Nhe I, utilize isocaudarner to connect the characteristic of the original restriction enzyme site disappearance in back, carrier or segmental two ends after enzyme is cut are different sticking section all the time; Solved the limited problem of carrier restriction enzyme site in the past; Simultaneously also avoid connecting certainly in the carrier connection procedure, need not to carry out again the phosphorylation processing of sticky end, also need not intermediate carrier and special engineering strain; Avoid most restriction endonuclease sites, only need carry out conventional steps such as enzyme is cut, connected, conversion.Simplified operation steps and reduced the experiment difficulty, technology maturation is reliable.With low cost, under common experiment condition, can accomplish, be convenient to promote, have bigger scientific research and actual application value.
The term definition that arrives involved in the present invention
Only if in addition definition, otherwise all technology used herein and scientific terminology all have with those skilled in the art and understand identical implication usually.Though in practice of the present invention or test, can use any method, device and the material similar or equivalent, describe preferred method, device and material now with person described herein.
Term " host cell " means the cell that comprises polynucleotide of the present invention, and no matter use which kind of method to insert, for example directly absorb to produce recombinant host cell, known other method in transduction, f pairing or the affiliated field.Exogenous polynucleotide for example can remain, and the nonconformity carrier of plasmid perhaps can be integrated in the host genome.
Term " polynucleotide " means deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Only if specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in said term, said analogue have the binding characteristic that is similar to reference nucleic acid and carry out metabolism with the mode of the Nucleotide that is similar to natural generation.Only if other specific limited, otherwise said term also means oligonucleotide analogs, it comprises PNA (PNAG3), used DNA analogue (thiophosphatephosphorothioate, phosphoramide acid esters or the like) in antisense technology.Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and clear and definite specified sequence.Specific; Can realize that through mixing base and/or the substituted sequence of Hypoxanthine deoxyriboside residue degenerate codon replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991) through producing one of them or selected more than one (or all) codon the 3rd; People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); People such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " foreign DNA " refers to that this dna sequence dna belongs to external source to this specific host cell, or if from identical primary source but this original series has been carried out modifying or transforms.
Term " conversion ": the heterology dna sequence dna is incorporated into host cell or organic method.
Term " expression ": endogenous gene or transgenic transcribing and/or translating in vegetable cell.
Term " encoding sequence ": the nucleotide sequence that is transcribed into RNA.
Term " plant expression vector ": one or more are used to realize the dna vector of Plant Transformation; These carriers often are called as binary vector in this area.Binary vector is to be usually used in agrobacterium-mediated conversion mostly together with the carrier with helper plasmid.Binary vector generally includes: T-DNA shifts needed cis acting sequence, handles so that the selectable marker that can in vegetable cell, express heterology dna sequence dna to be transcribed etc. through through engineering approaches.
Description of drawings
Fig. 1 cloning vector p6435x collection of illustrative plates.
Fig. 2 conversion carrier p209 collection of illustrative plates.
The synoptic diagram of Fig. 3 polygene plant conversion carrier of the present invention process.
The PCR of Fig. 4 carrier p2096871 identifies; M:maker; A: the specific amplified fragment of gene BtCryIIIA; B: the specific amplified fragment of gene BtCryIA.
Fig. 5 transformed plant (1-8) detects through PCR; The fragment with the identical size of positive control (CK+) occurs, but not transfer-gen plant (CK-) there is not this fragment; Tentatively prove transfer-gen plant.Proved that also plant expression vector of the present invention can be used for Plant Transformation.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Experiment material
1. plasmid: pUC18 (Dalian TaKaRa company), pBR322 (Dalian TaKaRa company), pUCm-T (worker is given birth in Shanghai), pET28a (Novagen company), pBI121 (Invitrogen company), pCAMBIA1302 (CambiaLabs)
2. bacterial strain: e. coli jm109 bacterial strain (Dalian TaKaRa company), DH10B bacterial strain (worker is given birth in Shanghai)
3. all ingredients and restriction endonuclease: give birth to the worker available from Dalian TaKaRa company, new England biolabs (NEB), Shanghai.
The structure of embodiment 1 cloning vector p6435x
1. carrier p1302x's is synthetic
Use the 26# primer:
F AGATCTCCACTTCTAAAATGGCTACAGGCATCGTGGTGT（SEQ?ID?No.1）
R GGTGACCCCACTTCTAAAATGGATTTTGCCTTCCTGTTTT（SEQ?ID?No.2）
With pUC18 is template, carries out pcr amplification, obtains a fragment about 500bp, and this fragment is connected into synthetic vectors p26 among the pUCm-T; Respectively p26 and pCAMBIA1302 are carried out double digestion with BglII and BstEII.Reclaim the small segment of p26 and the big fragment of pCAMBIA1302, with the two connection.Synthetic vectors p1302x.
2. carrier p1302x30's is synthetic
Use the 30# primer:
F aagcttagatctCTTGGATCAGATTGTCGT(SEQ?ID?No.3）
R ggatccAGAGTCCCCCGTGTTCT(SEQ?ID?No.4）
With pCAMBIA1302 is template, carries out pcr amplification, obtains the fragment of a 597bp, and this fragment is connected into synthetic vectors p30 among the pUCm-T.Respectively p30 and p1302x are carried out double digestion with HindIII and BamHI.Reclaim the small segment of p30 and the big fragment of p1302x, with the two connection, synthetic vectors p1302x30.
3. carrier p35x's is synthetic
Use the 4# primer:
F CTTGGATCAGATTGTCGT(SEQ?ID?No.5）
R TCCCGATCTAGTAACATA(SEQ?ID?No.6）
With pCAMBIA1302 is template, carries out pcr amplification, obtains the fragment of a 1622bp, and this fragment is connected into synthetic vectors p4 among the pUCm-T.Respectively p4 and p1302x30 are carried out double digestion with HindIII and BstEII.Reclaim the small segment of p1302x30 and the big fragment of p4, with the two connection, synthetic vectors p 35x.
4. carrier p2858's is synthetic
Use the 58# primer:
F ggatcCTACGGCTACACTAGAAGG(SEQ?ID?No.7）
R ggtcaccGAAACGCTGGTGAAAGT(SEQ?ID?No.8）
With pBR322 is template, carries out the pcr amplification, obtains the fragment of a 1122bp, and this fragment is connected into synthetic vectors p58 among the pUCm-T.Respectively p58 and pET28a are carried out double digestion with BamHI and BstEII.Reclaim the small segment of p58 and the big fragment of pET28a, with the two connection, synthetic vectors p2858.
5. carrier p285859's is synthetic
Use the 59# primer:
F gatatcgggcccagatctCTTCGGTTTCCGTGTT(SEQ?ID?No.9）
R CTGCAGTGATGCCTCCGTGTAA(SEQ?ID?No.10）
With pET28a is template, carries out pcr amplification, obtains a fragment about 300bp, and this fragment is connected into synthetic vectors p59 among the pUCm-T.Respectively p59 and p2858 are carried out double digestion with BglII and EcoRV.Reclaim the small segment of p59 and the big fragment of p2858, with the two connection, synthetic vectors p285859.
6. carrier p35N's is synthetic
Respectively p285859 and p35x are carried out double digestion with BglII and PstI.Reclaim the small segment of p35x and the big fragment of p285859, with the two connection, synthetic vectors p35N.
7. cloning vector p6435x's is synthetic
Use the 64# primer:
F GGTCACCgggccCTTCGGTTTCCGTGTT(SEQ?ID?No.11）
R gatatcGCTAGCactagTGATGCCTCCGTGTAA(SEQ?ID?No.12）
With pET28a is template, carries out pcr amplification, obtains a fragment about 300bp, and this fragment is connected into synthetic vectors p64 among the pUCm-T.Respectively p64 and p35N are carried out double digestion with BstEII and EcoRV.Reclaim the small segment of p64 and the big fragment of p35N, with the two connection, synthetic cloning vector p6435x.
The structure of embodiment 2 conversion carrier p209
1. carrier p88's is synthetic
Use the 88# primer:
F gaattctctagaCTTCGGTTTCCGTGTT(SEQ?ID?No.13）
R aagctTGATGCCTCCGTGTAA(SEQ?ID?No.14）
With pET28a is that template is carried out pcr amplification, obtains the fragment about a long 300bp.This fragment is connected into synthetic vectors p88 among the pUCm-T.
2. plant conversion carrier p209's is synthetic
Respectively p88 and pBI121 are carried out double digestion with HindIII and EcoRI.Reclaim the small segment of p88 and the big fragment of pBI121, with the two connection, synthetic plant conversion carrier p209.
1. the structure of plant conversion carrier p2096871
Adopting general PCR reaction system, is template with desinsection toxoprotein gene BtCryIA gene and BtCryIIIA gene respectively, makes PCR; The PCR response procedures is following: 94 ℃ of preparatory sex change 10min, and 94 ℃ of sex change 30s, 48-60 ℃ of annealing 40s, 72 ℃ are extended 1.5min, 20 circulations; 72 ℃ are extended 20min.Reaction finishes, and detects the amplified production fragment length that has that it's too late through the 1%TAE agarose gel electrophoresis, cuts glue and utilizes gel to reclaim test kit and reclaim suitable fragment, and primer is seen table 1.
Table 1 part primer
Extract the p6435x plasmid; Use restriction enzyme Eam1105I respectively, adopt 50 μ L endonuclease reaction systems, 37 ℃ of enzymes are cut 4h; Reaction finishes to add 6 * Loadingbuffer of 10 μ L; Check through the 1%TAE agarose gel electrophoresis, utilize gel to reclaim test kit and reclaim corresponding fragment, carry out next step research as the T carrier.
Amplified fragments is connected with the glue recovery fragment that the carrier enzyme is cut product, and reaction system is (10 μ L): the carrier enzyme is cut product 2 μ L, PCR product 4 μ L, ddH
2O 1 μ L, 50%PEG40001 μ L, 10 * T
4Lig buffer 1 μ L, T
4Lig 1 μ L.16 ℃ are spent the night.Through (utilizing Dalian Takara company to produce competence and prepare test kit preparation, the same specification sheets of method) in the heat shock method transformed into escherichia coli JM109 competence.Be coated on the LB solid medium of the Km that contains 50mg/l, cultivate 12h to strong point mono-clonal bacterium colony for 37 ℃.
35# primer according to CaMV35S termini of promoters sequences Design is a forward primer; With the segmental anti-primer to be measured detection primer that partners; Picking list bacterium colony is made bacterium colony PCR and is identified; If expanding fragment length conforms to goal gene length, think that then complete the and forward of amplified fragments is connected in the cloning vector, has been built into the ORFs (ORF) that comprises CaMV 35S promoter+goal gene+NOS terminator sequence.Qualified on inspection bacterium colony shakes bacterium, and bacterium liquid is served the Hai Shenggong order-checking, does further check.Through order-checking, the carrier that sequence does not become supplies further experiment.Cloning vector is called p6468 after carrying BtCryIA in the experiment, and cloning vector is called p6471 after carrying BtCryIIIA.
When structure contains the polygene conversion carrier of BtCryIA and BtCryIIIA, at first utilize Bsp120I and XbaI double digestion p209, utilize the two p6468 that cut of NotI and NheI.After reclaiming the ORF fragment of p209 endonuclease bamhi and goal gene BtCryIA; With the two connection; The NotI at the ORF two ends of original Bsp120I and XbaI site and goal gene BtCryIA and NheI site disappear on the p 209, and the Bsp120I and the SpeI site that have on the new synthetic plant conversion carrier p20968.
Use Bsp120I and SpeI double digestion p20968 again, utilize the two p 6471 that cut of NotI and NheI.After reclaiming the ORF fragment of p20968 endonuclease bamhi and goal gene BtCryIIIA; With the two connection; The NotI and the NheI site at the ORF two ends of the last original Bsp120I of p20968 and XbaI site and goal gene BtCryIIIA disappear once more, and the Bsp120I and the SpeI site that have on the new synthetic plant conversion carrier p2096871.P2096871 is exactly the plant conversion carrier of the needed BtCryIA of carrying and two desinsection toxoprotein genes of BtCryIIIA.
2. the evaluation of plant conversion carrier p2096871
Utilize round pcr to identify p2096871.With carrier to be detected is template, and the special primer that is adopted when using amplification BtCryIA and BtCryIIIA is respectively made PCR, and the PCR response procedures is following: 94 ℃ of preparatory sex change 10min; 94 ℃ of sex change 30s; 48-60 ℃ of annealing 40s, 72 ℃ are extended 1.5min, 20 circulations; 72 ℃ are extended 5min.Reaction finishes, and detects the amplified production fragment length that has that it's too late through the 1%TAE agarose gel electrophoresis.If expanding fragment length conforms to the goal gene theoretical length, can judge then to be connected into BtCryIA and BtCryIIIA gene in the carrier that carrier synthesizes successfully (Fig. 4).
3.2 the expression of individual foreign gene in transgene tobacco
Utilize enzyme linked immunological (ELISA) test kit to detect 2 expression of foreign gene in transgene tobacco.Transform BtCryIA and BtCryIIIA gene, transgene tobacco has two kinds of toxalbumin and expresses.
Detection method:
Specimen preparation: selecting 5 strain transgene tobaccos to make sample, is contrast with the non-transgenic tobacco.Get the fresh transgene tobacco blade of 100mg, add 1ml PBST and extract damping fluid, fully grind to form homogenate on ice, 4 ℃ of centrifugal 5min of 12000rpm are transferred in the new centrifugal bamboo of Eppendorf supernatant subsequent use.
The mensuration of total protein content:
(1) drafting of the typical curve bovine serum albumin solution of getting different concns adds water and supplies 1ml.Accurately draw institute and join each bamboo solution 0.1ml, add 5ml Xylene Brilliant Cyanine G G-250 reagent, mix repeatedly for several times, place 2min, measure the light absorption value under the 595nm on the spectrophotometer, the drawing standard curve.
(2) mensuration of protein concn is drawn sample extracting solution 0.1ml in the sample extracting solution; Add 5ml Xylene Brilliant Cyanine G G-250 reagent (establishing 2 repetitions), thorough mixing is placed behind the 2min colorimetric under 595nm; Write down absorbance, and check in protein contnt through typical curve.
The ELISA of Btl toxalbumin (BtCryIA) detects:
(1) prepares
1) with You Junshui 10g PBST is diluted to 1 * PBST.
2) ready reaction box will seal.
3) with the positive control that provides in the 2mL 1XPBST solubilising reagent box.
4) packing positive control, every pipe 120g1 places-20 ℃ of preservations.
5) design experiment sample arrangement figure remembers sequence number A-H, 1-120
(2) ELlsa reaction
1) every hole adds 100 μ l enzyme conjugate in respective aperture.
2) press schema, add positive control and ready sample in respective aperture, every hole 100 μ L establish 2 repetitions.
3) enzyme plate is transferred in the box that is covered with moist filter paper, at room temperature placed 2h or 4 ℃ of refrigerator overnight.
4) after insulation finishes, wash plate.Get rid of the sample on the enzyme plate, fill it up with each hole, place 3min (in box) and get rid of liquid in the hole then rapidly, do, notice that does not pollute each other in each hole, especially be not thrown in the blank at the filter paper arsis with 1 * PBST.Repeat 6-7 time (at room temperature).
5) add substrate material.Every hole adds 100 μ l TMB substrate materials, and enzyme plate is placed the box that is covered with moist filter paper, at room temperature leaves standstill, and waits for colour-change.
6) detect.About 5-15min produces colour-change, occurs blue positive result in the hole, if colourless or light blue then negative result.The 450nm place measures light absorption value (OD450) on ELIASA
7) do typical curve.Find sample BtCryIA concentration, calculate every gram leaf expression amount.The ELISA of Bt3 toxalbumin (BtCryIIIA) detects
(1) prepares
1) with 1L zero(ppm) water one bag of 20 * PBST dcq buffer liquid is diluted to 1 * PBST dcq buffer liquid.
2) join 100mLMEB sample extraction damping fluid
Nonfat?dried?milk 0.4g
Tween?20 0.5g
Add 1 * PBST to 100mL, slowly be stirred to dissolving fully under the room temperature.
3) join ECM 25mL
Nonfat?dried?milk 0.1g
Add 1 * PBST to 25mL, slowly be stirred to dissolving fully under the room temperature.
4) join the PNP damping fluid
Each PNP tablet preparation 5ml PNP solution, concentration 1mg/mL., hatch and finish preceding 15min preparation.
(2) El.lsa reaction
1) distributes sample according to last master drawing, add 10 μ l testing samples, contrast and standard model in suitable hole, establish 2 repetitions.
2) hatch plate plate has been put into moistening box, incubated at room 1h.
3) preparing enzyme conjugate hatches and finishes preceding youngster minute, preparation enzyme conjugate, every hole 100 μ l, 1 * ECM buffer.According to the hole count of test usefulness and the dilution method consumption of I I1X ECM buffer not on the bottle, calculate the consumption of enzyme conjugate.Use new, sterile pipette, prevent to pollute.
4) wash plate and hatch completion for the first time, wash plate, 1 * PBST is filled it up with in every hole, and liquid in the hole is toppled in upset fast, does not pollute each other between the hole, repeats 4-8 time.1 * PBST is filled it up with in every hole again, the static 3min of room temperature in moistening box.From box, take out plate, liquid in the emptying aperture that inclines fast raps on the folding again filter paper and removes raffinate in the hole.
5) add the every hole of enzyme conjugate and add 100 μ l enzyme conjugate.
6) hatch plate plate has been put into moistening box, incubated at room 1h.
7) prepare PNP solution
Each PNP tablet preparation 5ml PNP solution, concentration 1mg/mL, the band in enough 58 holes is used.Hatch and finish preceding 15min, each tablet for use gets 1 * PNPbuffer of 5ml room temperature, PNP tablet is added among the buffer mixing.Annotate: do not contact tablet or PNP solution is exposed under the high light, light or pollution can cause the negative control background to deepen.
8) wash plate and wash plate 4-8 time repeatedly with 1 * PBST.Inspection window has or not bubble, washes plate repeatedly, and clappers are to remove bubble.
9) add the every hole of PNP solution and add 100 μ l PNP solution.
10) hatching plate has put into moistening box with plate and has hatched 1h.
11) detected result detects at 405nm with ELIASA, and the hole that has color to take place is positive, does not have the negative of obvious colour-change.
The toxalbumin detected result
Detected result is seen table 2.In 5 transgenic tobacco plants, all there are two kinds of toxalbumin to produce, nontoxic protein is synthetic in the non-transgenic tobacco.Explain that the plant conversion carrier p2096871 that has BtCryIA and BtCryIIIA that utilizes this carrier system to make up can carry foreign gene and transform plant, and can in transfer-gen plant, express.
Toxalbumin accounts for total protein ratio in table 2 transfer-gen plant and the contrast
Claims (10)
1. carry the plant expression vector of single or a plurality of foreign genes, it is characterized in that, comprising: plant conversion carrier p209, cloning vector p6435x and one or more goal gene to be transformed.
2. according to the described plant expression vector of claim 1; It is characterized in that: said plant conversion carrier p209 is a Ti-plasmids; Be derived from carrier pBI121, between the Hind of original pBI121 carrier III and two restriction enzyme sites of EcoR I, introduce a fragment that has Xba I restriction enzyme site; Two restriction endonuclease sites of Bsp120 I and Xba I are arranged on p 209 carriers; P209 has foliage filter screening gene npt II;
Said cloning vector p6435x originates from prokaryotic expression carrier pET28a; The p6435x carrier has CaMV 35S promoter and NOS terminator sequence, and 2 Eam1105 I restriction enzyme sites are arranged between the two; The p6435x carrier carries Not I and Bsp120 I, Spe I and Nhe I restriction enzyme site.
3. according to claim 1 or 2 described plant expression vectors, it is characterized in that: described goal gene is that desinsection toxoprotein gene BtCryIA is or/and desinsection toxoprotein gene BtCryIIIA.
4. one kind makes up claim 1 or 2 said methods of carrying the plant expression vector of single or a plurality of foreign genes, may further comprise the steps:
(1) makes up plant conversion carrier p209 and cloning vector p6435x respectively;
(2) produce 23 ' overhangs that contain T with restriction endonuclease Eam1105 I enzyme cutting clone carrier p6435x; Form the T carrier; Link to each other with the 1st target gene PCR product that has the 3 ' overhang that contains A; Complete and forward is connected in the cloning vector with the 1st goal gene amplified fragments, is built into the ORFs that comprises CaMV35S promotor+goal gene+NOS terminator sequence; Utilize Not I and Nhe I pair to cut the cloning vector that carries the destination gene expression ORFs, reclaim the 1st destination gene expression ORFs fragment, subsequent use;
(3) utilize the two p209 that cut of Bsp120 I and Xba I, reclaim the p209 endonuclease bamhi; The p209 endonuclease bamhi is connected with the 1st the destination gene expression ORFs fragment that step (2) reclaims; The Bsp120 I that has on the new synthetic plant expression vector cuts through enzyme with Spe I site and has Not I and link to each other with the destination gene expression ORFs fragment in NheI site with the 2nd; So constantly repeat, construct the plant expression vector that carries a plurality of goal gene.
5. according to the described method of claim 4, it is characterized in that the construction process of described plant conversion carrier p209 comprises: between the Hind of pBI121 carrier III and two restriction enzyme sites of EcoR I, introduce a fragment that has Xba I restriction enzyme site, promptly get.
6. according to the described method of claim 4; It is characterized in that the construction process of said cloning vector p6435x comprises: cloned the fragment that has CaMV 35S promoter, gfp green fluorescence protein gene and NOS terminator sequence and introduced new restriction enzyme site from carrier pCAMBIA1302 earlier; The fragment that all has Eam1105 I site with two ends replaces the gfp green fluorescence protein gene, forms a new segment, links new segment and obtains cloning vector p6435x on the pET28a.
7. the host cell that contains claim 1 or 2 described plant expression vectors.
8. according to the described host cell of claim 7, it is characterized in that: described host cell is a vegetable cell.
9. claim 1 or 2 described plant expression vectors are transformed into the application in vegetable cell or the tissue with foreign gene.
10. according to the described application of claim 9, it is characterized in that: described foreign gene comprises desinsection toxoprotein gene BtCryIA or/and desinsection toxoprotein gene BtCryIIIA.
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| WO2005047512A2 (en) * | 2003-11-12 | 2005-05-26 | Shering Corporation | Plasmid system for multigene expression |
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| CN103114102A (en) * | 2013-01-29 | 2013-05-22 | 华南农业大学 | Polygene vector assembly method and application |
| CN103114102B (en) * | 2013-01-29 | 2014-12-03 | 华南农业大学 | Polygene vector assembly method and application |
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